| 1. |
Melckebeke HV,
Vreuls C,
Gans P,
Filée P,
Llabres G,
Joris B,
Simorre JP,
( 2003 ) Solution structural study of BlaI: implications for the repression of genes involved in beta-lactam antibiotic resistance. PMID : 14568532 : DOI : 10.1016/j.jmb.2003.09.005 Abstract >>
beta-Lactamase and penicillin-binding protein PBP2' mediate staphylococcal resistance to beta-lactam antibiotics, which are otherwise highly clinically effective. Two repressors (BlaI and MecI) regulate expression of these inducible proteins. Here, we present the first solution structure of the 82 amino acid residue DNA-binding domain of Bacillus licheniformis BlaI which is very similar in primary sequence to the medically significant Staphyloccocal BlaI and MecI proteins. This structure is composed of a compact core of three alpha-helices and a three-stranded beta-sheet typical of the winged helix protein (WHP) family. The protein/DNA complex was studied by NMR chemical shift comparison between the free and complexed forms of BlaI. Residues involved in DNA interaction were identified and a WHP canonical model of interaction with the operators is proposed. In this model, specific contacts occur between the base-pairs of the TACA motif and conserved amino acid residues of the repressor helix H3. These results help toward understanding the repression and induction mechanism of the genes coding for beta-lactamase and PBP2'.
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2. |
Kakudo S,
Kikuchi N,
Kitadokoro K,
Fujiwara T,
Nakamura E,
Okamoto H,
Shin M,
Tamaki M,
Teraoka H,
Tsuzuki H,
( 1992 ) Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580. PMID : 1429718 : Abstract >>
A glutamic acid-specific protease has been purified to homogeneity from Bacillus licheniformis ATCC 14580 utilizing Phe-Leu-D-Glu-OMe-Sepharose affinity chromatography and crystallized. The molecular weight of the protease was estimated to be approximately 25,000 by SDS-polyacrylamide gel electrophoresis. This protease, which we propose to call BLase (glutamic acid-specific protease from B. licheniformis ATCC 14580), was characterized enzymatically. Using human parathyroid hormone (13-34) and p-nitroanilides of peptidyl glutamic acid and aspartic acid, we found a marked difference between BLase and V8 protease, EC 3.4.21.9, although both proteases showed higher reactivity for glutamyl bonds than for aspartyl bonds. Diisopropyl fluorophosphate and benzyloxycarbonyl Leu-Glu chloromethyl ketone completely inhibited BLase, whereas EDTA reversibly inactivated the enzyme. The findings clearly indicate that BLase can be classified as a serine protease. To elucidate the complete primary structure and precursor of BLase, its gene was cloned from the genomic DNA of B. licheniformis ATCC 14580, and the nucleotide sequence was determined. Taking the amino-terminal amino acid sequence of the purified BLase into consideration, the clones encode a mature peptide of 222 amino acids, which follows a prepropeptide of 94 residues. The recombinant BLase was expressed in Bacillus subtilis and purified to homogeneity. Its key physical and chemical characteristics were the same as those of the wild-type enzyme. BLase was confirmed to be a protease specific for glutamic acid, and the primary structure deduced from the cDNA sequence was found to be identical with that of a glutamic acid-specific endopeptidase isolated from Alcalase (Svendsen, I., and Breddam, K. (1992) Eur. J. Biochem. 204, 165-171), being different from V8 protease and the Glu-specific protease of Streptomyces griseus which consist of 268 and 188 amino acids, respectively.
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3. |
Tschauder S,
Driessen AJ,
Freudl R,
( 1992 ) Cloning and molecular characterization of the secY genes from Bacillus licheniformis and Staphylococcus carnosus: comparative analysis of nine members of the SecY family. PMID : 1435726 : DOI : 10.1007/bf00286192 Abstract >>
SecY is a central component of the export machinery that mediates the translocation of secretory proteins across the plasma membrane of Escherichia coli. We have cloned and sequenced the secY genes from Bacillus licheniformis and Staphylococcus carnosus. The deduced amino acid sequences are highly homologous to those of other known SecY polypeptides, all having the potential to form 10 transmembrane segments. Comparative analysis of 9 SecY polypeptides, derived from different bacteria, revealed that 14 amino acid positions (2.7%) are identical in all SecY proteins and 89 (16.9%) show conservative changes. Clusters of conserved amino acid residues were found in 4 of the 10 transmembrane segments and 2 of the 6 cytoplasmic domains. It is suggested that the conserved regions might be involved in the translocation activity of SecY or might be required for the correct interaction of SecY with other components of the secretion apparatus.
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4. |
Craynest M,
Jørgensen S,
Sarvas M,
Kontinen VP,
( 2003 ) Enhanced secretion of heterologous cyclodextrin glycosyltransferase by a mutant of Bacillus licheniformis defective in the D-alanylation of teichoic acids. PMID : 12803561 : Abstract >>
To examine whether inactivation of the dlt operon and increased charge density of the wall enhances secretion of heterologous proteins in industrial strains of Bacillus licheniformis. The dltA gene of B. licheniformis was cloned, sequenced and mutated by inserting a chloramphenicol acetyl transferase (cat) gene cassette. The mutation facilitated growth in the late exponential growth phase, increased endogenous autolysis and decreased resistance to a cationic peptide, polylysine. It was observed that dltA mutation increased the production of cyclodextrin glycosyltransferase (CGTase) by 1.5- to sevenfold depending on the growth phase, but decreased the production of penicillinase by twofold. The results suggest that the d-alanylation of teichoic acids is an element that can be used to improve the production of some secretory proteins in industrial applications based on this important industrial microorganism.
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5. |
Planas A,
Juncosa M,
Lloberas J,
Querol E,
( 1992 ) Essential catalytic role of Glu134 in endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase from B. licheniformis as determined by site-directed mutagenesis. PMID : 1354172 : DOI : 10.1016/0014-5793(92)81262-k Abstract >>
Site-directed mutagenesis experiments designed to identify the active site of Bacillus licheniformis endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase (beta-glucanase) have been performed. Putative catalytic residues were chosen on the basis of sequence similarity analysis to viral and eukaryotic lysozymes. Four mutant enzymes were expressed and purified from recombinant E. coli and their kinetics analysed with barley beta-glucan. Replacement of Glu134 by Gln produced a mutant (E134Q) that retains less than 0.3% of the wild-type activity. The other mutants, D133N, E160Q and D179N, are active but show different kinetic parameters relative to wild-type indicative of their participation in substrate binding and transition-state complex stabilization. Glu134 is essential for activity; it is comprised in a region of high sequence similarity to the active site of T4 lysozyme and matches the position of the general acid catalyst. These results strongly support a lysozyme-like mechanism for this family of Bacillus beta-glucan hydrolases with Glu134 being the essential acid catalyst.
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6. |
Kim IC,
Cha JH,
Kim JR,
Jang SY,
Seo BC,
Cheong TK,
Lee DS,
Choi YD,
Park KH,
( 1992 ) Catalytic properties of the cloned amylase from Bacillus licheniformis. PMID : 1385394 : Abstract >>
A gene encoding a new amylolytic enzyme of Bacillus licheniformis (BLMA) has been cloned, and we characterized the enzyme expressed in Escherichia coli. The genomic DNA of B. licheniformis was double-digested with EcoRI and BamHI and ligated the pBR322. The transformed E. coli was selected by its amylolytic activity, which carries the recombinant plasmid pIJ322 containing a 3.5-kilobase fragment of B. licheniformis DNA. The purified enzyme encoded by pIJ322 was capable of hydrolyzing pullulan and cyclodextrin as well as starch. It was active over a pH range of 6-8 and its optimum temperature was 50 degrees C. The molecular weight of the enzyme was 64,000, and the isoelectric point was 5.4. It degraded soluble starch by cleaving maltose units preferentially but did not attack alpha-1,6-linkage. The enzyme also hydrolyzed pullulan to panose units exclusively. In the presence of glucose, however, it transferred the panosyl moiety to glucose with the formation of alpha-1,6-linkage. The specificity of transferring activity is evident from the result of the maltosyl-transferring reaction which produces isopanose from maltotriose and glucose. The molecular structure of the enzyme deduced from the nucleotide sequence of the clone maintains limited similarity in the conserved regions to the other amylolytic enzymes.
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7. |
Svendsen I,
Breddam K,
( 1992 ) Isolation and amino acid sequence of a glutamic acid specific endopeptidase from Bacillus licheniformis. PMID : 1346764 : DOI : 10.1111/j.1432-1033.1992.tb16619.x Abstract >>
An endopeptidase cleaving specifically at the carboxyl side of acidic amino acid residues, preferentially at glutamic acid, has been isolated from a commercial extract obtained by fermentation with Bacillus licheniformis. Using ion-exchange chromatography and affinity chromatography on bacitracin-Sepharose, it was possible, from 100 ml commercial extract, to isolate 100 mg homogeneous enzyme in a yield of 50%. It is the first description of a large-scale isolation of a Glu/Asp-specific enzyme. The preparation was essentially free of contaminating activities. The isolated enzyme consists of one peptide chain of 222 amino acid residues and has a calculated molecular mass of 23,589 Da. The determined amino acid sequence shows similarity to the Glu/Asp-specific enzymes previously isolated from Staphylococcus aureus V8, Actinomyces sp. and Streptomyces thermovulgaris. The substrate preference of the enzyme has been investigated. Although non-specific cleavages were observed after prolonged hydrolysis at high enzyme concentrations the enzyme appears to be essentially specific for Glu-Xaa and Asp-Xaa, with strong preference for the former. The isolated enzyme exhibits a bell-shaped pH/activity profile with an optimum at pH 7.5-8.0. The activity is adversely affected by high ionic strength and beneficially affected by the inclusion of calcium ions in the assay medium. The enzyme is completely inhibited by diisopropylfluorophosphate, suggesting that it is a serine endopeptidase. It is partially inhibited by EDTA.
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8. |
Declerck N,
Machius M,
Joyet P,
Wiegand G,
Huber R,
Gaillardin C,
( 2003 ) Hyperthermostabilization of Bacillus licheniformis alpha-amylase and modulation of its stability over a 50 degrees C temperature range. PMID : 12736372 : Abstract >>
Bacillus licheniformis alpha-amylase (BLA) is a highly thermostable starch-degrading enzyme that has been extensively studied in both academic and industrial laboratories. For over a decade, we have investigated BLA thermal properties and identified amino acid substitutions that significantly increase or decrease the thermostability. This paper describes the cumulative effect of some of the most beneficial point mutations identified in BLA. Remarkably, the Q264S-N265Y double mutation led to a rather limited gain in stability but significantly improved the amylolytic function. The most hyperthermostable variants combined seven amino acid substitutions and inactivated over 100 times more slowly and at temperatures up to 23 degrees C higher than the wild-type enzyme. In addition, two highly destabilizing mutations were introduced in the metal binding site and resulted in a decrease of 25 degrees C in the half-inactivation temperature of the double mutant enzyme compared with wild-type. These mutational effects were analysed by protein modelling based on the recently determined crystal structure of a hyperthermostable BLA variant. Our engineering work on BLA shows that the thermostability of an already naturally highly thermostable enzyme can be substantially improved and modulated over a temperature range of 50 degrees C through a few point mutations.
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9. |
Rivera MH,
López-Munguía A,
Soberón X,
Saab-Rincón G,
( 2003 ) Alpha-amylase from Bacillus licheniformis mutants near to the catalytic site: effects on hydrolytic and transglycosylation activity. PMID : 12915728 : Abstract >>
The alpha-amylase from Bacillus licheniformis is the most widely used enzyme in the starch industry owing to its hyperthermostability, converting starch to medium-sized oligosaccharides. Based on sequence alignment of homologous amylases, we found a semi-conserved sequence pattern near the active site between transglycosidic and hydrolytic amylases, which suggested that hydrophobicity may play a role in modifying the transglycosylation/hydrolysis ratio. Based on this analysis, we replaced residue Val286 by Phe and Tyr in Bacillus licheniformis alpha-amylase. Surprisingly, the two resultant mutant enzymes, Val286Phe and Val286Tyr, showed two different behaviors. Val286Tyr mutant was 5-fold more active for hydrolysis of starch than the wild-type enzyme. In contrast, the Val286Phe mutant, differing only by one hydroxyl group, was 3-fold less hydrolytic than the wild-type enzyme and apparently had a higher transglycosylation/hydrolysis ratio. These results are discussed in terms of affinity of subsites, hydrophobicity and electrostatic environment in the active site. The engineered enzyme reported here may represent an attractive alternative for the starch transformation industries as it affords direct and substantial material savings and requires no process modifications.
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10. |
Machius M,
Declerck N,
Huber R,
Wiegand G,
( 2003 ) Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface. PMID : 12540849 : DOI : 10.1074/jbc.M212618200 Abstract >>
It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis alpha-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a beta-sheet by introducing a residue with high beta-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.
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11. |
Tye AJ,
Siu FK,
Leung TY,
Lim BL,
( 2002 ) Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis. PMID : 12111145 : DOI : 10.1007/s00253-002-1033-5 Abstract >>
A novel phytase gene (phyL) was cloned from Bacillus licheniformis by multiple steps of degenerate and inverse PCR. The coding region of the phyL gene was 1,146 bp in size and a promoter region of approximately 300 bp was identified at the upstream sequence. This gene, together with a phytase gene (168phyA) identified in the B. subtilis strain 168 genome by a homology search, was cloned and over-expressed in B. subtilis using a phi105MU331 prophage vector system. Up to 35 units of phytase/ml were secreted into the culture media; and mature enzymes of around 44-47 kDa were purified for characterization. Both phytases exhibited broad temperature and pH optima and showed high thermostability. Of the two, the phytase encoded by phyL exhibited higher thermostability, even at a lower calcium concentration, as it was able to recover 80% of its original activity after denaturation at 95 degrees C for 10 min. With their neutral pH optima and good temperature stabilities, these Bacillus phytases are good candidates for animal feed applications and transgenic studies.
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12. |
Lapidus A,
Galleron N,
Andersen JT,
Jørgensen PL,
Ehrlich SD,
Sorokin A,
( 2002 ) Co-linear scaffold of the Bacillus licheniformis and Bacillus subtilis genomes and its use to compare their competence genes. PMID : 12007649 : DOI : 10.1111/j.1574-6968.2002.tb11104.x DOI : 10.1111/j.1574-6968.2002.tb11104.x Abstract >>
We have established the co-linear regions of Bacillus licheniformis, an industrially important bacterium, and Bacillus subtilis, a model bacterium. In the co-linear regions, revealed by PCR, gene content and order are presumed to be conserved. These regions constitute approximately 60% of the compared chromosomes. Sequencing of the competence genes of B. licheniformis allowed us to validate the approach, and to demonstrate how it can be used for the comparative analysis of complex genetic systems. A new insertion sequence, designated IS3Bli1, was discovered in the competence region of the analyzed B. licheniformis strain.
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13. |
Lapidus A,
Galleron N,
Andersen JT,
Jørgensen PL,
Ehrlich SD,
Sorokin A,
( 2002 ) Co-linear scaffold of the Bacillus licheniformis and Bacillus subtilis genomes and its use to compare their competence genes. PMID : 12007649 : DOI : 10.1111/j.1574-6968.2002.tb11104.x DOI : 10.1111/j.1574-6968.2002.tb11104.x Abstract >>
We have established the co-linear regions of Bacillus licheniformis, an industrially important bacterium, and Bacillus subtilis, a model bacterium. In the co-linear regions, revealed by PCR, gene content and order are presumed to be conserved. These regions constitute approximately 60% of the compared chromosomes. Sequencing of the competence genes of B. licheniformis allowed us to validate the approach, and to demonstrate how it can be used for the comparative analysis of complex genetic systems. A new insertion sequence, designated IS3Bli1, was discovered in the competence region of the analyzed B. licheniformis strain.
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14. |
Read TD,
Salzberg SL,
Pop M,
Shumway M,
Umayam L,
Jiang L,
Holtzapple E,
Busch JD,
Smith KL,
Schupp JM,
Solomon D,
Keim P,
Fraser CM,
( 2002 ) Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis. PMID : 12004073 : DOI : 10.1126/science.1071837 Abstract >>
Comparison of the whole-genome sequence of Bacillus anthracis isolated from a victim of a recent bioterrorist anthrax attack with a reference reveals 60 new markers that include single nucleotide polymorphisms (SNPs), inserted or deleted sequences, and tandem repeats. Genome comparison detected four high-quality SNPs between the two sequenced B. anthracis chromosomes and seven differences among different preparations of the reference genome. These markers have been tested on a collection of anthrax isolates and were found to divide these samples into distinct families. These results demonstrate that genome-based analysis of microbial pathogens will provide a powerful new tool for investigation of infectious disease outbreaks.
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15. |
Palmisano MM,
Nakamura LK,
Duncan KE,
Istock CA,
Cohan FM,
( 2001 ) Bacillus sonorensis sp. nov., a close relative of Bacillus licheniformis, isolated from soil in the Sonoran Desert, Arizona. PMID : 11594594 : DOI : 10.1099/00207713-51-5-1671 Abstract >>
Eight Bacillus strains isolated from Sonoran Desert soil were shown to belong to a previously unidentified species, for which the name Bacillus sonorensis sp. nov. is proposed. The type strain is strain L87-10T (= NRRL B-23154T). On the basis of phenotypic and genetic data, B. sonorensis is most closely related to Bacillus licheniformis. B. sonorensis can be distinguished from B. licheniformis by salt tolerance, pigmentation, multilocus enzyme electrophoresis, reassociation of genomic DNA and sequence differences in protein-coding genes and 16S rRNA.
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16. |
Fonzé E,
Vanhove M,
Dive G,
Sauvage E,
Frère JM,
Charlier P,
( 2002 ) Crystal structures of the Bacillus licheniformis BS3 class A beta-lactamase and of the acyl-enzyme adduct formed with cefoxitin. PMID : 11827533 : DOI : 10.1021/bi015789k Abstract >>
The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alpha-methoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 A resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges.
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17. |
Nthangeni MB,
Patterton H,
van Tonder A,
Vergeer WP,
Litthauer D,
( 2001 ) Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases. PMID : 11339956 : Abstract >>
The gene coding for an extracellular lipase of Bacillus licheniformis was cloned using PCR techniques. The sequence corresponding to the mature lipase was subcloned into the pET 20b(+) expression vector to construct a recombinant lipase protein containing 6 histidine residues at the C-terminal. High-level expression of the lipase by Escherichia coli cells harbouring the lipase gene-containing expression vector was observed upon induction with IPTG at 30 degrees C. A one step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified enzyme was 130 units/mg with p-nitrophenyl-palmitate as substrate. The enzyme showed maximum activity at pH 10-11.5 and was remarkably stable at alkaline pH values up to 12. The enzyme was active toward p-nitrophenyl esters of short to long chains fatty acids but with a marked preference for esters with C(6) and C(8) acyl groups. The amino acid sequence of the lipase shows striking similarities to lipases from Bacillus subtilis and Bacillus pumilus. Based on the amino acid identity and biochemical characteristics, we propose that Bacillus lipases be classified into two distinct subfamilies of their own.
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18. |
Chun J,
Bae KS,
( 2000 ) Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences. PMID : 11204764 : DOI : 10.1023/a:1026555830014 Abstract >>
Partial gyrA sequences were determined for twelve strains belonging to Bacillus amyloliquefaciens, B. atrophaeus, B. licheniformis, B. mojavensis, B. subtilis subsp. subtilis, B. subtilis subsp. spizizenii and B. vallismortis. The average nucleotide and translated amino acid similarities for the seven type strains were 83.7 and 95.1%, respectively, whereas the corresponding value for the 16S rRNA sequences was 99.1%. All of the type strains were sharply separated; the closest relationship was found between B. atrophaeus and B. mojavensis which shared a nucleotide similarity of 95.8%. Phylogenetic trees were inferred from gyrA nucleotide sequences using the neighbor-joining, Fitch-Margoliash and maximum parsimony algorithms. The test strains were divided into four groups, which generally reflected results previously reported in restriction digest and DNA-DNA hybridization studies. It is concluded from the comparative sequence analysis that the gyrA sequences provide a firm framework for the rapid and accurate classification and identification of Bacillus subtilis and related taxa.
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19. |
Evans KL,
Crowder J,
Miller ES,
( 2000 ) Subtilisins of Bacillus spp. hydrolyze keratin and allow growth on feathers. PMID : 11109488 : DOI : 10.1139/w00-085 Abstract >>
Keratinase is a serine protease produced by Bacillus licheniformis PWD-1 that effectively degrades keratin and confers the ability to grow on feathers to a protease-deficient B. subtilis strain. Studies presented herein demonstrate that B. licheniformis Carlsberg strain NCIMB 6816, which produces the well-characterized serine protease subtilisin Carlsberg, also degrades and grows on feathers. The PWD-1 and Carlsberg strains showed a similar time-course of enzyme production, and the purified serine proteases have similar enzymatic properties on insoluble azokeratin and soluble FITC-casein. Kinetic analysis of both enzymes demonstrated that they have high specificity for aromatic and hydrophobic amino acids in the P1 substrate position, although keratinase discriminates more than subtilisin Carlsberg against charged residues at this site. Nucleotide sequence analysis of the serine protease genes from B. licheniformis strains PWD-1, Carlsberg NCIMB 6816, ATCC 12759, and NCIMB 10689 showed that the kerA-encoded protease of PWD-1 differs from the others only by having V222, rather than A222, near the active site serine S220. Further, high-level expression of subE-encoded subtilisin from B. subtilis (78% similar to subtilisin Carlsberg) also confers growth on feathers on a protease-deficient B. subtilis strain. While strain PWD-1 and the kerA protease efficiently degrade keratin, keratin hydrolysis and growth on feathers is a property that can be conferred by appropriate expression of the major subtilisins, including the industrially produced enzymes.
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20. |
Svendsen A,
Borchert TV,
Bisgaard-Frantzen H,
Turkenburg JP,
Lawson DM,
Brzozowski AM,
( 2000 ) Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes. PMID : 10924103 : DOI : 10.1021/bi0000317 Abstract >>
Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.
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21. |
Petri R,
( 2000 ) The relationship of nitrate reducing bacteria on the basis of narH gene sequences and comparison of narH and 16S rDNA based phylogeny. PMID : 10879978 : DOI : 10.1016/S0723-2020(00)80045-4 Abstract >>
On the basis of available nitrate reductase gene sequences primer pairs were designed to specifically amplify gene stretches of the beta-subunit of the membrane-bound nitrate reductase (narH). Additional sequences of this gene were amplified and sequenced from pure cultures of reference species and new isolates. The distribution and phylogeny of this gene in denitrifying and nitrate-reducing bacteria was analysed. Comparison of phylogenetic trees based on 16S rDNA sequences with those based on narH sequences revealed highly similar relationships of both genes from most of the bacteria analysed. Since highly conserved functional cysteine clusters within bacterial and archaeal narH sequences support a linear evolution from one common progenitor a long evolutionary history of the respiratory membrane-bound nitrate reductase can be inferred from our phylogenetic data.
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22. |
Sievers J,
Errington J,
( 2000 ) Analysis of the essential cell division gene ftsL of Bacillus subtilis by mutagenesis and heterologous complementation. PMID : 10986263 : PMC : PMC111003 DOI : 10.1128/jb.182.19.5572-5579.2000 Abstract >>
The ftsL gene is required for the initiation of cell division in a broad range of bacteria. Bacillus subtilis ftsL encodes a 13-kDa protein with a membrane-spanning domain near its N terminus. The external C-terminal domain has features of an alpha-helical leucine zipper, which is likely to be involved in the heterodimerization with another division protein, DivIC. To determine what residues are important for FtsL function, we used both random and site-directed mutagenesis. Unexpectedly, all chemically induced mutations fell into two clear classes, those either weakening the ribosome-binding site or producing a stop codon. It appears that the random mutagenesis was efficient, so many missense mutations must have been generated but with no phenotypic effect. Substitutions affecting hydrophobic residues in the putative coiled-coil domain, introduced by site-directed mutagenesis, also gave no observable phenotype except for insertion of a helix-breaking proline residue, which destroyed FtsL function. ftsL homologues cloned from three diverse Bacillus species, Bacillus licheniformis, Bacillus badius, and Bacillus circulans, could complement an ftsL null mutation in B. subtilis, even though up to 66% of the amino acid residues of the predicted proteins were different from B. subtilis FtsL. However, the ftsL gene from Staphylococcus aureus (whose product has 73% of its amino acids different from those of the B. subtilis ftsL product) was not functional. We conclude that FtsL is a highly malleable protein that can accommodate a large number of sequence changes without loss of function.
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23. |
Declerck N,
Machius M,
Wiegand G,
Huber R,
Gaillardin C,
( 2000 ) Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase. PMID : 10966804 : DOI : 10.1006/jmbi.2000.4025 Abstract >>
Bacillus licheniformis alpha-amylase (BLA) is a starch-degrading enzyme that is highly thermostable although it is produced by a rather mesophilic organism. Over the last decade, the origin of BLA thermal properties has been extensively investigated in both academic and industrial laboratories, yet it is poorly understood. Here, we have used structure-based mutagenesis in order to probe the role of amino acid residues previously proposed as being important for BLA thermostability. Residues involved in salt-bridges, calcium binding or potential deamidation processes have been selected and replaced with various amino acids using a site-directed mutagenesis method, based on informational suppression. A total of 175 amylase variants were created and analysed in vitro. Active amylase variants were tested for thermostability by measuring residual activities after incubation at high temperature. Out of the 15 target residues, seven (Asp121, Asn126, Asp164, Asn192, Asp200, Asp204 and Ala269) were found to be particularly intolerant to any amino acid substitutions, some of which lead to very unstable mutant enzymes. By contrast, three asparagine residues (Asn172, Asn188 and Asn190) could be replaced with amino acid residues that significantly increase the thermostability compared to the wild-type enzyme. The highest stabilization event resulted from the substitution of phenylalanine in place of asparagine at position 190, leading to a sixfold increase of the enzyme's half-life at 80 degrees C (pH 5.6, 0.1 mM CaCl(2)). These results, combined with those of previous mutational analyses, show that the structural determinants contributing to the overall thermostability of BLA concentrate in domain B and at its interface with the central A domain. This region contains a triadic Ca-Na-Ca metal-binding site that appears extremely sensitive to any modification that may alter or reinforce the network of electrostatic interactions entrapping the metal ions. In particular, a loop spanning from residue 178 to 199, which undergoes pronounced conformational changes upon removal of calcium, appears to be the key feature for maintaining the enzyme structural integrity. Outside this region, most salt-bridges that were destroyed by mutations were found to be dispensable, except for an Asp121-Arg127 salt-bridge that contributes to the enhanced thermostability of BLA compared to other homologous bacterial alpha-amylases. Finally, our studies demonstrate that the natural resistance of BLA against high temperature is not optimized and can be enhanced further through various means, including the removal of possibly deamidating residues.
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24. |
Szabó L,
Ponyi T,
Orosz L,
Tran LS,
( 1999 ) Phage abortive infection of Bacillus licheniformis ATCC 9800; identification of the abiBL11 gene and localisation and sequencing of its promoter region. PMID : 10616719 : Abstract >>
The virulent bacteriophage BL11 infects almost all Bacillus licheniformis strains tested, including the industrial bacitracin-producing B. licheniformis 19. B. licheniformis ATCC 9800, however, was virtually insensitive to phage BL11 infection, and all of the few surviving progeny phages proved to be mutants. The phage-resistance mechanism was neither inhibition of adsorption, nor restriction or exclusion provided by a resident prophage, but was, instead, of another type. Phage BL11 adsorbed well on to ATCC 9800 cells, its DNA was injected, but replication of phage DNA was inhibited and the infected cells died. Thus, the mechanism of phage resistance was identified as abortive infection (AbiBL11). The so-called abiBL11 gene was identified on the chromosome of strain ATCC 9800 by Tn917PF1 transposon mutagenesis. Part of the abiBL11 gene from the phage-sensitive ATCC 9800::Tn917PFI was cloned. Gene-disruption analysis, based on Campbell-type integration, showed that a 0.3-kb EcoRI fragment contained the 5' end of abiBL11. The promoter region of abiBL11 was identified using promoter- and terminator-probe plasmids. The deduced sequence (206 amino acids) of the N-terminal part of abiBL11 showed no significant homology to known abortive-infection genes, but did show homology to a Saccharomyces cerevisiae gene coding for a serine/threonine protein kinase (RCK1).
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25. |
Majewski J,
( 1999 ) DNA sequence similarity requirements for interspecific recombination in Bacillus. PMID : 10581263 : PMC : PMC1460850 Abstract >>
Gene transfer in bacteria is notoriously promiscuous. Genetic material is known to be transferred between groups as distantly related as the Gram positives and Gram negatives. However, the frequency of homologous recombination decreases sharply with the level of relatedness between the donor and recipient. Several studies show that this sexual isolation is an exponential function of DNA sequence divergence between recombining substrates. The two major factors implicated in producing the recombinational barrier are the mismatch repair system and the requirement for a short region of sequence identity to initiate strand exchange. Here we demonstrate that sexual isolation in Bacillus transformation results almost exclusively from the need for regions of identity at both the 5' and 3' ends of the donor DNA strand. We show that, by providing the essential identity, we can effectively eliminate sexual isolation between highly divergent sequences. We also present evidence that the potential of a donor sequence to act as a recombinogenic, invasive end is determined by the stability (melting point) of the donor-recipient complex. These results explain the exponential relationship between sexual isolation and sequence divergence observed in bacteria. They also suggest a model for rapid spread of novel adaptations, such as antibiotic resistance genes, among related species.
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26. |
Wang LT,
Lee FL,
Tai CJ,
Kasai H,
( 2007 ) Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group. PMID : 17684269 : DOI : 10.1099/ijs.0.64685-0 Abstract >>
The Bacillus subtilis group comprises eight closely related species that are indistinguishable from one another by 16S rRNA gene sequence analysis. Therefore, the gyrB gene, which encodes the subunit B protein of DNA gyrase, was selected as an alternative phylogenetic marker. To determine whether gyrB gene sequence analysis could be used for phylogenetic analysis and species identification of members of the B. subtilis group, the congruence of gyrB grouping with both 16S rRNA gene sequencing and DNA-DNA hybridization data was evaluated. Ranges of gyrB nucleotide and translated amino acid sequence similarities among the eight type strains were 75.4-95.0 % and 88.5-99.2 %, respectively, whereas 16S rRNA gene sequence similarities were 98.1-99.8 %. Results showed that gyrB gene sequences provide higher resolution than 16S rRNA gene sequences. The classification achieved by gyrB sequence analysis was in agreement with results obtained with DNA-DNA hybridization. It is concluded that the gyrB gene may be an efficient alternative target for identification and taxonomic analysis of members of the B. subtilis group.
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27. |
Gloster TM,
Ibatullin FM,
Macauley K,
Eklöf JM,
Roberts S,
Turkenburg JP,
Bjørnvad ME,
Jørgensen PL,
Danielsen S,
Johansen KS,
Borchert TV,
Wilson KS,
Brumer H,
Davies GJ,
( 2007 ) Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12. PMID : 17376777 : DOI : 10.1074/jbc.M700224200 Abstract >>
The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed "hemicellulose." One such hemicellulose is xyloglucan, which displays a beta-1,4-linked d-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand-free and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (beta/alpha)(8) and beta-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the beta-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (beta/alpha)(8) GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.
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28. |
Voigt B,
Hoi le T,
Jürgen B,
Albrecht D,
Ehrenreich A,
Veith B,
Evers S,
Maurer KH,
Hecker M,
Schweder T,
( 2007 ) The glucose and nitrogen starvation response of Bacillus licheniformis. PMID : 17274076 : DOI : 10.1002/pmic.200600556 Abstract >>
The glucose and nitrogen starvation stimulons of Bacillus licheniformis were determined by transcriptome and proteome analyses. Under both starvation conditions, the main response of B. licheniformis was a switch to the usage of alternative nutrient sources. This was indicated by an induction of genes involved in the metabolism of C-2 substrates during glucose limitation. In addition, B. licheniformis seems to be using other organic substances like amino acids and lipids as carbon sources when subjected to glucose starvation. This observation is supported by the induction of a high number of genes coding for proteins involved in amino acid and lipid degradation. During nitrogen starvation, genes for several proteases and peptidases involved in nitrate and nitrite assimilation were induced, which enables this bacterium to recruit nitrogen from alternative sources. Both starvation conditions led to a down-regulation of transcription of most vegetative genes, which was subsequently reflected by a reduced synthesis of the corresponding proteins. A selected set of genes was induced by both starvation conditions. Among them were yvyD, citA and the putative methylcitrate shunt genes mmgD, mmgE and yqiQ. However, both starvation conditions did not induce a general SigmaB-dependent stress response.
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29. |
Yoshioka M,
Miwa T,
Horii H,
Takata M,
Yokoyama T,
Nishizawa K,
Watanabe M,
Shinagawa M,
Murayama Y,
( 2007 ) Characterization of a proteolytic enzyme derived from a Bacillus strain that effectively degrades prion protein. PMID : 17241357 : DOI : 10.1111/j.1365-2672.2006.03080.x Abstract >>
The purpose of this paper was to screen candidate bacterial strains for the production of proteases suitable for application to the degradation of pathogenic forms of prion protein (PrP(Sc)). This paper describes the biochemical characteristics and proteolytic activity of the isolated protease. After screening more than 200 bacterial proteases for keratinolytic activity, we identified a Bacillus stain that produced a protease exhibiting high-degradation activity against a scrapie PrP(Sc). Sequence analysis indicated that this serine-protease belonged to the Subtilisin family and had optimum pH and temperature ranges of 9-10 and 60-70 degrees C. Western blotting analysis revealed that the protease was also capable of decomposing bovine spongiform encephalopathy-infected brain homogenate. In addition, the protease was demonstrated to degrade dried PrP(Sc) that had become firmly attached to a plastic surface considerably more effectively than proteinase K or PWD-1, a previously reported keratinase. These results indicate that the isolated protease exhibited higher activity for PrP(Sc) degradation compared with other proteases examined. This protease could be used under moderate conditions for the decontamination of precision instruments that are susceptible to PrP(Sc) contamination.
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30. |
Siehl DL,
Castle LA,
Gorton R,
Keenan RJ,
( 2007 ) The molecular basis of glyphosate resistance by an optimized microbial acetyltransferase. PMID : 17272278 : DOI : 10.1074/jbc.M610267200 Abstract >>
GAT is an N-acetyltransferase from Bacillus licheniformis that was optimized by gene shuffling for acetylation of the broad spectrum herbicide, glyphosate, forming the basis of a novel mechanism of glyphosate tolerance in transgenic plants (Castle, L. A., Siehl, D. L., Gorton, R., Patten, P. A., Chen, Y. H., Bertain, S., Cho, H. J., Duck, N., Wong, J., Liu, D., and Lassner, M. W. (2004) Science 304, 1151-1154). The 1.6-A resolution crystal structure of an optimized GAT variant in ternary complex with acetyl coenzyme A and a competitive inhibitor, 3-phosphoglyerate, defines GAT as a member of the GCN5-related family of N-acetyltransferases. Four active site residues (Arg-21, Arg-73, Arg-111, and His-138) contribute to a positively charged substrate-binding site that is conserved throughout the GAT subfamily. Structural and kinetic data suggest that His-138 functions as a catalytic base via substrate-assisted deprotonation of the glyphosate secondary amine, whereas another active site residue, Tyr-118, functions as a general acid. Although the physiological substrate is unknown, native GAT acetylates D-2-amino-3-phosphonopropionic acid with a kcat/Km of 1500 min-1 mM-1. Kinetic data show preferential binding of short analogs to native GAT and progressively better binding of longer analogs to optimized variants. Despite a 200-fold increase in kcat and a 5.4-fold decrease in Km for glyphosate, only 4 of the 21 substitutions present in R7 GAT lie in the active site. Single-site revertants constructed at these positions suggest that glyphosate binding is optimized through substitutions that increase the size of the substrate-binding site. The large improvement in kcat is likely because of the cooperative effects of additional substitutions located distal to the active site.
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31. |
Kwak JH,
Choi EC,
Weisblum B,
( 1991 ) Transcriptional attenuation control of ermK, a macrolide-lincosamide-streptogramin B resistance determinant from Bacillus licheniformis. PMID : 1713206 : DOI : 10.1128/jb.173.15.4725-4735.1991 PMC : PMC208150 Abstract >>
ermK instructs bacteria to synthesize an erythromycin-inducible 23S rRNA methylase that confers resistance to the macrolide, lincosamide, and streptogramin B antibiotics. Expression of ermK is regulated by transcriptional attenuation, in contrast to other inducible erm genes, previously described, which are regulated translationally. The ermK mRNA leader sequence has a total length of 357 nucleotides and encodes a 14-amino-acid leader peptide together with its ribosome binding site. Additionally, the mRNA leader sequence can fold in either of two mutually exclusive conformations, one of which is postulated to form in the absence of induction and to contain two rho factor-independent terminators. Truncated transcription products ca. 210 and 333 nucleotides long were synthesized in the absence of induction, both in vivo and in vitro, as predicted by the transcriptional attenuation model; run-off transcription in vitro with rITP favored the synthesis of the full-length run-off transcript over that of the 210- and 333-nucleotide truncated products. Northern (RNA) blot analysis of transcripts synthesized in vivo in the absence of erythromycin indicated that transcription terminated at either of the two inverted complementary repeat sequences in the leader that were postulated to serve as rho factor-independent terminators; moreover, no full-length transcripts were detectable in the uninduced samples. In contrast, full-length (ca. 1,200-nucleotide) transcripts were only detected in RNA samples synthesized in vivo in the presence of erythromycin. Full-length transcripts formed in the absence of induction from transcriptional readthrough past the two proposed transcription terminators would fold in a way that would sequester the ribosome binding site together with the first two codons of the ErmK methylase, reducing its efficiency in translation. This feature could therefore provide additional control of expression in the absence of induction; however, such regulation, if operative, would act only secondarily, both in time and place, relative to transcriptional control. Analysis by reverse transcriptase mapping of in vivo transcripts from two primers that bracket the transcription terminator responsible for the 210-nucleotide truncated fragment supports the transcriptional attenuation model proposed and suggests further that the synthesis of the ermK message is initiated constitutively upstream of the proposed terminator but completed inductively downstream of this site.
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32. |
Youssef N,
Simpson DR,
Duncan KE,
McInerney MJ,
Folmsbee M,
Fincher T,
Knapp RM,
( 2007 ) In situ biosurfactant production by Bacillus strains injected into a limestone petroleum reservoir. PMID : 17172458 : DOI : 10.1128/AEM.02264-06 PMC : PMC1828672 Abstract >>
Biosurfactant-mediated oil recovery may be an economic approach for recovery of significant amounts of oil entrapped in reservoirs, but evidence that biosurfactants can be produced in situ at concentrations needed to mobilize oil is lacking. We tested whether two Bacillus strains that produce lipopeptide biosurfactants can metabolize and produce their biosurfactants in an oil reservoir. Five wells that produce from the same Viola limestone formation were used. Two wells received an inoculum (a mixture of Bacillus strain RS-1 and Bacillus subtilis subsp. spizizenii NRRL B-23049) and nutrients (glucose, sodium nitrate, and trace metals), two wells received just nutrients, and one well received only formation water. Results showed in situ metabolism and biosurfactant production. The average concentration of lipopeptide biosurfactant in the produced fluids of the inoculated wells was about 90 mg/liter. This concentration is approximately nine times the minimum concentration required to mobilize entrapped oil from sandstone cores. Carbon dioxide, acetate, lactate, ethanol, and 2,3-butanediol were detected in the produced fluids of the inoculated wells. Only CO(2) and ethanol were detected in the produced fluids of the nutrient-only-treated wells. Microbiological and molecular data showed that the microorganisms injected into the formation were retrieved in the produced fluids of the inoculated wells. We provide essential data for modeling microbial oil recovery processes in situ, including growth rates (0.06 +/- 0.01 h(-1)), carbon balances (107% +/- 34%), biosurfactant production rates (0.02 +/- 0.001 h(-1)), and biosurfactant yields (0.015 +/- 0.001 mol biosurfactant/mol glucose). The data demonstrate the technical feasibility of microbial processes for oil recovery.
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33. |
Waldeck J,
Daum G,
Bisping B,
Meinhardt F,
( 2006 ) Isolation and molecular characterization of chitinase-deficient Bacillus licheniformis strains capable of deproteinization of shrimp shell waste to obtain highly viscous chitin. PMID : 17028230 : DOI : 10.1128/AEM.00938-06 PMC : PMC1694268 Abstract >>
Proteolytic but chitinase-deficient microbial cultures were isolated from shrimp shell waste and characterized. The most efficient isolate was found to be a mixed culture consisting of two Bacillus licheniformis strains, which were first determined microscopically and physiologically. Molecular characterization was carried out by sequencing the 16S rRNA gene of both strains. According to the residual protein and ash content, the chitin obtained by fermentation of such a mixed culture was found to be comparable to a commercially available, chemically processed product. However, the strikingly high viscosity (80 versus 10 mPa of the commercially available sample) indicates its superior quality. The two strains differed in colony morphology and in their secretion capabilities for degradative extracellular enzymes. Sequencing of the loci encoding amylase, cellulase, chitinases, and proteases, as well as the degS/degU operon, which is instrumental in the regulation of degradative enzymes, and the pga operon, which is responsible for polyglutamic acid production, revealed no differences. However, a frameshift mutation in chiA, encoding a chitinase, was validated for both strains, providing an explanation for the ascertained absence of chitinolytic activities and the concomitant possibility of producing highly viscous chitin in a fermentational deproteinization process.
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34. |
Rueckert A,
Ronimus RS,
Morgan HW,
( 2006 ) Development of a real-time PCR assay targeting the sporulation gene, spo0A, for the enumeration of thermophilic bacilli in milk powder. PMID : 16943008 : DOI : 10.1016/j.fm.2005.05.003 Abstract >>
Thermophilic bacilli, such as Anoxybacillus, Geobacillus and Bacillus, are common contaminants growing within the processing lines of milk powder producing factories. These contaminants are used as indicator organisms for plant hygiene and specification limits based on their numbers have been implemented to ensure milk powder quality. In this study, we present a SYBR Green-based real-time PCR assay for the rapid detection and enumeration of these thermophilic bacilli in milk powder using the spo0A sporulation gene as quantification target. With this method the detection of thermophilic bacilli in milk powder can be accomplished within 1 h. The detection limit for reconstituted and inoculated milk was 80 vegetative cfu ml(-1) and 640 spores ml(-1), respectively.
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35. |
Iddar A,
Valverde F,
Assobhei O,
Serrano A,
Soukri A,
( 2005 ) Widespread occurrence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase among gram-positive bacteria. PMID : 16562377 : Abstract >>
The non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDHN, NADP+-specific, EC 1.2.1.9) is present in green eukaryotes and some Streptococcus strains. The present report describes the results of activity and immunoblot analyses, which were used to generate the first survey of bacterial GAPDHN distribution in a number of Bacillus, Streptococcus and Clostridium strains. Putative gapN genes were identified after PCR amplification of partial 700-bp sequences using degenerate primers constructed from highly conserved protein regions. Alignment of the amino acid sequences of these fragments with those of known sequences from other eukaryotic and prokaryotic GAPDHNs, demonstrated the presence of conserved residues involved in catalytic activity that are not conserved in aldehyde dehydrogenases, a protein family closely linked to GAPDHNs. The results confirm that the basic structural features of the members of the GAPDHN family have been conserved throughout evolution and that no identity exists with phosphorylating GAPDHs. Furthermore, phylogenetic trees generated from multiple sequence alignments suggested a close relationship between plant and bacterial GAPDHN families.
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36. |
Keenan RJ,
Siehl DL,
Gorton R,
Castle LA,
( 2005 ) DNA shuffling as a tool for protein crystallization. PMID : 15951425 : DOI : 10.1073/pnas.0502497102 PMC : PMC1149501 Abstract >>
The success of structural studies performed on an individual target in small scale or on many targets in the system-wide scale of structural genomics depends critically on three parameters: (i) obtaining an expression system capable of producing large quantities of the macromolecule(s) of interest, (ii) purifying this material in soluble form, and (iii) obtaining diffraction-quality crystals suitable for x-ray analysis. The attrition rate caused by these constraints is often quite high. Here, we present a strategy that addresses each of these three parameters simultaneously. Using DNA shuffling to introduce functional sequence variability into a protein of interest, we screened crude lysate supernatants for soluble variants that retain enzymatic activity. Crystallization trials performed on three WT and eight shuffled enzymes revealed two variants that crystallized readily. One of these was used to determine the high-resolution structure of the enzyme by x-ray analysis. The sequence diversity introduced through shuffling efficiently samples crystal packing space by modifying the surface properties of the enzyme. The approach demonstrated here does not require guidance as to the type of mutation necessary for improvements in expression, solubility, or crystallization. The method is scaleable and can be applied in situations where a single protein is being studied or in high-throughput structural genomics programs. Furthermore, it should be readily applied to structural studies of soluble proteins, membrane proteins, and macromolecular complexes.
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37. |
Nahrstedt H,
Waldeck J,
Gröne M,
Eichstädt R,
Feesche J,
Meinhardt F,
( 2005 ) Strain development in Bacillus licheniformis: construction of biologically contained mutants deficient in sporulation and DNA repair. PMID : 15951041 : DOI : 10.1016/j.jbiotec.2005.04.003 Abstract >>
By introducing defined deletions in recA and an essential sporulation gene (spoIV), stable mutant strains of Bacillus licheniformis were obtained which are totally asporogenous and severely affected in DNA repair, and thus being UV-hypersensitive. Studies on growth in various liquid media as well as on amylase production revealed no differences of the mutants when compared to the wild type. Hence, such genes appear to be suitable disruption targets for achieving passive biological containment in this industrially exploited species.
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38. |
Pattnaik P,
Grover S,
Batish VK,
( 2005 ) Effect of environmental factors on production of lichenin, a chromosomally encoded bacteriocin-like compound produced by Bacillus licheniformis 26L-10/3RA. PMID : 15881839 : Abstract >>
Effect of environmental factors on production of lichenin, a bacteriocin-like compound produced by Bacillus licheniformis 26L-10/3RA isolated from buffalo rumen was studied. Lichenin represents the first anaerobiosis-specific expression of broad-spectrum antibacterial compound effective only under anaerobic conditions. Production of lichenin by B. licheniformis 26L-10/3RA was found to be very high at 39 degrees C in L-10 medium supplemented with 0.5% glucose and 20% (w/v) inert thermocol beads. Lichenin production was highest at pH 6.8 after 72-96h of incubation. Our study also indicated that Lichenin is not a plasmid-linked characteristic and is encoded by chromosomal DNA. Results obtained can be used in large-scale production of Lichenin for potential application in manipulating rumen function intended for improving productivity of the ruminants.
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39. |
Akiyama K,
Mori K,
Takata R,
( 1999 ) Cloning and sequencing of the Pz-peptidase gene from Bacillus licheniformis N22. PMID : 16232456 : Abstract >>
Pz-peptidase is an endopeptidase that cleaves the synthetic substrate Pz-peptide (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg), which was originally developed for the assay of collagenase. The Pz-peptidase gene of Bacillus licheniformis N22 was cloned and sequenced. The gene consists of 628 amino acids with a motif for zinc-dependent metalloprotease, and shares 42% amino acid identity with the oligoendopeptidase of Lactococcus lactis. This is the first report on the gene structure of a Pz-peptidase.
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40. |
Nthangeni MB,
Ramagoma F,
Tlou MG,
Litthauer D,
( 2005 ) Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species. PMID : 15722149 : DOI : 10.1016/j.mimet.2004.11.021 Abstract >>
Since the invention of the PCR technology, adaptation techniques to clone DNA fragments flanking the known sequence continue to be developed. We describe a perfectly annealed cassette available in almost unlimited quantities with variable sticky-and blunt-end restriction enzyme recognition sites for efficient restriction and ligation with the restricted target genomic DNA. The cassette provides a 200-bp sequence, which is used to design a variety of cassette-specific primers. The dephosphorylation prevents cassette self-ligation and creates a nick at the cassette: target genome DNA ligation site suppressing unspecific PCR amplifications. We introduce the single-strand amplification PCR (SSA-PCR) technique where a lone known locus-specific primer is firstly used to enrich the targeted template DNA strand resulting in significant PCR product specificity during the second round conventional nested PCR. The distance between the known locus-specific primer and the nearest location of the restriction enzyme used determined the length of the obtained PCR product. We used this technique to walk downstream into the isochorismatase and upstream into the hypothetical conserved genes flanking the mature extracellular lipase gene from Bacillus licheniformis. We further demonstrated the potential of the technique as a cost-effective method during PCR-based prospecting for novel genes by designing "universal" degenerate primers that detected homologues of Family VII bacterial lipolytic genes in Bacillus species. The cassette ligation-mediated PCR was used to clone complete nucleotide sequences encoding functional lipolytic genes from B. licheniformis and Bacillus pumilus.
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41. |
Guglielmetti S,
Mora D,
Manachini PL,
Parini C,
( 2005 ) Genetic relationship among Bacillus licheniformis rolling-circle-replicating plasmids and complete nucleotide sequence of pBL63.1, an atypical replicon. PMID : 16122558 : DOI : 10.1016/j.plasmid.2005.01.002 Abstract >>
The degree of biodiversity among Bacillus licheniformis plasmids and their relation to other Bacillus subtilis group plasmids has been evaluated. To attain this goal we surveyed the diversity and linkage of replication modules in a collection of 21 naturally occurring plasmids of B. licheniformis strains, isolated from different geographical areas. On the basis of rep gene sequence analysis it was possible to group the B. licheniformis plasmids rep genes in two main cluster. Comparison with known rep genes from Bacillus rolling-circle-replicating (RCR) plasmids revealed the presence in B. licheniformis plasmids of replication genes with a DNA sequence peculiar to B. licheniformis species together with rep genes with a very high sequence similarity to B. subtilis plasmids. Furthermore, the molecular organization of an atypical replicon, pBL63.1, was shown. This plasmid did not display any significant similarity with known Bacillus RCR plasmids. The complete nucleotide sequence evidenced a replication module with an unexpected similarity with Rep proteins from RCR plasmids of bacterial species phylogenetically distantly related to Bacillus. pBL63.1 represents an exception to the low-level diversity hypothesis among Bacillus RC replicons.
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42. |
Parini C,
Guglielmetti S,
Mora D,
Ricci G,
( 2004 ) Homologies among three Bacillus licheniformis plasmids and molecular characterization of their replication module. PMID : 15462521 : DOI : 10.1016/j.micres.2004.04.003 Abstract >>
To assess to what extent three Bacillus licheniformis plasmids had the same molecular organization a physical map of the 9.34, 8.40 and 7.90 kb plasmids was achieved by using seventeen restriction enzymes. Southern hybridization was performed on plasmids using restriction fragments of the smallest plasmid as probes. Data from different hybridization patterns show a close homology among the three plasmids hypothesizing a similar molecular organization. The lack of plasmid diversity observed, seem to support the hypothesis of a similar phylogeny among these plasmids. This investigation provides more information concerning phylogeny, interrelationships and level of diversity among Bacillus plasmids and a molecular characterization of three plasmids useful for the construction of cloning vectors.
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43. |
Veith B,
Herzberg C,
Steckel S,
Feesche J,
Maurer KH,
Ehrenreich P,
Bäumer S,
Henne A,
Liesegang H,
Merkl R,
Ehrenreich A,
Gottschalk G,
( 2004 ) The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential. PMID : 15383718 : DOI : 10.1159/000079829 DOI : 10.1159/000079829 Abstract >>
The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.
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44. |
Rey MW,
Ramaiya P,
Nelson BA,
Brody-Karpin SD,
Zaretsky EJ,
Tang M,
Lopez de Leon A,
Xiang H,
Gusti V,
Clausen IG,
Olsen PB,
Rasmussen MD,
Andersen JT,
Jørgensen PL,
Larsen TS,
Sorokin A,
Bolotin A,
Lapidus A,
Galleron N,
Ehrlich SD,
Berka RM,
( 2004 ) Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species. PMID : 15461803 : DOI : 10.1186/gb-2004-5-10-r77 PMC : PMC545597 DOI : 10.1186/gb-2004-5-10-r77 PMC : PMC545597 Abstract >>
Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature. We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs. Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.
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45. |
Liu Y,
Zhang J,
Liu Q,
Zhang C,
Ma Q,
( 2004 ) Molecular cloning of novel cellulase genes cel9A and cel12A from Bacillus licheniformis GXN151 and synergism of their encoded polypeptides. PMID : 15386110 : DOI : 10.1007/s00284-004-4291-x Abstract >>
A thermophilic bacterial strain GXN151 which could degrade Avicel efficiently was isolated and identified as Bacillus licheniformis. A genomic library of GXN151 was constructed and two novel endoglucanase genes designated cel9A and cel12A were isolated by screening the library on carboxylmethyl cellulase indicator plates. The analysis of amino acid sequences deduced from the genes indicated that Cel9A consisted of a catalytic domain belonging to glycosyl hydrolase family 9, a linker domain, and a carbohydrate binding module family 3 from N-terminal to C-terminal; Cel12A had only one catalytic domain belonging to glycosyl hydrolase family 12. The combinations of Cel9A and Cel12A produced by the recombinant E. coli exhibited synergistic action against substrates of carboxylmethyl cellulose as well as Avicel.
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46. |
Ryttersgaard C,
Le Nours J,
Lo Leggio L,
Jørgensen CT,
Christensen LL,
Bjørnvad M,
Larsen S,
( 2004 ) The structure of endo-beta-1,4-galactanase from Bacillus licheniformis in complex with two oligosaccharide products. PMID : 15312766 : DOI : 10.1016/j.jmb.2004.05.017 Abstract >>
The beta-1,4-galactanase from Bacillus licheniformis (BLGAL) is a plant cell-wall-degrading enzyme involved in the hydrolysis of beta-1,4-galactan in the hairy regions of pectin. The crystal structure of BLGAL was determined by molecular replacement both alone and in complex with the products galactobiose and galactotriose, catching a first crystallographic glimpse of fragments of beta-1,4-galactan. As expected for an enzyme belonging to GH-53, the BLGAL structure reveals a (betaalpha)(8)-barrel architecture. However, BLGAL betaalpha-loops 2, 7 and 8 are long in contrast to the corresponding loops in structures of fungal galactanases determined previously. The structure of BLGAL additionally shows a calcium ion linking the long betaalpha-loops 7 and 8, which replaces a disulphide bridge in the fungal galactanases. Compared to the substrate-binding subsites predicted for Aspergillus aculeatus galactanase (AAGAL), two additional subsites for substrate binding are found in BLGAL, -3 and -4. A comparison of the pattern of galactan and galactooligosaccharides degradation by AAGAL and BLGAL shows that, although both are most active on substrates with a high degree of polymerization, AAGAL can degrade galactotriose and galactotetraose efficiently, whereas BLGAL prefers longer oligosaccharides and cannot hydrolyze galactotriose to any appreciable extent. This difference in substrate preference can be explained structurally by the presence of the extra subsites -3 and -4 in BLGAL.
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47. |
Parini C,
Guglielmetti S,
Mora D,
Ricci G,
( 2004 ) Complete sequence and structural organization of pFL5 and pFL7, two cryptic plasmids from Bacillus licheniformis. PMID : 15109826 : DOI : 10.1016/j.plasmid.2004.02.001 Abstract >>
The complete nucleotide sequences of two plasmids, pFL5 and pFL7, isolated from soil bacteria, Bacillus licheniformis FL5 and FL7, have been determined. The plasmids pFL5 and pFL7 were analyzed and found to be 9150 and 7853 bp in size with a G+C content of 41.0 and 43.6 mol%, respectively. Computer assisted analysis of sequence data revealed 11 possible ORFs in pFL5, four of which could be assigned no function from homology searches. Instead, eight putative ORFs were identified in pFL7, two of which appeared to have no biological function. All the ORFs were preceded by a ribosome binding site. The ORFs 9.5 and 6.7, each of 340 amino acids, were postulated to encode a replication protein similar to known replication proteins of rolling circle replicons, particularly those of the pC194 family. The structural organization of the two pFL plasmids is similar to the pTA plasmids family, with only a few putative coding regions that cannot be attributed to these plasmid backbone genes. In contrast to pTA plasmids, the majority of the genes have an orientation of transcription opposite to the direction of replication. The identified probable sso sequences seem to belong to a different group of those found in Bacillus plasmids; in fact, a significant level of homology was found with ssoA group sequences. These plasmids seem to be related to plasmids identified within the Bacillus subtilis group, confirming the low-level diversity among these replicons.
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48. |
Kuroda A,
Sugimoto Y,
Funahashi T,
Sekiguchi J,
( 1992 ) Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase. PMID : 1495475 : DOI : 10.1007/bf00272354 Abstract >>
A DNA fragment containing the gene for a cell wall hydrolase of Bacillus licheniformis was cloned into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame which encodes a polypeptide of 253 amino acids with a molecular mass of 27,513. The gene was designated as cwlM, for cell wall lysis. The deduced amino acid sequence indicated that there is a repeated sequence consisting of 33 amino acid residues in the C-terminal region. Deletion of the C-terminal region did not lead to any loss of cell wall lytic activity. The gene product purified from E. coli cells harboring a cwlM-bearing plasmid exhibited a M(r) value of 29 kDa on SDS-polyacrylamide gels, and characterization of the specific substrate bond cleaved by CWLM indicated that the enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28). The enzyme hydrolyzed the cell wall of Micrococcus luteus more efficiently than those of B. licheniformis and B. subtilis, but the truncated CWLM (lacking the C-terminal region) had lost this preference. CWLM prepared from B. subtilis cells harboring a plasmid containing cwlM had a similar M(r) value to that from E. coli. Amino acid sequence homologies between CWLM and other amidases, and their protein structures are discussed.
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49. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
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50. |
Berensmeier S,
Singh SA,
Meens J,
Buchholz K,
( 2004 ) Cloning of the pelA gene from Bacillus licheniformis 14A and biochemical characterization of recombinant, thermostable, high-alkaline pectate lyase. PMID : 14673544 : DOI : 10.1007/s00253-003-1446-9 Abstract >>
The pectate lyase gene pelA from alkaliphilic Bacillus licheniformis strain 14A was cloned and sequenced. The nucleotide sequence corresponded to an open reading frame of 1,026 bp that codes for a 39 amino acid signal peptide and a mature protein with a molecular mass of 33,451 Da. The mature PelA showed significant homology to other pectate lyases belonging to polysaccharide lyase family 1, such as enzymes from different Bacillus spp. and Erwinia chrysanthemi. The pelA gene was expressed in Escherichia coli as a recombinant fusion protein containing a C-terminal His-tag, allowing purification to near homogeneity in a one-step procedure. The values for the kinetic parameters K(m) and Vmax of the fusion protein were 0.56 g/l and 51 micromol/min, respectively. The activity of purified PelAHis was inhibited in the presence of excess substrate. Characterization of product formation revealed unsaturated trigalacturonate as the main product. The yields of unsaturated trigalacturonic acids were further examined for the substrates polygalacturonic acid, citrus pectin and sugar-beet pectin.
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51. |
Shahhoseini M,
Ziaee AA,
Ghaemi N,
( 2003 ) Expression and secretion of an alpha-amylase gene from a native strain of Bacillus licheniformis in Escherichia coli by T7 promoter and putative signal peptide of the gene. PMID : 14632998 : Abstract >>
The gene encoding a hyperthermostable alpha-amylase from a Bacillus licheniformis native strain was cloned in pET24d transcription vector containing T7 promoter, and expressed in Escherichia coli BL21(DE3) cells. Having confirmed the alpha-amylase activity through activity staining method on SDS-PAGE gel, the yields of production were determined in two separated intra and inter-cellular phases and compared using enzymatic assay methods. Extracellular production of the active recombinant enzyme implies the recognition of the putative signal peptide of this Bacillus sp. by E. coli secretory system. This may be because of the amino acid sequence of this signal peptide which covers all the structural parameters of a standard signal peptide processed by Lep B, the major signal peptidase in E. coli secretory system. This study recommends the use of this signal peptide for extracellular production of other foreign proteins in E. coli.
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52. |
Thorsen L,
Abdelgadir WS,
Rønsbo MH,
Abban S,
Hamad SH,
Nielsen DS,
Jakobsen M,
( 2011 ) Identification and safety evaluation of Bacillus species occurring in high numbers during spontaneous fermentations to produce Gergoush, a traditional Sudanese bread snack. PMID : 21429611 : DOI : 10.1016/j.ijfoodmicro.2011.02.028 Abstract >>
Gergoush is a naturally fermented Sudanese Bread snack produced in three fermentation steps (primary starter, adapted starter and final dough), followed by three baking steps for a half to one hour at above 200 �XC. This study examines the microbiota of two sets of fermentations performed at a traditional production site in Khartoum, Sudan in 2006 and 2009, respectively. In 2006 four different milk/legume based primary starters (faba bean, chick pea, lentil and white bean) were sampled in order to enumerate and identify the Bacillus at species or group level. In 2009 specific focus was on the enumeration and safety evaluation of the dominant Bacillus cereus group species occurring during various Gergoush productions (including the three fermentations steps and after baking). In 2006, the primary starters contained Bacillus spp. in the order of between 7.7 and 8.1 log(10) CFU/g. Species identifications were performed by M13-PCR typing using the Escherichia coli phage M13 derived primer PM13 combined with internally transcribed 16-23S rRNA PCR, 16S rRNA gene and gyrA or gyrB gene sequencing, and selected phenotypic tests. Depending on the legume used, 40-68% of the isolates were identified as B. cereus sensu lato, 16-27% as Bacillus licheniformis, 8-32% as Bacillus subtilis and 4-20% as Bacillus sonorensis. During the second set of fermentation trials performed in 2009, the Bacillus spp. and B. cereus occurred in numbers of between 7.7-9.9 and 6.1-7.8 log(10) CFU/g, respectively, while no bacteria were detected after baking. A total of 180 B. cereus sensu lato isolates from four different primary starters, adapted starters and final doughs were further identified as B. cereus sensu stricto (118 isolates) and Bacillus thuringiensis (62 isolates). The safety of Gergoush was evaluated based on the counts and toxin gene profiles of the dominant B. cereus species. Considering that no bacteria survived the baking process, and that the cereulide synthetase gene cesB involved in the production of the heat stable emetic toxin cereulide was not detected in any of the investigated B. cereus isolates, the results indicate, that Gergoush produced at the traditional production site is safe for human consumption. This study is the first to identify the Bacillus of Gergoush to species level, and it is the first to perform a safety evaluation of the product, based on the dominant B. cereus species.
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53. |
Shenoy RT,
Thangamani S,
Velazquez-Campoy A,
Ho B,
Ding JL,
Sivaraman J,
( 2011 ) Structural basis for dual-inhibition mechanism of a non-classical Kazal-type serine protease inhibitor from horseshoe crab in complex with subtilisin. PMID : 21541315 : DOI : 10.1371/journal.pone.0018838 PMC : PMC3082530 Abstract >>
Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 ? resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1?2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.
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54. |
Hill DE,
Aldape R,
Rozzell JD,
( 1990 ) Nucleotide sequence of a cyclodextrin glucosyltransferase gene, cgtA, from Bacillus licheniformis. PMID : 2137908 : DOI : 10.1093/nar/18.1.199 PMC : PMC330236 Abstract >>
N/A
|
55. |
Sekiguchi J,
Ohsu H,
Kuroda A,
Moriyama H,
Akamatsu T,
( 1990 ) Nucleotide sequences of the Bacillus subtilis flaD locus and a B. licheniformis homologue affecting the autolysin level and flagellation. PMID : 2121898 : DOI : 10.1099/00221287-136-7-1223 Abstract >>
A DNA fragment containing the flaD locus of Bacillus subtilis, which had been cloned into plasmid pAC3, was subcloned into an M13 phage and sequenced. The sequence contained five open reading frames (ORFs), of which ORF2 was the flaD gene. Unexpectedly, the sequence of the flaD locus was identical to that of sin [sporulation inhibition gene; Gaur, N. K., Dubnau, E. & Smith, I. (1986). Journal of Bacteriology 168, 860-869]. A B. licheniformis homologue (flaL) of the B. subtilis flaD locus was cloned into pUC19 and identified by colony hybridization. The B. licheniformis DNA was subcloned and sequenced. Two ORFs (ORF1, or L-ORF1; and ORF2, or flaL) were detected, encoding 58 and 111 amino acid residues, respectively. These are almost identical in length to ORF1 (D-ORF1; 57 amino acids) and flaD (111 amino acids) on the fragment of B. subtilis DNA. The overall interspecies differences between the nucleotide sequences of D-ORF1 and L-ORF1, and those of flaD and flaL, were 42% and 11%, respectively, and the differences in the predicted amino acid sequences were 50% and 7%, respectively. The regions 3' of the ORFs (flaL and flaD) in both species resemble rho-independent terminators of transcription. The characteristics of the amino acid sequences are also discussed.
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56. |
Caetano T,
Krawczyk JM,
Mösker E,
Süssmuth RD,
Mendo S,
( 2011 ) Heterologous expression, biosynthesis, and mutagenesis of type II lantibiotics from Bacillus licheniformis in Escherichia coli. PMID : 21276942 : DOI : 10.1016/j.chembiol.2010.11.010 Abstract >>
Lichenicidin is a class II two-component lantibiotic produced by Bacillus licheniformis. It is composed of the two peptides Bli�\ and Bli�], which act synergistically against various Gram-positive bacteria. The lichenicidin gene cluster was successfully expressed in Escherichia coli, thus constituting the first report to our knowledge of a full reconstitution of a lantibiotic biosynthetic pathway in vivo by a Gram-negative host. This system was further exploited to characterize and assign the function of proteins encoded in the biosynthetic gene cluster in the maturation of lichenicidin peptides. Moreover, a trans complementation system was developed for expression of Bli�\ and Bli�] variants in vivo. This contribution will spur future studies in the heterologous expression and engineering of lantibiotics.
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57. |
Bonnefond L,
Arai T,
Sakaguchi Y,
Suzuki T,
Ishitani R,
Nureki O,
( 2011 ) Structural basis for nonribosomal peptide synthesis by an aminoacyl-tRNA synthetase paralog. PMID : 21325056 : DOI : 10.1073/pnas.1019480108 PMC : PMC3053990 Abstract >>
Cyclodipeptides are secondary metabolites biosynthesized by many bacteria and exhibit a wide array of biological activities. Recently, a new class of small proteins, named cyclodipeptide synthases (CDPS), which are unrelated to the typical nonribosomal peptide synthetases, was shown to generate several cyclodipeptides, using aminoacyl-tRNAs as substrates. The Mycobacterium tuberculosis CDPS, Rv2275, was found to generate cyclodityrosine through the formation of an aminoacyl-enzyme intermediate and to have a structure and oligomeric state similar to those of the class Ic aminoacyl-tRNA synthetases (aaRSs). However, the poor sequence conservation among CDPSs has raised questions about the architecture and catalytic mechanism of the identified homologs. Here we report the crystal structures of Bacillus licheniformis CDPS YvmC-Blic, in the apo form and complexed with substrate mimics, at 1.7-2.4-? resolutions. The YvmC-Blic structure also exhibits similarity to the class Ic aaRSs catalytic domain. Our mutational analysis confirmed the importance of a set of residues for cyclodileucine formation among the conserved residues localized in the catalytic pocket. Our biochemical data indicated that YvmC-Blic binds tRNA and generates cyclodileucine as a monomer. We were also able to detect the presence of an aminoacyl-enzyme reaction intermediate, but not a dipeptide tRNA intermediate, whose existence was postulated for Rv2275. Instead, our results support a sequential catalytic mechanism for YvmC-Blic, with the successive attachment of two leucine residues on the enzyme via a conserved serine residue. Altogether, our findings suggest that all CDPS enzymes share a common aaRS-like architecture and a catalytic mechanism involving the formation of an enzyme-bound intermediate.
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58. |
Nijland R,
Hall MJ,
Burgess JG,
( 2010 ) Dispersal of biofilms by secreted, matrix degrading, bacterial DNase. PMID : 21179489 : DOI : 10.1371/journal.pone.0015668 PMC : PMC3001887 Abstract >>
Microbial biofilms are composed of a hydrated matrix of biopolymers including polypeptides, polysaccharides and nucleic acids and act as a protective barrier and microenvironment for the inhabiting microbes. While studying marine biofilms, we observed that supernatant produced by a marine isolate of Bacillus licheniformis was capable of dispersing bacterial biofilms. We investigated the source of this activity and identified the active compound as an extracellular DNase (NucB). We have shown that this enzyme rapidly breaks up the biofilms of both Gram-positive and Gram-negative bacteria. We demonstrate that bacteria can use secreted nucleases as an elegant strategy to disperse established biofilms and to prevent de novo formation of biofilms of competitors. DNA therefore plays an important dynamic role as a reversible structural adhesin within the biofilm.
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59. |
Tiwary E,
Gupta R,
( 2010 ) Extracellular expression of keratinase from Bacillus licheniformis ER-15 in Escherichia coli. PMID : 20597544 : DOI : 10.1021/jf100803g Abstract >>
Keratinase (ker BL) from Bacillus licheniformis ER-15 was cloned into vector pEZZ18 for extracellular expression in Escherichia coli HB101. Recombinant keratinase was secreted with high specific activity (75 units/mg) under non-inducible conditions after 36 h at 37 degrees C and 300 rpm in a shake flask. Protein was concentrated and, subsequently, purified by ion-exchange chromatography using Q-sepharose with 95.8% yield. The recombinant keratinase was a serine protease and most active in the pH range of 8-12 and at 60 degrees C. The enzyme was stable over a wide pH range of 4-12 for 3 h. ker BL degraded bovine serum albumin, casein, azocasein, gelatin, and feather. E. coli HB101 harboring pEZZ18 ker BL2 degraded chicken feather completely within 24 h at 37 degrees C.
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60. |
Hoffmann K,
Wollherr A,
Larsen M,
Rachinger M,
Liesegang H,
Ehrenreich A,
Meinhardt F,
( 2010 ) Facilitation of direct conditional knockout of essential genes in Bacillus licheniformis DSM13 by comparative genetic analysis and manipulation of genetic competence. PMID : 20543043 : DOI : 10.1128/AEM.00660-10 PMC : PMC2916460 Abstract >>
The genetic manageability of the biotechnologically important Bacillus licheniformis is hampered due to its poor transformability, whereas Bacillus subtilis efficiently takes up DNA during genetic competence, a quorum-sensing-dependent process. Since the sensor histidine kinase ComP, encoded by a gene of the quorum-sensing module comQXPA of B. licheniformis DSM13, was found to be inactive due to an insertion element within comP, the coding region was exchanged with a functional copy. Quorum sensing was restored, but the already-poor genetic competence dropped further. The inducible expression of the key regulator for the transcription of competence genes, ComK, in trans resulted in highly competent strains and facilitated the direct disruption of genes, as well as the conditional knockout of an essential operon. As ComK is inhibited at low cell densities by a proteolytic complex in which MecA binds ComK and such inhibition is antagonized by the interaction of MecA with ComS (the expression of the latter is controlled by cell density in B. subtilis), we performed an in silico analysis of MecA and the hitherto unidentified ComS, which revealed differences for competent and noncompetent strains, indicating that the reduced competence possibly is due to a nonfunctional coupling of the comQXPA-encoded quorum module and ComK. The obtained increased genetic tractability of this industrial workhorse should improve a wide array of scientific investigations.
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61. |
Thanh TN,
Jürgen B,
Bauch M,
Liebeke M,
Lalk M,
Ehrenreich A,
Evers S,
Maurer KH,
Antelmann H,
Ernst F,
Homuth G,
Hecker M,
Schweder T,
( 2010 ) Regulation of acetoin and 2,3-butanediol utilization in Bacillus licheniformis. PMID : 20524112 : DOI : 10.1007/s00253-010-2681-5 Abstract >>
The acoABCL and acuABC operons of Bacillus licheniformis DSM13 are strongly induced at the transcriptional level during glucose starvation conditions. Primer extension analyses of this study indicate that the acoABCL operon is controlled by a sigmaL-dependent promoter and the acuABC operon by a sigmaA-dependent promoter. Transcription at the acoA promoter is repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The acuA promoter shows a similar induction pattern, but its activity is independent from the presence of acetoin. It is demonstrated that the acoABCL operon is mainly responsible for acetoin and 2,3-butanediol degradation in B. licheniformis.
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62. |
Brown T,
Charlier P,
Herman R,
Schofield CJ,
Sauvage E,
( 2010 ) Structural basis for the interaction of lactivicins with serine beta-lactamases. PMID : 20593835 : DOI : 10.1021/jm100437u Abstract >>
Lactivicin (LTV) is a natural non-beta-lactam antibiotic that inhibits penicillin-binding proteins and serine beta-lactamases. A crystal structure of a BS3-LTV complex reveals that, as for its reaction with PBPs, LTV reacts with the nucleophilic serine and that cycloserine and lactone rings of LTV are opened. This structure, together with reported structures of PBP1b with lactivicins, provides a basis for developing improved lactivicin-based gamma-lactam antibiotics.
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63. |
Juajun O,
Nguyen TH,
Maischberger T,
Iqbal S,
Haltrich D,
Yamabhai M,
( 2011 ) Cloning, purification, and characterization of �]-galactosidase from Bacillus licheniformis DSM 13. PMID : 20852995 : DOI : 10.1007/s00253-010-2862-2 Abstract >>
The gene encoding homodimeric �]-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-�]-D: -galactoside (oNPG) and lactose hydrolysis, were 50�XC and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37�XC and over a wide range of temperatures (4-42�XC) at pH 6.5 for up to 1 month. The K(m) values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na(+) and K(+) in the concentration range of 1-100 mM as well as the divalent metal cations Mg?(+), Mn?(+), and Ca?(+) at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.
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64. |
Lloberas J,
Perez-Pons JA,
Querol E,
( 1991 ) Molecular cloning, expression and nucleotide sequence of the endo-beta-1,3-1,4-D-glucanase gene from Bacillus licheniformis. Predictive structural analyses of the encoded polypeptide. PMID : 2026156 : DOI : 10.1111/j.1432-1033.1991.tb15916.x Abstract >>
A Bacillus licheniformis gene coding for an endo-beta-1,3-1,4-D-glucanase have been cloned in Escherichia coli and sequenced. The open reading frame contains a sequence of 731 bp, encoding a polypeptide of 243 amino acid residues, with a molecular mass of 27404 Da (24418 Da without the putative signal peptide), which corresponds to the enzyme we had previously isolated and characterized. The signal peptide is functional in E. coli. More than 60% of the endo-beta-1,3-1,4-D-glucanase activity is extracellular or periplasmic. The polypeptide is highly similar to other reported Bacillus beta-glucanases. Several structural predictive analyses (secondary structure, hydropathic plots, similarity with other related enzymes, etc.) have been performed. From these analyses we assign a tentative three-functional-domain structure for the enzyme (signal peptide, substrate binding and catalytic domains) and a putative lysozyme-like active site.
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65. |
Lee JW,
Edwards CW,
Hulett FM,
( 1991 ) Identification of four unique clones encoding 10 kDa proteins from Bacillus that cause phenotypic complementation of a phoA mutant strain of Escherichia coli. PMID : 2033382 : DOI : 10.1099/00221287-137-3-667 Abstract >>
A number of clones have been isolated from two Bacillus species which complement the PhoA- phenotype of Escherichia coli mutants under conditions that induce the expression of alkaline phosphatase (APase). These clones were initially thought to carry XPases because the transformed host could hydrolyse a common APase substrate, XP (5-bromo-4-chloro-3-indolyl-phosphate). The sequences of the open reading frames responsible for the phenotypic complementation showed no sequence similarity to ATPases of E. coli, human (bone-liver-kidney, intestinal or placental) or Bacillus. Therefore, these clones were designated as XPA (for X Phosphatase Activity) clones. Four of the clones encoded small (10 kDa), basic, hydrophobic proteins. Two of these, xpaB from B. subtilis 168 and xpaL2 from B. licheniformis MC14, shared 62% identity at both the DNA and the predicted amino acid sequence level. The fact that homologues from two Bacillus strains were cloned indicated that the screen was specific, but not for APase genes. It is clear that phenotypic complementation with cloned DNA from another genus does not ensure the identification of an APase gene. Possible mechanisms for the abnormal phenotypic complementation are discussed.
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66. |
Connor N,
Sikorski J,
Rooney AP,
Kopac S,
Koeppel AF,
Burger A,
Cole SG,
Perry EB,
Krizanc D,
Field NC,
Slaton M,
Cohan FM,
( 2010 ) Ecology of speciation in the genus Bacillus. PMID : 20048064 : DOI : 10.1128/AEM.01988-09 PMC : PMC2832372 Abstract >>
Microbial ecologists and systematists are challenged to discover the early ecological changes that drive the splitting of one bacterial population into two ecologically distinct populations. We have aimed to identify newly divergent lineages ("ecotypes") bearing the dynamic properties attributed to species, with the rationale that discovering their ecological differences would reveal the ecological dimensions of speciation. To this end, we have sampled bacteria from the Bacillus subtilis-Bacillus licheniformis clade from sites differing in solar exposure and soil texture within a Death Valley canyon. Within this clade, we hypothesized ecotype demarcations based on DNA sequence diversity, through analysis of the clade's evolutionary history by Ecotype Simulation (ES) and AdaptML. Ecotypes so demarcated were found to be significantly different in their associations with solar exposure and soil texture, suggesting that these and covarying environmental parameters are among the dimensions of ecological divergence for newly divergent Bacillus ecotypes. Fatty acid composition appeared to contribute to ecotype differences in temperature adaptation, since those ecotypes with more warm-adapting fatty acids were isolated more frequently from sites with greater solar exposure. The recognized species and subspecies of the B. subtilis-B. licheniformis clade were found to be nearly identical to the ecotypes demarcated by ES, with a few exceptions where a recognized taxon is split at most into three putative ecotypes. Nevertheless, the taxa recognized do not appear to encompass the full ecological diversity of the B. subtilis-B. licheniformis clade: ES and AdaptML identified several newly discovered clades as ecotypes that are distinct from any recognized taxon.
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67. |
Shevtsov MB,
Chen Y,
Isupov MN,
Leech A,
Gollnick P,
Antson AA,
( 2010 ) Bacillus licheniformis Anti-TRAP can assemble into two types of dodecameric particles with the same symmetry but inverted orientation of trimers. PMID : 20138150 : DOI : 10.1016/j.jsb.2010.01.013 PMC : PMC2896485 Abstract >>
Anti-TRAP (AT) protein regulates expression of tryptophan biosynthetic genes by binding to the trp RNA-binding attenuation protein (TRAP) and preventing its interaction with RNA. Bacillus subtilis AT forms trimers that can either interact with TRAP or can further assemble into dodecameric particles. To determine which oligomeric forms are preserved in AT proteins of other Bacilli we studied Bacillus licheniformis AT which shares 66% sequence identity with the B. subtilis protein. We show that in solution B. licheniformis AT forms stable trimers. In crystals, depending on pH, such trimers assemble into two different types of dodecameric particles, both having 23 point group symmetry. The dodecamer formed at pH 6.0 has the same conformation as previously observed for B. subtilis AT. This dodecamer contains a large internal chamber with the volume of approximately 700 A(3), which is lined by the side chains of twelve valine residues. The presence of the hydrophobic chamber hints at the possibility that the dodecamer formation could be induced by binding of a ligand. Interestingly, in the dodecamer formed at pH 8.0 all trimers are turned inside out relatively to the form observed at pH 6.0.
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68. |
Irie K,
Kitagawa K,
Nagura H,
Imai T,
Shimomura T,
Fujiyoshi Y,
( 2010 ) Comparative study of the gating motif and C-type inactivation in prokaryotic voltage-gated sodium channels. PMID : 19959480 : DOI : 10.1074/jbc.M109.057455 PMC : PMC2823509 Abstract >>
Prokaryotic voltage-gated sodium channels (Na(V)s) are homotetramers and are thought to inactivate through a single mechanism, named C-type inactivation. Here we report the voltage dependence and inactivation rate of the NaChBac channel from Bacillus halodurans, the first identified prokaryotic Na(V), as well as of three new homologues cloned from Bacillus licheniformis (Na(V)BacL), Shewanella putrefaciens (Na(V)SheP), and Roseobacter denitrificans (Na(V)RosD). We found that, although activated by a lower membrane potential, Na(V)BacL inactivates as slowly as NaChBac. Na(V)SheP and Na(V)RosD inactivate faster than NaChBac. Mutational analysis of helix S6 showed that residues corresponding to the "glycine hinge" and "PXP motif" in voltage-gated potassium channels are not obligatory for channel gating in these prokaryotic Na(V)s, but mutations in the regions changed the inactivation rates. Mutation of the region corresponding to the glycine hinge in Na(V)BacL (A214G), Na(V)SheP (A216G), and NaChBac (G219A) accelerated inactivation in these channels, whereas mutation of glycine to alanine in the lower part of helix S6 in NaChBac (G229A), Na(V)BacL (G224A), and Na(V)RosD (G217A) reduced the inactivation rate. These results imply that activation gating in prokaryotic Na(V)s does not require gating motifs and that the residues of helix S6 affect C-type inactivation rates in these channels.
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69. |
Anthony T,
Chellappa GS,
Rajesh T,
Gunasekaran P,
( 2010 ) Functional analysis of a putative holin-like peptide-coding gene in the genome of Bacillus licheniformis AnBa9. PMID : 19967339 : DOI : 10.1007/s00203-009-0530-7 Abstract >>
BhlA, a putative holin-like protein of Bacillus licheniformis AnBa9 expressed in Escherichia coli BL21(DE3) showed antibacterial activity against several gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and Micrococcus luteus. Deletion analysis of bhlA suggests that a hydrophobic transmembrane domain, BhlATM is essential for antibacterial activity. Though the minimum inhibitory concentration (MIC) of BhlA was sevenfold lower than BhlATM, transmembrane domain deleted construct (BhlATM) had no antibacterial activity. The expression of BhlA in E. coli was found to be toxic to cells. Therefore, the bhlA was cloned in yeast surface display vector pYD1 and expressed in Saccharomyces cerevisiae. The surface displayed yeast showed inhibition of several gram-positive bacteria. This recombinant yeast expressing BhlA may be used as biodrug for efficient control of multiple drug-resistant bacterial infections.
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70. |
Begley M,
Cotter PD,
Hill C,
Ross RP,
( 2009 ) Identification of a novel two-peptide lantibiotic, lichenicidin, following rational genome mining for LanM proteins. PMID : 19561184 : DOI : 10.1128/AEM.00730-09 PMC : PMC2737927 Abstract >>
Lantibiotics are ribosomally synthesized peptide antimicrobials which contain considerable posttranslational modifications. Given their usually broad host range and their highly stable structures, there have been renewed attempts to identify and characterize novel members of the lantibiotic family in recent years. The increasing availability of bacterial genome sequences means that in addition to traditional microbiological approaches, in silico screening strategies may now be employed to the same end. Taking advantage of the highly conserved nature of lantibiotic biosynthetic enzymes, we screened publicly available microbial genome sequences for genes encoding LanM proteins, which are required for the posttranslational modification of type 2 lantibiotics. By using this approach, 89 LanM homologs, including 61 in strains not known to be lantibiotic producers, were identified. Of these strains, five (Streptococcus pneumoniae SP23-BS72, Bacillus licheniformis ATCC 14580, Anabaena variabilis ATCC 29413, Geobacillus thermodenitrificans NG80-2, and Herpetosiphon aurantiacus ATCC 23779) were subjected to a more detailed bioinformatic analysis. Four of the strains possessed genes potentially encoding a structural peptide in close proximity to the lanM determinants, while two, S. pneumoniae SP23-BS72 and B. licheniformis ATCC 14580, possess two potential structural genes. The B. licheniformis strain was selected for a proof-of-concept exercise, which established that a two-peptide lantibiotic, lichenicidin, which exhibits antimicrobial activity against all Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant enterococcus strains tested, was indeed produced, thereby confirming the benefits of such a bioinformatic approach when screening for novel lantibiotic producers.
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71. |
Scheler A,
Rygus T,
Allmansberger R,
Hillen W,
( 1991 ) Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus licheniformis encoded regulon for xylose utilization. PMID : 1953294 : DOI : 10.1007/bf00245345 DOI : 10.1007/bf00245345 Abstract >>
In this article we describe the cloning of the xyl regulon encoding xylose utilization from Bacillus licheniformis by complementation of a xyl mutant of B. subtilis. The xylose isomerase encoding gene, xylA, was sequenced and identified by its extensive homology to other xylose isomerases. The expression of xylA is regulated on the level of transcription by a repressor protein encoded by xylR. Its gene has the opposite orientation of xylA and the start codons are 181 bp apart. A deletion of xylR renders xylA expression constitutive. The xylR sequence was determined and is discussed with respect to its homology to other xylR structures. Primer extension analyses of the xylA and xylR transcripts under repressing and including conditions define their promoters and confirm the regulation of xylA transcription. Furthermore, some induction of the xylR transcript by xylose is also observed. The regulatory sequence of both genes consists of a bipolar promoter system and contains three palindromic sequence elements. Their potential functions with respect to xylA and xylR regulation are discussed. The primary structures of the genes, promoters and regulatory sequences are compared to the xyl regulons encoded by B. subtilis, B. megaterium, Staphylococcus xylosus and E. coli. Homology is greatest between the B. subtilis and B. megaterium encoded xyl genes while the B. licheniformis borne genes are clearly more distant. The next greater differences are found to the S. xylosus and the greatest to the E. coli encoded genes. These results are discussed with respect to the taxonomic relations of these bacteria.
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72. |
Scheler A,
Rygus T,
Allmansberger R,
Hillen W,
( 1991 ) Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus licheniformis encoded regulon for xylose utilization. PMID : 1953294 : DOI : 10.1007/bf00245345 DOI : 10.1007/bf00245345 Abstract >>
In this article we describe the cloning of the xyl regulon encoding xylose utilization from Bacillus licheniformis by complementation of a xyl mutant of B. subtilis. The xylose isomerase encoding gene, xylA, was sequenced and identified by its extensive homology to other xylose isomerases. The expression of xylA is regulated on the level of transcription by a repressor protein encoded by xylR. Its gene has the opposite orientation of xylA and the start codons are 181 bp apart. A deletion of xylR renders xylA expression constitutive. The xylR sequence was determined and is discussed with respect to its homology to other xylR structures. Primer extension analyses of the xylA and xylR transcripts under repressing and including conditions define their promoters and confirm the regulation of xylA transcription. Furthermore, some induction of the xylR transcript by xylose is also observed. The regulatory sequence of both genes consists of a bipolar promoter system and contains three palindromic sequence elements. Their potential functions with respect to xylA and xylR regulation are discussed. The primary structures of the genes, promoters and regulatory sequences are compared to the xyl regulons encoded by B. subtilis, B. megaterium, Staphylococcus xylosus and E. coli. Homology is greatest between the B. subtilis and B. megaterium encoded xyl genes while the B. licheniformis borne genes are clearly more distant. The next greater differences are found to the S. xylosus and the greatest to the E. coli encoded genes. These results are discussed with respect to the taxonomic relations of these bacteria.
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73. |
Strickler MA,
Hall JA,
Gaiko O,
Pajor AM,
( 2009 ) Functional characterization of a Na(+)-coupled dicarboxylate transporter from Bacillus licheniformis. PMID : 19840771 : DOI : 10.1016/j.bbamem.2009.10.008 PMC : PMC2787743 Abstract >>
The Na(+)-coupled dicarboxylate transporter, SdcL, from Bacillus licheniformis is a member of the divalent anion/Na(+) symporter (DASS) family that includes the bacterial Na(+)/dicarboxylate cotransporter SdcS (from Staphyloccocus aureus) and the mammalian Na(+)/dicarboxylate cotransporters, NaDC1 and NaDC3. The transport properties of SdcL produced in Escherichia coli are similar to those of its prokaryotic and eukaryotic counterparts, involving the Na(+)-dependent transport of dicarboxylates such as succinate or malate across the cytoplasmic membrane with a K(m) of approximately 6 microM. SdcL may also transport aspartate, alpha-ketoglutarate and oxaloacetate with low affinity. The cotransport of Na(+) and dicarboxylate by SdcL has an apparent stoichiometry of 2:1, and a K(0.5) for Na(+) of 0.9 mM. Our findings represent the characterization of another prokaryotic protein of the DASS family with transport properties similar to its eukaryotic counterparts, but with a broader substrate specificity than other prokaryotic DASS family members. The broader range of substrates carried by SdcL may provide insight into domains of the protein that allow a more flexible or larger substrate binding pocket.
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74. |
Gondry M,
Sauguet L,
Belin P,
Thai R,
Amouroux R,
Tellier C,
Tuphile K,
Jacquet M,
Braud S,
Courçon M,
Masson C,
Dubois S,
Lautru S,
Lecoq A,
Hashimoto S,
Genet R,
Pernodet JL,
( 2009 ) Cyclodipeptide synthases are a family of tRNA-dependent peptide bond-forming enzymes. PMID : 19430487 : DOI : 10.1038/nchembio.175 Abstract >>
Cyclodipeptides and their derivatives belong to the diketopiperazine (DKP) family, which is comprised of a broad array of natural products that exhibit useful biological properties. In the few known DKP biosynthetic pathways, nonribosomal peptide synthetases (NRPSs) are involved in the synthesis of cyclodipeptides that constitute the DKP scaffold, except in the albonoursin (1) pathway. Albonoursin, or cyclo(alpha,beta-dehydroPhe-alpha,beta-dehydroLeu), is an antibacterial DKP produced by Streptomyces noursei. In this pathway, the formation of the cyclo(Phe-Leu) (2) intermediate is catalyzed by AlbC, a small protein unrelated to NRPSs. We demonstrated that AlbC uses aminoacyl-tRNAs as substrates to catalyze the formation of the DKP peptide bonds. Moreover, several other bacterial proteins, presenting moderate similarity to AlbC, also use aminoacyl-tRNAs to synthesize various cyclodipeptides. Therefore, AlbC and these related proteins belong to a newly defined family of enzymes that we have named cyclodipeptide synthases (CDPSs).
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75. |
Islam SA,
Cho KM,
Hong SJ,
Math RK,
Kim JM,
Yun MG,
Cho JJ,
Heo JY,
Lee YH,
Kim H,
Yun HD,
( 2010 ) Chitinase of Bacillus licheniformis from oyster shell as a probe to detect chitin in marine shells. PMID : 19756579 : DOI : 10.1007/s00253-009-2215-1 Abstract >>
Bacillus licheniformis CBFOS-03 is a chitinase producing bacteria isolated from oyster (Crassostrea gigas) shell waste. We have cloned and expressed the chi18B gene of B. licheniformis CBFOS-03, which encodes a glycohydrolase family 18 chitinase (GH18). Chi18B is a predicted 598 amino acid protein that consists of a catalytic domain (GH18), a fibronectin type III domain (Fn3), and a chitin binding domain (CBD). Purified Chi18B showed optimum chitinase activity at pH 9 and 55 degrees C, and activity was stimulated with 25 mM Mn2+. In kinetic analysis, Chi18B showed Km values of 9.07 +/- 0.65 microM and 129.27 +/- 0.38 microM with the substrates 4-methylumbelliferyl-N-N'-diacetylchitobiose and alpha-chitin, respectively. Studies of C-terminal deletion constructs revealed that the GH18 domain with one amino acid in C-terminal region was sufficient for chitinase activity; however, fusions of full length and CBD-deleted constructs to green florescent protein (GFP) and yellow florescent protein (YFP) suggest that the C-terminus is supposedly important in binding to shell powder. Full length Chi18B with GFP showed green fluorescence with oyster shell powder, but GH18+Fn3 with GFP did not. Similarly, full length Chi18B with YFP showed yellow fluorescence with clam (Chamelea gallina) shell and disk abalone (Haliotis discus) shell powder, but GH18+Fn3 with YFP construct did not. So, the CBD domain of Chi18B appears to play an important role in binding of oyster and other marine shells. It is likely to be used as a probe to identify the presence of chitin in marine shells like oyster shell, clam shell, and disk abalone shell using fusions of Chi18B with fluorescent proteins.
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76. |
Xiao L,
Xie CC,
Cai J,
Lin ZJ,
Chen YH,
( 2009 ) Identification and characterization of a chitinase-produced bacillus showing significant antifungal activity. PMID : 19189178 : DOI : 10.1007/s00284-009-9363-5 Abstract >>
Strain MY75 is a gram-positive, aerobic, endospore-forming bacterium that can secrete high levels of extracellular chitinase (4.645 U/ml) when chitin powder exists as an inducer. This strain was identified as Bacillus licheniformis using the Biolog MicroLog microbial identification system and sequence analysis of 16S rDNA, gyrA and rpoB genes. Strain MY75 has the ability to inhibit the growth of Gibberella saubinetii and Aspergillus niger, two major pathogenic fungi in agriculture, and to restrain their spore germination completely. The chitinase was proved to play an important role in the strain's antifungal activity.
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77. |
van Dijl JM,
de Jong A,
Smith H,
Bron S,
Venema G,
( 1991 ) Lack of specific hybridization between the lep genes of Salmonella typhimurium and Bacillus licheniformis. PMID : 1916233 : DOI : 10.1016/0378-1097(91)90239-7 Abstract >>
This paper describes an attempt to clone the Bacillus licheniformis lep gene, encoding signal peptidase, using the Salmonella typhimurium lep gene as a hybridization probe. Although a hybridizing fragment was obtained, DNA sequence analysis indicated that it did not contain the lep gene. Instead, the protein encoded by the cloned fragment showed similarity with a variety of L-asparaginases.
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78. |
Madan B,
Mishra P,
( 2009 ) Overexpression, purification and characterization of organic solvent stable lipase from Bacillus licheniformis RSP-09. PMID : 19270444 : DOI : 10.1159/000208523 Abstract >>
The lipase gene (543 bp) from Bacillus licheniformis RSP-09, a thermophilic isolate, was overexpressed in Escherichia coli BL21 (DE3). It encodes a polypeptide of 181 residues and has 96% identity with Bacillus pumilus B26 lipase gene. The recombinant lipase was purified 19-fold to electrophoretic homogeneity by His-tag chromatography. The molecular mass of the purified recombinant B. licheniformis RSP-09 lipase was found to be 24 kDa. The purified recombinant B. licheniformis RSP-09 lipase exhibited optimal activity at pH 10.0 and 40 degrees C. The apparent K(m) and V(max) values for pNPP were found to be 453 +/- 118 microM and 288.5 +/- 33.67 micromol min(-1) mg protein(-1), respectively. The purified recombinant lipase had a wide range of substrate specificity and exhibited tolerance to both detergents and organic solvents. Thus, enzyme has potential to be employed in detergents and biocatalysis in nonaqueous solvents.
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79. |
Liu YH,
Lu FP,
Li Y,
Wang JL,
Gao C,
( 2008 ) Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations. PMID : 18626642 : DOI : 10.1007/s00253-008-1580-5 Abstract >>
This paper provided further understanding of the relationships between acid resistance and structural features of different mutants in Bacillus licheniformis alpha amylase (BLA) due to the changes of two crucial positions Leu134 and Ser320. In order to investigate effect of the two positions on the acid stability, we described the detailed characterization of wild-type and the single mutants L134R and S320A as well as the double mutant L134R/S320A. The highest k(cat)/Km with pH 4.5, approximately 14 times that of wild type, was observed in L134R/S320A. The k(cat)/Km corresponding to L134R and S320A were at an intermediate values between those for wild type and L134R/S320A. In addition, compared with wild type, which had a rapid decline of the activity, L134R/S320A could maintain its activity strongly in low pH. Meanwhile, lower tolerance of L134R and S320A in acidic conditions than that of L134R/S320A was determined. Surprisingly, the acid-resistant capability of L134R/S320A was significantly enhanced by directed evolution. These results, combined with three-dimensional structure analysis, show that the electrostatic effects play a significant role in determining the stability of BLA at two crucial positions, 134 and 320.
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80. |
Lee JK,
Edwards CW,
Hulett FM,
( 1991 ) Bacillus licheniformis APase I gene promoter: a strong well-regulated promoter in B. subtilis. PMID : 1907637 : DOI : 10.1099/00221287-137-5-1127 Abstract >>
The 5' regulatory region and the portion of the structural gene coding for the amino-terminal sequence of alkaline phosphatase I (APase I) were isolated from Bacillus licheniformis MC14 using a synthetic oligodeoxynucleotide deduced from the amino acid sequence of the enzyme. The DNA sequence analysis of this region revealed an open reading frame of 129 amino acids containing the amino-terminal sequence of the mature APase protein. The protein sequence was preceded by a putative signal sequence of 32 amino acid residues. The predicted amino acid sequence of the partial APase clone as well as the experimentally determined amino acid sequence of the enzyme indicated that B. licheniformis APase retains the important features conserved among other APases of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and various human tissues. Heterologous expression studies of the promoter using a fusion with the lacZ gene indicated that it functions as a very strong inducible promoter in B. subtilis that is tightly regulated by phosphate concentration.
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81. |
Moldover B,
Piggot PJ,
Yudkin MD,
( 1991 ) Identification of the promoter and the transcriptional start site of the spoVA operon of Bacillus subtilis and Bacillus licheniformis. PMID : 1903432 : DOI : 10.1099/00221287-137-3-527 Abstract >>
The region upstream of the coding sequence of the spoVA operon was studied by several techniques to identify the promoter and to determine the start point for transcription of spoVA. The results of plasmid integration analysis in Bacillus subtilis showed that no more than 119 bp upstream of the coding sequence is needed for expression. A comparison of the sequence of this upstream region with the corresponding sequence from Bacillus licheniformis showed four stretches that were perfectly conserved, interspersed with poorly conserved stretches; the second and third of these conserved stretches appeared to represent the '-35' and '-10' regions of a promoter recognized by RNA polymerase containing sigma G. Primer extension analysis in B. subtilis revealed a spoVA transcript which had apparently initiated 6 bp downstream of the putative '-10' heptanucleotide CATACTA, that is, 27 bp upstream of the coding sequence. This transcript was observed 4 h and 5 h after the initiation of sporulation, but not at earlier times.
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82. |
Lee CC,
Kibblewhite-Accinelli RE,
Smith MR,
Wagschal K,
Orts WJ,
Wong DW,
( 2008 ) Cloning of Bacillus licheniformis xylanase gene and characterization of recombinant enzyme. PMID : 18612683 : DOI : 10.1007/s00284-008-9193-x Abstract >>
Hemicellulose is a major component of lignocellulose biomass. Complete degradation of this substrate requires several different enzymatic activities, including xylanase. We isolated a strain of Bacillus licheniformis from a hot springs environment that exhibited xylanase activity. A gene encoding a 23-kDa xylanase enzyme, Xyn11, was cloned, and the recombinant protein was expressed in an Escherichia coli host and biochemically characterized. The optimum activity of the enzyme was at pH 5-7 and 40-50 degrees C. The enzyme was stable at temperatures up to 50 degrees C. Against birchwood xylan, the enzyme had an apparent K(m) of 6.7 mg/mL and V(max) of 379 micromol/min/mg.
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83. |
Knox JR,
Moews PC,
( 1991 ) Beta-lactamase of Bacillus licheniformis 749/C. Refinement at 2 A resolution and analysis of hydration. PMID : 1856867 : DOI : 10.1016/0022-2836(91)90023-y Abstract >>
The crystallographic and molecular structure of the class A beta-lactamase (penicillinase) of Bacillus licheniformis 749/C has been refined with X-ray diffraction data to 2 A resolution. For the 27,330 data with F greater than or equal to 3 sigma(F), the R factor is 0.15; for all 30,090 data, R is 0.16. The estimated co-ordinate error is 0.15 A. In the final model, the deviation of covalent bonds and angles from ideality is 0.012 A and 2.2 degrees, respectively. The model includes two molecules of 29,500 daltons each in the asymmetric unit of space group P2(1), 484 water molecules and two tetrahedral buffer anions. Overlay of the two protein molecules results in a root-mean-square difference of 0.17 A and 0.41 A for alpha-carbon atoms and for all atoms, respectively. Twenty-six water molecules fall within 0.25 A of matching water molecules associated with the second protein molecule. The reactive Ser70 is on a turn of 3(10) helix at the N terminus of a longer alpha-helix (72-83). The penicillin-binding site near this helix contains at least seven water molecules. Upon penicillin entry, a water molecule in the oxyanion hole, hydrogen-bonded between the N terminus of helix (80-83) and beta-strand (230-238), would be displaced by the oxygen atom of the beta-lactam carbonyl group. An unexpelled molecule of water is proposed to be the catalytic water required for penicillin hydrolysis. The water is hydrogen-bonded to Glu166, a conserved residue in all beta-lactamases, and it lies 3 A from the alpha-face of a previously modeled penicillin. The position of the water-Glu166 pair is stabilized in the active site by a cis peptide bond at Pro167.
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84. |
Zhu B,
Yu G,
Zhu J,
Shen S,
( 2000 ) Cloning and characterization of the glutamate dehydrogenase gene inBacillus licheniformis. PMID : 18726380 : DOI : 10.1007/BF02879284 Abstract >>
ThegdhA genes of IRC-3 GDH(-)strain and IRC-8 GDH(+) strain were cloned, and they both successfully complemented the nutritional lesion of anE. coli glutamate auxotroph, Q100 GDH(-). However, thegdhA gene from the mutant IRC-8 GDH(+) strain failed to complement the glutamate deficiency of the wild type strain IRC-3. ThegdhA genes of the wild type and mutant origin were sequenced separately. No nucleotide difference was detected between them. Further investigations indicated that thegdhA genes were actively expressed in both the wild type and the mutant. Additionally, no GDH inhibitor was found in the wild type strain IRC-3. It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of thegdhA expression. Examination of the deduced amino acid sequence ofBacillus licheniformis GDH revealed the presence of the motifs characteristic of the family I-type hexameric protein, while the GDH ofBacillus subtilis belongs to family II.
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85. |
Haakensen M,
Ziola B,
( 2008 ) Identification of novel horA-harbouring bacteria capable of spoiling beer. PMID : 18389005 : DOI : 10.1139/w08-007 Abstract >>
An ATP-binding cassette (ABC) multi-drug resistance (MDR) gene was found in 4 Gram-positive bacterial isolates of environmental origin and found capable of spoiling beer. The bacteria isolated were Bacillus cereus, Bacillus licheniformis, Paenibacillus humicus, and Staphylococcus epidermidis; all of which were previously unappreciated as beer-spoilage bacteria. The MDR gene found in these bacteria has less than 37% similarity to known ABC MDR proteins described for Bacillus and Staphylococcus, and this is the first finding of an ABC MDR gene in the genus Paenibacillus. The sequenced region of the gene was translated and compared phylogenetically with the closest GenBank matches of the respective species and the closest GenBank matches overall. The ABC MDR proteins from these isolates were found to cluster among known sequences of HorA, sharing 99.5% identity within the sequenced region. In the beer-spoilage-associated genera Lactobacillus and Pediococcus, the presence of the MDR gene horA correlates with the ability to grow in beer. As the unique horA-harbouring isolates described here are capable of growing in beer, it is likely that the presence of the horA gene likewise confers hop resistance to these organisms.
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86. |
Sareen R,
Mishra P,
( 2008 ) Purification and characterization of organic solvent stable protease from Bacillus licheniformis RSP-09-37. PMID : 18427806 : DOI : 10.1007/s00253-008-1429-y Abstract >>
A protease was purified from the cell-free supernatant of Bacillus licheniformis RSP-09-37, a mutant from a thermophilic bacterial strain, B. licheniformis RSP-09, using affinity chromatography with alpha-casein agarose resin. The protease was purified 85-fold to electrophoretic homogeneity. The apparent molecular mass of purified protease was 55 kDa using gel filtration in high-performance liquid chromatography, which is in agreement with the results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a monomeric nature of the protein. The purified protease revealed temperature optima of 50 degrees C and pH optima of 10.0 and was classified as serine protease based on its complete inhibition with phenyl methyl sulfonyl fluoride. The purified protease exhibited tolerance to both detergents and organic solvent. The synthetic activity of the protease was tested using the transesterification reaction between N-acetyl-L-phenylalanine-ethyl ester and n-propanol in organic solvents varying in their log P values and the kinetic parameters of the enzyme in these organic solvents were studied. The enzyme has potential to be employed for synthetic reactions and in detergent formulations.
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87. |
Meintanis C,
Chalkou KI,
Kormas KA,
Lymperopoulou DS,
Katsifas EA,
Hatzinikolaou DG,
Karagouni AD,
( 2008 ) Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus. PMID : 18266638 : DOI : 10.1111/j.1472-765X.2008.02328.x Abstract >>
To investigate the applicability of rpoB gene, which encodes the beta subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP- and BOX-polymerase chain reaction) were also used. rpoB DNA (458 bp) were amplified from 21 Geobacillus- and Bacillus type strains, producing different BOX- and REP-PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90-100% (16S rRNA) and 74-100% (rpoB) similarity among examined bacteria. BOX- and REP-PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.
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88. |
Koeppel A,
Perry EB,
Sikorski J,
Krizanc D,
Warner A,
Ward DM,
Rooney AP,
Brambilla E,
Connor N,
Ratcliff RM,
Nevo E,
Cohan FM,
( 2008 ) Identifying the fundamental units of bacterial diversity: a paradigm shift to incorporate ecology into bacterial systematics. PMID : 18272490 : DOI : 10.1073/pnas.0712205105 PMC : PMC2268166 Abstract >>
The central questions of bacterial ecology and evolution require a method to consistently demarcate, from the vast and diverse set of bacterial cells within a natural community, the groups playing ecologically distinct roles (ecotypes). Because of a lack of theory-based guidelines, current methods in bacterial systematics fail to divide the bacterial domain of life into meaningful units of ecology and evolution. We introduce a sequence-based approach ("ecotype simulation") to model the evolutionary dynamics of bacterial populations and to identify ecotypes within a natural community, focusing here on two Bacillus clades surveyed from the "Evolution Canyons" of Israel. This approach has identified multiple ecotypes within traditional species, with each predicted to be an ecologically distinct lineage; many such ecotypes were confirmed to be ecologically distinct, with specialization to different canyon slopes with different solar exposures. Ecotype simulation provides a long-needed natural foundation for microbial ecology and systematics.
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89. |
Meerak J,
Iida H,
Watanabe Y,
Miyashita M,
Sato H,
Nakagawa Y,
Tahara Y,
( 2007 ) Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from fermented soybean foods manufactured in Asian countries. PMID : 18187886 : Abstract >>
Natto-like fermented soybean products are manufactured and consumed in many Asian countries. In this study, we isolated thirty-four Bacillus strains capable of producing gamma-polyglutamic acid (PGA) from natto in mountainous areas of South Asia and Southeast Asia and from soils in Japan. To elucidate the phylogeny of these PGA-producing strains, phylogenetic trees based on sequences of 16S rDNA, housekeeping genes of rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) were constructed. A phylogenetic tree based on 16S rDNA sequences showed that twenty-one isolates were clustered in the same group of B. subtilis. The other thirteen isolates were located in the cluster of B. amyloliquefaciens. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. The results of the present study indicate that PGA-producing strains isolated from local natto in Asian countries and soil in Japan can be divided into two species, B. subtilis and B. amyloliquefaciens.
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90. |
Liu YH,
Lu FP,
Li Y,
Yin XB,
Wang Y,
Gao C,
( 2008 ) Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600. PMID : 18157528 : DOI : 10.1007/s00253-007-1287-z Abstract >>
Based on the original thermostable alpha-amylase gene from Bacillus licheniformis, two amino acids were site-directed mutagenised by polymerase chain reaction to obtain a new gene. This gene, with Leu134-->Arg and Ser320-->Ala, was substituted for acid-resistant capability previously. To favor purification of the product, high-level expression and secretion of mature, authentic and stable recombinant mutagenised alpha-amylase were achieved with protease-deficient strain Bacillus subtilis WB600 as the host. The recombinant mutagenised alpha-amylase with the activity of 4,700 U/mL was then purified by ammonium sulphate fractionation, anion exchange and gel filtration, consecutively. By multi-step purification, the specific activity of the recombinant protein was up to 916.7 U/mg with a 187.1-fold purification. The mutagenised protein was found to be more acid resistant than the native protein. The optimum pH and stable range of pH with the mutagenised protein was 4.5 and 4.0 to 6.5, respectively, compared with pH 6.5 and 5.5 to 7.0 as the favorite pH and pH stability range of the native protein.
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91. |
Yamabhai M,
Emrat S,
Sukasem S,
Pesatcha P,
Jaruseranee N,
Buranabanyat B,
( 2008 ) Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems. PMID : 17950946 : DOI : 10.1016/j.jbiotec.2007.09.005 Abstract >>
Bacillus spp. are Gram-positive bacteria that secrete a large number of extracellular proteins of industrial relevance. In this report, three Bacillus extracellular hydrolytic enzymes, i.e., alpha-amylase, mannanase and chitinase, were cloned and over-expressed in Gram-negative Escherichia coli. We found that both the native signal peptides and that of E. coli outer membrane protein, OmpA, could be used to direct the secretion of the recombinant enzymes. The expressed enzymes were observed as clearing zones on agar plates or in zymograms. Determination of enzyme activities in different cell compartments suggested that the ability of the enzymes to be secreted out into the culture medium depends on the time of induction, the type of the signal peptides and the molecular mass of the enzymes. After overnight induction, most of the enzyme activities (85-96%) could be harvested from the culture supernatant. Our results suggest that various signal peptides of Bacillus spp. can be recognized by the E. coli secretion machinery. It seems possible that other enzymes with similar signal peptide could be secreted equally well in E. coli expression systems. Thus, our finding should be able to apply for cloning and extracellular production of other Bacillus hydrolytic enzymes as well as other proteins.
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92. |
Nieminen T,
Rintaluoma N,
Andersson M,
Taimisto AM,
Ali-Vehmas T,
Seppälä A,
Priha O,
Salkinoja-Salonen M,
( 2007 ) Toxinogenic Bacillus pumilus and Bacillus licheniformis from mastitic milk. PMID : 17611049 : DOI : 10.1016/j.vetmic.2007.05.015 Abstract >>
To elucidate the occurrence of heat-stable toxin-producing strains among mastitic Bacillus isolates, 100 milk samples of mastitic cows from different parts of Finland were screened. Bacillus was identified as the major organism in 23 samples. Toxinogenic Bacillus isolates identified by sperm cell motility inhibition assay were isolated from six samples. Four isolates belonged to the species Bacillus pumilus and two to Bacillus licheniformis. The toxic substances were heat-stable and soluble to methanol thus being of non-protein nature. The methanol extracted substances disrupted the sperm cell plasma membrane permeability barrier at exposure concentrations of 1-15 microg ml(-1) (B. pumilus) or 20-30 microg ml(-1) (B. licheniformis). The toxic properties of the two mastitic B. licheniformis strains were similar to those of B. licheniformis strains known to produce the lipopeptide lichenysin A and the synthetase genes lchAA, lchAB and lchAC for lichenysin were found in the mastitic strains by PCR. Toxin synthetase genes for the syntheses of lichenysin or surfactin were searched but not found in the toxic B. pumilus strains. The ribopatterns of the mastitic B. pumilus and B. licheniformis isolates were similar to those of the toxinogenic strains described earlier from food poisoning incidents and contaminated indoor air. B. licheniformis and B. pumilus survive pasteurization and other heat treatments as spores. Toxin-producing strains of these species in the dairy production chain may thus be of food safety concern.
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93. |
Boudet J,
Duval V,
Van Melckebeke H,
Blackledge M,
Amoroso A,
Joris B,
Simorre JP,
( 2007 ) Conformational and thermodynamic changes of the repressor/DNA operator complex upon monomerization shed new light on regulation mechanisms of bacterial resistance against beta-lactam antibiotics. PMID : 17576674 : DOI : 10.1093/nar/gkm448 PMC : PMC1935004 Abstract >>
In absence of beta-lactam antibiotics, BlaI and MecI homodimeric repressors negatively control the expression of genes involved in beta-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to beta-lactam presence, BlaI/MecI is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA. These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.
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94. |
Radha S,
Gunasekaran P,
( 2007 ) Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. PMID : 17897234 : DOI : 10.1111/j.1365-2672.2007.03372.x Abstract >>
Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. The keratinase gene with and without leader sequence from the chromosomal DNA of Bacillus licheniformis MKU3 was amplified by PCR and cloned into pET30b and transferred into Escherichia coli BL21. The ker gene without leader sequence only expressed in E. coli and the recombinant strain produced an intracellular keratinase activity of 74.3 U ml(-1). The ker gene was further subcloned into E. coli-Bacillus shuttle vector, pWH1520. Bacillus megaterium ATCC 14945 carrying the recombinant plasmid pWHK3 expressed the ker gene placed under xylA promoter and produced an extracellular keratinase activity of 95 U ml(-1). Response surface methodology (RSM) was employed to optimize the fermentation condition and to improve the level of keratinase production by the recombinant strain. A maximum keratinolytic activity of 166.2 U ml(-1) (specific activity, 33.25 U mg(-1)) was obtained in 18 h of the fermentation carried out with an initial inoculum of 0.4 OD600 nm and xylose concentration of 0.75% w/v. Bacillus licheniformis keratinase was cloned and successfully expressed using T7 promoter in E. coli and xylose inducible expression system in B. megaterium. Response surface methodology was employed to optimize the process parameters, which resulted in a three-fold higher level of keratinase production by the recombinant B. megaterium (pWHK3) than the wild type strain B. licheniformis MKU3. This study suggests that B. megaterium is a suitable host for the expression of cloned genes from heterologous origin. Optimization of fermentation conditions improved the keratinase production by B. megaterium (pWHK3) and suggested that this recombinant strain could be used for the production of keratinase.
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95. |
Zhu YF,
Kobayashi T,
Lampen JO,
( 1988 ) A hypothetical protein (P20), homologous to Tn3 repressor is encoded downstream from the bla regulatory region in Bacillus licheniformis 749. PMID : 2838827 : DOI : 10.1093/nar/16.12.5691 PMC : PMC336798 Abstract >>
N/A
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96. |
Safary A,
Moniri R,
Hamzeh-Mivehroud M,
Dastmalchi S,
( 2016 ) Identification and Molecular Characterization of Genes Coding Pharmaceutically Important Enzymes from Halo-Thermo Tolerant Bacillus. PMID : 28101462 : DOI : 10.15171/apb.2016.069 PMC : PMC5241413 Abstract >>
Purpose: Robust pharmaceutical and industrial enzymes from extremophile microorganisms are main source of enzymes with tremendous stability under harsh conditions which make them potential tools for commercial and biotechnological applications. Methods: The genome of a Gram-positive halo-thermotolerant Bacillus sp. SL1, new isolate from Saline Lake, was investigated for the presence of genes coding for potentially pharmaceutical enzymes. We determined gene sequences for the enzymes laccase (CotA), l-asparaginase (ansA3, ansA1), glutamate-specific endopeptidase (blaSE), l-arabinose isomerase (araA2), endo-1,4-�] mannosidase (gmuG), glutaminase (glsA), pectate lyase (pelA), cellulase (bglC1), aldehyde dehydrogenase (ycbD) and allantoinases (pucH) in the genome of Bacillus sp. SL1. Results: Based on the DNA sequence alignment results, six of the studied enzymes of Bacillus sp. SL-1 showed 100% similarity at the nucleotide level to the same genes of B. licheniformis 14580 demonstrating extensive organizational relationship between these two strains. Despite high similarities between the B. licheniformis and Bacillus sp. SL-1 genomes, there are minor differences in the sequences of some enzyme. Approximately 30% of the enzyme sequences revealed more than 99% identity with some variations in nucleotides leading to amino acid substitution in protein sequences. Conclusion: Molecular characterization of this new isolate provides useful information regarding evolutionary relationship between B. subtilis and B. licheniformis species. Since, the most industrial processes are often performed in harsh conditions, enzymes from such halo-thermotolerant bacteria may provide economically and industrially appealing biocatalysts to be used under specific physicochemical situations in medical, pharmaceutical, chemical and other industries.
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97. |
Huang CH,
Huang L,
Chang MT,
Chen KL,
( 2016 ) Establishment and application of an analytical in-house database (IHDB) for rapid discrimination of Bacillus subtilis group (BSG) using whole-cell MALDI-TOF MS technology. PMID : 27507023 : DOI : 10.1016/j.mcp.2016.08.002 Abstract >>
Members of the Bacillus subtilis group (BSG) possess industrial applicability; unfortunately, B. subtilis and its phylogenetically closest species are indistinguishable from one another using 16S rDNA sequencing, physiological and biochemical tests. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively novel technique for the fast and reliable identification of microorganisms. The aim of this study was to construct a unique analytical in-house database (IHDB) for BSG discrimination based on whole-cell protein fingerprinting using MALDI-TOF MS, as well as to discover biomarkers from the MS peaks to generate a classification model for further differentiation using the ClinProTools software. Type strains of 12 species (included five subspecies) of the BSG were used to build a main spectrum profile (MSP) to create an IHDB under the optimized parameters. The BSG isolates obtained from partial recA gene sequencing were used for IHDB validation. A total of 84 (100%) isolates were correctly identified to the species level and had high score values (mean score: 2.52). However, the IHDB had ambiguous identification at the subspecies level of Bacillus amyloliquefaciens. After implementation of the classification models, the strains could be clearly differentiated. We have successfully developed a rapid, accurate and cost-effective platform for the species- and subspecies-level discrimination of BSG based on the implementation of the IHDB and coupled with ClinProTools, which can be employed as an alternative technology to DNA sequencing and applied for efficient quality control of the microbial agent.
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98. |
Liberato MV,
Silveira RL,
Prates ?T,
de Araujo EA,
Pellegrini VO,
Camilo CM,
Kadowaki MA,
Neto Mde O,
Popov A,
Skaf MS,
Polikarpov I,
( 2016 ) Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism. PMID : 27032335 : DOI : 10.1038/srep23473 PMC : PMC4817029 Abstract >>
Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.
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99. |
Zhang K,
Guo Y,
Yao P,
Lin Y,
Kumar A,
Liu Z,
Wu G,
Zhang L,
( 2016 ) Characterization and directed evolution of BliGO, a novel glycine oxidase from Bacillus licheniformis. PMID : 26920475 : DOI : 10.1016/j.enzmictec.2015.12.012 Abstract >>
Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtilis. The optimal activity of BliGO was observed at pH 8.5 and 40�XC. Interestingly, BliGO retained 60% of the maximum activity at 0�XC, suggesting it is a cold-adapted enzyme. The kinetic parameters on glyphosate (Km, kcat and k(cat)/K(m)) of BliGO were 11.22 mM, 0.08 s(-1), and 0.01 mM(-1) s(-1), respectively. To improve the catalytic activity to glyphosate, the BliGO was engineered by directed evolution. With error-prone PCR and two rounds of DNA shuffling, the most evolved mutant SCF-4 was obtained from 45,000 colonies, which showed 7.1- and 8-fold increase of affinity (1.58 mM) and catalytic efficiency (0.08 mM(-1) s(-1)) to glyphosate, respectively. In contrast, its activity to glycine (the natural substrate of GO) decreased by 113-fold. Structure modeling and site-directed mutation study indicated that Ser51 in SCF-4 involved in the binding of enzyme with glyphosate and played a crucial role in the improvement of catalytic efficiency.
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100. |
Lin MG,
Chi MC,
Naveen V,
Li YC,
Lin LL,
Hsiao CD,
( 2016 ) Bacillus licheniformis trehalose-6-phosphate hydrolase structures suggest keys to substrate specificity. PMID : 26894535 : DOI : 10.1107/S2059798315020756 Abstract >>
Trehalose-6-phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to yield glucose and glucose 6-phosphate. The products of this reaction can be further metabolized by the energy-generating glycolytic pathway. Here, crystal structures of Bacillus licheniformis TreA (BlTreA) and its R201Q mutant complexed with p-nitrophenyl-�\-D-glucopyranoside (R201Q-pPNG) are presented at 2.0 and 2.05 ? resolution, respectively. The overall structure of BlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site of BlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions of Bacillus cereus oligo-1,6-glucosidase (BcOgl) and Erwinia rhapontici isomaltulose synthase (NX-5), the surface potential of the BlTreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity of BlTreA. Strikingly, the (281)HHLK(284) motif and Lys292 play critical roles in substrate discrimination by BlTreA.
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101. |
Bourbouli M,
Katsifas EA,
Papathanassiou E,
Karagouni AD,
( 2015 ) The Kolumbo submarine volcano of Santorini island is a large pool of bacterial strains with antimicrobial activity. PMID : 25627249 : DOI : 10.1007/s00203-015-1086-3 Abstract >>
Microbes in hydrothermal vents with their unique secondary metabolism may represent an untapped potential source of new natural products. In this study, samples were collected from the hydrothermal field of Kolumbo submarine volcano in the Aegean Sea, in order to isolate bacteria with antimicrobial activity. Eight hundred and thirty-two aerobic heterotrophic bacteria were isolated and then differentiated through BOX-PCR analysis at the strain level into 230 genomic fingerprints, which were screened against 13 different type strains (pathogenic and nonpathogenic) of Gram-positive, Gram-negative bacteria and fungi. Forty-two out of 176 bioactive-producing genotypes (76 %) exhibited antimicrobial activity against at least four different type strains and were selected for 16S rDNA sequencing and screening for nonribosomal peptide (NRPS) and polyketide (PKS) synthases genes. The isolates were assigned to genus Bacillus and Proteobacteria, and 20 strains harbored either NRPS, PKS type I or both genes. This is the first report on the diversity of culturable mesophilic bacteria associated with antimicrobial activity from Kolumbo area; the extremely high proportion of antimicrobial-producing strains suggested that this unique environment may represent a potential reservoir of novel bioactive compounds.
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102. |
Hu H,
Li L,
Ding S,
( 2015 ) An organic solvent-tolerant phenolic acid decarboxylase from Bacillus licheniformis for the efficient bioconversion of hydroxycinnamic acids to vinyl phenol derivatives. PMID : 25547838 : DOI : 10.1007/s00253-014-6313-3 Abstract >>
A new phenolic acid decarboxylase gene (blpad) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli. The full-length blpad encodes a 166-amino acid polypeptide with a predicted molecular mass and pI of 19,521 Da and 5.02, respectively. The recombinant BLPAD displayed maximum activity at 37 �XC and pH 6.0. This enzyme possesses a broad substrate specificity and is able to decarboxylate p-coumaric, ferulic, caffeic, and sinapic acids at the relative ratios of specific activities 100:74.59:34.41:0.29. Kinetic constant K m values toward p-coumaric, ferulic, caffeic, and sinapic acids were 1.64, 1.55, 1.93, and 2.45 mM, and V max values were 268.43, 216.80, 119.07, and 0.78 U mg(-1), respectively. In comparison with other phenolic acid decarboxylases, BLPAD exhibited remarkable organic solvent tolerance and good thermal stability. BLPAD showed excellent catalytic performance in biphasic organic/aqueous systems and efficiently converted p-coumaric and ferulic acids into 4-vinylphenol and 4-vinylguaiacol. At 500 mM of p-coumaric and ferulic acids, the recombinant BLPAD produced a total 60.63 g l(-1) 4-vinylphenol and 58.30 g l(-1) 4-vinylguaiacol with the conversion yields 97.02 and 70.96 %, respectively. The low yield and product concentration are the crucial drawbacks to the practical bioproduction of vinyl phenol derivatives using phenolic acid decarboxylases. These unusual properties make BLPAD a desirable biocatalyst for commercial use in the bioconversion of hydroxycinnamic acids to vinyl phenol derivatives via enzymatic decarboxylation in a biphasic organic/aqueous reaction system.
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103. |
De Bellis P,
Minervini F,
Di Biase M,
Valerio F,
Lavermicocca P,
Sisto A,
( 2015 ) Toxigenic potential and heat survival of spore-forming bacteria isolated from bread and ingredients. PMID : 25555227 : DOI : 10.1016/j.ijfoodmicro.2014.12.017 Abstract >>
Fifty-four spore-forming bacterial strains isolated from bread ingredients and bread, mainly belonging to the genus Bacillus (including Bacillus cereus), together with 11 reference strains were investigated to evaluate their cytotoxic potential and heat survival in order to ascertain if they could represent a risk for consumer health. Therefore, we performed a screening test of cytotoxic activity on HT-29 cells using bacterial culture filtrates after growing bacterial cells in Brain Heart Infusion medium and in the bread-based medium Bread Extract Broth (BEB). Moreover, immunoassays and PCR analyses, specifically targeting already known toxins and related genes of B. cereus, as well as a heat spore inactivation assay were carried out. Despite of strain variability, the results clearly demonstrated a high cytotoxic activity of B. cereus strains, even if for most of them it was significantly lower in BEB medium. Cytotoxic activity was also detected in 30% of strains belonging to species different from B. cereus, although, with a few exceptions (e.g. Bacillus simplex N58.2), it was low or very low. PCR analyses detected the presence of genes involved in the production of NHE, HBL or CytK toxins in B. cereus strains, while genes responsible for cereulide production were not detected. Production of NHE and HBL toxins was also confirmed by specific immunoassays only for B. cereus strains even if PCR analyses revealed the presence of related toxin genes also in some strains of other species. Viable spore count was ascertained after a heat treatment simulating the bread cooking process. Results indicated that B. amyloliquefaciens strains almost completely survived the heat treatment showing less than 2 log-cycle reductions similarly to two strains of B. cereus group III and single strains belonging to Bacillus subtilis, Bacillus mojavensis and Paenibacillus spp. Importantly, spores from strains of the B. cereus group IV exhibited a thermal resistance markedly lower than B. cereus group III with high values of log-cycle reductions. In conclusion, our results indicate that spore-forming bacteria contaminating bread ingredients and bread could represent a source of concern for consumer health related to the presence of strains, such as strains of B. cereus group III and single strains of other species, showing the ability to produce toxic substances associated to a thermal resistance enough to survive the bread cooking conditions.
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104. |
Hebecker S,
Krausze J,
Hasenkampf T,
Schneider J,
Groenewold M,
Reichelt J,
Jahn D,
Heinz DW,
Moser J,
( 2015 ) Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine. PMID : 26261323 : DOI : 10.1073/pnas.1511167112 PMC : PMC4553816 Abstract >>
The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.
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105. |
Sudhir AP,
Dave BR,
Prajapati AS,
Panchal K,
Patel D,
Subramanian RB,
( 2014 ) Characterization of a recombinant glutaminase-free L-asparaginase (ansA3) enzyme with high catalytic activity from Bacillus licheniformis. PMID : 25224912 : DOI : 10.1007/s12010-014-1200-z Abstract >>
L-Asparaginase (3.5.1.1) is an enzyme widely used to treat the acute lymphoblastic leukemia. Two genes coding for L-asparaginase (ansA1 and ansA3) from Bacillus licheniformis MTCC 429 were cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were purified to homogeneity by one-step purification process and further characterized for various biochemical parameters. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that both the enzymes are monomers of ?37 kDa. Recombinant ansA1 was found to be highly unstable, and recombinant ansA3 was catalytically active and stable, which showed an optimum activity of 407.65 IU/mg at 37 �XC and pH 8. Recombinant ansA3 showed higher substrate specificity for L-asparagine with negligible glutaminase activity. Kinetic parameters like K m , V max, k cat, and k cat/K m were calculated for recombinant ansA3.
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106. |
Yudkin MD,
Appleby L,
Smith AJ,
( 1989 ) Nucleotide sequence of the Bacillus licheniformis homologue of the sporulation locus spoIIA of Bacillus subtilis. PMID : 2513372 : DOI : 10.1099/00221287-135-4-767 Abstract >>
The spoIIA locus of Bacillus licheniformis was cloned into the phage vector phi 105J9; selection was based on the ability of the clone to complement mutations in both the first and the last of the spoIIA genes of B. subtilis. The B. licheniformis DNA was subcloned into M13 and sequenced; it includes three genes of identical lengths to those of B. subtilis. The average interspecies difference in nucleotide sequence is 24%; C occurs at the third position of codons 55% more often in the B. licheniformis than in the B. subtilis sequence. The average difference in predicted amino acid sequence is 11%, but the distribution of these differences is far from random, and there are several long stretches of amino acid sequence that are identical in the two organisms. The distribution of non-conservative amino acid changes between the species is also strikingly non-random; no such changes are found in those regions in which missense mutations are known in B. subtilis. Each species has an open reading frame 5' of the spoIIAA gene, but the predicted amino acid sequence, and the distribution of differences between the two species in both nucleotides and predicted amino acids, suggest that these open reading frames are not expressed. Both species have regions 3' of the open reading frames which resemble rho-independent terminators of transcription, but that in B. licheniformis is longer and could form a more elaborate secondary structure than that in B. subtilis.
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107. |
Kpikpi EN,
Thorsen L,
Glover R,
Dzogbefia VP,
Jespersen L,
( 2014 ) Identification of Bacillus species occurring in Kantong, an acid fermented seed condiment produced in Ghana. PMID : 24747716 : DOI : 10.1016/j.ijfoodmicro.2014.03.028 Abstract >>
Kantong is a condiment produced in Ghana by the spontaneous fermentation of kapok tree (Ceiba pentandra) seeds with cassava flour as an additive. Fermentation is over a 48h period followed by a drying and a kneading process. Although lactic acid bacteria (LAB) have previously been identified other micro-organisms may also be involved in the fermentation process. In this study we examined the occurrence of aerobic endospore-forming bacteria (AEB) in raw materials, during fermentation and in the final product at 2 production sites in Northern Ghana. Total aerobic mesophilic bacterial counts increased from 5.4��0.1log10CFU/g in the raw materials to 8.9��0.1log10CFU/g in the final products, with the AEB accounting for between 23% and 80% of the total aerobic mesophilic (TAM) counts. A total of 196 AEB were identified at a species/subspecies level by the use of phenotypic tests and genotypic methods including M13-PCR typing, 16S rRNA and gyrA gene sequencing. Bacillus subtilis subsp. subtilis (63% of the AEB), Bacillus safensis (26% of the AEB) and Bacillus amyloliquefaciens subsp. plantarum/Bacillus methylotrophicus (9% of the AEB) were the predominant Bacillus species during fermentation and in the final products. B. amyloliquefaciens/B. methylotrophicus originated from cassava flour, B. safensis from seeds and cassava flour, while the origin of B. subtilis was less clear. Brevibacillus agri and Peanibacillus spp. occurred sporadically. Further investigations are required to elucidate the role of AEB occurring in high numbers, in the fermentation of Kantong.
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108. |
Laoide BM,
Chambliss GH,
McConnell DJ,
( 1989 ) Bacillus licheniformis alpha-amylase gene, amyL, is subject to promoter-independent catabolite repression in Bacillus subtilis. PMID : 2540150 : DOI : 10.1128/jb.171.5.2435-2442.1989 PMC : PMC209918 Abstract >>
Expression of the Bacillus licheniformis alpha-amylase gene, amyL, was temporally activated and subject to catabolite repression both in its natural host and when cloned on a 3.55-kilobase fragment in Bacillus subtilis. A subclone from which the promoter region of amyL and sequences upstream from the promoter were deleted had a low level of amylase activity. Expression of the promoterless gene was still subject to repression by glucose when the gene was present either on a multicopy plasmid or integrated into the B. subtilis chromosome. Catabolite repression occurred independently of the amylase promoter and irrespective of the distance of the promoterless amyL gene from the promoter which transcribed it. The transcriptional start sites of amyL activated by its own promoter and by a vector sequence promoter were determined by S1 mapping. alpha-Amylase-specific mRNA levels were measured in repressing and nonrepressing media, and catabolite repression was found to act at the level of transcription.
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109. |
Kumar V,
Sangwan P,
Verma AK,
Agrawal S,
( 2014 ) Molecular and biochemical characteristics of recombinant �]-propeller phytase from Bacillus licheniformis strain PB-13 with potential application in aquafeed. PMID : 24687556 : DOI : 10.1007/s12010-014-0871-9 Abstract >>
Phytic acid is the major storage form of organic phosphorus in nature- and plant-based animal feed. It forms insoluble complexes with nutritionally important metals and proteins that are unavailable for monogastric or agastric animals. Phytases initiate the stepwise hydrolysis of phytic acid and release inorganic orthophosphate. In the present investigation, the phytase gene from a phytase producing Bacillus licheniformis strain PB-13 was successfully expressed in Escherichia coli BL21. Recombinant phytase 'rPhyPB13' was found to be catalytically active, with an activity of 0.97 U/mL and specific activity of 0.77 U/mg. The rPhyPB13 was purified to 14.10-fold using affinity chromatography. Similar to other �]-propeller phytases, purified rPhyPB13 exhibited maximal activity at pH 6.0-6.5 and 60 �XC in the presence of 1 mM Ca(2+) and was highly active over a wider pH range (pH 4.0-8.0) and high temperature (80 �XC). It has shown maximum activity towards Na-phytate as substrate. The observed K m , V max and k cat of purified rPhyPB13 were 1.064 mM, 1.32 �gmol/min/mg and 27.46 s(-1), respectively. PhyPB13 was resistant to trypsin inactivation, activated in presence of Ca(2+) and inhibited in presence of EDTA. Crude rPhyPB13 has good digestion efficiency for commercial feed and soybean meal. These results indicate that PhyPB13 is a �]-propeller phytase that has application potential in aquaculture feed.
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110. |
Tian J,
Zhang Y,
Liu B,
Zuo D,
Jiang T,
Guo J,
Zhang W,
Wu N,
Fan Y,
( 2013 ) Presep: predicting the propensity of a protein being secreted into the supernatant when expressed in Pichia pastoris. PMID : 24278168 : DOI : 10.1371/journal.pone.0079749 PMC : PMC3836778 Abstract >>
Pichia pastoris is commonly used for the production of recombinant proteins due to its preferential secretion of recombinant proteins, resulting in lower production costs and increased yields of target proteins. However, not all recombinant proteins can be successfully secreted in P. pastoris. A computational method that predicts the likelihood of a protein being secreted into the supernatant would be of considerable value; however, to the best of our knowledge, no such tool has yet been developed. We present a machine-learning approach called Presep to assess the likelihood of a recombinant protein being secreted by P. pastoris based on its pseudo amino acid composition (PseAA). Using a 20-fold cross validation, Presep demonstrated a high degree of accuracy, with Matthews correlation coefficient (MCC) and overall accuracy (Q2) scores of 0.78 and 95%, respectively. Computational results were validated experimentally, with six �]-galactosidase genes expressed in P. pastoris strain GS115 to verify Presep model predictions. A strong correlation (R(2) = 0.967) was observed between Presep prediction secretion propensity and the experimental secretion percentage. Together, these results demonstrate the ability of the Presep model for predicting the secretion propensity of P. pastoris for a given protein. This model may serve as a valuable tool for determining the utility of P. pastoris as a host organism prior to initiating biological experiments. The Presep prediction tool can be freely downloaded at http://www.mobioinfor.cn/Presep.
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111. |
Jakobs M,
Hoffmann K,
Grabke A,
Neuber S,
Liesegang H,
Volland S,
Meinhardt F,
( 2014 ) Unravelling the genetic basis for competence development of auxotrophic Bacillus licheniformis 9945A strains. PMID : 25009236 : DOI : 10.1099/mic.0.079236-0 Abstract >>
Bacterial natural genetic competence - well studied in Bacillus subtilis - enables cells to take up and integrate extracellularly supplied DNA into their own genome. However, little is known about competence development and its regulation in other members of the genus, although DNA uptake machineries are routinely encoded. Auxotrophic Bacillus licheniformis 9945A derivatives, obtained from repeated rounds of random mutagenesis, were long known to develop natural competence. Inspection of the colony morphology and extracellular enzyme secretion of two of these derivatives, M28 and M18, suggested that regulator genes are collaterally hit. M28 emerged as a 14 bp deletion mutant concomitantly displaying a shift in the reading frame of degS that encodes the sensor histidine kinase, which is part of the molecular switch that directs cells to genetic competence, the synthesis of extracellular enzymes or biofilm formation, while for M18, sequencing of the suspected gene revealed a 375 bp deletion in abrB, encoding the major transition state regulator. With respect to colony morphology, enzyme secretion and competence development, both of the mutations, when newly generated on the wild-type B. licheniformis 9945A genetic background, resulted in phenotypes resembling M28 and M18, respectively. All of the known naturally competent B. licheniformis representatives, hitherto thoroughly investigated in this regard, carry mutations in regulator genes, and hence genetic competence observed in domesticated strains supposedly results from deregulation.
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112. |
Yuuki T,
Nomura T,
Tezuka H,
Tsuboi A,
Yamagata H,
Tsukagoshi N,
Udaka S,
( 1985 ) Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences. PMID : 2418011 : DOI : 10.1093/oxfordjournals.jbchem.a135381 Abstract >>
The gene coding for the heat-stable and pH-stable alpha-amylase of Bacillus licheniformis 584 (ATCC 27811) was cloned in Escherichia coli and the nucleotide sequence of a DNA fragment of 1,948 base pairs containing the entire amylase gene was determined. As inferred from the DNA sequence, the B. licheniformis alpha-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55,200. The amino acid sequence of B. licheniformis alpha-amylase showed 65.4% and 80.3% homology with those of heat-stable Bacillus stearothermophilus alpha-amylase and relatively heat-unstable Bacillus amyloliquefaciens alpha-amylase, respectively. Nevertheless, several regions of the alpha-amylases appeared to be clearly distinct from one another when their hydropathy profiles were compared.
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113. |
Tsvetanova F,
Petrova P,
Petrov K,
( 2014 ) 2,3-butanediol production from starch by engineered Klebsiella pneumoniae G31-A. PMID : 24323288 : DOI : 10.1007/s00253-013-5418-4 Abstract >>
2,3-Butanediol (2,3-BD) is an organic compound, which is widely used as a fuel and fuel additive and applied in chemical, food, and pharmaceutical industries. Contemporary strategies for its economic synthesis include the development of microbial technologies that use starch as cheap and renewable feedstock. The present work encompasses the metabolic engineering of the excellent 2,3-BD producer Klebsiella pneumoniae G31. In order to perform direct starch conversion into 2,3-BD, the amyL gene encoding quite active, liquefying �\-amylase in Bacillus licheniformis was cloned under lac promoter control in the recombinant K. pneumoniae G31-A. The enhanced extracellular over-expression of amyL led to the highest extracellular amylase activity (68 U/ml) ever detected in Klebsiella. The recombinant strain was capable of simultaneous saccharification and fermentation (SSF) of potato starch to 2,3-BD. In SSF batch process by the use of 200 g/l starch, the amount of total diols produced was 60.9 g/l (53.8 g/l 2,3-BD and 7.1 g/l acetoin), corresponding to 0.31 g/g conversion rate. The presented results are the first to show successful starch conversion to 2,3-BD by K. pneumoniae in a one-step process.
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114. |
Silva IR,
Jers C,
Otten H,
Nyffenegger C,
Larsen DM,
Derkx PM,
Meyer AS,
Mikkelsen JD,
Larsen S,
( 2014 ) Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations. PMID : 24419797 : DOI : 10.1007/s00253-013-5483-8 Abstract >>
Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of �\-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by �]-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM 13/ATCC14580) was examined by using a combinatorial protein engineering approach exploring additive effects of single amino acid substitutions. These were selected by using a consensus approach together with assessing protein stability changes (PoPMuSiC) and B-factor iterative test (B-FIT). The second-generation mutants involved combinations of two to seven individually favorable single mutations. Thermal stability was examined as half-life at 60 �XC and by recording of thermal transitions by circular dichroism. Surprisingly, the biggest increment in thermal stability was achieved by producing the wild-type RGI lyase in Bacillus subtilis as opposed to in Pichia pastoris; this effect is suggested to be a negative result of glycosylation of the P. pastoris expressed enzyme. A ~ twofold improvement in thermal stability at 60 �XC, accompanied by less significant increases in T m of the enzyme mutants, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino acids to hydrophobic ones in surface-exposed loops produced favorable thermal stability effects.
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115. |
Beri? T,
Stankovi? S,
Dragani? V,
Koji? M,
Lozo J,
Fira D,
( 2014 ) Novel antilisterial bacteriocin licheniocin 50.2 from Bacillus licheniformis VPS50.2 isolated from soil sample. PMID : 24238327 : DOI : 10.1111/jam.12393 Abstract >>
To isolate and characterize bacteriocin, licheniocin 50.2, from soil bacteria identified as Bacillus licheniformis. The strain B. licheniformis VPS50.2 was identified as bacteriocin producer, effective against Gram-positive bacteria, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and �]-haemolytic streptococci. The start of bacteriocin production coincides with the beginning of sporulation. Ammonium sulfate precipitation, chloroform extraction and ultrafiltration were used for bacteriocin purification. MALDI TOF/TOF mass spectrometry of purified sample detected the protein with molecular mass of 3253�P209 Da. N-terminal sequencing recognized first 15 amino acids with the sequence: W E E Y N I I X Q L G N K G Q. We named the newly characterized bacteriocin as subclass II.3 bacteriocin, licheniocin 50�P2. The bacteriocin activity was insensitive to lysozyme and proteinase K, heat stable after incubation at 100�XC for 30 min and over wide range of pH (2-12). MICs of crude bacteriocin extract were determined for L. monocytogenes and MRSA. Time-kill study showed that licheniocin had bactericidal effect to L. monocytogenes. A novel, thermostable, pH-tolerant bacteriocin active against Gram-positive bacteria was isolated. Attributes of new, stable licheniocin 50.2 make it a promising agent for application as biopreservative in food industry.
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116. |
Martínez-Gómez AI,
Soriano-Maldonado P,
Andújar-Sánchez M,
Clemente-Jiménez JM,
Rodríguez-Vico F,
Neira JL,
Las Heras-Vázquez FJ,
Martínez-Rodríguez S,
( 2014 ) Biochemical and mutational studies of allantoinase from Bacillus licheniformis CECT 20T. PMID : 24333989 : DOI : 10.1016/j.biochi.2013.12.002 Abstract >>
Allantoinases (allantoin amidohydrolase, E.C. 3.5.2.5) catalyze the hydrolysis of the amide bond of allantoin to form allantoic acid, in those organisms where allantoin is not the final product of uric acid degradation. Despite their importance in the purine catabolic pathway, sequences of microbial allantoinases with proven activity are scarce, and only the enzyme from Escherichia coli (AllEco) has been studied in detail in the genomic era. In this work, we report the cloning, purification and characterization of the recombinant allantoinase from Bacillus licheniformis CECT 20T (AllBali). The enzyme was a homotetramer with an apparent Tm of 62 �� 1 �XC. Optimal parameters for the enzyme activity were pH 7.5 and 50 �XC, showing apparent Km and kcat values of 17.7 �� 2.7 mM and 24.4 �� 1.5 s(-1), respectively. Co(2+) proved to be the most effective cofactor, inverting the enantioselectivity of AllBali when compared to that previously reported for other allantoinases. The common ability of different cyclic amidohydrolases to hydrolyze distinct substrates to the natural one also proved true for AllBali. The enzyme was able to hydrolyze hydantoin, dihydrouracil and 5-ethyl-hydantoin, although at relative rates 3-4 orders of magnitude lower than with allantoin. Mutagenesis experiments suggest that S292 is likely implicated in the binding of the allantoin ring through the carbonyl group of the polypeptide main chain, which is the common mechanism observed in other members of the amidohydrolase family. In addition, our results suggest an allosteric effect of H2O2 toward allantoinase.
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117. |
Cao M,
Geng W,
Zhang W,
Sun J,
Wang S,
Feng J,
Zheng P,
Jiang A,
Song C,
( 2013 ) Engineering of recombinant Escherichia coli cells co-expressing poly-�^-glutamic acid (�^-PGA) synthetase and glutamate racemase for differential yielding of �^-PGA. PMID : 23919316 : DOI : 10.1111/1751-7915.12075 PMC : PMC3815934 Abstract >>
Poly-�^-glutamic acid (�^-PGA) is a promising environmental-friendly material with outstanding water solubility, biocompatibility and degradability. However, it is tough to determine the relationship between functional synthetic enzyme and the strains' yield or substrate dependency. We cloned �^-PGA synthetase genes pgsBCA and glutamate racemase gene racE from both L-glutamate-dependent �^-PGA-producing Bacillus licheniformis NK-03 and L-glutamate-independent B. amyloliquefaciens LL3 strains. The deduced RacE and PgsA from the two strains shared the identity of 84.5% and 78.53%, while PgsB and PgsC possessed greater similarity with 93.13% and 93.96%. The induced co-expression of pgsBCA and racE showed that the engineered Escherichia coli strains had the capacity of synthesizing �^-PGA, and LL3 derived PgsBCA had higher catalytic activity and enhanced productivity than NK-03 in Luria-Bertani medium containing glucose or L-glutamate. However, the differential effect was weakened when providing sufficient immediateness L-glutamate substrate, that is, the supply of substrate could be served as the ascendance upon �^-PGA production. Furthermore, RacE integration could enhance �^-PGA yield through improving the preferred d-glutamate content. This is the first report about co-expression of pgsBCA and racE from the two Bacillus strains, which will be of great value for the determination of the biosynthetic mechanism of �^-PGA.
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118. |
Lin LL,
Chen YY,
Chi MC,
Merlino A,
( 2014 ) Low resolution X-ray structure of �^-glutamyltranspeptidase from Bacillus licheniformis: opened active site cleft and a cluster of acid residues potentially involved in the recognition of a metal ion. PMID : 24780583 : DOI : 10.1016/j.bbapap.2014.04.016 DOI : 10.1016/j.bbapap.2014.04.016 Abstract >>
�^-Glutamyltranspeptidases (�^-GTs) cleave the �^-glutamyl amide bond of glutathione and transfer the released �^-glutamyl group to water (hydrolysis) or acceptor amino acids (transpeptidation). These ubiquitous enzymes play a key role in the biosynthesis and degradation of glutathione, and in xenobiotic detoxification. Here we report the 3? resolution crystal structure of Bacillus licheniformis �^-GT (BlGT) and that of its complex with l-Glu. X-ray structures confirm that BlGT belongs to the N-terminal nucleophilic hydrolase superfamily and reveal that the protein possesses an opened active site cleft similar to that reported for the homologous enzyme from Bacillus subtilis, but different from those observed for human �^-GT and for �^-GTs from other microorganisms. Data suggest that the binding of l-Glu induces a reordering of the C-terminal tail of BlGT large subunit and allow the identification of a cluster of acid residues that are potentially involved in the recognition of a metal ion. The role of these residues on the conformational stability of BlGT has been studied by characterizing the autoprocessing, enzymatic activity, chemical and thermal denaturation of four new Ala single mutants. The results show that replacement of Asp568 with an Ala affects both the autoprocessing and structural stability of the protein.
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119. |
Lin LL,
Chen YY,
Chi MC,
Merlino A,
( 2014 ) Low resolution X-ray structure of �^-glutamyltranspeptidase from Bacillus licheniformis: opened active site cleft and a cluster of acid residues potentially involved in the recognition of a metal ion. PMID : 24780583 : DOI : 10.1016/j.bbapap.2014.04.016 DOI : 10.1016/j.bbapap.2014.04.016 Abstract >>
�^-Glutamyltranspeptidases (�^-GTs) cleave the �^-glutamyl amide bond of glutathione and transfer the released �^-glutamyl group to water (hydrolysis) or acceptor amino acids (transpeptidation). These ubiquitous enzymes play a key role in the biosynthesis and degradation of glutathione, and in xenobiotic detoxification. Here we report the 3? resolution crystal structure of Bacillus licheniformis �^-GT (BlGT) and that of its complex with l-Glu. X-ray structures confirm that BlGT belongs to the N-terminal nucleophilic hydrolase superfamily and reveal that the protein possesses an opened active site cleft similar to that reported for the homologous enzyme from Bacillus subtilis, but different from those observed for human �^-GT and for �^-GTs from other microorganisms. Data suggest that the binding of l-Glu induces a reordering of the C-terminal tail of BlGT large subunit and allow the identification of a cluster of acid residues that are potentially involved in the recognition of a metal ion. The role of these residues on the conformational stability of BlGT has been studied by characterizing the autoprocessing, enzymatic activity, chemical and thermal denaturation of four new Ala single mutants. The results show that replacement of Asp568 with an Ala affects both the autoprocessing and structural stability of the protein.
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120. |
Zhu YF,
Curran IH,
Joris B,
Ghuysen JM,
Lampen JO,
( 1990 ) Identification of BlaR, the signal transducer for beta-lactamase production in Bacillus licheniformis, as a penicillin-binding protein with strong homology to the OXA-2 beta-lactamase (class D) of Salmonella typhimurium. PMID : 2404938 : DOI : 10.1128/jb.172.2.1137-1141.1990 PMC : PMC208548 Abstract >>
The blaR gene of Bacillus licheniformis encodes the signal transducer for induction of the class A beta-lactamase. The protein product, BlaR, has a hydrophilic carboxy region that binds beta-lactams and shows high sequence homology to the class D beta-lactamases, particularly the OXA-2 beta-lactamase of Salmonella typhimurium. The BlaR-beta-lactam complex is stable and may provide the continuing stimulus needed for the prolonged production of the enzyme.
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121. |
Yan S,
Wang N,
Chen Z,
Wang Y,
He N,
Peng Y,
Li Q,
Deng X,
( 2013 ) Genes encoding the production of extracellular polysaccharide bioflocculant are clustered on a 30-kb DNA segment in Bacillus licheniformis. PMID : 23990282 : DOI : 10.1007/s10142-013-0333-4 Abstract >>
Bioflocculants are special high-molecular weight polymers produced by microorganisms. Despite the fact that several types of bioflocculants from different species of bacteria have been reported, there is a large gap in our knowledge regarding the molecular machine responsible for the production of bioflocculants. To investigate genes involved in bioflocculant synthesis, a fosmid library was generated from Bacillus licheniformis genomic DNA and screened for the production of bioflocculant. Four positive clones with distinct flocculation were isolated by a two-pooling scheme. The clone with 662 U ml(-1) flocculating activity was sequenced. As a result, a 30-kb fragment with 26 hypothetical genes was identified in the bioflocculant-producing clone. Most of the predicted proteins encoded by the inserted genes showed significant homology with enzymes involved in the biosynthesis of polysaccharide. Based on these homologies, a biosynthesis pathway and two gene clusters involved in the production of the polysaccharide bioflocculant were proposed with the integration of functional descriptions of individual genes by metabolic databases, and a glucose-sensitive glycosidases was predicted. This research supplied significant data for potential application of bioflocculant-producing strains in wastewater refining and industrial downstream treatments.
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122. |
Madslien EH,
Rønning HT,
Lindbäck T,
Hassel B,
Andersson MA,
Granum PE,
( 2013 ) Lichenysin is produced by most Bacillus licheniformis strains. PMID : 23844764 : DOI : 10.1111/jam.12299 Abstract >>
The aim of this study was to elucidate the prevalence of lichenysin production in Bacillus licheniformis and to see whether this feature was restricted to certain genotypes. Secondly, we wanted to see whether cytotoxicity reflected the measured levels of lichenysin. Fifty-three genotyped strains of B. licheniformis, representing a wide variety of sources, were included. lchAA gene fragments were detected in all strains by polymerase chain reaction (PCR). All 53 strains produced lichenysins with four molecular masses as confirmed by LC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis. The amounts of lichenysin varied more than two orders of magnitude between strains and were irrespective of genotype. Finally, there was a strong association between lichenysin concentrations and toxicity towards boar spermatozoa, erythrocytes and Vero cells. Lichenysin synthesis was universal among the 53 B. licheniformis strains examined. The quantities varied considerably between strains, but were not specifically associated with genotype. Cytotoxicity was evident at lichenysin concentrations above 10 �gg ml(-1) , which is in accordance with previous studies. This study might be of interest to those working on B. licheniformis for commercial use as well as for authorities who make risk assessments of B. licheniformis when used as a food and feed additive.
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123. |
Zhang H,
You C,
Ren J,
Xu D,
Han M,
Liao W,
( 2014 ) A simple one-step PCR walking method and its application of bacterial rRNA for sequencing identification. PMID : 24126601 : DOI : 10.1007/s00284-013-0462-y Abstract >>
There are many PCR walking methods applied currently, and they all have examples of successful application in organisms which are more complex than bacteria. However, to a certain extent, it will be more convenient for researchers if the complicated operation and poor specificity for bacteria can be improved. Here, we introduced an improved one-step PCR walking method of bacteria. Using a specific primer of the known sequence together with a universal semi-random primer, the unknown sequence adjacent to a known sequence can be obtained easily by just one ordinary round PCR. The products can be gel-purified and directly sequenced. Specific primers were designed according to the gene sequence of bacterial rRNA, and the variable and adjacent gene sequences were obtained by this method. The sequence analysis of the product showed that it can improve the resolution of bacterial identification to the species level.
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124. |
Li L,
Zhang L,
Li K,
Wang Y,
Gao C,
Han B,
Ma C,
Xu P,
( 2013 ) A newly isolated Bacillus licheniformis strain thermophilically produces 2,3-butanediol, a platform and fuel bio-chemical. PMID : 23981315 : DOI : 10.1186/1754-6834-6-123 PMC : PMC3766113 Abstract >>
2,3-Butanediol (2,3-BD), a platform and fuel bio-chemical, can be efficiently produced by Klebsiella pneumonia, K. oxytoca, and Serratia marcescens. However, these strains are opportunistic pathogens and not favorable for industrial application. Although some generally regarded as safe (GRAS) microorganisms have been isolated in recent years, there is still a demand for safe 2,3-BD producing strains with high productivity and yield under thermophilic fermentation. Bacillus licheniformis strain 10-1-A was newly isolated for 2,3-BD production. The optimum temperature and medium pH were 50�XC and pH 7.0 for 2,3-BD production by strain 10-1-A. The medium composition was optimized through Plackett-Burman design and response surface methodology techniques. With a two-stage agitation speed control strategy, 115.7 g/L of 2,3-BD was obtained from glucose by fed-batch fermentation in a 5-L bioreactor with a high productivity (2.4 g/L�Ph) and yield (94% of its theoretical value). The 2,3-BD produced by strain 10-1-A comprises (2R,3R)-2,3-BD and meso-2,3-BD with a ratio of nearly 1:1. The bdh and gdh genes encoding meso-2,3-butanediol dehydrogenase (meso-BDH) and glycerol dehydrogenase (GDH) of strain 10-1-A were expressed in Escherichia coli and the proteins were purified. meso-2,3-BD and (2R,3R)-2,3-BD were transformed from racemic acetoin by meso-BDH and GDH with NADH, respectively. Compared with the reported GRAS 2,3-BD producers, B. licheniformis 10-1-A could thermophilically produce 2,3-BD with a high concentration, productivity and yield. Thus, the newly isolated GRAS strain 10-1-A might be a promising strain for industrial production of 2,3-BD. Two key enzymes for meso-2,3-BD and (2R,3R)-2,3-BD production were purified and further studied, and this might be helpful to understand the mechanism for 2,3-BD stereoisomers forming in B. licheniformis.
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125. |
Sadeghi A,
Mortazavi SA,
Bahrami AR,
Sadeghi B,
( 2012 ) Design of multiplex PCR for simultaneous detection of rope-forming Bacillus strains in Iranian bread dough. PMID : 22555872 : DOI : 10.1002/jsfa.5681 Abstract >>
The objective of this study was optimisation of multiplex polymerase chain reaction (PCR) by a new primer set for simultaneous detection of ropiness agents as the main bacterial spoilage of Iranian bread. After inoculation of bread dough with activated Bacillus licheniformis (ATCC 9789) and Bacillus subtilis (ATCC 6633), DNA was extracted from the dough and subjected to PCR. Then simplex and multiplex PCR tests were optimised. From the results obtained, the optimum PCR conditions for simultaneous detection of the target genes in Bacillus species were an annealing temperature of 59 �XC and an MgCl(2) concentration of 2.5 mmol L(-1) . To design primers for these two bacteria, owing to close homology and the existence of similar conserved sequences in their 16S rRNA genes, lpaL and aprE genes (497 and 744 bp target sequences) respectively were chosen. Finally, by sequencing of PCR products, accurate and specific detection of the two desired Bacillus species was confirmed. The results were registered with GenBank under accession numbers HQ877873 and HQ871154. Compared with culture-dependent methods, this procedure offers significantly higher accuracy and speed, which are crucial criteria when it comes to food safety and high volumes of referred samples respectively.
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126. |
Lima LJ,
van der Velpen V,
Wolkers-Rooijackers J,
Kamphuis HJ,
Zwietering MH,
Nout MJ,
( 2012 ) Microbiota dynamics and diversity at different stages of industrial processing of cocoa beans into cocoa powder. PMID : 22327588 : DOI : 10.1128/AEM.07691-11 PMC : PMC3318835 Abstract >>
We sampled a cocoa powder production line to investigate the impact of processing on the microbial community size and diversity at different stages. Classical microbiological methods were combined with 16S rRNA gene PCR-denaturing gradient gel electrophoresis, coupled with clone library construction, to analyze the samples. Aerobic thermoresistant spores (ThrS) (100�XC; 10 min) were also isolated and characterized (identity, genetic diversity, and spore heat resistance), in view of their relevance to the quality of downstream heat-treated cocoa-flavored drinks. In the nibs (broken, shelled cocoa beans), average levels of total aerobic microorganisms (TAM) (4.4 to 5.6 log CFU/g) and aerobic total spores (TS) (80�XC; 10 min; 4.3 to 5.5 log CFU/g) were significantly reduced (P < 0.05) as a result of alkalizing, while fungi (4.2 to 4.4 log CFU/g) and Enterobacteriaceae (1.7 to 2.8 log CFU/g) were inactivated to levels below the detection limit, remaining undetectable throughout processing. Roasting further decreased the levels of TAM and TS, but they increased slightly during subsequent processing. Molecular characterization of bacterial communities based on enriched cocoa samples revealed a predominance of members of the Bacillaceae, Pseudomonadaceae, and Enterococcaceae. Eleven species of ThrS were found, but Bacillus licheniformis and the Bacillus subtilis complex were prominent and revealed great genetic heterogeneity. We concluded that the microbiota of cocoa powder resulted from microorganisms that could have been initially present in the nibs, as well as microorganisms that originated during processing. B. subtilis complex members, particularly B. subtilis subsp. subtilis, formed the most heat-resistant spores. Their occurrence in cocoa powder needs to be considered to ensure the stability of derived products, such as ultrahigh-temperature-treated chocolate drinks.
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127. |
Diderichsen B,
Jørgensen L,
( 1990 ) In vivo genetic engineering: homologous recombination as a tool for plasmid construction. PMID : 2265757 : DOI : 10.1016/0378-1119(90)90338-r Abstract >>
This paper describes a novel method for creating exact DNA fusions between any two points in a plasmid carried in Bacillus subtilis. It exploits the homologous in vivo recombination between directly repeated sequences that can be established by insertion of a synthetic oligodeoxyribonucleotide. The method was used to enhance the productivity in B. subtilis of a cloned alpha-amylase (Amy)-encoding gene originating from Bacillus stearothermophilus. Thus, an exact fusion between nucleotide sequences encoding the expression signals, including the signal peptide, of a Bacillus licheniformis Amy-encoding gene and the mature Amy of B. stearothermophilus, was created. The resulting hybrid translational product was processed correctly in B. subtilis during secretion, giving rise to an Amy identical to the mature Amy secreted by B. stearothermophilus.
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128. |
Kudan S,
Kuttiyawong K,
Pichyangkura R,
( 2011 ) Carboxy-terminus truncations of Bacillus licheniformis SK-1 CHI72 with distinct substrate specificity. PMID : 21699749 : DOI : 10.5483/BMBRep.2011.44.6.375 Abstract >>
Bacillus licheniformis SK-1 naturally produces chitinase 72 (CHI72) with two truncation derivatives at the C-terminus, one with deletion of the chitin binding domain (ChBD), and the other with deletions of both fibronectin type III domain (FnIIID) and ChBD. We constructed deletions mutants of CHI72 with deletion of ChBD (CHI72�GChBD) and deletions of both FnIIID and ChBD (CHI72�GFnIIID�GChBD), and studied their activity on soluble, amorphous and crystalline substrates. Interestingly, when equivalent amount of specific activity of each enzyme on soluble substrate was used, the product yield from CHI72- �GChBD and CHI72�GFnIIID�GChBD on colloidal chitin was 2.5 and 1.6 fold higher than CHI72, respectively. In contrast, the product yield from CHI72�GChBD and CHI72�GFnIIID- �GChBD on �E-chitin reduced to 0.7 and 0.5 fold of CHI72, respectively. These results suggest that CHI72 can modulate its substrate specificities through truncations of the functional domains at the C-terminus, producing a mixture of enzymes with elevated efficiency of hydrolysis.
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129. |
Simpson DR,
Natraj NR,
McInerney MJ,
Duncan KE,
( 2011 ) Biosurfactant-producing Bacillus are present in produced brines from Oklahoma oil reservoirs with a wide range of salinities. PMID : 21562978 : DOI : 10.1007/s00253-011-3326-z Abstract >>
Nine wells producing from six different reservoirs with salinities ranging from 2.1% to 15.9% were surveyed for presence of surface-active compounds and biosurfactant-producing microbes. Degenerate primers were designed to detect the presence of the surfactin/lichenysin (srfA3/licA3) gene involved in lipopeptide biosurfactant production in members of Bacillus subtilis/licheniformis group and the rhlR gene involved in regulation of rhamnolipid production in pseudomonads. Polymerase chain reaction amplification, cloning, and sequencing confirmed the presence of the srfA3/licA3 genes in brines collected from all nine wells. The presence of B. subtilis/licheniformis strains was confirmed by sequencing two other genes commonly used for taxonomic identification of bacteria, gyrA (gyrase A) and the 16S rRNA gene. Neither rhlR nor 16S rRNA gene related to pseudomonads was detected in any of the brines. Intrinsic levels of surface-active compounds in brines were low or not detected, but biosurfactant production could be stimulated by nutrient addition. Supplementation with a known biosurfactant-producing Bacillus strain together with nutrients increased biosurfactant production. The genetic potential to produce lipopeptide biosurfactants (e.g., srfA3/licA3 gene) is prevalent, and nutrient addition stimulated biosurfactant production in brines from diverse reservoirs, suggesting that a biostimulation approach for biosurfactant-mediated oil recovery may be technically feasible.
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130. |
( 1997 ) Unexpected influence of a C-terminal-fused His-tag on the processing of an enzyme and on the kinetic and folding parameters. PMID : 9280280 : DOI : 10.1016/s0014-5793(97)00908-3 Abstract >>
The addition of a poly-His C-terminal extension, designed to facilitate the purification of the protein, to the beta-lactamase of a thermophilic Bacillus licheniformis strain modified the site of action of the signal peptidase. This resulted in the secretion of a protein with a different N-terminus, showing that this type of protein engineering might not always be as 'neutral' as generally assumed.
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131. |
( 1997 ) Modulator protein RsbR regulates environmental signalling in the general stress pathway of Bacillus subtilis. PMID : 9179850 : DOI : 10.1046/j.1365-2958.1997.3631732.x Abstract >>
Bacillus subtilis responds to signals of environmental and metabolic stress by inducing over 40 general stress genes under the control of the sigma B transcription factor. sigma B activity is regulated post-translationally by a multi-component network composed of two coupled partner-switching modules, RsbX-RsbS-RsbT and RsbU-RsbV-RsbW, each containing a serine phosphatase (X or U), an antagonist protein (S or V), and a switch protein/serine kinase (T or W). The upstream module (X-S-T) is required to transmit signals of environmental stress. In contrast, the downstream module (U-V-W) is required to transmit signals of energy stress as well as the environmental signals conveyed to it from the upstream module. Until now the function of the rsbR gene product was unknown. RsbR shares significant sequence similarity with the RsbS and RsbV antagonist proteins whose phosphorylation states control key protein-protein interactions within their respective modules. Here we present evidence that RsbR is associated with RsbS in the upstream, environmental-sensing module. To investigate RsbR function, we constructed deletion and point mutations within rsbR and tested their effects on expression of sigma B-dependent reporter fusions, both singly and in combination with other rsb mutations. To determine the possible interaction of RsbR with other Rsb proteins, we tested the ability of wild-type or mutant RsbR to activate transcription in the yeast two-hybrid system in conjunction with other Rsb regulators. On the basis of this genetic analysis, we conclude that RsbR is a positive regulator which modulates sigma B activity in response to salt and heat stress. Our data further suggest that: (i) RsbR influences the antagonist function of RsbS by direct protein-protein interaction; and (ii) this interaction with RsbS is likely controlled by the phosphorylation state of RsbR.
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132. |
( 1995 ) Hyperthermostable mutants of Bacillus licheniformis alpha-amylase: multiple amino acid replacements and molecular modelling. PMID : 8771184 : Abstract >>
We have identified previously two critical positions for the thermostability of the highly thermostable alpha-amylase from Bacillus licheniformis. We have now introduced all 19 possible amino acid residues to these two positions, His133 and Ala209. The most favourable substitutions were to Ile and Val, respectively, which both increased the half-life of the enzyme at 80 degrees C by a factor of approximately 3. At both positions a stabilizing effect of hydrophobic residues was observed, although only in the case of position 133 could a clear correlation be drawn between the hydrophobicity of the inserted amino acid and the gain in protein stability. The construction of double mutants showed a cumulative effect of the most favourable and/or deleterious substitutions. Computer modelling was used to generate a 3-D structure of the wild-type protein and to model substitutions at position 209, which lies in the conserved (alpha/beta)8 barrel domain of alpha-amylase; Ala209 would be located at the beginning of the third helix of the barrel, in the bottom of a small cavity facing the fourth helix. The model suggests that replacement by, for example, a valine could fill this cavity and therefore increase intra- and interhelical compactness and hydrophobic interactions.
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133. |
( 1996 ) Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor. PMID : 8824586 : DOI : 10.1101/gad.10.18.2265 Abstract >>
The general stress response of the bacterium Bacillus subtilis is governed by a signal transduction network that regulates activity of the sigma(B) transcription factor. We show that this network comprises two partner-switching modules, RsbX-RsbS-RsbT and RsbU-RsbV-RsbW, which contribute to regulating sigma(B). Each module consists of a phosphatase (X or U), an antagonist protein (S or V), and a switch protein/kinase (T or W). In the downstream module, the W anti-sigma factor is the primary regulator of sigma(B) activity. If the V antagonist is phosphorylated, the W switch protein binds and inhibits sigma(B). If V is unphosphorylated, it complexes W, freeing sigma(B) to interact with RNA polymerase and promote transcription. The phosphorylation state of V is controlled by opposing kinase (W) and phosphatase (U) activities. The U phosphatase is regulated by the upstream module. The T switch protein directly binds U, stimulating phosphatase activity. The T-U interaction is governed by the phosphorylation state of the S antagonist, controlled by opposing kinase (T) and phosphatase (X) activities. This partner-switching mechanism provides a general regulatory strategy in which linked modules sense and integrate multiple signals by protein-protein interaction.
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134. |
( 1997 ) Nucleotide sequence of the Bacillus licheniformis ATCC 10716 dat gene and comparison of the predicted amino acid sequence with those of other bacterial species. PMID : 9003455 : DOI : 10.1016/s0167-4781(96)00204-7 Abstract >>
The gene encoding the D-aminotransferase from Bacillus licheniformis was cloned and the complete DNA sequence was determined. The deduced D-aminotransferase protein sequence, consists of 283 amino acids and shows a high degree of homology with other Bacillus D-aminotransferases, branched chain aminotransferase of Escherichia coli and the 4-amino-benzoate-4-deoxychorismate lyase of Bacillus subtilis and Escherichia coli.
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135. |
( 1996 ) The complete nucleotide sequence of the Bacillus licheniformis NM105 S-layer-encoding gene. PMID : 8964497 : DOI : 10.1016/0378-1119(96)00233-8 Abstract >>
A protein present on the cell surface of Bacillus licheniformis (Bl) NM105 was identified as an S-layer (OlpA in this paper), a protein present on many bacterial cell surfaces. Purification, SDS-PAGE and isoelectrofocusing showed one 94-kDa, slightly acidic (pI 6.5) protein band (defined as OlpA). The pure protein OlpA, has a tetragonal symmetry of its morphological subunits. Following Edman degradation, three 17-mer oligodeoxyribonucleotide (oligo) probes corresponding to the N-terminal sequence of Olpa were synthesized and used for gene cloning. The nucleotide (nt) sequence of the cloned gene (olpA) showed an ORF and encoded an 874 amino acid (aa) protein. In the promoter region of olpA, there appear to be -10 and -35 sigmaA-binding sites, as well as -10 and -35 regions specific for sigmaH. The existence of these two potential promoters suggests that OlpA would be produced during both the vegetative and sporulating stages of growth. The ribosome-binding site (RBS) sequence perfectly matched its consensus sequence, suggesting a high efficiency of translation of olpA. A typical 29-aa leader peptide, characteristic of secretory proteins in Bacilli, is present in the OlpA pre-protein sequence. In olpA, there are two stem-loop structures in tandem, downstream from the stop codon. These stem-loops are probably involved in prolonged olpA expression, by extending the half life of the mRNA.
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136. |
( 1995 ) Gene cloning, purification, and characterization of thermostable and halophilic leucine dehydrogenase from a halophilic thermophile, Bacillus licheniformis TSN9. PMID : 8597545 : Abstract >>
A halophilic and thermophilic isolate from the sand of Tottori Dune was found to produce a thermostable and halophilic leucine dehydrogenase (EC 1.4.1.9). It was identified to be a new strain of Bacillus licheniformis. The enzyme gene was cloned into Escherichia coli JM109 with a vector plasmid pUC18. The enzyme was purified to homogeneity from the clone cell extract by ion-exchange column chromatography with a yield of 31%. The enzyme was found to be composed of eight subunits identical in relative molecular mass (43,000). The amino acid sequence of the enzyme, deduced from the nucleotide sequence of the gene, showed an identity of 84.6% with that of the B. stearothermophilus enzyme [Nagata S, Tanizawa K, Esaki N, Sakamoto Y, Oshima T, Tanaka H, Soda K (1988) Biochemistry 27:9056-9062], although both enzymes were similar to each other in various enzymological properties such as thermostability, substrate and coenzyme specificities, and stereospecificity for hydrogen transfer from the C-4 of NADH. However, they were markedly distinct from each other in halophilicity; the B. licheniformis enzyme was much more stable than the other in the presence of high concentrations of salts.
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137. |
( 1996 ) Cloning and sequencing of a new holin-encoding gene of Bacillus licheniformis. PMID : 8626066 : DOI : 10.1016/0378-1119(95)00690-7 Abstract >>
A Bacillus licheniformis DNA fragment which exhibits homology with the upstream region of the cell-wall hydrolase-encoding gene, cwlL, was cloned into Escherichia coli (Ec). Nucleotide sequencing indicated that there are two open reading frames (tentatively designated as xpaG1 and xpaG2) which encode polypeptides of 89 and 88 amino acids (aa) (10044 and 9764 Da, respectively). Ec cells harboring two compatible plasmids (pMWB1 and pHSGKH) containing the Bacillus subtilis cell-wall hydrolase-encoding gene, cwlA, and xpaG1-G2, respectively, exhibited higher extra-cellular cell-wall hydrolase activity than did cells harboring pMWB1 and a control plasmid, pHSG398. The aa sequence homology of XpaG2 with other polypeptides indicated that xpaG2 is a holin-encoding gene. Moreover, Ec C600 harboring a plasmid containing xpaG1-xpaG2 led to leakage of beta-galactosidase into the extracellular fraction.
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138. |
( 1994 ) Identification of active site carboxylic residues in Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase by site-directed mutagenesis. PMID : 8182059 : Abstract >>
Active site residues of 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from Bacillus licheniformis have been identified by site-directed mutagenesis. Previous work revealed that Glu-134 was essential for enzymatic activity, and it was proposed as the catalytic nucleophile by affinity labeling of the highly homologous Bacillus amyloliquefaciens enzyme. To search for the general acid catalyst, the Asp and Glu residues conserved among the Bacillus isozymes have been mutated to Asn and Gln, respectively. Out of the 14 positions studied, only the E138Q mutation yielded an inactive enzyme, whereas the E134Q and D136N mutants retained less than 0.5% of the wild type activity. Based on the three-dimensional structure of a hybrid B. amyloliquefaciens-Bacillus macerans 1,3-1,4-beta-D-glucan 4-glucanohydrolase, Glu-134, Asp-136, and Glu-138 are the only carboxylic acid residues that are properly located into the active site cleft to participate in catalysis. Glu-138 appears as the most likely candidate to function as the general acid catalyst, while Asp-136 may affect the pK alpha of the catalytic residues.
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139. |
( 1994 ) Conservation of the 168 divIB gene in Bacillus subtilis W23 and B. licheniformis, and evidence for homology to ftsQ of Escherichia coli. PMID : 8088553 : DOI : 10.1016/0378-1119(94)90043-4 Abstract >>
The chromosomal regions of Bacillus subtilis (Bs) W23 and Bacillus licheniformis (Bl), which span the sequence encoding the homolog of the division initiation gene, divIB, of Bs168 were cloned and sequenced. The high level of conservation of the amino acid (aa) sequence of the DivIB protein (99 and 68% identity for BsW23 and Bl, respectively) was consistent with a significant role for this protein in the cell cycle of the two species. The hydropathy profile for DivIB of Bl was almost identical to that of Bs168 and consistent with a membrane location, as previously established for the latter. The higher than average level of identity (87%) of the 31-aa N-terminal cytoplasmic domain of DivIB between Bs168 and Bl raised the possibility of a special role for this domain. Database analyses using the Bl DivIB sequence and similarity analyses also strongly suggested that DivIB, of Bl and Bs, is a homolog of FtsQ of Escherichia coli. The flanking sequences extending into the unidentified orfs both upstream and downstream from divIB were highly conserved between Bs168 and Bl at both the nucleotide and aa levels. It was confirmed that orf4 of Bs168 is dispensable.
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140. |
( 1993 ) Crystal structure of selenosubtilisin at 2.0-A resolution. PMID : 8512925 : DOI : 10.1021/bi00075a007 Abstract >>
The three-dimensional structure of selenosubtilisin, an artificial selenoenzyme, has been solved at 2.0-A resolution by the method of molecular replacement. Selenosubtilisin is a chemical derivative of the bacterial serine protease subtilisin in which the catalytically essential serine residue has been replaced with a selenocysteine. Its unique hydrolytic and redox properties reflect the intrinsic chemical reactivity of the selenium prosthetic group. Structural analysis of the modified protein reveals that the selenium moiety is selectively incorporated into the side chain of residue 221 and confirms the seleninic acid oxidation state expected from treatment of the enzyme with hydrogen peroxide prior to crystallization. Although the seleninic acid replaces the essential nucleophile in the enzyme's catalytic triad and introduces a negative charge into the active site, the interaction between His64 and Asp32 is not altered by the modification. Hydrogen bonds from the oxygen atoms of the seleninic acid to His64 and to Asn155 in the oxyanion hole confine the prosthetic group to a single well-defined conformation within the active site. These interactions thus provide a structural basis for understanding the seleninic acid's unusually low pKa, the enzyme's relatively sluggish rate of reaction with thiols, and its much more efficient peroxidase activity. Aside from the active site region, the structure of the protein is essentially the same as that previously reported for native subtilisin Carlsberg, indicating the viability of chemical modification strategies for incorporating site-specific changes into the protein backbone. Comparison of the three-dimensional structures of selenosubtilisin and glutathione peroxidase, an important naturally occurring selenoenzyme, provides the means to evaluate how the function of the selenium prosthetic group varies with molecular context.
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141. |
( 1994 ) Identification of genes encoding ribosomal protein L33 from Bacillus licheniformis, Thermus thermophilus and Thermotoga maritima. PMID : 8112583 : DOI : 10.1016/0378-1119(94)90537-1 Abstract >>
Three previously unrecognized genes from Bacillus licheniformis, Thermus thermophilus and Thermotoga maritima, encoding ribosomal protein L33, have been identified and designated as rpmG genes. Their sequence and context have been compared with the three known rpmG genes from Escherichia coli, Lactococcus lactis and B. subtilis. Previously unrecognized open reading frames homologous to secE are located immediately downstream from rpmG in B. licheniformis, T. thermophilus and Ta. maritima: beyond that lie previously identified nusG and rplK genes. Thus, the genes located downstream from rpmG show similar organization across three of the four bacterial phyla represented.
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142. |
( 1994 ) Nucleotide sequence and features of the Bacillus licheniformis gnt operon. PMID : 8535972 : DOI : 10.1093/dnares/1.4.157 Abstract >>
Bacillus licheniformis was able to utilize gluconate as the sole carbon source as efficiently as Bacillus subtilis did. Southern analysis indicated that B. licheniformis likely possesses only one gnt determinant. The nucleotide sequence (6278 bp) of the B. licheniformis DNA containing the gnt operon was determined, revealing the five complete open reading frames (ORF; genes). The putative product of the first gene, oug, did not show any significant homology to known proteins, but those of the second to fifth genes exhibited striking homology to the gntRKPZ genes of B. subtilis, respectively, indicating that they are the corresponding gnt genes of B. licheniformis. Not only is the organization of the gnt genes of these two Bacilli highly conserved, but so are the cis regulatory elements of their gnt operon. Sequence analysis of the upstream regions of these two gnt operons implied that a chromosome rearrangement in B. subtilis might have occurred immediately upstream of the gnt operon during evolution, causing it to diverge from a common ancestor into B. licheniformis and B. subtilis.
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143. |
Lin X,
Kelemen DW,
Miller ES,
Shih JC,
( 1995 ) Nucleotide sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1. PMID : 7747965 : PMC : PMC167403 Abstract >>
Bacillus licheniformis PWD-1 (ATCC 53757) secretes keratinase, a proteolytic enzyme which is active on whole feathers. By amino acid sequence similarity and phenylmethylsulfonyl fluoride inhibition, the keratinase was demonstrated to be a serine protease. The entire nucleotide sequence of the coding and flanking regions of the keratinase structure gene, kerA, was determined. A fixed oligonucleotide primer derived from the N-terminal sequence of the purified enzyme and a second random oligonucleotide primer were used in a procedure called PCR walking, which was developed to amplify and sequence the upstream and downstream regions of kerA. Another method, PCR screening, was conducted with a lambda phage vector with inserted PWD-1 genomic DNA fragments as templates and with the known sequences of the vector arms and the N-terminal sequence of the enzyme as primers. PCR amplification and sequence analysis of the lambda library completed the entire kerA sequence and established a set of gene deletions. The kerA gene shares a 97% sequence identity with the gene encoding subtilisin Carlsberg from B. licheniformis NCIMB 6816. The putative promoters, ribosome binding sites, and transcriptional terminators are also similar in these two bacteria. The deduced amino acid sequences indicate only three amino acid differences between the two mature proteases. Northern (RNA) analysis demonstrates that transcriptional regulation controls kerA expression on different growth media.
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144. |
( 1993 ) Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwlL, of Bacillus licheniformis. PMID : 7902527 : DOI : 10.1007/bf00284691 Abstract >>
We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38,994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-L-alanine amidase, which has a M(r) value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.
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145. |
Turgay K,
Marahiel MA,
( N/A ) A general approach for identifying and cloning peptide synthetase genes. PMID : 7849417 : Abstract >>
A wide variety of bioactive peptides are synthesized nonribosomally by large multienzyme complexes employing the thiotemplate mechanism. Based on the known and highly conserved structures of several genes encoding multifunctional peptide synthetases, we developed a universal polymerase chain reaction (PCR) approach for amplifying, cloning and identifying parts of putative peptide synthetase genes. We showed, by cloning fragments of peptide synthetase genes from the phaseolotoxin-producing strain Pseudomonas syringe pv. phaseolica and from Bacillus licheniformis, which produces the branched peptide antibiotic bacitracin, that this approach is applicable. It gives a new and potentially general access to the biosynthetic genes of many nonribosomally synthesized peptides.
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146. |
Machius M,
Wiegand G,
Huber R,
( 1995 ) Crystal structure of calcium-depleted Bacillus licheniformis alpha-amylase at 2.2 A resolution. PMID : 7877175 : DOI : 10.1006/jmbi.1994.0106 Abstract >>
The three-dimensional structure of the calcium-free form of Bacillus licheniformis alpha-amylase (BLA) has been determined by multiple isomorphous replacement in a crystal of space group P4(3)2(1)2 (a = b = 119.6 A, c = 85.4 A). The structure was refined using restrained crystallographic refinement to an R-factor of 0.177 for 28,147 independent reflections with intensities FObs > 0 at 2.2 A resolution, with root mean square deviations of 0.008 A and 1.4 degrees from ideal bond lengths and bond angles, respectively. The final model contains 469 residue, 237 water molecules, and one chloride ion. The segment between Trp182 and Asn192 could not be located in the electron density, nor could the N and C termini. Cleavage of the calcium-free form of BLA was observed after Glu189, due to a Glu-C endopeptidase present in trace amounts in the preparation. BLA did not crystallize without this cleavage under the conditions applied. BLA exhibits the characteristic overall topological fold observed for other alpha-amylases and related amylolytic enzymes: a central domain A containing an alpha/beta-barrel with a large protrusion between beta-strand 3 and alpha-helix 3 (domain B) and a C-terminal greek key motif (domain C). Unlike in the other enzymes, domain B possesses a beta-sheet made up of six loosely connected, twisted beta-strands forming a kind of a barrel with a large hole in the interior. Topological comparisons to TAKA-amylase, pig pancreatic alpha-amylase and cyclodextrin glycosyltransferase reveal a very high structural equivalence for large portions of the proteins and an exceptionally pronounced structural similarity for calcium binding, chloride binding and the active site. None of the theories proposed to explain the enhanced thermostability of BLA showed a satisfactory correlation with the three-dimensional structure. Instead, sequence comparisons to the less thermostable bacterial alpha-amylase from Bacillus amyloliquefaciens (BAA) indicate that some ionic interactions present in BLA, but which cannot be formed in BAA, might be responsible for the enhanced thermostability of BLA.
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147. |
Meadway RJ,
( 1969 ) The amino acid sequence of penicillinase from Bacillus licheniformis. PMID : 5353499 : DOI : 10.1042/bj1150012pb PMC : PMC1185161 Abstract >>
N/A
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148. |
Ramakrishna N,
Dubnau E,
Smith I,
( 1984 ) The complete DNA sequence and regulatory regions of the Bacillus licheniformis spoOH gene. PMID : 6322121 : DOI : 10.1093/nar/12.4.1779 PMC : PMC318620 Abstract >>
We have determined the sequence of a 1228 base-pair cloned DNA fragment from Bacillus licheniformis capable of specifically complementing mutations in the spoOH gene, which is required for the early stage of sporulation in B. subtilis. The sequence has only one long open reading frame consisting of 168 codons. In vivo and in vitro transcription mapping studies indicate the size of complementary RNA to be around 1 kb with the 5' initiation site at base 79 and the 3' termination site in the area of base 1138. This indicates the presence of a 5' untranslated RNA and a fairly long 3' extension. The promoter sequence of this gene is 5'TATAAT3' at -10, and 5'TTGACG3' at -35, a typical E. coli-like promoter sequence, and is transcribed in vitro specifically only by RNA polymerase containing delta 55 and not delta 37-containing holoenzyme.
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149. |
Kuhn H,
Fietzek PP,
Lampen JO,
( ) PMID : 6172418 : PMC : PMC216633 Abstract >>
The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.
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150. |
Hahn M,
Pons J,
Planas A,
Querol E,
Heinemann U,
( 1995 ) Crystal structure of Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase at 1.8 A resolution. PMID : 7589539 : DOI : 10.1016/0014-5793(95)01111-q Abstract >>
The crystal structure of the 1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis is solved at a resolution of 1.8 A and refined to R = 16.5%. The protein has a similar beta-sandwich structure as the homologous enzyme from Bacillus macerans and the hybrid H(A16-M). This demonstrates that the jellyroll fold of these proteins is remarkably rigid and only weakly influenced by crystal contacts. The crystal structure permits to extend mechanistic considerations derived for the B. licheniformis enzyme to the entire class of bacterial 1,3-1,4-beta-D-glucan 4-glucanohydrolases.
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151. |
Nedkov P,
Oberthür W,
Braunitzer G,
( 1983 ) [Primary structure of subtilisin DY]. PMID : 6420308 : Abstract >>
The complete amino-acid sequence of subtilisin DY, an extracellular alkaline proteinase produced by Bacillus subtilis, strain DY, is presented. The enzyme's primary structure was elucidated using peptides obtained by tryptic hydrolysis and peptides released from BrCN, tryptophan and Asn-Gly cleavage (using hydroxylamine). The peptides were isolated by gelfiltration and by reversed phase high performance liquid chromatography and were degradated automatically in the sequenator. The complete sequence has been verified by peptide overlapping. The subtilisin DY polypeptide chain, like that of subtilisin Carlsberg, consists of 274 amino-acid residues. 32 Amino-acid replacements were found between these two molecules (37 nucleotide mutations, 5 of them two-point mutations). Between the subtilisins DY and Novo 82 amino-acid residue replacements (106 nucleotide mutations, 24 two-point mutations) and one deletion were found. The polypeptide chains of the three subtilisins mentioned were compared and some differences discussed.
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152. |
Stephens MA,
Ortlepp SA,
Ollington JF,
McConnell DJ,
( 1984 ) Nucleotide sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene: comparison with the B. amyloliquefaciens gene. PMID : 6609154 : PMC : PMC215428 Abstract >>
The DNA sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene is reported. Comparison of the inferred amino acid sequence of the B. licheniformis alpha-amylase gene with that of the Bacillus amyloliquefaciens gene shows that whereas the amino acid sequences of the mature proteins have considerable homology, the sequences for the signal peptides are distinct.
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153. |
Podlesek Z,
Comino A,
Herzog-Velikonja B,
Zgur-Bertok D,
Komel R,
Grabnar M,
( 1995 ) Bacillus licheniformis bacitracin-resistance ABC transporter: relationship to mammalian multidrug resistance. PMID : 7476193 : DOI : 10.1111/j.1365-2958.1995.tb02322.x Abstract >>
The nucleotide sequence of the Bacillus licheniformis bacitracin-resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP-binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydrophobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram-negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide-binding sites, BcrA protein and mammalian multidrug transporter or P-glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide-binding protein to determine the importance of various regions in transport.
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154. |
Sibakov M,
Palva I,
( 1984 ) Isolation and the 5'-end nucleotide sequence of Bacillus licheniformis alpha-amylase gene. PMID : 6334606 : DOI : 10.1111/j.1432-1033.1984.tb08594.x Abstract >>
We have isolated and determined the 5'-end nucleotide sequence of the alpha-amylase gene from Bacillus licheniformis ATCC 14580. The alpha-amylase produced by this strain is thermostable and of liquefying type. The gene was originally cloned in a bacteriophage lambda 1059 vector. A subclone containing a 5.3 X 10(3)-base insert in pBR322 was further characterized. The nucleotide sequence coding for the 5' end of the structural gene together with the sequence coding for the upstream control regions was determined. The deduced N-terminal amino acid sequence was identical with the previously published amino acid sequence of B. licheniformis alpha-amylase. There was also very strong homology to the N-terminal sequence of Bacillus amyloliquefaciens alpha-amylase. The Mr of the thermostable alpha-amylase, as determined in vitro in a cell-free transcription/translation system of Escherichia coli, was about 55 000.
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155. |
Neugebauer K,
Sprengel R,
Schaller H,
( 1981 ) Penicillinase from Bacillus licheniformis: nucleotide sequence of the gene and implications for the biosynthesis of a secretory protein in a Gram-positive bacterium. PMID : 6269055 : DOI : 10.1093/nar/9.11.2577 PMC : PMC326873 Abstract >>
The gene for the penicillinase from B. licheniformis has been cloned in a functional stat on a 1.5 kb DNA fragment and its nucleotide sequence has been determined. A sequence of 307 amino acid residues is infered for the penicillinase precursor. Of these 34 amino acids precede the sequence of the secreted form of the enzyme. This peptide extension shows the features of a signal for secretion and also provides the hydrophobic anchor for the membrane-bound form of the enzyme.
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156. |
Kroyer J,
Chang S,
( 1981 ) The promoter-proximal region of the Bacillus licheniformis penicillinase gene: Nucleotide sequence and predicted leader peptide sequence. PMID : 6977472 : DOI : 10.1016/0378-1119(81)90177-3 Abstract >>
Penicillinase (beta-lactamase) is a major species of secreted protein produced by Bacillus licheniformis 749. From the pTB2 recombinant plasmid containing the cloned entire penicillinase (penP) gene, we have isolated and sequenced a 446-bp HpaII fragment carrying the beginning of penP. The 3'-end coding region of 216-bp on this DNA fragment codes for the first 72 amino acids of the prepenicillinase protein. The deduced structure of the leader peptide consists of a 34 amino acid signal sequence with a hydrophilic N-terminal region and a central hydrophobic core.
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157. |
Izui K,
Nielsen JB,
Caulfield MP,
Lampen JO,
( 1980 ) Large exopenicillinase, initial extracellular form detected in cultures of Bacillus licheniformis. PMID : 6966510 : DOI : 10.1021/bi00550a023 Abstract >>
N/A
|
158. |
Gryczan T,
Israeli-Reches M,
Del Bue M,
Dubnau D,
( 1984 ) DNA sequence and regulation of ermD, a macrolide-lincosamide-streptogramin B resistance element from Bacillus licheniformis. PMID : 6429477 : DOI : 10.1007/bf00425543 Abstract >>
The DNA sequence of ermD , a macrolide-lincosamide-streptogramin B (MLS) resistance determinant cloned from the chromosome of Bacillus licheniformis, has been determined. ermD encodes an erythromycin inducible protein of molecular weight 32,796. S1 nuclease mapping of the ermD promoter has revealed the presence of an approximately 354 base leader sequence on the ermD transcript. This leader contains a short open reading frame sufficient to encode a 14 amino acid peptide, which is preceded by a potential ribosomal binding site. The leader sequence has the potential to fold into several base paired structures, in some of which the ribosomal binding site for the ermD product would be sequestered. Deletion analysis demonstrated that the leader contains regulatory sequences. Removal of the ermD promoter and fusion to an upstream promoter did not interfere with induction, strongly suggestion that ermD regulation is posttranscriptional. Based on these features it appears likely that ermD is regulated by a translational attenuation mechanism, analogous to that suggested for ermC , a resistance element from Staphylococcus aureus (Gryczan et al. 1980; Horinouchi and Weisblum 1980). Comparison of the ermD sequence and that of its product to two other sequenced MLS determinants reveals substantial phylogenetic relatedness, although the three genes are not homologous by the criterion of Southern blot hybridization.
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159. |
Smith EL,
DeLange RJ,
Evans WH,
Landon M,
Markland FS,
( 1968 ) Subtilisin Carlsberg. V. The complete sequence; comparison with subtilisin BPN'; evolutionary relationships. PMID : 4967581 : Abstract >>
N/A
|
160. |
Tanaka K,
Sakai H,
Ohta T,
Matsuzawa H,
( 1995 ) Molecular cloning of the genes for pyruvate kinase of two bacilli, Bacillus psychrophilus and Bacillus licheniformis, and comparison of the properties of the enzymes produced in Escherichia coli. PMID : 7549104 : DOI : 10.1271/bbb.59.1536 Abstract >>
The genes for the pyruvate kinases of a psychrophile, Bacillus psychrophilus, and a mesophile, Bacillus licheniformis, have been cloned in Escherichia coli, and all their nucleotides were sequenced. The two bacterial enzymes each had an extra C-terminal sequence consisting of about 110 amino acid residues, which has been found in the B. stearothermophilus enzyme. Both enzymes were overexpressed in E. coli and the properties of the purified enzymes were compared to those of the B. stearothermophilus enzyme. Both enzymes were less stable than the B. stearothermophilus one. The B. psychrophilus enzyme was more stable than the B. licheniformis one. Similarly to the B. licheniformis and B. stearothermophilus pyruvate kinases, the B. psychrophilus enzyme was activated by AMP or ribose 5-phosphate, and inhibited by ATP or fructose 1,6-bisphosphate. Thus, these enzymes were very similar in the sigmoidal saturation curve for phosphoenolpyruvate and allosteric effectors, but their optimum temperatures and thermostabilities were very different.
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161. |
Nicholls NJ,
Lampen JO,
( 1987 ) Repressor gene, blaI, for Bacillus licheniformis 749 beta-lactamase. PMID : 3305074 : DOI : 10.1016/0014-5793(87)80375-7 Abstract >>
The repressor gene, blaI, for the beta-lactamase of Bacillus licheniformis 749 was functional when cloned in Escherichia coli, but addition of a beta-lactam did not lead to induction. One plasmid contained fragments from the inducible strain (source of repressor), the other carried fragments from the blaI- mutant 749/C (target). blaI lies just 5' to the promoter for the structural gene, blaP, and the target is the promoter region between the two genes. Interaction with both promoters seemed necessary for full repression. BlaI is a hydrophilic protein (Mr 15036) with the some structural similarities to repressors from Gram-negative bacteria.
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162. |
Himeno T,
Imanaka T,
Aiba S,
( 1986 ) Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor. PMID : 3096969 : DOI : 10.1128/jb.168.3.1128-1132.1986 PMC : PMC213612 Abstract >>
Bacillus licheniformis penicillinase genes, penP and penI, are coded on a 4.2-kilobase EcoRI fragment of pTTE21 (T. Imanaka, T. Tanaka, H. Tsunekawa, and S. Aiba, J. Bacteriol. 147:776-186, 1981). The EcoRI fragment was subcloned in a low-copy-number plasmid pTB522 in Bacillus subtilis. B. subtilis carrying the recombinant plasmid pPTB60 (Tcr penP+ penI+) was chemically mutagenized. Of about 150,000 colonies, two penI(Ts) mutant plasmids, pPTB60D13 and pPTB60E24, were screened by the plate assay at 30 and 48 degrees C for penicillinase. By constructing recombinant plasmids between wild-type and mutant plasmids, the mutation points were shown to be located in a 1.7-kilobase EcoRI-PstI fragment. The EcoRI-PstI fragments of the wild-type plasmid and two mutant plasmids were sequenced. A large open reading frame, composed of 384 bases and 128 amino acid residues (molecular weight, 14,983), was found. Since the mutation points were located at different positions in the protein coding region (Ala to Val for pPTB60D13 and Pro to Leu for pPTB60E24), the coding region was concluded to be the penI gene. A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site (ATG). A probable promoter sequence which is very similar to the consensus sequence was also found upstream of the penP promoter, but in the opposite direction. A consensus twofold symmetric sequence (AAAGTATTA CATATGTAAGNTTT) which might have been used as a repressor binding region was found downstream and in the midst of the penP promoter and also downstream of the penI promoter. The regulation of penP and penI by the repressor is discussed.
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163. |
?zdemir F,
Arslan S,
( 2019 ) Molecular Characterization and Toxin Profiles of Bacillus spp. Isolated from Retail Fish and Ground Beef. PMID : 30690739 : DOI : 10.1111/1750-3841.14445 Abstract >>
Bacillus species are common in the environment due to their spore-forming ability and nutritional versatility and cause food contamination. Bacilli play a significant role in foodborne illnesses and food spoilage. In this study, 52 Bacillus isolates from retail fish and ground beef were identified and differentiated based on 16S rRNA, gyrB, and rpoB gene sequencing. The presence of genes encoding emetic toxin (ces), hemolytic enterotoxin hemolysin BL (hbl), nonhemolytic enterotoxin (nhe) and cytotoxin K (cytK1) was assessed in all Bacillus isolates. The ability of the Bacillus isolates to produce several extracellular enzymes that contribute to pathogenicity and food spoilage was investigated. The 16S rRNA, rpoB, and gyrB gene sequence similarities of the Bacillus isolates tested were 96.1%, 83.2%, and 77.5%, respectively. The gyrB gene demonstrated a higher degree of sequence variation than the 16S rRNA and rpoB genes. The prevalence of Bacillus isolates producing at least two of the genes of the HBL and NHE complexes was 23.1% and 15.4%, respectively. Of the B. cereus isolates, 10 (41.7%) possessed two or more enterotoxin genes. None of the isolates carried the ces and cytK1 genes. All isolates were positive for the production of enzymes such as protease, lipase, gelatinase, and DNase. However, only 92.3% of the tested isolates were positive for amylase. In conclusion, our results revealed that the presence of genes involved in toxin production and enzyme production in meat-originated B. cereus and other Bacillus isolates may cause spoilage of food and pose a health risk for consumers. PRACTICAL APPLICATION: Bacillus species can be found in various foods due to their ubiquitous nature. Bacillus spp., especially B. cereus, are associated with food poisoning and other infections in humans. Toxins and many extracellular enzymes produced by Bacillus spp. are the causative agents of foodborne outbreaks, food spoilage, and low-quality food with significantly reduced edibility. This study highlights the characterization of Bacillus spp. and presence of potentially pathogenic Bacillus species in meats.
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164. |
Grossman MJ,
Lampen JO,
( 1987 ) Purification and DNA binding properties of the blaI gene product, repressor for the beta-lactamase gene, blaP, of Bacillus licheniformis. PMID : 3498148 : DOI : 10.1093/nar/15.15.6049 PMC : PMC306067 Abstract >>
The location of the repressor gene, blaI, for the beta-lactamase gene blaP of Bacillus licheniformis 749, on the 5' side of blaP, was confirmed by sequencing the bla region of the constitutive mutant 749/C. An amber stop codon, likely to result in a nonfunctional truncated repressor, was found at codon 32 of the 128 codon blaI open reading frame (ORF) located 5' to blaP. In order to study the DNA binding activity of the repressor, the structural gene for blaI, from strain 749, with its ribosome binding site was expressed using a two plasmid T7 RNA polymerase/promotor system (S. Tabor and C. C. Richardson. Proc. Natl. Acad. Sci. 82, 1074-1078 (1985). Heat induction of this system in Escherichia coli K38 resulted in the production of BlaI as 5-10% of the soluble cell protein. Repressor protein was then purified by ammonium sulfate fractionation and cation exchange chromatography. The sequence of the N-terminal 28 amino acid residues was determined and was as predicted from the DNA. Binding of BlaI to DNA was detected by the slower migration of protein DNA complexes during polyacrylamide gel electrophoresis. BlaI was shown to selectively bind DNA fragments carrying the promoter regions of blaI and blaP.
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165. |
Wittman V,
Wong HC,
( 1988 ) Regulation of the penicillinase genes of Bacillus licheniformis: interaction of the pen repressor with its operators. PMID : 3260234 : DOI : 10.1128/jb.170.7.3206-3212.1988 PMC : PMC211270 Abstract >>
The synthesis of the inducible enzyme penicillinase of Bacillus licheniformis is negatively controlled by a repressor (D.A. Dubnau and M.R. Pollock, J. Gen. Microbiol. 41:7-21, 1965; D. J. Sherratt and J. F. Collins, J. Gen. Microbiol. 76:217-230,1973). The molecular organization of the genes coding for penicillinase (penP) and its repressor (penI) has recently been determined (T. Himeno, T. Imanaka, and S. Aiba, J. Bacteriol. 168:1128-1132, 1986). These two genes are transcribed divergently from within a 364-nucleotide region separating the coding sequences. We cloned and sequenced the repressor gene (penIc) from strain 749/C that constitutively produces penicillinase. The penIc and penI+ (wild-type) genes were expressed in Escherichia coli. Complementation analysis indicated that the repressor is the only trans-acting protein required to regulate the expression of the penI and penP genes. We purified the wild-type repressor protein, used it in gel retardation and DNase I protection experiments, and identified three operators positioned in the region between the penP and penI coding sequences. The spatial arrangement of the operators and the hierarchy in repressor binding seen in the protection experiments indicate that (i) the penI gene product represses the expression of the penP gene by physically blocking the RNA polymerase-binding site and (ii) the penI gene is autoregulated.
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166. |
Zanphorlin LM,
de Morais MAB,
Diogo JA,
Domingues MN,
de Souza FHM,
Ruller R,
Murakami MT,
( 2019 ) Structure-guided design combined with evolutionary diversity led to the discovery of the xylose-releasing exo-xylanase activity in the glycoside hydrolase family 43. PMID : 30556897 : DOI : 10.1002/bit.26899 Abstract >>
Rational design is an important tool for sculpting functional and stability properties of proteins and its potential can be much magnified when combined with in vitro and natural evolutionary diversity. Herein, we report the structure-guided design of a xylose-releasing exo-�]-1,4-xylanase from an inactive member of glycoside hydrolase family 43 (GH43). Structural analysis revealed a nonconserved substitution (Lys247) that results in the disruption of the hydrogen bond network that supports catalysis. The mutation of this residue to a conserved serine restored the catalytic activity and crystal structure elucidation of the mutant confirmed the recovery of the proper orientation of the catalytically relevant histidine. Interestingly, the tailored enzyme can cleave both xylooligosaccharides and xylan, releasing xylose as the main product, being the first xylose-releasing exo-�]-1,4-xylanase reported in the GH43 family. This enzyme presents a unique active-site topology when compared with closely related �]-xylosidases, which is the absence of a hydrophobic barrier at the positive-subsite region, allowing the accommodation of long substrates. Therefore, the combination of rational design for catalytic activation along with naturally occurring differences in the substrate binding interface led to the discovery of a novel activity within the GH43 family. In addition, these results demonstrate the importance of solvation of the �]-propeller hollow for GH43 catalytic function and expand our mechanistic understanding about the diverse modes of action of GH43 members, a key and polyspecific carbohydrate-active enzyme family abundant in most plant cell-wall-degrading microorganisms.
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167. |
Amory A,
Kunst F,
Aubert E,
Klier A,
Rapoport G,
( 1987 ) Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis. PMID : 3098732 : DOI : 10.1128/jb.169.1.324-333.1987 PMC : PMC211771 Abstract >>
The sacQ gene from Bacillus licheniformis was cloned and expressed in Bacillus subtilis. Deletion analysis shows that it encodes a 46-amino-acid polypeptide homologous to the B. subtilis sacQ gene product. The polypeptide, when it is overexpressed, activates the expression of a number of target genes in B. subtilis, all encoding secreted enzymes: alkaline protease, levansucrase, beta-glucanase(s), xylanase, and alpha-amylase. The maximum stimulations measured for alkaline protease and levansucrase were by a factor of 70 and 50, respectively, when the sacQ gene from B. licheniformis was present on a multicopy plasmid in B. subtilis. The sacQ genes from B. subtilis and B. licheniformis, cloned in the same multicopy plasmid, were compared under the same conditions. The sacQ gene from B. licheniformis was more efficient than the sacQ gene from B. subtilis in producing the hypersecretion phenotype. The sacQ structural genes from B. subtilis and B. licheniformis were placed under the control of the same inducible promoter. Hypersecretion was specifically obtained under conditions of full induction of the promoter. The target site of levansucrase regulation by sacQ was identified as a 440-base-pair fragment located in the 5' noncoding region of sacB, suggesting transcriptional control.
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168. |
Phetcharat T,
Dawkrajai P,
Chitov T,
Mhuantong W,
Champreda V,
Bovonsombut S,
( 2019 ) Biosurfactant-Producing Capability and Prediction of Functional Genes Potentially Beneficial to Microbial Enhanced Oil Recovery in Indigenous Bacterial Communities of an Onshore Oil Reservoir. PMID : 30734843 : DOI : 10.1007/s00284-019-01641-8 Abstract >>
Microbial enhanced oil recovery (MEOR) is a bio-based technology with economic and environmental benefits. The success of MEOR depends greatly on the types and characteristics of indigenous microbes. The aim of this study was to evaluate the feasibility of applying MEOR at Mae Soon Reservoir, an onshore oil reservoir experiencing a decline in its production rate. We investigated the capability of the reservoir's bacteria to produce biosurfactants, and evaluated the potentials of uncultured indigenous bacteria to support MEOR by means of prediction of MEOR-related functional genes, based on a set of metagenomic 16s rRNA gene data. The biosurfactant-producing bacteria isolated from the oil-bearing sandstones from the reservoir belonged to one species: Bacillus licheniformis, with one having the ability to decrease surface tension from 72 to 32 mN/m. Gene sequences responsible for biosurfactant (licA3), lipase (lipP1) and catechol 2,3-dioxygenase (C23O) were detected in these isolates. The latter two, and other genes encoding MEOR-related functional proteins such as enoyl-CoA hydratase and alkane 1-monooxygenase, were predicted in the bacterial communities residing the reservoir's sandstones. Exposure of these sandstones to nutrients, consisting of KNO3 and NaH2PO4, resulted in an increase in the proportions of some predicted functional genes. These results indicated the potentials of MEOR application at Mae Soon site. Using the approaches demonstrated in this study would also assist evaluation of the feasibility of applying MEOR in oil reservoirs, which may be enhanced by an appropriate nutrient treatment.
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169. |
Kobayashi T,
Zhu YF,
Nicholls NJ,
Lampen JO,
( 1987 ) A second regulatory gene, blaR1, encoding a potential penicillin-binding protein required for induction of beta-lactamase in Bacillus licheniformis. PMID : 3040663 : DOI : 10.1128/jb.169.9.3873-3878.1987 PMC : PMC213680 Abstract >>
A second regulatory locus (blaR1) required for the induction of beta-lactamase synthesis in Bacillus licheniformis 749 was cloned and sequenced. The gene was located on a 5.2-kilobase-pair SphI DNA fragment which also contained the beta-lactamase (blaP) and repressor (blaI) genes. Bacillus subtilis BD224 carrying these three genes synthesized beta-lactamase on exposure to cephalosporin C, whereas Escherichia coli HB101 carrying the genes did not show any detectable induction of the enzyme. An open reading frame of 1,803 bases was identified as the blaR1 gene by subcloning and DNA sequencing. The gene started 2 bases downstream of the termination codon of bla1 and was preceded by a putative Shine-Dalgarno sequence (AAGGA) with a spacing of 5 bases. The deduced blaR1 product (601 amino acids) had a molecular weight of 68,425. Five transmembrane regions were predicted from the hydrophobicity profile. The region around Phe-Ala-Pro-Ala-Ser-Thr-Tyr-Lys (amino acids 398 to 405), which appeared to be located outside the membrane, was homologous to the binding regions of penicillin-binding proteins, including the beta-lactamases. The segment of 22 amino acids from 400 to 421 showed more than 70% homology to the penicillin-binding region of PBP 2 of E. coli. The blaR1 gene encodes a potential penicillin receptor which is required for the induction of beta-lactamase in B. licheniformis 749.
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170. |
Dubnau E,
Weir J,
Nair G,
Carter L,
Moran C,
Smith I,
( 1988 ) Bacillus sporulation gene spo0H codes for sigma 30 (sigma H). PMID : 3277943 : DOI : 10.1128/jb.170.3.1054-1062.1988 PMC : PMC210873 Abstract >>
The DNA sequences of the spo0H genes from Bacillus licheniformis and B. subtilis are described, and the predicted open reading frames code for proteins of 26,097 and 25,447 daltons, respectively. The two spo0H gene products are 91% identical to one another and about 25% identical to most of the procaryotic sigma factors. The predicted proteins have a conserved 14-amino-acid sequence at their amino terminal end, typical of sigma factors. Antibodies raised against the spo0H gene product of B. licheniformis specifically react with RNA polymerase sigma factor protein, sigma 30, purified from B. subtilis. We conclude that the spo0H genes of B. licheniformis and B. subtilis code for sigma 30, now known as sigma H.
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171. |
Imanaka T,
Himeno T,
Aiba S,
( 1987 ) Cloning and nucleotide sequence of the penicillinase antirepressor gene penJ of Bacillus licheniformis. PMID : 3040662 : DOI : 10.1128/jb.169.9.3867-3872.1987 PMC : PMC213679 Abstract >>
The penicillinase antirepressor gene, penJ, of Bacillus licheniformis ATCC 9945a was cloned in Escherichia coli by using pMB9 as a vector plasmid. The penicillinase gene, penP, its repressor gene, penI, and penJ were encoded on the cloned 5.2-kilobase HindIII fragment of the recombinant plasmid pTTE71. The penJ open reading frame was composed of 1,803 bases and 601 amino acid residues (molecular weight, 68,388). A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site. Since this sequence was located in the 3'-terminal region of the penI gene, penJ might be transcribed together with penI as a polycistronic mRNA from the penI promoter. Frameshift mutations of penJ were constructed in vitro from pTTE71, and the penJ mutant gene was introduced into B. licheniformis by chromosomal recombination. The transformant B. licheniformis U173 (penP+ penI+ penJ) turned out to be uninducible for penicillinase production, whereas the wild-type strain (penP+ penI+ penJ+) was inducible. Only when these three genes (penP, penI, and PenJ) were simultaneously subcloned in Bacillus subtilis did the plasmid carrier exhibit inducible penicillinase production, as did wild-type B. licheniformis. It was concluded that penJ is involved in the penicillinase induction. The regulation of penP expression by penI and penJ is discussed.
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172. |
Gray GL,
Mainzer SE,
Rey MW,
Lamsa MH,
Kindle KL,
Carmona C,
Requadt C,
( 1986 ) Structural genes encoding the thermophilic alpha-amylases of Bacillus stearothermophilus and Bacillus licheniformis. PMID : 3009417 : DOI : 10.1128/jb.166.2.635-643.1986 PMC : PMC214652 Abstract >>
The genes encoding the thermostable alpha-amylases of Bacillus stearothermophilus and B. licheniformis were cloned in Escherichia coli, and their DNA sequences were determined. The coding and deduced polypeptide sequences are 59 and 62% homologous to each other, respectively. The B. stearothermophilus protein differs most significantly from that of B. licheniformis in that it possesses a 32-residue COOH-terminal tail. Transformation of E. coli with vectors containing either gene resulted in the synthesis and secretion of active enzymes similar to those produced by the parental organisms. A plasmid was constructed in which the promoter and the NH2-terminal two-thirds of the B. stearothermophilus coding sequence was fused out of frame to the entire mature coding sequence of the B. licheniformis gene. Approximately 1 in 5,000 colonies transformed with this plasmid was found to secrete an active amylase. Hybridization analysis of plasmids isolated from these amylase-positive colonies indicated that the parental coding sequences had recombined by homologous recombination. DNA sequence analysis of selected hybrid genes revealed symmetrical, nonrandom distribution of loci at which the crossovers had resolved. Several purified hybrid alpha-amylases were characterized and found to differ with respect to thermostability and specific activity.
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173. |
Jacobs M,
Eliasson M,
Uhlén M,
Flock JI,
( 1985 ) Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis. PMID : 3001653 : DOI : 10.1093/nar/13.24.8913 PMC : PMC318961 Abstract >>
The gene encoding subtilisin Carlsberg from Bacillus licheniformis has been isolated by molecular cloning using a mixture of synthetic oligonucleotides. The entire nucleotide sequence of the coding sequence as well as 5' and 3' flanking sequences have been determined. The deduced amino acid sequence reveals an N-terminal signal peptide consisting of 29 residues, a pro-peptide of 76 residues followed by the mature protein comprising 274 residues. The ATG initiator codon is preceded by two putative overlapping ribosomal binding sequences. A palindromic sequence typical for transcription termination is found downstream from the TAA stop codon. Structural comparisons between different known subtilisin genes reveal extensive homology, particularly in the parts coding for the pro-region and the mature protein. Expression studies in Bacillus subtilis show that the cloned fragment produces a functional enzyme when inserted after a B. subtilis promoter.
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174. |
Baslé A,
Hewitt L,
Koh A,
Lamb HK,
Thompson P,
Burgess JG,
Hall MJ,
Hawkins AR,
Murray H,
Lewis RJ,
( 2018 ) Crystal structure of NucB, a biofilm-degrading endonuclease. PMID : 29165717 : DOI : 10.1093/nar/gkx1170 PMC : PMC5758888 Abstract >>
Bacterial biofilms are a complex architecture of cells that grow on moist interfaces, and are held together by a molecular glue of extracellular proteins, sugars and nucleic acids. Biofilms are particularly problematic in human healthcare as they can coat medical implants and are thus a potential source of disease. The enzymatic dispersal of biofilms is increasingly being developed as a new strategy to treat this problem. Here, we have characterized NucB, a biofilm-dispersing nuclease from a marine strain of Bacillus licheniformis, and present its crystal structure together with the biochemistry and a mutational analysis required to confirm its active site. Taken together, these data support the categorization of NucB into a unique subfamily of the �]�]�\ metal-dependent non-specific endonucleases. Understanding the structure and function of NucB will facilitate its future development into an anti-biofilm therapeutic agent.
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175. |
Farro EGS,
Leite AET,
Silva IA,
Filgueiras JG,
de Azevedo ER,
Polikarpov I,
Nascimento AS,
( 2018 ) GH43 endo-arabinanase from Bacillus licheniformis: Structure, activity and unexpected synergistic effect on cellulose enzymatic hydrolysis. PMID : 29800670 : DOI : 10.1016/j.ijbiomac.2018.05.157 Abstract >>
The hydrolysis of the plant biomass provides many interesting opportunities for the generation of building blocks for the green chemistry industrial applications. An important progress has been made for the hydrolysis of the cellulosic component of the biomass while, for the hemicellulosic components, the advances are less straightforward. Here, we describe the cloning, expression and biochemical and structural characterization of BlAbn1, a GH43 arabinanase from Bacillus licheniformis. This enzyme is selective for linear arabinan and efficiently hydrolyzes this substrate, with a specific activity of 127 U/mg. The enzyme has optimal conditions for activity at pH 8.0 and 45 �XC and its activity is only partially dependent of a bound calcium ion since 70% of the maximal activity is preserved even when 1 mM EDTA is added to the reaction medium. BlAbn1 crystal structure revealed a typical GH43 fold and narrow active site, which explains the selectivity for linear substrates. Unexpectedly, the enzyme showed a synergic effect with the commercial cocktail Accellerase 1500 on cellulose hydrolysis. Scanning Electron Microscopy, Solid-State NMR and relaxometry data indicate that the enzyme weakens the interaction between cellulose fibers in filter paper, thus providing an increased access to the cellulases of the cocktail.
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176. |
Tang W,
Dong SH,
Repka LM,
He C,
Nair SK,
van der Donk WA,
( 2015 ) Applications of the class II lanthipeptide protease LicP for sequence-specific, traceless peptide bond cleavage. PMID : 30090246 : DOI : 10.1039/c5sc02329g PMC : PMC6054071 Abstract >>
The final step of lanthipeptide biosynthesis involves the removal of leader peptides by dedicated proteases. In vitro characterization of LicP, a class II LanP protease involved in the biosynthesis of the lantibiotic lichenicidin, revealed a self-cleavage step that removes 100 amino acids from the N-terminus. The 2.35 ? resolution crystal structure provides insights into the active site geometry and substrate specificity, and unveiled an unusual calcium-independent maturation mechanism of a subtilisin family member. LicP processes LicA2 peptides with or without post-translational modifications, but dehydrated and cyclized LicA2 is favored. Investigation of its substrate specificity demonstrated that LicP can serve as an efficient sequence-specific traceless protease and may have great utility in basic research and biotechnology. Encouraged by these findings for LicP, we identified 13 other class II LanPs, ten of which were previously unknown, and suggest that these proteins may serve as a pool of proteases with diverse recognition sequences for general traceless tag removal applications, expanding the current toolbox of proteases.
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177. |
Hadjidj R,
Badis A,
Mechri S,
Eddouaouda K,
Khelouia L,
Annane R,
El Hattab M,
Jaouadi B,
( 2018 ) Purification, biochemical, and molecular characterization of novel protease from Bacillus licheniformis strain K7A. PMID : 29605249 : DOI : 10.1016/j.ijbiomac.2018.03.167 Abstract >>
A novel extracellular alkaline protease, called SAPHM, from Bacillus licheniformis strain K7A was purified by four steps procedure involving heat treatment (30 min at 70 �XC) followed by ammonium sulfate precipitation (40-70%)-dialysis, UNO Q-12 FPLC, and ZORBAX PSM 300 HPLC, and submitted to biochemical characterization assays. The purified enzyme is a monomer of molecular mass of 30,325.12 Da. It was completely inhibited by phenylmethanesulfonyl fluoride (PMSF)and diiodopropyl fluorophosphates (DFP), which strongly suggested its belonging to the serine protease family. Its sequence of the 26 NH2-terminal residues showed high homology with those of Bacillus proteases. The purified enzyme was optimally active at pH 10 and temperature 70 �XC. Its catalytic efficiency was higher than those of Alcalase and Thermolysin. SAPHM exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively. Data suggest also that SAPHM may be considered as potential candidate for future applications in non-aqueous peptide biocatalysis because it possesses an elevated organic solvent resistance. The sapHM gene encoding SAPHM was cloned, sequenced, and expressed in Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rSAPHM) were similar to those of native one. The deduced amino acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with APRMP1 protease from Bacillus licheniformis strain MP1, with only 9 aa of difference.
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178. |
( 2013 ) Biodiversity of aerobic endospore-forming bacterial species occurring in Yanyanku and Ikpiru, fermented seeds of Hibiscus sabdariffa used to produce food condiments in Benin. PMID : 23571124 : DOI : 10.1016/j.ijfoodmicro.2013.02.008 Abstract >>
Yanyanku and Ikpiru made by the fermentation of Malcavene bean (Hibiscus sabdariffa) are used as functional additives for Parkia biglobosa seed fermentations in Benin. A total of 355 aerobic endospore-forming bacteria (AEFB) isolated from Yanyanku and Ikpiru produced in northern and southern Benin were identified using phenotypic and genotypic methods, including GTG5-PCR, M13-PCR, 16S rRNA, gyrA and gyrB gene sequencing. Generally, the same 5-6 species of the genus Bacillus predominated: Bacillus subtilis (17-41% of isolates), Bacillus cereus (8-39%), Bacillus amyloliquefaciens (9-22%), Bacillus licheniformis (3-26%), Bacillus safensis (8-19%) and Bacillus altitudinis (0-19%). Bacillus aryabhattai, Bacillus flexus, and Bacillus circulans (0-2%), and species of the genera Lysinibacillus (0-14%), Paenibacillus (0-13%), Brevibacillus (0-4%), and Aneurinibacillus (0-3%) occurred sporadically. The diarrheal toxin encoding genes cytK-1, cytK-2, hblA, hblC, and hblD were present in 0%, 91% 15%, 34% and 35% of B. cereus isolates, respectively. 9% of them harbored the emetic toxin genetic determinant, cesB. This study is the first to identify the AEFB of Yanyanku and Ikpiru to species level and perform a safety evaluation based on toxin gene detections. We further suggest, that the gyrA gene can be used for differentiating the closely related species Bacillus pumilus and B. safensis.
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( 2012 ) Antimicrobial susceptibility of Bacillus strains isolated from primary starters for African traditional bread production and characterization of the bacitracin operon and bacitracin biosynthesis. PMID : 22941078 : DOI : 10.1128/AEM.00730-12 PMC : PMC3485967 Abstract >>
Bacillus spp. are widely used as feed additives and probiotics. However, there is limited information on their resistance to various antibiotics, and there is a growing concern over the transfer of antibiotic resistance genes. The MIC for 8 antibiotics was determined for 85 Bacillus species strains, Bacillus subtilis subsp. subtilis (n = 29), Bacillus licheniformis (n = 38), and Bacillus sonorensis (n = 18), all of which were isolated from starters for Sudanese bread production. All the strains were sensitive to tetracycline (8.0 mg/liter), vancomycin (4.0 mg/liter), and gentamicin (4.0 mg/liter) but resistant to streptomycin. Sensitivity to clindamycin, chloramphenicol, and kanamycin was species specific. The erythromycin resistance genes ermD and ermK were detected by PCR in all of the erythromycin-resistant (MIC, ?16.0 mg/liter) B. licheniformis strains and one erythromycin-sensitive (MIC, 4.0 mg/liter) B. licheniformis strain. Several amino acid changes were present in the translated ermD and ermK nucleotide sequences of the erythromycin-sensitive strain, which could indicate ErmD and ErmK protein functionalities different from those of the resistance strains. The ermD and ermK genes were localized on an 11.4-kbp plasmid. All of the B. sonorensis strains harbored the bacitracin synthetase gene, bacA, and the transporter gene bcrA, which correlated with their observed resistance to bacitracin. Bacitracin was produced by all the investigated species strains (28%), as determined by ultra-high-definition quadrupole time-of-flight liquid chromatography-mass spectrometry (UHD-QTOF LC/MS). The present study has revealed species-specific variations in the antimicrobial susceptibilities of Bacillus spp. and provides new information on MIC values, as well as the occurrence of resistance genes in Bacillus spp., including the newly described species B. sonorensis.
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( 2013 ) Co-production of surfactin and a novel bacteriocin by Bacillus subtilis subsp. subtilis H4 isolated from Bikalga, an African alkaline Hibiscus sabdariffa seed fermented condiment. PMID : 23466466 : DOI : 10.1016/j.ijfoodmicro.2013.01.013 Abstract >>
Bikalga is a Hibiscus sabdariffa seed fermented condiment widely consumed in Burkina Faso and neighboring countries. The fermentation is dominated by Bacillus subtilis group species. Ten B. subtilis subsp. subtilis (six isolates) and Bacillus licheniformis (four isolates) isolated from traditional Bikalga were examined for their antimicrobial activity against a panel of 36 indicator organisms including Gram-positive and Gram-negative bacteria and yeasts. The Bacillus spp. isolates showed variable inhibitory abilities depending on the method used. Both Gram-positive and Gram-negative bacteria were inhibited in the agar spot assay while only Gram-positive pathogens were inhibited in the agar well diffusion assay. Cell free supernatants (CFS) of pure cultures of 3 B. subtilis subsp. subtilis (G2, H4 and F1) strains inhibited growth of Listeria monocytogenes, Micrococcus luteus, Staphylococcus aureus and Bacillus cereus, while CFS of 2 B. licheniformis (E3 and F9) strains only inhibited M. luteus. The antimicrobial substance(s) produced by B. subtilis subsp. subtilis H4 was further characterized. The antimicrobial substance(s) produced by H4 was detected from mid-exponential growth phase. The activity was sensitive to protease and trypsin, but resistant to the proteolytic action of proteinase K and papain. Treatment with �\-amylase and lipase II resulted in a complete loss of antimicrobial effect, indicating that a sugar moiety and lipid moiety are necessary for the activity. Treatment with mercapto-ethanol resulted in a significant loss, indicative of the presence of disulfide bridges. The antimicrobial activity of H4 was heat resistant and active at pH3-10. PCR detection of yiwB, sboA, spoX, albA and spaS, etnS genes and genes coding for surfactins and plipastatins (fengycins) indicated a potential for subtilosin, subtilin and lipopeptide production, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out and a single band of approximately 4kDa had antimicrobial activity. Ultra high performance liquid chromatography-time of flight mass spectrometry (UHPLC-TOFMS) analysis of the 4kDa band allowed identification of surfactin and a protein with a monoisotopic mass of 3346.59Da, which is dissimilar in size to subtilosin and subtilin. Surfactin is a cyclic lipoheptapeptide, which contains a �]-hydroxy fatty acid, but no di-sulfide bridges or sugar residues. The complete loss of activity upon amylase treatment indicates that surfactin was not responsible for the observed antimicrobial effect. However, it cannot completely be ruled out that surfactin acts synergistically with the detected protein, though further investigations are needed to confirm this.
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( 2012 ) The structure of L-amino-acid ligase from Bacillus licheniformis. PMID : 23090402 : DOI : 10.1107/S0907444912038103 Abstract >>
L-Amino-acid ligases (LALs) are enzymes which catalyze the formation of dipeptides by linking two L-amino acids. Although many dipeptides are known and expected to have medical and nutritional benefits, their practical use has been limited owing to their low availability and high expense. LALs are potentially desirable tools for the efficient production of dipeptides; however, the molecular basis of substrate recognition by LAL has not yet been sufficiently elucidated for the design of ideal LALs for the desired dipeptides. This report presents the crystal structure of the LAL BL00235 derived from Bacillus licheniformis NBRC 12200 determined at 1.9 ? resolution using the multi-wavelength anomalous dispersion method. The overall structure of BL00235 is fairly similar to that of YwfE, the only LAL with a known structure, but the structure around the catalytic site contains some significant differences. Detailed structural comparison of BL00235 with YwfE sheds some light on the molecular basis of the substrate specificities.
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( 2012 ) Crystal structure of greglin, a novel non-classical Kazal inhibitor, in complex with subtilisin. PMID : 23075397 : DOI : 10.1111/febs.12033 DOI : 10.1111/febs.12033 Abstract >>
Greglin is an 83-residue serine protease inhibitor purified from the ovaries of the locust Schistocerca gregaria. Greglin is a strong inhibitor of subtilisin and human neutrophil elastase, acting at sub-nanomolar and nanomolar concentrations, respectively; it also inhibits neutrophil cathepsin G, �\-chymotrypsin and porcine pancreatic elastase, but to a lesser extent. In the present study, we show that greglin resists denaturation at high temperature (95 �XC) and after exposure to acetonitrile and acidic or basic pH. Greglin is composed of two domains consisting of residues 1-20 and 21-83. Mass spectrometry indicates that the N-terminal domain (1-20) is post-translationally modified by phosphorylations at three sites and probably contains a glycosylation site. The crystal structure of the region of greglin comprising residues 21-78 in complex with subtilisin was determined at 1.75 ? resolution. Greglin represents a novel member of the non-classical Kazal inhibitors, as it has a unique additional C-terminal region (70-83) connected to the core of the molecule via a supplementary disulfide bond. The stability of greglin was compared with that of an ovomucoid inhibitor. The thermostability and inhibitory specificity of greglin are discussed in light of its structure. In particular, we propose that the C-terminal region is responsible for non-favourable interactions with the autolysis loop (140-loop) of serine proteases of the chymotrypsin family, and thus governs specificity. The atomic coordinates and structure factors for the greglin-subtilisin complex have been deposited with the RCSB Protein Data Bank under accession number 4GI3. Greglin and Subtilisin Carlsberg bind by X-ray crystallography (View interaction).
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( 1990 ) Beta-lactamase of Bacillus licheniformis 749/C at 2 A resolution. PMID : 2326252 : DOI : 10.1002/prot.340070205 Abstract >>
Two crystal forms (A and B) of the 29,500 Da Class A beta-lactamase (penicillinase) from Bacillus licheniformis 749/C have been examined crystallographically. The structure of B-form crystals has been solved to 2 A resolution, the starting model for which was a 3.5 A structure obtained from A-form crystals. The beta-lactamase has an alpha + beta structure with 11 helices and 5 beta-strands seen also in a penicillin target DD-peptidase of Streptomyces R61. Atomic parameters of the two molecules in the asymmetric unit were refined by simulated annealing at 2.0 A resolution. The R factor is 0.208 for the 27,330 data greater than 3 sigma (F), with water molecules excluded from the model. The catalytic Ser-70 is at the N-terminus of a helix and is within hydrogen bonding distance of conserved Lys-73. Also interacting with the Lys-73 are Asn-132 and the conserved Glu-166, which is on a potentially flexible helix-containing loop. The structure suggests the binding of beta-lactam substrates is facilitated by interactions with Lys-234, Thr-235, and Ala-237 in a conserved beta-strand peptide, which is antiparallel to the beta-lactam's acylamido linkage; an exposed cavity near Asn-170 exists for acylamido substituents. The reactive double bond of clavulanate-type inhibitors may interact with Arg-244 on the fourth beta-strand. A very similar binding site architecture is seen in the DD-peptidase.
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( 2012 ) phoR sequences as a phylogenetic marker to differentiate the species in the Bacillus subtilis group. PMID : 23145827 : DOI : 10.1139/w2012-106 Abstract >>
Bacillus subtilis and its closely related species are indistinguishable from one another by morphological characteristics and 16S rDNA sequences. In this study, the partial phoR sequence was tested to determine the phylogenetic relationship of species in the B. subtilis group. Degenerate primers were developed according to the relatively conserved nucleotide sequences of phoR and the linked gene phoP in the B. subtilis group. The primers amplified a 1100 bp phoR fragment from strains representative of 6 species in the B. subtilis group. Based on the sequenced fragments, 26 type strains comprising these 6 species were clearly distinguished. At the intraspecies level, the phoR sequence similarities were 90%-100%, but at the interspecies level, the phoR sequence similarities were 32.8%-75%. Compared with the gyrB sequence, the phoR sequences showed a larger divergence especially at the interspecies levels. Therefore, the phoR sequence may be an efficient alternative marker for phylogenetic and taxonomic analysis of species in the B. subtilis group. Twenty-three Bacillus undomesticated isolates were tested for identification and phylogenetic analysis based on the phoR and gyrB sequences. The 23 isolates could be clearly delineated into 4 distinct groups, 10 as B. subtilis, 3 as B. mojavensis, 2 as B. atrophaeus, and 8 as B. amyloliquefaciens.
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( 2012 ) Characterization of Xyn30A and Axh43A of Bacillus licheniformis SVD1 identified by its genomic analysis. PMID : 22883553 : DOI : 10.1016/j.enzmictec.2012.06.003 Abstract >>
The genome sequence of Bacillus licheniformis SVD1, that produces a cellulolytic and hemi-cellulolytic multienzyme complex, was partially determined, indicating that the glycoside hydrolase system of this strain is highly similar to that of B. licheniformis ATCC14580. All of the fifty-six genes encoding glycoside hydrolases identified in B. licheniformis ATCC14580 were conserved in strain SVD1. In addition, two new genes, xyn30A and axh43A, were identified in the B. licheniformis SVD1 genome. The xyn30A gene was highly similar to Bacillus subtilis subsp. subtilis 168 xynC encoding for a glucuronoarabinoxylan endo-1,4-�]-xylanase. Xyn30A, produced by a recombinant Escherichia coli, had high activity toward 4-O-methyl-D-glucurono-D-xylan but showed definite activity toward oat-spelt xylan and unsubstituted xylooligosaccharides. Recombinant Axh43A, consisting of a family-43 catalytic module of the glycoside hydrolases and a family-6 carbohydrate-binding module (CBM), was an arabinoxylan arabinofuranohydrolase (�\-L-arabinofuranosidase) classified as AXH-m23 and capable of releasing arabinosyl residues, which are linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan or arabinoxylan oligomers. The isolated CBM polypeptide had an affinity for soluble and insoluble xylans and removal of the CBM from Axh43A abolished the catalytic activity of the enzyme, indicating that the CBM plays an essential role in hydrolysis of arabinoxylan.
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( 2013 ) Expression and characterization of extreme alkaline, oxidation-resistant keratinase from Bacillus licheniformis in recombinant Bacillus subtilis WB600 expression system and its application in wool fiber processing. PMID : 23264133 : DOI : 10.1007/s11274-012-1237-5 Abstract >>
A keratin-degrading bacterium of Bacillus licheniformis BBE11-1 was isolated and its ker gene encoding keratinase with native signal peptide was cloned and expressed in Bacillus subtilis WB600 under the strong P HpaII promoter of the pMA0911 vector. In the 3-L fermenter, the recombinant keratinase was secreted with 323 units/mL when non-induced after 24 h at 37 �XC. And then, keratinase was concentrated and purified by hydrophobic interaction chromatography using HiTrap Phenyl-Sepharose Fast Flow. The recombinant keratinase had an optimal temperature and the pH at 40 �XC and 10.5, respectively, and was stable at 10-50 �XC and pH 7-11.5. We found this enzyme can retained 80 % activity after treated 5 h with 1 M H2O2, it was activated by Mg(2+), Co(2+) and could degraded broad substrates such as degraded feather, bovine serum albumin, casein, gelatin, the keratinase was considered to be a serine protease. Coordinate with Savinase, the keratinase could efficient prevent shrinkage and eliminate fibres of wool, which showed its potential in textile industries and detergent industries.
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( 1998 ) Crystal structure of subtilisin DY, a random mutant of subtilisin Carlsberg. PMID : 9826175 : DOI : 10.1046/j.1432-1327.1998.2570309.x Abstract >>
The crystal structure of subtilisin DY inhibited by N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone has been solved by molecular replacement with subtilisin Carlsberg as the starting model. The model has been refined to a crystallographic R factor (= sigma absolute value [(absolute value Fo) - (absolute value Fc)] / sigma (absolute value of Fo) of 15.1% using X-ray diffraction data to 0.175 nm resolution. Subtilisin DY is an alkaline proteinase from the X-irradiated Japanese strain DY of Bacillus licheniformis, which normally produces subtilisin Carlsberg. It has very similar properties to subtilisin Carlsberg, with a slightly enhanced resistance to heat and guanidine hydrochloride-induced denaturation, in spite of the fact that the sequences of the two enzymes differ in 31 positions out of 274 residues. The close similarity in overall three-dimensional structure of subtilisins DY and Carlsberg and also their physicochemical properties, such as activity and stability, shows that nature aided by X-irradiation for rapid 'evolution' is able to accommodate considerable changes in sequence without substantial changes in property.
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( 1998 ) Bacillus licheniformis sigB operon encoding the general stress transcription factor sigma B. PMID : 9661670 : DOI : 10.1016/s0378-1119(98)00140-1 Abstract >>
The general stress response of the Gram-positive soil bacterium Bacillus subtilis is controlled by the sigma B transcription factor. sigma B activity is regulated by the newly discovered partner switching mechanism of signal transduction, which integrates the two different classes of challenges which posttranslationally activate sigma B: environmental stress and energy stress. Our investigation of a possible sigma B homologue in the related soil bacterium B. licheniformis had two goals. First, this study would contribute to understanding the distribution of the sigma B general stress system among Gram-positive bacteria. Second, a phylogenetic comparison of regulatory systems can supplement genetic and biochemical analysis by revealing conserved features that are critical for function. We report here that (1) B. licheniformis cells contain a protein that closely resembles B. subtilis sigma B in size and antigenic properties; (2) the level of this potential sigma B homologue rapidly increases following environmental or energy stress; and (3) the B. licheniformis genome encodes a homologue of the sigB general stress operon, including the sigma B structural gene and seven rsb regulatory genes. Based on these results, B. licheniformis possesses a general stress system likely regulated by two coupled partner switching modules that sense and integrate the two broad classes of activating stress signals.
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( 1998 ) The arcABDC gene cluster, encoding the arginine deiminase pathway of Bacillus licheniformis, and its activation by the arginine repressor argR. PMID : 9851988 : PMC : PMC107747 PMC : PMC107747 Abstract >>
The arginine deiminase pathway enables Bacillus licheniformis to grow anaerobically on arginine. Both the presence of arginine and anaerobiosis are needed to trigger induction of the pathway. In this study we have cloned and sequenced the arc genes encoding the pathway. They appear clustered in an operon-like structure in the order arcA (arginine deiminase), arcB (ornithine carbamoyltransferase), arcD (putative arginine-ornithine antiporter), arcC (carbamate kinase). It was found that B. licheniformis has an arginine repressor, ArgR, homologous to the B. subtilis arginine repressor AhrC. Mutants affected in argR were isolated. These mutants have lost both repression by arginine of the anabolic ornithine carbamoyltransferase and induction of the arginine deiminase pathway. Electrophoretic band shift experiments and DNase I footprinting revealed that in the presence of arginine, ArgR binds to a site upstream from the arc promoter. The binding site is centered 108 nucleotides upstream from the transcription start point and contains a single Arg box.
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( 1998 ) The arcABDC gene cluster, encoding the arginine deiminase pathway of Bacillus licheniformis, and its activation by the arginine repressor argR. PMID : 9851988 : PMC : PMC107747 PMC : PMC107747 Abstract >>
The arginine deiminase pathway enables Bacillus licheniformis to grow anaerobically on arginine. Both the presence of arginine and anaerobiosis are needed to trigger induction of the pathway. In this study we have cloned and sequenced the arc genes encoding the pathway. They appear clustered in an operon-like structure in the order arcA (arginine deiminase), arcB (ornithine carbamoyltransferase), arcD (putative arginine-ornithine antiporter), arcC (carbamate kinase). It was found that B. licheniformis has an arginine repressor, ArgR, homologous to the B. subtilis arginine repressor AhrC. Mutants affected in argR were isolated. These mutants have lost both repression by arginine of the anabolic ornithine carbamoyltransferase and induction of the arginine deiminase pathway. Electrophoretic band shift experiments and DNase I footprinting revealed that in the presence of arginine, ArgR binds to a site upstream from the arc promoter. The binding site is centered 108 nucleotides upstream from the transcription start point and contains a single Arg box.
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( 1999 ) Molecular and biochemical characterization of the protein template controlling biosynthesis of the lipopeptide lichenysin. PMID : 9864322 : PMC : PMC103541 Abstract >>
Lichenysins are surface-active lipopeptides with antibiotic properties produced nonribosomally by several strains of Bacillus licheniformis. Here, we report the cloning and sequencing of an entire 26.6-kb lichenysin biosynthesis operon from B. licheniformis ATCC 10716. Three large open reading frames coding for peptide synthetases, designated licA, licB (three modules each), and licC (one module), could be detected, followed by a gene, licTE, coding for a thioesterase-like protein. The domain structure of the seven identified modules, which resembles that of the surfactin synthetases SrfA-A to -C, showed two epimerization domains attached to the third and sixth modules. The substrate specificity of the first, fifth, and seventh recombinant adenylation domains of LicA to -C (cloned and expressed in Escherichia coli) was determined to be Gln, Asp, and Ile (with minor Val and Leu substitutions), respectively. Therefore, we suppose that the identified biosynthesis operon is responsible for the production of a lichenysin variant with the primary amino acid sequence L-Gln-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Ile, with minor Leu and Val substitutions at the seventh position.
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( 1998 ) Activation of Bacillus licheniformis alpha-amylase through a disorder-->order transition of the substrate-binding site mediated by a calcium-sodium-calcium metal triad. PMID : 9551551 : Abstract >>
The structural basis as to how metals regulate the functional state of a protein by altering or stabilizing its conformation has been characterized in relatively few cases because the metal-free form of the protein is often partially disordered and unsuitable for crystallographic analysis. This is not the case, however, for Bacillus licheniformis alpha-amylase (BLA) for which the structure of the metal-free form is available. BLA is a hyperthermostable enzyme which is widely used in biotechnology, for example in the breakdown of starch or as a component of detergents. The determination of the structure of BLA in the metal-containing form, together with comparisons to the apo enzyme, will help us to understand the way in which metal ions can regulate enzyme activity. We report here the crystal structure of native, metal-containing BLA. The structure shows that the calcium-binding site which is conserved in all alpha-amylases forms part of an unprecedented linear triadic metal array, with two calcium ions flanking a central sodium ion. A region around the metal triad comprising 21 residues exhibits a conformational change involving a helix unwinding and a disorder-->order transition compared to the structure of metal-free BLA. Another calcium ion, not previously observed in alpha-amylases, is located at the interface between domains A and C. We present a structural description of a major conformational rearrangement mediated by metal ions. The metal induced disorder-->order transition observed in BLA leads to the formation of the extended substrate-binding site and explains on a structural level the calcium dependency of alpha-amylases. Sequence comparisons indicate that the unique Ca-Na-Ca metal triad and the additional calcium ion located between domains A and C might be found exclusively in bacterial alpha-amylases which show increased thermostability. The information presented here may help in the rational design of mutants with enhanced performance in biotechnological applications.
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( 1998 ) The fnr gene of Bacillus licheniformis and the cysteine ligands of the C-terminal FeS cluster. PMID : 9642208 : PMC : PMC107310 Abstract >>
In the facultatively anaerobic bacterium Bacillus licheniformis a gene encoding a protein of the fumarate nitrate reductase family of transcriptional regulators (Fnr) was isolated. Unlike Fnr proteins from gram-negative bacteria, but like Fnr from Bacillus subtilis, the protein contained a C-terminal cluster of cysteine residues. Unlike in Fnr from B. subtilis, this cluster (Cys226-X2-Cys229-X4-Cys234) is composed of only three Cys residues, which are supposed to serve together with an internal residue (Cys71) as the ligands for an FeS center. Transfer of the B. licheniformis gene to an fnr mutant of B. subtilis complemented the ability for synthesis of nitrate reductase during anaerobic growth.
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( 1998 ) Purification and characterization of a Bacillus licheniformis phosphatase specific for D-alpha-glycerophosphate. PMID : 9439579 : DOI : 10.1006/abbi.1997.0433 Abstract >>
Bacillus licheniformis ("Ford's type") was found to contain a novel enzyme, D-alpha-glycerophosphatase. The enzyme is highly specific for D-alpha-glycerophosphate, effecting little or no hydrolysis of L-alpha- or beta-glycerophosphate or other similar compounds. All other known alpha-glycerophosphatases preferentially hydrolyze the L isomer. The products of the D-alpha-glycerophosphatase reaction were identified as glycerol and inorganic phosphate. The enzyme is a monomer with an apparent molecular mass of approximately 25 kDa. As with most phosphatases, it requires divalent magnesium for activity, but unlike the nonspecific acid and alkaline phosphatases, its optimum pH is around neutral. Its K(m) for D-alpha-glycerophosphate in the presence of 1 mM Mg2+ was found to be 4.3 mM. D-alpha-glycerophosphatase was produced in B. licheniformis fermentations whether or not high levels of phosphate were present; the same was true of glycerol formation. D-alpha-glycerophosphatase is not strongly inhibited by inorganic phosphate and would therefore be capable of catalyzing the formation of glycerol in the presence of high levels of phosphate. The D-alpha-glycerophosphatase of B. licheniformis is similar in characteristics to L-alpha-glycerophosphatases known to synthesize glycerol in vivo, suggesting that D-alpha-glycerophosphatase may be the final enzyme in the fermentative glycerol formation pathway of B. licheniformis.
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( 1997 ) The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases. PMID : 9427658 : Abstract >>
The branched cyclic dodecylpeptide antibiotic bacitracin, produced by special strains of Bacillus, is synthesized nonribosomally by a large multienzyme complex composed of the three bacitracin synthetases BA1, BA2 and BA3. These enzymes activate and incorporate the constituent amino acids of bacitracin by a thiotemplate mechanism in a pathway driven by a protein template. The biochemical features of these enzymes have been studied intensively but little is known about the molecular organization of their genes. The entire bacitracin synthetase operon containing the genes bacA-bacC was cloned and sequenced, identifying a modular structure typical of peptide synthetases. The bacA gene product (BA1, 598kDa) contains five modules, with an internal epimerization domain attached to the fourth; bacB encodes BA2 (297kDa), and has two modules and a carboxy-terminal epimerization domain; bacC encodes BA3, five modules (723kDa) with additional internal epimerization domains attached to the second and fourth. A carboxy-terminal putative thioesterase domain was also detected in BA3. A putative cyclization domain was found in BA1 that may be involved in thiazoline ring formation. The adenylation/thioester-binding domains of the first two BA1 modules were overproduced and the detected amino-acid specificity coincides with the first two amino acids in bacitracin. Disruption of chromosomal bacB resulted in a bacitracin-deficient mutant. The genes encoding the bacitracin synthetases BA1, BA2 and BA3 are organized in an operon, the structure of which reflects the modular architecture expected of peptide synthetases. In addition, a putative thiazoline ring formation domain was identified in the BA1 gene.
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( 1998 ) Bacillus licheniformis MC14 alkaline phosphatase I gene with an extended COOH-terminus. PMID : 9485594 : DOI : 10.1111/j.1574-6968.1998.tb12840.x Abstract >>
Bacterial alkaline phosphatases (APases), except those isolated from Bacillus licheniformis, are approximately 45-kDa proteins while eucaryotic alkaline phosphatases are 60 kDa. To answer the question of whether the apparent 60-kDa alkaline phosphatase from Bacillus licheniformis accurately reflected the size of the protein, the entire gene was analyzed. DNA sequence analysis of the alkaline phosphatase I (APaseI) gene of B. licheniformis MC14 indicated that the gene could code for a 60-kDa protein of 553 amino acids. The deduced protein sequence of APaseI showed about 32% identity to those of B. subtilis APase III and IV and had apparent sequence homologies in the core structure and active sites that are conserved among APases of various sources. The extra carboxy-terminal sequence of APaseI, which made the enzyme bigger than other procaryotic APases, was not homologous to those of eucaryotic APases. The amino acid composition of APaseI was most similar to that of salt-dependent APase among the isozymes of B. licheniformis MC14. Another open reading frame of 261 amino acids was present 142 nucleotide upstream of the APaseI gene and its predicted amino acid sequence showed 68% identity to that of glucose dehydrogenase of B. megaterium.
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( 1998 ) Differences in binding modes of enantiomers of 1-acetamido boronic acid based protease inhibitors: crystal structures of gamma-chymotrypsin and subtilisin Carlsberg complexes. PMID : 9425066 : DOI : 10.1021/bi971166o Abstract >>
In order to probe the structural basis of stereoselectivity in the serine protease family, a series of enantiomeric boronic acids RCH2CH(NHCOCH3)B(OH)2 has been synthesized and kinetically characterized as transition-state analog inhibitors using alpha-chymotrypsin and subtilisin Carlsberg as model systems. When the R-substituent in this series was changed from a p-chlorophenyl to a 1-naphthyl group, alpha-chymotrypsin, but not subtilisin, reversed its usual preference for l-enantiomers and bound more tightly to the D-enantiomer [Martichonok, V., & Jones, J. B. (1996) J. Am. Chem. Soc. 118, 950-958]. The structural factors responsible for the differences in stereoselectivity between the two enzymes have been explored by X-ray crystallographic examination of subtilisin Carlsberg and gamma-chymotrypsin complexes of the L- and D-enantiomers of p-chlorophenyl and 1-naphthyl boronic acid derivatives. In both enzymes, the L-isomers of the inhibitors, which are more closely related to the natural L-amino acid substrates, form tetrahedral adducts, covalently linking the central boron atom and Ogamma of the catalytic serine. The d-isomers, however, differ in the way they interact with subtilisin or gamma-chymotrypsin. With subtilisin, both the D-p-chlorophenyl and D-1-naphthyl inhibitor complexes form covalent Ser Ogamma-to-boron bonds, but with gamma-chymotrypsin, the same inhibitors lead to novel tetrahedral adducts covalently linking both Ser195 Ogamma and His57 Nepsilon2 covalently via the boron atom.
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