| 1. |
Nakamura A,
Koide Y,
Miyazaki H,
Kitamura A,
Masaki H,
Beppu T,
Uozumi T,
( 1992 ) Gene cloning and characterization of a novel extracellular ribonuclease of Bacillus subtilis. PMID : 1396690 : DOI : 10.1111/j.1432-1033.1992.tb17268.x Abstract >>
An extracellular nuclease gene of Bacillus subtilis was cloned in the same organism by detecting the amplified enzyme activity, which was secreted from the transformant cells on an RNA-containing agar medium. An open reading frame encoding 289 amino acids was identified within the cloned fragment. The transcriptional initiation site was determined by nuclease S1 mapping and the promoter region showed similarity to the conserved recognition sequences for the E sigma A and/or E sigma E RNA polymerases. The production of the nuclease by the B. subtilis transformants greatly depends on the liquid medium used. SDS/PAGE analysis of the purified enzyme showed two adjoining bands of molecular mass about 32 kDa, and the NH2-terminal amino acid sequence analysis suggested that the NH2-terminal portion of the nuclease was subjected to a limited proteolysis after or during secretion. The nuclease was uniquely characterized as a Mg(2+)-activated ribonuclease which hydrolyzes RNA apparently nonspecifically into oligonucleotides with 5'-terminal phosphate. The deduced amino acid sequence of this enzyme shows no obvious similarity with other nuclease sequences.
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2. |
Peng Y,
Huang Q,
Zhang RH,
Zhang YZ,
( 2003 ) Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. PMID : 12524032 : Abstract >>
Bacillus amyloliquefaciens DC-4, which produces a strongly fibrinolytic enzyme, was isolated from douchi, a traditional Chinese soybean-fermented food. A fibrinolytic enzyme (subtilisin DFE) was purified from the supernatant of B. amyloliquefaciens DC-4 culture broth and displayed thermophilic, hydrophilic and strong fibrinolytic activity. Subtilisin DFE was demonstrated to be homogeneous by SDS-PAGE and isoelectric focusing electrophoresis, and has molecular mass of 28000 Da and a pI of 8.0. The optimal reaction pH value and temperature were 9.0 and 48 degrees C, respectively. Subtilisin DFE not only hydrolyzed fibrin but also several synthetic substrates, particularly Suc-Ala-Ala-Pro-Phe-pNA, and phenylmethylsulfony fluoride can completely inhibit its fibrinolytic activity. These results indicated that subtilisin DFE is a subtilisin-family serine protease, similar to nattokinase from Bacillus natto. The first 24 amino acid residues of the N-terminal sequence of subtilisin DFE were AQSVPYGVSQIKAPALHSQGFTGS, which is identical to that of subtilisin K-54, and different from that of NK and CK. Results from subtilisin DFE gene sequence analysis showed that subtilisin DFE is a novel fibrinolytic enzyme.
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3. |
Idriss EE,
Makarewicz O,
Farouk A,
Rosner K,
Greiner R,
Bochow H,
Richter T,
Borriss R,
( 2002 ) Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant-growth-promoting effect. PMID : 12101298 : DOI : 10.1099/00221287-148-7-2097 Abstract >>
Several Bacillus strains belonging to the B. subtilis/amyloliquefaciens group isolated from plant-pathogen-infested soil possess plant-growth-promoting activity [Krebs, B. et al. (1998) J Plant Dis Prot 105, 181-197]. Three out of the four strains investigated were identified as B. amyloliquefaciens and were able to degrade extracellular phytate (myo-inositol hexakisphosphate). The highest extracellular phytase activity was detected in strain FZB45, and diluted culture filtrates of this strain stimulated growth of maize seedlings under phosphate limitation in the presence of phytate. The amino acid sequence deduced from the phytase phyA gene cloned from FZB45 displayed a high degree of similarity to known Bacillus phytases. Weak similarity between FZB45 phytase and B. subtilis alkaline phosphatase IV pointed to a possible common origin of these two enzymes. The recombinant protein expressed by B. subtilis MU331 displayed 3(1)-phytase activity yielding D/L-Ins(1,2,4,5,6)P5 as the first product of phytate hydrolysis. A phytase-negative mutant strain, FZB45/M2, whose phyA gene is disrupted, was generated by replacing the entire wild-type gene on the chromosome of FZB45 with a km::phyA fragment, and culture filtrates obtained from FZB45/M2 did not stimulate plant growth. In addition, the growth of maize seedlings was promoted in the presence of purified phytase and the absence of culture filtrate. These genetic and biochemical experiments provide strong evidence that phytase activity of B. amyloliquefaciens FZB45 is important for plant growth stimulation under phosphate limitation.
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4. |
Anlezark GM,
Vaughan T,
Fashola-Stone E,
Michael NP,
Murdoch H,
Sims MA,
Stubbs S,
Wigley S,
Minton NP,
( 2002 ) Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954. PMID : 11782522 : DOI : 10.1099/00221287-148-1-297 Abstract >>
A nitroreductase with distinct properties that can activate the prodrug 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) was isolated from Bacillus amyloliquefaciens. The encoding gene was identified as a homologue of the ywrO of Bacillus subtilis, and was obtained as a PCR product by reverse genetics, cloned and the entire nucleotide sequence determined. The gene was found to reside between homologues of the B. subtilis alsD and yswB genes; however, the ywrO and yswB genes of B. amyloliquefaciens were not separated by a fourth gene, ywsA. The B. amyloliquefaciens ywrO gene was overexpressed, the recombinant protein purified and its properties were compared with those of two CB 1954-activating enzymes, Escherichia coli B nitroreductase (NTR) and Walker DT-diaphorase (DTD). In common with these enzymes menadione was an electron acceptor (K(m) 3 microM) and activity with this substrate was inhibited by the presence of dicoumarol (K(i) 1.0 microM). In contrast, YwrO showed a marked preference for NADPH as a cofactor (K(m) 40 microM) and therefore could not be classified as a DTD (EC 1.6.99.2). The flavin FMN was an acceptor with high affinity. B. amyloliquefaciens YwrO was shown to be a flavoprotein with a monomeric molecular mass of 21.5 kDa by calculation and SDS-PAGE. The cytotoxic 4-hydroxylamine derivative was the single CB 1954 reduction product, but B. amyloliquefaciens YwrO was inactive with the bischloroethyl analogue of CB 1954, SN 23862. In both of these properties B. amyloliquefaciens YwrO more closely resembles DTD than NTR. Its K(m) for CB 1954 was lower than that of NTR (617 microM compared to 862 microM). Enhanced in vitro cytotoxicity of CB 1954 was demonstrated on incubation of V79 cells with prodrug, NADPH and B. amyloliquefaciens YwrO. The work has led to the identification of a previously unknown nitroreductase, B. amyloliquefaciens YwrO, with distinct properties which will aid the rational selection of appropriate genes for applications in directed enzyme prodrug therapy (DEPT).
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5. |
Wilson GA,
Young FE,
( 1975 ) Isolation of a sequence-specific endonuclease (BamI) from Bacillus amyloliquefaciens H. PMID : 1177312 : DOI : 10.1016/s0022-2836(75)80028-3 Abstract >>
N/A
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6. |
Vaughan CK,
Harryson P,
Buckle AM,
Fersht AR,
( 2002 ) A structural double-mutant cycle: estimating the strength of a buried salt bridge in barnase. PMID : 11914482 : DOI : 10.1107/s0907444902001567 Abstract >>
Double-mutant cycles are widely used in the field of protein engineering to measure intermolecular and intramolecular interactions. Ideally, there should be no structural rearrangement of the protein on making the two single mutations and the double mutation within the cycle. However, structural pertubation on mutation does not preclude the use of this method, providing the sum of the changes in the single mutants equals the change in the double mutant. In this way, the energy associated with any structural rearrangement cancels in the double-mutant cycle. Previously, the contribution of a buried salt bridge between Arg69 and Asp93 in barnase to the stability of the folded protein has been determined by double-mutant cycle analysis. In order to determine whether the measured interaction of -14.0 kJ mol(-1) represents the true interaction energy, the crystal structure of each mutant within the double-mutant cycle was solved. Although mutation results in structural shifts, the majority of those in the single mutants are also found in the double mutant; their energetic effects in the double-mutant cycle are therefore cancelled. This study highlights the robust nature of the double-mutant cycle analysis.
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7. |
Chu HH,
Hoang V,
Kreutzmann P,
Hofemeister B,
Melzer M,
Hofemeister J,
( 2002 ) Identification and properties of type I-signal peptidases of Bacillus amyloliquefaciens. PMID : 11856304 : DOI : 10.1046/j.0014-2956.2001.02669.x Abstract >>
The use of Bacillus amyloliquefaciens for enzyme production and its exceptional high protein export capacity initiated this study where the presence and function of multiple type I signal peptidase isoforms was investigated. In addition to type I signal peptidases SipS(ba) [Meijer, W.J.J., de Jong, A., Bea, G., Wisman, A., Tjalsma, H., Venema, G., Bron, S. & van Dijl, J.M. (1995) Mol. Microbiol. 17, 621-631] and SipT(ba) [Hoang, V. & Hofemeister, J. (1995) Biochim. Biophys. Acta 1269, 64-68] which were previously identified, here we present evidence for two other Sip-like genes in B. amyloliquefaciens. Same map positions as well as sequence motifs verified that these genes encode homologues of Bacillus subtilis SipV and SipW. SipU-encoding DNA was not found in B. amyloliquefaciens. SipW-encoding DNA was also found for other Bacillus strains representing different phylogenetic groups, but not for Bacillus stearothermophilus and Thermoactinomyces vulgaris. The absence of these genes, however, could have been overlooked due to sequence diversity. Sequence alignments of 23 known Sip-like proteins from Bacillus origin indicated further branching of the P-group signal peptidases into clusters represented by B. subtilis SipV, SipS-SipT-SipU and B. anthracis Sip3-Sip5 proteins, respectively. Each B. amyloliquefaciens sip(ba) gene was expressed in an Escherichia coli LepBts mutant and tested for genetic complementation of the temperature sensitive (TS) phenotype as well as pre-OmpA processing. Although SipS(ba) as well as SipT(ba) efficiently restored processing of pre-OmpA in E. coli, only SipS(ba) supported growth at TS conditions, indicating functional diversity. Changed properties of the sip(ba) gene disruption mutants, including cell autolysis, motility, sporulation, and nuclease activities, seemed to correlate with specificities and/or localization of B. amyloliquefaciens SipS, SipT and SipV isoforms.
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8. |
Chu HH,
Hoang V,
Hofemeister J,
Schrempf H,
( 2001 ) A Bacillus amyloliquefaciens ChbB protein binds beta- and alpha-chitin and has homologues in related strains. PMID : 11429457 : DOI : 10.1099/00221287-147-7-1793 Abstract >>
A small (19.8 kDa) protein was identified in Bacillus amyloliquefaciens ALKO 2718 cultures during growth in the presence of yeast extract and chitin, but not with glucose. The protein targets beta-chitin best, then alpha-chitin, but barely any other polysaccharide. This described chitin-binding protein (ChbB) is the first of its type from a Bacillus strain and cross-reacts with antibodies raised against the Streptomyces alpha-chitin-binding protein CHB1. Using reverse genetics, the chromosomal chbB gene of strain ALKO 2718 was identified, cloned and sequenced. ChbB shares several motifs with the alpha-chitin-binding proteins CHB1 and CHB2 of Streptomyces and CBP21 of Serratia marcescens predominantly targeting beta-chitin. Synthesis was repressed by glucose and the presence of cre boxes suggests catabolite control. Using PCR, Southern hybridization and anti-ChbB antibodies, the presence of a chbB gene, as well as of a ChbB protein homologue, was ascertained in several tested B. amyloliquefaciens strains, but not in Bacillus subtilis 168. Contrary to B. subtilis 168, all B. amyloliquefaciens strains secreted varying amounts of enzymic activity, degrading carboxymethyl chitin coupled with Remazol brilliant violet.
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9. |
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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10. |
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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11. |
Clardy J,
Sato M,
Tsuru D,
Yoshimoto T,
Ito K,
Kabashima T,
Hirotsu K,
( 1999 ) The crystal structure of pyroglutamyl peptidase I from Bacillus amyloliquefaciens reveals a new structure for a cysteine protease. PMID : 10196127 : Abstract >>
The N-terminal pyroglutamyl (pGlu) residue of peptide hormones, such as thyrotropin-releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH), confers resistance to proteolysis by conventional aminopeptidases. Specialized pyroglutamyl peptidases (PGPs) are able to cleave an N-terminal pyroglutamyl residue and thus control hormonal signals. Until now, no direct or homology-based three-dimensional structure was available for any PGP. The crystal structure of pyroglutamyl peptidase I (PGP-I) from Bacillus amyloliquefaciens has been determined to 1.6 A resolution. The crystallographic asymmetric unit of PGP-I is a tetramer of four identical monomers related by noncrystallographic 222 symmetry. The protein folds into an alpha/beta globular domain with a hydrophobic core consisting of a twisted beta sheet surrounded by five alpha helices. The structure allows the function of most of the conserved residues in the PGP-I family to be identified. The catalytic triad comprises Cys144, His168 and Glu81. The catalytic site does not have a conventional oxyanion hole, although Cys144, the sidechain of Arg91 and the dipole of an alpha helix could all stabilize a negative charge. The catalytic site has an S1 pocket lined with conserved hydrophobic residues to accommodate the pyroglutamyl residue. Aside from the S1 pocket, there is no clearly defined mainchain substrate-binding region, consistent with the lack of substrate specificity. Although the overall structure of PGP-I resembles some other alpha/beta twisted open-sheet structures, such as purine nucleoside phosphorylase and cutinase, there are important differences in the location and organization of the active-site residues. Thus, PGP-I belongs to a new family of cysteine proteases.
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12. |
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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13. |
Koumoutsi A,
Chen XH,
Vater J,
Borriss R,
( 2007 ) DegU and YczE positively regulate the synthesis of bacillomycin D by Bacillus amyloliquefaciens strain FZB42. PMID : 17827323 : DOI : 10.1128/AEM.00565-07 PMC : PMC2074971 Abstract >>
Environmental strain Bacillus amyloliquefaciens FZB42 differs from the domesticated model organism of the same genus, Bacillus subtilis 168, in its ability to promote plant growth and suppress plant-pathogenic organisms present in the rhizosphere. This behavior is exerted mainly through the production of several nonribosomal cyclic lipopeptides and polyketides, which exhibit a broad range of action against phytopathogenic bacteria, fungi, and nematodes. Here, we provide evidence that the synthesis of the main antifungal agent of B. amyloliquefaciens FZB42, bacillomycin D, is regulated in multiple layers. Expression of the bacillomycin D operon (bmy) is dependent on a single sigma(A)-dependent promoter, P(bmy) and is favored in its natural host by the small regulatory protein DegQ. The global regulators DegU and ComA are required for the full transcriptional activation of bmy. DegU retains a key role since it binds directly to two sites located upstream of the bacillomycin D promoter. Moreover, both DegU and a transmembrane protein of unknown function, YczE, act on a later level of gene expression, exerting their posttranscriptional effects in a hitherto-unknown manner.
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14. |
Franke P,
Scholz R,
Schneider K,
Koumoutsi A,
Hitzeroth G,
Grammel N,
Strittmatter AW,
Gottschalk G,
Süssmuth RD,
Borriss R,
Chen XH,
Vater J,
Piel J,
( 2006 ) Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB 42. PMID : 16707694 : DOI : 10.1128/JB.00052-06 PMC : PMC1482889 Abstract >>
Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in Bacillus spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for the first time complete polyketide synthase (PKS) gene clusters to Bacillus antibiotics. Three giant modular PKS systems of the trans-acyltransferase type were identified in Bacillus amyloliquefaciens FZB 42. One of them, pks1, is an ortholog of the pksX operon with a previously unknown function in the sequenced model strain Bacillus subtilis 168, while the pks2 and pks3 clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry revealed that the pks1 (bae) and pks3 (dif) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and difficidin or oxydifficidin, respectively. In addition, B. subtilis OKB105 (pheA sfp(0)), a transformant of the B. subtilis 168 derivative JH642, was shown to produce bacillaene, demonstrating that the pksX gene cluster directs the synthesis of that polyketide. The GenBank accession numbers for gene clusters pks1(bae), pks2, and pks3(dif) are AJ 634060.2, AJ 6340601.2, and AJ 6340602.2, respectively.
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15. |
Makarewicz O,
Dubrac S,
Msadek T,
Borriss R,
( 2006 ) Dual role of the PhoP approximately P response regulator: Bacillus amyloliquefaciens FZB45 phytase gene transcription is directed by positive and negative interactions with the phyC promoter. PMID : 16980498 : DOI : 10.1128/JB.00681-06 PMC : PMC1595534 Abstract >>
Several Bacillus strains secrete phytase, an enzyme catalyzing dephosphorylation of myo-inositol hexakisphosphate (phytate). We identified the phyC (phytase) gene from environmental Bacillus amyloliquefaciens FZB45 as a member of the phosphate starvation-inducible PhoPR regulon. In vivo and in vitro assays revealed that PhoP approximately P is essential for phyC transcription. The transcriptional start site was identified downstream of a sigmaA-like promoter region located 27 bp upstream of the probable translation ATG start codon. Inspection of the phyC promoter sequence revealed an unusual structure. The -35 and -10 regions are separated by a window of 21 bp. A pair of tandemly repeated PhoP TT(T/A/C)ACA binding boxes was located within and upstream of the -35 consensus promoter region. A single PhoP box was found within the -10 consensus promoter region. DNase I footprinting experiments performed with isolated PhoP confirmed that PhoP approximately P binds at two sites overlapping with the phyC -35 and -10 consensus promoter region. While binding of dimeric PhoP approximately P at -35 is essential for activation of the phyC promoter, binding of PhoP approximately P at -10 suppresses promoter activity. A sixfold enhancement of phyC gene expression was registered after T:G substitution of nucleotide -13 (mutant MUT13), which eliminates PhoP binding at the single PhoP box without impairing the -10 consensus sequence. Moreover, MUT13 also expressed phyC during phosphate-replete growth, suggesting that the repressing effect due to binding of PhoP approximately P at -10 was abolished. A model is presented in which transcription initiation of phyC is positively and negatively affected by the actual concentration of the PhoP approximately P response regulator.
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16. |
Chao SH,
Cheng TH,
Shaw CY,
Lee MH,
Hsu YH,
Tsai YC,
( 2006 ) Characterization of a novel PepF-like oligopeptidase secreted by Bacillus amyloliquefaciens 23-7A. PMID : 16391147 : DOI : 10.1128/AEM.72.1.968-971.2006 PMC : PMC1352185 Abstract >>
An oligopeptidase from Bacillus amyloliquefaciens 23-7A was characterized along with its biochemical activities and structural gene. The protein's amino acid sequence and enzymatic activities were similar to those of other bacterial PepFs, which belong to metallopeptidase family M3. While most bacterial PepFs are cytoplasmic endopeptidases, the identified PepFBa oligopeptidase is a secreted protein and may facilitate the process of sporulation.
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17. |
Nahrstedt H,
Schröder C,
Meinhardt F,
( 2005 ) Evidence for two recA genes mediating DNA repair in Bacillus megaterium. PMID : 15758224 : DOI : 10.1099/mic.0.27626-0 Abstract >>
Isolation and subsequent knockout of a recA-homologous gene in Bacillus megaterium DSM 319 resulted in a mutant displaying increased sensitivity to mitomycin C. However, this mutant did not exhibit UV hypersensitivity, a finding which eventually led to identification of a second functional recA gene. Evidence for recA duplicates was also obtained for two other B. megaterium strains. In agreement with potential DinR boxes located within their promoter regions, expression of both genes (recA1 and recA2) was found to be damage-inducible. Transcription from the recA2 promoter was significantly higher than that of recA1. Since a recA2 knockout could not be achieved, functional complementation studies were performed in Escherichia coli. Heterologous expression in a RecA null mutant resulted in increased survival after UV irradiation and mitomycin C treatment, proving both recA gene products to be functional in DNA repair. Thus, there is evidence for an SOS-like pathway in B. megaterium that differs from that of Bacillus subtilis.
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18. |
Guillet V,
Lapthorn A,
Hartley RW,
Mauguen Y,
( 1993 ) Recognition between a bacterial ribonuclease, barnase, and its natural inhibitor, barstar. PMID : 16100951 : Abstract >>
Protein-protein recognition is fundamental to most biological processes. The information we have so far on the interfaces between proteins comes largely from several protease-inhibitor and antigen-antibody complexes. Barnase, a bacterial ribonuclease, and barstar, its natural inhibitor, form a tight complex which provides a good model for the study and design of protein-protein non-covalent interactions. Here we report the structure of a complex between barnase and a fully functional mutant of barstar determined by X-ray analysis. Barstar is composed of three parallel alpha-helices stacked against a three-stranded parallel, beta-sheet, and sterically blocks the active site of the enzyme with an alpha-helix and adjacent loop. The buried surface in the interface between the two molecules totals 1630 A2. The barnase-barstar complex is predominantly stabilized by charge interactions involving positive charges in the active site of the enzyme. Asp39 of barstar binds to the phosphate-binding site of barnase, mimicking enzyme-substrate interactions. The phosphate-binding site of the enzyme is the anchor point for inhibitor binding. We propose that this is also likely to be the case for other ribonuclease inhibitors.
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19. |
Sancho J,
Neira JL,
Fersht AR,
( 1992 ) An N-terminal fragment of barnase has residual helical structure similar to that in a refolding intermediate. PMID : 1569554 : DOI : 10.1016/0022-2836(92)90559-3 Abstract >>
A fragment of barnase comprising amino acids 1 to 36 (B(1-36)) that encompasses the region containing the two large helices (residues 6-18 and 26-34) of the native protein has been obtained by cleavage of the barnase mutant Val36----Met with cyanogen bromide. The circular dichroism (c.d.) spectrum of B(1-36) in the far ultraviolet indicates that the fragment is only weakly structured in water at neutral pH. The two-dimensional 1H nuclear magnetic resonance spectrum of B(1-36) shows, however, that a fraction of the population does have helical structure, spanning amino acid residues 8 to 18. B(1-36) becomes more helical in 35% trifluoroethanol. This is indicated by the c.d. spectrum and the increase from 6.6 to 7.0 in the pKa of His18, which is known to interact with the dipole of helix 6-18 in native barnase. The helical region of B(1-36) in 35% trifluoroethanol extends to residue 6. It is calculated from extrapolation of a trifluoroethanol titration of the ellipticity at 222 nm that B(1-36) exhibits in water approximately 6% of helical structure, calculated for a 36 residue alpha-helical peptide. This corresponds to approximately 20% of that expected for an 11-residue alpha-helical region. In trifluoroethanol, c.d. measurements indicate that approximately 30% of the 36-residue peptide is helical. It has been shown from extensive studies of the refolding of barnase that there is a folding intermediate that contains residues 8 to 18 in a helical conformation and that residue 6 is mainly unfolded. The experiments on the conformation of B(1-36) show that a small, but significant fraction, of its population in water adopts the conformation of the major alpha-helix during the barnase folding pathway, in the absence of tertiary interactions. Thus, in the folding of native barnase, secondary structure formation can precede the docking of the major alpha-helix onto the beta-sheet.
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20. |
Xiao L,
Zhang RH,
Peng Y,
Zhang YZ,
( 2004 ) Highly efficient gene expression of a fibrinolytic enzyme (subtilisin DFE) in Bacillus subtilis mediated by the promoter of alpha-amylase gene from Bacillus amyloliquefaciens. PMID : 15604765 : DOI : 10.1023/B:BILE.0000045634.46909.2b Abstract >>
Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. The promoter and signal peptide-coding sequence of alpha-amylase gene from B. amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE. This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4. Recombinant subtilisin DFE gene was successfully expressed in B. subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).
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21. |
Steinborn G,
Hajirezaei MR,
Hofemeister J,
( 2005 ) bac genes for recombinant bacilysin and anticapsin production in Bacillus host strains. PMID : 15609023 : DOI : 10.1007/s00203-004-0743-8 Abstract >>
The genes encoding the biosynthesis of the dipeptide bacilysin and its antibiotic constituent anticapsin were isolated from several strains of Bacillus subtilis as well as B. amyloliquefaciens and B. pumilus. The ywfBCDEF genes of B. subtilis 168 were shown to carry the biosynthetic core functions and were renamed bacABCDE. Mutation of the bacD gene or transformation of the bacABC genes into a B. subtilis Delta (ywfA-bacABCDE) deletion mutant led to the accumulation of anticapsin, which was fourfold higher after transformation of the bacABC genes into a bacD mutant. The genes bacD and bacE proved to encode the functions of amino acid ligation and self-protection to bacilysin, respectively. Amplification of the bacABCDE gene cluster in a bacAB gene-deficient host strain of B. amyloliquefaciens resulted in a tenfold bacilysin overproduction. Some host strains required distinct glucosamine and yeast extract supplements in order to prevent suicidal effects of the recombinant antibiotic production. The bac genes from different Bacillus species revealed the same arrangement and 72.6-88.6% of sequence identity.
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22. |
Gallagher T,
Oliver J,
Bott R,
Betzel C,
Gilliland GL,
( 1996 ) Subtilisin BPN' at 1.6 A resolution: analysis for discrete disorder and comparison of crystal forms. PMID : 15299573 : DOI : 10.1107/S0907444996007500 Abstract >>
The three-dimensional structure of the serine protease subtilisin BPN' (SBT) has been refined at 1.6 A resolution in space group C2 to a final R value of 0.17. 17 regions of discrete disorder have been identified and analyzed. Two of these are dual-conformation peptide units; the remainder involve alternate rotamers of side chains either alone or in small clusters. The structure is compared with previously reported high-resolution models of SBT in two other space groups, P2(1)2(1)2(1) and P2(1). Apart from the surface, there are no significant variations in structure among the three crystal forms. Structural variations observed at the protein surface occur predominantly in regions of protein-protein contact. The crystal packing arrangements in the three space groups are compared.
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23. |
Peng Y,
Yang XJ,
Xiao L,
Zhang YZ,
( 2004 ) Cloning and expression of a fibrinolytic enzyme (subtilisin DFE) gene from Bacillus amyloliquefaciens DC-4 in Bacillus subtilis. PMID : 15059629 : DOI : 10.1016/j.resmic.2003.10.004 Abstract >>
A strong fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4, subtilisin DFE, was isolated from douchi, a traditional Chinese soybean-fermented food. Based on the high homology between the N-terminal sequence of subtilisin DFE and that of subtilisin BPN, PCR primers were designed that allowed for the amplification and cloning of the intact subtilisin DFE gene. Sequence analysis indicated the presence of a 1149-bp open reading frame encoding 382 amino acid residues. The enzyme was actively expressed by the Escherichia coli-Bacillus subtilis shuttle expression vector pSUGV4 in the protease-deficient strain B. subtilis WB600, and its biochemical characteristics were the same as those of the original subtilisin DFE isolated from the donor strain, i.e., its molecular weight is approximately 28 kDa and it is a serine protease.
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24. |
Koumoutsi A,
Chen XH,
Henne A,
Liesegang H,
Hitzeroth G,
Franke P,
Vater J,
Borriss R,
( 2004 ) Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42. PMID : 14762003 : DOI : 10.1128/jb.186.4.1084-1096.2004 PMC : PMC344220 Abstract >>
The environmental strain Bacillus amyloliquefaciens FZB42 promotes plant growth and suppresses plant pathogenic organisms present in the rhizosphere. We sampled sequenced the genome of FZB42 and identified 2,947 genes with >50% identity on the amino acid level to the corresponding genes of Bacillus subtilis 168. Six large gene clusters encoding nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) occupied 7.5% of the whole genome. Two of the PKS and one of the NRPS encoding gene clusters were unique insertions in the FZB42 genome and are not present in B. subtilis 168. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis revealed expression of the antibiotic lipopeptide products surfactin, fengycin, and bacillomycin D. The fengycin (fen) and the surfactin (srf) operons were organized and located as in B. subtilis 168. A large 37.2-kb antibiotic DNA island containing the bmy gene cluster was attributed to the biosynthesis of bacillomycin D. The bmy island was found inserted close to the fen operon. The responsibility of the bmy, fen, and srf gene clusters for the production of the corresponding secondary metabolites was demonstrated by cassette mutagenesis, which led to the loss of the ability to produce these peptides. Although these single mutants still largely retained their ability to control fungal spread, a double mutant lacking both bacillomycin D and fengycin was heavily impaired in its ability to inhibit growth of phytopathogenic fungi, suggesting that both lipopeptides act in a synergistic manner.
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25. |
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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26. |
Geetha I,
Manonmani AM,
Prabakaran G,
( 2011 ) Bacillus amyloliquefaciens: a mosquitocidal bacterium from mangrove forests of Andaman & Nicobar islands, India. PMID : 21810402 : DOI : 10.1016/j.actatropica.2011.07.006 Abstract >>
Samples collected from the mangrove forests of Andaman & Nicobar islands yielded a mosquitocidal bacterium, whose extracellular metabolite(s) exhibited mosquito larvicidal and pupicidal activity. The bacterium was isolated using standard microbiological methods and identified using classical biochemical tests and rpoB gene sequences. The mosquitocidal bacterium was identified as Bacillus amyloliquefaciens. Mosquitocidal metabolite(s) was separated from the culture supernatant of the bacterium and its efficacy against the larval and pupal stages of different species of mosquitoes was determined in terms of LC(50) and LC(90). Mosquito larvicidal activity in terms of LC(50) against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti was respectively, 26.4�gg, 22.2�gg and 20.5�gg/ml and its pupicidal activity was 4.4�gg, 8.2�gg and 14.5�gg/ml respectively. The mosquitocidal metabolite(s) was found to be a biosurfactant. This is the first report of the mosquitocidal activity of B. amyloliquefaciens and it is a new weapon which can be added to the array of microbial agents for use against mosquitoes.
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27. |
Yomantas YA,
Abalakina EG,
Golubeva LI,
Gorbacheva LY,
Mashko SV,
( 2011 ) Overproduction of Bacillus amyloliquefaciens extracellular glutamyl-endopeptidase as a result of ectopic multi-copy insertion of an efficiently-expressed mpr gene into the Bacillus subtilis chromosome. PMID : 21819557 : DOI : 10.1186/1475-2859-10-64 PMC : PMC3166918 Abstract >>
Plasmid-less, engineered Bacillus strains have several advantages over plasmid-carrier variants. Specifically, their stability and potential ecological safety make them of use in industrial applications. As a rule, however, it is necessary to incorporate many copies of a key gene into a chromosome to achieve strain performance that is comparable to that of cells carrying multiple copies of a recombinant plasmid. A plasmid-less B. subtilis JE852-based strain secreting glutamyl-specific protease (GSP-the protein product of the mpr gene from B. amyloliquefaciens) was constructed that exhibits decreased levels of other extracellular proteases. Ten copies of an mprB.amy cassette in which the GSP gene was placed between the promoter of the B. amyloliquefaciens rplU-rpmA genes and the Rho-independent transcription terminator were ectopically inserted into designated (3 copies) and random (7 copies) points in the recipient chromosome. The resulting strain produced approximately 0.5 g/L of secreted GSP after bacterial cultivation in flasks with starch-containing media, and its performance was comparable to an analogous strain in which the mprB.amy cassette was carried on a multi-copy plasmid. A novel strategy for ectopically integrating a cassette into multiple random locations in the B. subtilis chromosome was developed. This new method is based on the construction of DNA fragments in which the desired gene, marked by antibiotic resistance, is sandwiched between front" and "back" portions of random chromosomal DNA restriction fragments. These fragments were subsequently inserted into the targeted sites of the chromosome using double-cross recombination. The construction of a marker-free strain was achieved by gene conversion between the integrated marked gene and a marker-less variant carried by plasmid DNA, which was later removed from the cells."
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28. |
Herzner AM,
Dischinger J,
Szekat C,
Josten M,
Schmitz S,
Yakéléba A,
Reinartz R,
Jansen A,
Sahl HG,
Piel J,
Bierbaum G,
( 2011 ) Expression of the lantibiotic mersacidin in Bacillus amyloliquefaciens FZB42. PMID : 21811596 : DOI : 10.1371/journal.pone.0022389 PMC : PMC3141056 Abstract >>
Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent. The aim of these studies was to test if the production of mersacidin could be transferred to a naturally competent Bacillus strain employing genomic DNA of the producer strain. Bacillus amyloliquefaciens FZB42 was chosen for these experiments because it already harbors the mersacidin immunity genes. After transfer of the biosynthetic part of the gene cluster by competence transformation, production of active mersacidin was obtained from a plasmid in trans. Furthermore, comparison of several DNA sequences and biochemical testing of B. amyloliquefaciens FZB42 and B. sp. HIL Y-85,54728 showed that the producer strain of mersacidin is a member of the species B. amyloliquefaciens. The lantibiotic mersacidin can be produced in B. amyloliquefaciens FZB42, which is closely related to the wild type producer strain of mersacidin. The new mersacidin producer strain enables us to use the full potential of the biosynthetic gene cluster for genetic manipulation and downstream modification approaches.
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29. |
Kubo Y,
Rooney AP,
Tsukakoshi Y,
Nakagawa R,
Hasegawa H,
Kimura K,
( 2011 ) Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production. PMID : 21764950 : DOI : 10.1128/AEM.00448-11 PMC : PMC3187134 Abstract >>
Spore-forming Bacillus strains that produce extracellular poly-�^-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.
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30. |
Rückert C,
Blom J,
Chen X,
Reva O,
Borriss R,
( 2011 ) Genome sequence of B. amyloliquefaciens type strain DSM7(T) reveals differences to plant-associated B. amyloliquefaciens FZB42. PMID : 21262282 : DOI : 10.1016/j.jbiotec.2011.01.006 Abstract >>
The complete genome sequence of Bacillus amyloliquefaciens type strain DSM7(T) is presented. A comparative analysis between the genome sequences of the plant associated strain FZB42 (Chen et al., 2007) with the genome of B. amyloliquefaciens DSM7(T) revealed obvious differences in the variable part of the genomes, whilst the core genomes were found to be very similar. The strains FZB42 and DSM7(T) have in common 3345 genes (CDS) in their core genomes; whilst 547 and 344 CDS were found to be unique in DSM7(T) and FZB42, respectively. The core genome shared by both strains exhibited 97.89% identity on amino acid level. The number of genes representing the core genome of the strains FZB42, DSM7(T), and Bacillus subtilis DSM10(T) was calculated as being 3098 and their identity was 92.25%. The 3,980,199 bp genome of DSM7(T) contains numerous genomic islands (GI) detected by different methods. Many of them were located in vicinity of tRNA, glnA, and glmS gene copies. In contrast to FZB42, but similar to B. subtilis DSM10(T), the GI were enriched in prophage sequences and often harbored transposases, integrases and recombinases. Compared to FZB42, B. amyloliquefaciens DSM7(T) possessed a reduced potential to non-ribosomally synthesize secondary metabolites with antibacterial and/or antifungal action. B. amyloliquefaciens DSM7(T) did not produce the polyketides difficidin and macrolactin and was impaired in its ability to produce lipopeptides other than surfactin. Differences established within the variable part of the genomes, justify our proposal to discriminate the plant-associated ecotype represented by FZB42 from the group of type strain related B. amyloliquefaciens soil bacteria.
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31. |
Qiao JQ,
Tian DW,
Huo R,
Wu HJ,
Gao XW,
( 2011 ) Functional analysis and application of the cryptic plasmid pBSG3 harboring the RapQ-PhrQ system in Bacillus amyloliquefaciens B3. PMID : 21118702 : DOI : 10.1016/j.plasmid.2010.11.008 Abstract >>
This work sequenced and characterized a cryptic plasmid called pBSG3 from wild-type Bacillus amyloliquefaciens B3--a powerful agent for suppression of plant pathogenic organisms. It is an 8439 bp circular molecule, with G+C content of 40.3%. We provide evidence that pBSG3 replicates via the rolling-circle (RC) mechanism and, sequence comparisons place it in the pC194 family of rolling-circle-replicons. The plasmid contains seven putative open reading frames (ORFs), including genes repB3, mobB3, rapQ, phrQ, pgsR, and two unknown ORFs (orf1c and orf2). Our observations reveal that the RapQ-PhrQ (response regulator aspartate phosphatase-phosphatase regulator) system is involved in sporulation and RapQ can delay the onset of sporulation. Two Escherichia coli and Bacillus potential shuttle vectors, pTRD (containing the minimal replicon) and pTRDS (containing the minimal replicon and the single-strand origin) were developed from pBSG3 and tested the stability. Moreover, HpaG(xooc) protein, which can induce disease and insect resistance in plants, was tried to express with the stable vector pTRDS in Bacillus subtilis. In summary, the pBSG3 plasmid containing various genes is not only a candidate tool for vector development in Bacillus genus research but also a potential vehicle for the exchange of genetic elements among Bacillus populations that contributes to the survival of bacilli in natural environments.
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32. |
Liu J,
He D,
Ma X,
Wu H,
Gao X,
( 2011 ) Identification of up-regulated genes of Bacillus amyloliquefaciens B55 during the early stage of direct surface contact with rice R109 root. PMID : 20625734 : DOI : 10.1007/s00284-010-9701-7 Abstract >>
The early stage of plant-rhizobacteria interaction, affected by plant root exudates and plant-rhizobacteria surface contact, is considered to be critical for plant growth-promoting rhizobacteria colonizing plant roots and initiating the beneficial effects on plant growth. However, little is known about the mechanisms of plant-rhizobacteria surface contact involved in early stage of plant-rhizobacteria interaction. In order to reveal the molecular mechanisms of the surface contact, a rhizobacterium Bacillus amyloliquefaciens B55 was interacted with plant roots of rice R109 and used to perform a cDNA-based suppression-subtractive hybridization. Seven differentially expressed DNA fragments were identified. Except for the two fragments showing no matches to any known sequences in the Genbank, the other five fragments were found to have high homologies with the genes encoding 2-oxoglutarate dehydrogenase E1 component OdhA, aspartate ammonia-lyase AnsB, and hypothetical protein proposed to be involved in surface adhesion, acetolactate decarboxylase AlsD, and DNA mismatch repair protein MutL, respectively. The induced RNA expression levels of two putative genes ansB and odhA and an unmatched DNA fragment BD33 were verified by RT-PCR analysis.
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33. |
Sheremet AS,
Gronskiy SV,
Akhmadyshin RA,
Novikova AE,
Livshits VA,
Shakulov RS,
Zakataeva NP,
( 2011 ) Enhancement of extracellular purine nucleoside accumulation by Bacillus strains through genetic modifications of genes involved in nucleoside export. PMID : 20814730 : DOI : 10.1007/s10295-010-0829-z Abstract >>
Using a simple method to introduce genetic modifications into the chromosome of naturally nontransformable Bacillus, a set of marker-free inosine-producing and 5-aminoimidazole-4-carboxamide (AICA) ribonucleoside-producing Bacillus amyloliquefaciens strains has been constructed. These strains differ in expression levels of the genes responsible for nucleoside export. Overexpression of B. amyloliquefaciens pbuE and heterologous expression of Escherichia coli nepI, which encode nucleoside efflux transporters, each notably enhanced inosine production by a B. amyloliquefaciens nucleoside-producing strain. pbuE overexpression was found to increase AICA ribonucleoside accumulation, indicating that the substrate specificity of the PbuE pump extends to this nucleoside. These results demonstrate that identifying genes whose products facilitate transport of a desired nucleoside out of cells and enhancing their expression can improve the performance of strains used for industrial production.
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34. |
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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35. |
Alikhajeh J,
Khajeh K,
Ranjbar B,
Naderi-Manesh H,
Lin YH,
Liu E,
Guan HH,
Hsieh YC,
Chuankhayan P,
Huang YC,
Jeyaraman J,
Liu MY,
Chen CJ,
( 2010 ) Structure of Bacillus amyloliquefaciens alpha-amylase at high resolution: implications for thermal stability. PMID : 20124706 : DOI : 10.1107/S1744309109051938 PMC : PMC2815676 Abstract >>
The crystal structure of Bacillus amyloliquefaciens alpha-amylase (BAA) at 1.4 A resolution revealed ambiguities in the thermal adaptation of homologous proteins in this family. The final model of BAA is composed of two molecules in a back-to-back orientation, which is likely to be a consequence of crystal packing. Despite a high degree of identity, comparison of the structure of BAA with those of other liquefying-type alpha-amylases indicated moderate discrepancies at the secondary-structural level. Moreover, a domain-displacement survey using anisotropic B-factor and domain-motion analyses implied a significant contribution of domain B to the total flexibility of BAA, while visual inspection of the structure superimposed with that of B. licheniformis alpha-amylase (BLA) indicated higher flexibility of the latter in the central domain A. Therefore, it is suggested that domain B may play an important role in liquefying alpha-amylases, as its rigidity offers a substantial improvement in thermostability in BLA compared with BAA.
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36. |
Baudet S,
Janin J,
( 1991 ) Crystal structure of a barnase-d(GpC) complex at 1.9 A resolution. PMID : 2023257 : DOI : 10.1016/0022-2836(91)90862-z Abstract >>
The ribonuclease excreted by Bacillus amyloliquefaciens, Barnase, was co-crystallized with the deoxy-dinucleotide d(GpC). The crystal structure was determined by molecular replacement from a model of free Barnase previously derived by Mauguen et al. Refinement was carried out using data to 1.9 A resolution. The final model, which has a crystallographic R factor of 22%, includes 869 protein atoms, 38 atoms from d(GpC), a sulfate ion and 73 water molecules. Only minor differences from free Barnase are seen in the protein moiety, the root-mean-square C alpha movement being 0.45 A. The dinucleotide has a folded conformation. It is located near the active site of the enzyme, but outside the protein molecule and making crystal packing contacts with neighboring molecules. The guanine base is stacked on the imidazole ring of active site His102, rather than binding to the so-called recognition loop as it does in other complexes of guanine nucleotides with microbial nucleases. The deoxyguanosine is syn, with the sugar ring in C-2'-endo conformation; the deoxycytidine is anti and C-4'-exo. In addition to the stacking interaction, His102 hydrogen bonds to the free 5' hydroxyl, which is located near the position where the 3' phosphate group is found in other inhibitors of microbial ribonucleases. While the mode of binding observed with d(GpC) and Barnase would be non-productive for a dinucleotide substrate, it may define a site for the nucleotide product on the 3' side of the hydrolyzed bond.
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37. |
Lee AR,
Kim GM,
Kwon GH,
Lee KW,
Park JY,
Chun J,
Cha J,
Song YS,
Kim JH,
( 2010 ) Cloning of aprE86-1 gene encoding 27 kDa mature fibrinolytic enzyme from Bacillus amyloliquefaciens CH86-1. PMID : 20208443 : Abstract >>
A gene, encoding the major secreted fibrinolytic protein of Bacillus amyloliquefaciens CH86-1, was cloned from the genomic DNA. DNA sequencing showed that the gene, aprE86-1, could direct the synthesis of a mature protein of 275 amino acids long after processing. When aprE86-1 was introduced into B. subtilis, 27 kDa mature protein was produced as expected. The fibrinolytic activity of B. subtilis transformant (TF) was higher than that of B. amyloliquefaciens CH86-1, showing the possibility of increasing fibrinolytic activities of Bacillus strains through genetic engineering.
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38. |
Rooney AP,
Price NP,
Ehrhardt C,
Swezey JL,
Bannan JD,
( 2009 ) Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov. PMID : 19622642 : DOI : 10.1099/ijs.0.009126-0 Abstract >>
The Bacillus subtilis species complex is a tight assemblage of closely related species. For many years, it has been recognized that these species cannot be differentiated on the basis of phenotypic characteristics. Recently, it has been shown that phylogenetic analysis of the 16S rRNA gene also fails to differentiate species within the complex due to the highly conserved nature of the gene, yet DNA-DNA hybridization values fall well below 70 % for the same species comparisons. As a complementary approach, we propose that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Indeed, our phylogenetic analyses revealed the existence of a previously unknown group of strains closely related to, but distinct from, Bacillus subtilis subsp. spizizenii. Results of matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the group produces a novel surfactin-like lipopeptide with mass m/z 1120.8 that is not produced by the other currently recognized subspecies. In addition, the group displayed differences in the total cellular content of the fatty acids C(16 : 0) and iso-C(17 : 1)omega10c that distinguish it from the closely related B. subtilis subsp. spizizenii. Consequently, the correlation of these novel phenotypic traits with the phylogenetic distinctiveness of this previously unknown subspecies group showed that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Therefore, we propose that this new group should be recognized as representing a novel taxon, Bacillus subtilis subsp. inaquosorum subsp. nov., with the type strain NRRL B-23052(T) (=KCTC 13429(T)=BGSC 3A28(T)).
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39. |
Rairakhwada D,
Seo JW,
Seo MY,
Kwon O,
Rhee SK,
Kim CH,
( 2010 ) Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens. PMID : 19916084 : DOI : 10.1007/s10295-009-0664-2 Abstract >>
Although levan produced by Bacillus amyloliquefaciens is known to have efficient immunostimulant property which gives 100% survival of common carp when infected with Aeromonas hydrophila, no detailed reports are available describing kinetic studies of D: -glucose production and levan formation. In this study, we cloned and characterized the enzymatic kinetics using levansucrase expressed in Escherichia coli. Optimum pH for D: -glucose production and levan formation was 6.0 and 8.0, respectively, whereas optimum temperature was 30 degrees C and 4 degrees C, respectively. The K (m) and V (max) values for levansucrase were calculated to be 47.81 mM sucrose and 57.47 1mole/min mg protein, respectively. Prominent expression of levansucrase was obtained through xylose induction in Bacillus megaterium, where most of the His(6)-tagged protein was secreted into the culture broth, giving levansucrase activity of 12,906 U/l. Response-surface methodology (RSM) was further employed to optimize the fermentation conditions and improve the level of levansucrase production. Maximum levansucrase activity of 20,251 U/l was obtained in 12 h of fermentation carried out at 28 degrees C, starting induction with 0.735% xylose when A (600) was 1.2, which was 1.6- and 62-fold higher than those obtained in the nonoptimized conditions for the recombinant strain and the native strain, respectively.
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40. |
Kim GM,
Lee AR,
Lee KW,
Park JY,
Park AY,
Chun J,
Cha J,
Song YS,
Kim JH,
( 2009 ) Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from cheonggukjang. PMID : 19809258 : Abstract >>
Bacillus amyloliquefancies CH51 isolated from cheonggukjang, a traditional Korean fermented soy food, has strong fibrinolytic activity and produces several fibrinolytic enzymes. Among four different growth media, tryptic soy broth was the best in terms of supporting cell growth and fibrinolytic activity of this strain. A protein with fibrinolytic activity was partially purified from the culture supernatant by CMSephadex and Phenyl Sepharose column chromatographies. Tandem mass spectrometric analysis showed that this protein is a homolog of AprE from B. subtilis and it was accordingly named AprE51. The optimum pH and temperature for partially purified AprE51 activity were 6.0 and 45 degrees , respectively. A gene encoding AprE51, aprE51, was cloned from B. amyloliquefaciens CH51 genomic DNA. The aprE51 gene was overexpressed in heterologous B. subtilis strains deficient in fibrinolytic activity using an E.colo-Bacillus Shuttle vector, pHY300PLK.
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41. |
Arrebola E,
Jacobs R,
Korsten L,
( 2010 ) Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens. PMID : 19674188 : DOI : 10.1111/j.1365-2672.2009.04438.x Abstract >>
A Bacillus amyloliquefaciens strain, surviving epiphytically on the surface of fruit, was isolated while searching for naturally occurring biological control agents. This bacterial strain was characterized for its antifungal activity against seven selected fungal postharvest pathogens of citrus. To understand the antifungal activity, seven postharvest fungal pathogens were screened for growth inhibition by B. amyloliquefaciens strain. Assays using B. amyloliquefaciens lipopeptide extracts showed a strong inhibitive activity. The inhibitory effect was observed in abnormal conidial germination and germ tube development when conidia were treated with different lipopeptide extract concentrations. Further analysis using PCR and chromatography confirmed the presence of fengycin, iturin and surfactine, of which iturin A showed the strongest and most common inhibitory effect. The results are supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of iturin A production. Fruit trials confirmed disease development inhibition when the antagonist was applied 1 day prior to or 1 day after fungal application. We conclude that the iturin family of lipopeptides are vital in the antagonism of B. amyloliquefaciens against the seven citrus postharvest pathogenic fungi tested. We elucidated the principal mechanism used by B. amyloliquefaciens PPCB004 to suppress postharvest disease development on stored fruits.
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42. |
Brooks JE,
Nathan PD,
Landry D,
Sznyter LA,
Waite-Rees P,
Ives CL,
Moran LS,
Slatko BE,
Benner JS,
( 1991 ) Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts. PMID : 1901989 : DOI : 10.1093/nar/19.4.841 PMC : PMC333720 Abstract >>
The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.
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43. |
Bycroft M,
Ludvigsen S,
Fersht AR,
Poulsen FM,
( 1991 ) Determination of the three-dimensional solution structure of barnase using nuclear magnetic resonance spectroscopy. PMID : 1888730 : DOI : 10.1021/bi00099a030 Abstract >>
The solution conformation of the ribonuclease barnase has been determined by using 1H nuclear magnetic resonance (NMR) spectroscopy. The 20 structures were calculated by using 853 interproton distance restraints obtained from analyses of two-dimensional nuclear Overhauser spectra, 72 phi and 53 chi 1 torsion angle restraints, and 17 hydrogen-bond distance restraints. The calculated structures contain two alpha-helices (residues 6-18 and 26-34) and a five-stranded antiparallel beta-sheet (residues 50-55, 70-75, 85-91, 94-101, and 105-108). The core of the protein is formed by the packing of one of the alpha-helices (residues 6-18) onto the beta-sheet. The average RMS deviation between the calculated structures and the mean structure is 1.11 A for the backbone atoms and 1.75 A for all atoms. The protein is least well-defined in the N-terminal region and in three large loops. When these regions are excluded, the average RMS deviation between the calculated structures and the mean structure for residues 5-34, 50-56, 71-76, 85-109 is 0.62 A for the backbone atoms and 1.0 A for all atoms. The NMR-derived structure has been compared with the crystal structure of barnase [Mauguen et al. (1982) Nature (London) 297, 162-164].
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44. |
Wang LT,
Lee FL,
Tai CJ,
Kuo HP,
( 2008 ) Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens. PMID : 18319476 : DOI : 10.1099/ijs.0.65191-0 DOI : 10.1099/ijs.0.65191-0 Abstract >>
Strain BCRC 14193, isolated from soil, shared more than 99 % 16S rRNA gene sequence similarity with Bacillus amyloliquefaciens BCRC 11601(T) and Bacillus velezensis BCRC 17467(T). This strain was previously identified as B. amyloliquefaciens, based on DNA-DNA hybridization, but its DNA relatedness value with B. velezensis BCRC 17467(T) was 89 %. To investigate the relatedness of strain BCRC 14193, B. amyloliquefaciens and B. velezensis, the partial sequence of the gene encoding the subunit B protein of DNA gyrase (gyrB) was determined. B. velezensis BCRC 17467(T) shared high gyrB gene sequence similarity with B. amyloliquefaciens BCRC 14193 (98.4 %) and all of the B. amyloliquefaciens strains available (95.5-95.6 %). DNA-DNA hybridization experiments revealed high relatedness values between B. velezensis BCRC 17467(T) and B. amyloliquefaciens BCRC 11601(T) (74 %) and the B. amyloliquefaciens reference strains (74-89 %). Based on these data and the lack of phenotypic distinctive characteristics, we propose Bacillus velezensis as a later heterotypic synonym of Bacillus amyloliquefaciens.
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45. |
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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46. |
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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47. |
Sutyak KE,
Wirawan RE,
Aroutcheva AA,
Chikindas ML,
( 2008 ) Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens. PMID : 17976171 : DOI : 10.1111/j.1365-2672.2007.03626.x PMC : PMC2322860 Abstract >>
To purify and characterize an antimicrobial protein (bacteriocin) isolated from the dairy product-derived Bacillus amyloliquefaciens. An unknown bacterial species cultured from the Yogu Farm probiotic dairy beverage was identified through 16S ribosomal RNA analysis as B. amyloliquefaciens, a phylogenetically close relative of Bacillus subtilis. The cell-free supernatant (CFS) of overnight cultures was active against Listeria monocytogenes and also against clinical isolates of Gardnerella vaginalis and Streptococcus agalactiae. At the same time, several isolates of vaginal probiotic Lactobacilli were resistant to the CFS. The nature of the compound causing inhibitory activity was confirmed as proteinaceous by enzymatic digestion. The protein was isolated using ammonium sulfate precipitation, and further purified via column chromatography. PCR analysis was conducted to determine relatedness to other bacteriocins produced by Bacillus spp. The antimicrobial protein isolated from B. amyloliquefaciens was shown to be subtilosin, a bacteriocin previously reported as produced only by B. subtilis. This is the first report of intra-species horizontal gene transfer for subtilosin and the first fully characterized bacteriocin isolated from B. amyloliquefaciens. Finally, this is the first report on subtilosin's activity against bacterial vaginosis-associated pathogens.
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48. |
Wang LT,
Lee FL,
Tai CJ,
Yokota A,
Kuo HP,
( 2007 ) Reclassification of Bacillus axarquiensis Ruiz-Garcia et al. 2005 and Bacillus malacitensis Ruiz-Garcia et al. 2005 as later heterotypic synonyms of Bacillus mojavensis Roberts et al. 1994. PMID : 17625213 : DOI : 10.1099/ijs.0.64808-0 Abstract >>
The Bacillus subtilis group encompasses the taxa Bacillus subtilis subsp. subtilis, B. licheniformis, B. amyloliquefaciens, B. atrophaeus, B. mojavensis, B. vallismortis, B. subtilis subsp. spizizenii, B. sonorensis, B. velezensis, B. axarquiensis and B. malacitensis. In this study, the taxonomic relatedness between the species B. axarquiensis, B. malacitensis and B. mojavensis was investigated. Sequence analysis of the 16S rRNA gene and the gene for DNA gyrase subunit B (gyrB) confirmed the very high similarities between these three type strains and a reference strain of B. mojavensis (>99 and >97 %, respectively). DNA-DNA hybridization experiments revealed high relatedness values between the type strains of B. axarquiensis, B. malacitensis and B. mojavensis and between these strains and a reference strain of B. mojavensis (83-98 %). Based on these molecular taxonomic data and the lack of phenotypic distinctive characteristics, Bacillus axarquiensis and Bacillus malacitensis should be reclassified as later heterotypic synonyms of Bacillus mojavensis.
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49. |
Zakataeva NP,
Gronskiy SV,
Sheremet AS,
Kutukova EA,
Novikova AE,
Livshits VA,
( N/A ) A new function for the Bacillus PbuE purine base efflux pump: efflux of purine nucleosides. PMID : 17935948 : DOI : 10.1016/j.resmic.2007.08.003 Abstract >>
The pbuE (ydhL) gene from Bacillus subtilis is known to encode the purine base efflux pump, and its expression is controlled by an adenine-dependent riboswitch. We cloned the pbuE gene from Bacillus amyloliquefaciens and examined gene expression by its own cis-acting regulatory elements in Escherichia coli. Regulation of pbuE expression, previously found in B. subtilis, was retained in this heterologous expression: it was induced by adenine and activated by a mutation in the 5' untranslated region, which disrupted transcription termination. This observation supports the model that the adenine-dependent riboswitch directly regulates pbuE expression, without requiring additional factors. Overexpression of the PbuE pump conferred upon the E. coli strain resistance to higher concentrations of inosine, adenosine and guanosine, and increased exogenous inosine accumulation by E. coli cells deficient in purine nucleoside phosphorylase. Overexpression of the PbuE pump also enhanced hypoxanthine excretion by the E. coli hypoxanthine-producing strain and inosine excretion both by the E. coli and B. amyloliquefaciens nucleoside-producing strains. Thus, for the first time, we obtained direct evidence for the involvement of PbuE in efflux of not only purine bases, but also purine ribonucleosides. A possible new role for the pump in cell physiology is discussed.
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50. |
Ruohonen L,
Hackman P,
Lehtovaara P,
Knowles JK,
Keränen S,
( 1987 ) Efficient secretion of Bacillus amyloliquefaciens alpha-amylase by [corrected] its own signal peptide from Saccharomyces cerevisiae host cells [corrected]. PMID : 2830166 : DOI : 10.1016/0378-1119(87)90324-6 Abstract >>
The expression and secretion of Bacillus amyloliquefaciens alpha-amylase was studied in yeast Saccharomyces cerevisiae. The Bacillus promoter was removed by BAL 31 digestion and three forms of the alpha-amylase gene were constructed: the Bacillus signal sequence was either complete (YEp alpha a1), partial (YEp alpha a2) or missing (YEp alpha a3). Secretion of alpha-amylase into the culture medium was obtained with the complete signal sequence only. The secreted alpha-amylase was glycosylated and its signal peptide was apparently processed. The glycosylated alpha-amylase remained active. The enzyme produced by the other constructions was not glycosylated and thus probably remained in the cytoplasm.
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51. |
Lee JM,
Lee J,
Nam GH,
Son BS,
Jang MU,
Lee SW,
Hurh BS,
Kim TJ,
( 2017 ) Heterologous expression and enzymatic characterization of �^-glutamyltranspeptidase from Bacillus amyloliquefaciens. PMID : 28120195 : DOI : 10.1007/s12275-017-6638-6 Abstract >>
�^-Glutamyltranspeptidase (GGT) catalyzes the cleavage of �^-glutamyl compounds and the transfer of �^-glutamyl moiety to water or to amino acid/peptide acceptors. GGT can be utilized for the generation of �^-glutamyl peptides or glutamic acid, which are used as food taste enhancers. In the present study, Bacillus amyloliquefaciens SMB469 with high GGT activity was isolated from Doenjang, a traditional fermented soy food of Korea. The gene encoding GGT from B. amyloliquefaciens SMB469 (BaGGT469) was cloned from the isolate, and heterologously expressed in E. coli and B. subtilis. For comparison, three additional GGT genes were cloned from B. subtilis 168, B. licheniformis DSM 13, and B. amyloliquefaciens FZB42. The BaGGT469 protein was composed of 591 amino acids. The final protein comprises two separate polypeptide chains of 45.7 and 19.7 kDa, generated via autocatalytic cleavage. The specific activity of BaGGT469 was determined to be 17.8 U/mg with �^-L-glutamyl-p-nitroanilide as the substrate and diglycine as the acceptor. GGTs from B. amyloliquefaciens showed 1.4- and 1.7-fold higher transpeptidase activities than those from B. subtilis and B. licheniformis, respectively. Especially, recombinant B. subtilis expressing BaGGT469 demonstrated 11- and 23-fold higher GGT activity than recombinant E. coli and the native B. amyloliquefaciens, respectively, did. These results suggest that BaGGT469 can be utilized for the enzymatic production of various �^-glutamyl compounds.
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52. |
Liao JH,
Chen PY,
Yang YL,
Kan SC,
Hsieh FC,
Liu YC,
( 2016 ) Clarification of the Antagonistic Effect of the Lipopeptides Produced by Bacillus amyloliquefaciens BPD1 against Pyricularia oryzae via In Situ MALDI-TOF IMS Analysis. PMID : 27918491 : DOI : 10.3390/molecules21121670 PMC : PMC6273258 Abstract >>
This study tried to clarify the antagonistic effect of the lipopeptides secreted by Bacillus amyloliquefaciens strain BPD1 (Ba-BPD1) against Pyricularia oryzae Cavara (PO). To determine the major antifungal lipopeptides effective against PO, single and dual cultures were carried out in solid-state media. The matrix-assisted laser desorption/ionization-time of flight imaging mass spectrometry (MALDI-TOF IMS) was used to identify the most effective lipopeptide in situ. Meanwhile, the morphology of pathogen fungi treated with lipopeptides was observed via the SEM. Of the three lipopeptide families, surfactin, iturin, and fengycin, the last was identified as the most effective for inhibiting mycelium growth and conidial germination of PO. The conidia and hyphae of fengycin-treated PO were shown to become deformed and tumorous under exposure. This study provides insights into the antagonistic effect of Ba-BPD1 against fungal phytopathogens. Such insights are helpful in the development of reagents for biological control applications.
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53. |
Hartley RW,
( 1989 ) Barnase and barstar: two small proteins to fold and fit together. PMID : 2696173 : Abstract >>
Barnase and barstar are the extracellular ribonuclease and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Both are small single-chain proteins and thus are suitable for application to the study of how a protein's sequence directs its fold. Barnase has neither disulfide bonds nor non-peptide components and unfolds reversibly in what closely approximates a two-state reaction. The genes for both these proteins have been cloned in E. coli. Expression of barstar is necessary to counter the lethal effect of expressed active barnase. Site-directed mutagenesis is being used to answer specific and general questions relating to protein folding and protein-protein interaction.
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54. |
Zalila-Kolsi I,
Ben Mahmoud A,
Ali H,
Sellami S,
Nasfi Z,
Tounsi S,
Jamoussi K,
( 2016 ) Antagonist effects of Bacillus spp. strains against Fusarium graminearum for protection of durum wheat (Triticum turgidum L. subsp. durum). PMID : 27664733 : DOI : 10.1016/j.micres.2016.06.012 Abstract >>
Bacillus species are attractive due to their potential use in the biological control of fungal diseases. Bacillus amyloliquefaciens strain BLB369, Bacillus subtilis strain BLB277, and Paenibacillus polymyxa strain BLB267 were isolated and identified using biochemical and molecular (16S rDNA, gyrA, and rpoB) approaches. They could produce, respectively, (iturin and surfactin), (surfactin and fengycin), and (fusaricidin and polymyxin) exhibiting broad spectrum against several phytopathogenic fungi. In vivo examination of wheat seed germination, plant height, phenolic compounds, chlorophyll, and carotenoid contents proved the efficiency of the bacterial cells and the secreted antagonist activities to protect Tunisian durum wheat (Triticum turgidum L. subsp. durum) cultivar Om Rabiia against F. graminearum fungus. Application of single bacterial culture medium, particularly that of B. amyloliquefaciens, showed better protection than combinations of various culture media. The tertiary combination of B. amyloliquefaciens, B. subtilis, and P. polymyxa bacterial cells led to the highest protection rate which could be due to strains synergistic or complementary effects. Hence, combination of compatible biocontrol agents could be a strategic approach to control plant diseases.
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55. |
van Staden Adu P,
Heunis T,
Smith C,
Deane S,
Dicks LM,
( 2016 ) Efficacy of Lantibiotic Treatment of Staphylococcus aureus-Induced Skin Infections, Monitored by In Vivo Bioluminescent Imaging. PMID : 27067340 : DOI : 10.1128/AAC.02938-15 PMC : PMC4914678 Abstract >>
Staphylococcus aureus is a bacterial pathogen responsible for the majority of skin and soft tissue infections. Antibiotics are losing their efficacy as treatment for skin and soft tissue infections as a result of increased resistance in a variety of pathogens, including S. aureus It is thus imperative to explore alternative antimicrobial treatments to ensure future treatment options for skin and soft tissue infections. A select few lantibiotics, a group of natural defense peptides produced by bacteria, inhibit the growth of numerous clinical S. aureus isolates, including methicillin-resistant strains. In this study, the antimicrobial activities of nisin, clausin, and amyloliquecidin, separately administered, were compared to that of a mupirocin-based ointment, which is commonly used as treatment for S. aureus-induced skin infections. Full-thickness excisional wounds, generated on the dorsal surfaces of mice, were infected with a bioluminescent strain of S. aureus (strain Xen 36). The infections were monitored in real time using in vivo bioluminescent imaging. Lantibiotic treatments significantly reduced the bioluminescence of S. aureus Xen 36 to a level similar to that recorded with mupirocin treatment. Wound closure, however, was more pronounced during lantibiotic treatment. Lantibiotics thus have the potential to be used as an alternative treatment option for S. aureus-induced skin infections.
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56. |
Li X,
Zhang Y,
Wei Z,
Guan Z,
Cai Y,
Liao X,
( 2016 ) Antifungal Activity of Isolated Bacillus amyloliquefaciens SYBC H47 for the Biocontrol of Peach Gummosis. PMID : 27583463 : DOI : 10.1371/journal.pone.0162125 PMC : PMC5008826 Abstract >>
The gummosis disease is caused by Botryosphaeria dothidea (Moug. ex. Fr) Ces. et de Not., and it is one of the most important diseases of stone fruits worldwide. The use of biocontrol as an alternative approach to synthetic chemical fungicides has aroused general concern about how to control plant diseases that are caused by phytopathogens. The aim of this study is to isolate Bacillus strains from raw honeys with the capacity to inhibit B. dothidea and to explore the mechanisms by which they could be used in the biocontrol of peach gummosis. Bacillus amyloliquefaciens SYBC H47 was isolated and identified on the basis of its physiological and biochemical characteristics and its 16S rRNA and gyrB gene sequences. The cell suspension and the cell-free supernatant of its culture showed significant antifungal activity against Aspergillus niger, Mucor racemosus, Fusarium oxysporum, Penicillium citrinum, and Candida albicans by agar-diffusion assays. The primary antifungal substances were bacillomycin L, fengycin, and surfactin, which were analyzed by HPLC LC/ESI-MS/MS. Bacillomycin L showed the best inhibitory effect against conidial germination of B. dothidea, followed by fengycin and surfactin. Surfactin had limited effects on mycelial growth, contrary to those of bacillomycin L and fengycin. However, a mixture of the three lipopeptides had a synergistic effect that disrupted the structure of the conidia and mycelia. In order to reduce the production cost, the use of waste frying peanut oil and soy oil as the sole carbon source increased the lipopeptide yield levels by approximately 17% (2.42 g/L) and 110% (4.35 g/L), respectively. In a field trial, the decreases in the infected gummosis rate (IGR) and the disease severity index (DSI) through cell suspension treatments were 20% and 57.5% (in 2014), respectively, and 40% and 57.5% (in 2015), respectively, in comparison with the control. In conclusion, B. amyloliquefaciens SYBC H47 could inhibit the germination of conidia and the growth of mycelia from B. dothidea; therefore, this strain behaves as a potential biocontrol agent against the gummosis disease.
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57. |
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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58. |
Castaneda-Alvarez C,
Prodan S,
Rosales IM,
Aballay E,
( 2016 ) Exoenzymes and metabolites related to the nematicidal effect of rhizobacteria on Xiphinema index Thorne & Allen. PMID : 26541369 : DOI : 10.1111/jam.12987 Abstract >>
To identify enzymes and metabolites in the rhizobacteria filtrates that have a nematicidal effect on Xiphinema index and perform molecular characterization of the strains evaluated. A series of four bacteria selected for their nematicidal potential were considered for in vitro, biochemical and molecular studies. The direct effect of the bacterial filtrates was evaluated in vitro on X. index juveniles and adults. Hydrogen sulphide and hydrogen cyanide liberation and protease, chitinase, collagenase and lipase activity were verified in the strains. Up to five housekeeping genes and one ITS 16S-23S rRNA were analysed. All bacterial filtrates presented 54-100% mortality when evaluated during up to 72 h of nematode exposure. Strains presented protease activity; two of them (strains FB833T and FR203A) showed reliable collagenase and chitinase activities, respectively, and three of them showed strong lipolytic activity (FB833T, FR203A and FS213P). Strain Bacillus megaterium FB133M had no lipase activity and presented the lowest nematicidal effect. Bacillus amyloliquefaciens FR203A had the largest lethal effect. The rhizobacteria strains evaluated in this study possess nematicidal compounds, which may offer an interesting alternative for X. index control. This is the first report of exoenzymes and metabolites associated with nematicidal effect of rhizobacteria on X. index, which can be a possible alternative for control of this plant-parasitic nematode.
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59. |
Ben Abdallah D,
Frikha-Gargouri O,
Tounsi S,
( 2015 ) Bacillus amyloliquefaciens strain 32a as a source of lipopeptides for biocontrol of Agrobacterium tumefaciens strains. PMID : 25764969 : DOI : 10.1111/jam.12797 Abstract >>
A Bacillus amyloliquefaciens strain, designated 32a, was used to identify new compounds active against Agrobacterium tumefaciens and to evaluate their efficiency to control crown gall on carrot discs. Based on PCR-assays, four gene clusters were shown to direct the synthesis of the cyclic lipopeptides surfactin, iturin A, bacillomycin D and fengycin. Mass spectrometry analysis of culture supernatant led to the identification of these secondary metabolites, except bacillomycin, with heterogeneous mixture of homologues. Antimicrobial assays using lipopeptides-enriched extract showed a strong inhibitory activity against several bacterial and fungal strains, including Ag. tumefaciens. Biological control assays on carrot discs using both 32a spores and extract resulted in significant protection against crown gall disease, similar to that provided by the reference antagonistic strain Agrobacterium rhizogenes K1026. In contrast to all active compounds against Ag. tumefaciens that are of proteinaceous nature, this work enables for the first time to correlate the strong protective effect of B. amyloliquefaciens strain 32a towards crown gall disease with the production of a mixture of lipopeptides. The findings could be useful for growers and nursery men who are particularly interested in the biocontrol of the crown gall disease.
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60. |
Wang H,
Yang L,
Ping Y,
Bai Y,
Luo H,
Huang H,
Yao B,
( 2016 ) Engineering of a Bacillus amyloliquefaciens Strain with High Neutral Protease Producing Capacity and Optimization of Its Fermentation Conditions. PMID : 26752595 : DOI : 10.1371/journal.pone.0146373 PMC : PMC4708984 Abstract >>
The neutral protease has high potential for industrial applications, and attempts to improve enzyme expression level have important application values. In the present study, a neutral protease-encoding gene, Banpr, was cloned from Bacillus amyloliquefaciens strain K11, and a genetic manipulation method specific for this difficult-to-transform strain was developed for the high-level expression of neutral protease. The recombinant plasmid pUB110-Banpr was constructed in Bacillus subtilis strain WB600 and then transformed into strain K11 under optimized conditions. A positive transformant 110N-6 with the highest protease secreting capacity on skim milk plates and great genetic stability for more than 100 generations was selected for further study. Optimization of the fermentation conditions increased the enzyme activity of strain 110N-6 to 8995 �� 250 U/ml in flask culture and 28084 �� 1282 U/ml in 15-l fermentor, which are significantly higher than that of the native strain K11 and industrial strain B. subtilis AS.1398, respectively. The high expression level and extreme genetic stability make B. amyloliquefaciens strain 110N-6 more favorable for mass production of neutral protease for industrial uses.
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61. |
Gay DC,
Wagner DT,
Meinke JL,
Zogzas CE,
Gay GR,
Keatinge-Clay AT,
( 2016 ) The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex. PMID : 26724270 : DOI : 10.1016/j.jsb.2015.12.011 PMC : PMC4738151 Abstract >>
Polyketides such as the clinically-valuable antibacterial agent mupirocin are constructed by architecturally-sophisticated assembly lines known as trans-acyltransferase polyketide synthases. Organelle-sized megacomplexes composed of several copies of trans-acyltransferase polyketide synthase assembly lines have been observed by others through transmission electron microscopy to be located at the Bacillus subtilis plasma membrane, where the synthesis and export of the antibacterial polyketide bacillaene takes place. In this work we analyze ten crystal structures of trans-acyltransferase polyketide synthases ketosynthase domains, seven of which are reported here for the first time, to characterize a motif capable of zippering assembly lines into a megacomplex. While each of the three-helix LINKS (Laterally-INteracting Ketosynthase Sequence) motifs is observed to similarly dock with a spatially-reversed copy of itself through hydrophobic and ionic interactions, the amino acid sequences of this motif are not conserved. Such a code is appropriate for mediating homotypic contacts between assembly lines to ensure the ordered self-assembly of a noncovalent, yet tightly-knit, enzymatic network. LINKS-mediated lateral interactions would also have the effect of bolstering the vertical association of the polypeptides that comprise a polyketide synthase assembly line.
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62. |
Pantoliano MW,
Whitlow M,
Wood JF,
Dodd SW,
Hardman KD,
Rollence ML,
Bryan PN,
( 1989 ) Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding. PMID : 2684274 : DOI : 10.1021/bi00444a012 Abstract >>
Six individual amino acid substitutions at separate positions in the tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to increase the stability of this enzyme, as judged by differential scanning calorimetry and decreased rates of thermal inactivation. These stabilizing changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered through the use of five different investigative approaches: (1) random mutagenesis; (2) design of buried hydrophobic side groups; (3) design of electrostatic interactions at Ca2+ binding sites; (4) sequence homology consensus; and (5) serendipity. Individually, the six amino acid substitutions increase the delta G of unfolding between 0.3 and 1.3 kcal/mol at 58.5 degrees C. The combination of these six individual stabilizing mutations together into one subtilisin BPN' molecule was found to result in approximately independent and additive increases in the delta G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C). Thermodynamic stability was also shown to be related to resistance to irreversible inactivation, which included elevated temperatures (65 degrees C) or extreme alkalinity (pH 12.0). Under these denaturing conditions, the rate of inactivation of the combination variant is approximately 300 times slower than that of the wild-type subtilisin BPN'. A comparison of the 1.8-A-resolution crystal structures of mutant and wild-type enzymes revealed only independent and localized structural changes around the site of the amino acid side group substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)
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63. |
Geraskina NV,
Butov IA,
Yomantas YA,
Stoynova NV,
( 2015 ) The dtd gene from Bacillus amyloliquefaciens encodes a putative D-tyrosyl-tRNATyr deacylase and is a selectable marker for Bacillus subtilis. PMID : 25441601 : DOI : 10.1016/j.micres.2014.11.001 Abstract >>
Genetically engineered microbes are of high practical importance due to their cost-effective production of valuable metabolites and enzymes, and the search for new selectable markers for genetic manipulation is of particular interest. Here, we revealed that the soil bacterium Bacillus amyloliquefaciens A50 is tolerant to the non-canonical amino acid D-tyrosine (D-Tyr), in contrast to the closely related Bacillus strain B. subtilis 168, which is a widely used "domesticated" laboratory strain. The gene responsible for resistance to D-Tyr was identified. The resistance was associated with the activity of a potential D-tyrosyl-tRNA(Tyr) deacylase. Orthologs of this enzyme are capable of hydrolyzing the ester bond and recycling misacetylated D-aminoacyl-tRNA molecules into free tRNAs and D-amino acids. This gene, yrvI (dtd), is applicable as a convenient, small selectable marker for non-antibiotic resistance selection in experiments aimed at genome editing of D-Tyr-sensitive microorganisms.
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64. |
Kim JD,
Jeon BJ,
Han JW,
Park MY,
Kang SA,
Kim BS,
( 2016 ) Evaluation of the endophytic nature of Bacillus amyloliquefaciens strain GYL4 and its efficacy in the control of anthracnose. PMID : 26518268 : DOI : 10.1002/ps.4181 Abstract >>
Endophytic bacteria are viewed as a potential new source of biofungicides because they have beneficial characteristics as control agents for plant disease. This study was performed to examine the endophytic feature and disease control efficacy of Bacillus amyloliquefaciens strain GYL4 and to identify the antifungal compounds produced by this strain. B. amyloliquefaciens strain GYL4 was isolated from leaf tissue of pepper plants (Capsicum annuum L.). Anthracnose symptoms were markedly reduced in the leaves of pepper plants colonised by GYL4. An egfp-expressing strain of GYL4 (GYL4-egfp) was constructed and reintroduced into pepper plants, which confirmed its ability to colonise the internal tissues of pepper plants. GYL4-egfp was observed in the root and stem tissues 4 days after treatment and abundantly found in the internal leaf tissue 9 days after treatment. Bacillomycin derivatives purified from the culture extract of GYL4 displayed control efficacy on anthracnose development in cucumber (Cucumis sativus L. cv. Chunsim). The present study is the first report on evaluation of the endophytic and systemic nature of B. amyloliquefaciens strain GYL4 and its potential as a biocontrol agent for anthracnose management. ? 2015 Society of Chemical Industry.
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65. |
Chakraborty K,
Thilakan B,
Raola VK,
( 2014 ) Polyketide family of novel antibacterial 7-O-methyl-5'-hydroxy-3'-heptenoate-macrolactin from seaweed-associated Bacillus subtilis MTCC 10403. PMID : 25420039 : DOI : 10.1021/jf504845m Abstract >>
Seaweed-associated heterotrophic bacterial communities were screened to isolate potentially useful antimicrobial strains, which were characterized by phylogenetic analysis. The bacteria were screened for the presence of metabolite genes involved in natural product biosynthetic pathway, and the structural properties of secondary metabolites were correlated with the genes. Bioactivity-guided isolation of polyene antibiotic 7-O-methyl-5'-hydroxy-3'-heptenoate-macrolactin from Bacillus subtilis MTCC10403 associated with seaweed Anthophycus longifolius using mass spectrometry and extensive 2D-NMR studies was carried out. The newly isolated macrolactin compound is a bactericidal antibiotic with broad spectrum activity against human opportunistic clinical pathogens. The biosynthetic pathway of 7-O-methyl-5'-hydroxy-3'-heptenoate-macrolactin by means of a stepwise, decarboxylative condensation pathway established the PKS-assisted biosynthesis of the parent macrolactin and the side-chain 5-hydroxyhept-3-enoate moiety attached to the macrolactin ring system at C-7. Antimicrobial activity analysis combined with the results of amplifying genes encoding for polyketide synthetase and nonribosomal peptide synthetase showed that seaweed-associated bacteria had broad-spectrum antimicrobial activity. The present work may have an impact on the exploitation of macrolactins for pharmaceutical and biotechnological applications.
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66. |
Wang M,
Xu M,
Rao Z,
Yang T,
Zhang X,
( 2015 ) Construction of a highly efficient Bacillus subtilis 168 whole-cell biocatalyst and its application in the production of L-ornithine. PMID : 26314414 : DOI : 10.1007/s10295-015-1672-z Abstract >>
L-Ornithine, a non-protein amino acid, is usually extracted from hydrolyzed protein as well as produced by microbial fermentation. Here, we focus on a highly efficient whole-cell biocatalyst for the production of L-ornithine. The gene argI, encoding arginase, which catalyzes the hydrolysis of L-arginine to L-ornithine and urea, was cloned from Bacillus amyloliquefaciens B10-127 and expressed in GRAS strain Bacillus subtilis 168. The recombinant strain exhibited an arginase activity of 21.9 U/mg, which is 26.7 times that of wild B. subtilis 168. The optimal pH and temperature of the purified recombinant arginase were 10.0 and 40 �XC, respectively. In addition, the recombinant arginase exhibited a strong Mn(2+) preference. When using whole-cell biocatalyst-based bioconversion, a hyper L-ornithine production of 356.9 g/L was achieved with a fed-batch strategy in a 5-L reactor within 12 h. This whole-cell bioconversion study demonstrates an environmentally friendly strategy for L-ornithine production in industry.
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67. |
Cai X,
Ma J,
Wei DZ,
Lin JP,
Wei W,
( 2014 ) Functional expression of a novel alkaline-adapted lipase of Bacillus amyloliquefaciens from stinky tofu brine and development of immobilized enzyme for biodiesel production. PMID : 25199563 : DOI : 10.1007/s10482-014-0274-5 Abstract >>
Using enrichment procedures, a lipolytic strain was isolated from a stinky tofu brine and was identified as Bacillus amyloliquefaciens (named B. amyloliquefaciens Nsic-8) by morphological, physiological, biochemical tests and 16S rDNA sequence analysis. Meanwhile, the key enzyme gene (named lip BA) involved in ester metabolism was obtained from Nsic-8 with the assistance of homology analysis. The novel gene has an open reading frame of 645 bp, and encodes a 214-amino-acid lipase (LipBA). The deduced amino acid sequence shows the highest identity with the lipase from B. amyloliquefaciens IT-45 (NCBI database) and belongs to the family of triacylglycerol lipase (EC 3.1.1.3). The lipase gene was expressed in Escherichia coli BL21(DE3) using plasmid pET-28a. The enzyme activity and specific activity were 250 �� 16 U/ml and 1750 �� 153 U/mg, respectively. The optimum pH and temperature of the recombinant enzyme were 9.0 and 40 �XC respectively. LipBA showed much higher stability under alkaline conditions and was stable at pH 7.0-11.0. The Km and Vmax values of purified LipBA using 4-nitrophenyl palmitate as the substrate were 1.04 �� 0.06 mM and 119.05 �� 7.16 �gmol/(ml min), respectively. After purification, recombinant lipase was immobilized with the optimal conditions (immobilization time 3 h at 30 �XC, with 92 % enzyme recovery) and the immobilized enzyme was applied in biodiesel production. This is the first report of the lipase activity and lipase gene obtained from B. amyloliquefaciens (including wild strain and recombinant strain) and the recombinant LipBA with the detailed enzymatic properties. Also the preliminary study of the transesterification shows the potential value in biodiesel production applications.
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68. |
Ait-Kaki A,
Kacem-Chaouche N,
Ongena M,
Kara-Ali M,
Dehimat L,
Kahlat K,
Thonart P,
( 2014 ) In vitro and in vivo characterization of plant growth promoting Bacillus strains isolated from extreme environments of Eastern Algeria. PMID : 24258791 : DOI : 10.1007/s12010-013-0617-0 Abstract >>
This report is to our knowledge the first to study plant growth promotion and biocontrol characteristics of Bacillus isolates from extreme environments of Eastern Algeria. Seven isolates of 14 (50 %) were screened for their ability to inhibit growth of some phytopathogenic fungi on PDA and some roots exudates. The bacteria identification based on 16S r-RNA and gyrase-A gene sequence analysis showed that 71 % of the screened isolates belonged to Bacillus amyloliquefaciens and the rest were closely related to B. atrophaeus and B. mojavensis. Most of them had high spore yields (22 �� 10(8)-27 �� 10(8) spores/ml). They produced protease and cellulase cell wall-degrading enzymes while the chitinase activity was only observed in the B. atrophaeus (6SEL). A wide variety of lipopeptides homologous was detected by liquid chromatography-electrospray ionization-mass spectrometry analysis. Interestingly, some additional peaks with new masses were characterized, which may correspond to new fengycin classes. The isolates produced siderophores and indole-3- acetic acid phytohormone. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. atrophaeus (6SEL) significantly increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05). These results suggest that these isolates may be used further as bio-inoculants to improve crop systems.
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69. |
Kurniasih SD,
Alfi A,
Natalia D,
Radjasa OK,
Nurachman Z,
( N/A ) Construction of individual, fused, and co-expressed proteins of endoglucanase and �]-glucosidase for hydrolyzing sugarcane bagasse. PMID : 24598011 : DOI : 10.1016/j.micres.2014.02.002 Abstract >>
At least a combination of endoglucanase (EglII) and �]-glucosidase (BglZ) is required for hydrolyzing crystalline cellulose. To understand the catalytic efficiency of combination enzymes for converting biomass to sugars, EglII and BglZ were constructed in the form of individual, fused as well as co-expression proteins, and their activities for hydrolyzing sugarcane bagasse were evaluated. The genes, eglII isolated from Bacillus amyloliquefaciens PSM3.1 earlier and bglZ from B. amyloliquefaciens ABBD, were expressed extracellularly in Bacillus megaterium MS941. EglII exhibited both exoglucanase and endoglucanase activities, and BglZ belonging to the glycoside hydrolase 1 family (GH 1) showed �]-glucosidase activity. A combination of EglII and BglZ showed activity on substrates Avicel, CMC and sugarcane bagasse. Specifically for hydrolyzing sugarcane bagasse, fused protein (fus-EglII+BglZ), co-expression protein (coex-BglZ+EglII), and mixed-individual protein (mix-EglII+BglZ) produced cellobiose as the main product, along with a small amount of glucose. The amount of reducing sugars released from the hydrolyzing bleached sugarcane bagasse (BSB) using fus-EglII+BglZ and mix-EglII+BglZ was 2.7- and 4.2-fold higher, respectively, than steamed sugarcane bagasse (SSB), indicating the synergetic enzymes worked better on treated sugarcane bagasse. Compared with fus-EglII+BglZ and mix-EglII+BglZ, coex-BglZ+EglII released more mol reducing sugars from SSB, indicating the enzymes were potential for biomass conversion. Additionally, coex-BglZ+EglII acted on BSB 2.5-fold faster than fus-EglII+BglZ. Thus, coex-bglZ+eglII expression system was the best choice to produce enzymes for hydrolyzing sugarcane baggase.
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70. |
Lee CK,
Kim GM,
Shin JH,
Kim JS,
Kim JH,
Heo K,
Cho KM,
( 2013 ) Characterization of a fibrinolytic enzyme secreted by Bacillus amyloliquefaciens CB1 and its gene cloning. PMID : 23727811 : Abstract >>
Bacillus amyloliquefaciens CB1 was isolated from cheonggukjang, a Korean fermented soy food. B. amyloliquefaciens CB1 secretes proteases with fibrinolytic activities. A gene homologous to aprE of Bacillus subtilis, aprECB1, was cloned from B. amyloliquefaciens CB1, and DNA sequencing showed that aprECB1 can encode a prepro-type serine protease consisting of 382 amino acids. When aprECB1 was introduced into B. subtilis WB600 using an E. coli-Bacillus shuttle vector, pHY300PLK, transformants showed fibrinolytic activity and produced a 28 kDa protein, the size expected for the mature enzyme. The 28 kDa fibrinolytic enzyme was purified from the culture supernatant of B. subtilis WB600 transformant. AprECB1 was completely inhibited by phenylmethylsulfonyl fluoride and almost completely inhibited by EDTA and EGTA, indicating that it is a serine metalloprotease. AprECB1 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, a known substrate for �\-chymotrypsin. A�\ and B�] chains of fibrinogen were quickly degraded by AprECB1, but the �^-chain was resistant.
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71. |
Sen R,
Tripathy S,
Padhi SK,
Mohanty S,
Maiti NK,
( 2014 ) Sequence polymorphism of GroEL gene in natural population of Bacillus and Brevibacillus spp. that showed variation in thermal tolerance capacity and mRNA expression. PMID : 24894903 : DOI : 10.1007/s00284-014-0614-8 Abstract >>
GroEL, a class I chaperonin, plays an important role in the thermal adaptation of the cell and helps to maintain the viability of the cell under heat shock condition. Function of groEL in vivo depends on the maintenance of proper structure of the protein which in turn depends on the nucleotide and amino acid sequence of the gene. In this study, we investigated the changes in nucleotide and amino acid sequences of the partial groEL gene that may affect the thermotolerance capacity as well as mRNA expression of bacterial isolates. Sequences among the same species having differences in the amino acid level were identified as different alleles. The effect of allelic variation on the groEL gene expression was analyzed by comparison and relative quantification in each allele under thermal shock condition by RT-PCR. Evaluation of K a/K s ratio among the strains of same species showed that the groEL gene of all the species had undergone similar functional constrain during evolution. The strains showing similar thermotolerance capacity was found to carry same allele of groEL gene. The isolates carrying allele having amino acid substitution inside the highly ATP/ADP or Mg(2+)-binding region could not tolerate thermal stress and showed lower expression of the groEL gene. Our results indicate that during evolution of these bacterial species the groEL gene has undergone the process of natural selection, and the isolates have evolved with the groEL allelic sequences that help them to withstand the thermal stress during their interaction with the environment.
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72. |
Lon?ar N,
Boži? N,
Lopez-Santin J,
Vuj?i? Z,
( 2013 ) Bacillus amyloliquefaciens laccase--from soil bacteria to recombinant enzyme for wastewater decolorization. PMID : 23994699 : DOI : 10.1016/j.biortech.2013.08.056 Abstract >>
One hundred wild type strains of Bacillus sp. were isolated from industrial and agricultural soil across Serbia and screened for laccase activity. Three strains showed high laccase activity temperature optimum of 65 and 80 �XC towards ABTS. A new laccase gene from the strain with highest temperature optimum, namely Bacillus amyloliquefaciens 12B was cloned and expressed in Escherichia coli. Recombinant laccase degraded dye Reactive blue 52 at pH 7.0 and pH 4.0 and at elevated temperature, while fungal laccases was unable to act on this substrate at pH higher than 4.0 and was quickly inactivated at temperatures higher than 45 �XC. Degradation of dye was monitored by HPLC-DAD and resulting precipitate was analyzed by FTIR spectroscopy. Single product peak without chromophore was detected in solution, while water insoluble aggregate, presumably dye polymer is formed retaining blue color.
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73. |
Lee J,
Hao Y,
Blair PM,
Melby JO,
Agarwal V,
Burkhart BJ,
Nair SK,
Mitchell DA,
( 2013 ) Structural and functional insight into an unexpectedly selective N-methyltransferase involved in plantazolicin biosynthesis. PMID : 23878226 : DOI : 10.1073/pnas.1306101110 PMC : PMC3740862 Abstract >>
Plantazolicin (PZN), a polyheterocyclic, N(�\),N(�\)-dimethylarginine-containing antibiotic, harbors remarkably specific bactericidal activity toward strains of Bacillus anthracis, the causative agent of anthrax. Previous studies demonstrated that genetic deletion of the S-adenosyl-L-methionine-dependent methyltransferase from the PZN biosynthetic gene cluster results in the formation of desmethylPZN, which is devoid of antibiotic activity. Here we describe the in vitro reconstitution, mutational analysis, and X-ray crystallographic structure of the PZN methyltransferase. Unlike all other known small molecule methyltransferases, which act upon diverse substrates in vitro, the PZN methyltransferase is uncharacteristically limited in substrate scope and functions only on desmethylPZN and close derivatives. The crystal structures of two related PZN methyltransferases, solved to 1.75 ? (Bacillus amyloliquefaciens) and 2.0 ? (Bacillus pumilus), reveal a deep, narrow cavity, putatively functioning as the binding site for desmethylPZN. The narrowness of this cavity provides a framework for understanding the molecular basis of the extreme substrate selectivity. Analysis of a panel of point mutations to the methyltransferase from B. amyloliquefaciens allowed the identification of residues of structural and catalytic importance. These findings further our understanding of one set of orthologous enzymes involved in thiazole/oxazole-modified microcin biosynthesis, a rapidly growing sector of natural products research.
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74. |
Singh S,
Moholkar VS,
Goyal A,
( 2013 ) Isolation, Identification, and Characterization of a Cellulolytic Bacillus amyloliquefaciens Strain SS35 from Rhinoceros Dung. PMID : 23762763 : DOI : 10.1155/2013/728134 PMC : PMC3676922 Abstract >>
Cellulose hydrolyzing bacteria were isolated from rhinoceros dung and tested for clear zone formation around the colonies on the agar plates containing the medium amended with carboxymethylcellulose as a sole carbon source. Isolates were further screened on the basis of carboxymethylcellulase production in liquid medium. Out of 36 isolates, isolate no. 35 exhibited maximum enzyme activity of 0.079 U/mL and was selected for further identification by using conventional biochemical tests and phylogenetic analyses. This was a Gram-positive, spore forming bacterium with rod-shaped cells. The isolate was identified as Bacillus amyloliquefaciens SS35 based on nucleotide homology and phylogenetic analysis using 16S rDNA and gyrase A gene sequences.
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75. |
Connaughton JF,
Kaloss WD,
Vanek PG,
Nardone GA,
Chirikjian JG,
( 1990 ) The complete sequence of the Bacillus amyloliquefaciens proviral H2, BamHI methylase gene. PMID : 2374727 : DOI : 10.1093/nar/18.13.4002 PMC : PMC331118 Abstract >>
N/A
|
76. |
Tang LB,
Nagarajan V,
( 1990 ) Isolation and characterization of levansucrase-encoding gene from Bacillus amyloliquefaciens. PMID : 2265762 : DOI : 10.1016/0378-1119(90)90345-r Abstract >>
The gene encoding levansucrase (LVS) from Bacillus amyloliquefaciens (sacB[BamP]) was isolated, sequenced and expressed in Bacillus subtilis. Analysis of the nucleotide sequence of sacB[BamP] reveals extensive homology with that of the B. subtilis LVS-encoding gene in the promoter and coding region. The sacB[BamP] gene cloned in a multicopy plasmid is induced by sucrose in B. subtilis.
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77. |
Siriphongphaew A,
Pisnupong P,
Wongkongkatep J,
Inprakhon P,
Vangnai AS,
Honda K,
Ohtake H,
Kato J,
Ogawa J,
Shimizu S,
Urlacher VB,
Schmid RD,
Pongtharangkul T,
( 2012 ) Development of a whole-cell biocatalyst co-expressing P450 monooxygenase and glucose dehydrogenase for synthesis of epoxyhexane. PMID : 22555910 : DOI : 10.1007/s00253-012-4039-7 Abstract >>
Oxygenases-based Escherichia coli whole-cell biocatalyst can be applied for catalysis of various commercially interesting reactions that are difficult to achieve with traditional chemical catalysts. However, substrates and products of interest are often toxic to E. coli, causing a disruption of cell membrane. Therefore, organic solvent-tolerant bacteria became an important tool for heterologous expression of such oxygenases. In this study, the organic solvent-tolerant Bacillus subtilis 3C5N was developed as a whole-cell biocatalyst for epoxidation of a toxic terminal alkene, 1-hexene. Comparing to other hosts tested, high level of tolerance towards 1-hexene and a moderately hydrophobic cell surface of B. subtilis 3C5N were suggested to contribute to its higher 1,2-epoxyhexane production. A systematic optimization of reaction conditions such as biocatalyst and substrate concentration resulted in a 3.3-fold increase in the specific rate. Co-expression of glucose dehydrogenase could partly restored NADPH-regenerating ability of the biocatalyst (up to 38 % of the wild type), resulting in approximately 53 % increase in specific rate representing approximately 22-fold increase in product concentration comparing to that obtained prior to an optimization.
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78. |
Liu W,
Wang X,
Wu L,
Chen M,
Tu C,
Luo Y,
Christie P,
( 2012 ) Isolation, identification and characterization of Bacillus amyloliquefaciens BZ-6, a bacterial isolate for enhancing oil recovery from oily sludge. PMID : 22365392 : DOI : 10.1016/j.chemosphere.2012.01.059 Abstract >>
Over 100 biosurfactant-producing microorganisms were isolated from oily sludge and petroleum-contaminated soil from Shengli oil field in north China. Sixteen of the bacterial isolates produced biosurfactants and reduced the surface tension of the growth medium from 71 to <30 mN m(-1) after 72 h of growth. These bacteria were used to treat oily sludge and the recovery efficiencies of oil from oily sludge were determined. The oil recovery efficiencies of different isolates ranged from 39% to 88%. Bacterial isolate BZ-6 was found to be the most efficient strain and the three phases (oil, water and sediment) were separated automatically after the sludge was treated with the culture medium of BZ-6. Based on morphological, physiological characteristics and molecular identification, isolate BZ-6 was identified as Bacillus amyloliquefaciens. The biosurfactant produced by isolate BZ-6 was purified and analyzed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry. There were four ion peaks representing four different fengycin A homologues.
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79. |
Kwon GH,
Park JY,
Kim JS,
Lim J,
Park CS,
Kwon DY,
Kim JH,
( 2011 ) Cloning and expression of a bpr gene encoding Bacillopeptidase F from Bacillus amyloliquefaciens CH86-1. PMID : 21617349 : Abstract >>
A gene encoding bacillopeptidase F, bpr86-1, was cloned from B. amyloliquefaciens CH86-1 isolated from cheonggukjang. This gene could encode a preproenzyme of 1,431 amino acids. When bpr86-1 was introduced into B. subtilis WB600 via pHY300PLK, an E. coli-Bacillus shuttle vector, the transformant showed fibrinolytic activity. During growth on LB, the fibrinolytic activity of cells increased sharply when they entered the stationary phase. The highest activity (761.4 mU/mg protein) was observed at 96 h of cultivation.
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80. |
Vanek PG,
Connaughton JF,
Kaloss WD,
Chirikjian JG,
( 1990 ) The complete sequence of the Bacillus amyloliquefaciens strain H, cellular BamHI methylase gene. PMID : 2235511 : DOI : 10.1093/nar/18.20.6145 PMC : PMC332434 Abstract >>
N/A
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81. |
Zakataeva NP,
Romanenkov DV,
Skripnikova VS,
Vitushkina MV,
Livshits VA,
Kivero AD,
Novikova AE,
( 2012 ) Wild-type and feedback-resistant phosphoribosyl pyrophosphate synthetases from Bacillus amyloliquefaciens: purification, characterization, and application to increase purine nucleoside production. PMID : 22083279 : DOI : 10.1007/s00253-011-3687-3 Abstract >>
Bacillus strains are used for the industrial production of the purine nucleosides inosine and guanosine, which are raw materials for the synthesis of the flavor enhancers disodium inosinate and disodium guanylate. An important precursor of purine nucleosides is 5-phospho-�\-D: -ribosyl-1-pyrophosphate, which is synthesized by phosphoribosyl pyrophosphate synthetase (PRS, EC 2.7.6.1). Class I PRSs are widespread in bacteria and mammals, are highly conserved among different organisms, and are negatively regulated by two end products of purine biosynthesis, adenosine 5'-diphosphate (ADP) and guanosine 5'-diphosphate (GDP). The D52H, N114S, and L129I mutations in the human PRS isozyme I (PRS1) have been reported to cause uric acid overproduction and gout due to allosteric deregulation and enzyme superactivity. In this study, to find feedback-resistant Bacillus amyloliquefaciens PRS, the influence of the D58H, N120S, and L135I mutations (corresponding to the D52H, N114S, and L129I mutations in PRS1, respectively) on PRS enzymatic properties has been studied. Recombinant histidine-tagged wild-type PRS and three mutant PRSs were expressed in Escherichia coli, purified, and characterized. The N120S and L135I mutations were found to release the enzyme from ADP and GDP inhibition and significantly increase its sensitivity to inorganic phosphate (P(i)) activation. In contrast, PRS with the D58H mutation exhibited nearly identical sensitivity to ADP and GDP as the wild-type protein and had a notably greater P(i) requirement for activation. The N120S and L135I mutations improved B. amyloliquefaciens and Bacillus subtilis purine nucleoside-producing strains.
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82. |
Jo HD,
Lee HA,
Jeong SJ,
Kim JH,
( 2011 ) Purification and characterization of a major fibrinolytic enzyme from Bacillus amyloliquefaciens MJ5-41 isolated from Meju. PMID : 22127128 : Abstract >>
Meju is a traditional Korean fermented soy product used as a key element for soy sauce and doenjang. Bacilli with antimicrobial activity were isolated from meju prepared by traditional methods at Sunchang county, Jeollabukdo, Korea. Six isolates were identified as Bacillus amyloliquefaciens by recA gene sequencing and RAPD-PCR. One isolate, B. amyloliquefaciens MJ5-41, showed the strongest fibrinolytic activity. A 27 kDa active fibrinolytic enzyme, AprE5-41, was purified from the culture supernatant of MJ5-41 grown on LB by chromatographic methods. The optimum pH and temperature for purified AprE5-41 were 7.0 and 45�XC, respectively. AprE5-41 quickly degraded A�\ and B�] chains but not the gamma-chain of fibrinogen. AprE5-41 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe pnitroanilide, a known substrate for �\-chymotrypsin, cathepsin G, and subtilisin BPN'. The structural gene, aprE5-41, was cloned by PCR and successfully expressed in B. subtilis.
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83. |
Alvarez F,
Castro M,
Príncipe A,
Borioli G,
Fischer S,
Mori G,
Jofré E,
( 2012 ) The plant-associated Bacillus amyloliquefaciens strains MEP2 18 and ARP2 3 capable of producing the cyclic lipopeptides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease. PMID : 22017648 : DOI : 10.1111/j.1365-2672.2011.05182.x Abstract >>
This work was conducted to identify the antifungal compounds produced by two previously isolated Bacillus sp. strains: ARP(2) 3 and MEP(2) 18. Both strains were subjected to further analysis to determine their taxonomic position and to identify the compounds responsible for their antifungal activity as well as to evaluate the efficiency of these strains to control sclerotinia stem rot in soybean. The antifungal compounds were isolated by acid precipitation of cell-free supernatants, purified by RP-HPLC and then tested for antagonistic activity against Sclerotinia sclerotiorum. Mass spectra from RP-HPLC eluted fractions showed the presence of surfactin C(15) , fengycins A (C(16) -C(17)) and B (C(16)) isoforms in supernatants from strain ARP(2) 3 cultures, whereas the major lipopeptide produced by strain MEP(2) 18 was iturin A C(15) . Alterations in mycelial morphology and sclerotial germination were observed in the presence of lipopeptides-containing supernatants from Bacillus strains cultures. Foliar application of Bacillus amyloliquefaciens strains on soybean plants prior to S. sclerotiorum infection resulted in significant protection against sclerotinia stem rot compared with noninoculated plants or plants inoculated with a nonlipopeptide-producing B. subtilis strain. Both strains, renamed as B. amyloliquefaciens ARP(2) 3 and MEP(2) 18, were able to produce antifungal compounds belonging to the cyclic lipopeptide family. Our data suggest that the foliar application of lipopeptide-producing B. amyloliquefaciens strains could be a promising strategy for the management of sclerotinia stem rot in soybean. Sclerotinia stem rot was ranked as one of the most severe soybean disease in Argentina and worldwide. The results of this study showed the potential of B. amyloliquefaciens strains ARP(2) 3 and MEP(2) 18 to control plant diseases caused by S. sclerotiorum.
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84. |
( N/A ) [Riboflavin biosynthetic genes in Bacillus amyloliquefaciens: primary structure, organization and regulation of activity]. PMID : 9297088 : Abstract >>
N/A
|
85. |
( 1993 ) Assignment of the backbone 1H and 15N NMR resonances and secondary structure characterization of barstar. PMID : 8405454 : DOI : 10.1016/0014-5793(93)80489-h Abstract >>
Barstar, a polypeptide inhibitor of ribonucleases, has been studied by 2D and 3D NMR techniques using uniformly 15N-labeled protein. Backbone (15NH-C alpha H-C beta H) resonances were assigned for all but 5 of the 89 residues. Dihedral angle and deuterium exchange studies were used in conjunction with medium range inter-proton NOEs to characterize the secondary structure of barstar. The protein is composed of four alpha-helices and three short stretches of extended strand. By further analysis of the NOE data three of the helices were found to be parallel to each other with the single disulphide bond linking the second and fourth helices at their C-terminal ends.
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86. |
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.
|
87. |
( 1995 ) The endogenous Bacillus subtilis (natto) plasmids pTA1015 and pTA1040 contain signal peptidase-encoding genes: identification of a new structural module on cryptic plasmids. PMID : 8801417 : DOI : 10.1111/j.1365-2958.1995.mmi_17040621.x Abstract >>
Various strains of Bacillus subtilis (natto) contain small cryptic plasmids that replicate via the rolling-circle mechanism. Like plasmids from other Gram-positive bacteria, these plasmids are composed of several distinct structural modules. A new structural module was identified on the B. subtilis plasmids pTA1015 and pTA1040. It is composed of two genes: one specifies an unidentified protein with a putative signal peptide; and the other (sipP) specifies a functional type 1 signal peptidase (SPase). The homologous, but non-identical, sipP genes of the two plasmids are the first identified plasmid-specific SPase-encoding genes. With respect to structure and activity, the corresponding enzymes (denoted SipP) are highly similar to the chromosomally encoded SPase, SipP, of B. subtillis and several newly identified SPases of other bacilli. Our findings suggest that plasmid-encoded SPases have evolved because, of under certain conditions, SPase can be a limiting factor for protein secretion in B. subtilis.
|
88. |
( 1994 ) Structure of restriction endonuclease BamHI and its relationship to EcoRI. PMID : 8145855 : DOI : 10.1038/368660a0 Abstract >>
Type II restriction endonucleases are characterized by the remarkable specificity with which they cleave specific DNA sequences. Surprisingly, their protein sequences are in most cases unrelated, and no recurring structural motif has yet been identified. We have determined the structure of restriction endonuclease BamHI at 1.95 A resolution. BamHI shows striking resemblance to the structure of endonuclease EcoRI (refs 3, 4), despite the lack of sequence similarity between them. We also observe some curious differences between the two structures, and propose an evolutionary scheme that may explain them. The active site of BamHI is structurally similar to the active sites of EcoRI and EcoRV (ref. 5), but the mechanism by which BamHI activates a water molecule for nucleophilic attack may be different.
|
89. |
( 1994 ) Three-dimensional solution structure and 13C assignments of barstar using nuclear magnetic resonance spectroscopy. PMID : 8043574 : Abstract >>
We present the high-resolution solution structure and 13C assignments of wild-type barstar, an 89 amino acid residue polypeptide inhibitor of barnase, derived from heteronuclear NMR techniques. These were obtained from measurements on unlabeled, uniformly 15N- and 13C/15N-labeled, and 10% 13C-labeled barstar samples that have both cysteines (at positions 40 and 82) fully reduced. In total, 30 structures were calculated by hybrid distance geometry-dynamical simulated annealing calculations. The atomic rms distribution about the mean coordinate positions is 0.42 A for all backbone atoms and 0.90 A for all atoms. The structure is composed of three parallel alpha-helices packed against a three-stranded parallel beta-sheet. A more poorly defined helix links the second beta-strand and the third major alpha-helix. The loop involved in binding barnase is extremely well defined and held rigidly by interactions from the main body of the protein to both ends and the middle of the loop. This structure will be used to aid protein engineering studies currently taking place on the free and bound states of barstar and barnase.
|
90. |
( 1977 ) Recognition sequence of specific endonuclease BamH.I from Bacillus amyloliquefaciens H. PMID : 834250 : DOI : 10.1038/265082a0 Abstract >>
N/A
|
91. |
( 1993 ) Pyroglutamyl peptidase gene from Bacillus amyloliquefaciens: cloning, sequencing, expression, and crystallization of the expressed enzyme. PMID : 8095933 : DOI : 10.1093/oxfordjournals.jbchem.a124005 Abstract >>
The pyroglutamyl peptidase [EC 3.4.11.8] gene from Bacillus amyloliquefaciens was cloned and expressed in Escherichia coli DH1. The transformant of E. coli DH1 harboring plasmid pBPG 1 with a 2.1 kb chromosomal DNA fragment showed 80-fold higher activity than B. amyloliquefaciens. The nucleotide sequence of a 0.9 kb fragment that contains the promoter and the mature protein coding region was determined by the dideoxy chain-termination method. An open reading frame of 648 bp starting with an ATG methionine codon was found, which encodes a protein of 215 amino acid residues with a deduced molecular weight of 23,286. The enzyme has two cysteine residues (Cys68 and Cys144) per subunit molecule. Substitution of Cys144 with Ser by site-directed mutagenesis resulted in a complete loss of the activity, while that of Cys68 with Ser did not affect the activity at all. This result and titration with DTNB suggest that Cys144 is concerned in the catalytic action and Cys68 is located inside the enzyme. The expressed enzyme was purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with an activity recovery of 42.7%. The enzyme was most active at pH 6.5 and stable at pH 7.0-9.0. Its molecular weight was estimated to be 51,000 by gel filtration, suggesting it to be a dimer. Big crystals of the wild and PCMB-modified enzymes were obtained by the hanging drop method.
|
92. |
Markland FS,
Shaw E,
Smith EL,
( 1968 ) Identification of histidine 64 in the active site of subtilisin. PMID : 5249818 : DOI : 10.1073/pnas.61.4.1440 PMC : PMC225275 Abstract >>
N/A
|
93. |
Yoshimura K,
Uemura J,
Seki T,
Oshima Y,
( 1984 ) Construction of a promoter-probe vector for a Bacillus subtilis host by using the trpD+ gene of Bacillus amyloliquefaciens. PMID : 6090398 : PMC : PMC215745 Abstract >>
The trp gene cluster of Bacillus amyloliquefaciens was found to be structurally similar to that of the Enterobacteriaceae. The translation termination codon of the putative trpE gene and the initiation codon for the putative trpD gene overlap at the trpE-trpD junction, and a promoter for the putative trpC gene is suggested to exist. A promoter-probe vector of Bacillus subtilis, pFTB281, was constructed with a DNA fragment of B. amyloliquefaciens, complementing the trpC and trpD mutations of B. subtilis, a 42-base-pair DNA fragment of M13mp7, and the larger EcoRI-PvuII fragment of pUB110, which confers an autonomous replication function and the kanamycin-resistance phenotype to the chimeric plasmid. pFTB281 has BamHI, EcoRI, and SalI cloning sites in the 5'-upstream portion of the protein-coding region of the putative trpD gene, and the insertion of a certain DNA fragment at any of these sites allowed the plasmid to transform a trpD mutant of B. subtilis to the TrpD+ phenotype. DNA fragments showing the promoter function for the trpD gene were obtained from B. amyloliquefaciens and Saccharomyces cerevisiae chromosomes and rho 11 and lambda phage DNAs, but rarely from the DNAs of Escherichia coli and pBR322.
|
94. |
Hartley RW,
Barker EA,
( 1972 ) Amino-acid sequence of extracellular ribonuclease (barnase) of Bacillus amyloliquefaciens. PMID : 4553460 : Abstract >>
N/A
|
95. |
Sachdev O,
Friedberg F,
( ) PMID : 6176566 : Abstract >>
Alpha amylase (Sigma type IIA) was cleaved by cyanogen bromide. The sequences of two fragments, D which contains 88 amino acids and E which contains 56, are presented. By applying the Chou-Fasman rules, D is predicted to have 32% beta sheet and 37% alpha helix, and E is predicted to exhibit 11% beta sheet and 71% alpha helix.
|
96. |
Palva I,
Pettersson RF,
Kalkkinen N,
Lehtovaara P,
Sarvas M,
Söderlund H,
Takkinen K,
Kääriäinen L,
( 1981 ) Nucleotide sequence of the promoter and NH2-terminal signal peptide region of the alpha-amylase gene from Bacillus amyloliquefaciens. PMID : 6170539 : DOI : 10.1016/0378-1119(81)90103-7 Abstract >>
We have isolated and partially sequenced the gene coding for alpha-amylase (EC 3.2.1.1) from Bacillus amyloliquefaciens by molecular cloning in the plasmid pUB110 using Bacillus subtilis as a host. The nucleotide sequence of the NH2-terminal region of the cloned gene was determined and found to contain a 31-residue-long stretch of amino acids preceding the NH2-terminal sequence of the extracellular alpha-amylase. Within this sequence there is a 15-residue-long stretch of uncharged amino acids similar to that found at the NH2 terminus of other precursors to exported proteins. This "signal sequence" is probably removed in conjunction with the translocation of alpha-amylase through the cytoplasmic membrane. In vitro labeling of alpha-amylase with radioactive amino acids in a coupled transcription-translation system followed by partial sequencing established the exact location of the NH2 terminus of the alpha-amylase gene. The nucleotide sequence preceding the NH2 terminus has properties resembling the RNA-polymerase- and ribosome-binding sites found at the 5' terminus of many prokaryotic genes.
|
97. |
Hoang V,
Hofemeister J,
( 1995 ) Bacillus amyloliquefaciens possesses a second type I signal peptidase with extensive sequence similarity to other Bacillus SPases. PMID : 7578273 : DOI : 10.1016/0167-4889(95)00101-w Abstract >>
A second sipS2(BA) gene was PCR cloned from Bacillus amyloliquefaciens. The deduced aa sequence is similar to those of the SPases of B. subtilis, B. amyloliquefaciens, and B. licheniformis and the domain structure of the gene has been preserved. A low level of monocistronic gene transcription could be shown using Northern analysis. The sipS2(BA) gene was mapped to a region downstream of an E. coli fruA gene homologue and shown to express a 21 kDa protein in Escherichia coli.
|
98. |
Hirono S,
Akagawa H,
Mitsui Y,
Iitaka Y,
( 1984 ) Crystal structure at 2.6 A resolution of the complex of subtilisin BPN' with streptomyces subtilisin inhibitor. PMID : 6387152 : DOI : 10.1016/0022-2836(84)90150-5 Abstract >>
The crystal structure of the complex of a bacterial alkaline serine proteinase, subtilisin BPN', with its proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was solved at 2.6 A resolution. Compared with other similar complexes involving serine proteinases of the trypsin family, the present structure is unique in several respects. (1) In addition to the usual antiparallel beta-sheet involving the P1, P2 and P3 residues of the inhibitor, the P4, P5 and P6 residues form an antiparallel beta-sheet with a previously unnoticed chain segment (residues 102 through 104, which was named the S4-6 site) of subtilisin BPN'. (2) The S4-6 site does not exist in serine proteinases of the trypsin family, whether of mammalian or microbial origin. (3) Global induced-fit movement seems to occur on SSI: a channel-like structure in SSI where hydrophobic side-chains are sandwiched between two lobes becomes about 2 A wider upon complexing with subtilisin. (4) The complex is most probably a Michaelis complex, as in most of the other complexes. (5) The main role of the "secondary contact region" of SSI seems to be to support the reactive site loop ("primary contact region"). Steric homology of the two contact regions between the inhibitors of the SSI family and the pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family is so high that it seems to indicate divergent evolutionary processes and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forward by Doolittle (1978).
|
99. |
Markland FS,
Smith EL,
( 1967 ) Subtilisin BPN. VII. Isolation of cyanogen bromide peptides and the complete amino acid sequence. PMID : 6065094 : Abstract >>
N/A
|
100. |
Chung H,
Friedberg F,
( 1980 ) Sequence of the N-terminal half of Bacillus amyloliquefaciens alpha-amylase. PMID : 6156671 : DOI : 10.1042/bj1850387 PMC : PMC1161365 Abstract >>
Bacillus amyloliquefaciens alpha-amylase (1,4-alpha-D-glucan glucanohydrolase. EC 3.2.1.1), which is commercially supplied as 'Bacillus subtilis alpha-amylase' does not cross-react immunologically with B. subtilis alpha-amylase. This enzyme (from B. amyloliquefaciens) was cleaved by treatment with CNBr into seven fragments. Peptide A was selected for sequence determination. It is the longest one, containing 185 amino acids (i.e. approx. 50% of the total molecule) and connects to the hexapeptide of the N-terminus. Its primary structure was aligned by use of various proteolytic enzymes. The sequence of amino acids 181-184 is identical with that of amino acids 14-17 of the alpha-amylase isolated from B. subtilis (except that amino acid 183 is asparagine rather than aspartic acid).
|
101. |
Vasantha N,
Thompson LD,
Rhodes C,
Banner C,
Nagle J,
Filpula D,
( 1984 ) Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein. PMID : 6090391 : PMC : PMC215730 Abstract >>
The genes for alkaline protease (apr[BamP]) and neutral protease (npr[BamP]) from Bacillus amyloliquefaciens have been isolated and expressed in Bacillus subtilis. The DNA sequences of apr[BamP] and npr[BamP] revealed, in each case, the presence of a large open reading frame. The inferred amino acid sequence of either gene contained a signal sequence and an additional polypeptide sequence ('pro' sequence) preceding the mature protein. Based on DNA sequence, the start point of translation has been identified as amino acid residue - 107 for apr[BamP] and -221 for npr[BamP]. To demonstrate that the start point of translation of apr[BamP] in vivo is probably at codon -107, codon -103 (AAA) was changed to an ochre (TAA) by site-directed mutagenesis. Alkaline protease was produced from this ochre mutant derivative of apr[BamP] only when the host strain was Su+. The presence of a pro sequence may be common to all of the secreted proteases from bacilli.
|
102. |
Takkinen K,
Pettersson RF,
Kalkkinen N,
Palva I,
Söderlund H,
Kääriäinen L,
( ) PMID : 6185474 : Abstract >>
We have isolated by molecular cloning the gene coding for the alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1.) from Bacillus amyloliquefaciens and determined its complete nucleotide sequence. The gene cloned in the plasmid pUB110 using Bacillus subtilis as a host, was contained in a 2.3-kilobase insert. Starting from an ATG initiator codon, an open reading frame comprising a total of 514 amino acids (1542 base pairs) was found within the cloned DNA fragment. The gene region encoding the COOH terminus of alpha-amylase was located by direct COOH-terminal analysis of the purified exoenzyme. The NH2-terminal portion of the gene encodes a 31 amino acid-long signal peptide (Palva, L., Pettersson, R. F., Kalkkinen, N., Lehtovaara, P., Sarvas, M., S?derlund, H., Takkinen, K., and K??ri?inen, L. (1981) Gene 15, 43-51). Since the signal peptide is correctly cleaved in the new host, as shown here by direct NH2-terminal sequence analysis, the exoamylase consists of 483 amino acid residues, corresponding to a molecular weight of 54,778. The reading frame used to deduce the amino acid sequence was found to be correct by comparison with partial amino acid sequence data published previously (Detera, S. D., and Friedberg, F. (1979) Int. J. Peptide Protein Res. 14, 364-372; Chung, H., and Friedberg, F. (1980) Biochem. J. 185, 387-395). Several differences between the sequence presented here and the partial ones published previously, however, were found. The nucleotide sequences both 5' and 3' to the alpha-amylase gene revealed palindromic structures including a stretch of six T-residues, suggesting transcription termination signals on both sides of the gene. Thus, it appears that alpha-amylase is translated from a monocistronic mRNA.
|
103. |
Wells JA,
Ferrari E,
Henner DJ,
Estell DA,
Chen EY,
( 1983 ) Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. PMID : 6316278 : DOI : 10.1093/nar/11.22.7911 PMC : PMC326549 Abstract >>
The subtilisin gene from B. amyloliquefaciens has been cloned and expressed under its own promoter on a high copy plasmid, pBS42, in Bacillus subtilis I-168 (Marburg strain). Greater than 95 percent of the expressed protease activity is secreted, and the activity is sensitive to inhibition by phenylmethylsulfonyl fluoride as expected for subtilisin. Bacillus subtilis transformants carrying the Bacillus amyloliquefaciens subtilisin gene in pBS42 (called pS4) secreted large amounts of a protein not seen in control pBS42 transformants. This protein migrated in SDS gels near the position of authentic subtilisin. The complete nucleotide sequence of the cloned gene has been determined using dideoxy sequencing methods. Ba131 exonuclease digestion studies at the 5' end of the gene have defined a 31 base pair stretch necessary for efficient expression of subtilisin. In addition to this putative promoter region, sequences have been assigned for ribosome binding, translation initiation, a signal peptide, the mature enzyme, and translation and transcription termination. A most interesting feature of the gene is a sequence of unknown function coding for roughly 75 amino acids between the signal sequence and the mature enzyme. It is proposed that this region serve as a pro-peptide as is commonly found in eukaryotic secreted proteases.
|
104. |
Alden RA,
Wright CS,
Kraut J,
( 1970 ) A hydrogen-bond network at the active site of subtilisin BPN'. PMID : 4399039 : DOI : 10.1098/rstb.1970.0014 Abstract >>
N/A
|
105. |
Detera SD,
Friedberg F,
( 1981 ) Sequence of the CNBr peptide containing the putative essential tyrosine of Bacillus amyloliquefaciens alpha amylase. PMID : 6164657 : Abstract >>
The amino acid sequence of the cyanogen bromide peptide (peptide B) containing the putative essential tyrosine residue in Bacillus amyloliquefaciens alpha-amylase (EC 3.2.1.1.) was determined. It is composed of 73 amino acids and the "active" tyrosine residue is the N-terminus of the peptide. Upon iodination of the whole enzyme by means of a lactoperoxidase-catalyzed reaction, a minimum of eight tyrosine residues are iodinated. Four of these belong to peptide B. Among the cyanogen bromide peptides, B is the most readily iodinated one. Hence, it is predicted that peptide B is an exposed segment of the amylase molecule.
|
106. |
Murai M,
Miyashita H,
Araki H,
Seki T,
Oshima Y,
( 1987 ) Molecular structure of the replication origin of a Bacillus amyloliquefaciens plasmid pFTB14. PMID : 3481020 : DOI : 10.1007/bf00337763 Abstract >>
The structure of a 1.5-kb DNA sequence that is necessary and sufficient for the replication of an 8.2-kb cryptic plasmid, pFTB14, isolated from a strain of Bacillus amyloliquefaciens has been characterized. The 1.5-kb DNA sequence contains an open reading frame, rep, stretching for 1017 bp, a promoter region for rep expression, and a possible replication origin for the plasmid upstream of the promoter. The rep product is trans-active and essential for plasmid replication. The predicted rep protein is a basic protein, as are the RepC protein of pT181, RepB of pUB110 and protein A of pC194 (all these found in staphylococci) and the pi protein of the R6K plasmid of Escherichia coli. The predicted rep protein has highly homologous amino acid sequences with protein A of pC194 and RepB of pUB110 throughout the protein molecule, but not with RepC of pT181, pi of R6K or protein RepH encoded by and initiating the replication of pC194.
|
107. |
Surova IA,
Ianonis VV,
Revina LP,
Levin ED,
Stepanov VM,
( 1988 ) [Primary structure of intracellular serine proteinase from Bacillus amyloliquefaciens. I. Isolation of the enzyme and amino acid sequence of peptides of tryptic hydrolysate]. PMID : 3190767 : Abstract >>
Method of isolation of intracellular serine protease was modified. Gramicidin S-sepharose CL-4B with a higher content of the ligand, synthesized through a modified procedure, was used as an affinity sorbent which simplified the purification and led to the pure enzyme with high specific activity and 90% yield. Trypsin hydrolyzate of the protease was separated by ion-exchange chromatography on a sulphocationite resin followed by paper chromatography and paper electrophoresis to yield twenty-five individual peptides. Their complete or partial sequences, corresponding in total to 146 amino acid residues, were determined by the manual Edman procedure.
|
108. |
Yang M,
Ferrari E,
Chen E,
Henner DJ,
( 1986 ) Identification of the pleiotropic sacQ gene of Bacillus subtilis. PMID : 3007431 : DOI : 10.1128/jb.166.1.113-119.1986 PMC : PMC214565 Abstract >>
The sacQ gene of Bacillus subtilis, a pleiotropic gene affecting the expression of a number of secreted gene products, has been identified as a small 46-amino-acid polypeptide. The increased expression of this polypeptide in strains carrying the sacQ36 allele, or in strains carrying the sacQ gene on a high copy plasmid, appears to be responsible for the phenotype of higher levels of proteases seen in these strains. A deletion of the sacQ gene had no apparent phenotype, indicating that it is not an essential gene.
|
109. |
Turner SM,
Mandelstam J,
( 1986 ) Cloning and sequencing of a gene from Bacillus amyloliquefaciens that complements mutations of the sporulation gene spoIID in Bacillus subtilis. PMID : 3114424 : DOI : 10.1099/00221287-132-11-3025 Abstract >>
A segment of DNA from Bacillus amyloliquefaciens, which complemented a mutant sporulation gene, spoIID68, in Bacillus subtilis, was cloned into a derivative of the temperate bacteriophage phi 105. The segment of DNA included an entire structural gene and complemented the mutation spoIID298, in addition to spoIID68, in B. subtilis. The nucleotide sequence of the gene from B. amyloliquefaciens was determined and compared with that of the B. subtilis gene; 74% homology was found in the coding region. Amino acid primary sequences derived from the nucleotide sequences of the two genes were also compared. The gene from B. amyloliquefaciens coded for a protein of 344 amino acid residues, one more than the protein coded by the corresponding gene from B. subtilis. Comparison of the primary amino acid sequences of the two genes showed that 78% of the residues were completely conserved and 8% were semi-conserved. Variation, however, was not random, i.e. some segments were much more highly conserved than others. Both proteins had a hydrophobic region at the N-terminus.
|
110. |
Hartley RW,
( 1988 ) Barnase and barstar. Expression of its cloned inhibitor permits expression of a cloned ribonuclease. PMID : 3050134 : DOI : 10.1016/0022-2836(88)90568-2 Abstract >>
Barnase is the extracellular ribonuclease of Bacillus amyloliquefaciens and barstar its specific intracellular inhibitor. The gene for barstar has now been cloned and sequenced. When the wild-type gene for barnase is reconstructed from its previously cloned parts on the same plasmid as the barstar gene, the lethal effect of its expression is suppressed. A plasmid has been devised which directs the secretion of 100 mg per active barnase liter by Escherichia coli and another which provides large (500 to 1000 mg/l) yields of barstar. The structure of these plasmids and the derived 89 amino acid sequence of barstar are reported.
|
111. |
Hofemeister J,
Kurtz A,
Borriss R,
Knowles J,
( 1986 ) The beta-glucanase gene from Bacillus amyloliquefaciens shows extensive homology with that of Bacillus subtilis. PMID : 3106158 : DOI : 10.1016/0378-1119(86)90278-7 Abstract >>
The nucleotide sequence of a 1583-bp DNA fragment containing gene bg1A for endo-beta-1,3-1,4-glucanase (EC 3.2.1.73) of Bacillus amyloliquefaciens strain BE20/78, a high producer of secreted enzymes, has been determined. The gene bg1A comprises an open reading frame (ORF) of 717 bp (= 239 codons) starting with ATG at 469 up to the translation stop codon TAA at 1188. Upstream from the translation initiation codon ATG, the ribosome-binding sequence 5'-AAAAAAGGGGG-3' and two putative bglA promoters have been identified. A box of eleven AT out of twelve base pairs (bp) precedes the -35 region of promoter P1. Beyond the translation stop codon UAA, a sequence of 69 bp can be folded into a hook-like stem-loop structure which probably functions as a transcriptional terminator. The ORF region of the gene bglA reveals about 90% homology with another beta-glucanase gene, bglS of Bacillus subtilis C120 sequenced by Murphy et al. (1984). Three regions of frequent amino acid (aa) changes are indicated. However, the major difference between these is a set of deletions within the non-coding region separating the bglA gene from an unknown preceding ORF and by one deletion shortening the proposed signal peptide by three aa (Pro-Tyr-Leu-). The putative transcription terminator of gene bglA completely lacks homology with a B. subtilis bglS gene. The signification of deletions erasing the 'sacR-homology region' in B. amyloliquefaciens, which have been detected in proximity of the beta-glucanase gene of B. subtilis by Steinmetz and Aymerich (1986), is discussed.
|
112. |
Yang J,
Gao MY,
Li M,
Li ZZ,
Li H,
Li HY,
( 2018 ) Bacillus amyloliquefaciens CotA degradation of the lignin model compound guaiacylglycerol-�]-guaiacyl ether. PMID : 30091245 : DOI : 10.1111/lam.13060 Abstract >>
The cotA gene from Bacillus amyloliquefaciens MN-13 was cloned and expressed in Escherichia coli Transetta. Nucleotide sequence analysis showed an open reading frame of 1542 bp encoding a polypeptide comprised of 513 amino acids. The degradation of lignin model compounds by recombinant CotA was investigated by HPLC-MS with guaiacylglycerol-�]-guaiacyl ether as the substrate. The compounds including guaiacol, 3-(4-hydro-3-methoxyphenyl)-3-oxo-propanol and 4-hydro-3-methoxy acetophenone detected by HPLC-MS verified the rupture of �]-O-4 bond and oxidation C�\ bond of guaiacylglycerol-�]-guaiacyl ether by CotA. 4-vinylguaiacol and 1-(4-hydroxy-3-methoxyl phenyl)-1-(2-methoxyl) phenoxyl ethylene were first time found in the degradation products of guaiacylglycerol-�]-guaiacyl ether. The appearance of 4-vinylguaiacol and 4-hydro-3-methoxy acetophenone confirmed the cleavage of C�]-C�^ bond. 1-(4-hydroxy-3-methoxyl phenyl)-2-(2-methoxyl) phenoxyl ethylene was coupled by the radical reaction of 4-vinylguaiacol with guaiacol. Otherwise, no corresponding degradation product was found to give a proof of cleavage of C�\-C�] bond in guaiacylglycerol-�]-guaiacyl ether by CotA.
|
113. |
Paddon CJ,
Hartley RW,
( 1985 ) Cloning, sequencing and transcription of an inactivated copy of Bacillus amyloliquefaciens extracellular ribonuclease (barnase). PMID : 3007290 : DOI : 10.1016/0378-1119(85)90045-9 Abstract >>
The gene for Bacillus amyloliquefaciens extracellular RNase (barnase) has been cloned in an inactive form in Escherichia coli following insertional mutagenesis by transposon Tn917. The nucleotide (nt) sequence of the gene was determined and the deduced amino acid (aa) sequence found to correspond almost precisely to the previously determined sequence. An open reading frame (ORF) of 72 codons precedes the mature sequence. The probable translation start site is 46 or 47 codons before the N-terminal alanine of the mature protein, 11 (or 14) bp from a putative ribosome-binding site (RBS). Within this leader sequence is a hydrophobic 15 aa core preceded by three basic residues which is characteristic of a secretory signal sequence. A pro-barnase protein with four extra aa at the N-terminus has been detected extracellularly indicating that the signal peptidase-cutting site lies before the mature protein. An inverted repeat that may act as a transcription terminator was found at the 3' end of the gene. The gene is maintained in E. coli with a short inverted repeat from the termini of Tn917 inserted into the coding sequence. Northern blot analysis of RNA from B. amyloliquefaciens shows an approx. 780-nt transcript produced during exponential and stationary growth phases. The inactive cloned gene produces an approx. 480-nt transcript in E. coli and two transcripts of approx. 480 and 780 nt in Bacillus subtilis.
|
114. |
( 2014 ) Bacillus amyloliquefaciens Q-426 as a potential biocontrol agent against Fusarium oxysporum f. sp. spinaciae. PMID : 23553741 : DOI : 10.1002/jobm.201200414 Abstract >>
In recent years, Bacillus species have received considerable attention for the biological control of many fungal diseases. In this study, Bacillus amyloliquefaciens Q-426 was tested for its potential use against a variety of plant pathogens. Our screen for genes involved in the biosynthesis of antifungal agents revealed that the fen and bmy gene clusters are present in the Q-426 genome. Lipopeptides such as bacillomycin D, fengycin A, and fengycin B were purified from the bacterial culture broth and subsequently identified by ESI-mass spectrometry. The minimal inhibitory concentration of fengycin A against Fusarium oxysporum f. sp. spinaciae W.C. Snyder & H.N. Hansen O-27 was determined to be 31.25 �gg ml(-1) . However, exposure of fungal cells to 50 �gg ml(-1) of fengycin A did not allow permeation of fluorescein diacetate into the cytoplasm through the cell membrane. Moreover, leakage of intracellular inorganic cations, nucleic acid and protein were also not detected, indicating that the fungal cell membrane is not the primary target of action for fengycin A. Profound morphological changes were observed in the F. oxysporum strain and spore germination was completely inhibited, suggesting that 50 �gg ml(-1) of fengycin A acts, at least, as a fungistatic agent.
|
115. |
Yang Y,
Li Y,
Gao T,
Zhang Y,
Wang Q,
( N/A ) C-di-GMP turnover influences motility and biofilm formation in Bacillus amyloliquefaciens PG12. PMID : 29859892 : DOI : 10.1016/j.resmic.2018.04.009 Abstract >>
Bis-(3'��5') cyclic dimeric guanosine monophosphate (c-di-GMP) is defined as a highly versatile secondary messenger in bacteria, coordinating diverse aspects of bacterial growth and behavior, including motility and biofilm formation. Bacillus amyloliquefaciens PG12 is an effective biocontrol agent against apple ring rot caused by Botryosphaeria dothidea. In this study, we characterized the core regulators of c-di-GMP turnover in B. amyloliquefaciens PG12. Using bioinformatic analysis, heterologous expression and biochemical characterization of knockout and overexpression derivatives, we identified and characterized two active diguanylate cyclases (which catalyze c-di-GMP biosynthesis), YhcK and YtrP and one active c-di-GMP phosphodiesterase (which degrades c-di-GMP), YuxH. Furthermore, we showed that elevating c-di-GMP levels up to a certain threshold inhibited the swimming motility of B. amyloliquefaciens PG12. Although yhcK, ytrP and yuxH knockout mutants did not display defects in biofilm formation, significant increases in c-di-GMP levels induced by YtrP or YuxH overexpression stimulated biofilm formation in B. amyloliquefaciens PG12. Our results indicate that B. amyloliquefaciens possesses a functional c-di-GMP signaling system that influences the bacterium's motility and ability to form biofilms. Since motility and biofilm formation influence the efficacy of biological control agent, our work provides a basis for engineering a more effective strain of B. amyloliquefaciens PG12.
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Mendis HC,
Thomas VP,
Schwientek P,
Salamzade R,
Chien JT,
Waidyarathne P,
Kloepper J,
De La Fuente L,
( 2018 ) Strain-specific quantification of root colonization by plant growth promoting rhizobacteria Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 in non-sterile soil and field conditions. PMID : 29447287 : DOI : 10.1371/journal.pone.0193119 PMC : PMC5814090 Abstract >>
Bacillus amyloliquefaciens QST713 and B. firmus I-1582 are bacterial strains which are used as active ingredients of commercially-available soil application and seed treatment products Serenade? and VOTiVO?, respectively. These bacteria colonize plant roots promoting plant growth and offering protection against pathogens/pests. The objective of this study was to develop a qPCR protocol to quantitate the dynamics of root colonization by these two strains under field conditions. Primers and TaqMan? probes were designed based on genome comparisons of the two strains with publicly-available and unpublished bacterial genomes of the same species. An optimized qPCR protocol was developed to quantify bacterial colonization of corn roots after seed treatment. Treated corn seeds were planted in non-sterile soil in the greenhouse and grown for 28 days. Specific detection of bacteria was quantified weekly, and showed stable colonization between ~104-105 CFU/g during the experimental period for both bacteria, and the protocol detected as low as 103 CFU/g bacteria on roots. In a separate experiment, streptomycin-resistant QST713 and rifampicin-resistant I-1582 strains were used to compare dilution-plating on TSA with the newly developed qPCR method. Results also indicated that the presence of natural microflora and another inoculated strain does not affect root colonization of either one of these strains. The same qPCR protocol was used to quantitate root colonization by QST713 and I-1582 in two corn and two soybean varieties grown in the field. Both bacteria were quantitated up to two weeks after seeds were planted in the field and there were no significant differences in root colonization in either bacteria strain among varieties. Results presented here confirm that the developed qPCR protocol can be successfully used to understand dynamics of root colonization by these bacteria in plants growing in growth chamber, greenhouse and the field.
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117. |
( 2013 ) Isolation of the putative biosynthetic gene cluster of 1-deoxynojirimycin by Bacillus amyloliquefaciens 140N, its production and application to the fermentation of soybean paste. PMID : 23391926 : DOI : 10.1271/bbb.120753 Abstract >>
The 1-deoxynojirimycin (DNJ) biosynthetic gene cluster of Bacillus amyloliquefaciens 140N isolated from traditional Korean fermented food was isolated by PCR screening. It showed 78.9% inhibitory activity against �\-glucosidase and produced 0.8 g/L of DNJ in an optimized medium containing 2% soluble starch, 1% tryptone, 0.05% KH(2)PO(4), and 0.05% (NH(2))(4)SO(4). Soybean paste fermented with B. amyloliquefaciens 140N produced DNJ with 84.4% inhibitory activity.
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118. |
( 2013 ) Control of postharvest soft rot caused by Erwinia carotovora of vegetables by a strain of Bacillus amyloliquefaciens and its potential modes of action. PMID : 23117674 : DOI : 10.1007/s11274-012-1193-0 Abstract >>
Erwinia carotovora subsp. carotovora (Ecc), the causal agent of bacterial soft rot, is one of the destructive pathogens of postharvest vegetables. In this study, a bacterial isolate (BGP20) from the vegetable farm soil showed strong antagonistic activity against Ecc in vitro, and its twofold cell-free culture filtrate showed excellent biocontrol effect in controlling the postharvest bacterial soft rot of potatoes at 25 �XC. The anti-Ecc metabolites produced by the isolate BGP20 had a high resistance to high temperature, UV-light and protease K. Based on the colonial morphology, cellular morphology, sporulation, and partial nucleotide sequences of 16S rRNA and gyrB gene, the isolate BGP20 was identified as Bacillus amyloliquefaciens subsp. plantarum. Further in vivo assays showed that the BGP20 cell culture was more effective in controlling the postharvest bacterial soft rot of green peppers and Chinese cabbages than its twofold cell-free culture filtrate. In contrast, the biocontrol effect and safety of the BGP20 cell culture were very poor on potatoes. In the wounds of potatoes treated with both the antagonist BGP20 and the pathogen Ecc, the viable count of Ecc was 31,746 times that of BGP20 at 48 h of incubation at 25 �XC. But in the wounds of green peppers, the viable count of BGP20 increased 182.3 times within 48 h, and that of Ecc increased only 51.3 %. In addition, the treatment with both BGP20 and Ecc induced higher activity of phenylalanine ammonia-lyase (PAL) than others in potatoes. But the same treatment did not induce an increase of PAL activity in green peppers. In conclusion, the present study demonstrated that the isolate BGP20 is a promising candidate in biological control of postharvest bacterial soft rot of vegetables, but its main mode of action is different among various vegetables.
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119. |
( 2013 ) Bacillus amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for the production of Bikalga, an alkaline fermented food. PMID : 23565829 : DOI : 10.1111/jam.12214 Abstract >>
To identify and screen dominant Bacillus spp. strains isolated from Bikalga, fermented seeds of Hibiscus sabdariffa for their antimicrobial activities in brain heart infusion (BHI) medium and in a H. sabdariffa seed-based medium. Further, to characterize the antimicrobial substances produced. The strains were identified by gyrB gene sequencing and phenotypic tests as B. amyloliquefaciens ssp. plantarum. Their antimicrobial activity was determined by the agar spot and well assay, being inhibitory to a wide range of Gram-positive and Gram-negative pathogenic bacteria and fungi. Antimicrobial activity against Bacillus cereus was produced in H. sabdariffa seed-based medium. PCR results revealed that the isolates have potential for the lipopeptides iturin, fengycin, surfactin, the polyketides difficidin, macrolactin, bacillaene and the dipeptide bacilysin production. Ultra-high-performance liquid chromatography-time of flight mass spectrometry analysis of antimicrobial substance produced in BHI broth allowed identification of iturin, fengycin and surfactin. The Bacillus amyloliquefaciens ssp. plantarum exhibited broad-spectrum antifungal and antibacterial properties. They produced several lipopeptide antibiotics and showed good potential for biological control of Bikalga. Pathogenic bacteria often occur in spontaneous food fermentations. This is the first report to identify indigenous B. amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for safeguarding Bikalga.
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120. |
( 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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121. |
( 2013 ) Enhanced root colonization and biocontrol activity of Bacillus amyloliquefaciens SQR9 by abrB gene disruption. PMID : 23196984 : DOI : 10.1007/s00253-012-4572-4 Abstract >>
Root colonization by antagonistic bacteria is a prerequisite for successful biological control, and the instability of colonization under varying environmental conditions has accentuated the need to improve the colonization activity. Root colonization by Bacillus spp. is mainly determined by chemotaxis and biofilm formation, and both functions are negatively controlled by the global transcription regulator AbrB. Here, we disrupted the gene abrB in Bacillus amyloliquefaciens SQR9, which has been proven to be a promising biocontrol agent of cucumber and watermelon wilt disease. Chemotaxis, biofilm formation, and colonization activities as well as biocontrol efficiency were measured and compared between the wild-type strain of SQR9 and the abrB mutant. The data presented in this article demonstrate that the colonization and biocontrol activity of B. amyloliquefaciens SQR9 could be significantly improved by abrB gene disruption. The results offer a new strategy to enhance the biocontrol efficacy of B. amyloliquefaciens SQR9.
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122. |
( 2013 ) Development of a specific real-time PCR assay targeting the poly-�^-glutamic acid synthesis gene, pgsB, for the quantification of Bacillus amyloliquefaciens in solid-state fermentation. PMID : 23266849 : DOI : 10.1016/j.biortech.2012.11.092 Abstract >>
A TaqMan real-time PCR procedure was developed for specific detection and quantification of strains belonging to Bacillus amyloliquefaciens group. The primer pair pgsB726-f/pgsB791-r and the pgsB-probe were designed from one of the poly-�^-glutamic acid synthesis gene (pgsB) of B. amyloliquefaciens. The detection limit was approximately between 10(2)-10(3) cells/mL. A linear correlation between the log10 input pMD-pgsB plasmid DNA copies and the threshold cycle values were observed with a magnitude of linearity in the range of 9.415��10(3)-10(7) copies/mL for the standard curve, which exhibited a slope of -3.35 and an R2 value of 99.8%. Results of validation of this method with artificially contaminated and natural solid-state fermentation samples showed that it was suitable for specific and sensitive detection and quantification for the target strains in solid-state fermentation samples. This could be more useful to understand the fermentation starting strain and the final microbiological properties of fermentation products.
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( 1998 ) The role of metals in catalysis by the restriction endonuclease BamHI. PMID : 9783752 : DOI : 10.1038/2352 Abstract >>
Type II restriction enzymes are characterized by their remarkable specificity and simplicity. They require only divalent metals (such as Mg2+ or Mn2+) as cofactors to catalyze the hydrolysis of DNA. However, most of the structural work on endonucleases has been performed in the absence of metals, leaving unanswered questions about their mechanisms of DNA cleavage. Here we report structures of the endonuclease BamHI-DNA complex, determined in the presence of Mn2+ and Ca2+, that describe the enzyme at different stages of catalysis. Overall, the results support a two-metal mechanism of DNA cleavage for BamHI which is distinct from that of EcoRV.
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124. |
( 1998 ) Three-dimensional structure of binase in solution. PMID : 9708913 : DOI : 10.1016/s0014-5793(98)00765-0 Abstract >>
We present the spatial structure of binase, a small extracellular ribonuclease, derived from 1H-NMR* data in aqueous solution. The total of 20 structures were obtained via torsion angle dynamics using DYANA program with experimental NOE and hydrogen bond distance constraints and phi and chi1 dihedral angle constraints. The final structures were energy minimised with ECEPP/2 potential in FANTOM program. Binase consists of three alpha-helices in N-terminal part (residues 6-16, 26-32 and 41-44), five-stranded antiparallel beta-sheet in C-terminal part (residues 51-55, 70-75, 86-90, 94-99 and 104-108) and two-stranded parallel beta-sheet (residues 22-24 and 49-51). Three loops (residues 36-39, 56-67 and 76-83), which play significant role in biological functioning of binase, are flexible in solution. The differences between binase and barnase spatial structures in solution explain the differences in thermostability of binase, barnase and their hybrids.
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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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126. |
( 2013 ) Isolation and characterization of antagonistic Bacillus strains capable to degrade ethylenethiourea. PMID : 23143288 : DOI : 10.1007/s00284-012-0263-8 Abstract >>
In this study, more than 150 bacteria showing antagonistic properties against bacterial and fungal pathogens of the tomato plant were isolated and characterized. The most efficient agents against these phytopathogenic microorganisms belong to the genus Bacillus: the best biocontrol isolates were representatives of Bacillus subtilis, B. mojavensis and B. amyloliquefaciens species. They intensively produced fengycin or/and surfactin depsipeptide antibiotics and also proved to be excellent protease secretors. It was proved, that the selected strains were able to use ethylenethiourea (ETU) as sole nitrogen source. These antagonistic and ETU-degrading Bacillus strains can be applied as biocontrol and also as bioremediation agents.
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127. |
( 1998 ) Replication terminator protein-based replication fork-arrest systems in various Bacillus species. PMID : 9642188 : PMC : PMC107290 Abstract >>
The replication terminator protein (RTP) of Bacillus subtilis interacts with its cognate DNA terminators to cause replication fork arrest, thereby ensuring that the forks approaching one another at the conclusion of a round of replication meet within a restricted terminus region. A similar situation exists in Escherichia coli, but it appears that the fork-arrest systems in these two organisms have evolved independently of one another. In the present work, RTP homologs in four species closely related to B. subtilis (B. atrophaeus, B. amyloliquefaciens, B. mojavensis, and B. vallismortis) have been identified and characterized. An RTP homolog could not be identified in another closely related species, B. licheniformis. The nucleotide and amino acid changes from B. subtilis among the four homologs are consistent with the recently established phylogenetic tree for these species. The GC contents of the rtp genes raise the possibility that these organisms arose within this branch of the tree by horizontal transfer into a common ancestor after their divergence from B. licheniformis. Only 5 amino acid residue positions were changed among the four homologs, despite an up to 17.2% change in the nucleotide sequence, a finding that highlights the importance of the precise folded structure to the functioning of RTP. The absence of any significant change in the proposed DNA-binding region of RTP emphasizes the importance of its high affinity for the DNA terminator in its functioning. By coincidence, the single change (E30K) found in the B. mojavensis RTP corresponds exactly to that purposefully introduced by others into B. subtilis RTP to implicate a crucial role for E30 in the fork-arrest mechanism. The natural occurrence of this variant is difficult to reconcile with such an implication, and it was shown directly that RTP.E30K functions normally in fork arrest in B. subtilis in vivo. Additional DNA terminators were identified in the new RTP homolog-containing strains, allowing the definition of a Bacillus terminator consensus and identification of two more terminators in the B. subtilis 168 genome sequence to bring the total to nine.
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( 1998 ) Genes encoding thymidylate synthases A and B in the genus Bacillus are members of two distinct families. PMID : 9648749 : DOI : 10.1007/pl00008625 Abstract >>
Bacillus subtilis strain 168 is known to possess two genes that encode thymidylate synthases, thyA and thyB. We have identified genes similar to the thyA and thyB genes in several Bacillus strains by Southern hybridization and by DNA amplification with sequence-specific primers. Analysis of thyA genes cloned from B. subtilis W23 strain 2A6, B. subtilis ATCC6633, B. amyloliquefaciens S18 and B. atrophaeus S223 reveals that they are very similar to the thyA genes from B. subtilis 168 and its phage phi3T, but differ considerably from the majority of known prokaryotic and eukaryotic thymidylate synthases.
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129. |
( 1998 ) Recognition of RNase Sa by the inhibitor barstar: structure of the complex at 1.7 A resolution. PMID : 9757110 : DOI : 10.1107/s0907444998004429 Abstract >>
We report the 1.7 A resolution structure of RNase Sa complexed with the polypeptide inhibitor barstar. The crystals are in the hexagonal space group P65 with unit-cell dimensions a = b = 56.9, c = 135.8 A and the asymmetric unit contains one molecule of the complex. RNase Sa is an extracellular microbial ribonuclease produced by Streptomyces aureofaciens. Barstar is the natural inhibitor of barnase, the ribonuclease of Bacillus amyloliquefaciens. It inhibits RNase Sa and barnase in a similar manner by steric blocking of the active site. The structure of RNase Sa is very similar to that observed in crystals of the native enzyme and its complexes with nucleotides. Barstar retains the structure found in its complex with barnase. The accessible surface area of protein buried in the complex is about 300 A2 smaller and there are fewer hydrogen bonds in the enzyme-inhibitor interface in RNase Sa-barstar than in barnase-barstar, providing an explanation of the reduced binding affinity in the former. Previous studies of barstar complexes have used mutants of the inhibitor and this is the first structure which includes wild-type barstar.
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