| 1. |
Grundy FJ,
Henkin TM,
( 1992 ) Characterization of the Bacillus subtilis rpsD regulatory target site. PMID : 1400226 : DOI : 10.1128/jb.174.21.6763-6770.1992 PMC : PMC207351 Abstract >>
The Bacillus subtilis rpsD gene, which encodes ribosomal protein S4, is subject to autogenous regulation. Repression of rpsD expression by excess S4 protein was previously shown to be affected by mutations in the leader region of the gene. A large number of deletion and point mutations in the leader region were generated, and their effect on repression by S4 in vivo was tested. These studies indicated that the required region was within positions +30 to +190 relative to the transcription start point. Replacement of the rpsD promoter with a lac promoter derivative which is expressed in B. subtilis had no effect, indicating that repression by S4 occurs at a level subsequent to transcription initiation. The rpsD leader region was isolated from several Bacillus species. Members of the B. subtilis group, as defined by analysis of 16S rRNA sequence, contained a leader region target site very closely related in structure to that of B. subtilis, despite considerable primary sequence variation; the B. brevis rpsD leader contained some but not all of the structural features found in the regulatory target sites of the other Bacillus species. Very little similarity to the Escherichia coli alpha operon S4 target site was found at either the primary-sequence or the secondary-structure level. Mutagenic and phylogenetic data indicate that the secondary structure of the leader region regulatory target site contains two large stem-loop domains. The first of these helices has a side loop which is essential for autoregulation, is highly conserved among Bacillus rpsD genes, and is similar to a region of 16S rRNA important in S4 binding.
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2. |
Ren B,
Tibbelin G,
Kajino T,
Asami O,
Ladenstein R,
( 2003 ) The multi-layered structure of Dps with a novel di-nuclear ferroxidase center. PMID : 12767829 : DOI : 10.1016/s0022-2836(03)00466-2 Abstract >>
The crystallization of cellular components represents a unique survival strategy for bacterial cells under stressed conditions. A highly ordered, layered structure is often formed in such a process, which may involve one or more than one type of bio-macromolecules. The main advantage of biocrystallization has been attributed to the fact that it is a physical process and thus is independent of energy consumption. Dps is a protein that crystallizes to form a multi-layered structure in starved cells in order to protect DNA against oxidative damage and other detrimental factors. The multi-layered crystal structure of a Dps protein from Bacillus brevis has been revealed for the first time at atomic resolution in the absence of DNA. Inspection of the structure provides the first direct evidence for the existence of a di-nuclear ferroxidase center, which possesses unique features among all the di-iron proteins identified so far. It constitutes the structural basis for the ferroxidase activity of Dps in the crystalline state as well as in solution. This finding proves that the enzymatic process of detoxification of metal ions, which may cause severe oxidative damage to DNA, is the other important aspect of the defense mechanism performed by Dps. In the multi-layered structure, Dps dodecamers are organized in a highly ordered manner. They adopt the classic form of hexagonal packing in each layer of the structure. Such arrangement results in reinforced structural features that would facilitate the attraction and absorption of metal ions from the environment. The highly ordered layered structure may provide an ideal basis for the accommodation of DNA between the layers so that it can be isolated and protected from harmful factors under stress conditions.
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3. |
Hanagata H,
Ebisu S,
Koh M,
( 2000 ) Use of Bacillus brevis for synthesis and secretion of Des-B30 single-chain human insulin precursor. PMID : 10879487 : DOI : 10.1271/bbb.64.1079 Abstract >>
A synthetic gene encoding a single chain human insulin precursor [B-chain (1-29)-A-chain] linked to the C-terminal lysine of human epidermal growth factor (1-28) (EGF-SCI) was constructed. This gene was expressed using Bacillus brevis. EGF-SCI was isolated from the supernatant of the culture broth. Treatment of EGF-SCI with lysyl endopeptidase resulted in the formation of des-B30 human insulin. The identification of the formed des-B30 human insulin was made by the measurement of molecular weight and amino acid analysis. The binding coefficient to anti-human insulin antibody was comparable to that of human insulin.
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4. |
Johansson P,
( 1999 ) Organization of genes for tetrapyrrole biosynthesis in gram--positive bacteria. PMID : 10217486 : DOI : 10.1099/13500872-145-3-529 Abstract >>
Clusters of genes encoding enzymes for tetrapyrrole biosynthesis were cloned from Bacillus sphaericus, Bacillus stearothermophilus, Brevibacillus brevis and Paenibacillus macerans. The sequences of all hemX genes found, and of a 6.3 kbp hem gene cluster from P. macerans, were determined. The structure of the hem gene clusters was compared to that of other Gram-positive bacteria. The Bacillus and Brevibacillus species have a conserved organization of the genes hemAXCDBL, required for biosynthesis of uroporphyrinogen III (UroIII) from glutamyl-tRNA. In P. macerans, the hem genes for UroIII synthesis are also closely linked but their organization is different: there is no hemX gene and the gene cluster also contains genes, cysG8 and cysG(A)-hemD, encoding the enzymes required for synthesis of sirohaem from UroIII. Bacillus subtilis contains genes for three proteins, NasF, YInD and YInF, with sequence similarity to Escherichia coli CysG, which is a multi-functional protein catalysing sirohaem synthesis from UroIII. It is shown that YInF is required for sirohaem synthesis and probably catalyses the precorrin-2 to sirohaem conversion. YInD probably catalyses precorrin-2 synthesis from UroIII and NasF seems to be specific for nitrite reduction.
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5. |
Zhang XZ,
Yan X,
Cui ZL,
Li SP,
( 2006 ) [Construction of the shuttle expression-secretion vector with the promoters and signal peptide-encoding sequence from Brevibacillus brevis]. PMID : 17037048 : Abstract >>
The multiple and tandem promoters and signal peptide-encoding sequence of cell wall protein encoding gene was amplified from Brevibacillus brevis B15 total DNA, the PCR fragment was cloned, sequenced and analyzed, then was submitted to GenBank with a Accession No. AY956423. Another pair of primers were designed to amplify the fragment again, BamHI and Pstl sites was introduced flanking the PCR production. BamHI/Pstl digested fragment was cloned into the corresponding site of shuttle vector pP43NMK to generate the expression-secretion vector pP15MK. The inserted fragment was upstream of mpd gene and the signal peptide-encoding sequence was fused in frame with the mpd gene, which its own signal peptide-encoding sequence was deleted. The recombinant vector was transformed into Bacillus subtilis 1A751, under the control of the promoters and signal peptide from Brevibacillus brevis B15, mpd gene was continuously expressed and secreted at a high efficiency throughout the exponential growth phase and into the late stationary phase, the expression production methyl parathion hydrolase (MPH) was attached on the outside of the cell membrane. MPH activity accumulated to a maximum level of 7.79 U/mL after 48 h of cultivation at the late stationary phase, which was 8.1-fold higher than the expression level of the original Plesiomonas strain M6.
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6. |
Heiter DF,
Lunnen KD,
Wilson GG,
( 2005 ) Site-specific DNA-nicking mutants of the heterodimeric restriction endonuclease R.BbvCI. PMID : 15826660 : DOI : 10.1016/j.jmb.2005.02.034 Abstract >>
The restriction enzyme R.BbvCI cleaves duplex DNA within a seven base-pair asymmetric recognition sequence, thus: CCTCAGC/GCTGAGG-->CC--TCAGC/GC--TGAGG. We show that R.BbvCI comprises two different subunits, R(1) and R(2); that each subunit contains a catalytic site for DNA strand hydrolysis; and that these sites act independently and strand-specifically. In turn, each catalytic site was inactivated by mutagenesis to form dimeric enzymes in which only one site remained functional. The altered enzymes hydrolyzed just one strand of the recognition sequence, nicking the DNA rather than cleaving it. Enzymes in which the catalytic site in the R(1) subunit remained functional nicked the bottom strand of the sequence, producing CCTCAGC/GC--TGAGG, while those in which the catalytic site in the R(2) subunit remained functional nicked the top strand, producing CC--TCAGC/GCTGAGG. These DNA-nicking enzymes could prove useful for investigation of DNA repair, recombination, and replication, and for laboratory procedures that initiate from nicks, such as DNA degradation, synthesis, and amplification.
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7. |
Kaĭdalova NV,
Akimkina TV,
Khodova OD,
Kostrov SV,
Strongin AIa,
( N/A ) [Analysis of the structure of Bacillus brevis neutral proteinase and its biosynthesis in Bacillus subtilis cells]. PMID : 2127074 : Abstract >>
Amino acid sequence of neutral metalloprotease from Bac. brevis has been compared with that of Bac. amyloloquefaciens, Bac. cereus, Bac. subtilis, Bac. stearothermophilis, Bac. thermoproteolyticus (thermolysine). A sequence region from N-40 to N-1 with a significant degree of homology allowed to predict the processing site of the propart of Bac. brevis enzyme. The sequence comparison allows to put Bac. brevis enzyme within the evolutionary branch of enzymes, which includes thermolysin and proteases of Bac. cereus and Bac. stearothermophilus. Using automated Edman degradation the N-terminal sequence of Bac. brevis protease has been determined. It does not differ from the sequence predicted from the nucleotide sequence of the gene. It was shown that, when Bac. brevis gene coding for thermostable protease is expressed on a plasmid vector in Bac. subtilis cells at 37 degrees C, enzyme forms possessing low activity are secreted. The enzyme may be significantly activated without an additional cleavage or processing and the activation includes numerous conformation transition states of the protein molecule.
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8. |
Hori K,
Yamamoto Y,
Tokita K,
Saito F,
Kurotsu T,
Kanda M,
Okamura K,
Furuyama J,
Saito Y,
( 1991 ) The nucleotide sequence for a proline-activating domain of gramicidin S synthetase 2 gene from Bacillus brevis. PMID : 1939016 : DOI : 10.1093/oxfordjournals.jbchem.a123528 Abstract >>
A fragment encoding proline-activating domain (grs 2-pro) of gramicidin S synthetase 2 (GS 2) was found in an 8.1-kilobase pairs (kb) DNA fragment of Bacillus brevis Nagano, which contained the full length of GS 1 gene (grs 1). The clones designated GS719 and GS708, which expressed gramicidin S synthetase 1, were elucidated to express immunoreactive proteins to GS 2 antibodies with approximate molecular weights of 115,000, 105,000 (GS719), and 110,000 (GS708). The partial purification of the gene products of these clones was carried out using DEAE-Sepharose CL-6B column chromatography. The immunoreactive proteins to GS 2 antibodies were separated from gramicidin S synthetase 1 protein and had specific proline-dependent ATP-32PPi exchange activity. The nucleotide sequence for the proline-activating domain in the 8.1-kb insert was determined. This fragment was 2,879 base pairs long, and encoded 959 amino acids. The calculated molecular weight of 111,671 was consistent with the apparent molecular weight of 115,000 found in SDS-PAGE of the immunoreactive products to GS 2 antibodies. The open reading frame for this protein followed grs 1 gene, though two were separated by a 73-base pair noncoding sequence, and remained open to the end.(ABSTRACT TRUNCATED AT 250 WORDS)
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9. |
Hu XF,
Li SX,
Wu JG,
Wang JF,
Fang QL,
Chen JS,
( 2010 ) Transfer of Bacillus mucilaginosus and Bacillus edaphicus to the genus Paenibacillus as Paenibacillus mucilaginosus comb. nov. and Paenibacillus edaphicus comb. nov. PMID : 19643872 : DOI : 10.1099/ijs.0.008532-0 Abstract >>
Bacillus mucilaginosus and Bacillus edaphicus were reclassified based on their 16S rRNA and gyrB gene sequences, DNA-DNA hybridization, fatty acid methyl esters and other taxonomic characteristics. Phylogenetic analysis based on 16S rRNA and gyrB gene sequences indicated that strains of B. mucilaginosus and B. edaphicus were members of the genus Paenibacillus, with over 90.4 % and 70.3 % sequence similarity, respectively. Their DNA G+C contents were 54.5-56.8 mol%. The DNA-DNA relatedness values of B. edaphicus VKPM B-7517(T) with B. mucilaginosus KNP414 and B. mucilaginosus CGMCC 1.236 were 89.2 % and 88.7 %, respectively. The major isoprenoid quinone of B. mucilaginosus and B. edaphicus was MK-7 (94.1-95.7 %). The peptidoglycan type was A1gamma (meso-diaminopimelic acid) and the major polar lipids were phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids were anteiso-C(15 : 0), C(16 : 1)omega11c and C(16 : 0). Phenotypic features and fatty acid profiles supported the similarity of B. mucilaginosus and B. edaphicus to Paenibacillus validus CCTCC 95016(T) and confirmed their relationship with members of the genus Paenibacillus. Therefore, it is proposed that Bacillus mucilaginosus and Bacillus edaphicus be transferred to the genus Paenibacillus as Paenibacillus mucilaginosus comb. nov. (type strain HSCC 1605(T)=VKPM B-7519(T)=VKM B-1480D(T)=CIP 105815(T)=KCTC 3870(T)) and Paenibacillus edaphicus comb. nov. (type strain VKPM B-7517(T)=DSM 12974(T)=CIP 105814(T)), respectively.
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10. |
Moreno B,
Vivas A,
Nogales R,
Benitez E,
( 2009 ) Solvent tolerance acquired by Brevibacillus brevis during an olive-waste vermicomposting process. PMID : 19665790 : DOI : 10.1016/j.ecoenv.2009.06.011 Abstract >>
In this work, a cultivable, Gram-positive, solvent-resistant bacterium was isolated from vermicomposted olive wastes (VOW). The highest 16S rRNA sequence similarity (99%) was found in Brevibacillus brevis. The genome of the isolate, selected for trichloroethylene (TCE)-tolerance, contained a nucleotide sequence encoding a conserved protein domain (ACR_tran) ascribable to the HAE1-RND family. Members of this family are hydrophobic/amphiphilic efflux pumps largely restricted to Gram-negative bacteria. No DNA sequences of HAE1 transporters were detected in the genome of a reference B. brevis strain isolated from natural soil. Since no cultivable solvent-tolerant bacterium was detected in the unvermicomposted olive waste, a transfer of solvent-resistance genes from Gram-negative bacteria during the vermicomposting process could explain the presence of HAE1 transporters in B. brevis isolated from the vermicompost. Under TCE stress conditions, the acquired nucleotide sequence could be translated into proteins, and the tolerance to solvents is conferred to the bacterium. The isolate was designated as strain BEA1 (EF079071).
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11. |
Kurotsu T,
Hori K,
Kanda M,
Saito Y,
( 1991 ) Characterization and location of the L-proline activating fragment from the multifunctional gramicidin S synthetase 2. PMID : 1917901 : DOI : 10.1093/oxfordjournals.jbchem.a123454 Abstract >>
Gramicidin S synthetase 2 (GS2) derived from Bacillus brevis is a multifunctional single polypeptide (Mr 280,000) with a 4'-phosphopantetheine residue covalently bound to the enzyme. When GS2 was treated with trypsin or chymotrypsin, fragments with some activity were liberated. The molecular mass of the L-proline activating fragment was 114 kDa on SDS-PAGE. This fragment, when incubated with gramicidin S synthetase 1 (GS1) in the presence of phenylalanine and proline, produced D-Phe-L-Pro dipeptide. The fragment accepted D-phenylalanine from GS1 in the absence of L-proline. The L-proline activating fragment was shown to lack pantothenic acid by microbiological assay. On the other hand, the L-leucine activating fragment, which was partially purified, contained a large amount of pantothenic acid, although it did not form the D-Phe-L-Pro dipeptide. These results indicate that the L-proline activating site is located near an acceptor site for D-phenylalanine on GS2, but that it is not adjacent to a 4'-phosphopantetheine group. The N-terminal sequence (15 amino acid residues) of the L-proline activating fragment obtained by trypsin treatment was identical with that of GS2, indicating that the L-proline activating site is located at the N-terminus of the native synthetase. The N-terminal sequence of GS2 has been matched with the amino acid sequence deduced from the nucleotide sequence 71 bp downstream of the stop codon of the GS1 gene except that the first initiator methionine was not detected.
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12. |
Mittenhuber G,
Weckermann R,
Marahiel MA,
( 1989 ) Gene cluster containing the genes for tyrocidine synthetases 1 and 2 from Bacillus brevis: evidence for an operon. PMID : 2768192 : DOI : 10.1128/jb.171.9.4881-4887.1989 PMC : PMC210293 Abstract >>
From a genomic library of the tyrocidine producer Bacillus brevis ATCC 8185 constructed in the bacteriophage vector EMBL3, a recombinant phage which contains the structural genes coding for tyrocidine synthetases 1 and 2, TycA and TycB, was identified. The location of the tycA gene within the 16-kilobase insert of this clone, EMBL25-1, was mapped by hybridization studies by using the previously isolated tycA DNA as a probe. Restriction analyses, the construction of subclones, and the analysis of proteins encoded by the subclones located the tycB gene at the 3' end of the tycA gene and revealed that the two genes are transcribed in the same direction. Nuclease S1 protection studies and DNA sequencing studies of the intergenic region indicated that tycA and tycB are separated by a 94-base-pair noncoding region and suggested that these genes are organized as an operon.
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13. |
Hori K,
Yamamoto Y,
Minetoki T,
Kurotsu T,
Kanda M,
Miura S,
Okamura K,
Furuyama J,
Saito Y,
( 1989 ) Molecular cloning and nucleotide sequence of the gramicidin S synthetase 1 gene. PMID : 2691508 : DOI : 10.1093/oxfordjournals.jbchem.a122909 Abstract >>
The entire gene for gramicidin S synthetase 1 (GS 1) was cloned into the plasmid vector pUC18, and the nucleotide sequences of the GS 1 gene and its flanking region were determined. The full-length clone was 4,539 base pairs long and had an open reading frame of 3,294 nucleotides coding for 1,098 amino acids. The calculated molecular weight of 123,474 agreed with the apparent molecular weight of 120,000 found in SDS-PAGE of GS 1 from B. brevis. The nucleotide sequence of GS 1 gene was highly homologous to that of tyrocidine synthetase 1. The overall similarity between the deduced amino acid sequences of the two genes was 57.5%. The gene product of clone GS309 was easily purified to an essentially homogeneous state by ammonium sulfate fractionation followed by DEAE-Sepharose CL-6B, Ultrogel AcA-34, and second DEAE-Sepharose CL-6B column chromatography. The purified protein catalyzed the D-phenylalanine-dependent ATP-32PPi exchange reaction which is specific for GS 1 activity, and the specific activity of the purified product was nearly the same as the purified GS 1 from B. brevis. The product also showed a weak phenylalanine racemase activity.
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14. |
Hou Q,
Wang C,
Hou X,
Xia Z,
Ye J,
Liu K,
Liu H,
Wang J,
Guo H,
Yu X,
Yang Y,
Du B,
Ding Y,
( 2015 ) Draft Genome Sequence of Brevibacillus brevis DZQ7, a Plant Growth-Promoting Rhizobacterium with Broad-Spectrum Antimicrobial Activity. PMID : 26294619 : DOI : 10.1128/genomeA.00831-15 PMC : PMC4543497 Abstract >>
Brevibacillus brevis DZQ7 is a plant growth-promoting rhizobacterium (PGPR) isolated from tobacco rhizosphere. Here, we report the draft genome sequence of B. brevis DZQ7. Several functional genes related to antimicrobial activity were identified in the genome.
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15. |
Samel SA,
Czodrowski P,
Essen LO,
( 2014 ) Structure of the epimerization domain of tyrocidine synthetase A. PMID : 24816112 : DOI : 10.1107/S1399004714004398 Abstract >>
Tyrocidine, a macrocyclic decapeptide from Bacillus brevis, is nonribosomally assembled by a set of multimodular peptide synthetases, which condense two D-amino acids and eight L-amino acids to produce this membrane-disturbing antibiotic. D-Phenylalanine, the first amino acid incorporated into tyrocidine, is catalytically derived from enzyme-bound L-Phe by the C-terminal epimerization (E) domain of tyrocidine synthetase A (TycA). The 1.5 ? resolution structure of the cofactor-independent TycA E domain reveals an intimate relationship to the condensation (C) domains of peptide synthetases. In contrast to the latter, the TycA E domain uses an enlarged bridge region to plug the active-site canyon from the acceptor side, whereas at the donor side a latch-like floor loop is suitably extended to accommodate the �\III helix of the preceding peptide-carrier domain. Additionally, E domains exclusively harbour a conserved glutamate residue, Glu882, that is opposite the active-site residue His743. This active-site topology implies Glu882 as a candidate acid-base catalyst, whereas His743 stabilizes in the protonated state a transient enolate intermediate of the L?D isomerization.
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16. |
Westman EL,
Yan M,
Waglechner N,
Koteva K,
Wright GD,
( 2013 ) Self resistance to the atypical cationic antimicrobial peptide edeine of Brevibacillus brevis Vm4 by the N-acetyltransferase EdeQ. PMID : 23891151 : DOI : 10.1016/j.chembiol.2013.06.010 Abstract >>
Edeines are atypical cationic peptides produced by Brevibacillus brevis Vm4 with broad-spectrum antimicrobial activity. These linear nonribosomal peptides bind to the 30S ribosomal subunit and block t-RNA binding to the P-site. To identify the mechanism of high-level self-resistance in the producing organism, the B. brevis Vm4 genome was sequenced and the edeine biosynthetic cluster discovered. A potential edeine-modifying enzyme, EdeQ, showed similarity to spermidine N-acetyltransferases. EdeQ was purified and shown to convert edeine to N-acetyledeine, which is inactive against cells in vivo and against cell-free extracts. Unexpectedly, tandem mass spectroscopy and nuclear magnetic resonance demonstrate that N-acylation occurs on the free amine of the internal diaminopropionic acid rather than the N-terminal spermidine polyamine. Acetylation of edeine by EdeQ abolishes its ability to inhibit translation, thus conferring resistance to the antibiotic in the producing organism.
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17. |
Diderichsen B,
Wedsted U,
Hedegaard L,
Jensen BR,
Sjøholm C,
( 1990 ) Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis. PMID : 2198252 : DOI : 10.1128/jb.172.8.4315-4321.1990 PMC : PMC213256 Abstract >>
A gene for alpha-acetolactate decarboxylase (ALDC) was cloned from Bacillus brevis in Escherichia coli and in Bacillus subtilis. The 1.3-kilobase-pair nucleotide sequence of the gene, aldB, encoding ALDC and its flanking regions was determined. An open reading frame of 285 amino acids included a typical N-terminal signal peptide of 24 or 27 amino acids. A B. subtilis strain harboring the aldB gene on a recombinant plasmid processed and secreted ALDC. In contrast, a similar enzyme from Enterobacter aerogenes is intracellular.
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18. |
( 1997 ) Molecular cloning and nucleotide sequence of the arginase gene of Bacillus brevis TT02-8 and its expression in Escherichia coli. PMID : 9339546 : DOI : 10.1271/bbb.61.1459 Abstract >>
The gene from Bacillus brevis TT02-8 encoding arginase was cloned into Escherichia coli, and its nucleotide sequence was identified. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 298 amino acid residues with a predicted molecular weight of 31,891, which was consistent with that previously calculated for arginase purified from this bacterium. Comparison of the deduced amino acid sequence of the B. brevis TT02-8 arginase with that of the prokaryotic and eukaryotic arginases of Bacillus caldovelox, Bacillus subtilis, Agrobacterium Ti plasmid C58, Saccharomyces cerevisiae, Coccidioides immitis, Xenopus laevis, Rana catesbeiana, rat liver, and human liver, showed 33-66% of the sequences to be similar; there were several highly conserved regions. Arginase activity was detected in Escherichia coli cells transformed with an expression plasmid of the cloned arginase gene.
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19. |
( 1997 ) The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. PMID : 9352938 : DOI : 10.1128/jb.179.21.6843-6850.1997 PMC : PMC179617 Abstract >>
The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His6-tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PPi exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The Km values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3' end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.
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20. |
Louw ME,
Reid SJ,
James DM,
Watson TG,
( 1994 ) Cloning and sequencing the degS-degU operon from an alkalophilic Bacillus brevis. PMID : 7765823 : Abstract >>
The sacU region from an alkalophilic Bacillus brevis was cloned and sequenced. The two open reading frames of the degS-degU operon encode polypeptides that gave calculated molecular masses of 43.8 kDa and 27.0 kDa, respectively. Sequence comparisons at the amino acid level to the B. subtilis degS-degU genes showed 74% and 84% similarity, respectively. On a multicopy vector the B. brevis degS-degU genes were found to cause hypersecretion of several extracellular enzymes in a B. subtilis rec- strain as well as in a B. subtilis sacU(HY) strain.
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21. |
Louw ME,
Reid SJ,
Watson TG,
( 1993 ) Characterization, cloning and sequencing of a thermostable endo-(1,3-1,4) beta-glucanase-encoding gene from an alkalophilic Bacillus brevis. PMID : 7763386 : Abstract >>
A Bacillus brevis gene coding for an endo-(1,3-1,4)-beta-glucanase was cloned in Escherichia coli and sequenced. The open reading frame contains a sequence of 759 nucleotides encoding a polypeptide of 252 amino acid residues. The amino acid sequence of the beta-glucanase gene showed only a 50% similarity to previously published data for Bacillus endo-(1,3-1,4)-beta-glucanases. The optimum temperature and pH for enzyme activity were 65-70 degrees C and 8-10, respectively. When held at 75 degrees C for 1 h, 75% residual activity was measured. The molecular mass was estimated to be about 29 kDa on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and the enzyme was found to be resistant to SDS.
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22. |
Saito F,
Hori K,
Kanda M,
Kurotsu T,
Saito Y,
( 1994 ) Entire nucleotide sequence for Bacillus brevis Nagano Grs2 gene encoding gramicidin S synthetase 2: a multifunctional peptide synthetase. PMID : 7822255 : DOI : 10.1093/oxfordjournals.jbchem.a124532 Abstract >>
Bacillus brevis Nagano grs2 gene, which encodes gramicidin S synthetase 2 (GS2) catalyzing activation and combination of four constituent amino acids of gramicidin S, namely, proline, valine, ornithine, and leucine, has been sequenced. The open reading frame of grs2 gene specifies a 4,450-amino acid protein with a calculated molecular weight of 508,658. There are four domains with a mean of 1,042 amino acid residues containing a repeated sequence of about 600 amino acids, which is highly homologous to the amino-terminal half of gramicidin S synthetase 1 (GS1) (about 40-50% identity). Three domains of grs2 protein, excluding the first one, show homology over the entire sequences of 1,042 amino acids, but the first domain only shows homology in the conserved 600-amino acid sequence. The last 300-amino acid sequence of grs2 protein following the fourt domain has no homology with any of the above sequences. Translation products of subcloned fragments containing the third or the fourth domain catalyzed ornithine- or leucine-dependent ATP-32Pi exchange, respectively. These results, together with a previous report on a proline-activation domain indicated that the repeated and conserved domains are the individual activation sites of the constituent amino acids; the activation sites are arranged in the order of peptide elongation on GS2. Several motifs of grs2 protein are conserved among the multiple domains of peptide synthetases and aminoacyl or acyl adenylate-forming enzymes.
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23. |
Takechi M,
Kanda M,
Hori K,
Kurotsu T,
Saito Y,
( 1994 ) Purification and properties of L-ornithine delta-aminotransferase from gramicidin S-producing Bacillus brevis. PMID : 7534759 : DOI : 10.1093/oxfordjournals.jbchem.a124652 Abstract >>
In gramicidin S-producing Bacillus brevis, the addition of L-ornithine to the minimal medium with L-glutamate as the sole carbon and nitrogen source caused an 8-fold induction of L-ornithine delta-aminotransferase [EC 2.6.1.13]. The enzyme was purified to homogeneity. The native enzyme had a molecular weight of about 88,000 after gel filtration and consisted of two subunits with an identical in molecular weight of about 45,000. The enzyme was specific for L-ornithine (Km = 1.05 mM) as an amino donor and for 2-oxoglutarate (Km = 6.25 mM) as an amino acceptor, and catalyzed the conversion of L-ornithine and 2-oxoglutarate, respectively, to glutamic-gamma-semialdehyde, which is spontaneously cyclized to delta 1-pyrroline-5-carboxylate and L-glutamate. The enzyme exhibits an absorption maximum at 425 nm at neutral pH, and 1 mol of pyridoxal phosphate is bound per subunit. The enzyme activity was irreversibly inhibited by gabaculine, and L-ornithine protected the enzyme from the inhibition. The N-terminal amino acid sequence revealed a noteworthy similarity between human and yeast L-ornithine delta-aminotransferases in residues 17-28 of the B. brevis enzyme.
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24. |
Shen BW,
Doyle L,
Bradley P,
Heiter DF,
Lunnen KD,
Wilson GG,
Stoddard BL,
( 2019 ) Structure, subunit organization and behavior of the asymmetric Type IIT restriction endonuclease BbvCI. PMID : 30395313 : DOI : 10.1093/nar/gky1059 PMC : PMC6326814 Abstract >>
BbvCI, a Type IIT restriction endonuclease, recognizes and cleaves the seven base pair sequence 5'-CCTCAGC-3', generating 3-base, 5'-overhangs. BbvCI is composed of two protein subunits, each containing one catalytic site. Either site can be inactivated by mutation resulting in enzyme variants that nick DNA in a strand-specific manner. Here we demonstrate that the holoenzyme is labile, with the R1 subunit dissociating at low pH. Crystallization of the R2 subunit under such conditions revealed an elongated dimer with the two catalytic sites located on opposite sides. Subsequent crystallization at physiological pH revealed a tetramer comprising two copies of each subunit, with a pair of deep clefts each containing two catalytic sites appropriately positioned and oriented for DNA cleavage. This domain organization was further validated with single-chain protein constructs in which the two enzyme subunits were tethered via peptide linkers of variable length. We were unable to crystallize a DNA-bound complex; however, structural similarity to previously crystallized restriction endonucleases facilitated creation of an energy-minimized model bound to DNA, and identification of candidate residues responsible for target recognition. Mutation of residues predicted to recognize the central C:G base pair resulted in an altered enzyme that recognizes and cleaves CCTNAGC (N = any base).
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( N/A ) [Structure of the Bacillus brevis metalloprotease gene]. PMID : 2290427 : Abstract >>
Primary structure of DNA fragment of 2355 b.p., encoding metalloprotease gene of Bac. brevis, had been determined. Open reading frame for a protein with size of 528 amino acid residues was found in this sequence. The encoding protein is homologous to metalloproteases of Bac. stearothermophilus, Bac. cereus, Bac. subtilis and Bac. amyloliquefaciens. The structure of Bac. brevis metalloprotease gene reveals that this enzyme is synthesised as pre-pro-protease with signal peptide and pro-region, which are cut during its synthesis. The proposed size of mature protease is 304 amino acid residues. The residues, essential for catalysis, binding of Zn ion and Ca ions were found on the basis of Bacilli metalloproteases structures comparison.
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( 2013 ) Overexpression of the formaldehyde dehydrogenase gene from Brevibacillus brevis to enhance formaldehyde tolerance and detoxification of tobacco. PMID : 23160947 : DOI : 10.1007/s12010-012-9957-4 Abstract >>
The faldh gene coding for a putative Brevibacillus brevis formaldehyde dehydrogenase (FALDH) was isolated and then transformed into tobacco. A total of three lines of transgenic plants were generated, with each showing 2- to 3-fold higher specific formaldehyde dehydrogenase activities than wild-type tobacco, a result that demonstrates the functional activity of the enzyme in formaldehyde (HCHO) oxidation. Overexpression of faldh in tobacco confers a high tolerance to exogenous HCHO and an increased ability to take up HCHO. A (13)C-nuclear magnetic resonance technique revealed that the transgenic plants were able to oxidize more aqueous HCHO to formate than the wild-type (WT) plants. When treated with gaseous HCHO, the transgenic tobacco exhibited an enhanced ability to transform more HCHO into formate, citrate acid, and malate but less glycine than the WT plants. These results indicate that the increased capacity of the transgenic tobacco to take up, tolerate, and metabolize higher concentrations of HCHO was due to the overexpression of B. brevis FALDH, revealing the essential function of this enzyme in HCHO detoxification. Our results provide a potential genetic engineering strategy for improving the phytoremediation of HCHO pollution.
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27. |
( 1998 ) Cloning and sequencing of a cyclodextrin glycosyltransferase gene from Brevibacillus brevis CD162 and its expression in Escherichia coli. PMID : 9682490 : DOI : 10.1111/j.1574-6968.1998.tb13117.x Abstract >>
A cyclodextrin glycosyltransferase (CGTase) gene of Brevibacillus brevis CD162 was cloned into Escherichia coli using pUC19 as a vector. Determination of the nucleotide sequence showed the presence of an open reading frame of 2079 bp encoding a polypeptide of 693 amino acid residues, composed of a 20-amino acid signal sequence and a 673-amino acid mature enzyme. Neither a TATA- nor a TTGA-like sequence was observed within the cloned DNA fragment. However, the fragment was expressed in Escherichia coli by the lac promoter of pUC19 and 74% of the total activity was secreted into the fermentation medium. The amino acid sequence of the mature CGTase showed the highest homology of 86% to that of Bacillus sp. KC201. The CGTase purified to homogeneity from the recombinant E. coli exhibited the same properties as those of native CGTase from Brevibacillus brevis CD162 in terms of molecular mass, reaction conditions, stability and the production of cyclodextrins.
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