Cloning and sequencing of a gene from Bacillus amyloliquefaciens that complements mutations of the sporulation gene spoIID in Bacillus subtilis

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.     

Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci.

The rnc gene of Bacillus subtilis, which has 36% amino acid identity with the gene that encodes Escherichia coli RNase III endonuclease, was cloned in E. coli and shown by functional assays to encode B. subtilis RNase III (Bs-RNase III). The cloned B. subtilis rnc gene could complement an E. coli rnc strain that is deficient in rRNA processing, suggesting that Bs-RNase III is involved in rRNA processing in B. subtilis. Attempts to construct a B. subtilis rnc null mutant were unsuccessful, but a strain was constructed in which only a carboxy-terminal truncated version of Bs-RNase III was expressed. The truncated Bs-RNase III showed virtually no activity in vitro but was active in vivo. Analysis of expression of a copy of the rnc gene integrated at the amy locus and transcribed from a p(spac) promoter suggested that expression of the B. subtilis rnc is under regulatory control.     

The recE(A)+ gene of B subtilis and its gene product: further characterization of this universal protein

Although the SOS system of E coli and the SOB system of B subtilis share many similarities, there are distinct differences with respect to the regulation and specificity of the phenomena that constitute these global regulons. One of these differences resides in the regulation of the respective RecA and RecA-like proteins. In B subtilis the RecA-like protein, the RecE protein, shares 60% amino acid homology with its E coli counterpart. The E coli recA gene can complement most, but not all, of the functions that are lost in strains of B subtilis that do not produce a functional RecE protein. The DNA sequence of the recE+ gene as well as the sequence of the recE4 allele and the recA73 allele of B subtilis has demonstrated that mutants of the recE and recA loci of this bacterium actually represent alleles of the same complex gene. Accordingly, the major recombination protein of B subtilis should be referred to as RecA and the gene that encodes this protein as recA+.     

透明颤菌vgb基因在产核黄素B.subtilis中的整合表达研究

目的:将透明颤菌血红蛋白vgb基因应用于核黄素的工业化生产。方法:以枯草芽孢杆菌整合载体pAmyE构建了vgb基因的整合表达载体pAudV,采用化学转化法将vgb基因整合到枯草芽孢杆菌GJ08的染色体上,并通过发酵摇瓶实验检测核黄素的产量。结果:得到产核黄素枯草芽孢杆菌GJ09,摇瓶试验结果表明,在限氧条件下核黄素的产量分别提高了5.23%和3.42%。结论:透明颤菌血红蛋白vgb基因能够促进核黄素产量的提高,可以应用于核黄素的工业化生产中。     

Expression of the staphylococcal protein A gene in Bacillus subtilis by gene fusions utilizing the promoter from a Bacillus amyloliquefaciens alpha-amylase gene.

Gene fusions of DNA sequences encoding protein A from Staphylococcus aureus (spa) with expression elements from an alpha-amylase gene from Bacillus amyloliquefaciens (amyEBamP) directed the synthesis and efficient secretion of protein A in Bacillus subtilis. The fusions were established on multicopy pUB110-based plasmid vectors, in contrast to the intact spa gene, which could not be stably established on plasmids in B. subtilis. Some of the resulting B. subtilis strains secreted protein A at levels in excess of 1 g/liter, demonstrating that a foreign protein encoded by an engineered gene can be secreted by B. subtilis at levels comparable to endogenous exoproteins.     

Cloning and mapping of the dihydrofolate reductase gene of Bacillus subtilis

The structural gene for dihydrofolate reductase (dfrA) from the Bacillus subtilis 168 chromosome has been cloned, along with the thyB gene, on a 4.5-kb insert contained on chimeric plasmid pER1. The presence of the dfrA gene on pER1 was demonstrated by showing that: (i) transformation of Escherichia coli strains RUE10(Thy-) and RUE11(Thy+) with pER1 resulted in a 60 to 130-fold increase in dihydrofolate reductase (DFRase) activity with a turnover number characteristic of that of B. subtilis and (ii) pER1-mediated transformation of trimethoprim-resistant E. coli strain D05, which overproduced a DFRase with a decreased affinity for trimethoprim, resulted in a 41-fold increase in DFRase activity with an affinity for trimethoprim similar to that of the B. subtilis enzyme. The dfrA gene was mapped to the 200 degrees region of the B. subtilis chromosome, and the gene order was established as thyB dfrA ilvA. Furthermore, the dfrA gene was shown to be linked closely (95-99% cotransformation) to the thyB gene.     

Expression of Bacillus circulans Teri-42 xylanase gene in Bacillus subtilis

The xylanase gene of Bacillus circulans Teri-42 was cloned in both B. subtilis and Escherichia coli. The enzyme activity was almost 87% higher in B. subtilis (pBA7) than in E. coli (pAQ4). No cellulase activity was detected in the clones, B. subtilis (pBA7) and E. coli (pAQ4). Approximately 1120 U (80%) of the xylanase was secreted extracellularly by the clone B. subtilis (pBA7) as compared to 79 U (88%) excreted in E. coli (pAQ4). In B. subtilis (pBA7) the optimal xylanase activity was at pH 7.0 and 50 degrees C, which was the same as that of the parent B. circulans Teri-42. The recombinant xylanase in B. subtilis was more stable at higher temperatures than the parent B. circulans Teri-42. Purification of xylanase from the clone B. subtilis (pBA7) showed a 71 kDa polypeptide similar to that observed in B. circulans Teri-42.     

Cloning and strong expression of a Bacillus subtilis WL-3 mannanase gene in B. subtilis

A gene encoding the mannanase of Bacillus subtilis WL-3, which had been isolated from Korean soybean paste, was cloned into Escherichia coli and the nucleotide sequence of a 2.7-kb DNA fragment containing the mannanase gene was subsequently determined. The mannanase gene, designated manA, consisted of 1,080 nucleotides encoding polypeptide of 360 amino acid residues. The deduced amino acid sequence was highly homologous to those of mannanases belonging to glycosyl hydrolase family 26. The manA gene was strongly expressed in B. subtilis 168 by cloning the gene downstream of a strong B. subtilis promoter of plasmid pJ27Delta 88U. In flask cultures, the production of mannanase by recombinant B. subtilis 168 reached maximum levels of 300 units/ml and 450 units/ml in LB medium and LB medium containing 0.3% locust bean gum, respectively. Based on the zymogram of the mannanase, it was found that the mannanase produced by recombinant B. subtilis could be maintained stably without proteolytic degradation during the culture time.     

Glutamine synthetase gene of Bacillus subtilis

The glutamine synthetase gene (glnA) of Bacillus subtilis was purified from a library of B. subtilis DNA cloned in phage lambda. By mapping the locations of previously identified mutations in the glnA locus it was possible to correlate the genetic and physical maps. Mutations known to affect expression of the glnA gene and other genes were mapped within the coding region for glutamine synthetase, as determined by measuring the sizes of truncated, immunologically cross-reacting polypeptides coded for by various sub-cloned regions of the glnA gene. When the entire B. subtilis glnA gene was present on a plasmid it was capable of directing synthesis in Escherichia coli of B. subtilis glutamine synthetase as judged by enzymatic activity, antigenicity, and ability to allow growth of a glutamine auxotroph. By use of the cloned B. subtilis glnA gene as a hybridization probe, it was shown that the known variability of glutamine synthetase specific activity during growth in various nitrogen sources is fully accounted for by changes in glnA mRNA levels.     

枯草芽孢杆菌ccpA基因敲除及对其核黄素产量的影响

CcpA蛋白是介导枯草芽孢杆菌碳分解代谢物阻遏(CCR)的全局调控因子,由ccpA基因编码。CCR效应的存在影响B.subtilis对葡萄糖的利用,降低B.subtilis生产发酵产品的效率。采用基因重组技术敲除了核黄素发酵菌株B.subtilis24/pMX45的ccpA基因,构建了CcpA缺陷株B.subtilis24A1/pMX45。发酵结果显示:B.subtilis24A1/pMX45能够在70h内基本耗尽10%的葡萄糖,生物量达到1.5×109个细胞/mL,溢流代谢产物积累量减少,在8%和10%葡萄糖浓度下,B.subtilis24A1/pMX45核黄素产量分别比B.subtilis24/pMX45提高了62%和95%。CcpA的缺陷,可以缓解葡萄糖引起的CCR效应,显著提高菌株的核黄素产量。     

In vivo and in vitro characterization of the secA gene product of Bacillus subtilis.

The putative amino acid sequence from the wild-type Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, shares a 50% identity with the sequence from Escherichia coli secA (Y. Sadaie, H. Takamatsu, K. Nakamura, and K. Yamane, Gene 98:101-105, 1991). The B. subtilis div-341 mutant accumulated the precursor proteins of alpha-amylase and beta-lactamase at 45 degrees C as in the case of sec mutants of E. coli. The div-341 mutation is a transition mutation causing an amino acid replacement from Pro to Leu at residue 431 of the putative amino acid sequence. The B. subtilis div+ gene was overexpressed in E. coli under the control of the tac promoter, and its product was purified to homogeneity. The Div protein consists of a homodimer of 94-kDa subunits which possesses ATPase activity, and the first 7 amino acids of the putative Div protein were found to be subjected to limited proteolysis in the purified protein. The antiserum against B. subtilis Div weakly cross-reacted with E. coli SecA. On the other hand, B. subtilis Div could not replace E. coli SecA in an E. coli in vitro protein translocation system. The temperature-sensitive growth of the E. coli secA mutant could not be restored by the introduction of B. subtilis div+, which is expressed under the control of the spac-1 promoter, and vice versa. The B. subtilis div+ gene is the B. subtilis counterpart of E. coli secA, and we propose that the div+ gene be referred to as B. subtilis secA, although Div did not function in the protein translocation system of E. coli.     

Isolation and characterization of levansucrase-encoding gene from Bacillus amyloliquefaciens

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.     

Cloning and characterization of a Bacillus subtilis gene homologous to E. coli secY

A 3.5-kb HindIII DNA fragment containing the secY gene of Bacillus subtilis has been cloned into plasmid pUC13 using the Escherichia coli secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained five open reading frames, and their order in the region, given by the gene product, was suggested to be L30-L15-SecY-Adk-Map by their similarity to the products of the E. coli genes. The region was similar to a part of the spc operon of the E. coli chromosome, although the genes for Adk and Map were not included. The gene product of the B. subtilis secY homologue was composed of 423 amino acids and its molecular weight was calculated to be 46,300. The distribution of hydrophobic amino acids in the gene product suggested that the protein is a membrane integrated protein with ten transmembrane segments. The total deduced amino acid sequence of the B. subtilis SecY homologue shows 41.3% homology with that of E. coli SecY, but remarkably higher homologous regions (more than 80% identity) are present in the four cytoplasmic domains.     

Bacillus sporulation gene spo0H codes for sigma 30 (sigma H).

The DNA sequences of the spo0H genes from Bacillus licheniformis and B. subtilis are described, and the predicted open reading frames code for proteins of 26,097 and 25,447 daltons, respectively. The two spo0H gene products are 91% identical to one another and about 25% identical to most of the procaryotic sigma factors. The predicted proteins have a conserved 14-amino-acid sequence at their amino terminal end, typical of sigma factors. Antibodies raised against the spo0H gene product of B. licheniformis specifically react with RNA polymerase sigma factor protein, sigma 30, purified from B. subtilis. We conclude that the spo0H genes of B. licheniformis and B. subtilis code for sigma 30, now known as sigma H.     

Cloning and expression of subtilisin amylosacchariticus gene

The gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosacchariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1,426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-cellulose.     

Identification of a putative Bacillus subtilis rho gene.

Transposon Tn917 mutagenesis of Bacillus subtilis BD99 followed by selection for protonophore resistance led to the isolation of strain MS119, which contained a single Tn917 insertion in an open reading frame whose deduced amino acid sequence was 56.6% identical to that of the Escherichia coli rho gene product. The insertional site was near the beginning of the open reading frame, which was located in a region of the B. subtilis chromosome near the spoOF gene; new sequence data for several open reading frames surrounding the putative rho gene are presented. The predicted B. subtilis Rho protein would have 427 amino acids and a molecular weight of 48,628. The growth of the mutant strain was less than that of the wild type on defined medium at 30 degrees C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type on defined medium at 30 degrees C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type at 30 degrees C but was much slower at lower temperatures; sporulation occurred and competence was developed in cells of the mutant grown at 30 degrees C. To determine whether the protonophore resistance and sensitivity to low growth temperature resulted from the insertion, a chloramphenicol resistance cassette was inserted into the wild-type B. subtilis rho gene of strain BD170; the resulting derivative displayed the same phenotype as MS119.