Inter- and intraspecies conjugal transfer of different plasmids in bacilli

Conjugal transfer of plasmid pUB110 between different strains of bacilli was studied. The plasmid transfer was possible not only between various strains of B. subtilis, but also when many other species of bacilli served as recipients. Conjugation of a donor strain B. subtilis 19 (p19pUB110) was accompanied by a transfer of plasmid p19 along with plasmid pUB110 to the B. subtilis recipient strains lacking a large plasmid p19. If, like the donor cells, the recipient B. subtilis strain carried plasmid p19, the frequency of conjugation decreased. The small plasmid pBC16 was also capable of conjugative transfer. However, if this plasmid carried the mob gene with an inverted region, the frequency of its transmission dramatically decreased. If the donor strain contained another small plasmid, pV, which also carried the mob gene, the efficiency of transmission was partially restored.     

Growth and sporulation of entrapped Bacillus subtilis cells

Growing Bacillus subtilis cells were immobilized in k-carrageenan gel beads. Growth and sporulation of entrapped cells were studied by two different methods: cell enumeration and transmission electron microscopy. Immobilized growing cells had a shorter generation time than free cells did, whereas sporulation timing was unchanged. When steady state was reached, cell density was very high in gel beads.     

Immunocytochemical Localization of a Calmodulinlike Protein in Bacillus subtilis Cells

To determine possible functions of the calmodulinlike protein of Bacillus subtilis, the time course of its expression during sporulation and its cellular localization were studied. The protein was expressed in a constitutive manner from the end of logarithmic growth through 8 h of sporulation as determined by antibody cross-reactivity immunoblots and enzyme-linked immunosorbent assays (ELISAs). In partially purified extracts, the immunopositive protein comigrated upon electrophoresis with a protein which selectively bound [(45)Ca]CaCl(2), ruthenium red, and Stains-all. Previous studies showed increased extractability of the calmodulinlike protein from B. subtilis cells when urea and 2-mercaptoethanol were used in breakage buffers, implying that the protein might be partially associated with the membrane fraction. This was confirmed by demonstrating that isolated membrane vesicles of B. subtilis also gave positive immunological tests with Western blotting and ELISAs. To more precisely locate the protein in cells, thin sections of late-log-phase cells, sporulating cells, and free spores were reacted first with bovine brain anticalmodulin specific antibodies and then with gold-conjugated secondary antibodies; the thin sections were examined by transmission electron microscopy. The calmodulinlike protein was found almost exclusively associated with the cell envelope of these fixed, sectioned cells. A possible function of the calmodulinlike protein in sensing calcium ions or regulating calcium ion transport is suggested.     

Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector

The Bacillus subtilis GenoMe (BGM) vector was designed as a versatile vector for the cloning of giant DNA segments. Cloned DNA in the BGM can be retrieved to a plasmid using our Bacillus recombinational transfer (BReT) method that takes advantage of competent cell transformation. However, delivery of the plasmid to a different B. subtilis strain by the normal transformation method is hampered by DNA size-related inefficiency. Therefore, we designed a novel method, conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector, and investigated conjugational transmission to traverse DNA between cells to circumvent the transformation-induced size limitation. pLS20, a 65-kb plasmid capable of conjugational transfer between B. subtilis strains, was modified to retrieve DNA cloned in the BGM vector by homologous recombination during normal culture. As the plasmid copy number was estimated to be 3, the retrieval plasmid was selected using increased numbers of marker genes derived from the retrieved DNA. We applied this method to retrieve Synechocystis genome segments up to 90 kb in length. We observed retrieved plasmid transfers between B. subtilis strains by conjugation in the absence of structural alterations in the DNA fragment. Our observations extend DNA transfer protocols over previously exploited size ranges.     

Plasmid and chromosomal gene transfer in Bacillus subtilis and Escherichia coli in natural transformation

The transfer of hDHFR gene by natural transformation between Escherichia coli and Bacillus subtilis cells has been studied, as well as intrageneric gene transfer in Bacillus subtilis. The gene was transferred by natural transformation in bacterial cells and included into the chromosome or the plasmid.     

Mapping and cloning of the gene coding for beta-glucanase from various bacilli

Beta-glucanase gene from Bacillus subtilis 168 has been mapped by bacteriophage pBS1 transduction technique between sacA and purA genes. The stimulating effect of pleiotropic mutations pap, amyB and sacUh on beta-glucanase production in Bacillus subtilis and Bacillus amyloliquefaciens has been described. Beta-glucanase gene from Bacillus amyloliquefaciens has been cloned ona Charon 4A vector. Expression of the gene in E. coli cells depended on the orientation of the cloned DNA on a pBR322 vector plasmid. Maximal enzymatic activity was registered in periplasm. Beta-glucanase gene was recloned in Bacillus subtilis cells. Bacillus subtilis strain, harbouring pBG1, produces 500 times more beta-glucanase as compared with the wild type strain of Bacillus subtilis.     

Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes.     

Associated roles of hemolysin and p60 protein for the intracellular growth of Bacillus subtilis

Hemolysin expressing Bacillus subtilis strain (B. subtilis ble/hlA) was used as a carrier for listerial protein p60 to study the impact of this protein on bacterial virulence independent of other gene products of Listeria monocytogenes. Bacillus subtilis ble/hlyA exhibited longer cell chains than B. subtilis ble/hlyA/iap. Recombinant Bacillus strains are able to adhere to the mouse macrophage-like J774 and human epithelial-like Int407 cell lines. The bacterial number of B. subtilis ble/hlyA/iap strain that adhered to the Int407 cell lines was 2.52-fold higher, and its invasion level strain was 2.66-fold higher than that observed for the hemolytic strain. Microscopy analysis of infected monolayers showed that recombinant B. subtilis cells were localized inside the cytoplasm of epithelial cells, near to the nuclei, in cellular compartments with low internal pH. Furthermore, in cells infected with bacteria, the actin structures rapidly changed and accumulation of a fat, wide actin layer around the nucleus zone was observed.     

Isolation and characteristics of a plasmid from the thermotolerant strain of Bacillus licheniformis

A 8.3 kb cryptic plasmid was isolated from the thermotolerant strain of Bacillus licheniformis 28KA and designated pLT83. The replicative (rep) region was localized on the plasmid map. The pLT83 plasmid labelled in vitro with an antibiotic resistance determinant is able to replicate in B. subtilis cells. The pLT83 plasmid replicates stably in B. licheniformis strain at higher temperatures (37-60 degrees C) than in B. subtilis cells (37-50 degrees C). The plasmid and its derivatives may be used as vectors for gene cloning in B. subtilis and B. licheniformis cells.     

Cloning and expression in Bacillus subtilis of the gene for neutral protease of Bacillus brevis

Plasmids pCB20 and pCB22 were used for cloning and expression of the Bac brevis 7882 neutral protease gene in Bac. subtilis cells. The protease-containing fragments of 13 and 14 kb were cloned in pCB20 plasmid based on replication region of Streptococci plasmid pSM19035. Expression of the gene was shown to take place in Bac. subtilis. Application of vegetative promoters of the previously identified expression unit EU19035 greatly increases the expression of the protease in Bac. subtilis. Bac. subtilis cells, expressing the gene of Bac. brevis neutral protease, do not sporulate, are considerably larger than the cells which do not contain the gene and form multicellular structures.     

Cloning of lysozyme and lysostaphin genes of Staphylococcus aureus and their expression in Bacillus subtilis cells

The gene of microbial lysozyme (lyz) of S. aureus 118 and the gene of lysostaphin (lzf) of S. aureus RN 3239 were cloned and their expression in B. subtilis cells was shown. Lysozyme production in B. subtilis recombinant clone pLF14-Lyz, obtained as the result of cloning, was 2.5-fold greater than lysozyme production in S. aureus wild strain 118. Lysostaphin production in B. subtilis recombinant strain pLF14-Lzf which had inherited the cloned genes was approximately equal to lysostaphin production observed in S. aureus initial strain RN 3239. The production of lysozyme and lysostaphin in the cells of B. subtilis recombinant strains was observed at 30 degrees C and pH 5.5, while in S. aureus initial strains 118 and RN 3239 bacteria produced lysozyme and lysostaphin at 37 degrees C and pH 7.5 respectively.     

Cloning the alpha-amylase gene of Streptococcus bovis and its expression in Bacillus subtilis cells

The gene coding for alpha-amylase from the ruminant bacterium Streptococcus bovis was cloned on the plasmid pMX39 in Bacillus subtilis cells. An alpha-amylase positive colony was isolated in the initial screening of 3900 colonies on the medium containing insoluble starch. The size of the insert was approximately 2.8 kb. The recombinant plasmid was stably maintained in Bacillus subtilis cells under the nonselective conditions.     

Expression of Escherichia coli Min system in Bacillus subtilis and its effect on cell division

In both rod-shaped Bacillus subtilis and Escherichia coli cells, Min proteins are involved in the regulation of division septa formation. In E. coli , dynamic oscillation of MinCD inhibitory complex and MinE, a topological specificity protein, prevents improper polar septation. However, in B. subtilis no MinE is present and no oscillation of Min proteins can be observed. The function of MinE is substituted by that of an unrelated DivIVA protein, which targets MinCD to division sites and retains them at the cell poles. We inspected cell division when the E. coli Min system was introduced into B. subtilis cells. Expression of these heterologous Min proteins resulted in cell elongation. We demonstrate here that E. coli MinD can partially substitute for the function of its B. subtilis protein counterpart. Moreover, E. coli MinD was observed to have similar helical localization as B. subtilis MinD.     

3-甾酮-1-脱氢酶基因的表达及甾体转化分析

本文利用带有P43启动子的表达分泌载体pWB980,实现了简单节杆菌3-甾酮-1-脱氢酶在枯草芽孢杆菌中的表达,表达出的目的蛋白的分子量为55KDa。利用分光光度法检测得到胞内和胞外可溶性部分的酶活分别为110±0.5mU和15±0.6mU每毫克蛋白, 比出发菌株简单节杆菌提高了将近30倍。重组芽孢杆菌对甾体底物4-AD的转化率为45.3％,比出发菌株简单节杆菌提高了近10倍。利用枯草芽孢杆菌对甾体底物进行脱氢为甾体药物的生产开辟了一个新的途径。     

Factor of salinity and adaptive capacity of recombinant strains of Escherichia coli and Bacillus subtilis

Effect of different concentrations of salts on natural and recombinant strains of Bacillus subtilis and Escherichia coli was studied. The recombinant strain of B. subtilis was found to be more osmotolerant than the wild-type strain of this bacterium, whereas the opposite situation was observed for the recombinant and wild-type strains of E. coli. Some salts exerted a bacteriostatic effect on E. coli and B. subtilis. The adaptive capacity of recombinant strains depended on the number of plasmid copies in the cells. The introduction of recombinant bacteria into model ecosystems resulted in the generation of their variants with increased osmotolerance.     

SepF increases the assembly and bundling of FtsZ polymers and stabilizes FtsZ protofilaments by binding along its length

SepF (Septum Forming) protein has been recently identified through genetic studies, and it has been suggested to be involved in the division of Bacillus subtilis cells. We have purified functional B. subtilis SepF from the inclusion bodies overexpressed in Escherichia coli. Far-UV circular dichroism and fluorescence spectroscopic analysis involving the extrinsic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid suggested that the purified SepF had characteristics of folded proteins. SepF was found to promote the assembly and bundling of FtsZ protofilaments using three complimentary techniques, namely 90 degrees light scattering, sedimentation, and transmission electron microscopy. SepF also decreased the critical concentration of FtsZ assembly, prevented the dilution-induced disassembly of FtsZ protofilaments, and suppressed the GTPase activity of FtsZ. Further, thick bundles of FtsZ protofilaments were observed using fluorescein isothiocyanate-labeled SepF (FITC-SepF). Interestingly, FITC-SepF was found to be uniformly distributed along the length of the FtsZ protofilaments, suggesting that SepF copolymerizes with FtsZ. SepF formed a stable complex with FtsZ, as evident from the gel filtration analysis. Using a C-terminal tail truncated FtsZ (FtsZDelta16) and a C-terminal synthetic peptide of B. subtilis FtsZ (366-382); we provided evidence indicating that SepF binds primarily to the C-terminal tail of FtsZ. The present work in concert with the available in vivo data support a model in which SepF plays an important role in regulating the assembly dynamics of the divisome complex; therefore, it may have an important role in bacterial cell division.     

Regulation of autolysins in teichuronic acid-containing Bacillus subtilis cells

Bacillus subtilis cells grown under phosphate starvation induce teichuronic acid (TUA) synthesis while simultaneously repressing teichoic acid synthesis (TA). The turnover rates of TA-containing and TUA-containing walls are similar, indicating that autolysin function is similar and suggesting that modulation of autolytic function may be similar. In this study, it is demonstrated, utilizing fluorescein isothiocyanate (FITC)-dextran to probe the wall pH, that a low pH exists in the wall matrix. A second probe, cationized ferritin (CF), was used to observe cell surface protonation. Suspensions of B. subtilis cells containing either TA or TUA were aggregated with CF only after the addition of a proton-motive-force-dissipating agent. Respiring B. subtilis TUA-containing cells labelled with FITC-dextran exhibited little fluorescence. Conversely, fluorescence intensities exhibited by cells de-energized with nitrogen gas were significantly greater. The effects of protonmotive force on autolytic activity were studied by adding cell wall protein extract containing concentrated autolysin to exponentially growing TA-containing and TUA-containing B. subtilis cells. Both TUA-containing and TA-containing cells were lysed only after the addition of sodium azide. These data suggest that during normal growth the wall of TUA-containing B. subtilis cells is protonated, and proton-motive force influences autolytic regulation in both TUA-containing and TA-containing B. subtilis cells.     

Expression of ksdD gene encoding 3-ketosteroid-Delta1-dehydrogenase from Arthrobacter simplex in Bacillus subtilis

AIMS: To improve KSDH enzyme activity and the transformation level for androst-4-ene-3,17-dione. METHODS AND RESULTS: 3-ketosteroid-Delta(1)-dehydrogenase gene from Arthrobacter simplex was expressed in Bacillus subtilis under the control of P43 promoter. The molecular weight of expressed enzyme was about 55 kDa by SDS-PAGE analysis. The activities of intracellular and extracellular soluble enzymes examined by spectrophotometrical method were 110 +/- 0.5 mU mg(-1) and 15 +/- 0.6 mU mg(-1) of protein, respectively. The transformation rate of androst-4-ene-3,17-dione was 45.3% in the B. subtilis recombinant cells. CONCLUSIONS: The enzyme activity of KSDH expressed in B. subtilis was improved about 30-fold compared with that of Arthrobacter simplex, and the transformation level of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was improved about 10-fold. SIGNIFICANCE AND IMPACT OF THE STUDY: The recombinant B. subtilis cells used for biotransformation of steroids provide a new method for production of steroid medicine. The time required for transformation of B. subtilis is much shorter than that of other bacteria, which means it will have wider usage in biopharmaceutical industry.     

Secretion activities of Bacillus subtilis alpha-amylase signal peptides of different lengths in Escherichia coli cells

The B. subtilis alpha-amylase promoter and signal peptide are functional in E. coli cells. DNA fragments coding for signal peptides with different lengths (28, 31, 33 and 41 amino acids from the translation initiator Met) were prepared and fused with the E. coli beta-lactamase structural gene. In B. subtilis cells, the sequences of 31, 33 and 41 amino acids were able to secrete beta-lactamase into the surrounding media, but the 28 amino acid sequence was not. In contrast, all of the four sequences were able to export beta-lactamase into the periplasmic space of E. coli cells. Thus, the recognition of the B. subtilis alpha-amylase signal peptide in E. coli cells seems to be different from that in B. subtilis cells.