Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX.

Bacillus subtilis mutants with lesions in PBSX prophage genes have been isolated. One of these appears to be a regulatory mutant and is defective for mitomycin C-induced derepression of PBSX; the others are defective for phage capsid formation. All of the PBSX structural proteins are synthesized during induction of the capsid defective mutants; however, several of these proteins exhibit abnormal serological reactivity with anti-PBSX antiserum. The two head proteins X4 and X7 are not immunoprecipitable in a mutant which fails to assemble phage head structures. In the tail mutant, proteins X5 and X6 are not immunoprecipitable, tails are not assembled, and a possible tail protein precursor remains uncleaved. The noninducible mutant does not synthesize any PBSX structural proteins after exposure to mitomycin C. The mutation is specific for PBSX since ø105 and SPO2 lysogens of the mutant are inducible. All of the known PBSX-specific mutations were shown to be clustered between argC and metC on the host chromosome. In addition, the metC marker was shown to be present in multiple copies in cells induced for PBSX replication. This suggests that the derepressed prophage replicates while still integrated and that replication extends into the adjacent regions of the host chromosome.     

DNA packaging by the Bacillus subtilis defective bacteriophage PBSX.

Defective bacteriophage PBSX, a resident of all Bacillus subtilis 168 chromosomes, packages fragments of DNA from all portions of the host chromosome when induced by mitomycin C. In this study, the physical process for DNA packaging of both chromosomal and plasmid DNAs was examined. Discrete 13-kilobase (kb) lengths of DNA were packaged by wild-type phage, and the process was DNase I resistant and probably occurred by a head-filling mechanism. Genetically engineered isogenic host strains having a chloramphenicol resistance determinant integrated as a genetic flag at two different regions of the chromosome were used to monitor the packaging of specific chromosomal regions. No dramatic selectivity for these regions could be documented. If the wild-type strain 168 contains autonomously replicating plasmids, especially pC194, the mitomycin C induces an increase in size of resident plasmid DNA, which is then packaged as 13-kb pieces into phage heads. In strain RB1144, which lacks substantial portions of the PBSX resident phage region, mitomycin C treatment did not affect the structure of resident plasmids. Induction of PBSX started rolling circle replication on plasmids, which then became packaged as 13-kb fragments. This alteration or cannibalization of plasmid replication resulting from mitomycin C treatment requires for its function some DNA within the prophage deletion of strain RB1144.     

Heterologous expression of Bacillus intermedius gene of glutamyl endopeptidase in Bacillus subtilis strains defective in regulatory proteins

Expression of the gene of glutamyl endopeptidase from Bacillus intermedius (gseBi) cloned on the plasmid pV has been studied in Bacillus subtilis recombinant strains with mutations of the regulatory proteins involved in sporogenesis and spore germination. It has been established that inactivation of the regulatory protein Spo0A involved in sporulation initiation resulted in a decrease in the expression of the gseBi gene by 65% on average. A mutation in the gene of the sensor histidine kinase kinA had no effect on the biosynthesis of the enzyme. Inactivation of Ger proteins regulating bacterial spore germination resulted in a 1.5-5-fold decrease in glutamyl endopeptidase activity. It has been concluded that expression of the B. intermedius glutamyl endopeptidase gene from plasmid pV in recombinant cells of B. subtilis is under impaired control by the regulatory system of Spo0F/Spo0A phosphorelay, which participates in sporulation initiation. The regulatory Ger proteins responsible for spore germination also affect expression of the gene of this enzyme.     

Temperature-sensitive sporulation caused by a mutation in the Bacillus subtilis secY gene.

A thermosensitive sporulation mutant of Bacillus subtilis containing a mutation in the secY gene was isolated and characterized. No asymmetric septum specific to the sporulation was detected by electron microscopy at the nonpermissive temperature, indicating that the block occurred at a very early stage of sporulation. Furthermore, competence development in the mutant cell was affected even at the sporulation-proficient temperature. It is assumed that the SecY protein of B. subtilis has multiple roles both in the regulation of spore formation and in stationary-phase-associated phenomena.     

Induction of prophage SPO2 in Bacillus subtilis: isolation of excised prophage DNA as a covently closed circle.

Bacillus subtilis tryC2, thyA, thyB, lysogenic for the phage DNA polymerase negative mutant SPO2 susL244, was induced under conditions preventing phage and bacterial DNA synthesis. The biological activity of DNA from induced cells and from uninduced controls was assayed by transformation and transfection, respectively. About 50% of the phage DNA biological activity in DNA extracted from induced cells was resistant to exposure to pH 11.8 TO 11.9. This DNA was operationally defined as alkali-resistant phage DNA. Transforming bacterial DNA from uninduced or induced cells and transfecting DNA from uninduced cells were more than 95% inactivated after exposure to high pH. The alkali-resistant phage DNA was characterized by sucrose gradient centrifugation, by centrifugation in cesium chloride-propidium iodide, and by electron microscopy. It was found to consist of a majority of covalently closed circular DNA molecules. Length measurements of a few relaxed circular molecules indicate a molecular weight of these similar to that previously found for mature SPO2DNA. Attempts to isolate similar covalently closed circular phage DNA from induced bacteria lysogenic for SPO2 phage with a functional DNA polymerase gene were unsuccessful. The gene order in mature and prophage SPO2 was determined by rescue of single and double markers from the respective type of DNA. The data obtained show that prophage DNA is (genetically) permuted relative to mature DNA. The phage attachment site is suggested to be located between genes I and J.     

Localization of UvrA and Effect of DNA Damage on the Chromosome of Bacillus subtilis

We found that the nucleotide excision repair protein UvrA, which is involved in DNA damage recognition, localizes to the entire chromosome both before and after damage in living Bacillus subtilis cells. We suggest that the UvrA(2)B damage recognition complex is constantly scanning the genome, searching for lesions in the DNA. We also found that DNA damage induces a dramatic reconfiguration of the chromosome such that it no longer fills the entire cell as it does during normal growth. This reconfiguration is reversible after low doses of damage and is dependent on the damage-induced SOS response. We suggest that this reconfiguration of the chromosome after damage may be either a reflection of ongoing DNA repair or an active mechanism to protect the cell's genome. Similar observations have been made in Escherichia coli, indicating that the alteration of chromosome structure after DNA damage may be a widespread phenomenon.     

High-frequency elimination of SP02 prophage from Bacillus subtilis by plasmid transformation

Transformation of competent Bacillus subtilis lysogenic for SP02 with any of three plasmids (pCM194, pUB110, pAM77) generates drug-resistant transformants of which 5 to 20% have lost the infectivity and immunity associated with the SP02 prophage. Such cured derivatives can be again lysogenized with SP02 and again cured by introduction of a different plasmid. Elimination of the SP02 prophage was not detected when plasmids were introduced by PBS1 transduction or by transformation of protoplasts. Similarly, transformants of B. subtilis selected for chromosome markers retained the prophage. The phi 105 prophage was not eliminated from competent B. subtilis transformed with plasmids.     

The expression of the serine proteinase gene of Bacillus intermedius in Bacillus subtilis

The gene encoding for Bacillus intermedius serine proteinase was cloned and the complete nucleotide sequence was determined. Gene expression was explored in the protease-deficient strain Bacillus subtilis AJ73 during different stages of growth. Catabolite repression involved in control of proteinase expression during transition state and onset of sporulation was not efficient at the late stationary phase. Salt stress leads to induction of serine proteinase production during B. subtilis AJ73(pCS9) post-exponential growth. Expression of proteinase in B. subtilis deg-mutants may be controlled by DegU regulator. B. subtilis spo0-mutants failed to accomplish B. intermedius proteinase production. These data suggest complex network regulation of B. intermedius serine proteinase expression, including the action of spo0, degU, catabolite repression and demonstrate changes in control of enzyme biosynthesis at different stages of growth.     

拆分获得(S)-酮基布洛芬脂肪酶基因在枯草芽孢杆菌中的克隆与表达

【目的】筛选具有不对称拆分消旋酮基布洛芬氯乙酯能力的脂肪酶基因,构建其表达分泌型工程菌,并进一步提高该脂肪酶的立体选择性。【方法】以自筛选出的一株具有不对称拆分消旋酮基布洛芬氯乙酯能力的菌株NK13为材料,通过构建其基因组文库,筛选具有不对称拆分消旋酮基布洛芬氯乙酯能力的脂肪酶基因。通过构建该脂肪酶基因的分泌型诱导表达载体pHY300-plk-sacR-gene,将其转入枯草芽孢杆菌WB600,获得基因重组菌WB600(pHY300-plk-sacR-gene)。用SDS-PAGE检测其表达和转化情况,采用非变性聚丙烯酰胺凝胶电泳的方法纯化脂肪酶;并利用TLC和HPLC检测该酶的立体选择专一性。【结果】得到了具有专一性拆分获得(S)-酮基布洛芬能力、长度为633bp的脂肪酶基因(GenBank登录号为:EU381317)。该脂肪酶在枯草芽孢杆菌WB600中得到了分泌表达。TLC和HPLC检测结果显示,纯化的脂肪酶对底物转化40h时转化率为30%,生成(S)-酮基布洛芬的e.e.%值最高,达60.02%,与未加Tween-80的枯草芽孢杆菌转化子体系相同。而在含Tween-80的环境下,枯草芽孢杆菌表达重组菌对底物转化36h时转化率约为45%,生成(S)-酮基布洛芬的e.e.%值最高,达93.64%,是野生菌NK13的16倍。【结论】从NK13号菌株中筛选得到的新的脂肪酶具有很高的不对称拆分获得(S)-酮基布洛芬的能力,实现了NK13菌中633bp脂肪酶基因在枯草芽孢杆菌中的分泌表达,研究证明Tween-80能提高该脂肪酶的拆分专一性。     

The forespore line of gene expression in Bacillus subtilis

Endospore formation by Bacillus subtilis involves three differentiating cell types, the predivisional cell, the mother cell, and the forespore. Here we report the program of gene expression in the forespore, which is governed by the RNA polymerase sigma factors sigma(F) and sigma(G) and the DNA-binding proteins RsfA and SpoVT. The sigma(F) factor turns on about 48 genes, including the gene for RsfA, which represses a gene in the sigma(F) regulon, and the gene for sigma(G). The sigma(G) factor newly activates 81 genes, including the gene for SpoVT, which turns on (in nine cases) or stimulates (in 11 cases) the expression of 20 genes that had been turned on by sigma(G) and represses the expression of 27 others. The forespore line of gene expression consists of many genes that contribute to morphogenesis and to the resistance and germination properties of the spore but few that have metabolic functions. Comparative genomics reveals a core of genes in the sigma(F) and sigma(G) regulons that are widely conserved among endospore-forming species but are absent from closely related, but non-spore-forming Listeria spp. Two such partially conserved genes (ykoU and ykoV), which are members of the sigma(G) regulon, are shown to confer dry-heat resistance to dormant spores. The ykoV gene product, a homolog of the non-homologous end-joining protein Ku, is shown to associate with the nucleoid during germination. Extending earlier work on gene expression in the predivisional cell and the mother cell, we present an integrated overview of the entire program of sporulation gene expression.     

Mutants of Bacillus subtilis defective in protein export

We have isolated a set of strains with mutations (designated prs) that decrease secretion of alpha-amylase and have a pleiotropic effect on secretion of other exoproteins. The seven mutants were selected in a strain of Bacillus subtilis which overproduces alpha-amylase due to the presence of an alpha-amylase gene on a multicopy plasmid. The mutations were mapped to four different chromosomal loci. The phenotype of the mutants, especially their pleiotropic effects and the accumulation of alpha-amylase precursor, indicated that they have defects in the mechanism of protein export. Double mutants with certain pairwise combinations of mutations in different loci had additive effects on secretion, suggesting that these prs genes encode different components of the secretion pathway.     

Patterns of gene expression in Bacillus subtilis colonies.

Bacillus subtilis 5:7, a derivative of macrofiber-producing strain FJ7, carries the lacZ reporter gene within Tn917 at an unknown location in the host genome. Expression of the host gene carrying lacZ within colonies of 5:7 was observed by examining growth under different conditions in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). At a high plating density small colonies arose that expressed the host gene early and throughout the colony, whereas at a low density large colonies were produced that expressed the host gene late in development and only in cells forming a ring pattern close to the colony periphery. A highly regulated spatial and temporal gene expression pattern was observed in growth from cross-streaks, suggesting that gene expression is responsive to concentration gradient fields established by neighboring growth. Colonies cultured on agar blocks revealed that expression was governed by depletion of a medium component and also by the geometry of the substrate upon which the colonies grew. At least three factors influenced the control of expression: (i) the concentration of a diffusible component of the medium exhausted by cell growth, (ii) a spatial-temporal factor related to growth within the colony, and (iii) the geometry of the growth substrate.     

Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.