Two cistrons of the gerC operon of Bacillus subtilis encode the two subunits of heptaprenyl diphosphate synthase.

The two proteins (GerC1 and GerC3) encoded by the gerC locus of Bacillus subtilis, which has been shown to be involved in vegetative cell growth and spore germination, were identified as dissociable heterodimers of the heptaprenyl diphosphate synthase involved in the biosynthesis of the side chain of menaquinone-7.     

Isolation of the ERG12 gene of Saccharomyces cerevisiae encoding mevalonate kinase

The ERG12 gene of Saccharomyces cerevisiae has been cloned by complementation of an erg12-1 mutation affecting mevalonate kinase. From the 2.8-kb insert isolated, the functional gene has been localized on a DNA fragment of 2.1 kb. The mRNA is 1.45 kb long. Gene disruption shows that the ERG12 gene is essential in yeast, both for spore germination and vegetative growth.     

A C-methyltransferase involved in both ubiquinone and menaquinone biosynthesis: isolation and identification of the Escherichia coli ubiE gene.

Strains of Escherichia coli with mutations in the ubiE gene are not able to catalyze the carbon methylation reaction in the biosynthesis of ubiquinone (coenzyme Q) and menaquinone (vitamin K2), essential isoprenoid quinone components of the respiratory electron transport chain. This gene has been mapped to 86 min on the chromosome, a region where the nucleic acid sequence has recently been determined. To identify the ubiE gene, we evaluated the amino acid sequences encoded by open reading frames located in this region for the presence of sequence motifs common to a wide variety of S-adenosyl-L-methionine-dependent methyltransferases. One open reading frame in this region (o251) was found to encode these motifs, and several lines of evidence that confirm the identity of the o251 product as UbiE are presented. The transformation of a strain harboring the ubiE401 mutation with o251 on an expression plasmid restored both the growth of this strain on succinate and its ability to synthesize both ubiquinone and menaquinone. Disruption of o251 in a wild-type parental strain produced a mutant with defects in growth on succinate and in both ubiquinone and menaquinone synthesis. DNA sequence analysis of the ubiE401 allele identified a missense mutation resulting in the amino acid substitution of Asp for Gly142. E. coli strains containing either the disruption or the point mutation in ubiE accumulated 2-octaprenyl-6-methoxy-1,4-benzoquinone and demethylmenaquinone as predominant intermediates. A search of the gene databases identified ubiE homologs in Saccharomyces cerevisiae, Caenorhabditis elegans, Leishmania donovani, Lactococcus lactis, and Bacillus subtilis. In B. subtilis the ubiE homolog is likely to be required for menaquinone biosynthesis and is located within the gerC gene cluster, known to be involved in spore germination and normal vegetative growth. The data presented identify the E. coli UbiE polypeptide and provide evidence that it is required for the C methylation reactions in both ubiquinone and menaquinone biosynthesis.     

Transitory germinative excision repair in Bacillus subtilis.

Bacillus subtilis strains UVSSP-42-1 (hcr42 ssp1) and UVSSP-1-1 (hcr1 ssp1) are ultraviolet (UV) radiation sensitive both as dormant spores and as vegetative cells. These strains are unable to excise cyclobutane-type dimers from the deoxyribonucleic acid (DNA) of irradiated vegetative cells and fail to remove spore photoproduct from the DNA of irradiated spores either by excision (controlled by gene hcr) or by spore repair (controlled by gene ssp1). When irradiated soon after spore germination, these strains excise dimers, but not spore photoproduct, from their DNA. This process, termed germinative excision repair, functions only transiently in the germination phase and is responsible for the high UV resistance of germinated spores and for their temporary capacity to host cell reactivate irradiated phages infecting them. The recA1 mutation confers higher UV sensitivity to the germinated spores, but does not interfere with dimer removal by germinative excision repair.     

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.     

Survival of Microorganisms in a Simulated Martian Environment: II. Moisture and Oxygen Requirements for Germination of Bacillus cereus and Bacillus subtilis var. niger Spores1

The effects of moisture and oxygen concentration on germination of Bacillus cereus and B. subtilis var. niger spores were investigated in a simulated Martian environment. Less moisture was required for germination than for vegetative growth of both organisms. A daily freeze-thaw cycle lowered moisture requirements for spore germination and vegetative growth of both organisms, as compared with a constant 35 C environment. Oxygen had a synergistic effect by lowing the moisture requirements for vegetative growth, and possibly germination, of both organisms. Oxygen was not required for spore germination of either organism, but was required for vegetative growth of B. subtilis and for sporulation of both organisms.     

Complete nucleotide sequence of the fumarase gene (citG) of Bacillus subtilis 168.

The nucleotide sequence of a 2.14 kb fragment of Bacillus subtilis DNA containing the citG gene encoding fumarase was determined using the dideoxy chain termination method. The citG coding region of 1392 base pairs (464 codons) was identified, and the deduced Mr (50425) is in good agreement with that of the protein identified from expression in Escherichia coli maxicells. There is no sequence homology between the B. subtilis and E. coli fumarases. Overlapping potential promoter sequences have been identified for sigma 28, sigma 37 and sigma 55 RNA polymerase holoenzymes. The DNA fragment also contains the proximal part of the gerA locus, responsible for L-alanine-sensitive spore germination.     

Identification of the nonA and nonB loci of Bacillus subtilis Marburg permitting the growth of SP10 phage

Mutational inactivation of both nonA and nonB genes are required for the permissiveness of Bacillus subtilis Marburg cells to infection by phage SP10. By transformational analysis of the nonA strain with DNAs from gently lysed protoplasts carrying the integrative plasmid pMUTIN (em) insertions in every 20 kb along the whole chromosome, we have identified the nonA to be the cured state of endogenous prophage SPbeta. Direct DNA sequencing, on the other hand, revealed one nonsense mutation of nonB in ydiR, which is a component gene of the intrinsic restriction system BsuMR of B.subtilis Marburg. Introduction of the wild type ydiR into the nonB strain at aprE locus resulted in complementation of nonB. Furthermore, as the SP10 genome was found to possess multiple BsuM target sites, it is considered that SP10 can infect and multiply in B.subtilis cells, which are SPbeta free and possess a defective BsuMR restriction system.     

苏云金芽孢杆菌芽孢萌发相关基因gerM的克隆及功能研究

依照蜡状芽孢杆菌gerM基因的保守序列设计引物,从苏云金芽孢杆菌中扩增出640bp的DNA片段。以此为探针,从苏云金芽孢杆菌部分基因组酶切文库中成功地克隆到了一个4·5kb的DNA片段。序列分析表明,该片段包含一个完整的开放阅读框,其预测的编码产物与枯草芽孢杆菌GerM蛋白具有很高的同源性,将该基因命名为gerM。RT-PCR分析表明,gerM基因仅在芽孢形成的过程中表达。通过同源重组的策略构建了gerM基因的阻断突变株。研究表明,gerM基因的破坏影响苏云金芽孢杆菌芽孢萌发的速率和比例。     

The spoIIN279(ts) mutation affects the FtsA protein of Bacillus subtilis.

The spo-279(ts) mutation, originally thought to be located in the spoIIG operon of Bacillus subtilis, has been mapped in close proximity but outside of the spoIIG locus. This mutation defines a new gene, spoIIN, located midway between the spoIIG and the spoVE loci, and whose product is required for successful completion of the asymmetric septation step. The spoIIN locus was cloned using a combination of 'walking steps' upstream from the spoIIG region and hybridization screening of a bacteriophage lambda library. Sequencing of DNA fragments able to rescue the spoIIN279(ts) mutation revealed that the spoIIN locus is identical with the B subtilis counterpart of the Escherichia coli ftsA gene. After cloning the ftsA region from a strain containing the spoIIN279(ts) mutation we found that this mutation converts the ninth residue of the FtsA protein from serine to asparagine. The spoIIN279(ts) mutation, which is recessive, leads to filamentation during growth at 42 degrees C and causes defective formation of the sporulation septum at this non-permissive temperature. The FtsA protein is therefore required for proper cell septation, both during vegetative growth and sporulation. Possible additional roles of FtsA during sporulation are discussed.     

H2, a temperate bacteriophage isolated from Bacillus amyloliquefaciens strain H

Bacillus amyloliquefaciens strain H is lysogenic for a large temperate phage we call H2. H2 has a polyhedral head 85 nm in diameter and a tail of about 17 x 434 nm. H2 lysogenizes Bacillus subtilis between the tyrA and metB genes, and gives specialized transduction of metB and, at lower frequencies, of ilvD and ilvA. The phage carries a thymidylate synthase gene and converts thymine auxotrophs of B. subtilis to prototrophy. The H2 genome is a linear DNA molecule about 129 kb in length. DNA extracted from phage particles grown in B. subtilis is not cut by the restriction endonucleases HaeIII, Fnu4HI, Bsp1286I, and BamHI; the latter enzyme is produced by B. amyloliquefaciens strain H. The prophage in lysogenic B. subtilis cells can be cut by these enzymes. We have isolated H2 mutants that carry the transposon Tn917, or a mutation resulting in clear-plaque morphology, or both.     

Molecular cloning and characterization of the Bacillus subtilis spore photoproduct lyase (spl) gene, which is involved in repair of UV radiation-induced DNA damage during spore germination.

Upon UV irradiation, Bacillus subtilis spore DNA accumulates the novel thymine dimer 5-thyminyl-5,6-dihydrothymine. Spores can repair this "spore photoproduct" (SP) upon germination either by the uvr-mediated general excision repair pathway or by the SP-specific spl pathway, which involves in situ monomerization of SP to two thymines by an enzyme named SP lyase. Mutants lacking both repair pathways produce spores that are extremely sensitive to UV. For cloning DNA that can repair a mutation in the spl pathway called spl-1, a library of EcoRI fragments of chromosomal DNA from B. subtilis 168 was constructed in integrative plasmid pJH101 and introduced by transformation into a mutant B. subtilis strain that carries both the uvrA42 and spl-1 mutations, and transformants whose spores exhibited UV resistance were selected by UV irradiation. With a combination of genetic and physical mapping techniques, the DNA responsible for the restoration of UV resistance was shown to be present on a 2.3-kb EcoRI-HindIII fragment that was mapped to a new locus in the metC-pyrD region of the B. subtilis chromosome immediately downstream from the pstI gene. The spl coding sequence was localized on the cloned fragment by analysis of in vitro-generated deletions and by nucleotide sequencing. The spl nucleotide sequence contains an open reading frame capable of encoding a 40-kDa polypeptide that shows regional amino acid sequence homology to DNA photolyases from a number of bacteria and fungi.     

Ultraviolet Sensitivity of Bacillus subtilis Spores upon Germination and Outgrowth

A strain of Bacillus subtilis, UVSSP-42-1, which produces ultraviolet (UV)-sensitive spores and vegetative cells, was found to possess germinated spores 25 times more UV resistant than the resting spores. This relative resistance achieved upon germination was associated with the transition of the heat-resistant refractile spores to the heat-sensitive phase-dark forms. Several generations of outgrowth were required before the cells attained the level of UV sensitivity characteristic of the vegetative cell. The UV sensitivity of germinated spores was compared with other strains with various combinations of mutations affecting deoxyribonucleic acid repair capabilities. The presence of hcr and ssp mutations which are known to abolish the removal of photoproducts from deoxyribonucleic acid did not alter significantly the sensitivity of the germinated forms. However, the addition of the recA mutation and, to some extent, the pol mutation increased the UV sensitivity of the germinated spores. These results indicate that deoxyribonucleic acid repair mechanisms dependent on the recA gene are active in the germinated spores. The chemical nature of the damage repaired by the recA gene product is not known. This study indicates that the life cycle of sporulating bacilli consists of at least three photobiologically distinct forms: spore, germinated spore, and vegetative cell.     

The isolation, cloning and identification of a vegetative catalase gene from Bacillus subtilis.

A Bacillus subtilis library of Tn917::lacZ insertions was screened for mutants that were unable to grow in the presence of normally sublethal concentrations of hydrogen peroxide. The identification and subsequent analysis of one mutant strain, YB2003, which carried the mutation designated kat-19, revealed that this strain was deficient in the expression of a vegetative catalase. Regions of the chromosome both 5' and 3' to the site of the Tn917 insertion, as well as the gene without the insertion (kat-19+) were cloned. The presence of the functional kat-19+ gene on a high-copy plasmid restored catalase activity to the kat-19::Tn917 strain as well as to strains of B. subtilis that carried the katA 1 mutation. While the katA+ locus is believed to represent the structural gene for the vegetative catalase of B. subtilis [Loewen and Switala, J. Bacteriol. 169 (1987) 5848-5851], the sequence analysis of the cloned kat-19+ DNA fragments revealed an open reading frame that showed significant homology between the deduced amino acid sequence of this gene product and that of known eukaryotic catalases.     

Modulation of deoxyribonucleic acid polymerase III level during the life cycle of Bacillus subtilis.

Deoxyribonucleic acid (DNA) polymerase III is not detectable in Bacillus subtilis spores; the enzyme activity appears 20 to 30 min after spore activation and rapidly increases just before the onset of the first round of DNA replication (30 min later); the level of polymerase III further increases and reaches its maximum (on a per-genome basis) when the cells enter the vegetative phase of growth; this level is six- to eightfold higher than the one observed during germination. In the stationary phase, the polymerase III drops to levels comparable to those found in germinating spores at the first round of replication. On the contrary, DNA polymerase I is present at appreciable levels in the dormant spore; it increases during vegetative growth by a factor of three and, during the stationary phase, reaches its maximum level which is sixfold higher than that observed in the spores. The block of protein synthesis during vegetative growth does not cause an appreciable reduction of the two enzymes (in absolute terms), showing that the regulation of their levels is probably not due to a balance between synthesis and breakdown. These results indicate that polymerase III is probably one of the factors controlling the initiation of DNA synthesis during spore germination.     

Cloning of an unstable spoIIA-tyrA fragment from Bacillus subtilis

A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spoVA or spoIVA (spoIIA+) strains, subclones of pRC12, lacking a functional spoIIA gene, did complement these mutations. pRC12 inhibited sporulation in a spo+ recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.     

PCR detection of potentially pathogenic aeromonads in raw and cold-smoked freshwater fish

AIMS: To determine the mechanism of killing of spores of Bacillus subtilis by ortho-phthalaldehyde (OPA), an aromatic dialdehyde currently in use as an antimicrobial agent. METHODS AND RESULTS: OPA is sporicidal, although spores are much more OPA resistant than are vegetative cells. Bacillus subtilis mutants deficient in DNA repair, spore DNA protection and spore coat assembly have been used to show that (i) the coat appears to be a major component of spore OPA resistance, which is acquired late in sporulation of B. subtilis at the time of spore coat maturation, and (ii) B. subtilis spores are not killed by OPA through DNA damage but by elimination of spore germination. Furthermore, OPA-treated spores that cannot germinate are not recovered by artificial germinants or by treatment with NaOH or lysozyme. CONCLUSIONS: OPA appears to kill spores by blocking the spore germination process. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides information on the mechanism of spore resistance to, and spore killing by, the disinfectant, OPA.     

Identification of the sporulation gene spoOA product of Bacillus subtilis

A 2.4-kilobase fragment of the Bacillus subtilis chromosome containing the wild-type spoOA gene derived from the phi 105dspoOA+-Bc-1 transducing phage was cloned onto plasmid pBR322 in Escherichia coli. A recombinant plasmid harboring the mutant spoOA12 allele on the 2.4-kilobase insert was also constructed from the phi 105dspoOA12-1 phage DNA and pBR322. Protein products synthesized in response to plasmid DNA in a DNA-directed cell-free system derived from E. coli were analyzed by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. A protein of approximately 27,500 daltons synthesized with the recombinant plasmid DNA harboring the wild-type spoOA gene as template was not formed with the recombinant plasmid DNA harboring the spoOA12 allele. Since the spoOA12 mutation is a nonsense mutation, we conclude that the 27.5-kilodalton protein is the product of the spoOA gene.