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PubMed1987

A new mutator strain of Bacillus subtilis.

Bacillus subtilis strain SB1207, widely used in our laboratory, was found to be highly temperature-sensitive and to exhibit a strong SOS-independent mutator phenotype at elevated temperatures. Both chromosomal and plasmid-borne genes were affected by the mutator. Lethality and mutator phenotype could not be attributed to a replication shut off or to thymine starvation. Due to the high frequency of base misincorporation, the mutator phenotype probably results from an editing defect rather than from a post-replication defect (mismatch repair).

Bacillus subtilis↗

PubMed1978

Further studies on the competence development in exponentially growing cultures of Bacillus subtilis.

Bacillus subtilis, growing in Bott and Wilson's medium, develops two peaks of competence in batch cultures. The first maximum developed during the exponential growth phase and it has been studied comparatively with the competence level reached in continuous culture at Dt = 2.5 h. In both cases, 100 microgram/ml of arginine inhibited competence. Continuous cultures treated with arginine recovered competence specifically after the addition of Mn2+. In addition, a reduction in the synthesis of aconitase and fumarase was observed in the arginine-inhibited continuous cultures.

Aconitate Hydratase↗

PubMed1977

Purification and comparative properties of the delta and sigma subunits of RNA polymerase from Bacillus subtilis.

Bacillus subtilis delta protein is a 21 500-Mr polypeptide that can be isolated in association with RNA polymerase holoenzyme from uninfected bacteria and with modified forms of RNA polymerase from cells infected with phage SP01 [Pero, J., Nelson, J. and Fox, T. (1975) Proc. Natl Acad. Sci. U.S.A. 72,1589]. Although no function has been assigned to delta protein in uninfected cells, this host polypeptide enhances the specificity of transcription by phage-modified forms of RNA polymerase that contain SP01-coded regulatory subunits. This report describes the purification of delta and sigma proteins from uninfected B. subtilis and examines the comparative effects of these polypeptides on transcription by core RNA polymerase. Purified sigma polypeptide was found to stimulate the transcription of phage DNA while having little effect on RNA synthesis with the synthetic DNA poly(dA-dT) as template. In contrast, purified delta protein markedly depressed the transcription of poly(dA-dT) while having little effect on enzyme activity with phage DNA as template. The inhibitory effect of delta protein on poly (dA-dT) transcription was strongly dependent on the presence of KC1 in the RNA synthesis reaction mixture.

Bacillus subtilis↗

PubMed1976

Effect of methionine on chemotaxis by Bacillus subtilis.

Bacillus subtilis, like Escherichia coli and Salmonella typhimurium, carries out chemotaxis by modulating the relative frequency of smooth swimming and tumbling. Like these enteric bacteria, methionine auxotrophs starved for methionine show an abnormally long-period of smooth swimming after addition of attractant. This "hypersensitive" state requires an hour of starvation for its genesis, which can be hastened by including alanine, a strong attractant, in starvation medium. Susceptibility to repellent, which causes transient tumbling when added, if anything, increases slightly by starvation for methionine. The results are interpreted by postulating the existence of a methionine-derived structure that hastens recovery of attractant-stimulated bacteria back to normal.

Alanine↗

PubMedDate not supplied

Utilization of one promoter by two forms of RNA polymerase from Bacillus subtilis.

Bacillus subtilis possesses several forms of RNA polymerase, each differing in its sigma subunit and its specificity of promoter recognition. The sequential appearance of sigma subunits, which change the promoter recognition specificity of RNA polymerase, may have a key role in controlling the temporal pattern of gene expression required for endospore development in B. subtilis. Several genes that are expressed over relatively long periods of time during the developmental cycle are transcribed by more than one form of RNA polymerase, which initiate transcription from either tandem or overlapping promoter. The promoter region for the ctc gene is interesting because transcription is initiated at or near the same position by both sigma 37 RNA polymerase (E sigma 37), a minor form in growing cells, and sigma 29 RNA polymerase (E sigma 29), a form which appears approximately 2 h after the initiation of sproulation. Here we report that several base substitutions in the ctc promoter differentially affect the utilization of the promoter by E sigma 37 or E sigma 29.

Bacillus subtilis↗

PubMed1989

Influence of spo mutations on sigma E synthesis in Bacillus subtilis.

Bacillus subtilis mutants blocked at the same stage of development (stage II) as strains with mutations in the structural gene for sigma E (sigE[spoIIGB]) were analyzed immunologically for sigma E and its precursor protein, P31. Mutations at spoIIL, spoIIN, and spoIIJ loci but not at the spoIIM locus significantly reduced P31 formation. Mutations at the spoIIAA, spoIIAC, spoIIEA, spoIIEB, and spoIIEC loci did not affect P31 synthesis but blocked its processing into sigma E. These results demonstrate a requirement for at least eight stage II gene products in the developmental pathway which leads to sigma E and brings to 11 the number of stage II genes (including spoIIGA, spoIIGB, and spoIIF) now known to be needed for sigma E formation.

Bacillus subtilis↗

PubMed1987

Studies on cellulase production by a Bacillus subtilis.

Bacillus subtilis AU-1 was found to produce carboxymethylcellulase (CMCase) and Avicelase activities in the culture supernatant when grown on a variety of carbohydrates as major carbon source. Maximum CMCase production was obtained in a liquid medium containing 0.2% D (+) raffinose as inducer, 0.5% each of yeast extract, casamino acids and proteose peptone at 50 degrees C and at an initial pH of 6.0. CMCase activity was detected at early log phase of growth, and reached the maximum level at early stationary phase of growth which occurred at the 10th hour of cultivation. The optimal temperature for CMCase activity was 65 degrees C, and the enzyme was highly stable up to 60 degrees C. CMCase synthesis was subjected to catabolite repression by glucose and cellobiose.

Bacillus subtilis↗

PubMed1992

Purification and characterization of recombinant pyrrolidone carboxyl peptidase of Bacillus subtilis.

Bacillus subtilis pyrrolidone carboxyl peptidase (Pcp) overexpressed in Escherichia coli was purified to homogeneity in less than 12 h using ammonium sulphate precipitation and hydrophobic interaction chromatography. The enzyme, which removes amino-terminal L-pyroglutamic acid from peptides, appears to be a tetramer of 25,200 molecular mass subunits. The protein cross-reacted with polyclonal antibodies raised against Pcp from Streptococcus pyogenes. The overexpressed enzyme exhibits an absolute substrate specificity towards N-terminal pyroglutamyl residues with a Michaelis constant of 1.04 mM for L-pyroglutamyl-beta-naphthylamide. The enzyme could be used for the removal of pyroglutamyl residues that block amino termini of proteins and peptides before performing Edman sequential degradation.

Amino Acid Sequence↗

PubMed1975

Identification of poly-gamma-glutamyl chain lengths in folates of Bacillus subtilis.

Bacillus subtilis strains 168 met ile leu and 23 thy contain folates which differ from one another in the number of glutamyl residues. The folate species were identified by reductive cleavage to the corresponding p-aminobenzoylglutamyl poly-gamma-glutamates and chromatography on diethylaminoethyl-cellulose. Pteroyltriglutamate is the predominant folate type, accounting for 86 to 88% of the total. Pteroyltetraglutamate is the only other type present in appreciable quantities, accounting for 5 to 6% of the total folates. Pteroyldiglutamate and pteroylpentaglutamate are present in small amounts, accounting for 1 to 3% and 1% of the total folates, respectively. Strain 168 met ile leu contains a very small amount of pteroylmonoglutamate (less than 0.5% of the total folates), but the other strain contains none.

Bacillus subtilis↗

PubMed1991

Regulation of the oxidative stress response by the hpr gene in Bacillus subtilis.

Bacillus subtilis mutants with null mutations in the spo0 A gene are resistant to oxidative stress during the exponential phase of growth. This resistance phenotype can be suppressed by mutations in the abrB gene, or in the hpr gene. Both of these gene products are negative regulatory proteins which are over-produced in a spo0 A strain, and the over-production of the hpr gene product results from over-production of the abrB gene product. The results suggested that the resistance to oxidative stress in a spo0 A strain is due to the lack of a protein directly controlled by the hpr negative regulator. Other mutations in the spo0 A gene conferring resistance to ethanol stress (eth) or suppressors of sporulation phenotypes (sof) had no effect on the sensitivity to oxidative stress of strains bearing them.

Bacillus subtilis↗

PubMed1992

Stress proteins and cross-protection by heat shock and salt stress in Bacillus subtilis.

Bacillus subtilis induced a set of general stress proteins in response to a salt or heat stress. Cells subjected to a mild heat stress showed a protective response which enabled them to survive otherwise lethal temperatures (e.g. 52 degrees C). In a similar way bacteria were enabled to survive toxic concentrations of NaCl by pretreatment with lower salt concentrations. A mild heat shock induced a cross-protection against lethal salt stress. The pretreatment of cells with low salt, however, was less effective in the induction of thermotolerance than a preceding mild heat stress. Three stress proteins were identified on the basis of their N-terminal amino acid sequences as homologues of GroEL, DnaK and ClpP of Escherichia coli. The role of general and specific stress proteins in the induction of thermotolerance/salt tolerance and cross-protection is discussed.

ATP-Dependent Proteases↗

PubMed1977

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.

Bacillus subtilis↗

PubMed1987

Evidence for an intermediate methyl-acceptor for chemotaxis in Bacillus subtilis.

Bacillus subtilis responds to chemotactic attractants by demethylating certain membrane-bound proteins, termed methyl-accepting chemotaxis proteins (MCPs) and by augmenting the evolution of methanol. We propose that the methanol comes from a methylated intermediate rather than directly from the MCPs themselves. First, repellent blocks attractant-induced smooth swimming and methanol formation, but not MCP demethylation. Second, prior treatment of cells with much attractant to reduce radiolabeling of MCPs and increase that of the putative intermediate caused increased, rather than decreased, production of methanol upon addition and then removal of the repellent. Third, such cells also produced much, rather than little, methanol upon addition of less attractant than during the pretreatment. We speculate that unmethylated intermediate causes tumbling; attractant causes its methylation and hence absence of tumbling (smooth swimming). Its demethylation during the period of smooth swimming affords adaptation.

Aspartic Acid↗

PubMed1986

Determination of the signal peptidase cleavage site in the preprosubtilisin of Bacillus subtilis.

Bacillus subtilis subtilisin is predicted to be synthesized as a preproenzyme according to the sequence analysis of its gene. We have synthesized the [35S] methionine-labeled preprosubtilisin in vitro and processed the precursor to prosubtilisin by the addition of membrane vesicles derived from vegetative cells of B. subtilis and Triton X-100. Radiosequencing of the prosubtilisin allowed the precise determination of the signal peptidase cleavage site. The preprosubtilisin was found to have a 29-amino-acid-long signal peptide with the signal peptidase cleavage sequence of AlaGln-AlaAla. Fusion of the signal peptide sequence to the mature TEM beta-lactamase structural gene allowed the production of an active and secreted form of beta-lactamase in vivo. An N-terminal sequence analysis of this product indicated that the observed in vivo signal peptidase cleavage site was exactly the same as that determined by in vitro analysis. During the development of the in vitro processing system, we demonstrated that the replacement of the subtilisin transcription regulatory sequence by a vegetative promoter allowed the vegetative expression and secretion of subtilisin. Thus, the late expression of the native subtilisin gene is mainly controlled at the transcription level and the secretion/processing systems are available for vegetative production of subtilisin.

Amino Acid Sequence↗

PubMed1992

The spo0A gene is implicated in the maintenance of non-complementing diploids in Bacillus subtilis.

Bacillus subtilis can exist in a diploid state in which two genetically distinct chromosomes co-exist in the same cell and yet only one of them is expressed, thereby determining the phenotype. Such cells are called non-complementing diploids (Ncds). In this study, two types of experiments are reported which indicate that a previously known pleiotropic gene, spo0A, plays a role in the maintaining the diploid state, as follows. (i) When protoplasts of two Spo0A mutant strains were fused, the resulting products continued to segregate cells of both parental phenotypes for many more divisions than had been reported previously. (ii) When a stable Ncd (an Ncd in which the unexpressed markers are not spontaneously activated at a detectable level) harbouring a chloramphenicol acetyltransferase gene on the silent chromosome was transformed with spo0A null alleles the transformants often expressed chloramphenicol acetyltransferase activity. Together these results indicate that the spo0A gene is involved in maintenance of the diploid state in both unstable and stable Ncds.

Bacillus subtilis↗

PubMed1987

Kinetic characterization of cytochrome c oxidase from Bacillus subtilis.

Bacillus subtilis aa3-type cytochrome c oxidase is capable of oxidizing cytochrome c from different origins. The kinetic properties of the enzyme are influenced by ionic strength. The affinity for Saccharomyces cerevisiae cytochrome c declines with increasing ionic strength whereas the Vmax remains almost constant. An increase of Vmax is observed when the enzyme is incorporated in artificial membranes. Negatively charged phospholipids allow high turnover rates of the aa3-type oxidase. The effect of ionic strength on oxidation of horse heart cytochrome c results in significant changes of both Km and Vmax. These effects can be explained by disturbances of enzyme-substrate interactions and are not related to changes in the aggregation state of the enzyme. The respiration control index of the enzyme reconstituted in artificial membranes appeared to be dependent on phospholipid composition, protein/lipid ratios and also on the external pH. The action of the ionophores nigericin and valinomycin, at various pH values, on the enzyme activity and proton-permeability measurements of the membranes indicate that both components of the proton-motive force, the membrane potential and the pH gradient, can in principle regulate enzyme activity in the reconstituted state.

Animals↗

PubMed1987

Plasmid marker rescue transformation proceeds by breakage-reunion in Bacillus subtilis.

Bacillus subtilis carrying a plasmid which replicates with a copy number of about 1 was transformed with linearized homologous plasmid DNA labeled with the heavy isotopes 2H and 15N, in the presence of 32Pi and 6-(p-hydroxyphenylazo)-uracil to inhibit DNA replication. Plasmid DNA was isolated from the transformed culture and fractionated in cesium chloride density gradients. The distribution of total and donor plasmid DNA was examined, using specific hybridization probes. The synthesis of new DNA, associated with the integration of donor moiety, was also monitored. Donor-specific sequences were present at a density intermediate between that of light and hybrid DNA. This recombinant DNA represented 1.4% of total plasmid DNA. The latter value corresponded well with the transforming activity (1.7%) obtained for the donor marker. Newly synthesized material associated with plasmid DNA at the recombinant density amounted to a minor portion of the recombinant plasmid DNA. These data suggest that, like chromosomal transformation, plasmid marker rescue transformation does not require replication for the integration of donor markers and, also like chromosomal transformation, proceeds by a breakage-reunion mechanism. The extent of donor DNA replacement of recipient DNA per plasmid molecule of 54 kilobases (27 kilobase pairs) was estimated as 16 kilobases.

Bacillus subtilis↗

PubMed1988

Gene encoding sigma E is transcribed from a sigma A-like promoter in Bacillus subtilis.

Bacillus subtilis produces several RNA polymerase sigma factors. At least two of these factors are essential for endospore formation, sigma H, which is present in vegetative cells, and sigma E, which is produced exclusively after the start of endospore formation. The structural gene that encodes sigma E is part of the spoIIG operon, which is transcribed after the onset of sporulation. We have determined the starting point of transcription and the nucleotide sequence of the spoIIG promoter. This promoter contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by E sigma A, the primary form of RNA polymerase in vegetative cells. The unusual feature of this promoter is that these putative sigma A contact sites are separated by 22 base pairs, rather than the typical 17 or 18 base pairs. Single-base substitutions in the -10-like sequence reduced utilization of the spoIIG promoter in vivo. Furthermore, E sigma A, but not E sigma H and other secondary forms of RNA polymerase, accurately initiated transcription from the spoIIG promoter in an in vitro assay; therefore, we suggest that E sigma A transcribes the spoIIG operon in vivo. A base substitution in the -35-like sequence caused constitutive transcription from the promoter in vegetative cells; therefore, regulation of this sporulation-specific transcription may involve a novel mechanism.

Bacillus subtilis↗