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PubMedDate not supplied

Spore formation in Bacillus subtilis.

Bacillus subtilis is the best studied of the bacteria that make heat-resistant spores in response to starvation. The process of sporulation results in the formation of a cell type which is quite different in morphology from that of bacteria during normal growth. Sporulation requires the activation, in an ordered sequence, of many genes that are kept silent during vegetative growth. It also requires that these genes be activated differentially in two sister cells that are genetically identical. The sophisticated mechanisms responsible for both the temporal and the spatial regulation of gene expression are now understood in a fair amount of detail. They are likely to provide models which will make a valuable contribution to studies of development and differentiation in higher cells.

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↗

PubMed1993

Genetic mapping of bfmA mutation causing fatty acid deficiency in Bacillus subtilis.

Bacillus subtilis strain 626, defective in the bfmA gene, is a derivative derivatives of B. subtilis strain 168 and requires branched short-chain carboxylic acids for growth. Branched-chain 2-keto acid decarboxylase activity and fatty acid synthesis in B. subtilis strain 626 were 14 and 7%, respectively, of their levels in strain 626-2R, a spontaneously reverted strain. These results indicate that the bfmA mutation is in either the branched-chain 2-keto acid decarboxylase gene itself or its controlling gene. Thus, primer synthesis from the 2-keto acid substrate in strain 626 is defective, causing a deficiency in fatty acid synthesis. A bfmA mutation was transferred to a suitable genetic background and analysed. The bfmA strain, CAC1, was competent and motile, and required 50 microM CaCl2 or 5 microM FeCl3 for growth on glucose minimal agar plates. The bfmA gene was mapped between glyC (320 degrees) and ctrA (325 degrees) and estimated to be at 320 degrees by protoplast fusion and PBS1 phage transduction.

Bacillus subtilis↗

PubMed1980

Capacity for postreplication repair correlated with transducibility in Rec- mutants of Bacillus subtilis.

Bacillus subtilis strains deficient in transduction, transformation, or both were examined for the ability to remove pyrimidine dimers and to convert deoxyribonucleic acid newly synthesized after ultraviolet irradiation to high molecular weight. In one strain deficient in both recombination processes, short pieces of deoxyribonucleic acid synthesized after irradiation were not converted to high molecular weight. Two transformable strains deficient in transduction were also deficient in postreplication repair (i.e., joining of newly synthesized DNA fragments), whereas a nontransformable strain that was normal in transduction was proficient in postreplication repair. None of the transformable strains showed deficiencies in repair resynthesis or ligase activity. Our results suggest that some recombinational events may be common to transduction and postreplication repair but not to transformation, emphasizing the difference between these two pathways for genetic exchange.

Bacillus subtilis↗

PubMed2005

Genome-wide transcriptional analysis of the phosphate starvation stimulon of Bacillus subtilis.

Bacillus subtilis responds to phosphate starvation stress by inducing the PhoP and SigB regulons. While the PhoP regulon provides a specific response to phosphate starvation stress, maximizing the acquisition of phosphate (P(i)) from the environment and reducing the cellular requirement for this essential nutrient, the SigB regulon provides nonspecific resistance to stress by protecting essential cellular components, such as DNA and membranes. We have characterized the phosphate starvation stress response of B. subtilis at a genome-wide level using DNA macroarrays. A combination of outlier and cluster analyses identified putative new members of the PhoP regulon, namely, yfkN (2',3' cyclic nucleotide 2'-phosphodiesterase), yurI (RNase), yjdB (unknown), and vpr (extracellular serine protease). YurI is thought to be responsible for the nonspecific degradation of RNA, while the activity of YfkN on various nucleotide phosphates suggests that it could act on substrates liberated by YurI, which produces 3' or 5' phosphoribonucleotides. The putative new PhoP regulon members are either known or predicted to be secreted and are likely to be important for the recovery of inorganic phosphate from a variety of organic sources of phosphate in the environment.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

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↗

PubMed2002

Impact of the Mg(2+)-citrate transporter CitM on heavy metal toxicity in Bacillus subtilis.

Bacillus subtilis possesses a secondary transporter, CitM, that is specific for the complex of citrate and Mg(2+) but is also capable of transporting citrate in complex with the heavy metal ions Zn(2+), Ni(2+) and Co(2+). We report on the impact of CitM activity on the toxicity of Zn(2+), Ni(2+) and Co(2+) in B. subtilis. In a citM deletion mutant or under conditions in which CitM is not expressed, the toxic effects of the metals were reduced by the presence of citrate in the medium. In contrast, the presence of citrate dramatically enhanced toxicity when the Mg(2+)-citrate transporter was present in the membrane. It is demonstrated that the complex of Ni(2+) and citrate is transported into the cell and that the uptake is responsible for the enhanced toxicity. At toxic concentrations of the metal ions, the cultures adapted by developing tolerance against these ions. Tolerant cells isolated by exposure to one of the metal ions remained tolerant after growth in the absence of toxic metal ions and were cross-tolerant against the other two toxic ions. Tolerant strains were shown to contain point mutations in the citM gene, which resulted in premature termination of translation.

Adaptation, Physiological↗

PubMed1969

Phospholipid composition of Bacillus subtilis.

Bacillus subtilis contained at least five phospholipids, four of which have been isolated and identified as a polyglycerol phospholipid, probably cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, and lysylphosphatidylglycerol. Further purification of the latter phosphoglyceride was obtained by high-voltage electrophoresis, and it was shown that this treatment removed amino acid-containing, nonlipidic material from the phosphoglyceride. This associated material, which is not covalently linked to the lipid, gave rise to minor amounts of a number of amino acids, other than lysine, in acid hydrolysates of the lysylphosphatidylglycerol. The phospholipid composition of B. subtilis appeared to depend on the growth conditions. Addition of glucose to the medium lowered the pH during growth; this was accompanied by an increase in the amount of lysylphosphatidylglycerol and a decrease in the phosphatidylglycerol content, when compared with growth at neutral pH. The amount of the other phospholipids and the total amount of phospholipid remained constant under the different conditions. The shape and the osmotic susceptibility of the protoplasts of this organism appeared to depend on the growth conditions. Cells harvested from a neutral growth medium gave spherical protoplasts which lysed rapidly, whereas cells grown in an acidic medium maintained their rod-shaped form to a great extent after the cell wall had been removed, even after being suspended in a hypotonic medium. The latter observation suggests the presence of a more rigid membranous structure in cells which have been exposed to a low environmental pH during growth.

Bacillus subtilis↗

PubMed1980

Action of uncouplers of oxidative phosphorylation as chemotactic repellents of Bacillus subtilis.

Bacillus subtilis alternately swims smoothly and tumbles; when administered repellent it only tumbles, but later resumes normal swimming and tumbling. Repellents of B. subtilis include membrane-active agents like uncouplers of oxidative phosphorylation and local anaesthetics and have previously been found to act in a fundamentally different way compared with attractants. It has been suggested previously that uncouplers act as repellents as a result of their ability to depolarize the membrane and that depolarization might effect flagellar function by causing a flux of Ca2+ into the cell. However, we found that there is no correlation between membrane depolarization and chemotaxis and no detectable flux of Ca2+ following tactic stimulation by uncouplers. Experiments with analogues of the uncoupler pentachlorophenol, all of which are weaker acids than pentachlorophenol, indicated that the anionic form of the uncoupler is the potent form and we propose that it binds to a certain membrane protein to cause release into the cytoplasm of the substance (ion, metabolite or protein) that controls tumbling frequency. Adaptation is assumed to occur when this excess is removed by active transport or metabolism.

Bacillus subtilis↗

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↗

PubMed1994

The oxidative stress response in Bacillus subtilis.

Bacillus subtilis undergoes a typical bacterial stress response when exposed to low concentrations (0.1 mM) of hydrogen peroxide. Protection is thereby induced against otherwise lethal, challenge concentrations (10 mM) of this oxidant and a number of proteins are induced including the scavenging enzymes, catalase and alkyl hydroperoxide reductase, and a putative DNA binding and protecting protein. Induced protection against higher concentrations (10-30 mM) of hydrogen peroxide is eliminated in a catalase-deficient mutant. Both RecA and Spo0A influence the basal but not the induced resistance to hydrogen peroxide. A regulatory mutation has been characterized that affects the inducible phenotype and is constitutively resistant to high concentrations of hydrogen peroxide. This mutant constitutively overexpresses the proteins induced by hydrogen peroxide in the wild-type. The resistance of spores to hydrogen peroxide is partly attributable to binding of small acid soluble proteins by the spore DNA and partly to a second step which coincides with the depletion of the NADH pool, which may inhibit the generation of hydroxyl radicals from hydrogen peroxide.

Bacillus subtilis↗