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PubMed1999

Microbiological efficacy of superheated steam. I. Communication: results with spores of Bacillus subtilis and Bacillus stearothermophilus and with spore earth.

For the spores of Bacillus subtilis and Bacillus stearothermophilus as well as for spore earth (acc. DIN 58,946 Part 4 of August 1982), the dependence of resistance on the superheating of the steam used to kill germs was determined. A material (glass fibre fleece) was used as the germ carrier which does not superheat on contact with steam. The temperature of the saturated steam was 100 degrees C (B. subtilis) and 120 degrees C (B. stearothermophilus and spore earth). The yardstick for the resistance of the spores or bioindicators was the exposure period of the saturated or superheated steam at which 50% of the treated test objects no longer showed any viable test germs. The spores of Bacillus subtilis were far more sensitive to superheating of steam and reacted far more than the spores of Bacillus stearothermophilus and the germs in the spore earth. When superheating by 4 Kelvin the spores of Bacillus subtilis were approximately 2.5 times more resistant than they were to saturated steam. The resistance of Bacillus stearothermophilus and spore earth was only slightly higher up to superheating by 10 Kelvin. The spores of Bacillus subtilis had the highest resistance during superheating by 29 Kelvin; they were 119 times more resistant than they were to saturated steam. The resistance maximum of the spores of Bacillus stearothermophilus was at an superheating by around 22 Kelvin. However, the spores were only 4.1 times more resistant than they were to saturated steam. When using steam to kill germs, we must expect superheated steam. This raises the question whether the spores of Bacillus stearothermophilus, with their weaker reaction to the superheating of steam, are suitable as test germs for sterilisation with steam in all cases.

Bacillus subtilis↗
PubMed2003

Recognition of DNA by three ferric uptake regulator (Fur) homologs in Bacillus subtilis.

Bacillus subtilis contains three Fur homologs: Fur, PerR, and Zur. Despite significant sequence similarities, they respond to different stimuli and regulate different sets of genes. DNA target site comparisons indicate that all three paralogs recognize operators with a core 7-1-7 inverted repeat. The corresponding consensus sequences are identical at five or more of the seven defined positions. Using site-directed mutagenesis, the Per box at the mrgA promoter was altered to mimic the core 7-1-7 motif of the Fur and Zur boxes. In vitro, the mrgA promoter containing a Zur box was only recognized by Zur, as demonstrated by DNase I footprinting assays. In contrast, both Fur and PerR bound to the mrgA promoter region containing a consensus Fur box. Expression analysis of these promoters is consistent with the in vitro data demonstrating as few as 1 or 2 base changes per half-site are sufficient to alter regulation. Similarly, the Fur box at the feuA promoter can be converted into a Per or a Zur box by appropriate mutations. While both Fur and PerR could recognize some of the same synthetic operator sequences, no naturally occurring sites are known that are subject to dual regulation. However, the PerR-regulated zosA gene is controlled from a regulatory region that contains both Per and Fur boxes. Although purified Fur protein bound to the candidate Fur boxes, Fur has little effect on zosA expression-possibly due to the location of the Fur boxes relative to the zosA promoter. Together, our results identify two nucleotide positions that are important for the ability of PerR, Fur, and Zur to distinguish among the many closely related operator sites present in the B. subtilis genome.

Bacillus subtilis↗
PubMed1988

Molecular cloning of a tetracycline-resistance determinant from Bacillus subtilis chromosomal DNA and its expression in Escherichia coli and B. subtilis.

Bacillus subtilis GSY908 DNA fragments (5.1 and 4.4 kilobase pairs (kb)) containing a tetracycline-resistance determinant were cloned in Escherichia coli using a shuttle plasmid vector pLS353. Restriction endonucelase analysis showed that the 4.4 kb fragment is a spontaneous deletion derivative of the 5.1 kb fragment. E. coli tetracycline-resistance transformants carrying pLS353 with the 5.1 kb fragment (named pTBS1) and that with 4.4 kb fragment (pTBS1.1) could grow at tetracycline concentrations up to 80 and 50 micrograms per ml, respectively. B. subtilis MI112 and RM125 were transformed by pTBS1, resulting in isolation of transformants of MI112 maintaining pTBS1 and RM125 maintaining either pTBS1 or pTBS1.1. Maximum tetracycline concentrations permitting growth of plasmidless MI112 and MI112 with pTBS1 were 4 and 10 micrograms per ml, respectively, while those of plasmidless RM125, RM125 with pTBS1 and RM125 with pTBS1.1 were 7, 50 and 80 micrograms per ml, respectively. It was interesting to note that the tetracycline-resistance level in E. coli conferred by the 5.1 kb fragment is higher than that conferred by the 4.4 kb fragment, but in B. subtilis the 4.4 kb fragment, in contrast, confers a higher level of tetracycline resistance. The level of tetracycline resistance in B. subtilis conferred by the cloned determinant clearly depends on the host strain. The tetracycline resistance conferred by the cloned determinant was associated with decreased accumulation of the drug into the cells. However, it was constitutive in E. coli, but inducible in B. subtilis. The cloned tetracycline-resistance determinant was detected specifically on the chromosome of B. subtilis Marburg 168 derivatives.

Bacillus subtilis↗
PubMed1976

Uptake of branched-chain alpha-keto acids in Bacillus subtilis.

Bacillus subtilis has a constitutive system for the uptake of alpha-keto-beta-methylvalerate, alpha-ketoisovalerate, and (probably) alpha-ketoisocaproate. A mutation, kauA1, which blocks the uptake of alpha-keto-beta-methylvalerate and alpha-ketoisovalerate, is located between metB and citK on the B. subtilis chromosome.

Bacillus subtilis↗
PubMed1990

Heat-inducible translational coupling in Bacillus subtilis.

Bacillus subtilis plasmid pGR71 is a promoter-probe shuttle vector derived from pUB110. The expression of the cat gene on pGR71 in B. subtilis requires the insertion of a Bacillus promoter and a ribosomal binding site (RBS) into the HindIII cloning site immediately upstream from the cat gene. A recombinant plasmid of pGR71, named pGR71-369, was obtained by a spontaneous deletion of a fragment containing most of the inserted HindIII fragment and the replication origin necessary for multiplication in Escherichia coli. The expression of the cat gene in B. subtilis cells carrying this plasmid was inducible by heat. Nucleotide sequence analysis of the upstream region of the cat gene, deletion analysis, and dot blot hybridization analysis of mRNA in various conditions revealed that the cat gene was expressed by heat-inducible translational coupling and that the regulatory region of heat inducibility was present in the upstream region of the cat gene.

Bacillus subtilis↗
PubMed1994

DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis.

Bacillus subtilis growing at 37 degrees C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37 degrees C is transferred to 20 degrees C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20 degrees C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20 degrees C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37 degrees C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.

Bacillus subtilis↗
PubMed1998

Molecular cloning and nucleotide sequence of the superoxide dismutase gene and characterization of its product from Bacillus subtilis.

Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2, 600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designated sodA, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region of sodA. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutant Escherichia coli sodA sodB, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of B. subtilis sodA.

Amino Acid Sequence↗
PubMed1994

Decrease in spermidine content during logarithmic phase of cell growth delays spore formation of Bacillus subtilis.

Bacillus subtilis 168M contained a large amount of spermidine during the logarithmic phase of growth, but the amount decreased drastically during the stationary phase. The extracts, prepared from B. subtilis cells harvested in the logarithmic phase, contained activity of arginine decarboxylase (ADC) rather than the activity of ornithine decarboxylase. In the presence of alpha-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of ADC, the amount of spermidine in B. subtilis during the logarithmic phase decreased to about 25% of the control cells. Under these conditions, spore formation of B. subtilis 168M delayed greatly without significant inhibition of cell growth. The decrease in spermidine content in the logarithmic phase rather than in the stationary phase was involved in the delay of sporulation. Electron microscopy of cells at 24 hrs. of culture confirmed the delay of spore formation by the decrease of spermidine content. Furthermore, the delay of sporulation was negated by the addition of spermidine. These data suggest that a large amount of spermidine existing during the logarithmic phase plays an important role in the sporulation of B. subtilis.

Arginine↗
PubMed1984

Promoter recognition by sigma-37 RNA polymerase from Bacillus subtilis.

Bacillus subtilis possesses at least five different forms of RNA polymerase holoenzyme which are distinguished by their sigma subunit and their promoter recognition specificity. Sigma-37 RNA polymerase, a minor form of RNA polymerase, recognizes a class of promoters, which includes promoters for genes transcribed early during endospore formation. We have used site-directed bisulfite mutagenesis to construct a series of single and multiple base substitutions in a promoter recognized by sigma-37 RNA polymerase. In vitro transcription analysis of this series of mutant promoters demonstrated that single base substitutions at positions -36, -16, -15 and -14 most dramatically reduced the efficiency of promoter utilization by sigma-37 RNA polymerase. These results support a model in which sigma-37 RNA polymerase recognizes its cognate promoters by interacting with a sequence of nucleotides near the -10 region and the -35 region of the promoter--a sequence not recognized by B. subtilis sigma-55 RNA polymerase or Escherichia coli RNA polymerase.

Bacillus subtilis↗
PubMed1993

Heme b (protoheme IX) is a precursor of heme a and heme d in Bacillus subtilis.

Bacillus subtilis can synthesise cytochromes containing a-, b-, c- and d-type heme. The biosynthetic pathways of these heme prosthetic groups were investigated by using strains blocked in uroporphyrinogen III synthesis from porphobilinogen or in heme b (protoheme IX) synthesis from uroporphyrinogen III. The results strongly suggest that heme a and heme d are both synthesised from heme b (protoheme IX). They also indicate that B. subtilis contains a novel ferrochelatase involved in the synthesis of siroheme.

Bacillus subtilis↗
PubMed1985

Isolation and characterization of a cis-acting mutation conferring catabolite repression resistance to alpha-amylase synthesis in Bacillus subtilis.

Bacillus subtilis 168GR10 was shown to contain a mutation, gra-10, which allowed normal temporal activation of alpha-amylase synthesis in the presence of a concentration of glucose that is inhibitory to activation of amylase synthesis in the parent strain, 168. The gra-10 mutation was mapped by phage PBS-1-mediated transduction and by transformation to a site between lin-2 and aroI906, very tightly linked to amyE, the alpha-amylase structural gene. The gra-10 mutation did not pleiotropically affect catabolite repression of sporulation or of the synthesis of extracellular proteases or RNase and was unable to confer glucose-resistance to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 gene driven by the amyE promoter region (amyR1) inserted into the promoter-probe plasmid pPL603B. It therefore appears that gra-10 defines a cis-regulatory site for catabolite repression, but not for temporal activation, of amyE expression. The evidence shows that temporal activation and glucose-mediated repression of alpha-amylase synthesis in B. subtilis 168 are distinct phenomena that can be separated by mutation.

Bacillus subtilis↗
PubMed1996

Two-component regulatory proteins ResD-ResE are required for transcriptional activation of fnr upon oxygen limitation in Bacillus subtilis.

Bacillus subtilis can grow anaerobically in the presence of nitrate as a terminal electron acceptor. The two component regulatory proteins, ResD and ResE, and an anaerobic gene regulator, FNR, were previously shown to be indispensable for nitrate respiration in B. subtilis. Unlike Escherichia coli fnr, B. subtilis fnr transcription was shown to be highly induced by oxygen limitation. fnr is transcribed from its own promoter as well as from a promoter located upstream of narK, the first gene in the narK-fnr dicistronic operon. DNA fragments containing the narK promoter, the fnr promoter, and both of the promoters were used to construct three lacZ fusions to examine the transcriptional regulation of the narK-fnr operon. ResDE was found to be required for transcriptional activation of fnr from the fnr-specific promoter, and FNR was required for activation of narK-fnr transcription from the FNR-dependent narK operon promoter under anaerobiosis. In order to determine if the requirement for ResDE in nitrate respiration is solely to activate fnr transcription, fnr was placed under control of the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter, Pspac. The observed defect in anaerobic growth of a Pspac-fnr delta resDE mutant in the presence of IPTG indicated that resDE has an additional role in B. subtilis anaerobic gene regulation.

Anaerobiosis↗
PubMed1999

Identification and characterization of a stress-responsive promoter in the macromolecular synthesis operon of Bacillus subtilis.

Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant. Induction of sigmaA has been observed exclusively in this mutant harbouring extra copies of the plasmid-borne Ts sigA gene transcriptionally controlled by the P1P2 promoters of the B. subtilis macromolecular synthesis (MMS; rpoD or sigA) operon. Investigation of the mechanisms leading to the induction has allowed us to identify a sigmaB-type promoter, P7, in the MMS operon for the first time. Therefore, at least seven promoters in total are responsible for the regulation of the B. subtilis MMS operon, including the four known sigmaA- and sigmaH-type promoters, as well as two incompletely defined promoters. The P7 promoter was activated in B. subtilis after the imposition of heat, ethanol and salt stresses, indicating that the MMS operon of B. subtilis is subjected to the control of general stress. The significant heat induction of P7 in B. subtilis DB1005 harbouring a plasmid-borne Ts sigA gene can be explained by a model of competition between sigmaA and sigmaB for core binding; very probably, the sigmaB factor binds more efficiently to core RNA polymerase under heat shock. This mechanism may provide a means for the expression of the B. subtilis MMS operon when sigmaA becomes defective in core binding.

Bacillus subtilis↗
PubMed1994

Purification and characterisation of a water-soluble ferrochelatase from Bacillus subtilis.

Bacillus subtilis ferrochelatase is encoded by the hemH gene of the hemEHY gene cluster and catalyses the incorporation of Fe2+ into protoporphyrin IX. B. subtilis ferrochelatase produced in Escherichia coli was purified. It was found to be a monomeric, water-soluble enzyme of molecular mass 35 kDa which in addition to Fe2+ can incorporate Zn2+ and Cu2+ into protoporphyrin IX. Chemical modification experiments indicated that the single cysteine residue in the ferrochelatase is required for enzyme activity although it is not a conserved residue compared to other ferrochelatases. In growing B. subtilis, the ferrochelatase constitutes approximately 0.05% (by mass) of the total cell protein, which corresponds to some 600 ferrochelatase molecules/cell. The turnover number of isolated ferrochelatase, 18-29 min-1, was found to be consistent with the rate of haem synthesis in exponentially growing cells (0.2 mol haem formed/min/mol enzyme). It is concluded that the B. subtilis ferrochelatase has enzymic properties which are similar to those of other characterised ferrochelatases of known primary structure, i.e. ferrochelatases of the mitochondrial inner membrane of yeast and mammalian cells. However, in contrast to these enzymes the B. subtilis enzyme is a water-soluble protein and should be more amenable to structural analysis.

Amino Acid Sequence↗
PubMed1984

In vitro methylation and demethylation of methyl-accepting chemotaxis proteins in Bacillus subtilis.

Bacillus subtilis responds to attractants by demethylating a group of integral membrane proteins referred to as methyl-accepting chemotaxis proteins (MCPs). We have studied the methylation and demethylation of these proteins in an in vitro system, consisting of membrane vesicles, and purified methyltransferase and methylesterase. The chemoattractant aspartate was found to inhibit methylation and stimulate demethylation of MCPs. Escherichia coli radiolabeled membranes in the presence of B. subtilis enzyme do not respond to aspartate by an increase demethylation rate. We also report that B. subtilis MCPs are multiply methylated, demethylation resulting in slower migrating proteins on sodium dodecyl sulfate-polyacrylamide gels.

Bacillus subtilis↗
PubMed2004

Purification, characterization and functional analysis of an endo-arabinanase (AbnA) from Bacillus subtilis.

Bacillus subtilis synthesizes at least one arabinanase encoded by the abnA gene that is able to degrade the polysaccharide arabinan. Here, we report the expression in Escherichia coli of the full-length abnA coding region with a His6-tag fused to the C-terminus. The recombinant protein was secreted to the periplasmic space and correctly processed by the E. coli signal peptidase. The substrate specificity of purified AbnA, the physico-chemical properties and kinetic parameters were determined. Functional analysis studies revealed Glu 215 as a key residue for AbnA hydrolytic activity and indicated that in addition to AbnA B. subtilis secretes other enzyme(s) able to degrade linear 1,5-alpha-l-arabinan.

Amino Acid Sequence↗
PubMed1998

Regulation of a new cell wall hydrolase gene, cwlF, which affects cell separation in Bacillus subtilis.

Bacillus subtilis produces a 35-kDa cell wall hydrolase, CwlF, during vegetative growth. The CwlF protein was extracted from B. subtilis cwlB sigD mutant cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequencing revealed that its sequence is completely identical to that of the internal region of the papQ gene product. Disruption of the papQ gene in the B. subtilis chromosome led to the complete loss of CwlF, indicating that papQ is identical to cwlF. CwlF exhibits high sequence similarity to the p60 proteins of Listeria species, NlpC proteins of Escherichia coli and Haemophilus influenzae, and Enp2 protein of Bacillus sphaericus. The beta-galactosidase activity of the cwlF-lacZ transcriptional fusion and Northern blot analysis of the cwlF gene indicated that the gene is expressed as a monocistronic operon during the exponential growth phase, and primer extension analysis suggested that the cwlF gene is transcribed mainly by EsigmaA RNA polymerase and weakly by EsigmaH RNA polymerase. While the cells of the cwlF-deficient mutant were about twice as long as those of the wild-type strain, the cwlF sigD double mutant cells exhibited extraordinary microfiber formation, in contrast to the filamentation of the sigD mutant. The CwlF production was not affected by the pleiotropic mutations flaD1 and degU32(Hy), which endow cells with the ability of extensive filamentation.

Amino Acid Sequence↗
PubMed1993

Regulation of the synthesis of unsaturated fatty acids by growth temperature in Bacillus subtilis.

Bacillus subtilis synthesizes, almost exclusively, saturated fatty acids, when grown at 37 degrees C. When cultures were transferred from 37 degrees C to 20 degrees C, a chloramphenicol- and rifampicin-sensitive synthesis of a C-16 unsaturated fatty acid was observed. Synthesis of this compound reached a plateau after 5 h at 20 degrees C, reaching levels of 20% of the total fatty acid content. [14C]-labelled fatty acids attached as thioesters to acyl-carriers compounds, such as coenzyme A (CoA) or acyl-carrier protein (ACP) synthesized de novo by glycerol-requiring auxotrophs deprived of glycerol to arrest phospholipid synthesis, could not be desaturated at 20 degrees C. Desaturation of these fatty acids was readily observed when glycerol was restored to the cultures allowing resumption of transfer of acyl-moieties from acyl-thioesters to phospholipid. It was also observed that depletion of the pools of CoA and ACP by starvation of pantothenate auxotrophs had no effect on the observed synthesis of unsaturated fatty acid at 20 degrees C. The overall results indicate that synthesis of unsaturated fatty acids in B. subtilis is a cold-inducible process and that phospholipids are obligate intermediates in this fatty acid desaturation pathway.

Acetyl Coenzyme A↗