Abstract: In Escherichia coli, the main player in transcription regulation of fatty acid metabolism is the FadR protein, which is involved in negative regulation of fatty acid degradation and in positive and negative regulation of the cellular processes related to it, as well as in positive regulation of the biosynthesis of unsaturated fatty acids in a concerted manner with negative regulation of FabR. On the other hand, Bacillus subtilis possesses two global transcriptional regulators, FadR (YsiA) and FapR. B. subtilis FadR represses fatty acid degradation, whereas FapR represses almost all the processes in the biosynthesis of saturated fatty acids and phospholipids. Furthermore, Streptococcus pneumoniae FabT represses the genes of fatty acid biosynthesis that are clustered in its genome. Long-chain acyl-CoAs appear to be metabolic signals for fatty acid degradation by bacteria in general, and antagonize the FadR protein from either E. coli or B. subtilis. However, malonyl-CoA is a metabolic signal for fatty acid and phospholipid biosynthesis by Gram-positive low-GC bacteria, and it antagonizes FapR. These would be the primary aspects for understanding the elaborate and complex regulation of fatty acid metabolism in bacteria to maintain membrane lipid homeostasis.
Abstract: The sigma gene, sigI, of Bacillus subtilis belongs to the group IV heat-shock response genes and has many orthologues in the bacterial phylum Firmicutes. The B. subtilis sigI gene is considered to constitute an operon with rsgI (regulation of sigI, formerly ykrI). As little is known about either the structure and function of the sigI-rsgI operon or the SigI regulons, the role of RsgI in heat-inducible transcription of the sigI-rsgI operon was investigated, using Northern analysis and a heat-stable beta-galactosidase reporter assay. Heat-inducible, SigI-dependent transcription of the sigI-rsgI operon was stimulated greatly by disrupting rsgI. Yeast two-hybrid analysis showed direct interaction between the N-terminal portion of the presumed RsgI protein and SigI. Without RsgI function, induction of transcription of the sigI-rsgI operon upon transient heat stress depended on dnaK activity. However, transcription of the operon was induced during growth at prolonged higher temperature even without DnaK function. Without RsgI function, sigI-rsgI operon transcription was induced after the end of growth independent of any temperature shift in a sporulation medium and toward the end of growth in a rich complex medium. Furthermore, glucose addition resulted in a strong suppression of sigI-rsgI transcription. Therefore it is hypothesized that transcription of the sigI-rsgI operon of B. subtilis is negatively regulated by the putative transmembrane protein RsgI, which moderates SigI's sensitivity to heat shock or nutritional stress.
Abstract: The organization and function of the Bacillus subtilis YsiA regulon involved in fatty acid degradation were investigated. Northern and primer extension analyses indicated that this regulon comprises five operons, i.e. lcfA-ysiA-B-etfB-A, ykuF-G, yhfL, yusM-L-K-J, and ywjF-acdA-rpoE. YusJ and AcdA, YsiB and YusL, and YusK presumably encode acyl-CoA dehydrogenases, 3-hydroxyl-CoA dehydrogenase/enoyl-CoA hydratase complexes, and acetyl-CoA C-acyltransferase, respectively, which are directly involved in the fatty acid beta-oxidation cycle. In addition, LcfA and YhfL are likely to encode long chain acyl-CoA ligases. On gel retardation and footprinting analyses involving the purified YsiA protein, we identified cis-sequences for YsiA binding (YsiA boxes) in the promoter regions upstream of ysiA, ykuF, yusL, yhfL, and ywjF, the equilibrium dissociation constants (K(d)) for YsiA binding being 20, 21, 37, 43, and 65 nm, respectively. YsiA binding was specifically inhibited by long chain acyl-CoAs with 14-20 carbon atoms, acyl-CoAs with 18 carbon atoms being more effective; out of long chain acyl-CoAs tested, monounsaturated oleoyl-CoA, and branched chain 12-metyltetradecanoyl-CoA were most effective. These in vitro findings were supported by the in vivo observation that the knock-out of acyl-CoA dehydrogenation through yusJ, etfA, or etfB disruption resulted in YsiA inactivation, probably because of the accumulation of long chain acyl-CoAs in the cells. Furthermore, the disruption of yusL, yusK, yusJ, etfA, etfB, or ykuG affected the utilization of palmitic acid, a representative long chain fatty acid. Based on this work, ysiA, ysiB, ykuF, ykuG, yhfL, yusM, yusL, yusK, yusJ, and ywjF can be renamed fadR, fadB, fadH, fadG, lcfB, fadM, fadN, fadA, fadE, and fadF.
Abstract: Bacillus subtilis LmrA is known to be a repressor that regulates the lmrAB and yxaGH operons; lmrB and yxaG encode a multidrug resistance pump and quercetin 2,3-dioxygenase, respectively. DNase I footprinting analysis revealed that LmrA and YxaF, which are paralogous to each other, bind specifically to almost the same cis sequences, LmrA/YxaF boxes, located in the promoter regions of the lmrAB operon, the yxaF gene, and the yxaGH operon for their repression and containing a consensus sequence of AWTATAtagaNYGgTCTA, where W, Y, and N stand for A or T, C or T, and any base, respectively (three-out-of-four match [in lowercase type]). Gel retardation analysis indicated that out of the eight flavonoids tested, quercetin, fisetin, and catechin are most inhibitory for LmrA to DNA binding, whereas quercetin, fisetin, tamarixetin, and galangin are most inhibitory for YxaF. Also, YxaF bound most tightly to the tandem LmrA/YxaF boxes in the yxaGH promoter region. The lacZ fusion experiments essentially supported the above-mentioned in vitro results, except that galangin did not activate the lmrAB and yxaGH promoters, probably due to its poor incorporation into cells. Thus, the LmrA/YxaF regulon presumably comprising the lmrAB operon, the yxaF gene, and the yxaGH operon is induced in response to certain flavonoids. The in vivo experiments to examine the regulation of the synthesis of the reporter beta-galactosidase and quercetin 2,3-dioxgenase as well as that of multidrug resistance suggested that LmrA represses the lmrAB and yxaGH operons but that YxaF represses yxaGH more preferentially.
Abstract: The Bacillus subtilis genome encodes eleven Rap proteins, which are conserved tetratricopeptide-containing regulatory proteins. Of those characterized to date, all except RapI negatively regulate response regulators, including Spo0F, ComA and DegU, via protein-protein interactions. RapD has not yet been fully characterized. It was examined whether RapD inhibits the expression of spoIIE, srfA and aprE, which are Spo0F-, ComA- and DegU-regulated genes, respectively. It was observed that multicopy rapD inhibited srfA expression, which suggests that RapD inhibits ComA. This was reinforced by the fact that multicopy rapD also blocked the expression of rapC and rapF, which belong to the ComA regulon. The expression of rapD was reported to depend on the extracytoplasmic function sigma factor SigX. DNA microarray analysis and gel retardation assays revealed that rapD expression is directly repressed by RghR. Thus, the ComA regulon is regulated by rapD in a SigX- and RghR-dependent manner.
Abstract: Many bacterial genomes encode multiple metal-sensing ArsR-SmtB transcriptional repressors. There is interest in understanding and predicting their metal specificities. Here we analyse two arsR-smtB genes, ydeT and yozA (now aseR and czrA) from Bacillus subtilis. Purified AseR and CzrA formed complexes in gel-retardation and fluorescence-anisotropy assays with fragments of promoters that were derepressed in DeltaaseR and DeltaczrA cells. Candidate (i) partly thiolate, alpha3-helix (for AseR) and (ii) tetrahedral, non-thiolate, alpha5-helix (for CzrA) metal binding sites were predicted then tested in vitro and/or in vivo. The precedents are for such sites to sense arsenite/antimonite (alpha3) and zinc (alpha5). This correlated with the respective metal inducers of AseR and CzrA repressed promoters in B. subtilis and matched the metals that impaired formation of protein-DNA complexes in vitro. The putative sensory sites of 1024 ArsR-SmtB homologues are reported. Although AseR did not sense zinc in vivo, it bound zinc in vitro exploiting alpha3 thiols, but AseR DNA binding was not impaired by zinc. If selectivity relies on discriminatory triggering of allostery not just selective metal binding, then tight non-effector metal complexes could theoretically inhibit metal sensing. AseR remained arsenite-sensitive in equimolar zinc, while CzrA remained zinc-sensitive in equimolar arsenite in vitro. However, cupric ions did not impair CzrA-DNA complex formation but did inhibit zinc-mediated allostery in vitro and prevent zinc binding. Access to copper must be controlled in vivo to avoid formation of cupric CzrA.
Abstract: Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently iolE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second step in the pathway. Here we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol. An iolE ortholog was cloned from USDA191. USDA191 iolE was expressed in Escherichia coli as a His(6)-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 iolE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis iolE deficient mutant. These results suggest that S. fredii USDA191 utilizes a myo-inositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and iolE.
Abstract: The Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids is under negative regulation mediated by TnrA and CodY, which recognize and bind to their respective cis-elements located upstream of the ilv-leu promoter. This operon is known to be under CcpA-dependent positive regulation. We have currently identified a catabolite-responsive element (cre) for this positive regulation (bases -96 to -82; +1 is the ilv-leu transcription initiation base) by means of DNase I-footprinting in vitro, and deletion and base-substitution analyses of cre. Under nitrogen-rich growth conditions in glucose-minimal medium supplemented with glutamine and amino acids, CcpA and CodY exerted positive and negative regulation of ilv-leu, respectively, but TnrA did not function. Moreover, CcpA and CodY were able to function without their counteracting regulation of each other, although the CcpA-dependent positive regulation did not overcome the CodY-dependent negative regulation. Furthermore, under nitrogen-limited conditions in glucose-minimal medium with glutamate as the sole nitrogen source, CcpA and TnrA exerted positive and negative regulation, respectively, but CodY did not function. This CcpA-dependent positive regulation occurred without the TnrA-dependent negative regulation. However, the TnrA-dependent negative regulation did not occur without the CcpA-dependent positive regulation, raising the possibility that this negative regulation might decrease the CcpA-dependent positive regulation. The physiological role of this elaborate transcription regulation of the B. subtilis ilv-leu operon in overall metabolic regulation in this organism is discussed.
Abstract: During DNA microarray analysis, we discovered that the GlnK-GlnL (formerly YcbA-YcbB) two-component system positively regulates the expression of the glsA-glnT (formerly ybgJ-ybgH) operon in response to glutamine in the culture medium on Northern analysis. As a result of gel retardation and DNase I footprinting analyses, we found that the GlnL protein interacts with a region (bases -13 to -56; +1 is the transcription initiation base determined on primer extension analysis of glsA-glnT) in which a direct repeat, TTTTGTN4TTTTGT, is present. Furthermore, the glsA and glnT genes were biochemically verified to encode glutaminase and glutamine transporter, respectively.
Abstract: The myo-inositol catabolism pathway of Bacillus subtilis has not been fully characterized but was proposed to involve step-wise multiple reactions that finally yielded acetyl-CoA and dihydroxyacetone phosphate. It is known that the iolABCDEFGHIJ operon is responsible for the catabolism of inositol. IolG catalyses the first step of myo-inositol catabolism, the dehydrogenation of myo-inositol, producing 2-keto-myo-inositol (inosose). The second step was thought to be the dehydration of inosose. Genetic and biochemical analyses of the iol genes led to the identification of iolE, encoding the enzyme for the second step of inositol catabolism, inosose dehydratase. The reaction product of inosose dehydratase was identified as D-2,3-diketo-4-deoxy-epi-inositol.
Abstract: The SigD-regulated genes in Bacillus subtilis were systematically analyzed by comparing the pattern of transcripts derived from wild-type cells with those from sigD mutant cells using DNA microarray technology. One hundred and fifty-eight genes were found to be SigD-dependent candidates, 46 of which being known SigD-regulated genes. Northern blot analysis revealed that 18 of the remaining genes were SigD-dependent. The SigD consensus sequence was newly identified in the upstream regions of nine operons (11 genes): ybdO, yfmT-yfmS, hemAT, yjcP-yjcQ, yjfB, ylqB, yoaH, yscB and yxkC, and the other seven genes were assumed to be indirectly affected by a SigD mutation. Furthermore, yviE-yviF are likely to be SigD-dependent genes, because three independent sets of array data for yviE and yviF indicated they are SigD-dependent, and these genes are neighbors of flgL and hag transcribed by SigD RNA polymerase.
Abstract: The Bacillus subtilis lmrAB operon is involved in multidrug resistance. LmrA is a repressor of its own operon, while LmrB acts as a multidrug efflux transporter. LmrA was produced in Escherichia coli cells and was shown to bind to the lmr promoter region, in which an LmrA-binding site was identified. Genome-wide screening involving DNA microarray analysis allowed us to conclude that LmrA also repressed yxaGH, which was not likely to contribute to the multidrug resistance. LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding sites were identified. The LmrA regulon was thus determined to comprise lmrAB and yxaGH. All three LmrA-binding sites contained an 18-bp consensus sequence, TAGACCRKTCWMTATAWT, which could play an important role in LmrA binding.
Abstract: The Bacillus subtilis ilv-leu operon is involved in the synthesis of branched-chain amino acids (valine, isoleucine, and leucine). The two- to threefold repression of expression of the ilv-leu operon during logarithmic-phase growth under nitrogen-limited conditions, which was originally detected by a DNA microarray analysis to compare the transcriptomes from the wild-type and tnrA mutant strains, was confirmed by lacZ fusion and Northern experiments. A genome-wide TnrA box search revealed a candidate box approximately 200 bp upstream of the transcription initiation base of the ilv-leu operon, the TnrA binding to which was verified by gel retardation and DNase I footprinting analyses. Deletion and base substitution of the TnrA box sequence affected the ilv-leu promoter activity in vivo, implying that TnrA bound to the box might be able to inhibit the promoter activity, possibly through DNA bending. The negative control of the expression of the ilv-leu operon by TnrA, which is considered to represent rather fine-tuning (two- to threefold), is a novel regulatory link between nitrogen and amino acid metabolism.
Abstract: Target gene candidates of the seven extracytoplasmic function (ECF) sigma factors of Bacillus subtilis have been surveyed using DNA microarray analysis of mRNA extracted from cells grown in Luria-Bertani broth, in which an ECF sigma factor gene was placed under the control of the spac promoter on multicopy plasmid pDG148 and overexpressed. The number of target candidates for each of the sigma factors varied greatly, and a total of 278 genes were selected. Interestingly, the above target gene candidates shared only one gene out of 94 target genes of the general stress sigma B that have been reported in the literature thus far. Furthermore, lacZ-fusion experiments based on the results of DNA microarray analysis indicated that each ECF sigma factor directs transcription of its own operon, with the exception of sigZ. The DNA microarray data collected in this study are available at the KEGG Expression Database web site (http://www.genome.ad.jp/kegg/expression/).
Abstract: Additional targets of CodY, a GTP-activated repressor of early stationary-phase genes in Bacillus subtilis, were identified by combining chromatin immunoprecipitation, DNA microarray hybridization, and gel mobility shift assays. The direct targets of CodY newly identified by this approach included regulatory genes for sporulation, genes that are likely to encode transporters for amino acids and sugars, and the genes for biosynthesis of branched-chain amino acids.
Abstract: To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.
Abstract: We investigated the organization and expression of the Bacillus subtilis sigY operon, the first gene of which codes for sigmaY, a member of the extracytoplasmic function (ECF) family of sigma factors. The sigY operon, comprising six genes (sigY, yxlC, D, E, F, and G), was induced upon nitrogen starvation; it was continuously transcribed from the 31st base upstream of sigY to a neighboring convergent gene, yxlH, resulting in a 4.2-kb mRNA. The expression of the sigY operon was also positively autoregulated through sigmaY, suggesting that its transcription is likely to be directed by sigmaY. Deletion analysis of the sigY promoter, which was localized by primer extension, revealed the promoter region of sigY with the "-10" and "-35" sequences of CGTC and TGAACG, respectively. The latter sequence was distinct from those recognized by sigmaW, sigmaX, and sigmaM. The sigmaY-directed transcription of sigY was under negative regulation involving YxlD. sigY disruption affected sporulation induced by nitrogen starvation, but sigY induction upon nitrogen starvation was not associated with the sporulation process. The organization and function of the sigY operon are significantly conserved in several microorganisms living in adverse living environments.
Abstract: We found that mannitol-1-phosphate dehydrogenase (MtlD), a component of the mannitol-specific phosphotransferase system, is required for glucitol assimilation in addition to GutR, GutB, and GutP in Bacillus subtilis. Northern hybridization of total RNA and microarray studies of RNA from cells cultured on glucose, mannitol, and glucitol indicated that mannitol as the sole carbon source induced hyperexpression of the mtl operon, whereas glucitol induced both mtl and gut operons. The B. subtilis mtl operon consists of mtlA (encoding enzyme IICBA(mt1)) and mtlD, and its transcriptional regulator gene, mtlR, is located 14.4 kb downstream from the mtl operon on the chromosome. The mtlA, mtlD, and mtlR mutants disrupted by the introduction of the pMUTin derivatives MTLAd, MTLDd, and MTLRd, respectively, could not grow normally on either mannitol or glucitol. However, the growth of MTLAd on glucitol was enhanced by IPTG (isopropyl-beta-D-thiogalactopyranoside). This mutant has an IPTG-inducible promoter (Pspac promoter) located in mtlA, and this site corresponds to the upstream region of mtlD. Insertion mutants of mtlD harboring the chloramphenicol resistance gene also could not grow on either mannitol or glucitol. In contrast, an insertion mutant of mtlA could grow on glucitol but not on mannitol in the presence or absence of IPTG. MtlR bound to the promoter region of the mtl operon but not to a DNA fragment containing the gut promoter region.
Abstract: A transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene. The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (kcat/Km 102 and 10 s(-1) mM(-1), respectively). However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression. Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. ywkA and yufL/yufM could thus be renamed maeA for malic enzyme and malK/malR for malate kinase sensor/malate response regulator, respectively.
Abstract: Bacillus subtilis TnrA is a global regulator that responds to the availability of nitrogen sources and both activates and represses many genes during nitrogen-limited growth. In order to obtain a holistic view of the gene regulation depending on TnrA, we performed a genome-wide screening for TnrA-regulated genes associated with a TnrA box. A combination of DNA microarray hybridization and a genome-wide search for TnrA boxes allowed us to find 36 TnrA-regulated transcription units associated with a putative TnrA box. Gel retardation assaying, using probes carrying at least one putative TnrA box and the deletion derivatives of each box, indicated that 17 out of 36 transcription units were likely TnrA targets associated with the TnrA boxes, two of which (nasA and nasBCDEF) possessed a common TnrA box. The sequences of these TnrA boxes contained a consensus one, TGTNANAWWWTMTNACA. The TnrA targets detected in this study were nrgAB, pucJKLM, glnQHMP, nasDEF, oppABCDF, nasA, nasBCDEF and ywrD for positive regulation, and gltAB, pel, ywdIJK, yycCB, yttA, yxkC, ywlFG, yodF and alsT for negative regulation, nrgAB and gltAB being well-studied TnrA targets. It was unexpected that the negatively regulated TnrA targets were as many as the positively regulated targets. The physiological role of the TnrA regulon is discussed.
Abstract: Among hundreds of mutants constructed systematically by the Japanese groups participating in the functional analysis of the Bacillus subtilis genome project, we found that a mutant with inactivation of iolT (ydjK) exhibited a growth defect on myo-inositol as the sole carbon source. The putative product of iolT exhibits significant similarity with many bacterial sugar transporters in the databases. In B. subtilis, the iolABCDEFGHIJ and iolRS operons are known to be involved in inositol utilization, and its transcription is regulated by the IolR repressor and induced by inositol. Among the iol genes, iolF was predicted to encode an inositol transporter. Inactivation of iolF alone did not cause such an obvious growth defect on inositol as the iolT inactivation, while simultaneous inactivation of the two genes led to a more severe defect than the single iolT inactivation. Determination of inositol uptake by the mutants revealed that iolT inactivation almost completely abolished uptake, but uptake by IolF itself was slightly detectable. These results, as well as the K(m) and V(max) values for the IolT and IolF inositol transporters, indicated that iolT and iolF encode major and minor inositol transporters, respectively. Northern and primer extension analyses of iolT transcription revealed that the gene is monocistronically transcribed from a promoter likely recognized by final sigma(A) RNA polymerase and negatively regulated by IolR as well. The interaction between IolR and the iolT promoter region was analyzed by means of gel retardation and DNase I footprinting experiments, it being suggested that the mode of interaction is quite similar to that found for the promoter regions of the iol divergon.
Abstract: The Bacillus subtilis competence transcription factor ComK is required for establishment of competence for genetic transformation. In an attempt to study the ComK factor further, we explored the genes regulated by ComK using the DNA microarray technique. In addition to the genes known to be dependent on ComK for expression, we found many genes or operons whose ComK dependence was not known previously. Among these genes, we confirmed the ComK dependence of 16 genes by using lacZ fusions, and three genes were partially dependent on ComK. Transformation efficiency was significantly reduced in an smf disruption mutant, although disruption of the other ComK-dependent genes did not result in significant decreases in transformation efficiency. Nucleotide sequences similar to that of the ComK box were found for most of the newly discovered genes regulated by ComK.
Abstract: With the completion of the determination of its entire genome sequence, one of the next major targets of Bacillus subtilis genomics is to clarify the whole gene regulatory network. To this end, the results of systematic experiments should be compared with the rich source of individual experimental results accumulated so far. Thus, we constructed a database of the upstream regulatory information of B.subtilis (DBTBS). The current version was constructed by surveying 291 references and contains information on 90 binding factors and 403 promoters. For each promoter, all of its known cis-elements are listed according to their positions, while these cis-elements are aligned to illustrate their consensus sequence for each transcription factor. All probable transcription factors coded in the genome were classified with the Pfam motifs. Using this database, we compared the character of B.subtilis promoters with that of Escherichia coli promoters. Our database is accessible at http://elmo.ims.u-tokyo.ac.jp/dbtbs/.
Abstract: We have analyzed the regulons of the Bacillus subtilis two-component regulators DegU, ComA and PhoP by using whole genome DNA microarrays. For these experiments we took the strategy that the response regulator genes were cloned downstream of an isopropyl-beta-D-thiogalactopyranoside-inducible promoter on a multicopy plasmid and expressed in disruptants of the cognate sensor kinase genes, degS, comP and phoR, respectively. The feasibility of this experimental design to detect target genes was demonstrated by the following two results. First, expression of lacZ fusions of aprE, srfA and ydhF, the target genes of DegU, ComA and PhoP, respectively, was stimulated in their cognate sensor kinase-deficient mutants upon overproduction of the regulators. Secondly, by microarray analysis most of the known target genes for the regulators were detected and, where unknown genes were found, the regulator dependency of several of them was demonstrated. As the mutants used were deficient in the kinase genes, these results show that target candidates can be detected without signal transduction. Using this experimental design, we identified many genes whose dependency on the regulators for expression had not been known. These results suggest the applicability of the strategy to the comprehensive transcription analysis of the B.subtilis two-component systems.
Abstract: We used 2D protein gel electrophoresis and DNA microarray technologies to systematically analyze genes under glucose repression in B:acillus subtilis. In particular, we focused on genes expressed after the shift from glycolytic to gluconeogenic at the middle logarithmic phase of growth in a nutrient sporulation medium, which remained repressed by the addition of glucose. We also examined whether or not glucose repression of these genes was mediated by CcpA, the catabolite control protein of this bacterium. The wild-type and ccpA1 cells were grown with and without glucose, and their proteomes and transcriptomes were compared. 2D gel electrophoresis allowed us to identify 11 proteins, the synthesis of which was under glucose repression. Of these proteins, the synthesis of four (IolA, I, S and PckA) was under CcpA-independent control. Microarray analysis enabled us to detect 66 glucose-repressive genes, 22 of which (glmS, acoA, C, yisS, speD, gapB, pckA, yvdR, yxeF, iolA, B, C, D, E, F, G, H, I, J, R, S and yxbF ) were at least partially under CcpA-independent control. Furthermore, we found that CcpA and IolR, a repressor of the iol divergon, were involved in the glucose repression of the synthesis of inositol dehydrogenase encoded by iolG included in the above list. The CcpA-independent glucose repression of the iol genes appeared to be explained by inducer exclusion.
Abstract: It has recently been shown through DNA microarray analysis of Bacillus subtilis two-component regulatory systems (DegS-DegU, ComP-ComA, and PhoR-PhoP) that overproduction of a response regulator of the two-component systems in the background of a deficiency of its cognate sensor kinase affects the regulation of genes, including its target ones. The genome-wide effect on gene expression caused by the overproduction was revealed by DNA microarray analysis. In the present work, we newly analyzed 24 two-component systems by means of this strategy, leaving out 8 systems to which it was unlikely to be applicable. This analysis revealed various target gene candidates for these two-component systems. It is especially notable that interesting interactions appeared to take place between several two-component systems. Moreover, the probable functions of some unknown two-component systems were deduced from the list of their target gene candidates. This work is heuristic but provides valuable information for further study toward a comprehensive understanding of the B. subtilis two-component regulatory systems. The DNA microarray data obtained in this work are available at the KEGG Expression Database website (http://www.genome.ad.jp/kegg/expression).
Abstract: The Bacillus subtilis inositol operon (iolABCDEFGHIJ) is involved in myo-inositol catabolism. Glucose repression of the iol operon induced by inositol is exerted through catabolite repression mediated by CcpA and the iol induction system mediated by IolR. In this study, we identified two iol catabolite-responsive elements (cre's), to which CcpA complexed with P-Ser-HPr or P-Ser-Crh probably binds. One is located in iolB (cre-iolB, nucleotides +2397 to +2411; +1 is the transcription initiation nucleotide), which was the only cre-iol found in the previous cre search of the B. subtilis genome using a query sequence of WTGNAANCGNWNNCW (W stands for A or T, and N stands for any base). Deletion and base substitution analysis of the iol region indicated that cre-iolB functions even if it is located far downstream of the iol promoter. Further deletion and base substitution analysis revealed another cre located between the iol promoter and the iolA gene (cre-iiolA, nucleotides +86 to +100); the prefix "i" indicates a location in the intergenic region. Both cre-iiolA and cre-iolB appeared to be recognized to almost the same extent by CcpA complexed with either P-Ser-HPr or P-Ser-Crh. Sequence alignment of the six known cre's, including cre-iiolA, which were not revealed in the previous cre search, exhibited another consensus sequence of WTGAAARCGYTTWWN (R stands for A or G, and Y stands for C or T); the right two thymines (TT) were found to be essential for the function of cre-iiolA by means of base substitution analysis. A cre search with this query sequence led to the finding of 14 additional putative cre's.
Abstract: The ytrABCDEF operon of Bacillus subtilis was deduced to encode a putative ATP-binding cassette (ABC) transport system. YtrB and YtrE could be the ABC subunits, and YtrC and YtrD are highly hydrophobic and could form a channel through the cell membrane, while YtrF could be a periplasmic lipoprotein for substrate binding. Expression of the operon was examined in cells grown in a minimal medium. The results indicate that the expression was induced only early in the stationary phase. The six ytr genes form a single operon, transcribed from a putative sigma(A)-dependent promoter present upstream of ytrA. YtrA, which possesses a helix-turn-helix motif of the GntR family, acts probably as a repressor and regulates its own transcription. Inactivation of the operon led to a decrease in maximum cell yield and less-efficient sporulation, suggesting its involvement in the growth in stationary phase and sporulation. It is known that B. subtilis produces acetoin as an external carbon storage compound and then reuses it later during stationary phase and sporulation. When either the entire ytr operon or its last gene, ytrF, was inactivated, the production of acetoin was not affected, but the reuse of acetoin became less efficient. We suggest that the Ytr transport system plays a role in acetoin utilization during stationary phase and sporulation.
Abstract: A global mechanism of catabolite repression of the genus Bacillus comprises negative regulation exerted through the binding of the CcpA protein to the catabolite-responsive elements (cres) of the target genes. We searched for cre sequences in the Bacillus subtilis genome using a query sequence, WTGNAANCGNWNNCW (N and W stand for any base and A or T, respectively), picking out 126 putative and known cre sequences. To examine their cre function, we integrated spac promoter (P spac )-cre-lacZ fusions into the amyE locus. Examination of catabolite repression of beta-galactosidase synthesis in the integrants led us to the following conclusions: (i) lower mismatching of cre sequences to the query sequence is required for their function; (ii) although cre sequences are partially palindromic, low mismatching in the same direction as that of transcription of the target genes is more critical for their function than that in the inverse direction; and (iii) yet, a more palindromic nature of cre sequences is desirable for a better function. Furthermore, the alignment of 22 cre s that function in vivo implicated a consensus sequence, WWTGNAARCGNWWWCAWW (R stands for G or A). Interestingly, in the case where cre sequences are located in the protein-coding regions of the target genes, their conserved bases are preferentially the third bases of codons where base degeneracy is allowed.
Abstract: Within the framework of the international project 'The functional analysis of the Bacillus subtilis genome' in Japan and Europe, the gene expression and transcription organization of the gntZ-ywaA region (160 kb) of the B. subtilis genome has been systematically analysed. First, all unanalysed genes comprising more than 80 amino acids (125 genes) in this region were inactivated through integration of plasmid pMUTIN. No essential gene was found which could not be inactivated. All the integrants grew normally in both nutrient sporulation medium and glucose minimal medium. But an integrant in the yxbG gene exhibited an oligosporogenic phenotype in the nutrient sporulation medium. The synthesis of beta-galactosidase was examined, as a reporter for expression of the inactivated genes, during growth and sporulation in the two media. The results indicated that 36% of the promoters were inactive when cells were grown in at least one of these two media. Furthermore, the transcription of the 119 genes in this region was analysed by Northern blotting, resulting in a transcription map. The results indicate that the gntZ-ywaA region contains at least 24 polycistronic operons, including several published ones. The operons newly found in this work are yxaAB, yxaGH, yxaJKL, yxbBA-yxnB-asnH-yxaM, yxbCD, yxcED, yxdJK, yxeFGH, yxeKLMNOPQ, yxeR-yxxB, hutPHUIGM, bgIPH-yxiE, wapA-yxxG, yxiM-deaD, katB-yxiS, yxjCDEF, yxjJI and yxkF-mmsX.
Abstract: Transcription of the Bacillus subtilis iol divergon is negatively regulated by a repressor encoded by iolR, which belongs to the DeoR family of bacterial regulators. Gel retardation analysis involving the IolR protein synthesized in Escherichia coli revealed that IolR bound specifically and independently to each of the iol and iolRS promoter regions, with higher affinity to iol. DNase I footprinting revealed that IolR affected DNase I sensitivity either in the iol promoter region between nucleotides -46 and +51 or in iolRS between -79 and -2 (+1 is the transcription initiation nucleotide of both iol and iolRS), indicating its interaction with the extended regions of the iol and iolRS promoters. Deletion analysis indicated that the iol region between -23 and +21 is involved mainly in IolR binding and negative regulation, while the iolRS region between -70 and -44 comprises at least part of the cis-acting sequences for IolR binding and negative regulation. Sequence examination of the extended regions revealed that a tandem direct repeat consisting of two relatively conserved 11-mer sequences, WRAYCAADARD (where D is A, G or T; R is A or G; W is A or T; and Y is C or T), found in each of the iol and iolRS regions might be a determinant sequence for the IolR-DNA interaction. Actual involvement of the direct repeats in the IolR-DNA interaction was shown by the deficiency of IolR-binding and negative regulation that was caused by substitution of the conserved bases within the conserved sequences. These results imply a unique mode of interaction of IolR with the target DNA.
Abstract: We have shown previously that induction of the stringent response in Bacillus subtilis resulted in the arrest of chromosomal replication between 100 and 200 kb either side of oriC at distinct stop sites, designated LSTer and RSTer, left and right stringent terminators respectively. This replication checkpoint was also shown to involve the RTP protein, normally active at the chromosomal terminus. In this study, we show that the replication block is absolutely dependent upon RelA, correlated with high levels of ppGpp, but that efficient arrest at STer sites also requires RTP. DNA-DNA hybridization data indicated that one or more such LSTer sites mapped to gene yxcC (-128 kb from oriC). A 7.75 kb fragment containing this gene was cloned into a theta replicating plasmid, and plasmid replication arrest, requiring both RelA and RTP, was demonstrated. This effect was polar, with plasmid arrest only detected when the fragment was orientated in the same direction with respect to replication, as in the chromosome. This LSTer2 site was further mapped to a 3.65 kb fragment overlapping the next40 probe. Remarkably, this fragment contains a 17 bp sequence (B'-1) showing 76% identity with an RTP binding site (B sequence) present at the chromosomal terminus. This B'-1 sequence, located in the gene yxcC, efficiently binds RTP in vitro, as shown by DNA gel retardation studies and DNase I footprinting. Importantly, precise deletion of this sequence abolished the replication arrest. We propose that this modified B site is an essential constituent of the LSTer2 site. The differences between arrest at the normal chromosomal terminus and arrest at LSTer site are discussed.
Abstract: Three asparagine synthetase genes, asnB, asnH, and asnO (yisO), were predicted from the sequence of the Bacillus subtilis genome. We show here that the three genes are expressed differentially during cell growth. In a rich sporulation medium, expression of asnB was detected only during exponential growth, that of asnH was drastically elevated at the transition between exponential growth and stationary phase, and that of asnO was seen only later in sporulation. In a minimal medium, both asnB and asnH were expressed constitutively during exponential growth and in stationary phase, while the expression of asnO was not detected in either phase. However, when the minimal medium was supplemented with asparagine, only the expression of asnH was partially repressed. Transcription analyses revealed that asnB was possibly cotranscribed with a downstream gene, ytnA, while the asnH gene was transcribed as the fourth gene of an operon comprising yxbB, yxbA, yxnB, asnH, and yxaM. The asnO gene is a monocistronic operon, the expression of which was dependent on one of the sporulation sigma factors, sigma-E. Each of the three genes, carried on a low-copy-number plasmid, complemented the asparagine deficiency of an Escherichia coli strain lacking asparagine synthetases, indicating that all encode an asparagine synthetase. In B. subtilis, deletion of asnO or asnH, singly or in combination, had essentially no effect on growth rates in media with or without asparagine. In contrast, deletion of asnB led to a slow-growth phenotype, even in the presence of asparagine. A strain lacking all three genes still grew without asparagine, albeit very slowly, implying that B. subtilis might have yet another asparagine synthetase, not recognized by sequence analysis. The strains lacking asnO failed to sporulate, indicating an involvement of this gene in sporulation.
Abstract: The Bacillus subtilis fbp gene encoding fructose-1,6-bisphosphatase (FBPase) was originally identified as yydE. The fbp gene was expressed at a fairly constant level in cells undergoing glycolysis or gluconeogenesis. fbp transcription was initiated 94 bp upstream of the translation initiation codon, resulting in a 2.4-kb monocistronic transcript. Interestingly, B. subtilis FBPase exhibited no significant similarity to other FBPases in protein sequence databases.
Abstract: Under aerobic conditions Bacillus subtilis utilizes a branched electron transport chain comprising various cytochromes and terminal oxidases. At present there is evidence for three types of terminal oxidases in B. subtilis: a caa3-, an aa3-, and a bd-type oxidase. We report here the cloning of the structural genes (cydA and cydB) encoding the cytochrome bd complex. Downstream of the structural genes, cydC and cydD are located. These genes encode proteins showing similarity to bacterial ATP-binding cassette (ABC)-type transporters. Analysis of isolated cell membranes showed that inactivation of cydA or deletion of cydABCD resulted in the loss of spectral features associated with cytochrome bd. Gene disruption experiments and complementation analysis showed that the cydC and cydD gene products are required for the expression of a functional cytochrome bd complex. Disruption of the cyd genes had no apparent effect on the growth of cells in broth or defined media. The expression of the cydABCD operon was investigated by Northern blot analysis and by transcriptional and translational cyd-lacZ fusions. Northern blot analysis confirmed that cydABCD is transcribed as a polycistronic message. The operon was found to be expressed maximally under conditions of low oxygen tension.
Abstract: Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting catabolite-responsive element (cre) by a complex of CcpA, which is a member of the GalR-Lacl family of bacterial regulatory proteins, and the seryl-phosphorylated form of HPr (P-ser-HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the cre in the first gene (gntR) of the gnt operon (credown), this operon contains another cre located in the promoter region (creup). A translational gntR'-'lacZ fusion expressed under the control of various combinations of wild-type and mutant credown and creup was integrated into the chromosomal amyE locus, and then catabolite repression of beta-galactosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by creup was probably independent of catabolite repression exerted by credown; both creup and credown catabolite repression involved CcpA. Catabolite repression exerted by creup was independent of P-ser-HPr, and catabolite repression exerted by credown was partially independent of P-ser-HPr. DNase I footprinting experiments indicated that a complex of CcpA and P-ser-HPr did not recognize creup, in contrast to its specific recognition of credown. However, CcpA complexed with glucose-6-phosphate specifically recognized creup as well as credown, but the physiological significance of this complexing is unknown.
Abstract: Previous determination of the nucleotide sequence of the iol region of the Bacillus subtilis genome allowed us to predict the structure of the iol operon for myo-inositol catabolism, consisting of 10 iol genes (iolA to iouJ); iolG corresponds to idh, encoding myo-inositol 2-dehydrogenase (Idh). Primer extension analysis suggested that an inositol-inducible promoter for the iol operon (iol promoter) might be a promoter-like sequence in the 5' region of iolA, which is probably recognized by sigmaA. S1 nuclease analysis implied that a rho-independent terminator-like structure in the 3' region of iolJ might be a terminator for iol transcription. Disruption of the iol promoter prevented synthesis of the iol transcript as well as that of Idh, implying that the iol operon is most probably transcribed as an 11.5-kb mRNA containing the 10 iol genes. Immediately upstream of the iol operon, two genes (iolR and iolS) with divergent orientations to the iol operon were found. Disruption of iolR (but not iolS) caused constitutive synthesis of the iol transcript and Idh, indicating that the iolR gene encodes a transcription-negative regulator (presumably a repressor) for the iol operon. Northern and S1 nuclease analyses revealed that the iolRS genes were cotranscribed from another inositol-inducible promoter, which is probably recognized by sigmaA. The promoter assignments of the iol and iolRS operons were confirmed in vivo with a lacZ fusion integrated into the amyE locus.
Abstract: Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, this paper communicates the sequencing of a chromosome region containing the lic and cel loci (65 kb), which creates a 177 kb contig covering the region from gnt to sacXY. This 65 kb region contains 64 ORFs (62 complete and two partial genes). The 14th, 15th and 17th genes correspond to licT, licS and katE, encoding the antiterminator for licS transcription, beta-glucanase (lichenase) and catalase 2, respectively. The 11th, 30th, 36th, 39th, 41st, 45th-48th, 51st and 58th genes are designated deaD, pepT, galE, aldY, msmX, cydABCD, sigY and katX because their products probably encode ATP-dependent RNA helicase, tripeptidase, UDP-glucose 4-epimerase, aldehyde dehydrogenase, multiple sugar-binding transport ATP-binding protein, the respective components of cytochrome d ubiquinol oxidase and ATP-binding cassette transporter, sigma-factor of RNA polymerase and catalase, respectively. The 60th-64th genes are celRABCD, which are probably involved in cellobiose utilization. Gene organization and gene features in the gnt-sacXY region are discussed.
Abstract: Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 29 kb chromosome segment, which contains the hut operon (335 degrees) and the wapA gene, has been cloned and sequenced. This region (28,954 bp) contains 21 complete ORFs and one partial one. The 5th, 6th and 17th genes correspond to hutH encoding histidase, hutP encoding the positive regulator for the hut operon and wapA encoding a precursor of three major wall-associated proteins, respectively. A homology search for their products deduced from the 21 complete ORFs revealed that nine of them exhibit significant homology to known proteins such as urocanase (Pseudomonas putida), a protein involved in clavulanic acid biosynthesis (Streptomyces griseus), amino acid permeases (lysine, Escherichia coli; histidine, Saccharomyces cerevisiae; and others), beta-glucoside-specific phosphotransferases (E. coli and Erwinia chrysanthemi) and 6-phospho-beta-glucosidases (E. coli and Erw. chrysanthemi). Based on the features of the determined sequence and the results of the homology search, as well as on genetic data and sequence of the hut genes reported by other groups, it is predicted that the B. subtilis hut operon may consist of the following six genes (6th-1st), the last of which is followed by a typical rho-independent transcription terminator: hutP, hutH, EE57A (hutU) encoding urocanase, EE57B (hutI) encoding imidazolone-5-propionate hydrolase, EE57C (hutG) encoding formiminoglutamate hydrolase and EE57D (tentatively designated as hutM) possibly encoding histidine permease. Interestingly, the direction of transcription of these hut genes is opposite to that of the movement of the replication fork.
Abstract: The Bacillus subtilis gnt operon is negatively regulated by GntR, which is antagonized by gluconate. Three GntR mutants with diminished gluconate-binding ability were obtained. Two were missense mutants (Met-209 to Ile and Ser-230 to Leu), whereas the third had a deletion of the C-terminal 23 amino acids. The mutant GntR proteins were unable to become properly detached from the gnt operator even in the presence of gluconate.
Abstract: Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 36-kb chromosome segment, which covers the region between the gnt and iol operons, has been cloned and sequenced. This region (36447 bp) contains 33 complete open reading frames (ORFs; genes) including the four gnt genes and one partial gene. A homology search for the products of the 33 complete ORFs revealed significant homology to known proteins in 16 of them such as tetracycline resistance protein (Clostridium perfringens), asparagine synthetase (Arabidopsis thaliana), aldehyde dehydrogenase (Pseudomonas oleovorans), 2,5-dichloro-2,5-cyclohexadiene-1, 4-diol dehydrogenase (P. paucimobilis), heat shock protein HtpG (Escherichia coli), galactose-proton symporter (E. coli), auxin-induced protein (common tobacco), glucitol operon repressor (E. coli) and methylmalonate-semialdehyde dehydrogenase (P. aeruginosa). Unlike the regions we sequenced so far, this region contained two short sequence multiplications: one was a tandem sequence duplication (409 and 410 bp), and the other a triplication consisting of two highly conserved 118-bp tandem sequences preceded by a less conserved similar sequence (129 bp). The reasons for the presence of these sequence multiplications in the gnt to iol region were deduced.
Abstract: Catabolite repression of various Bacillus subtilis catabolic operons which carry a cis-acting catabolite-responsive element (CRE), such as the gnt operon, is mediated by CcpA, a protein belonging to the GalR-Lacl family of bacterial transcriptional repressors/activators, and the seryl-phosphorylated form of HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. Footprinting experiments revealed that the purified CcpA protein interacted with P-ser-HPr to cause specific protection of the gnt CRE against DNase I digestion. The specific recognition of the gnt CRE was confirmed by the results of footprinting experiments using mutant gnt CREs carrying one of the following base substitutions within the CRE consensus sequence: G to T at position +149 or C to T at position +154 (+1 is the gnt transcription initiation nucleotide). The two mutant CREs causing a partial relief from catabolite repression were not protected by the CcpA/P-ser-HPr complex in footprinting experiments. Based on these and previous findings, we propose a molecular mechanism underlying catabolite repression in B. subtilis mediated by CcpA and P-ser-HPr.
Abstract: Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 23-kb chromosomal segment, which covers the region between the iol and hut operons, has been cloned and sequenced, creating a 99-kb contig from the gnt operon to the wapA locus. This region (23351 bp) contains 25 complete open reading frames (ORFs; genes) including deoR, dra, nupC and pdp and two partial ones. The region (5140 bp) containing these four genes, being also sequenced by H. H. Saxild et al., was sequenced by subjecting a long polymerase chain reaction product to random sequencing using phage M13mp19. However, we could detect no conflict, between two independently determined sequences, which could be attributed to our sequencing method. A homology search for the 24 newly identified gene products revealed significant homology to known proteins in 14 of them. It was notable that three proteins, encoded by the successive genes (yxeMNO), exhibited meaningful homology to the E. coli GlnHPQ products constituting a periplasmic ATP-dependent transport system for glutamine.
Abstract: The Bacillus subtilis gnt operon is negatively regulated via interaction of the gnt repressor (GntR) with an operator upstream of gntR, which is antagonized by gluconate. An 8 bp insertional operator mutation (gntOi) of the gnt operon was constructed which affected the expression level of this operon. Two suppressors of this gntOi mutation, exhibiting normal expression, were also isolated; one involved a threonine substitution for the Ala-48 residue (gntR48T) within the helix-turn-helix DNA-binding motif of GntR, and the other an adenine substitution for the guanine at nucleotide -4 within the gntOi operator (gntOiM4A) (+ 1 is the transcription initiation site). The gntR48T mutation by itself rendered the gnt operon partially constitutive. When the gntR43L mutation, which renders the gnt operon fully constitutive, was introduced into the gntOi or gntOiM4A mutant, the operator mutations were found not to affect the promoter activity of the gnt operon. These in vivo results indicate that the gntOi mutation affects the operator interaction with GntR, causing a low expression level even in the presence of gluconate. In vitro gel retardation and DNase I footprint analyses demonstrated that even when gluconate was present, GntR still bound to the gntOi operator region.
Abstract: Inducer exclusion was not important in catabolite repression of the Bacillus subtilis gnt operon. The CcpA protein (also known as AlsA) was found to be necessary for catabolite repression of the gnt operon, and a mutation (crsA47, which is an allele of the sigA gene) partially affected this catabolite repression.
Abstract: Bacillus licheniformis was able to utilize gluconate as the sole carbon source as efficiently as Bacillus subtilis did. Southern analysis indicated that B. licheniformis likely possesses only one gnt determinant. The nucleotide sequence (6278 bp) of the B. licheniformis DNA containing the gnt operon was determined, revealing the five complete open reading frames (ORF; genes). The putative product of the first gene, oug, did not show any significant homology to known proteins, but those of the second to fifth genes exhibited striking homology to the gntRKPZ genes of B. subtilis, respectively, indicating that they are the corresponding gnt genes of B. licheniformis. Not only is the organization of the gnt genes of these two Bacilli highly conserved, but so are the cis regulatory elements of their gnt operon. Sequence analysis of the upstream regions of these two gnt operons implied that a chromosome rearrangement in B. subtilis might have occurred immediately upstream of the gnt operon during evolution, causing it to diverge from a common ancestor into B. licheniformis and B. subtilis.
Abstract: Within the framework of an international project on the sequencing of the whole Bacillus subtilis genome, a 15 kb chromosome segment, which contains the iol operon involved in inositol utilization, has been cloned and sequenced. This region (14,974 bp) contains 12 complete open reading frames (ORFs; genes) and two partial ones; the seventh gene (E83G) is the idh gene encoding inositol dehydrogenase. All the genes identified are transcribed in the same direction as that of the movement of the replication fork. A homology search for their products deduced from the 12 complete ORFs revealed that eight of them exhibit significant homology to known proteins such as fructokinase, acetolactate synthase, fructose-1,6-bisphosphate aldolase (B. subtilis), and PhoB and FtsE proteins (Escherichia coli). It also implied that two genes (B65D and B65E) might encode a set of two-component regulatory proteins and that the B65F gene might encode a protein belonging to the ATP-binding cassette (ABC) family. Based on the features of the nucleotide sequence determined and the results of the homology search, the primary structure of the iol operon is predicted.
Abstract: The ccpA mutations alsA1 (alsA1 is allelic to ccpA) and ccpA::Tn917 completely abolished catabolite repression of gluconate kinase and sorbitol dehydrogenase synthesis in Bacillus subtilis, whereas they only partially affected the catabolite repression of inositol dehydrogenase, histidase and xylose isomerase synthesis. The alsA1 mutation also partially affected catabolite repression of sporulation. Analysis of revertants from the alsA1 mutant by direct sequencing indicated that this mutation comprises a base substitution of guanine at nucleotide -14 to adenine within the Shine-Dalgarno sequence of the ccpA gene (ccpA translation starts at nucleotide +1). A 1.37 kb EcoRI fragment carrying the ccpA gene was cloned into Escherichia coli plasmid pUC19 and B. subtilis plasmid pUB110, resulting in plasmids pCCPA19 and pCCPA110, respectively. The ccpA gene carried in pCCPA110 complemented the alsA1 mutation. Western blotting revealed that the level of the CcpA protein in B. subtilis cells, which seemed to be constitutively synthesized, was approximately 10 times lower for the alsA1 mutant than for the wild-type. The CcpA protein synthesized by either E. coli cells bearing pCCPA19 or B. subtilis cells bearing pCCPA110 was purified to over 90% homogeneity; the latter cells were grown in the presence of glucose. The molecular mass of the protein purified from E. coli was 74 kDa, suggesting that this protein exists as a dimer because its subunit molecular mass was 38 kDa as determined by SDS-PAGE. Gel retardation analysis indicated that the purified CcpA protein in both cases did not bind to the cis sequence for catabolite repression of the gnt operon, but it bound non-specifically to DNA.
Abstract: The Bacillus subtilis gnt operon is negatively regulated by the gnt repressor (GntR, 243 amino acids), which is antagonized by gluconate. The GntR protein belongs to a new family of bacterial regulatory proteins (GntR family). To locate the DNA-binding domain of the GntR protein, we obtained mutations of this protein, by hydroxylamine mutagenesis, which diminish its operator binding ability. Sequence analysis of these mutations indicated that the mutant GntR proteins (GntR43L, GntR66T, GntR74K and GntR75Q) had amino acid substitutions (Ser43 to Leu, Ala66 to Thr, Glu74 to Lys and Arg75 to Gln), respectively. They were all located within the N-terminal conserved region of the GntR family. In vivo and in vitro analysis of these GntR proteins indicated that their relative operator binding abilities became weaker in the order of GntR (wild type), GntR66T, GntR75Q, GntR74K and GntR43L. The equilibrium dissociation constants of GntR (wild type), GntR66T, GntR75Q and GntR74K as to operator binding were determined by gel retardation assays to be 0.43, 2.6, 4.2 and 8.8 M x 10(-10), respectively.
Abstract: The mechanism underlying catabolite repression in Bacillus species remains unknown. A recent study of the promoter-independent catabolite repression of the gnt operon implicated a consensus sequence (ATTGAAAG) in catabolite repression in the genus Bacillus. The introduction of base-substitutions into the ATTGAAAG sequence in the chromosomal gnt operon affected catabolite repression of the gnt operon. Deletion analysis indicated that the ATTGAAAG sequence is probably part of a cis sequence necessary for the promoter-independent catabolite repression of the gnt operon. Furthermore, we subjected gnt transcripts synthesized with and without glucose to S1 nuclease and slot blotting analyses. The results indicated that the gnt transcripts decreased in the region (+93 to +203; +1, the transcription initiation nucleotide) only in the presence of glucose. Mechanisms underlying this promoter-independent catabolite repression are discussed.
Abstract: The Bacillus subtilis inositol dehydrogenase (Idh)-encoding gene (idh) was cloned in the B. subtilis temperate phage, rho 11, and then in Escherichia coli plasmids (pBR322 and pUC118). The nucleotide sequence of the idh gene, which consists of 344 codons and whose product has an Mr of 38,351, was determined. E. coli, bearing pIOL05d15, in which expression of the idh gene is under the control of the lac promoter of pUC118, overproduced an active Idh to approx. 20% of total protein upon addition of isopropyl-beta-D-thiogalactopyranoside. This overproduced enzyme cross-reacted with an anti-Idh antibody, and exhibited the same Mr and substrate specificity as those of the B. subtilis enzyme.
Abstract: Transcription of the Bacillus subtilis gnt operon results in a polycistronic mRNA that encodes from the 5' end the Gnt repressor, gluconate kinase and permease. The RNA is drastically induced through inactivation of the Gnt repressor by gluconate. The results of deletion analysis of the gnt promoter region upstream of the repressor gene indicated that no other promoter except the gnt promoter was present for expression of the gluconate kinase gene. In contrast to the synthesis of gluconate kinase and permease, which was markedly induced by gluconate, the results of a radioimmunoassay for the Gnt repressor indicated that synthesis of the Gnt repressor from the induced mRNA was posttranscriptionally repressed.
Abstract: The mechanism underlying catabolite repression in Bacillus species remains unsolved. The gluconate (gnt) operon of Bacillus subtilis is one of the catabolic operons which is under catabolite repression. To identify the cis sequence involved in catabolite repression of the gnt operon, we performed deletion analysis of a DNA fragment carrying the gnt promoter and the gntR gene, which had been cloned into the promoter probe vector, pWP19. Deletion of the region upstream of the gnt promoter did not affect catabolite repression. Further deletion analysis of the gnt promoter and gntR coding region was carried out after restoration of promoter activity through the insertion of internal constitutive promoters of the gnt operon before the gntR gene (P2 and P3). These deletions revealed that the cis sequence involved in catabolite repression of the gnt operon is located between nucleotide positions +137 and +148. This DNA segment contains a sequence, ATTGAAAG, which may be implicated as a consensus sequence involved in catabolite repression in the genus Bacillus.
Abstract: The gluconate (gnt) operon contains genes for a repressor of the operon, gluconate kinase, and gluconate permease. A nonleaky kinase mutation (gntK4) induced the gnt operon constitutively through interaction of the repressor with an inducer of gluconate which had been endogenously formed and accumulated in the cell owing to the complete deficiency of the kinase even in the absence of gluconate in the medium. In contrast, a nonleaky permease mutation (gntP9) never induced the operon by gluconate likely because it cannot give rise to its inducing concentration in the cell even in the presence of gluconate in the medium.
Abstract: Expression of the Bacillus subtilis gluconate (gnt) operon is negatively regulated by the gnt repressor which is antagonized by D-gluconate or D-glucono-delta-lactone. The repressor specifically binds to a gnt operator near the gnt promoter. From the results of gel retardation assaying of the gnt repressor and various restriction enzyme digests of a fragment carrying the gnt promoter and the gntR gene, the gnt operator was found to be located within a 141-base pair region containing the gnt promoter. Deoxyribonuclease I protection experiments revealed that the gnt repressor protected a gnt promoter region, between -10 and +15, which contains a region showing dyad symmetry with a sequence showing half-symmetry, ATACTTGTA. A 35-base pair synthetic duplex DNA containing this region showing dyad symmetry specifically bound to the gnt repressor. Through cleavage at two AccI sites in the protected region an 8-base pair deletion was introduced into the region showing dyad symmetry, which made expression of the gnt promoter constitutive. A DNA fragment carrying this deletion did not bind to the repressor. These results clearly indicated that the gnt operator sequence is a sequence containing the region showing dyad symmetry located at the transcription initiation site of the gnt operon. It was also suggested that the gnt operon contains only one operator. The similarity in the recognition between the repressor-operator interaction of the gnt operon and those of Escherichia coli TrpR and its operators was discussed.
Abstract: The GntR protein is a negative regulator involved in gluconate-inducible expression of the Bacillus subtilis gnt operon which is responsible for gluconate metabolism. The GntR protein has been purified to homogeneity from an overproducing Escherichia coli strain harboring a gntR gene-carrying plasmid. The total amino acid composition and the NH2-terminal amino acid sequence of the purified protein were essentially the same as those deduced from the nucleotide sequence of the gntR gene. Molecular weight determination by gel filtration revealed that the purified protein is in a highly polymerized form, but it likely exists as a dimer when highly diluted. The purified GntR protein was found to be specifically bound to DNA fragments carrying the promoter of the gnt operon in an electrophoretic mobility shift assay. This binding was specifically inhibited by the addition of gluconate or glucono-delta-lactone. The purified protein repressed in vitro transcription from the promoter of the gnt operon. This repression was suppressed by gluconate or glucono-delta-lactone. These results indicate that the GntR protein is a repressor for the gnt operon and that gluconate and glucono-delta-lactone are inducers for this operon.
Abstract: A DNA fragment containing the promoter of the Bacillus subtilis gluconate (gnt) operon and its first gene (gntR) was cloned into Escherichia coli. E. coli recognized this promoter efficiently and precisely. Moreover, the gluconate-inducible system of this operon operated even in E. coli.
Abstract: The gluconate (gnt) operon of Bacillus subtilis consists of four gnt genes; the second and third genes code for gluconate kinase (gluconokinase, EC 2.7.1.12) and gluconate permease, respectively. A fragment carrying the promoter of this operon (gnt promoter) and the first gene (gntR) was subcloned into a promoter probe vector (pPL603B). Repression of the expression of cat-86 gene, encoded in the vector portion of a constructed plasmid (pgnt21), that is under the control of the gnt promoter was removed by gluconate. The results of deletion analysis and of insertional inactivation of the gntR gene cloned in pgnt21 suggested that the product of the gntR gene, actually synthesized as a 29-kDa protein in vivo, is involved in repression of the gnt promoter. A 4-base-pair insertional mutation within the gntR gene constructed in vitro was introduced into the B. subtilis chromosomal gnt operon by use of linkage of the 4 base pairs to gntK10 in transformation. The introduced mutation gntR1 caused the constitutive expression of the gluconate kinase and gluconate permease genes. S1 nuclease analysis indicated that the mRNA of this operon is synthesized in the gntR1 strain and amounts of mRNA are not changed very much by gluconate, which acts as an inducer in the wild-type gene. These results strongly indicate that the gntR gene codes for a transcriptional negative regulator for the gnt operon.
Abstract: The nucleotide sequence (742 bp) of the promoter region of the Bacillus subtilis gluconate (gnt) operon is presented. Nuclease Sl mapping revealed the start point of the transcription and suggested that the expression of this operon is probably regulated at the transcriptional level. The sequences of the -35 and -10 regions suggested that RNA polymerase possessing sigma-43 may recognize this structure. The 223 bp fragment containing 100 bp upstream from the transcription start site actually exhibited a promoter activity when cloned in a promoter probe vector of pPL603B. This promoter activity was highly derepressed and although still under catabolite repression. The fragment on a high copy plasmid could titrate a regulator of the gnt operon so that the expression of the operon on the host chromosome also became derepressed.
Abstract: The enzymes involved in gluconate utilization in Bacillus subtilis seemed to be gluconate permease and gluconate kinase. Several mutants unable to grow on gluconate were isolated. The mutations they harboured (gnt) were clustered between iol-6 and fdp-74 on the B. subtilis chromosome (a tentative map order of gnt-10, gnt-4, gnt-26, gnt-23 and gnt-9 was obtained). The gnt-10 mutation seemed to be located within the structural gene of the kinase, and the gnt-23 and gnt-26 mutations seemed to be within that of the permease. An EcoRI fragment (4.5 MDal) containing an intact gluconate (gnt) operon consisting of these two structural genes was cloned in phage phi 105 by prophage transformation and was mapped physically. The physical location of the mutations coincided with their order on the genetic map. The HindIII-A fragment (2.4 MDal), which corrects all the gnt mutations, was subcloned in plasmid pC194. The fragment contained the structural genes for the gluconate permease and kinase, but not the regulatory region of the gluconate operon.
Abstract: The gluconate (gnt) operon of Bacillus subtilis has been cloned and sequenced. Analysis of the sequence (5482 base pairs) revealed four open reading frames, each of which was preceded by a Shine-Dalgarno sequence. These four frames were designated from the 5'-end as gntR, gntK, gntP, and gntZ. The gntR and gntK genes overlapped by 5 bases. The gntK and gntP gene products (consisting of 513 and 448 amino acids) were identified as gluconate kinase and permease, respectively, by means of insertional inactivation and deletion analysis of these genes subcloned in plasmid pC194. The functions of the gntR and gntZ gene products (243 and 468 amino acids) are presently unknown. S1 nuclease mapping and subcloning in a promoter probe vector (pPL603B) provided evidence that the gnt operon was transcribed as a polycistronic mRNA. Besides the gnt promoter about 40 base pairs upstream of the gntR gene, we detected two overlapping internal promoters between the gntP and gntZ genes. The gnt transcripts terminate about 45 base pairs downstream of the gntZ gene.
Abstract: The regulation of induction of inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) was studied in Bacillus subtilis. Inositol dehydrogenase is induced by myo-inositol and gluconate kinase is induced by D-gluconate. Both inductions were strongly repressed by rapidly metabolizable carbohydrates such as D-glucose, D-mannose, D-fructose and glycerol (D-glucose had the strongest repressive effect) but they were weakly repressed by slowly metabolizable carbohydrates. Although each carbohydrate exerted a stronger effect on the induction of inositol dehydrogenase than that of gluconate kinase, it showed a similar tendency with respect to the degree of repression of each induction. This catabolite repression could not be diminished by addition of cyclic AMP to medium. In addition, non-metabolizable D-glucose analogues had no or weak repressive effects. On the assumption that rapidly metabolizable carbohydrates might be metabolized to repress both inductions, it was investigated whether several mutants blocked in the Embden-Meyerhof pathway could produce metabolite(s) (repressor) to repress them. A phosphoglycerate kinase (EC 2.7.2.3) deficient mutant could produce the repressor from D-glucose, D-mannose, D-fructose and glycerol but other mutants could not produce it from carbohydrates unable to be metabolized in each mutant. Thus, catabolite repression of both enzyme inductions seemed to be under similar regulation. The identification of the possible repressor of the induction of in of inositol dehydrogenase and gluconate kinase in vivo was discussed.
Abstract: A delta igf mutation of Bacillus subtilis (formerly called fdpAl) is a large deletion causing pleiotropic defects. The mapping of the delta igf deletion by phage PBS1 transduction revealed the following map order: sacA, thiC, hsrE, delta igf, ts199, purA. To analyze the pleiotropic nature of the delta igf mutation, mutants affected in each property of the pleiotropic mutation were isolated, and the mutations were mapped. iol and gnt mutants could not grow on inositol and gluconate, respectively, and fdp mutants were affected only in fructose-bisphosphatase. The map order from sacA to purA was as follows: sacA, thiC, hsrE, iol-6, gnt-4, fdp-74, hsrB, ts199, purA. The delta igf deletion covered loci from iol-6 to hsrB.
Abstract: A Bacillus subtilis mutation (gene symbol fdpA1), producing a deficiency of D-fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11, fructose-bisphosphatase), was isolated and genetically purified. An fdpA1-containing mutant did not produce cross-reacting material. It grew on any carbon source that allowed growth of the standard strain except myo-inositol and D-gluconate. Because the mutant could grow on D-fructose, glycerol, or L-malate as the sole carbon source, B. subtilis can produce fructose-6-phosphate and the derived cell wall precursors from these carbon sources in the absence of fructose-bisphosphatase. In other words, during gluconeogenesis B. subtilis must be able to bypass this reaction. Fructose-bisphosphatase is also not needed for the sporulation of B., subtilis. The fdpA1 mutation has the pleiotropic consequence that mutants carrying it cannot produce inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) under conditions that normally induce these enzymes.
Abstract: Inositol 2-dehydrogenase (EC 1.1.1.18) activity appears during growth of Bacillus subtilis (strain 60015) in nutrient sporulation medium. Its synthesis is induced by myo-inositol and repressed by D-glucose. The enzyme has an apparent molecular weight of 155,000 to 160,000 as determined by sucrose density gradient centrifugation, and it is comprised of four subunits, each having a molecular weight of 39,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme is 4.4 as determined by column isoelectric focusing. The enzyme shows the highest Vmax and lowest Km with myo-inositol as substrate but does not react with scyllo-inositol; it also reacts with the alpha anomer (but not the beta anomer) of D-glucose and with D-xylose. Apparently, the enzyme can remove only the single equatorial hydrogen of the cyclitol or pyranose ring. In contrast to the glucose dehydrogenase of spores, which reacts with D-glucose or 2-deoxy-D-glucose and with NAD or NADP, inositol dehydrogenase requires NAD and does not react with 2-deoxy-D-glucose.
Abstract: Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrase, EC 3.1.3.11) of Bacillus subtilis is a constitutive enzyme that was purified 1000-fold (30% yield) to 80% purity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis where it exhibits a band corresponding to 72,000 daltons. It sediments at 15 S in sucrose density gradients indicating a molecular weight of 380,000, but apparently is very asymmetric. Its activity is irreversibly inactivated in the absence of Mn2+. The enzyme specifically catalyzes dephosphorylation of D-fructose 1,6-bisphosphate with a pH optimum of 8.0. It has 40 to 60% of full activity in the absence of P-enolpyruvate; 20 microM P-enolpyruvate activates it maximally. High concentrations of monovalent cations also activate, NH4+ being most effective. Inhibitors fall into two groups. 1) Nucleoside monophosphates, phosphorylated coenzymes, and polynucleotides inhibit competitively with P-enolpyruvate (AMP (Ki = 2 microM) and dAMP are most effective). 2) The inhibition by nucleoside di- and triphosphates, PPi, and highly phosphorylated nucleotides (guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and adenosine 5'-triphosphate 3'-diphosphate are most effective) is not competed by P-enolpyruvate but is partially overcome by fructose 1,6-bisphosphate (2 microM). Therefore, highly phosphorylated nucleotides (pppGpp and others), produced in over 0.2 mM concentrations upon step down from fast to slow growth rates (Gallant, J., and Lazzarini, R.A. (1976) in Protein Synthesis (McConkey, E.H., ed) Vol. 2, pp. 309-349, Marcel Dekker, Inc., New York), can reduce the conversion rate of fructose 1,6-bisphosphate to fructose 6-phosphate during gluconeogenesis. Comparing glycolytic growth on D-glucose and gluconeogenic growth on L-malate, the intracellular concentrations of fructose 1,6-bisphosphate differ but are both above the Km (13 microM) of the enzyme, those of AMP are similar, whereas those of P-enolpyruvate (0.18 mM versus 1.3 mM) indicate that the enzyme has only 40% of its full activity during glycolysis; nucleotides other than AMP may inhibit additionally. Thus, the futile cycle of fructose 1,6-bisphosphate synthesis and degradation during glycolysis is partially avoided, but the cells are poised for rapid adaptation upon change to gluconeogenic growth conditions.
Abstract: Late during sporulation, Bacillus subtilis produces glucose dehydrogenase (GlcDH; EC 1.1.1.47), which can react with D-glucose or 2-deoxy-D-glucose and can use nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a cofactor. This enzyme is found mainly in the forespore compartment and is present in spores; it is probably made exclusively in the forespore. The properties of GlcDH were determined both in crude cell extracts and after purification. The enzyme is stable at pH 6.5 but labile at pH 8 or higher; the pH optimum of enzyme activity is 8. After inactivation at pH 8, the activity can be recovered in crude extracts, but not in solutions of the purified enzyme, by incubation with 3 M KCl and 5 mM NAD or NADP. As determined by gel filtration, enzymatically active GlcDH has a molecular weight of about 115,000 (if the enzyme is assumed to be globular). GlcDH is distinct from a catabolite-repressible inositol dehydrogenase (EC 1.1.1.18), which can also react with D-glucose, requires specifically NAD as a cofactor, and has an electrophoretic mobility different from that of GlcDH.
Abstract: The direct measurement of ultraviolet light-stimulated DNA synthesis in the permeable Bacillus subtilis cells was performed. Bacillus subtilis spores germinated in the presence of chloramphenicol were treated with Brij 58 and irradiated with ultraviolet light, and (3H)dTTP was incorporated into these cells by the DNA polymerase assay system. Characteristics of the incorporation were distinct from those into spores germinated in the absence of chloramphenicol and treated with Brij 58, in the respect that the former incorporation did not require ATP and only partially depended on the presence of all four deoxyribonucleoside triphosphates. The incorporation of (3H)dTTP into DNA was confirmed by CsCl density gradient centrifugation. A DNA polymerase I-deficient strain, JBl 49(59) had no (3H)sTTP incorporating activity induced by ultraviolet light irradiation when the germinated spores were treated with Brij 58. Analysis of alkaline sucrose gradient centrifugation revealed that fragmented DNA caused by ultraviolet light irradiation was rejoined to the size of DNA of non-irradiated cells by incubating irradiated cells in the DNA polymerase assay mixture containing NAD+. The results also suggested that a machinery of DNA repair probably pre-existed in the spore.
Abstract: The incorporation of (3)H-labeled thymidine triphosphate ((3)H-dTTP) into deoxyribonucleic acid (DNA) of germinated and then Brij 58-treated Bacillus subtilis spores was measured to study DNA replication activity of cells. The dTTP incorporation rate was very low in dormant spores, gradually increased as germination proceeded, and reached a level of the vegetative cell activity approximately 4 hr after the start of germination. This is in contrast to the DNA polymerase activity in the cell extract which remained at the same level throughout the germination period. The increase of the dTTP incorporation activity was inhibited by chloramphenicol or phenethyl alcohol. When these inhibitors were added after germination had proceeded, the elevated dTTP incorporation activity gradually decreased. Permeability to dTTP of spores germinated in the presence of chloramphenicol and then treated with Brij 58 was confirmed by (i) (3)H-dTTP incorporation into the treated spores following either electron or ultraviolet irradiation and (ii) release of radioactivity from the treated spores containing radioactively labeled DNA after deoxyribonuclease I treatment.
