Bacillus subtilis functional genomics: genome-wide analysis of the DegS-DegU regulon by transcriptomics and proteomics.

The DegS-DegU two-component regulatory system of Bacillus subtilis controls various processes that characterize the transition from the exponential to the stationary growth phase, including the induction of extracellular degradative enzymes, expression of late competence genes and down-regulation of the sigma(D) regulon. The degU32(Hy) mutation stabilizes the phosphorylated form of DegU (DegU-P), resulting in overproduction of several extracellular degradative enzymes. In this study, the pleiotropic DegS-DegU regulon was characterized by combining proteomic and transcriptomic approaches. A comparative analysis of wild-type B. subtilis and the degU32(Hy) mutant grown in complex medium was performed during the exponential and in the stationary growth phase. Besides genes already known to be under the control of DegU-P, novel putative members of this regulon were identified. Although the degU32(Hy) mutant is assumed to contain high levels of phosphorylated DegU in the exponential as well as in the stationary growth phase, many genes known to be positively regulated by DegU-P did not show enhanced expression in the mutant strain during exponential growth. This is consistent with the fact that most genes belonging to the DegS-DegU regulon are subject to multiple regulation; this is also reflected in the strong stationary-phase induction of these genes in the mutant strain. As expected, during the exponential growth phase, the sigma(D) regulon was expressed at significantly lower levels in the degU32(Hy) mutant than in the wild type.

Bacillus subtilis contains a cyclodextrin-binding protein which is part of a putative ABC-transporter.

Bacillus subtilis is able to grow on alpha-, beta- and gamma-cyclodextrins as a carbon source via a yet unknown metabolizing system. Sequence analysis of the B. subtilis genome reveals that the putative yvfK-yvfO operon seems to be involved in cyclodextrin utilization, containing the open reading frame yvfK, now termed cycB. The amino acid sequence derived from the DNA sequence bears high similarities to solute-binding proteins from B. subtilis, as well as to cymE from Klebsiella oxytoca and malE from Escherichia coli, both encoding solute-binding proteins able to interact with cyclodextrins. A [His](6)-tagged variant of CycB from B. subtilis was constructed, overproduced in E. coli and purified. The modified protein has been used to study its substrate specificity by surface plasmon resonance and fluorescence spectroscopy. From these data, CycB can be classified as a cyclodextrin-binding protein which interacts with all three natural cyclodextrins: alpha, beta and gamma, thereby showing the highest affinity to gamma-cyclodextrin.

Purification and enzymatic characterization of PgcM: a beta-phosphoglucomutase and glucose-1-phosphate phosphodismutase of Bacillus subtilis.

Phosphoglucomutases catalyze the reversible conversion of D-glucose 1-phosphate to D-glucose 6-phosphate, a key metabolic step in all cells. Two classes of phosphoglucomutases have been described so far, using either the alpha- or beta-forms of the phosphorylated sugars. The pgcM gene of Bacillus subtilis was cloned and used to construct a plasmid-based overexpression system for PgcM in Bacillus megaterium. The obtained protein was purified and its enzymatic activities were characterized. PgcM exhibits beta-phosphoglucomutase activity, transforming mainly beta-glucose 1-phosphate to beta-glucose 6-phosphate via the intermediate glucose 1,6-bisphosphate. Nevertheless, alpha-glucose 1-phosphate can also serve as a substrate, but with a seven-fold lower affinity than that observed for the beta-form. Additionally, PgcM exhibits a glucose-1-phosphate phosphodismutase activity using the alpha- and beta-forms as substrates, with affinities comparable to those observed for the phosphoglucomutase activity. Conformational changes of PgcM triggered by cofactors (MgCl2, glucose 1,6-bisphosphate) and substrate (glucose 1-phosphate) were detected by fluorescence spectra. Insertional mutagenesis of pgcM resulted in an inactivation of beta-phosphoglucomutase activity in B. subtilis. These mutants showed growth deficiency on minimal medium containing starch or maltodextrins (maltose to maltoheptaose) compared either to the wild-type or to growth on minimal medium containing glucose.

Mutations in catabolite control protein CcpA separating growth effects from catabolite repression.

Carbon catabolite repression in Bacillus megaterium is mediated by the transcriptional regulator CcpA. A chromosomal deletion of ccpA eliminates catabolite repression and reduces the growth rate on glucose. We describe four single-amino-acid mutations in CcpA which separate the growth effect from catabolite repression, suggesting distinct regulatory pathways for these phenotypes.

Properties of maltose-inducible alpha-glucosidase MalL (sucrase-isomaltase-maltase) in Bacillus subtilis: evidence for its contribution to maltodextrin utilization.

Recently, we identified the maltose inducible alpha-glucosidase MalL of Bacillus subtilis. The malL gene encodes a 561-residue protein with amino acid identities to several alpha-glucosidases and is located in a nine-gene spanning gene cluster, which is presumably organized in an operon. MalL was overproduced, purified, and its enzymatic characteristics were described in more detail. This characterization of the enzyme showed a protein stable up to 37 degrees C after temperature treatment for 15 min and exhibiting an optimal reaction temperature of 42 degrees C. Various disaccharides such as sucrose, maltose, and isomaltose were hydrolyzed with different efficiencies. MalL also hydrolyzes longer maltodextrins from maltotriose up to maltohexaose, but not maltoheptaose, palatinose, isomaltotriose, or isomaltotetraose. MalL expression is subject to both maltose induction and carbon catabolite repression. In this article, we present data demonstrating that induction of MalL expression also occurs when starch, amylose, or glycogen are present in the growth medium. The hydrolysis of these substrates by alpha-amylase presumably leads to products which, when taken up into the cytoplasm, trigger the initiation of maltose operon transcription. Furthermore, MalL expression varies temporally, showing a second induction in the stationary growth phase.

Molecular analysis of the interaction between the Bacillus subtilis trehalose repressor TreR and the tre operator.

The trehalose operon of Bacillus subtilis is subject to regulation by induction, mediated by the repressor TreR, and by carbon catabolite repression (CCR). For in vitro investigations, TreR from B. subtilis was overproduced and purified. Its molecular mass, as estimated by SDS-PAGE, is 27 kDa. Size fractionation under native conditions yielded a size estimate of 56 kDa, indicating that TreR exists as a dimer in its native state. Analysis of its interaction with various DNA fragments shows that TreR is able to recognize two tre operators with different efficiencies, and indicates cooperative binding. Previous results have suggested that CCR of the tre operon occurs by a mechanism in which the specific regulator, TreR, may be involved independently of the central component, CcpA. The data presented here indicate that the TreR-tre operator interaction is influenced by several effectors. Thus, the presence of trehalose-6-phosphate, as well as glucose-1-phosphate and sodium chloride, inhibits tre operator binding. Glucose-6-phosphate can act as an anti-inducer, which might reflect its additional role in CCR exerted by glucose.

The glucose kinase of Bacillus subtilis.

The open reading frame yqgR (now termed glcK), which had been sequenced as part of the genome project, encodes a glucose kinase of Bacillus subtilis. A 1.1-kb DNA fragment containing glcK complemented an Escherichia coli strain deficient in glucose kinase activity. Insertional mutagenesis of glcK resulted in a complete inactivation of glucose kinase activity in crude protein extracts, indicating that B. subtilis contains one major glucose kinase. The glcK gene encodes a 321-residue protein with a molecular mass of 33.5 kDa. The glucose kinase was overexpressed as a fusion protein to a six-His affinity tag and purified to homogeneity. The enzyme had K(m) values for ATP and glucose of 0.77 and 0.24 mM, respectively, and a Vmax of 93 mumol min-1 mg-1. A B. subtilis strain deficient for glucose kinase grew at the same rate on different carbon sources tested, including disaccharides such as maltose, trehalose, and sucrose.

Identification and enzymatic characterization of the maltose-inducible alpha-glucosidase MalL (sucrase-isomaltase-maltase) of Bacillus subtilis.

A gene coding for a putative alpha-glucosidase has been identified in the open reading frame yvdL (now termed malL), which was sequenced as part of the Bacillus subtilis genome project. The enzyme was overproduced in Escherichia coli and purified. Further analyses indicate that MalL is a specific oligo-1,4-1,6-alpha-glucosidase (sucrase-maltase-isomaltase). MalL expression in B. subtilis requires maltose induction and is subject to carbon catabolite repression by glucose and fructose. Insertional mutagenesis of malL resulted in a complete inactivation of the maltose-inducible alpha-glucosidase activity in crude protein extracts and a Mal- phenotype.

Analysis of DNA flanking the treA gene of Bacillus subtilis reveals genes encoding a putative specific enzyme IITre and a potential regulator of the trehalose operon.

Nucleotide sequencing revealed the genes treP encoding a putative specific enzyme IITre upstream from treA and treR encoding a potential regulator downstream from treA of Bacillus subtilis 168. The treP gene encodes a 470-amino acid (aa) protein (50 kDa) showing high similarities to several different specific enzymes II of phosphoenolpyruvate-dependent phosphotransferase systems. treR encodes a 238-aa protein (28 kDa) with high homologies in its N-terminal part to DNA-binding proteins including a helixturn-helix motif. Homologies in its C-terminal part place it in the family of FadR-GntR transcriptional regulators. The three genes, treP-treA-treR, are probably organized in one operon expressed by a sigma A-dependent promoter 53 bp upstream from treP and a rho-independent terminator 28 bp downstream from treR.

Expression of the tre operon of Bacillus subtilis 168 is regulated by the repressor TreR.

The tre locus from Bacillus subtilis containing the genes treP, treA, and treR has been analyzed for its regulation. We demonstrate that at least treP and treA form an operon whose expression is regulated at the transcriptional level. TreR activity has been investigated in in vivo and in vitro studies. An insertional inactivation of treR led to a constitutive expression of treP and treA. Upstream of treP we identified a 248-bp DNA fragment containing a potential sigmaA-dependent promoter and two palindromes reflecting potential tre operators which led to complex formation with TreR-containing protein extracts in DNA retardation experiments. This complex formation is abolished in the presence of trehalose-6-phosphate, which probably acts as an inducer. Therefore, we assume that treR encodes the specific Tre repressor involved in regulation of the expression of the tre operon.

Vectors using the phospho-alpha-(1,1)-glucosidase-encoding gene treA of Bacillus subtilis as a reporter.

The intracellular phospho-alpha-(1,1)-glucosidase, TreA, from Bacillus subtilis (Bs) hydrolyses trehalose 6-phosphate into glucose and glucose 6-phosphate. The enzyme is also able to cleave p-nitrophenyl alpha-D-glucopyranoside (PNPG). This enzymatic reaction can be easily monitored in a beta-galactosidase analogous enzyme assay. The vectors we have constructed can be used to study promoter activity in transcriptional treA fusions and may prove especially useful under high-salt conditions due to the halophilic character of TreA. The treA gene is useful as a reporter in either Bs or Escherichia coli (Ec). Such fusions can be integrated in the Bs amyE locus and selected on either kanamycin or chloramphenicol, or used as plasmids in Ec. As an example of the general utility, we demonstrate treA expression under xylA-operator-promoter control.

Glucose and glucose-6-phosphate interaction with Xyl repressor proteins from Bacillus spp. may contribute to regulation of xylose utilization.

The xyl operons of several gram-positive bacteria are regulated at the level of transcription by xylose-responsive repressor proteins (XylR). In addition, they are catabolite repressed. Here, we describe a mechanism by which glucose metabolism can affect both regulatory mechanisms. Glucose-6-phosphate appeared to be an anti-inducer of xyl operon transcription, since it could compete with xylose in interaction in vitro with XylR from Bacillus subtilis, B. megaterium, and B. licheniformis. On the other hand, glucose was a low-efficiency inactivator of XylR from B. subtilis and B. megaterium and a weak anti-inducer of XylR from B. licheniformis. Thus, the chemical nature of the substituent at C-5 of xylose and the primary structure of XylR determine the effect of these compounds on xyl operon transcription.

Purification and characterization of the phospho-alpha(1,1)glucosidase (TreA) of Bacillus subtilis 168.

The intracellular phospho-alpha(1,1)glucosidase TreA from Bacillus subtilis has been overproduced in Escherichia coli and purified by ion-exchange chromatography and gel filtration. The molecular mass, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 64 kDa. Isoelectric focusing indicated homogeneity of the protein, and its pI was determined to be 4.3. Characterization of the enzyme showed a protein which is stable up to 44 degrees C after temperature treatment for 15 min. The temperature optimum was found to be 37 degrees C, and the pH optimum was 4.5. TreA activity is stimulated by high salt concentrations with different efficiencies depending on the kind of salt. When increasing amounts of ammonium sulfate are used, the increase of TreA activity is correlated with a conformational change of the protein or dimerization. The substrate specificity of the purified enzyme was characterized, showing additionally that trehalose is also hydrolyzed, but to a much smaller extent than trehalose-6-phosphate. In vitro, the presence of glucose reduces TreA activity, indicating product inhibition of the enzyme.

Cleavage of trehalose-phosphate in Bacillus subtilis is catalysed by a phospho-alpha-(1-1)-glucosidase encoded by the treA gene.

A 2.5 kb DNA fragment contain a gene encoding a phospho-alpha-(1-1)-glucosidase (phosphotrehalase), designated treA, was isolated from a Bacillus subtilis chromosomal library by complementation of the tre-12 mutation. The major TreA activity was found in the cytoplasm. TreA exhibits high sequence similarity to thermostable oligo 1,6 beta-glucosidases of several species and the trehalose-6-phosphate hydrolase TreC of Escherichia coli. TreA activity is induced by trehalose and repressed by glucose, fructose or mannitol. Induction by trehalose and repression by glucose are concentration dependent. The highest activity of TreA occurs 90 min before the end of the exponential growth phase in crude cell extracts. The enzyme is able to cleave para-nitrophenyl-glucopyranoside and trehalose-6-phosphate but not trehalose. These results indicate that treA encodes a specific phospho-alpha-(1-1)-glucosidase which cleaves trehalose-6-phosphate in the cytoplasm after transport and phosphorylation of trehalose. The 5' flanking region of treA contains an open reading frame which was partially sequenced, whose product shows about 40% identity to sucrose Enzyme II of the phosphotransferase transport system from several organisms.

Transcription of the xyl operon is controlled in Bacillus subtilis by tandem overlapping operators spaced by four base-pairs.

The expression of xylose utilization in Bacillus subtilis is regulated at the level of transcription by xylose dependent Xyl repressor-xyl operator interaction. We have structurally and functionally characterized the binding sites of Xyl repressor in the xyl regulatory region. Methylation and hydroxyl radical protection and ethylation interference of binding suggests tandem overlapping xyl operators spaced by four base-pairs. A mutational inactivation of each and both operators was performed. DNA retardation experiments with these mutants confirmed the existance of two binding sites. They can be simultaneously occupied, despite their overlapping, intertwined organization. In vivo repressor titration and regulation of indicator gene expression by the xylO mutants confirmed that both binding sites contribute to regulation of the xyl operon. The protection and interference patterns of both sites are identical and indicate binding of a repressor oligomer to one side of B-form DNA of each operator. A tandem overlapping arrangement of two operators is also found in the xyl regulatory sequences of Bacillus megaterium, Staphylococcus xylosus and Lactobacillus pentosus. The xyl operon of Bacillus licheniformis contains a similar element in which the second operator is more diverged. This high degree of conservation among bacteria of different genera supports the conclusion that a tandem overlapping arrangement of xyl operators contributes to efficient regulation.

A series of integrative plasmids for Bacillus subtilis containing unique cloning sites in all three open reading frames for translational lacZ fusions.

Two sets of three plasmids each were constructed based on integrative plasmids for Bacillus subtilis. Each set encodes resistance to either chloramphenicol or kanamycin. The plasmids contain a cassette consisting of the resistance gene, BamHI and SmaI cloning sites, and the promoterless lacZ gene. The cassette is flanked by the 3' and 5' ends of the amyE gene (encoding amylase) allowing integration of the cassette into that locus in the B. subtilis chromosome. For propagation and selection in Escherichia coli, the plasmids contain the pBR322 origin of replication and the beta-lactamase-encoding gene. Within a set, each of the three plasmids carries the unique SmaI and BamHI restriction sites in a different reading frame relative to the 'lacZ gene. These sets of plasmids allow the easy construction of translational fusions with lacZ and single-copy integration at the amyE locus of B. subtilis.

The phosphorylation state of the DegU response regulator acts as a molecular switch allowing either degradative enzyme synthesis or expression of genetic competence in Bacillus subtilis.

Two classes of mutations were identified in the degS and degU regulatory genes of Bacillus subtilis, leading either to deficiency of degradative enzyme synthesis (degS or degU mutations) or to a pleiotropic phenotype which includes overproduction of degradative enzymes and the loss of genetic competence (degS(Hy) or degU(Hy) mutations). We have shown previously that the DegS protein kinase and the DegU response regulator form a signal transduction system in B. subtilis. We now demonstrate that the DegS protein kinase also acts as a DegU phosphatase. We present evidence that the DegU response regulator has two active conformations: a phosphorylated form which is necessary for degradative enzyme synthesis and a nonphosphorylated form required for expression of genetic competence. The degU146-encoded response regulator, allowing expression of genetic competence, has been purified and seems to be modified within the putative phosphorylation site (D56----N) since it is no longer phosphorylated by DegS. Both the degU146 mutation as well as the degS220 mutation, which essentially abolishes DegS protein kinase activity, lead to deficiency of degradative enzyme synthesis, indicating the requirement of phosphorylated DegU for the expression of this phenotype. We also purified the degU32(Hy)-encoded protein and showed that this response regulator is phosphorylated by the DegS protein kinase in vitro. In addition, the phosphorylated form of the degU32(Hy)-encoded protein presented a strongly increased stability as compared with the wild type DegU protein, thus leading to hyperproduction of degradative enzymes in vivo.

Mutational analysis of the Bacillus subtilis DegU regulator and its phosphorylation by the DegS protein kinase.

The DegS-DegU protein kinase-response regulator pair controls the expression of genes encoding degradative enzymes as well as other cellular functions in Bacillus subtilis. Both proteins were purified. The DegS protein was autophosphorylated and shown to transfer its phosphate to the DegU protein. Phosphoryl transfer to the wild-type DegU protein present in crude extracts was shown by adding 32P-labeled DegS to the reaction mixture. Under similar conditions, the modified proteins encoded by the degU24 and degU31 alleles presented a stronger phosphorylation signal compared with that of the wild-type DegU protein. This may suggest an increased phosphorylation of these modified proteins, responsible for the hyperproduction of degradative enzymes observed in the degU24 and degU31 mutants. However, the degU32 allele, which also leads to hyperproduction of degradative enzymes, encodes a modified DegU response regulator which seems not to be phosphorylatable. The expression of the hyperproduction phenotype of the degU32 mutant is still dependent on the presence of a functional DegS protein. DegS may therefore induce a conformational change of the degU32-encoded response regulator enabling this protein to stimulate degradative enzyme synthesis. Two alleles, degU122 and degU146, both leading to deficiency of degradative enzyme synthesis, seem to encode phosphorylatable and nonphosphorylatable DegU proteins, respectively.