The phosphoenolpyruvate:sugar phosphotransferase system is involved in sensitivity to the glucosylated bacteriocin sublancin.

The mode of action of a group of glycosylated antimicrobial peptides known as glycocins remains to be elucidated. In the current study of one glycocin, sublancin, we identified the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus species as a key player in bacterial sensitivity. Sublancin kills several Gram-positive bacteria, such as Bacillus species and Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). Unlike other classes of bacteriocins for which the PTS is involved in their mechanism of action, we show that the addition of PTS-requiring sugars leads to increased resistance rather than increased sensitivity, suggesting that sublancin has a distinct mechanism of action. Collectively, our present mutagenesis and genomic studies demonstrate that the histidine-containing phosphocarrier protein (HPr) and domain A of enzyme II (PtsG) in particular are critical determinants for bacterial sensitivity to sublancin.

The Bacillus subtilis catabolite control protein CcpA exerts all its regulatory functions by DNA-binding.

In Bacillus subtilis, carbon catabolite control is mediated by the regulatory protein CcpA. In addition to loss of catabolite repression, ccpA mutants exhibit a severe growth defect. They are not able to grow with glucose and ammonium as single sources of carbon and nitrogen, respectively. Only ccpA mutant strains carrying either secondary suppressor mutations or that are affected in specific amino acids in the DNA-binding domain of CcpA grow on minimal media. We addressed the importance of DNA-binding by CcpA for both carbon catabolite repression and growth of a mutant strain. A strain specifically deleted for the N-terminal DNA-binding domain of CcpA was constructed. This deletion resulted in complete loss of catabolite repression of beta-xylosidase synthesis and prevented bacteria from growing on minimal media, suggesting that DNA-binding by CcpA is required for both carbon catabolite repression and efficient growth on minimal media.

Transcription of glycolytic genes and operons in Bacillus subtilis: evidence for the presence of multiple levels of control of the gapA operon.

Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis. Although the biochemical activity of glycolytic enzymes has been studied in detail, no information about the expression of glycolytic genes has so far been available in this organism. Therefore, transcriptional analysis of all glycolytic genes was performed. The genes cggR, gapA, pgk, tpi, pgm and eno, encoding the enzymes required for the interconversion of triose phosphates, are transcribed as a hexacistronic operon as demonstrated by Northern analysis. This gapA operon is repressed by the regulator CggR. The presence of sugars and amino acids synergistically results in the induction of the gapA operon. The transcriptional start site upstream of cggR was mapped by primer extension. Transcripts originating upstream of cggR are processed near the 3' end of cggR. This endonucleolytic cleavage leads to differential stability of the resulting processing products: the monocistronic cggR message is very rapidly degraded, whereas the mRNA species encoding glycolytic enzymes exhibit much higher stability. An additional internal constitutive promoter was identified upstream of pgk. Thus, gapA is the most strongly regulated gene of this operon. The pfk pyk operon encoding phosphofructokinase and pyruvate kinase is weakly induced by glucose. In contrast, the genes pgi and fbaA, coding for phosphoglucoisomerase and fructose-1,6-bisphosphate aldolase, are constitutively expressed.

Characterization of glucose-repression-resistant mutants of Bacillus subtilis: identification of the glcR gene.

In Bacillus subtilis, carbon catabolite repression (CCR) is mediated by the pleiotropic repressor CcpA and by ATP-dependent phosphorylation of the HPr protein of the phosphotransferase system (PTS). In this study, we attempted to identify novel genes that are involved in the signal transduction pathway that ultimately results in CCR in the presence of repressing carbon sources such as glucose. Seven mutants resistant to glucose repression of the levanase operon were isolated and characterized. All mutations were trans-acting and pleiotropic as determined by analyzing CCR of beta-xylosidase and of the sacPA and bglPH operon. Moreover, all mutations specifically affected repression exerted by glucose but not by other sugars. The mutations were mapped to three different loci on the genetic map, ptsG, glcR, and pgi. These three genes encode proteins involved in glucose metabolism. A novel repressor gene, glcR (ywpI), defined by two mutations, was studied in more detail. The glcR mutants exhibit loss of glucose repression of catabolic operons, a deficiency in glucose transport, and absence of expression of the ptsG gene. The mutant GlcR proteins act as super-repressors of ptsG expression.

Regulation of carbon catabolism in Bacillus species.

The gram-positive bacterium Bacillus subtilisis capable of using numerous carbohydrates as single sources of carbon and energy. In this review, we discuss the mechanisms of carbon catabolism and its regulation. Like many other bacteria, B. subtilis uses glucose as the most preferred source of carbon and energy. Expression of genes involved in catabolism of many other substrates depends on their presence (induction) and the absence of carbon sources that can be well metabolized (catabolite repression). Induction is achieved by different mechanisms, with antitermination apparently more common in B. subtilis than in other bacteria. Catabolite repression is regulated in a completely different way than in enteric bacteria. The components mediating carbon catabolite repression in B. subtilis are also found in many other gram-positive bacteria of low GC content.

Specific interaction of the RNA-binding domain of the bacillus subtilis transcriptional antiterminator GlcT with its RNA target, RAT.

Expression of the Bacillus subtilis ptsGHI operon is controlled by transcriptional antitermination mediated by the antiterminator protein GlcT. The antiterminator is inactivated in the absence of glucose, presumably by phosphorylation. A conditional terminator in the ptsG mRNA leader region has been identified. Mutations in this terminator resulted in constitutive expression of the operon. The terminator is overlapped by an inverted repeat (called ribonucleic-antiterminator, RAT) which is thought to form a stem-loop structure upon binding of the antiterminator protein GlcT. The N-terminal 60 amino acid residues of GlcT are able to bind to the RAT and prevent transcriptional termination in vivo. Sequence-specific interaction between the RNA-binding domain and the RAT was demonstrated by surface plasmon resonance analysis. Mutations affecting the RNA-binding domain were isolated and will be discussed with respect to their consequences for dimerization and RNA binding.

Role of CcpA in regulation of the central pathways of carbon catabolism in Bacillus subtilis.

The Bacillus subtilis two-dimensional (2D) protein index contains almost all glycolytic and tricarboxylic acid (TCA) cycle enzymes, among them the most abundant housekeeping proteins of growing cells. Therefore, a comprehensive study on the regulation of glycolysis and the TCA cycle was initiated. Whereas expression of genes encoding the upper and lower parts of glycolysis (pgi, pfk, fbaA, and pykA) is not affected by the glucose supply, there is an activation of the glycolytic gap gene and the pgk operon by glucose. This activation seems to be dependent on the global regulator CcpA, as shown by 2D polyacrylamide gel electrophoresis analysis as well as by transcriptional analysis. Furthermore, a high glucose concentration stimulates production and excretion of organic acids (overflow metabolism) in the wild type but not in the ccpA mutant. Finally, CcpA is involved in strong glucose repression of almost all TCA cycle genes. In addition to TCA cycle and glycolytic enzymes, the levels of many other proteins are affected by the ccpA mutation. Our data suggest (i) that ccpA mutants are unable to activate glycolysis or carbon overflow metabolism and (ii) that CcpA might be a key regulator molecule, controlling a superregulon of glucose catabolism.

Regulation of the lic operon of Bacillus subtilis and characterization of potential phosphorylation sites of the LicR regulator protein by site-directed mutagenesis.

The lic operon of Bacillus subtilis is required for the transport and degradation of oligomeric beta-glucosides, which are produced by extracellular enzymes on substrates such as lichenan or barley glucan. The lic operon is transcribed from a sigma(A)-dependent promoter and is inducible by lichenan, lichenan hydrolysate, and cellobiose. Induction of the operon requires a DNA sequence with dyad symmetry located immediately upstream of the licBCAH promoter. Expression of the lic operon is positively controlled by the LicR regulator protein, which contains two potential helix-turn-helix motifs, two phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domains (PRDs), and a domain similar to PTS enzyme IIA (EIIA). The activity of LicR is stimulated by modification (probably phosphorylation) of both PRD-I and PRD-II by the general PTS components and is negatively regulated by modification (probably phosphorylation) of its EIIA domain by the specific EII(Lic) in the absence of oligomeric beta-glucosides. This was shown by the analysis of licR mutants affected in potential phosphorylation sites. Moreover, the lic operon is subject to carbon catabolite repression (CCR). CCR takes place via a CcpA-dependent mechanism and a CcpA-independent mechanism in which the general PTS enzyme HPr is involved.

Carbon catabolite repression in bacteria.

Carbon catabolite repression (CCR) is a regulatory mechanism by which the expression of genes required for the utilization of secondary sources of carbon is prevented by the presence of a preferred substrate. This enables bacteria to increase their fitness by optimizing growth rates in natural environments providing complex mixtures of nutrients. In most bacteria, the enzymes involved in sugar transport and phosphorylation play an essential role in signal generation leading through different transduction mechanisms to catabolite repression. The actual mechanisms of regulation are substantially different in various bacteria. The mechanism of lactose-glucose diauxie in Escherichia coli has been reinvestigated and was found to be caused mainly by inducer exclusion. In addition, the gene encoding HPr kinase, a key component of CCR in many bacteria, was discovered recently.

The catabolite control protein CcpA controls ammonium assimilation in Bacillus subtilis.

Carbon catabolite repression of several catabolic operons in Bacillus subtilis is mediated by the repressor CcpA. An inactivation of the ccpA gene has two distinct phenotypes: (i) catabolite repression of catabolic operons is lost and (ii) the growth of bacteria on minimal medium is severely impaired. We have analyzed the physiological properties of a ccpA mutant strain and show that the ccpA mutation does not affect sugar transport. We have isolated extragenic suppressors of ccpA that suppress the growth defect (sgd mutants). Catabolite repression of beta-xylosidase synthesis was, however, not restored suggesting that the suppressor mutations allow differentiation between the phenotypes of the ccpA mutant. A close inspection of the growth requirements of the ccpA mutant revealed the inability of the mutant to utilize inorganic ammonium as a single source of nitrogen. An intact ccpA gene was found to be required for expression of the gltAB operon encoding glutamate synthase. This enzyme is necessary for the assimilation of ammonium. In a sgd mutant, gltAB operon expression was no longer dependent on ccpA, suggesting that the poor expression of the gltAB operon is involved in the growth defect of the ccpA mutant.

Regulation of the Bacillus subtilis GlcT antiterminator protein by components of the phosphotransferase system.

Bacillus subtilis utilizes glucose as the preferred source of carbon and energy. The sugar is transported into the cell by a specific permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) encoded by the ptsGHI operon. Expression of this operon is induced by glucose and requires the action of a positive transcription factor, the GlcT antiterminator protein. Glucose availability is sensed by glucose-specific enzyme II (EIIGlc), the product of ptsG. In the absence of inducer, the glucose permease negatively controls the activity of the antiterminator. The GlcT antiterminator has a modular structure. The isolated N-terminal part contains the RNA-binding protein and acts as a constitutively acting antiterminator. GlcT contains two PTS regulation domains (PRDs) at the C terminus. One (PRD-I) is the target of negative control exerted by EIIGlc. A conserved His residue (His-104 in GlcT) is involved in inactivation of GlcT in the absence of glucose. It was previously proposed that PRD-containing transcriptional antiterminators are phosphorylated and concomitantly inactivated in the absence of the substrate by their corresponding PTS permeases. The results obtained with B. subtilis glucose permease with site-specific mutations suggest, however, that the permease might modulate the phosphorylation reaction without being the phosphate donor.

PRD--a protein domain involved in PTS-dependent induction and carbon catabolite repression of catabolic operons in bacteria.

Several operon-specific transcriptional regulators, including antiterminators and activators, contain a duplicated conserved domain, the PTS regulation domain (PRD). These duplicated domains modify the activity of the transcriptional regulators both positively and negatively. PRD-containing regulators are very common in Gram-positive bacteria. In contrast, antiterminators controlling beta-glucoside utilization are the only functionally characterized members of this family from gram-negative bacteria. PRD-containing regulators are controlled by PTS-dependent phosphorylation with different consequences: (i) In the absence of inducer, the phosphorylated EIIB component of the sugar permease donates its phosphate to a PRD, thereby inactivating the regulator. In the presence of the substrate, the regulator is dephosphorylated, and the phosphate is transferred to the sugar, resulting in induction of the operon. (ii) In gram-positive bacteria, a novel mechanism of carbon catabolite repression mediated by PRD-containing regulators has been demonstrated. In the absence of PTS substrates, the HPr protein is phosphorylated by enzyme I at His-15. This form of HPr can, in turn, phosphorylate PRD-containing regulators and stimulate their activity. In the presence of rapidly metabolizable carbon sources, ATP-dependent phosphorylation of HPr at Ser-46 by HPr kinase inhibits phosphorylation by enzyme I, and PRD-containing regulators cannot, therefore, be stimulated and are inactive. All regulators of this family contain two copies of PRD, which are functionally specialized in either induction or catabolite repression.

Antagonistic effects of dual PTS-catalysed phosphorylation on the Bacillus subtilis transcriptional activator LevR.

LevR, which controls the expression of the levoperon of Bacillus subtilis, is a regulatory protein containing an N-terminal domain similar to the NifA/NtrC transcriptional activator family and a C-terminal domain similar to the regulatory part of bacterial anti-terminators, such as BgIG and LicT. Here, we demonstrate that the activity of LevR is regulated by two phosphoenolpyruvate (PEP)-dependent phosphorylation reactions catalysed by the phosphotransferase system (PTS), a transport system for sugars, polyols and other sugar derivatives. The two general components of the PTS, enzyme I and HPr, and the two soluble, sugar-specific proteins of the lev-PTS, LevD and LevE, form a signal transduction chain allowing the PEP-dependent phosphorylation of LevR, presumably at His-869. This phosphorylation seems to inhibit LevR activity and probably regulates the induction of the lev operon. Mutants in which His-869 of LevR has been replaced with a non-phosphorylatable alanine residue exhibited constitutive expression from the lev promoter, as do levD or levE mutants. In contrast, PEP-dependent phosphorylation of LevR in the presence of only the general components of the PTS, enzyme I and HPr, regulates LevR activity positively. This phosphorylation most probably occurs at His-585. Mutants in which His-585 has been replaced with an alanine had lost stimulation of LevR activity and PEP-dependent phosphorylation by enzyme I and HPr. This second phosphorylation of LevR at His-585 is presumed to play a role in carbon catabolite repression.

A novel protein kinase that controls carbon catabolite repression in bacteria.

HPr(Ser) kinase is the sensor in a multicomponent phosphorelay system that controls catabolite repression, sugar transport and carbon metabolism in gram-positive bacteria. Unlike most other protein kinases, it recognizes the tertiary structure in its target protein, HPr, a phosphocarrier protein of the bacterial phosphotransferase system and a transcriptional cofactor controlling the phenomenon of catabolite repression. We have identified the gene (ptsK) encoding this serine/threonine protein kinase and characterized the purified protein product. Orthologues of PtsK have been identified only in bacteria. These proteins constitute a novel family unrelated to other previously characterized protein phosphorylating enzymes. The Bacillus subtilis kinase is shown to be allosterically activated by metabolites such as fructose 1,6-bisphosphate and inhibited by inorganic phosphate. In contrast to wild-type B. subtilis, the ptsK mutant is insensitive to transcriptional regulation by catabolite repression. The reported results advance our understanding of phosphorylation-dependent carbon control mechanisms in Gram-positive bacteria.

Coupling physiology and gene regulation in bacteria: the phosphotransferase sugar uptake system delivers the signals.

In many bacteria a crucial link between metabolism and regulation of catabolic genes is based on the phosphotransferase sugar uptake system (PTS). We summarize the mechanisms of the signaling pathways originating from PTS and leading to regulation of transcription. A protein domain, called PTS regulation domain (PRD), is linked to many antiterminators and transcriptional activators and regulates their activity depending on its state of phosphorylation. Two sites can be phosphorylated in most PRDs: HPr-dependent modification at one site leads to activation while enzyme II dependent phosphorylation of the other site renders it inactive. In addition, PTS components are used to generate cofactors for regulators of transcription. The paradigm is the enzyme II dependent activity of adenylate cyclase determining the cyclic AMP level in Escherichia coli and thereby the activity of the catabolite activator protein. In many gram-positive bacteria catabolite repression is mediated by the catabolite control protein CcpA, which requires HPr Ser-46 phosphate as a cofactor to regulate transcription of catabolic genes. HPr Ser-46 phosphate is produced by HPr kinase, the activity of which is under metabolic control via the concentrations of glycolytic intermediates. These recent results establish a multifaceted regulatory role for PTS in addition to its well-established function in active sugar uptake.

The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression.

Carbon catabolite repression (CCR) of several Bacillus subtilis catabolic genes is mediated by ATP-dependent phosphorylation of histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP): sugar phosphotransferase system. In this study, we report the discovery of a new B. subtilis gene encoding a HPr-like protein, Crh (for catabolite repression HPr), composed of 85 amino acids. Crh exhibits 45% sequence identity with HPr, but the active site His-15 of HPr is replaced with a glutamine in Crh. Crh is therefore not phosphorylated by PEP and enzyme I, but is phosphorylated by ATP and the HPr kinase in the presence of fructose-1,6-bisphosphate. We determined Ser-46 as the site of phosphorylation in Crh by carrying out mass spectrometry with peptides obtained by tryptic digestion or CNBr cleavage. In a B. subtilis ptsH1 mutant strain, synthesis of beta-xylosidase, inositol dehydrogenase, and levanase was only partially relieved from CCR. Additional disruption of the crh gene caused almost complete relief from CCR. In a ptsH1 crh1 mutant, producing HPr and Crh in which Ser-46 is replaced with a nonphosphorylatable alanyl residue, expression of beta-xylosidase was also completely relieved from glucose repression. These results suggest that CCR of certain catabolic operons requires, in addition to CcpA, ATP-dependent phosphorylation of Crh, and HPr at Ser-46.

Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT.

Glucose is the preferred carbon and energy source of Bacillus subtilis. It is transported into the cell by the glucose-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS) encoded by the ptsGHI locus. We show here that these three genes (ptsG, ptsH, and ptsI) form an operon, the expression of which is inducible by glucose. In addition, ptsH and ptsl form a constitutive ptsHI operon. The promoter of the ptsGHI operon was mapped and expression from this promoter was found to be constitutive. Deletion mapping of the promoter region revealed the presence of a transcriptional terminator as a regulatory element between the promoter and coding region of the ptsG gene. Mutations within the ptsG gene were characterized and their consequences on the expression of ptsG studied. The results suggest that expression of the ptsGHI operon is subject to negative autoregulation by the glucose permease, which is the ptsG gene product. A regulatory gene located upstream of the ptsGHI operon, termed glcT, was also identified. The GlcT protein is a novel member of the BglG family of transcriptional antiterminators and is essential for the expression of the ptsGHI operon. A deletion of the terminator alleviates the need for GlcT. The activity of GlcT is negatively regulated by the glucose permease.

Characterization of the presumptive phosphorylation sites of the Bacillus subtilis glucose permease by site-directed mutagenesis: implication in glucose transport and catabolite repression.

Bacillus subtilis utilizes glucose as the preferred source of carbon and energy. Glucose is transported and concomitantly phosphorylated by the glucose permease (PtsG) of the phosphoenolpyruvate:sugar phosphotransferase system. The phosphate is transferred from enzyme I via HPr and domains IIA and IIB of the glucose permease to the sugar. In this study mutants affected in the putative phosphorylation sites of glucose permease were constructed and the effect on sugar transport and glucose repression tested. Phosphorylation of both domains IIAGlc and IIBGlc is required for efficient glucose transport and repression of beta-xylosidase and the bglPH operon.

LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family.

Gene licS of Bacillus subtilis encodes an excreted Beta-1,3-1,4-endoglucanase necessary for lichenan utilization. Upstream of licS we found a gene (termed licT) together with its promoter which encodes a transcriptional antiterminator of the BglG family. Genes licT and licS are separated by a palindromic sequence (lic-t) reminiscent of transcriptional terminators recognized by the antiterminator proteins of the BglG family. The LicT protein can prevent termination at terminator lic-t and also at terminator t2 of the Escherichia coli bgl operon and BglG prevents termination at lic-t. The role of LicT in licS regulation by preventing termination at its terminator lic-t appears to be limited since expression of licS is inducible only two- to threefold. This limited regulation is mainly due to a high basal level of licS expression which can in part be attributed to the presence of a second promoter preceding licS and located downstream of lic-t. However, disruption of gene licT leads not only to loss of inducibility of licS but also to loss of growth on lichenan or on its degradation products, indicating its stringent role in beta-glucan utilization.

Two different mechanisms mediate catabolite repression of the Bacillus subtilis levanase operon.

There are two levels of control of the expression of the levanase operon in Bacillus subtilis: induction by fructose, which involves a positive regulator, LevR, and the fructose phosphotransferase system encoded by this operon (lev-PTS), and a global regulation, catabolite repression. The LevR activator interacts with its target, the upstream activating sequence (UAS), to stimulate the transcription of the E sigma L complex bound at the "-12, -24" promoter. Levanase operon expression in the presence of glucose was tested in strains carrying a ccpA gene disruption or a ptsH1 mutation in which Ser-46 of HPr is replaced by Ala. In a levR+ inducible genetic background, the expression of the levanase operon was partially resistant to catabolite repression in both mutants, indicating that the CcpA repressor and the HPr-SerP protein are involved in the glucose control of this operon. In addition, a cis-acting catabolite-responsive element (CRE) of the levanase operon was identified and investigated by site-directed mutagenesis. The CRE sequence TGAAAACGCTT(a)ACA is located between positions -50 and -36 from the transcriptional start site, between the UAS and the -12, -24 promoter. However, in a background constitutive for levanase, neither HPr, CcpA, nor CRE is involved in glucose repression, suggesting the existence of a different pathway of glucose regulation. Using truncated LevR proteins, we showed that this CcpA-independent pathway required the presence of the domain of LevR (amino acids 411 to 689) homologous to the BglG family of bacterial antiterminators.