Structural analysis of B. subtilis CcpA effector binding site.
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Bacillus subtilis↗
Biomedical subjects
Sandrine Poncet
Publications and source records attributed to Sandrine Poncet.
P-Ser-HPr--a link between carbon metabolism and the virulence of some pathogenic bacteria.
HPr kinase/phosphorylase phosphorylates HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system, at serine-46. P-Ser-HPr is the central regulator of carbon metabolism in Gram-positive bacteria, but also plays a role in virulence development of certain pathogens. In Listeria monocytogenes, several virulence genes, which depend on the transcription activator PrfA, are repressed by glucose, fructose, etc., in a catabolite repressor (CcpA)-independent mechanism. However, the catabolite co-repressor P-Ser-HPr was found to inhibit the activity of PrfA. In an hprKV267F mutant, in which most of the HPr is transformed into P-Ser-HPr, PrfA was barely active. The ptsH1 mutation (Ser-46 of HPr replaced with an alanine) prevented the inhibitory effect of the hprKV267F mutation. Interestingly, disruption of ccpA also inhibited PrfA activity. This effect is probably also mediated via P-Ser-HPr, since ccpA disruption leads to elevated amounts of P-Ser-HPr. Indeed, a ccpA ptsH1 double mutant exhibited normal PrfA activity. In S. pyogenes, the expression of several virulence genes depends on the transcription activator Mga. Interestingly, the mga promoter is preceded by an operator site, which serves as target for the CcpA/P-Ser-HPr complex. Numerous Gram-negative pathogens also contain hprK, which is often organised in an operon with transcription regulators necessary for the development of virulence, indicating that in these organisms P-Ser-HPr also plays a role in pathogenesis. Indeed, inactivation of Neisseria meningitidis hprK strongly diminished cell adhesion of this pathogen.
Bacterial Proteins↗
How seryl-phosphorylated HPr inhibits PrfA, a transcription activator of Listeria monocytogenes virulence genes.
Listeria monocytogenes PrfA, a transcription activator for several virulence genes, including the hemolysin-encoding hly, is inhibited by rapidly metabolizable carbon sources (glucose, fructose, etc.). This inhibition is not mediated via the major carbon catabolite repression mechanism of gram-positive bacteria, since inactivation of the catabolite control protein A (CcpA) did not prevent the repression of virulence genes by the above sugars. In order to test whether the catabolite co-repressor P-Ser-HPr might be involved in PrfA regulation, we used a Bacillus subtilis strain (BUG1199) containing L. monocytogenes prfA under control of pspac and the lacZ reporter gene fused to the PrfA-activated hly promoter. Formation of P-Ser-HPr requires the bifunctional HPr kinase/phosphorylase (HprK/P), which, depending on the concentration of certain metabolites, either phosphorylates HPr at Ser-46 or dephosphorylates P-Ser-HPr. The hprKV267F allele codes for an HprK/P leading to the accumulation of P-Ser-HPr, since it has normal kinase, but almost no phosphorylase activity. Interestingly, introducing hprKV267F into BUG1199 strongly inhibited transcription activation by PrfA. Preventing the accumulation of P-Ser-HPr in the hprKV267F mutant by replacing Ser-46 in HPr with an alanine restored PrfA activity, while ccpA inactivation had no effect. Interestingly, disruption of ccpA in the hprK wild-type strain BUG1199 also led to inhibition of PrfA. The lowered lacZ expression in the ccpA strain is probably also due to elevated amounts of P-Ser-HPr, since it disappeared when Ser-46 in HPr was replaced with an alanine. To carry out its catalytic function in sugar transport, HPr of the phosphotransferase system (PTS) is also phosphorylated by phosphoenolpyruvate and enzyme I at His-15. However, P-Ser-HPr is only very slowly phosphorylated by enzyme I, which probably accounts for PrfA inhibition. In agreement with this concept, disruption of the enzyme I- or HPr-encoding genes also strongly inhibited PrfA activity. PrfA activity therefore seems to depend on a fully functional PTS phosphorylation cascade.
Amino Acid Substitution↗
Is 2-phosphoglycerate-dependent automodification of bacterial enolases implicated in their export?
We observed that in vivo and in vitro a small fraction of the glycolytic enzyme enolase became covalently modified by its substrate 2-phosphoglycerate (2-PG). In modified Escherichia coli enolase, 2-PG was bound to Lys341, which is located in the active site. An identical reversible modification was observed with other bacterial enolases, but also with enolase from Saccharomyces cerevisiae and rabbit muscle. An equivalent of Lys341, which plays an important role in catalysis, is present in enolase of all organisms. Covalent binding of 2-PG to this amino acid rendered the enzyme inactive. Replacement of Lys341 of E.coli enolase with other amino acids prevented the automodification and in most cases strongly reduced the activity. As reported for other bacteria, a significant fraction of E.coli enolase was found to be exported into the medium. Interestingly, all Lys341 substitutions prevented not only the automodification, but also the export of enolase. The K341E mutant enolase was almost as active as the wild-type enzyme and therefore allowed us to establish that the loss of enolase export correlates with the loss of modification and not the loss of glycolytic activity.
Animals↗
HPr kinase/phosphorylase, a Walker motif A-containing bifunctional sensor enzyme controlling catabolite repression in Gram-positive bacteria.
Carbon catabolite repression (CCR) in Gram-positive bacteria is regulated by the bifunctional enzyme HPr kinase/phosphorylase (HprK/P). This enzyme catalyses the ATP- as well as the pyrophosphate-dependent phosphorylation of Ser-46 in HPr, a phosphocarrier protein of a sugar transport and phosphorylation system. HprK/P also catalyses the pyrophosphate-producing, inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr (P-Ser-HPr). P-Ser-HPr functions as catabolite co-repressor by interacting with the LacI/GalR-type repressor, catabolite control protein A (CcpA), and allowing it to bind to operator sites preceding catabolite-regulated transcription units. HprK/P thus indirectly controls the expression of about 10% of the genes of Gram-positive bacteria. The two antagonistic activities of HprK/P are regulated by intracellular metabolites, which change their concentration in response to the absence or presence of rapidly metabolisable carbon sources (glucose, fructose, etc.) in the growth medium. Biochemical and structural studies revealed that HprK/P exhibits no similarity to eukaryotic protein kinases and that it contains a Walker motif A (or P-loop) as nucleotide binding site. Interestingly, HprK/P has a structural fold resembling that in kinases phosphorylating certain low molecular weight substrates such as nucleosides, nucleotides or oxaloacetate. The structures of the complexes of HprK/P with HPr and P-Ser-HPr have also been determined, which allowed proposing a detailed mechanism for the kinase and phosphorylase functions of HprK/P.
Amino Acid Motifs↗
The Lactobacillus casei ptsHI47T mutation causes overexpression of a LevR-regulated but RpoN-independent operon encoding a mannose class phosphotransferase system.
A proteome analysis of Lactobacillus casei mutants that are affected in carbon catabolite repression revealed that a 15-kDa protein was strongly overproduced in a ptsHI47T mutant. This protein was identified as EIIA of a mannose class phosphotransferase system (PTS). A 7.1-kb DNA fragment containing the EIIA-encoding open reading frame and five other genes was sequenced. The first gene encodes a protein resembling the RpoN (sigma54)-dependent Bacillus subtilis transcription activator LevR. The following pentacistronic operon is oriented in the opposite direction and encodes four proteins with strong similarity to the proteins of the B. subtilis Lev-PTS and one protein of unknown function. The genes present on the 7.1-kb DNA fragment were therefore called levR and levABCDX. The levABCDX operon was induced by fructose and mannose. No "-12, -24" promoter typical of RpoN-dependent genes precedes the L. casei lev operon, and its expression was therefore RpoN independent but required LevR. Phosphorylation of LevR by P approximately His-HPr stimulates its activity, while phosphorylation by P approximately EIIBLev inhibits it. Disruption of the EIIBLev-encoding levB gene therefore led to strong constitutive expression of the lev operon, which was weaker in a strain carrying a ptsI mutation preventing phosphorylation by both P approximately EIIBLev and P approximately His-HPr. Expression of the L. casei lev operon is also subject to P-Ser-HPr-mediated catabolite repression. The observed slow phosphoenolpyruvate- and ATP-dependent phosphorylation of HPrI47T as well as the slow phosphoryl group transfer from the mutant P approximately His-HPr to EIIALev are assumed to be responsible for the elevated expression of the lev operon in the ptsHI47T mutant.
Amino Acid Sequence↗
Transmembrane modulator-dependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases.
Protein-tyrosine kinases regulating bacterial exopolysaccharide synthesis autophosphorylate on tyrosines located in a conserved C-terminal region. So far no other substrates have been identified for these kinases. Here we demonstrate that Bacillus subtilis YwqD not only autophosphorylates at Tyr-228, but that it also phosphorylates the two UDP-glucose dehydrogenases (UDP-glucose DHs) YwqF and TuaD at a tyrosine residue. However, phosphorylation of YwqF and TuaD occurs only in the presence of the transmembrane protein YwqC. The presumed intracellular C-terminal part of YwqC (last 50 amino acids) seems to interact with the tyrosine-kinase and to allow YwqD-catalysed phosphorylation of the two UDP-glucose DHs, which are key enzymes for the synthesis of acidic polysaccharides. However, only when phosphorylated by YwqD do the two enzymes exhibit detectable UDP-glucose DH activity. Dephosphorylation of P-Tyr-YwqF and P-Tyr-TuaD by the P-Tyr-protein phosphatase YwqE switched off their UDP-glucose DH activity. YwqE, which is encoded by the fourth gene of the B.subtilis ywqCDEF operon, also dephosphorylates P-Tyr-YwqD.
Amino Acid Sequence↗
CcpA regulation of aerobic and respiration growth in Lactococcus lactis.
The catabolic control protein CcpA is the highly conserved regulator of carbon metabolism in Gram-positive bacteria. We recently showed that Lactococcus lactis, a fermenting bacterium in the family of Streptococcaceae, is capable of respiration late in growth when haem is added to aerated cultures. As the start of respiration coincides with glucose depletion from the medium, we hypothesized that CcpA is involved in this metabolic switch and investigated its role in lactococcal growth under aeration and respiration conditions. Compared with modest changes observed in fermentation growth, inactivation of ccpA shifts metabolism to mixed acid fermentation under aeration conditions. This shift is due to a modification of the redox balance via derepression of NADH oxidase, which eliminates oxygen and decreases the NADH pool. CcpA also plays a decisive role in respiration metabolism. Haem addition to lag phase ccpA cells results in growth arrest and cell mortality. Toxicity is due to oxidative stress provoked by precocious haem uptake. We identify the repressor of the haem transport system and show that it is a target of CcpA activation. We propose that CcpA-mediated repression of haem uptake is a means of preventing oxidative damage at the start of exponential growth. CcpA thus appears to govern a regulatory network that coordinates oxygen, iron and carbon metabolism.
Aerobiosis↗
HPr kinase/phosphorylase, the sensor enzyme of catabolite repression in Gram-positive bacteria: structural aspects of the enzyme and the complex with its protein substrate.
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Bacterial Proteins↗
Transcription regulators potentially controlled by HPr kinase/phosphorylase in Gram-negative bacteria.
Phosphorylation and dephosphorylation at Ser-46 in HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is controlled by the bifunctional HPr kinase/phosphorylase (HprK/P). In Gram-positive bacteria, P-Ser-HPr controls (1) sugar uptake via the PTS; (2) catabolite control protein A (CcpA)-mediated carbon catabolite repression, and (3) inducer exclusion. Genome sequencing revealed that HprK/P is absent from Gram-negative enteric bacteria, but present in many other proteobacteria. These organisms also possess (1) HPr, the substrate for HprK/P; (2) enzyme I, which phosphorylates HPr at His-15, and (3) one or several enzymes IIA, which receive the phosphoryl group from P approximately His-HPr. The genes encoding the PTS proteins are often organized in an operon with HPRK. However, most of these organisms miss CcpA and a functional PTS, as enzymes IIB and membrane-integrated enzymes IIC seem to be absent. HprK/P and the rudimentary PTS phosphorylation cascade in Gram-negative bacteria must therefore carry out functions different from those in Gram-positive organisms. The gene organization in many HprK/P-containing Gram-negative bacteria as well as some preliminary experiments suggest that HprK/P might control transcription regulators implicated in cell adhesion and virulence. In alpha-proteobacteria, HPRK is located downstream of genes encoding a two-component system of the EnvZ/OmpR family. In several other proteobacteria, HPRK is organized in an operon together with genes from the RPON region of ESCHERICHIA COLI (RPON encodes a sigma54). We propose that HprK/P might control the phosphorylation state of HPr and EIIAs, which in turn could control the transcription regulators.
Amino Acid Sequence↗
X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr.
HPr kinase/phosphorylase (HprK/P) controls the phosphorylation state of the phosphocarrier protein HPr and regulates the utilization of carbon sources by Gram-positive bacteria. It catalyzes both the ATP-dependent phosphorylation of Ser-46 of HPr and its dephosphorylation by phosphorolysis. The latter reaction uses inorganic phosphate as substrate and produces pyrophosphate. We present here two crystal structures of a complex of the catalytic domain of Lactobacillus casei HprK/P with Bacillus subtilis HPr, both at 2.8-A resolution. One of the structures was obtained in the presence of excess pyrophosphate, reversing the phosphorolysis reaction and contains serine-phosphorylated HPr. The complex has six HPr molecules bound to the hexameric kinase. Two adjacent enzyme subunits are in contact with each HPr molecule, one through its active site and the other through its C-terminal helix. In the complex with serine-phosphorylated HPr, a phosphate ion is in a position to perform a nucleophilic attack on the phosphoserine. Although the mechanism of the phosphorylation reaction resembles that of eukaryotic protein kinases, the dephosphorylation by inorganic phosphate is unique to the HprK/P family of kinases. This study provides the structure of a protein kinase in complex with its protein substrate, giving insights into the chemistry of the phospho-transfer reactions in both directions.
Bacillus subtilis↗
Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: a relic of early life?
In most Gram-positive bacteria, serine-46-phosphorylated HPr (P-Ser-HPr) controls the expression of numerous catabolic genes ( approximately 10% of their genome) by acting as catabolite corepressor. HPr kinase/phosphorylase (HprK/P), the bifunctional sensor enzyme for catabolite repression, phosphorylates HPr, a phosphocarrier protein of the sugar-transporting phosphoenolpyruvate/glycose phosphotransferase system, in the presence of ATP and fructose-1,6-bisphosphate but dephosphorylates P-Ser-HPr when phosphate prevails over ATP and fructose-1,6-bisphosphate. We demonstrate here that P-Ser-HPr dephosphorylation leads to the formation of HPr and pyrophosphate. HprK/P, which binds phosphate at the same site as the beta phosphate of ATP, probably uses the inorganic phosphate to carry out a nucleophilic attack on the phosphoryl bond in P-Ser-HPr. HprK/P is the first enzyme known to catalyze P-protein dephosphorylation via this phospho-phosphorolysis mechanism. This reaction is reversible, and at elevated pyrophosphate concentrations, HprK/P can use pyrophosphate to phosphorylate HPr. Growth of Bacillus subtilis on glucose increased intracellular pyrophosphate to concentrations ( approximately 6 mM), which in in vitro tests allowed efficient pyrophosphate-dependent HPr phosphorylation. To effectively dephosphorylate P-Ser-HPr when glucose is exhausted, the pyrophosphate concentration in the cells is lowered to 1 mM. In B. subtilis, this might be achieved by YvoE. This protein exhibits pyrophosphatase activity, and its gene is organized in an operon with hprK.
Bacillus subtilis↗
Antitermination by GlpP, catabolite repression via CcpA and inducer exclusion triggered by P-GlpK dephosphorylation control Bacillus subtilis glpFK expression.
The Bacillus subtilis glpFK operon encoding the glycerol transport facilitator (GlpF) and glycerol kinase (GlpK) is induced by glycerol-3-P and repressed by rapidly metabolizable sugars. Carbon catabolite repression (CCR) of glpFK is partly mediated via a catabolite response element cre preceding glpFK. This operator site is recognized by the catabolite control protein A (CcpA) in complex with one of its co-repressors, P-Ser-HPr or P-Ser-Crh. HPr is a component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and Crh is an HPr homologue. The hprK-encoded HPr kinase phosphorylates HPr and Crh at Ser-46. But in neither ccpA nor hprK mutants was expression of a glpF'-lacZ fusion relieved from CCR, as a second, CcpA-independent CCR mechanism implying the terminator tglpFK, whose formation is prevented by the glycerol-3-P-activated antiterminator GlpP, is operative. Deletion of tglpFK led to elevated expression of the glpF'-lacZ fusion and to partial relief from CCR. CCR completely disappeared in DeltatglpFK mutants carrying a disruption of ccpA or hprK. The tglpFK-requiring CCR mechanism seems to be based on insufficient synthesis of glycerol-3-P, as CCR of glpFK was absent in ccpA mutants growing on glycerol-3-P or synthesizing H230R mutant GlpK. In cells growing on glycerol, glucose prevents the phosphorylation of GlpK by P-His-HPr. P-GlpK is much more active than GlpK, and the absence of P~GlpK formation in DeltaptsHI strains prevents glycerol metabolism. As a consequence, only small amounts of glycerol-3-P will be formed in glycerol and glucose-exposed cells (inducer exclusion). The uptake of glycerol-3-P via GlpT provides high concentrations of this metabolite in the ccpA mutant and allows the expression of the glpF'-lacZ fusion even when glucose is present. Similarly, despite the presence of glucose, large amounts of glycerol-3-P are formed in a glycerol-exposed strain synthesizing GlpKH230R, as this mutant GlpK is as active as P-GlpK.
Amino Acid Sequence↗
Glycerol transport and phosphoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of glycerol kinase in Thermus flavus.
The genes glpK and glpF, encoding glycerol kinase and the glycerol facilitator of Thermus flavus, a member of the Thermus/Deinococcus group, have recently been identified. The protein encoded by glpK exhibited an unusually high degree of sequence identity (80-6%) when compared to the sequence of glycerol kinase from Bacillus subtilis and a similar high degree of sequence identity (64.8%) was observed when the sequences of the glycerol facilitators of the two organisms were compared. The work presented in this paper demonstrates that T. flavus is capable of taking up glycerol, that glpF and glpK are expressed constitutively and that glucose exerts a repressive effect on the expression of these genes. T. flavus was found to possess the general components of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) enzyme I and histidine-containing protein (HPr). These proteins catalyse the phosphorylation of T. flavus glycerol kinase, which contains a histidyl residue equivalent to His-232, the site of PEP-dependent, PTS-catalysed phosphorylation in glycerol kinase of Enterococcus casseliflavus. Purified glycerol kinase from T. flavus could also be phosphorylated with enzyme I and HPr from B. subtilis. Similar to enterococcal glycerol kinases, phosphorylated T. flavus glycerol kinase exhibited an electrophoretic mobility on denaturing and non-denaturing polyacrylamide gels that is different from the electrophoretic mobility of non-phosphorylated glycerol kinase. However, in contrast to PEP-dependent phosphorylation of enterococcal glycerol kinases, which stimulated glycerol kinase activity about 10-fold, phosphorylation of T. flavus glycerol kinase caused only a slight increase in enzyme activity.
Amino Acid Sequence↗
Spo0A represses transcription of the cry toxin genes in Bacillus thuringiensis.
The DNA regions upstream from the genes encoding polypeptides of Bacillus thuringiensis subsp. israelensis larvicidal crystals (cry4A, cry4B, cry11A) contain sequences with similarities to the spo0A box of Bacillus subtilis (or '0A' box) and the promoter recognized by the sigma H-associated RNA polymerase of B. subtilis. Expression of cry-lacZ transcriptional fusions was analysed in various B. thuringiensis genetic backgrounds. The early transcription of the toxin genes was not sporulation-dependent, whereas the late-stage expression at t4-6 was sigma E-dependent. Primer extension analysis confirmed that the cry4- and cry11-type toxin genes were weakly transcribed during the transition phase; expression analysis of a cry11A'-lacZ transcriptional fusion in B. subtilis sporulation mutants confirmed the involvement of the sigma H-RNA polymerase. Primer extension analysis showed that in B. thuringiensis subsp. israelensis, the cry4A and cry11A gene transcription observed at the end of the growth stage was turned off at the beginning of the sporulation phase. The DNA region located upstream from the cry11A gene promoter including the putative '0A' box was deleted. This led to a derepression of the expression of the cry11A operon. These results suggest that the cry4A, cry4B and cry11A toxin genes of B. thuringiensis subsp. Israelensis are transcribed during the transition phase by the RNA polymerase associated with the sigma H factor and are subject to Spo0A repression.
Bacillus subtilis↗