Changes in Glycolytic Activity of Lactococcus lactis Induced by Low Temperature

The effects of low-temperature stress on the glycolytic activity of the lactic acid bacterium Lactococcus lactis were studied. The maximal glycolytic activity measured at 30°C increased approximately 2.5-fold following a shift from 30 to 10°C for 4 h in a process that required protein synthesis. Analysis of cold adaptation of strains with genes involved in sugar metabolism disrupted showed that both the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) subunit HPr and catabolite control protein A (CcpA) are involved in the increased acidification at low temperatures. In contrast, a strain with the PTS subunit enzyme I disrupted showed increased acidification similar to that in the wild-type strain. This indicates that the PTS is not involved in this response whereas the regulatory function of 46-seryl phosphorylated HPr [HPr(Ser-P)] probably is involved. Protein analysis showed that the production of both HPr and CcpA was induced severalfold (up to two- to threefold) upon exposure to low temperatures. The las operon, which is subject to catabolite activation by the CcpA-HPr(Ser-P) complex, was not induced upon cold shock, and no increased lactate dehydrogenase (LDH) activity was observed. Similarly, the rate-limiting enzyme of the glycolytic pathway under starvation conditions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not induced upon cold shock. This indicates that a factor other than LDH or GAPDH is rate determining for the increased glycolytic activity upon exposure to low temperatures. Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA-HPr(Ser-P) control circuit regulates this factor(s) and hence couples catabolite repression and cold shock response in a functional and mechanistic way.     

Phosphorylation state of HPr determines the level of expression and the extent of phosphorylation of the lactose transport protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus is composed of a translocator domain and a regulatory domain that is phosphorylated by HPr(His approximately P), the general energy coupling protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). Lactose transport is affected by the phosphorylation state of HPr through changes in the activity of the LacS protein as well as expression of the lacS gene. To address whether or not CcpA-HPr(Ser-P)-mediated catabolite control is involved, the levels of LacS were determined under conditions in which the cellular phosphorylation state of HPr greatly differed. It appears that HPr(Ser-P) is mainly present in the exponential phase of growth, whereas HPr(His approximately P) dominates in the stationary phase. The transition from HPr(Ser-P) to HPr(His approximately P) parallels an increase in LacS level, a drop in lactose and an increase in galactose concentration in the growth medium. Because the K(m)(out) for lactose is higher than that for galactose, the lactose transport capacity decreases as lactose concentration decreases and galactose accumulates in the medium. Our data indicate that S. thermophilus compensates for the diminished transport capacity by synthesizing more LacS and phosphorylating the protein, which results in increased transport activity. The link between transport capacity and lacS expression levels and LacS phosphorylation are discussed.     

Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram-positive bacteria

CcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram-positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag. CcpA-his immobilized on a Ni-NTA resin specifically interacted with HPr phosphorylated at seryl residue 46. HPr, a phosphocarrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His-15 in a PEP-dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser-46 in an ATP-dependent reaction catalysed by a metabolite-activated protein kinase. Neither unphosphorylated HPr nor HPr phosphorylated at His-15 nor the doubly phosphorylated HPr bound to CcpA. The interaction with seryl-phosphorylated HPr required the presence of fructose 1,6-bisphosphate. These findings suggest that carbon catabolite repression in Gram-positive bacteria is a protein kinase-triggered mechanism. Glycolytic intermediates, stimulating the corresponding protein kinase and the P-ser-HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His-15 also suggests a link between carbon catabolite repression and PTS transport activity.     

Significance of HPr in catabolite repression of alpha-amylase.

CcpA and HPr are presently the only two proteins implicated in Bacillus subtilis global carbon source catabolite repression, and the ptsH1 mutation in the gene for the HPr protein was reported to relieve catabolite repression of several genes. However, alpha-amylase synthesis by B. subtilis SA003 containing the ptsH1 mutation was repressed by glucose. Our results suggest HPr(Ser-P) may be involved in but is not required for catabolite repression of alpha-amylase, indicating that HPr(Ser-P) is not the sole signaling molecule for CcpA-mediated catabolite repression in B. subtilis.     

Effect of HPr phosphorylation on structure,dynamics, and interactions in the course of transcriptional control

The serine46-phosphorylated form of the bacterial protein HPr fulfils an essential function in carbon catabolite repression (CCR). Using molecular dynamics (MD) we studied the effect of Ser46 phosphorylation on the molecular properties of HPr and its capability to act as the co-repressor of carbon catabolite protein A (CcpA). The calculated pK (a) values for a representative set of HPr(Ser46P) structures indicate that the phosphate group of HPr(Ser46P) exists predominantly in the unprotonated form under neutral conditions. A hydrogen bond detected in HPr(Ser46P) between one phosphate-group oxygen and a side-chain hydrogen of Asn43-an amino acid conserved in all HPr proteins of Gram-positive bacteria that regulate their carbon consumption by CCR-might fulfil an important role in CcpA-HPr(Ser46P) complex formation. MD simulations show that the Ser46P-Asn43 hydrogen bond present in the unbound structure is replaced by intermolecular interactions upon complex formation. The degree to which amino acids in the CcpA-HPr(Ser46P) interface contribute to cofactor binding was analyzed by in silico alanine scanning. Lys307, Arg303, Asp296, Val300, and Tyr295 of CcpA were identified as important amino acids for the CcpA-HPr(Ser46P) interaction. Three of these residues are directly involved in sensing the correct phosphorylation state at His15(HPr) and Ser46(HPr). A substitution of interface residues Val319, Val314, Ser316, Leu321 and Gln320 by alanine showed that these amino acids, which contact helix alpha2 of HPr(Ser46P), play a less prominent role for complex formation.     

Carbon catabolite repression by the catabolite control protein CcpA in Staphylococcus xylosus

Carbon catabolic repression (CR) by the catabolite control protein CcpA has been analyzed in Staphylococcus xylosus. Genes encoding components needed to utilize lactose, sucrose, and maltose were found to be repressed by CcpA. In addition, the ccpA gene is under negative autogenous control. Among several tested sugars, glucose caused strongest CcpA-dependent repression. Glucose can enter S. xylosus in nonphosphorylated form via the glucose uptake protein GlcU. Internal glucose is then phosphorylated by the glucose kinase GlkA. Alternatively, glucose can be transported and concomitantly phosphorylated by glucose-specific permease(s) of the phosphotransferase system (PTS). S. xylosus mutant strains deficient in GlcU or GlkA showed partial relief of glucose-specific, CcpA-dependent repression. Likewise, blocking PTS activity completely by inactivation of the gene encoding the general PTS protein enzyme I resulted in diminished glucose-mediated repression. Thus, both glucose entry routes contribute to glucose-specific CR in S. xylosus. The sugar transport activity of the PTS is not required to trigger glucose-specific repression. The phosphocarrier protein HPr however, is absolutely essential for CcpA activity. Inactivation of the HPr gene led to a complete loss of CR. Repression is also abolished upon inactivation of the HPr kinase gene or by replacing serine at position 46 of HPr by alanine. These results clearly show that HPr kinase provides the signal, seryl-phosphorylated HPr, to activate CcpA in S. xylosus.     

Synthesis of HPr(Ser-P)(His-P) by enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus salivarius

HPr is a protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In Gram-positive bacteria, HPr can be phosphorylated on Ser(46) by HPr(Ser) kinase/phosphorylase (HPrK/P) and on His(15) by enzyme I (EI) of the PTS. In vitro studies have shown that phosphorylation on one residue greatly inhibits the second phosphorylation. However, streptococci contain significant amounts of HPr(Ser-P)(His approximately P) during exponential growth, and recent studies suggest that phosphorylation of HPr(Ser-P) by EI is involved in the recycling of HPr(Ser-P)(His approximately P). We report in this paper a study on the phosphorylation of Streptococcus salivarius HPr, HPr(Ser-P), and HPr(S46D) by EI. Our results indicate that (i) the specificity constant (k(cat)/K(m)) of EI for HPr(Ser-P) at pH 7.9 was approximately 5000-fold smaller than that observed for HPr, (ii) no metabolic intermediates were able to stimulate HPr(Ser-P) phosphorylation, (iii) the rate of HPr phosphorylation decreased at pHs below 6.5, while that of HPr(Ser-P) increased and was almost 10-fold higher at pH 6.1 than at pH 7.9, (iv) HPr(S46D), a mutated HPr alleged to mimic HPr(Ser-P), was also phosphorylated more efficiently under acidic conditions, and, lastly, (v) phosphorylation of Bacillus subtilis HPr(Ser-P) by B. subtilis EI was also stimulated at acidic pH. Our results suggest that the high levels of HPr(Ser-P)(His approximately P) in streptococci result from the combination of two factors, a high physiological concentration of HPr(Ser-P) and stimulation of HPr(Ser-P) phosphorylation by EI at acidic pH, an intracellular condition that occurs in response to the acidification of the external medium during growth of the culture.     

Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the Gram-positive bacteria?

Three components involved in catabolite repression (CR) of gene expression in Bacillus have been identified. The cis-acting catabolite responsive element (CRE), which is present in many genes encoding carbon catabolic enzymes in various species of the Gram-positive bacteria, mediates CR of several genes in Bacillus subtilis, Bacillus megaterium, and Staphylococcus xylosus. CR of most genes regulated via CRE is also affected by the trans-acting factors CcpA and HPr. Similarities between CcpA and Lac and Gal repressors suggest binding of CcpA to CRE. HPr, a component of the phosphoenol pyruvate:sugar phosphotransferase system, undergoes regulatory phosphorylation at a serine residue by a fcuctose-1,6-diphosphate-activated kinase. A mutant of HPr, which is not phosphorylatable at this position because of an exchange of serine to alanine, lacks CR of several catabolic activities. This mutant phenotype is similar to the one exhibited by a ccpA mutant. Direct protein-protein interaction between CcpA and HPr(Ser-P) was recently demonstrated and constitutes a link between metabolic activity and CR.     

Control of the glycolytic gapA operon by the catabolite control protein A in Bacillus subtilis: a novel mechanism of CcpA-mediated regulation

Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis. Expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase, the key enzyme of glycolysis from an energetic point of view, is induced by glucose and other sugars. Two regulators are involved in induction of the gapA operon, the product of the first gene of the operon, the CggR repressor, and catabolite control protein A (CcpA). CcpA is required for induction of the gapA operon by glucose. Genetic evidence has demonstrated that CcpA does not control the expression of the gapA operon by binding directly to a target in the promoter region. Here, we demonstrate by physiological analysis of the inducer spectrum that CcpA is required only for induction by sugars transported by the phosphotransferase system (PTS). A functional CcpA is needed for efficient transport of these sugars. This interference of CcpA with PTS sugar transport results from an altered phosphorylation pattern of HPr, a phosphotransferase of the PTS. In a ccpA mutant strain, HPr is nearly completely phosphorylated on a regulatory site, Ser-46, and is trapped in this state, resulting in its inactivity in PTS phosphotransfer. A mutation in HPr affecting the regulatory phosphorylation site suppresses both the defect in PTS sugar transport and the induction of the gapA operon. We conclude that a low-molecular effector derived from glucose that acts as an inducer for the repressor CggR is limiting in the ccpA mutant.     

Residues His-15 and Arg-17 of HPr participate differently in catabolite signal processing via CcpA

The carbon catabolite control protein A (CcpA) senses the physiological state of the cell by binding several effectors and responds with differential regulation of many genes in Bacilli. HPr-Ser46-P or Crh-Ser46-P interact with CcpA and stimulate binding to catabolite responsive elements. In addition, the glycolytic intermediates fructose 1,6-bisphosphate (FBP) and glucose 6-phosphate (Glc-6-P) stimulate HPr-Ser46-P but not Crh-Ser46-P binding to CcpA. The mechanisms by which coeffector binding to CcpA is linked to differential gene expression are unclear. To address this question we mutated residues participating in the interaction between HPr-Ser46-P or Crh-Ser46-P and CcpA and analyzed their effects on CcpA binding and stimulation of cre binding by surface plasmon resonance. The HPrH15A and CcpAD297A mutations do not affect complex formation but abolish FBP and Glc-6-P stimulation. Likewise, the CrhQ15H mutant becomes sensitive to these glycolytic intermediates. Hence, the contact of HPrHis-15 to Asp-297 in CcpA is a determinant for HPr specific FBP and Glc-6-P stimulation. The HPrR17A and -K mutants are both strongly impaired in stimulation of CcpA binding to cre, but only HPrR17A is defect in binding to CcpA indicating that these residues affect allostery of CcpA. Mutations of the residues of CcpA, which contact Arg-17 of HPr, exhibit differential effects on regulation of catabolic genes. Taken together, His-15 of HPr processes sensing information, while Arg-17 is involved in determining the genetic output.     

Streptococci and lactococci synthesize large amounts of HPr(Ser-P)(His~P)

HPr is a protein of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In gram-positive bacteria, HPr can be phosphorylated on Ser-46 by the kinase/phosphorylase HprK/P and on His-15 by phospho-enzyme I (EI~P) of the PTS. In vitro studies with purified HPrs from Bacillus subtilis, Enterococcus faecalis, and Streptococcus salivarius have indicated that the phosphorylation of one residue impedes the phosphorylation of the other. However, a recent study showed that while the rate of Streptococcus salivarius HPr phosphorylation by EI~P is reduced at acidic pH, the phosphorylation of HPr(Ser-P) by EI~P, generating HPr(Ser-P)(His~P), is stimulated. This suggests that HPr(Ser-P)(His~P) synthesis may occur in acidogenic bacteria unable to maintain their intracellular pH near neutrality. Consistent with this hypothesis, significant amounts of HPr(Ser-P)(His~P) have been detected in some streptococci. The present study was aimed at determining whether the capacity to synthesize HPr(Ser-P)(His~P) is common to streptococcal species, as well as to lactococci, which are also unable to maintain their intracellular pH near neutrality in response to a decrease in extracellular pH. Our results indicated that unlike Staphylococcus aureus, B. subtilis, and E. faecalis, all the streptococcal and lactococcal species tested were able to synthesize large amounts of HPr(Ser-P)(His~P) during growth. We also showed that Streptococcus salivarius IIABLMan, a protein involved in sugar transport by the PTS, could be efficiently phosphorylated by HPr(Ser-P)(His~P).     

Replacement of isoleucine-47 by threonine in the HPr protein of Streptococcus salivarius abrogates the preferential metabolism of glucose and fructose over lactose and melibiose but does not prevent the phosphorylation of HPr on serine-46

Phosphorylation of HPr on a serine residue at position 46 (Ser-46) by an ATP-dependent protein kinase has been reported in several Gram-positive bacteria, and the resulting intermediate, HPr(Ser-P), has been shown to mediate inducer exclusion in lactococci and lactobacilli and catabolite repression in Bacillus subtilis and Bacillus megaterium . We report here the phenotypic properties of an isogenic spontaneous mutant (G22.4) of Streptococcus salivarius ATCC 25975, in which a missense mutation results in the replacement of isoleucine at position 47 (Ile-47) by threonine (Thr) in HPr. This substitution did not prevent the phosphorylation of HPr on Ser-46, nor did it impede the phosphorylation of HPr on His-15 by EI or the transfer of the phosphoryl group from HPr(His∼P) to other PTS proteins. However, the I47T substitution did perturb, in glucose-grown but not in galactose-grown cells, the cellular equilibrium between the various forms of HPr, resulting in an increase in the amount of free HPr at the expense of HPr(His∼P)(Ser-P); the levels of HPr(His∼P) and HPr(Ser-P) were not affected. Growth on melibiose was virtually identical for the wild-type and mutant strains, whereas the generation time of the mutant on the other sugars tested (glucose, fructose, mannose, lactose and galactose) increased 1.2- to 1.5-fold. The preferential metabolism of PTS sugars (glucose and fructose) over non-PTS sugars (lactose and melibiose) that is observed in wild-type cells was abolished in cells of mutant G22.4. Moreover, α- and β-galactosidases were derepressed in glucose- and fructose-grown cells of the mutant. The data suggest that HPr regulates the preferential metabolism of PTS sugars over the non-PTS sugars, lactose and melibiose, through the repression of the pertinent catabolic genes. This HPr-dependent repression, however, seems to occur solely when cells are growing on a PTS sugar.     

CcpA-mediated repression of Clostridium difficile toxin gene expression

The presence of glucose or other rapidly metabolizable carbon sources in the bacterial growth medium strongly represses Clostridium difficile toxin synthesis independently of strain origin. In Gram-positive bacteria, carbon catabolite repression (CCR) is generally regarded as a regulatory mechanism that responds to carbohydrate availability. In the C. difficile genome all elements involved in CCR are present. To elucidate in vivo the role of CCR in C. difficile toxin synthesis, we used the ClosTron gene knockout system to construct mutants of strain JIR8094 that were unable to produce the major components of the CCR signal transduction pathway: the phosphotransferase system (PTS) proteins (Enzyme I and HPr), the HPr kinase/phosphorylase (HprK/P) and the catabolite control protein A, CcpA. Inactivation of the ptsI, ptsH and ccpA genes resulted in derepression of toxin gene expression in the presence of glucose, whereas repression of toxin production was still observed in the hprK mutant, indicating that uptake of glucose is required for repression but that phosphorylation of HPr by HprK is not. C. difficile CcpA was found to bind to the regulatory regions of the tcdA and tcdB genes but not through a consensus cre site motif. Moreover in vivo and in vitro results confirmed that HPr-Ser45-P does not stimulate CcpA-dependent binding to DNA targets. However, fructose-1,6-biphosphate (FBP) alone did increase CcpA binding affinity in the absence of HPr-Ser45-P. These results showed that CcpA represses toxin expression in response to PTS sugar availability, thus linking carbon source utilization to virulence gene expression in C. difficile.     

Quantitative determination of the intracellular concentration of the various forms of HPr, a phosphocarrier protein of the phosphoenolpyruvate: sugar phosphotransferase system in growing cells of oral streptococci.

A simple procedure for quantitative estimation of the different phosphorylated forms of the phosphocarrier protein HPr in growing cells of oral streptococci is described. The growth of the cells was rapidly stopped by acidification of the medium and concomitant addition of the ionophore Gramicidin D. This procedure inactivated Enzyme I, HPr(Ser) kinase, HPr(Ser-P) phosphatase, and the enzymes involved in the metabolism of the allosteric effectors as well as the substrates of HPr phosphorylation. The cellular concentrations of HPr (His approximately P), HPr (Ser-P), HPr (His approximately P) (Ser-P), and free HPr were then determined by crossed immunoelectrophoresis.