trans-acting factors affecting carbon catabolite repression of the hut operon in Bacillus subtilis

In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins. During growth in minimal medium, the ptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither the ptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, the ptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase (glnA). In addition, these glnA mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.     

The galU gene expression in Streptococcus pneumoniae

Bacillus subtilis possesses carbon-flux regulating histidine protein (Crh), a paralog of the histidine protein (HPr) of the phosphotransferase system (PTS). Like HPr, Crh becomes (de)phosphorylated in vitro at residue Ser46 by the metabolite-controlled HPr kinase/phosphorylase HPrK/P. Depending on its phosphorylation state, Crh exerts regulatory functions in connection with carbohydrate metabolism. So far, knowledge on phosphorylation of Crh in vivo has been limited and derived from indirect evidence. Here, we studied the dynamics of Crh phosphorylation directly by non-denaturing gel electrophoresis followed by Western analysis. The results confirm that HPrK/P is the single kinase catalyzing phosphorylation of Crh in vivo. Accordingly, phosphorylation of Crh is triggered by the carbon source as observed previously for HPr, but with some differences. Phosphorylation of both proteins occurred during exponential growth and disappeared upon exhaustion of the carbon source. During exponential growth, ~80% of the Crh molecules were phosphorylated when cells utilized a preferred carbon source. The reverse distribution, i.e. around 20% of Crh molecules phosphorylated, was obtained upon utilization of less favorable substrates. This clear-cut classification of the substrates into two groups has not previously been observed for HPr(Ser)~P formation. The likely reason for this difference is the additional PTS-dependent phosphorylation of HPr at His15, which limits accumulation of HPr(Ser)~P.     

Crh, the paralogue of the phosphocarrier protein HPr, controls the methylglyoxal bypass of glycolysis in Bacillus subtilis

The histidine protein HPr has a key role in regulation of carbohydrate utilization in low-GC Gram-positive bacteria. Bacilli possess the paralogue Crh. Like HPr, Crh becomes phosphorylated by kinase HPrK/P in response to high fructose-1,6-bisphosphate concentrations. However, Crh can only partially substitute for the regulatory functions of HPr leaving its role mysterious. Using protein co-purification, we identified enzyme methylglyoxal synthase MgsA as interaction partner of Crh in Bacillus subtilis. MgsA converts dihydroxyacetone-phosphate to methylglyoxal and thereby initiates a glycolytic bypass that prevents the deleterious accumulation of phospho-sugars under carbon overflow conditions. However, methylgyloxal is toxic and its production requires control. We show here that exclusively the non-phosphorylated form of Crh interacts with MgsA in vivo and inhibits MgsA activity in vitro. Accordingly, Crh inhibits methylglyoxal formation in vivo under nutritional famine conditions that favour a low HPr kinase activity. Thus, Crh senses the metabolic state of the cell, as reflected by its phosphorylation state, and accordingly controls flux through the harmful methylglyoxal pathway. Interestingly, HPr is unable to bind and regulate MgsA, making this a bona fide function of Crh. Four residues that differ in the interaction surfaces of HPr and Crh may account for this difference.     

Evidence for a dimerisation state of the Bacillus subtilis catabolite repression HPr-like protein, Crh

The Bacillus subtilis catabolite repression HPr (Crh) exhibits 45% sequence identity when compared to histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system. We report here that Crh preparations contain a mixture of monomers and homodimers, whereas HPr is known to be monomeric in solution. The dissociation rate of dimers is very slow (t1/2 of about 10 hours), and the percentage of dimers in Crh preparations increases with rising temperature or protein concentration. However, at temperatures above 25 degrees C and a protein concentration of 10 mg/ml, Crh dimers slowly aggregate. Typically, NMR spectra recorded at 25 degrees C showed the coexistence of both forms of Crh, while in Crh solutions kept at 35 degrees C, almost exclusively Crh monomers could be detected. Circular dichroism analysis revealed that the monomeric and dimeric forms of Crh are well folded and exhibit the same overall structure. The physiological significance of the slow Crh monomer/dimer equilibrium remains enigmatic.     

Drastic differences in Crh and HPr synthesis levels reflect their different impacts on catabolite repression in Bacillus subtilis

In Bacillus subtilis, carbon catabolite repression (CCR) of catabolic genes is mediated by ATP-dependent phosphorylation of HPr and Crh. Here we show that the different efficiencies with which these two proteins contribute to CCR may be due to the drastic differences in their synthesis rates under conditions that cause CCR.     

Solution structure and dynamics of Crh, the Bacillus subtilis catabolite repression HPr

The solution structure and dynamics of the Bacillus subtilis HPr-like protein, Crh, have been investigated using NMR spectroscopy. Crh exhibits high sequence identity (45 %) to the histidine-containing protein (HPr), a phospho-carrier protein of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system, but contains no catalytic His15, the site of PEP-dependent phosphorylation in HPr. Crh also forms a mixture of monomers and dimers in solution whereas HPr is known to be monomeric. Complete backbone and side-chain assignments were obtained for the monomeric form, and 60 % of the dimer backbone resonances; allowing the identification of the Crh dimer interface from chemical-shift mapping. The conformation of Crh was determined to a precision of 0.46(+/-0.06) A for the backbone atoms, and 1.01(+/-0.08) A for the heavy atoms. The monomer structure is similar to that of known HPr 2.67(+/-0.22) A (C(alpha) rmsd), but has a few notable differences, including a change in the orientation of one of the helices (B), and a two-residue shift in beta-sheet pairing of the N-terminal strand with the beta4 strand. This shift results in a shortening of the surface loop present in HPr and consequently provides a flatter surface in the region of dimerisation contact, which may be related to the different oligomeric nature of these two proteins. A binding site of phospho-serine(P-Ser)-Crh with catabolite control protein A (CcpA) is proposed on the basis of highly conserved surface side-chains between Crh and HPr. This binding site is consistent with the model of a dimer-dimer interaction between P-Ser-Crh and CcpA. (15)N relaxation measured in the monomeric form also identified differential local mobility in the helix B which is located in the vicinity of this site.     

Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis

In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli, the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA. The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.     

Insights into the functioning of Bacillus subtilis HPr kinase/phosphatase: affinity for its protein substrates and role of cations and phosphate

In Bacillus subtilis, carbon catabolite repression is mediated by the HPr kinase/phosphatase (HprK/P) which catalyzes both an ATP-dependent phosphorylation and a dephosphorylation on Ser-46 of either HPr (histidine-containing protein) or Crh (catabolite repression HPr). By using a surface plasmon resonance approach, it was shown here that the presence of magnesium is a prerequisite for the interaction of HprK/P with either HPr or Crh. HprK/P binds both protein substrates with a similar affinity (K(D) of about 40 nM), and addition of nucleotides increases by about 10-fold its affinity for each substrate. In addition, the specificity and the concentration of the cation required for the binding of protein substrates are different from that exhibited by the cation-binding site involved in the nucleotide binding, suggesting the presence of two cation-binding sites on HprK/P. The effects of phosphate on enzymatic activities of HprK/P were also investigated. Phosphate was able to unmask the phosphatase activity, especially in the presence of ATP or both ATP and fructose 1,6-bisphosphate whereas it was shown to inhibit the kinase activity of HprK/P. An apparent competition between phosphate and a fluorescent analogue of nucleotide led to the suggestion that phosphate mediates its effect by binding directly to the ATP-binding site of the enzyme.     

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.     

trans-Acting factors and cis elements involved in glucose repression of arabinan degradation in Bacillus subtilis

In Bacillus subtilis, the synthesis of enzymes involved in the degradation of arabinose-containing polysaccharides is subject to carbon catabolite repression (CCR). Here we show that CcpA is the major regulator of repression of the arabinases genes in the presence of glucose. CcpA acts via binding to one cre each in the promoter regions of the abnA and xsa genes and to two cres in the araABDLMNPQ-abfA operon. The contributions of the coeffectors HPr and Crh to CCR differ according to growth phase. HPr dependency occurs during both exponential growth and the transitional phase, while Crh dependency is detected mainly at the transitional phase. Our results suggest that Crh synthesis may increase at the end of exponential growth and consequently contribute to this effect, together with other factors.     

The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding

Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46. We report here that B. subtilis HprK forms homo-oligomers constituted most likely of eight subunits. Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235. It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations. A positive cooperativity was also detected for the binding of natural nucleotides or their 2'(3')-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments. Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations. This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK.     

Dimerization of Crh by reversible 3D domain swapping induces structural adjustments to its monomeric homologue Hpr

The crystal structure of the regulatory protein Crh from Bacillus subtilis was solved at 1.8A resolution and showed an intertwined dimer formed by N-terminal beta1-strand swapping of two monomers. Comparison with the monomeric NMR structure of Crh revealed a domain swap induced conformational rearrangement of the putative interaction site with the repressor CcpA. The resulting conformation closely resembles that observed for the monomeric Crh homologue HPr, indicating that the Crh dimer is the active form binding to CcpA. An analogous dimer of HPr can be constructed without domain swapping, suggesting that HPr may dimerize upon binding to CcpA. Our data suggest that reversible 3D domain swapping of Crh might be an efficient regulatory mechanism to modulate its activity.     

The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase

The HPr kinase of Gram-positive bacteria is an ATP-dependent serine protein kinase, which phosphorylates the HPr protein of the bacterial phosphotransferase system (PTS) and is involved in the regulation of carbohydrate metabolism. The hprK gene from Enterococcus faecalis was cloned via polymerase chain reaction (PCR) and sequenced. The deduced amino acid sequence was confirmed by microscale Edman degradation and mass spectrometry combined with collision-induced dissociation of tryptic peptides derived from the HPr kinase of E. faecalis . The gene was overexpressed in Escherichia coli , which does not contain any ATP-dependent HPr kinase or phosphatase activity. The homogeneous recombinant protein exhibits the expected HPr kinase activity as well as a P-Ser-HPr phosphatase activity, which was assumed to be a separate enzyme activity. The bifunctional HPr kinase/phosphatase acts preferentially as a kinase at high ATP levels of 2 mM occurring in glucose-metabolizing Streptococci . At low ATP levels, the enzyme hydrolyses P-Ser-HPr. In addition, high concentrations of phosphate present under starvation conditions inhibit the HPr kinase activity. Thus, a putative function of the enzyme may be to adjust the ratio of HPr and P-Ser-HPr according to the metabolic state of the cell; P-Ser-HPr is involved in carbon catabolite repression and regulates sugar uptake via the phosphotransferase system (PTS). Reinvestigation of the previously described Bacillus subtilis HPr kinase revealed that it also possesses P-Ser-HPr phosphatase activity. However, contrary to the E. faecalis enzyme, ATP alone was not sufficient to switch the phosphatase activity of the B. subtilis enzyme to the kinase activity. A change in activity of the B. subtilis HPr kinase was only observed when fructose-1,6-bisphosphate was also present.