Mycoplasma pneumoniae HPr kinase/phosphorylase.

HPr kinase/phosphorylase (HPrK/P) is the key regulator of carbon metabolism in many Gram-positive bacteria. It phosphorylates/dephosphorylates the HPr protein of the bacterial phosphotransferase system on a regulatory serine residue in response to the nutrient status of the cell. In Mycoplasma pneumoniae, HPrK/P is one of the very few regulatory proteins encoded in the genome. The regulation of this enzyme by metabolites is unique among HPrK/P proteins studied so far: it is active as a kinase at low ATP concentrations, whereas the proteins from other bacteria need high ATP concentrations as an indicator of a good nutrient supply for kinase activity. We studied the interaction of M. pneumoniae HPrK/P with ATP, Fru1,6P2 and Pi by fluorescence spectroscopy. In agreement with the previously observed unique regulation, we found a very high affinity for ATP (K(d)=5.4 microM) compared with the HPrK/P proteins from other bacteria. The Kd for Fru1,6P2 was three orders of magnitude higher, which explains why Fru1,6P2 has only a weak regulatory effect on M. pneumoniae HPrK/P. Mutations of two important regions in the active site of HPrK/P, the nucleotide binding P-loop and the HPrK/P family signature sequence, had different effects. P-loop region mutations strongly affect ATP binding and thus all enzymatic functions, whereas the signature sequence motif seems to be important for the catalytic mechanism rather than for nucleotide binding.     

Regulatory protein phosphorylation in Mycoplasma pneumoniae. A PP2C-type phosphatase serves to dephosphorylate HPr(Ser-P)

Among the few regulatory events in the minimal bacterium Mycoplasma pneumoniae is the phosphorylation of the HPr phosphocarrier protein of the phosphotransferase system. In the presence of glycerol, HPr is phosphorylated in an ATP-dependent manner by the HPr kinase/phosphorylase. The role of the latter enzyme was studied by constructing a M. pneumoniae hprK mutant defective in HPr kinase/phosphorylase. This mutant strain no longer exhibited HPr kinase activity but, surprisingly, still had phosphatase activity toward serine-phosphorylated HPr (HPr(Ser-P)). An inspection of the genome sequence revealed the presence of a gene (prpC) encoding a presumptive protein serine/threonine phosphatase of the PP2C family. The phosphatase PrpC was purified and its biochemical activity in HPr(Ser-P) dephosphorylation demonstrated. Moreover, a prpC mutant strain was isolated and found to be impaired in HPr(Ser-P) dephosphorylation. Homologues of PrpC are present in many bacteria possessing HPr(Ser-P), suggesting that PrpC may play an important role in adjusting the cellular HPr phosphorylation state and thus controlling the diverse regulatory functions exerted by the different forms of HPr.     

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.     

Characterization of an HPr kinase mutant of Staphylococcus xylosus

The Staphylococcus xylosus gene hprK, encoding HPr kinase (HPrK), has been isolated from a genomic library. The HPrK enzyme, purified as a His(6) fusion protein, phosphorylated HPr, the phosphocarrier protein of the bacterial phosphotransferase system, at a serine residue in an ATP-dependent manner, and it also catalyzed the reverse reaction. Therefore, the enzyme constitutes a bifunctional HPr kinase/phosphatase. Insertional inactivation of the gene in the genome of S. xylosus resulted in the concomitant loss of both HPr kinase and His serine-phosphorylated-HPr phosphatase activities in cell extracts, strongly indicating that the HPrK enzyme is also responsible for both reactions in vivo. HPrK deficiency had a profound pleiotropic effect on the physiology of S. xylosus. The hprK mutant strain showed a severe growth defect in complex medium upon addition of glucose. Glucose uptake in glucose-grown cells was strongly enhanced compared with the wild type. Carbon catabolite repression of three tested enzyme activities by glucose, sucrose, and fructose was abolished. These results clearly demonstrate the prominent role of HPr kinase in global control to adjust catabolic capacities of S. xylosus according to the availability of preferred carbon sources.     

High-resolution structure of the histidine-containing phosphocarrier protein (HPr) from Staphylococcus aureus and characterization of its interaction with the bifunctional HPr kinase/phosphorylase

A high-resolution structure of the histidine-containing phosphocarrier protein (HPr) from Staphylococcus aureus was obtained by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy on the basis of 1,766 structural restraints. Twenty-three hydrogen bonds in HPr could be directly detected by polarization transfer from the amide nitrogen to the carbonyl carbon involved in the hydrogen bond. Differential line broadening was used to characterize the interaction of HPr with the HPr kinase/phosphorylase (HPrK/P) of Staphylococcus xylosus, which is responsible for phosphorylation-dephosphorylation of the hydroxyl group of the regulatory serine residue at position 46. The dissociation constant Kd was determined to be 0.10 +/- 0.02 mM at 303 K from the NMR data, assuming independent binding. The data are consistent with a stoichiometry of 1 HPr molecule per HPrK/P monomer in solution. Using transversal relaxation optimized spectroscopy-heteronuclear single quantum correlation, we mapped the interaction site of the two proteins in the 330-kDa complex. As expected, it covers the region around Ser46 and the small helix b following this residue. In addition, HPrK/P also binds to the second phosphorylation site of HPr at position 15. This interaction may be essential for the recognition of the phosphorylation state of His15 and the phosphorylation-dependent regulation of the kinase/phosphorylase activity. In accordance with this observation, the recently published X-ray structure of the HPr/HPrK core protein complex from Lactobacillus casei shows interactions with the two phosphorylation sites. However, the NMR data also suggest differences for the full-length protein from S. xylosus: there are no indications for an interaction with the residues preceding the regulatory Ser46 residue (Thr41 to Lys45) in the protein of S. xylosus. In contrast, it seems to interact with the C-terminal helix of HPr in solution, an interaction which is not observed for the complex of HPr with the core of HPrK/P of L. casei in crystals.     

Crystal structure of HPr kinase/phosphatase from Mycoplasma pneumoniae

HPr kinase/phosphatase (HPrK/P) modifies serine 46 of histidine-containing protein (HPr), the phosphorylation state of which is the control point of carbon catabolite repression in low G+C Gram-positive bacteria. To understand the structural mechanism by which HPrK/P carries out its dual, competing activities we determined the structure of full length HPrK/P from Mycoplasma pneumoniae (PD8 ID, 1KNX) to 2.5A resolution. The enzyme forms a homo-hexamer with each subunit containing two domains connected by a short loop. The C-terminal domain contains the well-described P-loop (Walker A box) ATP binding motif and takes a fold similar to phosphoenolpyruvate carboxykinase (PEPCK) from Escherichia coli as recently described in other HPrK/P structures. As expected, the C-terminal domain is very similar to the C-terminal fragment of Lactobacillus casei HPrK/P and the C-terminal domain of Staphylococcus xylosus HPrK/P; the N-terminal domain is very similar to the N-terminal domain of S.xylosus HPrK/P. Unexpectedly, the N-terminal domain resembles UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase (MurE), yet the function of this domain is unclear. We discuss these observations as well as the structural significance of mutations in the P-loop and HPrK/P family sequence motif.     

Corynebacterium diphtheriae: a PTS view to the genome

We have surveyed the publicly available genome sequence of Corynebacterium diphtheriae (www.sanger.ac.uk) to identify components of the phosphotransferase system (PTS), which plays a central role in carbon metabolism in many bacteria. Three gene loci were found to contain putative pts genes. These comprise: (i) the genes of the general phosphotransferases enzyme I (ptsI) and HPr (ptsH), a fructose-specific enzyme IIABC permease (fruA), and a fructose 1-phosphate kinase (fruK); (ii) a gene that encodes an enzyme IIAB of the fructose/mannitol family, and a novel HPr-like gene, ptsF, that encodes an HPr domain fused to a domain of unknown function; (iii) and a gene for a glucose-specific enzyme IIBCA (ptsG). A search for genes that may be putative PTS-targets or that may operate in general carbon regulation revealed a possible regulatory gene encoding an antiterminator protein downstream from ptsG. Furthermore, genes were detected encoding glycerol kinase, glucose kinase, and a homologue of the global activator of carbon catabolite repression in Escherichia coli, CAP. The possible significance of these observations in carbon metabolism and the novel features of the detected genes are discussed.     

HPrK regulates succinate-mediated catabolite repression in the gram-negative symbiont Sinorhizobium meliloti

The HPrK kinase/phosphatase is a common component of the phosphotransferase system (PTS) of gram-positive bacteria and regulates catabolite repression through phosphorylation/dephosphorylation of its substrate, the PTS protein HPr, at a conserved serine residue. Phosphorylation of HPr by HPrK also affects additional phosphorylation of HPr by the PTS enzyme EI at a conserved histidine residue. Sinorhizobium meliloti can live as symbionts inside legume root nodules or as free-living organisms and is one of the relatively rare gram-negative bacteria known to have a gene encoding HPrK. We have constructed S. meliloti mutants that lack HPrK or that lack key amino acids in HPr that are likely phosphorylated by HPrK and EI. Deletion of hprK in S. meliloti enhanced catabolite repression caused by succinate, as did an S53A substitution in HPr. Introduction of an H22A substitution into HPr alleviated the strong catabolite repression phenotypes of strains carrying ΔhprK or hpr(S53A) mutations, demonstrating that HPr-His22-P is needed for strong catabolite repression. Furthermore, strains with a hpr(H22A) allele exhibited relaxed catabolite repression. These results suggest that HPrK phosphorylates HPr at the serine-53 residue, that HPr-Ser53-P inhibits phosphorylation at the histidine-22 residue, and that HPr-His22-P enhances catabolite repression in the presence of succinate. Additional experiments show that ΔhprK mutants overproduce exopolysaccharides and form nodules that do not fix nitrogen.     

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.     

Characterization of a bifunctional HPr kinase/phosphorylase from Leuconostoc mesenteroides SY1

The hprK gene encoding bifunctional HPrK/P (kinase/ phosphorylase) was cloned from L. mesenteroides SY1, a strain isolated from kimchi. hprK was transcribed as a monocistronic gene. His-tagged HPrH16A and HPrK/P were produced in E. coli BL21(DE3) using pET26b(+) and purified. HPrK/P phosphorylation assay with purified proteins showed that the kinase activity of HPrK/P increased at slightly acidic pHs. Divalent cations such as Mg2+ and Mn2+ and glycolytic intermediates such as fructose-1, 6-bisphosphate (FBP) and phosphoenolpyruvate (PEP) increased the kinase activity of HPrK/P, but inorganic phosphate strongly inhibited it. Kinetic studies for the kinase activity of HPrK/P showed that the apparent Km values were 0.18 and 14.57 microM for ATP and HPr, respectively. The Km value for the phosphorylase activity of HPrK/P was 14.16 microM for P-Ser-HPr (HPr phosphorylated at the serine residue).     

Sugar uptake and utilisation in Streptomyces coelicolor: a PTS view to the genome

Our research group is studying the phosphotransferase system (PTS) of Streptomyces coelicolor, which, in other bacteria, is centrally involved in carbon source uptake and regulation. We have surveyed the public available S. coelicolor genome sequence produced by the ongoing genome sequencing project for pts gene homologues (http://www.sanger.ac.uk/Projects/S_coelicolor/). Three genes encoding homologues of the general PTS components enzyme I (ptsI), HPr (ptsH), and enzyme IIA^Crr (crr; IIA^Glc-homologue) and six genes encoding homologues of sugar-specific PTS components were identified. The deduced primary sequences of the sugar-specific components shared significant similarities to PTS permeases of the mannitol/fructose family and of the glucose/sucrose family. A model is presented, in which possible functions of the novel described PTS homologues are discussed.     

An N-terminal half fragment of the histidine phosphocarrier protein,HPr, is disordered but binds to HPr partners and shows antibacterial properties

BackgroundThe phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. It is formed by a protein cascade in which the first two proteins are general (namely enzyme I, EI, and the histidine phosphocarrier protein, HPr) and the others are sugar-specific permeases; the active site of HPr is His15. The HPr kinase/phosphorylase (HPrK/P), involved in the use of carbon sources in Gram-positive, phopshorylates HPr at a serine. The regulator of sigma D protein (Rsd) also binds to HPr. We are designing specific fragments of HPr, which can be used to interfere with those protein-protein interactions (PPIs), where the intact HPr intervenes.MethodsWe obtained a fragment (HPr48) comprising the first forty-eight residues of HPr. HPr48 was disordered as shown by fluorescence, far-ultraviolet (UV) circular dichroism (CD), small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR).ResultsSecondary structure propensities, from the assigned backbone nuclei, further support the unfolded nature of the fragment. However, HPr48 was capable of binding to: (i) the N-terminal region of EI, EIN; (ii) the intact Rsd; and, (iii) HPrK/P, as shown by fluorescence, far-UV CD, NMR and biolayer interferometry (BLI). The association constants for each protein, as measured by fluorescence and BLI, were in the order of the low micromolar range, similar to those measured between the intact HPr and each of the other macromolecules.ConclusionsAlthough HPr48 is forty-eight-residue long, it assisted antibiotics to exert antimicrobial activity.General significanceHPr48 could be used as a lead compound in the development of new antibiotics, or, alternatively, to improve the efficiency of existing ones.     

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

SUMMARY

The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.     

Regulation of carbon metabolism in the mollicutes and its relation to virulence

The mollicutes are cell wall-less bacteria that live in close association with their eukaryotic hosts. Their genomes are strongly reduced and so are their metabolic capabilities. A survey of the available genome sequences reveals that the mollicutes are capable of utilizing sugars as source of carbon and energy via glycolysis. The pentose phosphate pathway is incomplete in these bacteria, and genes encoding enzymes of the tricarboxylic acid cycle are absent from the genomes. Sugars are transported by the phosphotransferase system. As in related bacteria, the phosphotransferase system does also seem to play a regulatory role in the mollicutes as can be concluded from the functionality of the regulatory HPr kinase/phosphorylase. In Mycoplasma pneumoniae, the activity of HPr kinase is triggered in the presence of glycerol. This carbon source may be important for the mollicutes since it is available in epithelial tissues and its metabolism results in the formation of hydrogen peroxide, the major virulence factor of several mollicutes. In plant-pathogenic mollicutes such as Spiroplasma citri, the regulation of carbon metabolism is crucial in the adaptation to life in plant tissues or the insect vectors. Thus, carbon metabolism seems to be intimately linked to pathogenicity in the mollicutes.     

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.     

Structural analysis of the bacterial HPr kinase/phosphorylase V267F mutant gives insights into the allosteric regulation mechanism of this bifunctional enzyme

The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phospho-carrier protein HPr, which regulates the utilization of carbon sources in Gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of Ser(P)-46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of nucleotide-binding protein with Walker A motif. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the x-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6A resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P.