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Enterococci are gram‐positive pathogens and lead to cause hospital‐acquired infections worldwide. Central carbon metabolism was shown as highly induced in Enterococcus faecalis during infection context. Metabolism of α‐polysaccharides was previously described as an important factor for host colonisation and biofilm formation. A better characterisation of the adaptation of this bacterium to carbohydrate availabilities may lead to a better understanding of the link between carbohydrate metabolism and the infection process of E. faecalis. Here we show that MalR, a LacI/GalR transcriptional regulator, is the main factor in the regulation of the two divergent operons involved in maltose metabolism in this bacterium. The malR gene is transcribed from the malP promoter, but also from an internal promoter inside the gene located upstream of malR. In the absence of maltose, MalR acts as a repressor and in the presence of glucose, it exerts efficient CcpA‐independent carbon catabolite repression. The central PTS protein P‐Ser‐HPr interacts directly with MalR and enhances its DNA binding capacity, which allows E. faecalis to adapt its metabolism to environmental conditions. [ABSTRACT FROM AUTHOR]

Summary: The bicistronic genBA operon (formerly named celBA) of the opportunistic pathogen Enterococcus faecalis, encodes a 6‐phospho‐β‐glucosidase (GenA) and a phosphotransferase system permease EIIC (GenB). It resembles the cel operon of Streptococcus pyogenes, which is implicated in the metabolism of cellobiose. However, genBA mutants grew normally on cellobiose, but not (genA) or only slowly (genB) on gentiobiose and amygdalin. The two glucosides were also found to be the main inducers of the operon, confirming that the encoded proteins are involved in the utilization of β‐1,6‐ rather than β‐1,4‐linked oligosaccharides. Expression of the genBA operon is regulated by the transcriptional activator GenR, which is encoded by the gene upstream from genB. Thermal shift analysis showed that it binds gentiobiose‐6′‐P with a Kd of 0.04 mM and with lower affinity also other phospho‐sugars. The GenR/gentiobiose‐6′‐P complex binds to the promoter region upstream from genB. The genBA promoter region contains a cre box and gel‐shift experiments demonstrated that the operon is under negative control of the global carbon catabolite regulator CcpA. We also show that the orphan EIIC (GenB) protein needs the EIIA component of the putative OG1RF_10750‐OG1RF_10755 operon situated elsewhere on the chromosome to form a functional PTS transporter. [ABSTRACT FROM AUTHOR]

Catabolite repression is a mechanism that enables bacteria to control carbon utilization. As part of this global regulatory network, components of the phosphoenolpyruvate:carbohydrate phosphotransferase system inhibit the uptake of less favorable sugars when a preferred carbon source such as glucose is available. This process is termed inducer exclusion. In bacteria belonging to the phylum Firmicutes, HPr, phosphorylated at serine 46 (P-Ser46-HPr) is the key player but its mode of action is elusive. To address this question at the level of purified protein components, we have chosen a homolog of the Escherichia coli maltose/maltodextrin ATP-binding cassette transporter from Lactobacillus casei (MalE1-MalF1G1K12) as a model system. We show that the solute binding protein, MalE1, binds linear and cyclic maltodextrins but not maltose. Crystal structures of MalE1 complexed with these sugars provide a clue why maltose is not a substrate. P-Ser46-HPr inhibited MalE1/maltotetraose-stimulated ATPase activity of the transporter incorporated in proteoliposomes. Furthermore, cross-linking experiments revealed that P-Ser46-HPr contacts the nucleotide-binding subunit, MalK1, in proximity to the Walker A motif. However, P-Ser46-HPr did not block binding of ATP to MalK1. Together, our findings provide first biochemical evidence that P-Ser-HPr arrests the transport cycle by preventing ATP hydrolysis at the MalK1 subunits of the transporter. [ABSTRACT FROM AUTHOR]

The bacterial phosphotransferase system (PTS) transports and phosphorylates sugars, but also carries out numerous regulatory functions. The β-proteobacterium Neisseria meningitidis possesses an incomplete PTS unable to transport carbon sources because it lacks a membrane component. Nevertheless, the residual phosphorylation cascade is functional and the meningococcal PTS was therefore expected to carry out regulatory roles. Interestingly, a Δ ptsH mutant (lacks the PTS protein HPr) exhibited reduced virulence in mice and after intraperitoneal challenge it was rapidly cleared from the bloodstream of BALB/c mice. The rapid clearance correlates with lower capsular polysaccharide production by the Δ ptsH mutant, which is probably also responsible for its increased adhesion to Hec-1-B epithelial cells. In addition, compared to the wild-type strain more apoptotic cells were detected when Hec-1-B cells were infected with the Δ ptsH strain. Coimmunoprecipitation revealed an interaction of HPr and P-Ser-HPr with the LysR type transcription regulator CrgA, which among others controls its own expression. Moreover, ptsH deletion caused increased expression of a Φ crgA-lacZ fusion. Finally, the presence of HPr or phospho-HPr's during electrophoretic mobility shift assays enhanced the affinity of CrgA for its target sites preceding crgA and pilE, but HPr did not promote CrgA binding to the sia and pilC1 promoter regions. [ABSTRACT FROM AUTHOR]

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate ( PEP):maltose phosphotransferase system ( PTS). The maltose-specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6-phospho-α-glucosidase, which in B. subtilis hydrolyses maltose 6′- P into glucose and glucose 6- P. Instead, an operon encoding a maltose phosphorylase ( MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6- P into glucose 1- P and glucose 6- P. However, purified MalP phosphorolyses maltose but not maltose 6′- P. We discovered that the gene downstream from malT encodes a novel enzyme ( MapP) that dephosphorylates maltose 6′- P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1- P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6′- P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6′- P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1- P. MapP therefore connects PTS-mediated maltose uptake to maltose phosphorylase-catalysed metabolism. Dephosphorylation assays with a wide variety of phospho-substrates revealed that MapP preferably dephosphorylates disaccharides containing an O-α-glycosyl linkage. [ABSTRACT FROM AUTHOR]

In most firmicutes expression of the mannitol operon is regulated by MtlR. This transcription activator is controlled via phosphorylation of its regulatory domains by components of the phosphoenolpyruvate : carbohydrate phosphotransferase system ( PTS). We found that activation of Bacillus subtilis MtlR also requires an interaction with the EIIBMtl domain of the mannitol permease MtlA ( EIICBMtl). The constitutive expression of the mtlAFD operon in an mtlF mutant was prevented when entire mtlA or only its 3′ part ( EIIBMtl) were deleted. Yeast two-hybrid experiments revealed a direct interaction of the EIIBMtl domain with the two C-terminal domains of MtlR. Complementation of the Δ3′- mtlA Δ mtlF or Δ mtlAFD mutants with mtlA restored constitutive MtlR activity, whereas complementation with only 3′- mtlA had no effect. Moreover, synthesis of EIIBMtl in strains producing constitutively active MtlR caused MtlR inactivation. Interestingly, EIIBMtl fused to the trans-membrane protein YwqC restored constitutive MtlR activity in the above mutants. Replacing the phosphorylatable Cys with Asp in MtlA or soluble EIIBMtl lowered MtlR activation, indicating that MtlR does not interact with phosphorylatyed EIIBMtl. Induction of the B. subtilis mtl operon therefore follows a novel regulation mechanism where the transcription activator needs to be sequestered to the membrane by unphosphorylated EIICBMtl in order to be functional. [ABSTRACT FROM AUTHOR]

Summary In sporulating Bacillus, major processes like virulence gene expression and sporulation are regulated by communication systems involving signalling peptides and regulators of the RNPP family. We investigated the role of one such regulator, NprR, in bacteria of the Bacillus cereus group. We show that NprR is a transcriptional regulator whose activity depends on the NprX signalling peptide. In association with NprX, NprR activates the transcription of an extracellular protease gene ( nprA) during the first stage of the sporulation process. The transcription start site of the nprA gene has been identified and the minimal region necessary for full activation has been characterized by promoter mutagenesis. We demonstrate that the NprX peptide is secreted, processed and then reimported within the bacterial cell. Once inside the cell, the mature form of NprX, presumably the SKPDIVG heptapeptide, directly binds to NprR allowing nprA transcription. Alignment of available NprR sequences from different species of the B. cereus group defines seven NprR clusters associated with seven NprX heptapeptide classes. This cell-cell communication system was found to be strain-specific with a possible cross-talk between some pherotypes. The phylogenic relationship between NprR and NprX suggests a coevolution of the regulatory protein and its signalling peptide. [ABSTRACT FROM AUTHOR]

Summary Listeria monocytogenes transports glucose/mannose via non-PTS permeases and phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS). Two mannose class PTS are encoded by the constitutively expressed mpoABCD and the inducible manLMN operons. The man operon encodes the main glucose transporter because manL or manM deletion significantly slows glucose utilization, whereas mpoA deletion has no effect. The PTS [ABSTRACT FROM AUTHOR]

Many bacteria transport mannitol via the mtlAF-encoded phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). In most firmicutes the transcriptional activator MtlR controls expression of the mtl operon. MtlR possesses an N-terminal DNA binding domain, two PTS regulation domains (PRDs), an EIIBGat- and EIIAMtl-like domain. These four regulatory domains contain one or two potential PTS phosphorylation sites. Replacement of His-342 or His-399 in PRD2 with Ala prevented the phosphorylation of Bacillus subtilis MtlR by PEP, EI and HPr. These mutations as well as EI inactivation caused a loss of MtlR function in vivo. In contrast, phosphomimetic replacement of His-342 with Asp rendered MtlR constitutively active. The absence of phosphorylation in PRD2 serves as catabolite repression mechanism. When EIIAMtl and the soluble EIIBMtl domain of the EIICBMtl permease were included in the phosphorylation mixture, His-599 in the EIIA-like domain of MtlR also became phosphorylated. Replacement of His-599 with Asp rendered MtlR inactive, while His599Ala replacement caused slightly constitutive, glucose-repressible MtlR activity. Doubly mutated His342Ala/His599Ala MtlR was still phosphorylated by EI, HPr and EIIAMtl at Cys-419 in the EIIBGat-like domain. Cys419Ala replacement and deletion of EIIAMtl caused strong constitutive glucose-repressible MtlR activity. This is the first report that Cys phosphorylation controls PRD-containing transcriptional activators. [ABSTRACT FROM AUTHOR]

TheBacillus subtilis ilv-leuoperon involved in the biosynthesis of branched-chain amino acids is under negative regulation mediated by TnrA and CodY, which recognize and bind to their respectivecis-elements located upstream of theilv-leupromoter. This operon is known to be under CcpA-dependent positive regulation. We have currently identified a catabolite-responsive element (cre) for this positive regulation (bases−96 to−82;+1 is theilv-leutranscription initiation base) by means of DNase I-footprintingin vitro, and deletion and base-substitution analyses ofcre. Under nitrogen-rich growth conditions in glucose-minimal medium supplemented with glutamine and amino acids, CcpA and CodY exerted positive and negative regulation ofilv-leu, respectively, but TnrA did not function. Moreover, CcpA and CodY were able to function without their counteracting regulation of each other, although the CcpA-dependent positive regulation did not overcome the CodY-dependent negative regulation. Furthermore, under nitrogen-limited conditions in glucose-minimal medium with glutamate as the sole nitrogen source, CcpA and TnrA exerted positive and negative regulation, respectively, but CodY did not function. This CcpA-dependent positive regulation occurred without the TnrA-dependent negative regulation. However, the TnrA-dependent negative regulation did not occur without the CcpA-dependent positive regulation, raising the possibility that this negative regulation might decrease the CcpA-dependent positive regulation. The physiological role of this elaborate transcription regulation of theB. subtilis ilv-leuoperon in overall metabolic regulation in this organism is discussed. [ABSTRACT FROM AUTHOR]

Summary 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 phos-photransferase 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 t glpFK , whose formation is prevented by the glycerol-3-P-activated antiterminator GlpP, is operative. Deletion of t glpFK led to elevated expression of the glpF ′ –lacZ fusion and to partial relief from CCR. CCR completely disappeared in Δ t glpFK mutants carrying a disruption of ccpA or hprK . The t glpFK -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 Δ ptsHI 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... [ABSTRACT FROM AUTHOR]

The Bacillus subtilis homologous transcriptional antiterminators LicT and SacY control the inducible expression of genes involved in aryl β-glucoside and sucrose utilization respectively. Their RNA-binding activity is carried by the N-terminal domain (CAT), and is regulated by two similar C-terminal domains (PRD1 and PRD2), which are the targets of phosphorylation reactions catalysed by the phosphoenolpyruvate: sugar phosphotransferase system (PTS). In the absence of the corresponding inducer, LicT is inactivated by BglP, the PTS permease (EII) specific for aryl β-glucosides, and SacY by SacX, a negative regulator homologous to the EII specific for sucrose. LicT, but not SacY, is also subject to a positive control by the general PTS components EI and HPr, which are thought to phosphorylate LicT in the absence of carbon catabolite repression. Construction of SacY/LicT hybrids and mutational analysis enabled the location of the sites of this positive regulation at the two phosphorylatable His207 and His269 within LicT-PRD2, and suggested that the presence of negative charges at these sites is sufficient for LicT activation in vivo. The BglP-mediated inhibition process was found to essentially involve His100 of LicT-PRD1, with His159 of the same domain playing a minor role in this regulation. In vitro experiments indicated that His100 could be phosphorylated directly by the general PTS proteins, this phosphorylation being stimulated by P∼BglP. We confirmed that, similarly, the corresponding conserved His99 residue in SacY is the major site of the negative control exerted by SacX on SacY activity. Thus, for both antiterminators, the EII-mediated inhibition process seems to rely primarily on the presence of a negative charge at the first conserved histidine of the PRD1. [ABSTRACT FROM AUTHOR]

The oligomeric bifunctional HPr kinase/P-Ser-HPr phosphatase (HprK/P) regulates many metabolic functions in Gram-positive bacteria by phosphorylating the phosphocarrier protein HPr at Ser46. We isolated Lactobacillus casei hprK alleles encoding mutant HprK/Ps exhibiting strongly reduced phosphatase, but almost normal kinase activity. Two mutations affected the Walker motif A of HprK/P and four a conserved C-terminal region in contact with the ATP-binding site of an adjacent subunit in the hexamer. Kinase and phosphatase activity appeared to be closely associated and linked to the Walker motif A, but dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr) is not simply a reversal of the kinase reaction. When the hprKV267F allele was expressed in Bacillus subtilis, the strongly reduced phosphatase activity of the mutant enzyme led to increased amounts of P-Ser- HPr. The hprKV267F mutant was unable to grow on carbohydrates transported by the phosphoenol- pyruvate:glycose phosphotransferase system (PTS) and on most non-PTS carbohydrates. Disrupting ccpA relieved the growth defect only on non PTS sugars, whereas replacing Ser46 in HPr with alanine also restored growth on PTS substrates. [ABSTRACT FROM AUTHOR]

HPr kinase/phosphatase (HprK/P) is a key regulatory enzyme controlling carbon metabolism in Gram- positive bacteria. It catalyses the ATP-dependent phosphorylation of Ser46 in HPr, a protein of the phosphotransferase system, and also its dephosphorylation. HprK/P is unrelated to eukaryotic protein kinases, but contains the Walker motif A characteristic of nucleotide-binding proteins. We report here the X-ray structure of an active fragment of Lactobacillus casei HprK/P at 2.8 Å resolution, solved by the multi- wavelength anomalous dispersion method on a seleniated protein (PDB code 1jb1). The protein is a hexamer, with each subunit containing an ATP-binding domain similar to nucleoside/nucleotide kinases, and a putative HPr-binding domain unrelated to the substrate-binding domains of other kinases. The Walker motif A forms a typical P-loop which binds inorganic phosphate in the crystal. We modelled ATP binding by comparison with adenylate kinase, and designed a tentative model of the complex with HPr based on a docking simulation. The results confirm that HprK/P represents a new family of protein kinases, first identified in bacteria, but which may also have members in eukaryotes. [ABSTRACT FROM AUTHOR]

Examines the phosphotransferase system gene from Bacillus thuringiensis israelensis. Formation of the phosphoryl bond; Replacement of histidine 14 with alanine; Characterization of the phosphorylation site on the phosphocarrier protein.

Examines the role of enzyme I and HPr from Lactobacillus casei on sugar transport, carbon catabolite repression and inducer exclusion. Components of phosphoenolpyruvate-carbohydrate phosphotransferase system; Phosphorylation of HPr at Ser-46; Sugar metabolism in Gram-positive bacteria.

The transcriptional antiterminator LicT regulates the induction and carbon catabolite repression of the Bacillus subtilis bglPH operon. LicT is inactive in mutants affected in one of the two general components of the phosphoenolpyruvate (PEP):glycose phosphotransferase system, enzyme I or histidine-containing protein (HPr). We demonstrate that LicT becomes phosphorylated in the presence of PEP, enzyme I and HPr. The phosphoryl group transfer between HPr and LicT is reversible. Phosphorylation of LicT with PEP, enzyme I and HPr led to the appearance of three additional LicT bands on polyacrylamide–urea gels. These bands probably correspond to one-, two- and threefold phosphorylated LicT. After phosphorylation of LicT with [32 P]-PEP, enzyme I and HPr, proteolytic digestion of [32 P]-P-LicT, separation of the peptides by reverse-phase chromatography, mass spectrometry and N-terminal sequencing of radiolabelled peptides, three histidyl residues were found to be phosphorylated in LicT. These three histidyl residues (His-159, His-207 and His-269) are conserved in most members of the BglG/SacY family of transcriptional antiterminators. Phosphorylation of LicT in the presence of seryl-phosphorylated HPr (P-Ser-HPr) was much slower compared with its phosphorylation in the presence of HPr. The slower phosphorylation in the presence of P-Ser-HPr leading to reduced LicT activity is presumed to play a role in a recently described LicT-mediated CcpA-independent carbon catabolite repression mechanism operative for the bglPH operon. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

LevR, which controls the expression of the lev operon 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 BglG 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. [ABSTRACT FROM AUTHOR]

Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting c̱atabolite-ṟesponsive e̱lement (cre) by a complex of CcpA, which is a member of the GalR-LacI family of bacterial regulatory proteins, and the seryl-phosphorylated form of HPr (P-ser-HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the cre in the first gene (gntR) of the gnt operon (credown), this operon contains another cre located in the promoter region (creup). A translational gntR'-'lacZ fusion expressed under the control of various combinations of wild-type and mutant credown and creup was integrated into the chromosomal amyE locus, and then catabolite repression of β-galactosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by creup was probably independent of catabolite repression exerted by credown; both creup and credown catabolite repression involved CcpA. Catabolite repression exerted by creup was independent of P-ser-HPr, and catabolite repression exerted by credown was partially independent of P-ser-HPr. DNase I footprinting experiments indicated that a complex of CcpA and P-ser-HPr did not recognize creup, in contrast to its specific recognition of credown. However, CcpA complexed with glucose-6-phosphate specifically recognized creup as well as credown, but the physiological significance of this complexing is unknown. [ABSTRACT FROM AUTHOR]

Catabolite repression of various Bacillus subtilis catabolic operons which carry a c/s-acting catabolite-responsive element (CRE), such as the gnt operon, is mediated by CcpA, a protein belonging to the GalR-Lacl family of bacterial transcriptional repressors/ activators, and the seryl-phosphorylated form of HPr, a phosphocarrier protein of the phosphoenol-pyruvate: sugar phosphotransferase system. Footprinting experiments revealed that the purified CcpA protein interacted with P-ser-HPr to cause specific protection of the gnt CRE against DNase I digestion. The specific recognition of the gnt CRE was confirmed by the results of footprinting experiments using mutant gnt CREs carrying one of the following base substitutions within the CRE consensus sequence: G to T at position +149 or C to T at position +154 (+1 is the gnt transcription initiation nucleotide). The two mutant CREs causing a partial relief from catabolite repression were not protected by the CcpA/Pser-HPr complex in footprinting experiments. Based on these and previous findings, we propose a molecular mechanism underlying catabolite repression in B. subtilis mediated by CcpA and P-ser-HPr. [ABSTRACT FROM AUTHOR]

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 phospho-carrier 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-phosphory-lated 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 cataboiite repression and PTS transport activity. [ABSTRACT FROM AUTHOR]

Enzyme-IIIgle is part of the glucose phosphotransferase system of Escherichia coli and Salmonella typhimturium and is phosphorylated by phosphoenolpyruvate in a reduction requiring enzyme 1 (phosphoeno/pyruvate-protein phospho- transferase), and the histidine-containing phospho-carrier protein HPr. In this paper we report the isolation of IIIgle from E. coil and the characterization of the activc center. Alkaline hydrolysis of [32P]P-IIIgle and chromatography of the hydrolysate suggested that the phosphoryl group is bound to a histidyl residue in P-IIIgle of S. typhimurium. Here we present 1H-NMR measurements of IIIgle and P-IIIgle from E. coli which further substantiate that the phosphoryl group in P-IIIgle is linked to the N-3 position of a histidyl residue. After phosporylation of IIIgle with [32P]Phosphoenolpyruvate, enzyme I and HPr, the phosphorylated protein was cleaved with either alkaline protease from Streptomyces griseus or subtilisin from Bacillus subtilis. According to amino acid analysis both proteases produced the same peptide carrying the phosphoryl group. The amino acid sequence of this peptide was found to be Val-His-Phe-Gly-Ile-Asp. The lower electrophoretic mobility of P-IIIgle on dodecylsulfute/polyacrylamide gels and its stronger binding to the hydrophobic matrix of a reversed-phase column compared to uuphosphorylated protein may indicate a structural change following phosphoenol/pyruvate-dependent phosphorylation. [ABSTRACT FROM AUTHOR]