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  • 1995-1999  (7)
  • 1
    Electronic Resource
    Electronic Resource
    Analysis of a ptsH homologue from Streptomyces coelicolor A3(2) (1999)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 177 (1999), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: A ptsH homologue of Streptomyces coelicolor A3(2) was identified in the emerging genome sequence, cloned in Escherichia coli and the S. coelicolor HPr over-produced and purified. The protein was phosphorylated in vitro in a phosphoenolpyruvate (PEP)-dependent manner by purified enzyme I (EI) from Bacillus subtilis, and much less efficiently in an ATP-dependent manner by purified HPr kinase, also from B. subtilis. There was no indication of ATP-dependent phosphorylation of the purified protein by cell extracts of either S. coelicolor or Streptomyces lividans. Deletion of the ptsH homologue from the S. coelicolor and S. lividans chromosomes had no effect on growth when fructose was supplied as sole carbon source, and in S. coelicolor it had no effect on glucose repression of agarase and galactokinase synthesis, suggesting that the HPr encoded by this gene does not play an essential role in fructose transport nor a general role in carbon catabolite repression.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.1999.tb13744.x
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  • 2
    Electronic Resource
    Electronic Resource
    Catabolite repression of the Bacillus subtilis gnt operon exerted by two catabolite-responsive elements (1997)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 23 (1997), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting catabolite-responsive element (cre) by a complex of CcpA, which is a member of the GalR-LacI family of bacterial regulatory proteins, and the seryl-phos-phorylated 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 ere in the first gene (gntR) of the gnt operon (credown), this operon contains another ere 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 p-galac-tosidase 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1997.2921662.x
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  • 3
    Electronic Resource
    Electronic Resource
    Specific recognition of the Bacillus subtilis gnt cis-acting catabolite-responsive element by a protein complex formed between CcpA and seryl-phosphorylated HPr (1995)
    Osney Mead, Oxford OX2 0EL, UK : Blackwell Scientific Publication
    Molecular microbiology 17 (1995), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Catabolite repression of various Bacillus subtilis catabolic operons which carry a cis-acting catabolite-responsive element (ORE), 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 Tat 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/P-ser-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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1995.mmi_17050953.x
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  • 4
    Electronic Resource
    Electronic Resource
    Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram-positive bacteria (1995)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 15 (1995), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1995.tb02280.x
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  • 5
    Electronic Resource
    Electronic Resource
    Antagonistic effects of dual PTS-catalysed phosphorylation on the Bacillus subtilis transcriptional activator LevR (1998)
    Oxford BSL : Blackwell Science Ltd, UK
    Molecular microbiology 28 (1998), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1998.00781.x
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  • 6
    Electronic Resource
    Electronic Resource
    Regulation of the activity of the Bacillus subtilis antiterminator LicT by multiple PEP-dependent, enzyme I- and HPr-catalysed phosphorylation (1999)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 31 (1999), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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 [32P]-PEP, enzyme I and HPr, proteolytic digestion of [32P]-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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01262.x
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  • 7
    Electronic Resource
    Electronic Resource
    The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase (1999)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 31 (1999), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01146.x
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