Cloning and Sequencing of two Enterococcal glpK Genes and Regulation of the Encoded Glycerol Kinases by Phosphoenolpyruvate-dependent, Phosphotransferase System-catalyzed Phosphorylation of a Single Histidyl Residue

Charrier, Véronique; Buckley, Ellen; Parsonage, Derek; Galinier, Anne; Darbon, Emmanuelle; Jaquinod, Michel; Forest, Éric; Deutscher, Josef; Claiborne, Al

doi:10.1074/jbc.272.22.14166

Cited by 84 publications

(110 citation statements)

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“…Although protein phosphorylation is implicated in both cases, the mechanisms of CCR and inducer control are quite different. In Gram-positive bacteria, PEP-dependent phosphorylation of HPr at His-15 is not only necessary for PTS-catalyzed sugar transport and phosphorylation, but is also implicated in phosphorylation and regulation of transcriptional effectors (36,37) and catabolic enzymes (29). By contrast, ATP-dependent phosphorylation of HPr at Ser-46 is involved in CCR (10) and inducer exclusion (35).…”

Section: Discussionmentioning

confidence: 99%

“…Tryptic fragments were obtained by incubating 100 g of unphosphorylated or phosphorylated protein for 7 h at 37°C in 100 l of 20 mM Tris⅐HCl (pH 8) containing 20 g of trypsin. Matrix-assisted laser desorption ionization mass spectra (MALDI-MS) of peptides derived from phosphorylated or unphosphorylated Crh(His) 6 were recorded on a RETOF (time of flight) instrument from Perseptive Biosystems (Framingham, MA) as described by Charrier et al (29).…”

Section: Methodsmentioning

confidence: 99%

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The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression

Galinier

Haiech

Kilhoffer

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Carbon catabolite repression (CCR) of several Bacillus subtilis catabolic genes is mediated by ATPdependent phosphorylation of histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP): sugar phosphotransferase system. In this study, we report the discovery of a new B. subtilis gene encoding a HPr-like protein, Crh (for catabolite repression HPr), composed of 85 amino acids. Crh exhibits 45% sequence identity with HPr, but the active site His-15 of HPr is replaced with a glutamine in Crh. Crh is therefore not phosphorylated by PEP and enzyme I, but is phosphorylated by ATP and the HPr kinase in the presence of fructose-1,6-bisphosphate. We determined Ser-46 as the site of phosphorylation in Crh by carrying out mass spectrometry with peptides obtained by tryptic digestion or CNBr cleavage. In a B. subtilis ptsH1 mutant strain, synthesis of ␤-xylosidase, inositol dehydrogenase, and levanase was only partially relieved from CCR. Additional disruption of the crh gene caused almost complete relief from CCR. In a ptsH1 crh1 mutant, producing HPr and Crh in which Ser-46 is replaced with a nonphosphorylatable alanyl residue, expression of ␤-xylosidase was also completely relieved from glucose repression. These results suggest that CCR of certain catabolic operons requires, in addition to CcpA, ATP-dependent phosphorylation of Crh, and HPr at Ser-46.The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) catalyzes the transport and concomitant phosphorylation of carbohydrates via a protein phosphorylation chain including PEP-dependent phosphorylation of His-15 in histidine-containing protein (HPr) by enzyme I (EI). P-His-HPr phosphorylates the sugar-specific EIIAs. In Gram-positive bacteria, the PTS regulates also induction and carbon catabolite repression (CCR) of numerous catabolic genes (1). The central regulatory protein involved in these various functions is HPr. In Gram-positive bacteria, this small phosphoryl transfer protein can be phosphorylated at a regulatory serine (Ser-46) by ATP and the HPr kinase (2, 3), in addition to phosphorylation at the catalytic His-15 by PEP and EI (4, 5). PEP-dependent and ATP-dependent phosphorylation of HPr interfere with each other-i.e., P-His-HPr is a poor substrate for the HPr kinase and P-Ser-HPr is a poor substrate for EI (6, 7). ATP-dependent phosphorylation of HPr is stimulated by glycolytic intermediates such as fructose-1,6-bisphosphate (FBP) in Enterococcus faecalis (6) and in Streptococcus pyogenes (7). It has been reported that FBP is also implicated in CCR of the Bacillus subtilis gnt and iol operons (8, 9), and a potential role of phosphorylation of HPr at Ser-46 in CCR has therefore been investigated (10). The gnt operon contains the genes gntRKPZ encoding the repressor GntR, gluconate kinase, gluconate permease, and a gluconate-6-Pdehydrogenase (11), whereas the iol operon is composed of 10 genes encoding enzymes presumably implicated in inositol metabolism, including iolG encoding inositol dehydro...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression

Galinier

Haiech

Kilhoffer

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

210

274

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“…In contrast, phosphorylated EIIA Glc had no inhibitory effect on glycerol kinase activity. In Grampositive bacteria such as Enterococcus faecalis and Enterococcus casseliflavus, glycerol kinase was found to be phosphorylated by PEP, enzyme I and HPr, and phosphorylation caused an " 10-fold increase of glycerol kinase activity (Deutscher, 1985 ;Deutscher & Sauerwald, 1986 ;Charrier et al, 1997). This phosphorylation is reversible, and phosphorylated glycerol kinase is dephosphorylated probably by transferring its phosphoryl group to HPr when a rapidly metabolizable PTS substrate is present in the growth medium leading to dephosphorylation of the PTS proteins (Deutscher et al, 1993).…”

Section: Abbreviationsmentioning

confidence: 99%

“…Recently, His-232 of glycerol kinase from Ent. casseliflavus has been identified as the site of PEPdependent phosphorylation (Charrier et al, 1997). In glycerol kinase of Bacillus subtilis, the equivalent His-230 is probably also phosphorylated.…”

Section: Abbreviationsmentioning

confidence: 99%

Glycerol transport and phosphoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of glycerol kinase in Thermus flavus

Darbon¹,

Itō

Huang

et al. 1999

Microbiology

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The genes glpK and glpF, encoding glycerol kinase and the glycerol facilitator of Thermus flavus, a member of the Thermus/Deinococcus group, have recently been identified. The protein encoded by glpK exhibited an unusually high degree of sequence identity (806 %) when compared to the sequence of glycerol kinase from Bacillus subtilis and a similar high degree of sequence identity (648 %) was observed when the sequences of the glycerol facilitators of the two organisms were compared. The work presented in this paper demonstrates that T. flavus is capable of taking up glycerol, that glpF and glpK are expressed constitutively and that glucose exerts a repressive effect on the expression of these genes. T. flavus was found to possess the general components of the phosphoenolpyruvate (PEP) : sugar phosphotransferase system (PTS) enzyme I and histidine-containing protein (HPr). These proteins catalyse the phosphorylation of T. flavus glycerol kinase, which contains a histidyl residue equivalent to His-232, the site of PEP-dependent, PTS-catalysed phosphorylation in glycerol kinase of Enterococcus casseliflavus. Purified glycerol kinase from T. flavus could also be phosphorylated with enzyme I and HPr from B. subtilis. Similar to enterococcal glycerol kinases, phosphorylated T. flavus glycerol kinase exhibited an electrophoretic mobility on denaturing and non-denaturing polyacrylamide gels that is different from the electrophoretic mobility of non-phosphorylated glycerol kinase. However, in contrast to PEPdependent phosphorylation of enterococcal glycerol kinases, which stimulated glycerol kinase activity about 10-fold, phosphorylation of T. flavus glycerol kinase caused only a slight increase in enzyme activity.

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“…PEPdependent phosphorylation by EI occurs at the His15 site and this phosphoryl group is transferred to the EII sugar (Postma et al, 1993). P-His15-HPr can also transfer its phosphoryl group to other non-PTS proteins, such as enzymes like glycerol kinase (Charrier et al, 1997) or regulators. These are antiterminators and transcriptional activators that possess PTS regulatory domains (PRD) which contain histidine residues phosphorylatable by P-His15-HPr and also by other components of the PTS (Stülke et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Phosphoenolpyruvate phosphotransferase system and N-acetylglucosamine metabolism in Bacillus sphaericus

2003

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Bacillus sphaericus, a bacterium of biotechnological interest due to its ability to produce mosquitocidal toxins, is unable to use sugars as carbon source. However, ptsHI genes encoding HPr and EI proteins belonging to a PTS were cloned, sequenced and characterized. Both HPr and EI proteins were fully functional for phosphoenolpyruvate-dependent transphosphorylation in complementation assays using extracts from Staphylococcus aureus mutants for one of these proteins. HPr(His 6 ) was purified from wild-type and a Ser46/Gln mutant of B. sphaericus, and used for in vitro phosphorylation experiments using extracts from either B. sphaericus or Bacillus subtilis as kinase source. The results showed that both phosphorylated forms, P-Ser46-HPr and P-His15-HPr, could be obtained. The findings also proved indirectly the existence of an HPr kinase activity in B. sphaericus. The genetic structure of these ptsHI genes has some unusual features, as they are co-transcribed with genes encoding metabolic enzymes related to N-acetylglucosamine (GlcNAc) catabolism (nagA, nagB and an undetermined orf2). In fact, this bacterium was able to utilize this amino sugar as carbon and energy source, but a ptsH null mutant had lost this characteristic. Investigation of GlcNAc uptake and streptozotocin inhibition in both a wild-type and a ptsH null mutant strain led to the proposal that GlcNAc is transported and phosphorylated by an EII Nag element of the PTS, as yet uncharacterized. In addition, GlcNAc-6-phosphate deacetylase and GlcN-6-phosphate deaminase activities were determined; both were induced in the presence of GlcNAc. These results, together with the authors' recent findings of the presence of a phosphofructokinase activity, are strongly indicative of a glycolytic pathway in B. sphaericus. They also open new possibilities for genetic improvements in industrial applications.

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Cloning and Sequencing of two Enterococcal glpK Genes and Regulation of the Encoded Glycerol Kinases by Phosphoenolpyruvate-dependent, Phosphotransferase System-catalyzed Phosphorylation of a Single Histidyl Residue

Cited by 84 publications

References 44 publications

The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression

The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression

Glycerol transport and phosphoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of glycerol kinase in Thermus flavus

Phosphoenolpyruvate phosphotransferase system and N-acetylglucosamine metabolism in Bacillus sphaericus

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