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

Abstract

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...

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…Inactivation of the crh alone does not affect CCR or CCA, but the residual CCR observed in pstH1 mutants disappears when the crh is disrupted or a crh1 mutation (replacement of Ser-46 with Ala) is introduced. 20,23,32,33,41,45,55,58,61,63,65,82,83) The question of how P-Ser-HPr and P-Ser-Crh contribute to CCR and CCA with different efficiencies has not been clearly answered yet, although a Crh-specific function in the regulation of expression during growth on substrates other than carbohydrates was recently revealed, 84) probably because of the drastically higher amount of HPr than of Crh during growth on carbohydrates. 85) Also, P-Ser-Crh displays altered binding to CcpA to effect catabolite control.…”

“…1, an HPr-like protein (Crh) is another corepressor of CcpA, found during the B. subtilis genome sequencing project. 81) While Crh contains conserved Ser-46, which can be phosphorylated with ATP and HPrK/P, it lacks the active site His-15, 82) so Crh is active only in catabolite control. Inactivation of the crh alone does not affect CCR or CCA, but the residual CCR observed in pstH1 mutants disappears when the crh is disrupted or a crh1 mutation (replacement of Ser-46 with Ala) is introduced.…”

A novel mode of control of Mycoplasma pneumoniae HPr kinase/phosphatase activity reflects its parasitic lifestyle

“…HPr-Ser-P and Crh-Ser-P serve as cofactors for the transcriptional regulator, CcpA. HPr-Ser-P is a poor substrate for phosphorylation by Enzyme I of the PTS (Galinier et al, 1997 ;Reizer et al, 1998 ;Deutscher et al, 1995). The regulatory consequences of HPr phosphorylation by HPrK\P have been reviewed in detail (Stu$ lke & Hillen, 1999).…”

“…Moreover, HPrK\P activity is also present in mollicutes such as Mycoplasma capricolum and Acholeplasma laidlawii (Hoischen et al, 1993 ;Zhu et al, 1997). In Bacillus subtilis, HPrK\P senses the metabolic state of the cell and reversibly phosphorylates HPr of the PTS and an HPr homologue, Crh, at seryl residues (Galinier et al, 1997(Galinier et al, , 1998Reizer et al, 1998). HPr-Ser-P and Crh-Ser-P serve as cofactors for the transcriptional regulator, CcpA.…”

Structural analysis of B. subtilis CcpA effector binding site

“…B. subtilis HPr was produced in E. coli with a N-terminal histidine tag as described. 13 ATP-dependent seryl-phosphorylation of HPr was carried out as described using the B. subtilis HPr kinase/phosphorylase. 14 Crystals were grown in sitting drops containing 70 M ⌬CcpA, 138.5 M PserHPr, 4 mM FBP, 0.5% (v/v) MPD, 50 mM Mes (pH 6.5), 1.75 M ammonium sulfate over pits containing 1% (v/v) MPD, 100 mM Mes (pH 6.5), and 3.5 M ammonium sulfate.…”