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...

In gram-positive bacteria, HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), is phosphorylated by an ATP-dependent, metabolite-activated protein kinase on seryl residue 46. In a Bacillus subtilis mutant strain in which Ser-46 of HPr was replaced with a nonphosphorylatable alanyl residue (ptsHl mutation), synthesis of gluconate kinase, glucitol dehydrogenase, mannitol-l-P dehydrogenase and the mannitol-specific PTS permease was completely relieved from repression by glucose, fructose, or mannitol, whereas synthesis of inositol dehydrogenase was partially relieved from catabolite repression and synthesis of oa-glucosidase and glycerol kinase was still subject to catabolite repression. When the S46A mutation in HPr was reverted to give S46 wild-type HPr, expression of gluconate kinase and glucitol dehydrogenase regained full sensitivity to repression by PTS sugars. These results suggest that phosphorylation of HPr at Ser-46 is directly or indirectly involved in catabolite repression. A strain deleted for the ptsGHI genes was transformed with plasmids expressing either the wild-type ptsH gene or various S46 mutant ptsH genes (S46A or S46D). Expression of the gene encoding S46D HPr, having a structure similar to that of P-ser-HPr according to nuclear magnetic resonance data, caused significant reduction of gluconate kinase activity, whereas expression of the genes encoding wild-type or S46A HPr had no effect on this enzyme activity. When the promoterless lacZ gene was put under the control of the gnt promoter and was subsequently incorporated into the amyE gene on the B. subtilis chromosome, expression of 1-galactosidase was inducible by gluconate and repressed by glucose. However, we observed no repression of I-galactosidase activity in a strain carrying the ptsHl mutation. Additionally, we investigated a ccpA mutant strain and observed that all of the enzymes which we found to be relieved from carbon catabolite repression in the ptsHl mutant strain were also insensitive to catabolite repression in the ccpA mutant. Enzymes that were repressed in the ptsHl mutant were also repressed in the ccpA mutant.Catabolite-repressible genes in the gram-positive bacterium Bacillus subtilis are controlled by multiple mechanisms rather than by a single global regulatory system. These mechanisms are known to differ from those operating in enteric bacteria such as Escherichia coli, but the molecular details are poorly understood (15,40,46

Solid state NMR sample preparation and resonance assignments of the U-[13C,15N] 2x10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra- and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all 15N, 13C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.