Cooperative and non-cooperative DNA binding modes of catabolite control protein CcpA from Bacillus megaterium result from sensing two different signals

Gösseringer, Roger; Küster, Elke; Galinier, Anne; Deutscher, Josef; Hillen, Wolfgang

doi:10.1006/jmbi.1996.0820

Cited by 123 publications

(161 citation statements)

References 46 publications

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“…P-Ser-Crh might interact with CcpA and allows its binding to the cre sequences. Multiple cre sites have recently been described for the gnt operon of B. subtilis (38) and for the xyl operon of Bacillus megaterium (39). These novel cre sequences were not recognized by the CcpA͞P-Ser-HPr complex, but were recognized by CcpA in the presence of glucose-6-P.…”

Section: Discussionmentioning

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.

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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%

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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208

274

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“…These motifs have been proposed to be the targets of regulatory proteins, but no evidence of their binding activity has been reported so far. On the other hand, these sequences have no similarity with the operator sites of the GN 4 CGN 4 C type, which are recognized by homologs of the LacI/GalR family and contain a central CG pair essential for their ability of protein binding (13,53), a G at the fifth position upstream of the CG pair, and a C at the fifth position downstream of the CG pair (15,20,52). Interestingly, several putative sites of this type could be identified in the upstream regions of the ␣-amylase genes regulated by Reg1.…”

Section: Discussionmentioning

confidence: 99%

Amylase and chitinase genes in Streptomyces lividans are regulated by reg1, a pleiotropic regulatory gene

et al. 1997

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A regulatory gene, reg1, was identified in Streptomyces lividans. It encodes a 345-amino-acid protein (Reg1) which contains a helix-turn-helix DNA-binding motif in the N-terminal region. Reg1 exhibits similarity with the LacI/GalR family members over the entire sequence. It displays 95% identity with MalR (the repressor of malE in S. coelicolor), 65% identity with ORF-Sl (a putative regulatory gene of ␣-amylase of S. limosus), and 31% identity with CcpA (the carbon catabolite repressor in Bacillus subtilis). In S. lividans, the chromosomal disruption of reg1 affected the expression of several genes. The production of ␣-amylases of S. lividans and that of the ␣-amylase of S. limosus in S. lividans were enhanced in the reg1 mutant strains and relieved of carbon catabolite repression. As a result, the transcription level of the ␣-amylase of S. limosus was noticeably increased in the reg1 mutant strain. Moreover, the induction of chitinase production in S. lividans was relieved of carbon catabolite repression by glucose in the reg1 mutant strain, while the induction by chitin was lost. Therefore, reg1 can be regarded as a pleiotropic regulatory gene in S. lividans.Streptomyces species, gram-positive soil bacteria with a high GC content, have the capacity to produce a vast array of secondary metabolites and extracellular proteins. The latter comprise many hydrolytic enzymes such as cellulases, chitinases, amylases, and xylanases allowing Streptomyces to grow on polymeric substrates (12). Genes encoding these enzymes are generally induced by degradation products of their polymeric substrates and repressed by readily metabolizable carbon sources such as glucose by a process known as catabolite repression. Several cis-acting sequences, displaying no apparent similarity, have been shown to be involved in the regulation of genes encoding such hydrolytic enzymes in various species of Streptomyces (10,11,44,49). However, no specific regulatory gene among those encoding extracellular proteins has so far been characterized for Streptomyces.␣-Amylases are secreted by several species of Streptomyces and catalyze the cleavage of the ␣-1,4 linkage of starch, yielding short linear maltodextrins which are finally converted to glucose. The highest ␣-amylase production was obtained when the ␣-amylase gene aml of Streptomyces limosus was cloned in S. lividans, which has a negligible ␣-amylase activity (43). In S. lividans, aml expression was induced by maltose and repressed by glucose. The regulation occurs at the level of transcription from the unique promoter. This led to the assumption that a regulatory protein(s) in S. lividans controlling the expression of aml might exist. A putative repressor, called open reading frame R (ORFR) in the present work, which is homologous to the regulatory proteins LacI and GalR of Escherichia coli has been located upstream of the ␣-amylase gene of S. venezuelae (45) and is also present upstream of the ␣-amylase genes of S. limosus (27) and S. griseus (42). Therefore, it was assumed that an ORF homo...

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“…In addition, a mutation at His-15 of HPr also conferred catabolite repression resistance to expression of the gnt operon (24), indicating that the two residues at Ser-46 and His-15 are linked to catabolite repression. It also reported that HPr-P (Ser-46) is essential for CcpA binding to the gnt and xyl cres in vitro (20,21). FDP, a key glycolytic intermediate, was reported to stimulate the phosphorylation of HPr at Ser-46 by an ATP-dependent HPr kinase (23).…”

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confidence: 97%

“…The relatively weak affinity of CcpA for its DNA-binding sites, the observation that CcpA is expressed constitutively in the presence and absence of glucose (18), and the presence of a conserved effector-binding domain in CcpA (3) together strongly suggest the necessity of an effector(s) such as a corepressor to activate CcpA. To date, HPr, a phosphocarrier protein in the phosphoenolpyruvate-:sugar phosphotransferase system, and two glycolytic metabolites, fructose-1,6-diphosphate (FDP) and glucose-6-phosphate, have been proposed as effectors of CcpA (19)(20)(21)(22).…”

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confidence: 99%

“…Therefore, it was suggested that FDP could stimulate CcpA binding to DNA through an interaction between FDP and HPr-P (Ser-46), not directly between CcpA and FDP. But, it was not clear whether FDP would stimulate CcpA binding to cres even in the presence of HPr-P (Ser-46) because the CcpA binding to the gnt and xyl cres triggered by HPr-P (Ser-46) did not require FDP nor was it stimulated by it (20)(21)(22).…”

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confidence: 99%

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NADP, corepressor for the Bacillus catabolite control protein CcpA

Kim

Voskuil

Chambliss

1998

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

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Expression of the ␣-amylase gene (amyE) of Bacillus subtilis is subject to CcpA (catabolite control protein A)-mediated catabolite repression, a global regulatory mechanism in Bacillus and other Gram-positive bacteria. To determine effectors of CcpA, we tested the ability of glycolytic metabolites, nucleotides, and cofactors to affect CcpA binding to the amyE operator, amyO. Those that stimulated the DNA-binding affinity of CcpA were tested for their effect on transcription. HPr-P (Ser-46), proposed as an effector of CcpA, also was tested. In DNase I footprint assays, the affinity of CcpA for amyO was stimulated 2-fold by fructose-1,6-diphosphate (FDP), 1.5-fold by oxidized or reduced forms of NADP, and 10-fold by HPr-P (Ser-46). However, the triple combinations, CcpA͞NADP͞HPr-P (Ser-46) and CcpA͞FDP͞ HPr-P (Ser-46) synergistically stimulated DNA-binding affinity by 120-and 300-fold, respectively. NADP added to CcpA specifically stimulated transcription inhibition of the amyE promoter by 120-fold. CcpA combined with HPr (Ser-46) inhibited transcription from the amyE promoter, but it also inhibited several control promoters. FDP did not stimulate transcription inhibition by CcpA nor did the triple combinations. The finding that NADP had little effect on CcpA DNA binding but increased the ability of CcpA to inhibit transcription suggests that catabolite repression is not simply caused by CcpA binding amyO but rather a result of interactions with the transcription machinery enhanced by NADP.

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Cooperative and non-cooperative DNA binding modes of catabolite control protein CcpA from Bacillus megaterium result from sensing two different signals

Cited by 123 publications

References 46 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

Amylase and chitinase genes in Streptomyces lividans are regulated by reg1, a pleiotropic regulatory gene

NADP, corepressor for the Bacillus catabolite control protein CcpA

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