Multiple mechanisms controlling carbon metabolism in bacteria

Catabolite repression is a universal phenomenon, found in virtually all living organisms. These organisms range from the simplest bacteria to higher fungi, plants, and animals. A mechanism involving cyclic AMP and its receptor protein (CRP) in Escherichia coli was established years ago, and this mechanism has been assumed by many to serve as the prototype for catabolite repression in all organisms. However, recent studies have shown that this mechanism is restricted to enteric bacteria and their close relatives. Cyclic AMP-independent mechanisms of catabolite repression occur in other bacteria, yeast, plants, and even E. coli. In fact, single-celled organisms such as E. coli, Bacillus subtilis, and Saccharomyces cerevisiae exhibit multiple mechanisms of catabolite repression, and most of these are cyclic AMP-independent. The mechanistic features of the best of such characterized processes are briefly reviewed, and references are provided that will allow the reader to delve more deeply into these subjects.     

Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram-positive bacteria

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.     

In vitro binding of the CcpA protein of Bacillus megaterium to cis-acting catabolite responsive elements (CREs) of Gram-positive bacteria

Abstract Using DNA band migration retardation assays, specific binding of the CcpA protein of Bacillus megaterium to the ds-acting catabolite responsive element (CRE) of the xyl operon of B. subtilis has been demonstrated. Binding of CcpA was specifically inhibited by addition of unlabeled DNA fragments containing CREs of other operons but not by DNA fragments lacking a CRE. Binding was stimulated by high concentrations of phosphate, pyrophosphate, and organic phosphate esters and specifically inhibited by serine phosphorylated HPr and its conformational analogue, S46D HPr. This report therefore documents the specific binding of CcpA to a target CRE and defines its regulation by HPr(ser-P) and phosphorylated metabolites.     

Analysis of the elements of catabolite repression in Clostridium acetobutylicum ATCC 824

The PTSH gene, encoding the phosphotransferase protein HPr, from Clostridium acetobutylicum ATCC 824 was identified from the genome sequence, cloned and shown to complement a PTSH mutant of Escherichia coli. The deduced protein sequence shares significant homology with HPr proteins from other low-GC gram-positive bacteria, although the highly conserved sequence surrounding the Ser-46 phosphorylation site is not well preserved in the clostridial protein. Nevertheless, the HPr was phosphorylated in an ATP-dependent manner in cell-free extracts of C. Acetobutylicum. Furthermore, purified His-tagged HPr from Bacillus Subtilis was also a substrate for the clostridial HPr kinase/phosphorylase. This phosphorylation reaction is a key step in the mechanism of carbon catabolite repression proposed to operate in B. Subtilis and other low-GC gram-positive bacteria. Putative genes encoding the HPr kinase/phosphorylase and the other element of this model, namely the catabolite control protein CcpA, were identified from the C. Acetobutylicum genome sequence, suggesting that a similar mechanism of carbon catabolite repression may operate in this industrially important organism.     

Carbon catabolite repression in bacteria.

Carbon catabolite repression (CCR) is a regulatory mechanism by which the expression of genes required for the utilization of secondary sources of carbon is prevented by the presence of a preferred substrate. This enables bacteria to increase their fitness by optimizing growth rates in natural environments providing complex mixtures of nutrients. In most bacteria, the enzymes involved in sugar transport and phosphorylation play an essential role in signal generation leading through different transduction mechanisms to catabolite repression. The actual mechanisms of regulation are substantially different in various bacteria. The mechanism of lactose-glucose diauxie in Escherichia coli has been reinvestigated and was found to be caused mainly by inducer exclusion. In addition, the gene encoding HPr kinase, a key component of CCR in many bacteria, was discovered recently.     

CcpA-mediated repression of Clostridium difficile toxin gene expression

The presence of glucose or other rapidly metabolizable carbon sources in the bacterial growth medium strongly represses Clostridium difficile toxin synthesis independently of strain origin. In Gram-positive bacteria, carbon catabolite repression (CCR) is generally regarded as a regulatory mechanism that responds to carbohydrate availability. In the C. difficile genome all elements involved in CCR are present. To elucidate in vivo the role of CCR in C. difficile toxin synthesis, we used the ClosTron gene knockout system to construct mutants of strain JIR8094 that were unable to produce the major components of the CCR signal transduction pathway: the phosphotransferase system (PTS) proteins (Enzyme I and HPr), the HPr kinase/phosphorylase (HprK/P) and the catabolite control protein A, CcpA. Inactivation of the ptsI, ptsH and ccpA genes resulted in derepression of toxin gene expression in the presence of glucose, whereas repression of toxin production was still observed in the hprK mutant, indicating that uptake of glucose is required for repression but that phosphorylation of HPr by HprK is not. C. difficile CcpA was found to bind to the regulatory regions of the tcdA and tcdB genes but not through a consensus cre site motif. Moreover in vivo and in vitro results confirmed that HPr-Ser45-P does not stimulate CcpA-dependent binding to DNA targets. However, fructose-1,6-biphosphate (FBP) alone did increase CcpA binding affinity in the absence of HPr-Ser45-P. These results showed that CcpA represses toxin expression in response to PTS sugar availability, thus linking carbon source utilization to virulence gene expression in C. difficile.     

Catabolite repression resistance of gnt operon expression in Bacillus subtilis conferred by mutation of His-15, the site of phosphoenolpyruvate-dependent phosphorylation of the phosphocarrier protein HPr.

Carbon catabolite repression of the gnt operon of Bacillus subtilis is mediated by the catabolite control protein CcpA and by HPr, a phosphocarrier protein of the phosphotransferase system. ATP-dependent phosphorylation of HPr at Ser-46 is required for carbon catabolite repression as ptsH1 mutants in which Ser-46 of HPr is replaced with an unphosphorylatable alanyl residue are resistant to carbon catabolite repression. We here demonstrate that mutation of His-15 of HPr, the site of phosphoenolpyruvate-dependent phosphorylation, also prevents carbon catabolite repression of the gnt operon. A strain which expressed two mutant HPrs (one in which Ser-46 is replaced by Ala [S46A HPr] and one in which His-15 is replaced by Ala [H15A HPr]) on the chromosome was barely sensitive to carbon catabolite repression, although the H15A mutant HPr can be phosphorylated at Ser-46 by the ATP-dependent HPr kinase in vitro and in vivo. The S46D mutant HPr which structurally resembles seryl-phosphorylated HPr has a repressive effect on gnt expression even in the absence of a repressing sugar. By contrast, the doubly mutated H15E,S46D HPr, which resembles the doubly phosphorylated HPr because of the negative charges introduced by the mutations at both phosphorylation sites, had no such effect. In vitro assays substantiated these findings and demonstrated that in contrast to the wild-type seryl-phosphorylated HPr and the S46D mutant HPr, seryl-phosphorylated H15A mutant HPr and H15E,S46D doubly mutated HPr did not interact with CcpA. These results suggest that His-15 of HPr is important for carbon catabolite repression and that either mutation or phosphorylation at His-15 can prevent carbon catabolite repression.     

Catabolite regulation of the cytochrome c550-encoding Bacillus subtilis cccA gene

In Gram-positive bacteria, catabolite control protein A (CcpA)-mediated catabolite repression or activation regulates not only the expression of a great number of catabolic operons, but also the synthesis of enzymes of central metabolic pathways. We found that a constituent of the Bacillus subtilis respiratory chain, the small cytochrome c550 encoded by the cccA gene, was also submitted to catabolite repression. Similar to most catabolite-repressed genes and operons, the Bacillus subtilis cccA gene contains a potential catabolite response element cre, an operator site recognized by CcpA. The presumed cre overlaps the -35 region of the cccA promoter. Strains carrying a cccA'-IacZ fusion formed blue colonies when grown on rich solid medium, whereas white colonies were obtained when glucose was present. beta-Galactosidase assays with cells grown in rich medium confirmed the repressive effect of glucose on cccA'-lacZ expression. Introduction of a ccpA or hprK mutation or of a mutation affecting the presumed cccA cre relieved the repressive effect of glucose during late log phase. An additional glucose repression mechanism was activated during stationary phase, which was not relieved by the ccpA, hprK or cre mutations. An interaction of the repressor/corepressor complex (CcpA/seryl-phosphorylated HPr (P-Ser-HPr)) with the cccA cre could be demonstrated by gel shift experiments. By contrast, a DNA fragment carrying mutations in the presumed cccA cre was barely shifted by the CcpA/P-Ser-HPr complex. In footprinting experiments, the region corresponding to the presumed cccA cre was specifically protected in the presence of the CcpA/P-Ser-HPr complex.     

Loss of protein kinase-catalyzed phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, by mutation of the ptsH gene confers catabolite repression resistance to several catabolic genes of Bacillus subtilis.

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 (ptsH1 mutation), synthesis of gluconate kinase, glucitol dehydrogenase, mannitol-1-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 alpha-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 beta-galactosidase was inducible by gluconate and repressed by glucose. However, we observed no repression of beta-galactosidase activity in a strain carrying the ptsH1 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 ptsH1 mutant strain were also insensitive to catabolite repression in the ccpA mutant. Enzymes that were repressed in the ptsH1 mutant were also repressed in the ccpA mutant.     

The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding

Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46. We report here that B. subtilis HprK forms homo-oligomers constituted most likely of eight subunits. Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235. It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations. A positive cooperativity was also detected for the binding of natural nucleotides or their 2'(3')-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments. Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations. This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK.