ccpA Mutant Research Articles

CcpA and LacD.1 affect temporal regulation of Streptococcus pyogenes virulence genes.

Production of H(2)O(2) follows a growth phase-dependent pattern that mimics that of many virulence factors of Streptococcus pyogenes. To gain greater insight into mechanisms coupling virulence factor expression to growth phase, we investigated the molecular basis for H(2)O(2) generation and its regulation. Deletion of the gene encoding lactate oxidase (lctO) or culture in the presence of glucose eliminated H(2)O(2) production, implicating carbohydrate regulation of lctO as a key element of growth phase control. In examining known carbohydrate-responsive regulators, deletion of the gene encoding CcpA but not that encoding LacD.1 resulted in both derepression and an uncoupling of lctO transcription from its growth phase pattern. Expanding this analysis to additional virulence factors demonstrated both negative (cfa, encoding CAMP factor) and positive (speB, encoding a cysteine protease) regulation by CcpA and that CcpA mutants were highly cytotoxic for cultured macrophages. This latter property resulted from enhanced transcription of the streptolysin S biogenesis operon. Examination of CcpA-promoter interactions using a DNA pull-down assay mimicking physiological conditions showed direct binding to the promoters of lctO and speB but not those of sagA. CcpA but not LacD.1 mutants were attenuated in a murine model of soft-tissue infection, and analysis of gene expression in infected tissue indicated that CcpA mutants had altered expression of lctO, cfa, and speB but not the indirectly regulated sagA gene. Taken together, these data show that CcpA regulates virulence genes via at least three distinct mechanisms and that disruption of growth phase regulation alters transcriptional patterns in infected tissues.

Identification and functional characterisation of cellobiose and lactose transport systems in Lactococcus lactis IL1403

Physiological, biochemical and macroarray analyses of Lactococcus lactis IL1403 and its ccpA and bglR single and double mutants engaged in lactose and beta-glucosides catabolism were performed. The kinetic analysis indicated the presence of different transport systems for salicin and cellobiose. The control of salicin catabolism was found to be mediated by the transcriptional regulator BglR and the CcpA protein. The transcriptional analysis by macroarray technology of genes from the PEP:PTS regions showed that several genes, like ybhE, celB, ptcB and ptcA, were expressed at higher levels both in wild type cells exposed to cellobiose and in the ccpA mutant. We also demonstrated that in L. lactis IL1403 cultured on medium with cellobiose and lactose as carbon sources, after the first phase of cellobiose consumption and then co-metabolism of the two sugars, when cellobiose is exhausted the strain uses lactose as the only carbon source. These data could indicate that lactose and cellobiose are transported by a unique system-a PTS carrier induced by the presence of cellobiose, and negatively controlled by the CcpA regulator.

An acetoin-regulated expression system of Bacillussubtilis

An expression system, which is based on the promoter of the acoABCL operon of Bacillus subtilis was developed and characterized. The acoABCL operon codes for the acetoin dehydrogenase complex, which is the major enzyme system responsible for the catabolism of acetoin in B. subtilis. Besides weak organic acids, the neutral overflow metabolite acetoin is metabolized by the cells in the early stationary phase. Transcription of reporter gene fusions with the acoA promoter of this operon is strongly repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The co-expression of an additional copy of the regulator gene acoR led to more than twofold higher activity of the acoA promoter. It is demonstrated that the induction of this promoter in growing cells with acetoin is possible with non-phosphotransferase system sugars as carbon and energy source and in a ccpA mutant background. Moreover, it could be shown that the activity of the acoA-directed expression system correlates with the level of acetoin in the medium. During glucose limitation, the utilization of the alternative energy source acetoin keeps the protein synthesis machinery of B. subtilis cells active and thus allows for a long lasting acoA-controlled expression of recombinant genes.

Knockout of the HprK Gene in Bacillus subtilis CcpA Mutant and Its Influence on Riboflavin Fermentation

In Bacillus subtilis, increasing the carbon catabolite content in vivo would lead to carbon catabolite repression (CCR) and restrain the absorption of glucose. By deleting CcpA, the CCR effect could be alleviated, while the absorption of glucose remains restrained. The phosphoenol-pyruvate (PEP)-sugar phosphotransferase system (PTS) is the main glucose transportation system in B. subtilis. HPr protein together with HprK/P participates in the glucose transportation. The HPr protein is phosphorylated at His-15 resulting in the formation of HPr-His-15-P, and the phosphate group is transferred from HPr to E II. The HprK/P phosphorylates HPr at Ser-46 form HPr-Ser-46-P. HPr-Ser-46-P cannot participate in the transportation of glucose. The knockout of ccpA gene increases the amount of fructose-1, 6-bisphosphate (FBP) in vivo and FBP could activate HPr kinase. So when CcpA is deleted, most part of HPr will be phosphorylated at Ser-46. Therefore, absorption of glucose is blocked. In this study, by disruption of the hprK gene, the obtained B. subtilis ZHc/pMX45 reaches the peak riboflavin production of 4.374 mg/mL at the optimum glucose concentration of 10%, 19.2% higher than that of B. subtilis 24A1/pMX45 at the optimum glucose concentration of 8%.

CcpA causes repression of the phoPR promoter through a novel transcription start site, P(A6).

The Bacillus subtilis PhoPR two-component system is directly responsible for activation or repression of Pho regulon genes in response to phosphate deprivation. The response regulator, PhoP, and the histidine kinase, PhoR, are encoded in a single operon with a complex promoter region that contains five known transcription start sites, which respond to at least two regulatory proteins. We report here the identification of another direct regulator of phoPR transcription, carbon catabolite protein A, CcpA. This regulator functions in the presence of glucose or other readily metabolized carbon sources. The maximum derepression of phoPR expression in a ccpA mutant compared to a wild-type stain was observed under excess phosphate conditions with glucose either throughout growth in a high-phosphate defined medium or in a low-phosphate defined medium during exponential growth, a growth condition when phoPR transcription is low in a wild-type strain due to the absence of autoinduction. Either HPr or Crh were sufficient to cause CcpA dependent repression of the phoPR promoter in vivo. A ptsH1 (Hpr) crh double mutant completely relieves phoPR repression during phosphate starvation but not during phosphate replete growth. In vivo and in vitro studies showed that CcpA repressed phoPR transcription by binding directly to the cre consensus sequence present in the promoter. Primer extension and in vitro transcription studies revealed that the CcpA regulation of phoPR transcription was due to repression of P(A6), a previously unidentified promoter positioned immediately upstream of the cre box. Esigma(A) was sufficient for transcription of P(A6), which was repressed by CcpA in vitro. These studies showed direct repression by CcpA of a newly discovered Esigma(A)-responsive phoPR promoter that required either Hpr or Crh in vivo for direct binding to the putative consensus cre sequence located between P(A6) and the five downstream promoters characterized previously.

Complementation of a Delta ccpA mutant of Lactobacillus casei with CcpA mutants affected in the DNA- and cofactor-binding domains.

In low-G+C Gram-positive bacteria, the regulatory protein CcpA has been shown to play a major part in the so-called carbon catabolite repression (CCR) process, as well as in the induction of basic metabolic genes, for which it is considered a global regulator. A strain of Lactobacillus casei that carried a complete deletion of ccpA has been constructed and used to test the effect of CCR on N-acetylglucosaminidase activity and growth performance of a collection of seven CcpA mutations obtained by site-directed mutagenesis. The replaced amino acids were located in the DNA- and cofactor (P-Ser-HPr)-binding domains. Mutations in the DNA-binding domain lacked CCR, as found in Bacillus megaterium. However, mutations in the cofactor-binding domain of L. casei CcpA had a different phenotype to that observed in the previous studies with B. megaterium. Two of them, S80L and T307I, displayed a significant hyper-repression, an effect never reported before for CcpA. Comparison of growth capabilities provided by the different mutants and their ability to sustain CCR demonstrated that CCR, at least on the enzymic activity tested, and the growth defect caused by the CcpA mutations are unrelated features.

Cloning and characterization of the catabolite control protein A gene from Bacillus stearothermophilus No. 236.

The gene encoding the catabolite control protein A (CcpA) of Bacillus stearothermophilus No. 236, a strong xylanolytic bacterium, was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the ccpA gene corresponded to an open reading frame of 1,005 bp that encodes a polypeptide of 334 amino acid residues with a calculated molecular mass of 36,902 kDa. The CcpA protein belonging to the LacI/GalR family of transcriptional regulators was produced by a recombinant E. coli strain expressing the B. stearothermophilus No. 236 ccpA gene and purified to apparent homogeneity. The transcription start site was mapped at a position 63 nucleotides upstream of the translation initiation codon, and a presumed promoter sequence was also identified. The deduced amino acid sequence of the ccpA gene product contained the helix-turn-helix motif found in many DNA-binding proteins, and showed the highest identity (62%) with CcpA from B. subtilis. The B. stearothermophilus No. 236 ccpA gene was demonstrated to be able to complement a B. subtilis ccpA mutant that exhibited two distinct mutant phenotypes: a growth defect and a release of carbon catabolite repression (CCR). These results indicate that the ccpA gene product of B. stearothermophilus No. 236 is functionally active also in B. subtilis. Electrophoretic mobility shift assay with the purified CcpA revealed that the CcpA of B. stearothermophilus No. 236 bound specifically to the xynA creB (catabolite responsive element B) sequence. Taken together, these results strongly suggest that the CcpA protein participates in CCR of B. stearothermophilus No. 236 xynA gene.

The regulatory link between carbon and nitrogen metabolism in Bacillus subtilis: regulation of the gltAB operon by the catabolite control protein CcpA.

Bacillus subtilis assimilates ammonium by the concerted action of glutamine synthetase and glutamate synthase. The expression of the gltAB operon encoding the latter enzyme is impaired in B. subtilis ccpA mutant strains. CcpA is a pleiotropic transcriptional regulator that is the key factor in the regulation of carbon metabolism. However, in addition to their defect in catabolite repression ccpA mutants are unable to grow on minimal media with glucose and ammonium as the single sources of carbon and nitrogen, respectively. In this work, the expression of the gltAB operon was analysed and its role in growth on minimal sugar/ammonium media was studied. Expression of gltAB requires induction by glucose or other glycolytically catabolized carbon sources. In ccpA mutants, gltAB cannot be induced by glucose due to the low activity of the phosphotransferase sugar transport system in these mutants. A mutation that allowed phosphotransferase system activity in a ccpA background simultaneously restored glucose induction of gltAB and growth on glucose/ammonium medium. Moreover, artificial induction of the gltAB operon in the ccpA mutant allowed the mutant strain to grow on minimal medium with glucose and ammonium. It may be concluded that expression of the gltAB operon depends on the accumulation of glycolytic intermediates which cannot occur in the ccpA mutant. The lack of gltAB induction is the bottleneck that prevents growth of the ccpA mutant on glucose/ammonium media. The control of expression of the gltAB operon by CcpA provides a major regulatory link between carbon and amino acid metabolism.

Insufficient Expression of theilv-leuOperon Encoding Enzymes of Branched-Chain Amino Acid Biosynthesis Limits Growth of aBacillus subtilis ccpAMutant

Bacillus subtilis ccpA mutant strains exhibit two distinct phenotypes: they are defective in catabolite repression, and their growth on minimal media is strongly impaired. This growth defect is largely due to a lack of expression of the gltAB operon. However, growth is impaired even in the presence of glutamate. Here, we demonstrate that the ccpA mutant strain needs methionine and the branched-chain amino acids for optimal growth. The control of expression of the ilv-leu operon by CcpA provides a novel regulatory link between carbon and amino acid metabolism.

Control of the glycolytic gapA operon by the catabolite control protein A in Bacillus subtilis: a novel mechanism of CcpA-mediated regulation.

Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis. Expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase, the key enzyme of glycolysis from an energetic point of view, is induced by glucose and other sugars. Two regulators are involved in induction of the gapA operon, the product of the first gene of the operon, the CggR repressor, and catabolite control protein A (CcpA). CcpA is required for induction of the gapA operon by glucose. Genetic evidence has demonstrated that CcpA does not control the expression of the gapA operon by binding directly to a target in the promoter region. Here, we demonstrate by physiological analysis of the inducer spectrum that CcpA is required only for induction by sugars transported by the phosphotransferase system (PTS). A functional CcpA is needed for efficient transport of these sugars. This interference of CcpA with PTS sugar transport results from an altered phosphorylation pattern of HPr, a phosphotransferase of the PTS. In a ccpA mutant strain, HPr is nearly completely phosphorylated on a regulatory site, Ser-46, and is trapped in this state, resulting in its inactivity in PTS phosphotransfer. A mutation in HPr affecting the regulatory phosphorylation site suppresses both the defect in PTS sugar transport and the induction of the gapA operon. We conclude that a low-molecular effector derived from glucose that acts as an inducer for the repressor CggR is limiting in the ccpA mutant.

Direct and indirect roles of CcpA in regulation of Bacillus subtilis Krebs cycle genes.

Carbon catabolite repression of the Bacillus subtilis citrate synthase (citZ) and aconitase (citB) genes, previously known to be regulated by CcpC, was shown to depend on CcpA as well. Transcription of the citZ gene was partially derepressed in ccpA and ccpC single mutants and fully derepressed in a ccpA ccpC double mutant. DNase I footprinting studies showed that CcpA binds to a catabolite-responsive element (cre) site located at positions +80 to +97 with respect to the transcription start site, whereas CcpC binds at positions -14 to +6 and +16 to +36. Mutations in the citZ cre site greatly altered CcpA binding and repression. A ccpA null mutation also caused partial derepression of citB. Disruption of citrate synthase activity, however, suppressed the effect of the ccpA mutation, suggesting that increased citrate accumulation in a ccpA mutant partially inactivates CcpC and causes partial derepression of citB. Therefore, CcpA controls expression of Krebs cycle genes directly by regulating transcription of citZ and in-directly by regulating availability of citrate, the inducer for CcpC.

Antitermination by GlpP, catabolite repression via CcpA and inducer exclusion triggered by P-GlpK dephosphorylation control Bacillus subtilis glpFK expression.

The Bacillus subtilis glpFK operon encoding the glycerol transport facilitator (GlpF) and glycerol kinase (GlpK) is induced by glycerol-3-P and repressed by rapidly metabolizable sugars. Carbon catabolite repression (CCR) of glpFK is partly mediated via a catabolite response element cre preceding glpFK. This operator site is recognized by the catabolite control protein A (CcpA) in complex with one of its co-repressors, P-Ser-HPr or P-Ser-Crh. HPr is a component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and Crh is an HPr homologue. The hprK-encoded HPr kinase phosphorylates HPr and Crh at Ser-46. But in neither ccpA nor hprK mutants was expression of a glpF'-lacZ fusion relieved from CCR, as a second, CcpA-independent CCR mechanism implying the terminator tglpFK, whose formation is prevented by the glycerol-3-P-activated antiterminator GlpP, is operative. Deletion of tglpFK led to elevated expression of the glpF'-lacZ fusion and to partial relief from CCR. CCR completely disappeared in DeltatglpFK mutants carrying a disruption of ccpA or hprK. The tglpFK-requiring CCR mechanism seems to be based on insufficient synthesis of glycerol-3-P, as CCR of glpFK was absent in ccpA mutants growing on glycerol-3-P or synthesizing H230R mutant GlpK. In cells growing on glycerol, glucose prevents the phosphorylation of GlpK by P-His-HPr. P-GlpK is much more active than GlpK, and the absence of P~GlpK formation in DeltaptsHI strains prevents glycerol metabolism. As a consequence, only small amounts of glycerol-3-P will be formed in glycerol and glucose-exposed cells (inducer exclusion). The uptake of glycerol-3-P via GlpT provides high concentrations of this metabolite in the ccpA mutant and allows the expression of the glpF'-lacZ fusion even when glucose is present. Similarly, despite the presence of glucose, large amounts of glycerol-3-P are formed in a glycerol-exposed strain synthesizing GlpKH230R, as this mutant GlpK is as active as P-GlpK.

The Bacillus subtilis catabolite control protein CcpA exerts all its regulatory functions by DNA-binding.

In Bacillus subtilis, carbon catabolite control is mediated by the regulatory protein CcpA. In addition to loss of catabolite repression, ccpA mutants exhibit a severe growth defect. They are not able to grow with glucose and ammonium as single sources of carbon and nitrogen, respectively. Only ccpA mutant strains carrying either secondary suppressor mutations or that are affected in specific amino acids in the DNA-binding domain of CcpA grow on minimal media. We addressed the importance of DNA-binding by CcpA for both carbon catabolite repression and growth of a mutant strain. A strain specifically deleted for the N-terminal DNA-binding domain of CcpA was constructed. This deletion resulted in complete loss of catabolite repression of beta-xylosidase synthesis and prevented bacteria from growing on minimal media, suggesting that DNA-binding by CcpA is required for both carbon catabolite repression and efficient growth on minimal media.

The Bacillus subtilis catabolite control protein CcpA exerts all its regulatory functions by DNA-binding

In Bacillus subtilis, carbon catabolite control is mediated by the regulatory protein CcpA. In addition to loss of catabolite repression, ccpA mutants exhibit a severe growth defect. They are not able to grow with glucose and ammonium as single sources of carbon and nitrogen, respectively. Only ccpA mutant strains carrying either secondary suppressor mutations or that are affected in specific amino acids in the DNA-binding domain of CcpA grow on minimal media. We addressed the importance of DNA-binding by CcpA for both carbon catabolite repression and growth of a mutant strain. A strain specifically deleted for the N-terminal DNA-binding domain of CcpA was constructed. This deletion resulted in complete loss of catabolite repression of β-xylosidase synthesis and prevented bacteria from growing on minimal media, suggesting that DNA-binding by CcpA is required for both carbon catabolite repression and efficient growth on minimal media.

Contribution of the phosphoenolpyruvate:mannose phosphotransferase system to carbon catabolite repression in Lactobacillus pentosus.

The role of the Lactobacillus pentosus phosphoenolpyruvate:mannose phosphotransferase system (mannose PTS) in sugar transport and control of sugar utilization was investigated. Growth experiments and measurements of PEP-dependent phosphorylation of sugars, of sugar transport and of catabolic enzyme activity were performed, to compare a wild-type strain with an EIIB(Man) mutant, LPE6, and a ccpA mutant, LPE4. Fructose uptake in wild-type bacteria demonstrated the presence of two fructose-specific PTSs: a high-affinity system, EII(Fru) (K:(m)=52 microM) which is inducible by fructose, and a low-affinity system (K:(m)=300 microM). The latter system was lacking in LPE6 and therefore corresponds to EII(Man). LPE6 was unable to phosphorylate glucose, mannose, N:-acetylglucosamine and 2-deoxyglucose in a PEP-dependent reaction, indicating that these sugars are substrates of EII(Man). Transport and phosphorylation of these compounds was the same in LPE4 and in wild-type bacteria, although growth of LPE4 on these sugars was impaired. In wild-type bacteria and in LPE4 the activity of EII(Fru) was lowered by the presence of EII(Man) substrates in the growth medium, but this decrease was not observed in LPE6. These results indicate that EII(Man) but not CcpA regulates the synthesis of EII(Fru). Mutations in EII(Man) or CcpA resulted in a relief of catabolite repression exerted by EII(Man) substrates on the activity of beta-galactosidase and beta-glucosidase, indicating that EII(Man) and CcpA are important components in catabolite repression in L. pentosus. Fructose-mediated repression of these two enzymes appeared to be correlated with the activity of EII(Fru).

Catabolite repression of dra-nupC-pdp operon expression in Bacillus subtilis.

Expression of the Bacillus subtilis dra-nupC-pdp operon is subject to catabolite repression by glucose. It was shown that a cis-acting catabolite-responsive element (CRE) sequence located 64 bp downstream of the transcription-start site mediated catabolite repression of the dra-nupC-pdp operon as it does for many other B. subtilis genes. Point mutations in the CRE sequence caused the loss of catabolite repression of the operon. Catabolite repression of dra-nupC-pdp expression was relieved in a ccpA mutant and was found to be dependent on both HPr and the HPr-like protein Crh. Furthermore, a transcription-repair coupling factor, Mfd, was also found to be involved in the glucose repression of dra-nupC-pdp expression. By the use of in vitro gel mobility shift analysis, a specific HPr-P dependent binding of CcpA to the dra CRE site was demonstrated.

Bacillus subtilis ccpA gene mutants specifically defective in activation of acetoin biosynthesis.

A large number of carbon source utilization pathways are repressed in Bacillus subtilis by the global regulator CcpA, which also acts as an activator of carbon excretion pathways during growth in media containing glucose. In this study, CcpA mutants defective in transcriptional activation of the alsSD operon, which is involved in acetoin biosynthesis, were identified. These mutants retained normal glucose repression of amyE, encoding alpha-amylase, and acsA, encoding acetyl-coenzyme A synthetase, and normal activation of ackA, which is involved in acetate excretion; in these ccpA mutants the CcpA functions of activation of the acetate and acetoin excretion pathways appear to be separated.

The CitST two-component system regulates the expression of the Mg-citrate transporter in Bacillus subtilis.

citS and citT genes encoding a new two-component system were identified in the 71 degrees region between the pel and citM loci on the Bacillus subtilis chromosome. citS- and citT-deficient strains were unable to grow on minimal plates including citrate as a sole carbon source. In addition, a strain deficient in citM, which encodes the secondary transporter of the Mg-citrate complex, exhibited the same phenotype on this medium. Northern blot analysis revealed that citM was polycistronically transcribed with the downstream yflN gene, and that CitS and CitT were necessary for transcription of the citM-yflN operon. Upon addition of 2 mM citrate to DSM, this operon was strongly induced after the middle of the exponential growth phase in the wild type, but not in the citST double null mutant. Moreover, the transcription of this operon was completely repressed in the presence of 1% glucose. We found a sequence exhibiting homology to a catabolite-responsive element (cre) in the citM promoter region. Glucose repression was lost in ccpA and citM-cre mutants. From the result of a citM-promoter deletion experiment, putative CitT target sequences were found to be located around two regions, from -62 to -74 and from -149 to -189, relative to the citM start point. Furthermore, DNase I footprinting assays revealed that these two CitT target regions extended maximally from -36 to -84 and from -168 to -194. From these findings, we concluded that the expression of citM is positively regulated by the CitST system and negatively regulated by CcpA.

Cloning and expression of the ccpA gene encoding catabolite control protein from Thermoactinomyces sp. E79.

The gene ccpA encoding the catabolite control protein A (CcpA) of Thermoactinomyces sp. E79 has been cloned and characterized. Nucleotide sequence analysis of the CcpA clone showed that the cloned fragment contained the full structural gene for a protein of 346 amino acids. The predicted amino acid sequence shows similarity to the transcriptional regulators of the Lacl-GalR family; a highly conserved helix-turn-helix motif, which might bind to DNA, was identified through comparison with regulator proteins in this family. The highest sequence identity was obtained when it was compared with the CcpA of Bacillus subtilis (60%) or Bacillus megaterium (60%). The expression of ccpA in Thermoactinomyces sp. E79 was dependent on glucose, which is contrast to the cases of B. subtilis, B. megaterium and S. xylosus. The complementation experiment with the B. megaterium ccpA mutant indicated that the cloned gene was a ccpA.

Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis.

In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli, the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA. The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.