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1.  CcpA-Mediated Repression of Streptolysin S Expression and Virulence in the Group A Streptococcus▿†  
Infection and Immunity  2008;76(8):3451-3463.
CcpA is the global mediator of carbon catabolite repression (CCR) in gram-positive bacteria, and growing evidence from several pathogens, including the group A streptococcus (GAS), suggests that CcpA plays an important role in virulence gene regulation. In this study, a deletion of ccpA in an invasive M1 GAS strain was used to test the contribution of CcpA to pathogenesis in mice. Surprisingly, the ΔccpA mutant exhibited a dramatic “hypervirulent” phenotype compared to the parental MGAS5005 strain, reflected as increased lethality in a model of systemic infection (intraperitoneal administration) and larger lesion size in a model of skin infection (subcutaneous administration). Expression of ccpA in trans from its native promoter was able to complement both phenotypes, suggesting that CcpA acts to repress virulence in GAS. To identify the CcpA-regulated gene(s) involved, a transcriptome analysis was performed on mid-logarithmic-phase cells grown in rich medium. CcpA was found to primarily repress 6% of the GAS genome (124 genes), including genes involved in sugar metabolism, transcriptional regulation, and virulence. Notably, the entire sag operon necessary for streptolysin S (SLS) production was under CcpA-mediated CCR, as was SLS hemolytic activity. Purified CcpA-His bound specifically to a cre within sagAp, demonstrating direct repression of the operon. Finally, SLS activity is required for the increased virulence of a ΔccpA mutant during systemic infection but did not affect virulence in a wild-type background. Thus, CcpA acts to repress SLS activity and virulence during systemic infection in mice, revealing an important link between carbon metabolism and GAS pathogenesis.
PMCID: PMC2493232  PMID: 18490461
2.  Control of Lactose Transport, β-Galactosidase Activity, and Glycolysis by CcpA in Streptococcus thermophilus: Evidence for Carbon Catabolite Repression by a Non-Phosphoenolpyruvate-Dependent Phosphotransferase System Sugar 
Journal of Bacteriology  2000;182(21):5982-5989.
Streptococcus thermophilus, unlike many other gram-positive bacteria, prefers lactose over glucose as the primary carbon and energy source. Moreover, lactose is not taken up by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) but by the dedicated transporter LacS. In this paper we show that CcpA plays a crucial role in the fine-tuning of lactose transport, β-galactosidase (LacZ) activity, and glycolysis to yield optimal glycolytic flux and growth rate. A catabolite-responsive element (cre) was identified in the promoter of the lacSZ operon, indicating a possible role for regulation by CcpA. Transcriptional analysis showed a sevenfold relief of repression in the absence of a functional CcpA when cells were grown on lactose. This CcpA-mediated repression of lacSZ transcription did not occur in wild-type cells during growth on galactose, taken up by the same LacS transport system. Lactose transport during fermentation was increased significantly in strains carrying a disrupted ccpA gene. Moreover, a ccpA disruption strain was found to release substantial amounts of glucose into the medium when grown on lactose. Transcriptional analysis of the ldh gene showed that expression was induced twofold during growth on lactose compared to glucose or galactose, in a CcpA-dependent manner. A reduced rate of glycolysis concomitant with an increased lactose transport rate could explain the observed expulsion of glucose in a ccpA disruption mutant. We propose that CcpA in S. thermophilus acts as a catabolic regulator during growth on the preferred non-PTS sugar lactose. In contrast to other bacteria, S. thermophilus possesses an overcapacity for lactose uptake that is repressed by CcpA to match the rate-limiting glycolytic flux.
PMCID: PMC94730  PMID: 11029416
3.  CcpA Ensures Optimal Metabolic Fitness of Streptococcus pneumoniae 
PLoS ONE  2011;6(10):e26707.
In Gram-positive bacteria, the transcriptional regulator CcpA is at the core of catabolite control mechanisms. In the human pathogen Streptococcus pneumoniae, links between CcpA and virulence have been established, but its role as a master regulator in different nutritional environments remains to be elucidated. Thus, we performed whole-transcriptome and metabolic analyses of S. pneumoniae D39 and its isogenic ccpA mutant during growth on glucose or galactose, rapidly and slowly metabolized carbohydrates presumably encountered by the bacterium in different host niches. CcpA affected the expression of up to 19% of the genome covering multiple cellular processes, including virulence, regulatory networks and central metabolism. Its prevalent function as a repressor was observed on glucose, but unexpectedly also on galactose. Carbohydrate-dependent CcpA regulation was also observed, as for the tagatose 6-phosphate pathway genes, which were activated by galactose and repressed by glucose. Metabolite analyses revealed that two pathways for galactose catabolism are functionally active, despite repression of the Leloir genes by CcpA. Surprisingly, galactose-induced mixed-acid fermentation apparently required CcpA, since genes involved in this type of metabolism were mostly under CcpA-repression. These findings indicate that the role of CcpA extends beyond transcriptional regulation, which seemingly is overlaid by other regulatory mechanisms. In agreement, CcpA influenced the level of many intracellular metabolites potentially involved in metabolic regulation. Our data strengthen the view that a true understanding of cell physiology demands thorough analyses at different cellular levels. Moreover, integration of transcriptional and metabolic data uncovered a link between CcpA and the association of surface molecules (e.g. capsule) to the cell wall. Hence, CcpA may play a key role in mediating the interaction of S. pneumoniae with its host. Overall, our results support the hypothesis that S. pneumoniae optimizes basic metabolic processes, likely enhancing in vivo fitness, in a CcpA-mediated manner.
PMCID: PMC3198803  PMID: 22039538
4.  Carbon Catabolite Repression in Lactobacillus pentosus: Analysis of the ccpA Region 
The catabolite control protein CcpA is a central regulator in low-G+C-content gram-positive bacteria. It confers carbon catabolite repression to numerous genes required for carbon utilization. It also operates as a transcriptional activator of genes involved in diverse phenomena, such as glycolysis and ammonium fixation. We have cloned the ccpA region of Lactobacillus pentosus. ccpA encodes a protein of 336 amino acids exhibiting similarity to CcpA proteins of other bacteria and to proteins of the LacI/GalR family of transcriptional regulators. Upstream of ccpA was found an open reading frame with similarity to the pepQ gene, encoding a prolidase. Primer extension experiments revealed two start sites of transcription for ccpA. In wild-type cells grown on glucose, mRNA synthesis occurred only from the promoter proximal to ccpA. In a ccpA mutant strain, both promoters were used, with increased transcription from the distant promoter, which overlaps a presumptive CcpA binding site called cre (for catabolite responsive element). This suggests that expression of ccpA is autoregulated. Determination of the expression levels of CcpA in cells grown on repressing and nonrepressing carbon sources revealed that the amounts of CcpA produced did not change significantly, leading to the conclusion that the arrangement of two promoters may ensure constant expression of ccpA under various environmental conditions. A comparison of the genetic structures of ccpA regions revealed that lactic acid bacteria possess the gene order pepQ-ccpA-variable while the genetic structure in bacilli and Staphylococcus xylosus is aroA-ccpA-variable-acuC.
PMCID: PMC91818  PMID: 10618236
5.  CcpA-Dependent Carbohydrate Catabolite Repression Regulates Galactose Metabolism in Streptococcus oligofermentans 
Journal of Bacteriology  2012;194(15):3824-3832.
Streptococcus oligofermentans is an oral commensal that inhibits the growth of the caries pathogen Streptococcus mutans by producing copious amounts of H2O2 and that grows faster than S. mutans on galactose. In this study, we identified a novel eight-gene galactose (gal) operon in S. oligofermentans that was comprised of lacABCD, lacX, and three genes encoding a galactose-specific transporter. Disruption of lacA caused more growth reduction on galactose than mutation of galK, a gene in the Leloir pathway, indicating that the principal role of this operon is in galactose metabolism. Diauxic growth was observed in cultures containing glucose and galactose, and a luciferase reporter fusion to the putative gal promoter demonstrated 12-fold repression of the operon expression by glucose but was induced by galactose, suggesting a carbon catabolite repression (CCR) control in galactose utilization. Interestingly, none of the single-gene mutations in the well-known CCR regulators ccpA and manL affected diauxic growth, although the operon expression was upregulated in these mutants in glucose. A double mutation of ccpA and manL eliminated glucose repression of galactose utilization, suggesting that these genes have parallel functions in regulating gal operon expression and mediating CCR. Electrophoretic mobility shift assays demonstrated binding of CcpA to the putative catabolite response element motif in the promoter regions of the gal operon and manL, suggesting that CcpA regulates CCR through direct regulation of the transcription of the gal operon and manL. This provides the first example of oral streptococci using two parallel CcpA-dependent CCR pathways in controlling carbohydrate metabolism.
PMCID: PMC3416513  PMID: 22609925
6.  Molecular Characterization of CcpA and Involvement of This Protein in Transcriptional Regulation of Lactate Dehydrogenase and Pyruvate Formate-Lyase in the Ruminal Bacterium Streptococcus bovis 
A ccpA gene that encodes global catabolite control protein A (CcpA) in Streptococcus bovis was identified and characterized, and the involvement of CcpA in transcriptional control of a gene (ldh) encoding lactate dehydrogenase (LDH) and a gene (pfl) encoding pyruvate formate-lyase (PFL) was examined. The ccpA gene was shown to be transcribed as a monocistronic operon. A catabolite-responsive element (cre) was found in the promoter region of ccpA, suggesting that ccpA transcription in S. bovis is autogenously regulated. CcpA required HPr that was phosphorylated at the serine residue at position 46 (HPr-[Ser-P]) for binding to the cre site, but glucose 6-phosphate, fructose 1,6-bisphosphate, and NADP had no effect on binding. Diauxic growth was observed when S. bovis was grown in a medium containing glucose and lactose, but it disappeared when ccpA was disrupted, which indicates that CcpA is involved in catabolite repression in S. bovis. The level of ccpA mRNA was higher when cells were grown on glucose than when they were grown on lactose, which was in line with the level of ldh mRNA. When cells were grown on glucose, the ldh mRNA level was lower but the pfl mRNA level was higher in a ccpA-disrupted mutant than in the parent strain, which suggests that ldh transcription is enhanced and pfl transcription is suppressed by CcpA. The ccpA-disrupted mutant produced less lactate and more formate than the parent, probably because the mutant had reduced LDH activity and elevated PFL activity. In the upper region of both ldh and pfl, a cre-like sequence was found, suggesting that the complex consisting of CcpA and HPr-[Ser-P] binds to the possible cre sites. Thus, CcpA appears to be involved in the global regulation of sugar utilization in S. bovis.
PMCID: PMC520867  PMID: 15345406
7.  Assessment of CcpA-mediated catabolite control of gene expression in Bacillus cereus ATCC 14579 
BMC Microbiology  2008;8:62.
The catabolite control protein CcpA is a transcriptional regulator conserved in many Gram-positives, controlling the efficiency of glucose metabolism. Here we studied the role of Bacillus cereus ATCC 14579 CcpA in regulation of metabolic pathways and expression of enterotoxin genes by comparative transcriptome analysis of the wild-type and a ccpA-deletion strain.
Comparative analysis revealed the growth performance and glucose consumption rates to be lower in the B. cereus ATCC 14579 ccpA deletion strain than in the wild-type. In exponentially grown cells, the expression of glycolytic genes, including a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase that mediates conversion of D-glyceraldehyde 3-phosphate to 3-phospho-D-glycerate in one single step, was down-regulated and expression of gluconeogenic genes and genes encoding the citric acid cycle was up-regulated in the B. cereus ccpA deletion strain. Furthermore, putative CRE-sites, that act as binding sites for CcpA, were identified to be present for these genes. These results indicate CcpA to be involved in the regulation of glucose metabolism, thereby optimizing the efficiency of glucose catabolism. Other genes of which the expression was affected by ccpA deletion and for which putative CRE-sites could be identified, included genes with an annotated function in the catabolism of ribose, histidine and possibly fucose/arabinose and aspartate. Notably, expression of the operons encoding non-hemolytic enterotoxin (Nhe) and hemolytic enterotoxin (Hbl) was affected by ccpA deletion, and putative CRE-sites were identified, which suggests catabolite repression of the enterotoxin operons to be CcpA-dependent.
The catabolite control protein CcpA in B. cereus ATCC 14579 is involved in optimizing the catabolism of glucose with concomitant repression of gluconeogenesis and alternative metabolic pathways. Furthermore, the results point to metabolic control of enterotoxin gene expression and suggest that CcpA-mediated glucose sensing provides an additional mode of control in moderating the expression of the nhe and hbl operons in B. cereus ATCC 14579.
PMCID: PMC2358912  PMID: 18416820
8.  Carbon Catabolite Repression of Type IV Pilus-Dependent Gliding Motility in the Anaerobic Pathogen Clostridium perfringens▿ †  
Journal of Bacteriology  2007;190(1):48-60.
Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium responsible for the production of severe histotoxic and gastrointestinal diseases in humans and animals. In silico analysis of the three available genome-sequenced C. perfringens strains (13, SM101, and ATCC13124) revealed that genes that encode flagellar proteins and genes involved in chemotaxis are absent. However, those strains exhibit type IV pilus (TFP)-dependent gliding motility. Since carbon catabolite regulation has been implicated in the control of different bacterial behaviors, we investigated the effects of glucose and other readily metabolized carbohydrates on C. perfringens gliding motility. Our results demonstrate that carbon catabolite regulation constitutes an important physiological regulatory mechanism that reduces the proficiencies of the gliding motilities of a large number of unrelated human- and animal-derived pathogenic C. perfringens strains. Glucose produces a strong dose-dependent inhibition of gliding development without affecting vegetative growth. Maximum gliding inhibition was observed at a glucose concentration (1%) previously reported to also inhibit other important behaviors in C. perfringens, such as spore development. The inhibition of gliding development in the presence of glucose was due, at least in part, to the repression of the genes pilT and pilD, whose products are essential for TFP-dependent gliding proficiency. The inhibitory effects of glucose on pilT and pilD expression were under the control of the key regulatory protein CcpA (catabolite control protein A). The deficiency in CcpA activity of a ccpA knockout C. perfringens mutant strain restored the expressions of pilT and pilD and gliding proficiency in the presence of 1% glucose. The carbon catabolite repression of the gliding motility of the ccpA mutant strain was restored after the introduction of a complementing plasmid harboring a wild-type copy of ccpA. These results point to a central role for CcpA in orchestrating the negative effect of carbon catabolite regulation on C. perfringens gliding motility. Furthermore, we discovered a novel positive role for CcpA in pilT and pilD expression and gliding proficiency in the absence of catabolite regulation. Carbon catabolite repression of gliding motility and the dual role of CcpA, either as repressor or as activator of gliding, are analyzed in the context of the different social behaviors and diseases produced by C. perfringens.
PMCID: PMC2223757  PMID: 17981974
9.  The CcpA Protein Is Necessary for Efficient Sporulation and Enterotoxin Gene (cpe) Regulation in Clostridium perfringens 
Journal of Bacteriology  2004;186(16):5221-5229.
Clostridium perfringens is the cause of several human diseases, including gas gangrene (clostridial myonecrosis), enteritis necroticans, antibiotic-associated diarrhea, and acute food poisoning. The symptoms of antibiotic-associated diarrhea and acute food poisoning are due to sporulation-dependent production of C. perfringens enterotoxin encoded by the cpe gene. Glucose is a catabolite repressor of sporulation by C. perfringens. In order to identify the mechanism of catabolite repression by glucose, a mutation was introduced into the ccpA gene of C. perfringens by conjugational transfer of a nonreplicating plasmid into C. perfringens, which led to inactivation of the ccpA gene by homologous recombination. CcpA is a transcriptional regulator known to mediate catabolite repression in a number of low-G+C-content gram-positive bacteria, of which C. perfringens is a member. The ccpA mutant strain sporulated at a 60-fold lower efficiency than the wild-type strain in the absence of glucose. In the presence of 5 mM glucose, sporulation was repressed about 2,000-fold in the wild-type strain and 800-fold in the ccpA mutant strain compared to sporulation levels for the same strains grown in the absence of glucose. Therefore, while CcpA is necessary for efficient sporulation in C. perfringens, glucose-mediated catabolite repression of sporulation is not due to the activity of CcpA. Transcription of the cpe gene was measured in the wild-type and ccpA mutant strains grown in sporulation medium by using a cpe-gusA fusion (gusA is an Escherichia coli gene encoding the enzyme β-glucuronidase). In the exponential growth phase, cpe transcription was two times higher in the ccpA mutant strain than in the wild-type strain. Transcription of cpe was highly induced during the entry into stationary phase in wild-type cells but was not induced in the ccpA mutant strain. Glucose repressed cpe transcription in both the wild-type and ccpA mutant strain. Therefore, CcpA appears to act as a repressor of cpe transcription in exponential growth but is required for efficient sporulation and cpe transcription upon entry into stationary phase. CcpA was also required for maximum synthesis of collagenase (kappa toxin) and acted as a repressor of polysaccharide capsule synthesis in the presence of glucose, but it did not regulate synthesis of the phospholipase PLC (alpha toxin).
PMCID: PMC490932  PMID: 15292123
10.  Regulators of the Bacillus subtilis cydABCD Operon: Identification of a Negative Regulator, CcpA, and a Positive Regulator, ResD▿  
Journal of Bacteriology  2007;189(9):3348-3358.
The cydABCD operon of Bacillus subtilis encodes products required for the production of cytochrome bd oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the cydABCD operon. Two additional regulatory proteins for the cydABCD operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the cydA promoter in vivo. ResD binding to the cydA promoter between positions −58 and −107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A ccpA mutant had increased expression from the full-length cydA promoter during stationary growth compared to the wild-type strain. Maximal expression in a ccpA mutant was observed from a 3′-deleted cydA promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the cydA promoter, protecting the region from positions −4 to −33, which contains sequences similar to the CcpA consensus binding sequence, the cre box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the cre box reduced CcpA binding 10-fold in vitro and increased cydA expression in vivo. Thus, CcpA and ResD, along with the previously identified cydA regulator Rex (YdiH), affect the expression of the cydABCD operon. Low-level induction of the cydA promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the cydA promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.
PMCID: PMC1855890  PMID: 17322317
11.  Catabolite Control Protein A (CcpA) Contributes to Virulence and Regulation of Sugar Metabolism in Streptococcus pneumoniae 
Journal of Bacteriology  2005;187(24):8340-8349.
We characterized the role of catabolite control protein A (ccpA) in the physiology and virulence of Streptococcus pneumoniae. S. pneumoniae has a large percentage of its genome devoted to sugar uptake and metabolism, and therefore, regulation of these processes is likely to be crucial for fitness in the nasopharynx and may play a role during invasive disease. In many bacteria, carbon catabolite repression (CCR) is central to such regulation, influencing hierarchical sugar utilization and growth rates. CcpA is the major transcriptional regulator in CCR in several gram-positive bacteria. We show that CcpA functions in CCR of lactose-inducible β-galactosidase activity in S. pneumoniae. CCR of maltose-inducible α-glucosidase, raffinose-inducible α-galactosidase, and cellobiose-inducible β-glucosidase is unaffected in the ccpA strain, suggesting that other regulators, possibly redundant with CcpA, control these systems. The ccpA strain is severely attenuated for nasopharyngeal colonization and lung infection in the mouse, establishing its role in fitness on these mucosal surfaces. Comparison of the cell wall fraction of the ccpA and wild-type strains shows that CcpA regulates many proteins in this compartment that are involved in central and intermediary metabolism, a subset of which are required for survival and multiplication in vivo. Both in vitro and in vivo defects were complemented by providing ccpA in trans. Our results demonstrate that CcpA, though not a global regulator of CCR in S. pneumoniae, is required for colonization of the nasopharynx and survival and multiplication in the lung.
PMCID: PMC1317011  PMID: 16321938
12.  Global transcriptional control by glucose and carbon regulator CcpA in Clostridium difficile 
Nucleic Acids Research  2012;40(21):10701-10718.
The catabolite control protein CcpA is a pleiotropic regulator that mediates the global transcriptional response to rapidly catabolizable carbohydrates, like glucose in Gram-positive bacteria. By whole transcriptome analyses, we characterized glucose-dependent and CcpA-dependent gene regulation in Clostridium difficile. About 18% of all C. difficile genes are regulated by glucose, for which 50% depend on CcpA for regulation. The CcpA regulon comprises genes involved in sugar uptake, fermentation and amino acids metabolism, confirming the role of CcpA as a link between carbon and nitrogen pathways. Using combination of chromatin immunoprecipitation and genome sequence analysis, we detected 55 CcpA binding sites corresponding to ∼140 genes directly controlled by CcpA. We defined the C. difficile CcpA consensus binding site (creCD motif), that is, ‘RRGAAAANGTTTTCWW’. Binding of purified CcpA protein to 19 target creCD sites was demonstrated by electrophoretic mobility shift assay. CcpA also directly represses key factors in early steps of sporulation (Spo0A and SigF). Furthermore, the C. difficile toxin genes (tcdA and tcdB) and their regulators (tcdR and tcdC) are direct CcpA targets. Finally, CcpA controls a complex and extended regulatory network through the modulation of a large set of regulators.
PMCID: PMC3510511  PMID: 22989714
13.  The EIIABMan Phosphotransferase System Permease Regulates Carbohydrate Catabolite Repression in Streptococcus gordonii▿ †  
Commensal oral streptococci play critical roles in oral biofilm formation and promote dental health by competing with, and antagonizing the growth of, pathogenic organisms, such as Streptococcus mutans. Efficient utilization of the spectrum of carbohydrates in the oral cavity by commensal streptococci is essential for their persistence, and yet very little is known about the regulation of carbohydrate catabolism by these organisms. Carbohydrate catabolite repression (CCR) in the abundant oral commensal Streptococcus gordonii strain DL-1 was investigated using the exo-β-d-fructosidase gene (fruA) and a fructose/mannose sugar:phosphotransferase (PTS) enzyme II operon (levDEFG) as model systems. Functional studies confirmed the predicted roles of FruA and LevD in S. gordonii. ManL, the AB domain of a fructose/mannose-type enzyme II PTS permease, contributed to utilization of glucose, mannose, galactose, and fructose and exerted primary control over CCR of the fruA and levD operons. Unlike in S. mutans, ManL-dependent CCR was not sugar specific, and galactose was very effective at eliciting CCR in S. gordonii. Inactivation of the apparent ccpA homologue of S. gordonii actually enhanced CCR of fruA and levD, an effect likely due to its demonstrated role in repression of manL expression. Thus, there are some similarities and fundamental differences in CCR control mechanisms between the oral pathogen S. mutans and the oral commensal S. gordonii that may eventually be exploited to enhance the competitiveness of health-associated commensals in oral biofilms.
PMCID: PMC3067331  PMID: 21239541
Molecular microbiology  2008;70(1):197-208.
Streptococcus mutans is particularly well-adapted for high-affinity, high-capacity catabolism of multiple carbohydrate sources. S. mutans EIILev, a fructose/mannose permease encoded by the levDEFG genes, and fruA, which encodes a hydrolase that releases fructose from fructan polymers, are transcriptionally regulated by the LevQRST four-component signal transduction system. Here, we demonstrate that (1) levDEFGX are co-transcribed and the levE/F intergenic region is required for optimal expression of levFGX; (2) D-mannose is a potent inducer of the levD and fruA operons; (3) CcpA regulates levD expression in a carbohydrate-specific manner; (4) deletion of the genes for the fructose/mannose-EII enzymes of S. mutans (manL, fruI, and levD) enhances levD expression; (5) repression of the LevQRST regulon by EII enzymes depends on the presence of their substrates and requires LevR, but not LevQST; and (6) CcpA inhibits expression of the manL and frul genes to indirectly control the LevQRST regulon. Further, the manL, ccpA, frul/fruCD and levD gene products differentially exert control over the cellobiose and lactose operons. Collectively, the results reveal the existence of a global regulatory network in S. mutans that governs the utilization of non-preferred carbohydrates in response to the availability and source of multiple preferred carbohydrates.
PMCID: PMC2583961  PMID: 18699864
Sugar:phosphotransferase system; β-D-fructosidase; Catabolite repression; CcpA; Gene regulation
15.  A Combination of Independent Transcriptional Regulators Shapes Bacterial Virulence Gene Expression during Infection 
PLoS Pathogens  2010;6(3):e1000817.
Transcriptional regulatory networks are fundamental to how microbes alter gene expression in response to environmental stimuli, thereby playing a critical role in bacterial pathogenesis. However, understanding how bacterial transcriptional regulatory networks function during host-pathogen interaction is limited. Recent studies in group A Streptococcus (GAS) suggested that the transcriptional regulator catabolite control protein A (CcpA) influences many of the same genes as the control of virulence (CovRS) two-component gene regulatory system. To provide new information about the CcpA and CovRS networks, we compared the CcpA and CovR transcriptomes in a serotype M1 GAS strain. The transcript levels of several of the same genes encoding virulence factors and proteins involved in basic metabolic processes were affected in both ΔccpA and ΔcovR isogenic mutant strains. Recombinant CcpA and CovR bound with high-affinity to the promoter regions of several co-regulated genes, including those encoding proteins involved in carbohydrate and amino acid metabolism. Compared to the wild-type parental strain, ΔccpA and ΔcovRΔccpA isogenic mutant strains were significantly less virulent in a mouse myositis model. Inactivation of CcpA and CovR alone and in combination led to significant alterations in the transcript levels of several key GAS virulence factor encoding genes during infection. Importantly, the transcript level alterations in the ΔccpA and ΔcovRΔccpA isogenic mutant strains observed during infection were distinct from those occurring during growth in laboratory medium. These data provide new knowledge regarding the molecular mechanisms by which pathogenic bacteria respond to environmental signals to regulate virulence factor production and basic metabolic processes during infection.
Author Summary
Group A Streptococcus (GAS) causes diverse infections in humans ranging from pharyngitis (strep throat) to necrotizing fasciitis (the flesh-eating disease). It is well known that GAS secretes a broad array of virulence factors that are critical to its ability to cause human infections, but how GAS coordinates virulence factor production during infection is poorly understood. We discovered that two GAS proteins, catabolite control protein A (CcpA) and control of virulence regulator (CovR), regulate production of many of the same virulence factor encoding genes, indicating that GAS uses these two regulatory proteins to modulate virulence factor production in response to environmental stimuli. We determined that CcpA and CovR are able to bind to DNA from co-regulated genes, indicating that the proteins control gene expression by directly interacting with DNA. Using a mouse model of muscle infection, we found that CcpA and CovR, alone and in combination, are critical to the ability of GAS to regulate expression of virulence factor encoding genes during infection. These findings increase understanding regarding the regulatory mechanisms critical to the ability of bacterial pathogens to cause infection.
PMCID: PMC2841617  PMID: 20333240
16.  Interference of Components of the Phosphoenolpyruvate Phosphotransferase System with the Central Virulence Gene Regulator PrfA of Listeria monocytogenes▿  
Journal of Bacteriology  2006;189(2):473-490.
Analysis of Listeria monocytogenes ptsH, hprK, and ccpA mutants defective in carbon catabolite repression (CCR) control revealed significant alterations in the expression of PrfA-dependent genes. The hprK mutant showed high up-regulation of PrfA-dependent virulence genes upon growth in glucose-containing medium whereas expression of these genes was even slightly down-regulated in the ccpA mutant compared to the wild-type strain. The ptsH mutant could only grow in a rich culture medium, and here the PrfA-dependent genes were up-regulated as in the hprK mutant. As expected, HPr-Ser-P was not produced in the hprK and ptsH mutants and synthesized at a similar level in the ccpA mutant as in the wild-type strain. However, no direct correlation was found between the level of HPr-Ser-P or HPr-His-P and PrfA activity when L. monocytogenes was grown in minimal medium with different phosphotransferase system (PTS) carbohydrates. Comparison of the transcript profiles of the hprK and ccpA mutants with that of the wild-type strain indicates that the up-regulation of the PrfA-dependent virulence genes in the hprK mutant correlates with the down-regulation of genes known to be controlled by the efficiency of PTS-mediated glucose transport. Furthermore, growth in the presence of the non-PTS substrate glycerol results in high PrfA activity. These data suggest that it is not the component(s) of the CCR or the common PTS pathway but, rather, the component(s) of subsequent steps that seem to be involved in the modulation of PrfA activity.
PMCID: PMC1797385  PMID: 17085572
17.  Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus 
BMC Microbiology  2009;9:95.
The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram-positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in Staphylococcus aureus, focussing on short-time effects of glucose under stable pH conditions.
The addition of glucose to exponentially growing S. aureus increased the expression of genes and enzymes of the glycolytic pathway, while genes and proteins of the tricarboxylic acid (TCA) cycle, required for the complete oxidation of glucose, were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetyl-CoA to acetate with a concomitant substrate-level phosphorylation, were neither regulated by glucose nor by CcpA. CcpA directly repressed genes involved in utilization of amino acids as secondary carbon sources. Interestingly, the expression of a larger number of genes was found to be affected by ccpA inactivation in the absence of glucose than after glucose addition, suggesting that glucose-independent effects due to CcpA may have a particular impact in S. aureus. In the presence of glucose, CcpA was found to regulate the expression of genes involved in metabolism, but also that of genes coding for virulence determinants.
This study describes the CcpA regulon of exponentially growing S. aureus cells. As in other bacteria, CcpA of S. aureus seems to control a large regulon that comprises metabolic genes as well as virulence determinants that are affected in their expression by CcpA in a glucose-dependent as well as -independent manner.
PMCID: PMC2697999  PMID: 19450265
18.  CcpA represses the expression of the divergent cit operons of Enterococcus faecalis through multiple cre sites 
BMC Microbiology  2011;11:227.
In Enterococcus faecalis the genes encoding the enzymes involved in citrate metabolism are organized in two divergent operons, citHO and oadHDB-citCDEFX-oadA-citMG (citCL locus). Expression of both operons is specifically activated by adding citrate to the medium. This activation is mediated by binding of the GntR-like transcriptional regulator (CitO) to the cis-acting sequences located in the cit intergenic region. Early studies indicated that citrate and glucose could not be co-metabolized suggesting some form of catabolite repression, however the molecular mechanism remained unknown.
In this study, we observed that the citHO promoter is repressed in the presence of sugars transported by the Phosphoenolpyruvate:carbohydrate Phosphotranserase System (PTS sugars). This result strongly suggested that Carbon Catabolic Repression (CCR) impedes the expression of the activator CitO and the subsequent induction of the cit pathway. In fact, we demonstrate that CCR is acting on both promoters. It is partially relieved in a ccpA-deficient E. faecalis strain indicating that a CcpA-independent mechanism is also involved in regulation of the two operons. Furthermore, sequence analysis of the citH/oadH intergenic region revealed the presence of three putative catabolite responsive elements (cre). We found that they are all active and able to bind the CcpA/P-Ser-HPr complex, which downregulates the expression of the cit operons. Systematic mutation of the CcpA/P-Ser-HPr binding sites revealed that cre1 and cre2 contribute to citHO repression, while cre3 is involved in CCR of citCL
In conclusion, our study establishes that expression of the cit operons in E. faecalis is controlled by CCR via CcpA-dependent and -independent mechanisms.
PMCID: PMC3198936  PMID: 21989394
19.  Pleiotropic functions of catabolite control protein CcpA in Butanol-producing Clostridium acetobutylicum 
BMC Genomics  2012;13:349.
Clostridium acetobutylicum has been used to produce butanol in industry. Catabolite control protein A (CcpA), known to mediate carbon catabolite repression (CCR) in low GC gram-positive bacteria, has been identified and characterized in C. acetobutylicum by our previous work (Ren, C. et al. 2010, Metab Eng 12:446–54). To further dissect its regulatory function in C. acetobutylicum, CcpA was investigated using DNA microarray followed by phenotypic, genetic and biochemical validation.
CcpA controls not only genes in carbon metabolism, but also those genes in solvent production and sporulation of the life cycle in C. acetobutylicum: i) CcpA directly repressed transcription of genes related to transport and metabolism of non-preferred carbon sources such as d-xylose and l-arabinose, and activated expression of genes responsible for d-glucose PTS system; ii) CcpA is involved in positive regulation of the key solventogenic operon sol (adhE1-ctfA-ctfB) and negative regulation of acidogenic gene bukII; and iii) transcriptional alterations were observed for several sporulation-related genes upon ccpA inactivation, which may account for the lower sporulation efficiency in the mutant, suggesting CcpA may be necessary for efficient sporulation of C. acetobutylicum, an important trait adversely affecting the solvent productivity.
This study provided insights to the pleiotropic functions that CcpA displayed in butanol-producing C. acetobutylicum. The information could be valuable for further dissecting its pleiotropic regulatory mechanism in C. acetobutylicum, and for genetic modification in order to obtain more effective butanol-producing Clostridium strains.
PMCID: PMC3507653  PMID: 22846451
CcpA; Pleiotropic regulator; Acidogenesis and solventogenesis; Sporulation
20.  NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718 
Journal of Bacteriology  2015;197(20):3339-3353.
Clostridium perfringens type D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011, that BMC202, a nanI null mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due to nanI gene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions in codY and ccpA gene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A double codY ccpA null mutant produced even less ETX than a codY or ccpA single null mutant. CcpA bound directly to sequences upstream of the etx or codY start codon, and bioinformatics identified putative CcpA-binding cre sites immediately upstream of both the codY and etx start codons, suggesting possible direct CcpA regulatory effects. A ccpA mutation also decreased codY transcription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects on codY transcription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease.
IMPORTANCE Clostridium perfringens NanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators. Results obtained using single ccpA or codY null mutants and a ccpA codY double null mutant showed that codY and ccpA regulate ETX production independently of one another but that ccpA also affects codY transcription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulate etx transcription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producing C. perfringens strains, via CcpA and CodY, to upregulate ETX production and cause disease.
PMCID: PMC4573732  PMID: 26260460
21.  Distinct Time-Resolved Roles for Two Catabolite-Sensing Pathways during Streptococcus pyogenes Infection▿ †  
Infection and Immunity  2010;79(2):812-821.
Many Gram-positive pathogens link the expression of virulence genes to the presence of carbon source substrates using overlapping pathways for global control of carbon catabolite regulation. However, how these pathways are integrated to control the behavior of the transcriptome in time- and compartment-specific patterns is typically not well understood. In the present study, global transcriptome profiling was used to determine the extent to which glucose alters gene expression in Streptococcus pyogenes (group A streptococcus) and the contributions of the CcpA and LacD.1 catabolite control pathways to the regulation of this response in vitro. This analysis revealed that the expression of as many as 15% of the genes examined was regulated and that CcpA and LacD.1 together contribute to the regulation of 60% of this subset. However, numerous patterns were observed, including both CcpA- and LacD.1-specific and independent regulation, coregulation, and regulation of genes by these pathways independently of glucose. In addition, CcpA and LacD.1 had antagonistic effects on most coregulated genes. To resolve the roles of these regulators during infection, the expression of selected transcripts representative of different regulatory patterns was examined in a murine model of soft tissue infection. This revealed distinct patterns of misregulation with respect to time in CcpA− versus LacD.1− mutants. Taken together, these data support an important role for carbohydrate in the regulation of the transcriptome in tissue and suggest that the CcpA and LacD.1 pathways are organized to function at different times during the course of an infection.
PMCID: PMC3028826  PMID: 21098101
22.  Analysis of Catabolite Control Protein A-Dependent Repression in Staphylococcus xylosus by a Genomic Reporter Gene System 
Journal of Bacteriology  2001;183(2):580-586.
A single-copy reporter system for Staphylococcus xylosus has been developed, that uses a promoterless version of the endogenous β-galactosidase gene lacH as a reporter gene and that allows integration of promoters cloned in front of lacH into the lactose utilization gene cluster by homologous recombination. The system was applied to analyze carbon catabolite repression of S. xylosus promoters by the catabolite control protein CcpA. To test if lacH is a suitable reporter gene, β-galactosidase activities directed by two promoters known to be subject to CcpA regulation were measured. In these experiments, repression of the malRA maltose utilization operon promoter and autoregulation of the ccpA promoters were confirmed, proving the applicability of the system. Subsequently, putative CcpA operators, termed catabolite-responsive elements (cres), from promoter regions of several S. xylosus genes were tested for their ability to confer CcpA regulation upon a constitutive promoter, PvegII. For that purpose, cre sequences were placed at position +3 or +4 within the transcribed region of PvegII. Measurements of β-galactosidase activities in the presence or absence of glucose yielded repression ratios between two- and eightfold. Inactivation of ccpA completely abolished glucose-dependent regulation. Therefore, the tested cres functioned as operator sites for CcpA. With promoters exclusively regulated by CcpA, signal transduction leading to CcpA activation in S. xylosus was examined. Glucose-dependent regulation was measured in a set of isogenic mutants showing defects in genes encoding glucose kinase GlkA, glucose uptake protein GlcU, and HPr kinase HPrK. GlkA and GlcU deficiency diminished glucose-dependent CcpA-mediated repression, but loss of HPr kinase activity abolished regulation. These results clearly show that HPr kinase provides the essential signal to activate CcpA in S. xylosus. Glucose uptake protein GlcU and glucose kinase GlkA participate in activation, but they are not able to trigger CcpA-mediated regulation independently from HPr kinase.
PMCID: PMC94913  PMID: 11133951
23.  Functional Genomics Approach to Identifying Genes Required for Biofilm Development by Streptococcus mutans 
Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxSSm) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.
PMCID: PMC123778  PMID: 11872468
24.  CcpA Causes Repression of the phoPR Promoter through a Novel Transcription Start Site, PA6 
Journal of Bacteriology  2006;188(4):1266-1278.
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 PA6, a previously unidentified promoter positioned immediately upstream of the cre box. EσA was sufficient for transcription of PA6, which was repressed by CcpA in vitro. These studies showed direct repression by CcpA of a newly discovered EσA-responsive phoPR promoter that required either Hpr or Crh in vivo for direct binding to the putative consensus cre sequence located between PA6 and the five downstream promoters characterized previously.
PMCID: PMC1367233  PMID: 16452408
25.  Catabolite Repression and Activation in Bacillus subtilis: Dependency on CcpA, HPr, and HprK 
Journal of Bacteriology  2005;187(22):7826-7839.
Previous studies have suggested that the transcription factor CcpA, as well as the coeffectors HPr and Crh, both phosphorylated by the HprK kinase/phosphorylase, are primary mediators of catabolite repression and catabolite activation in Bacillus subtilis. We here report whole transcriptome analyses that characterize glucose-dependent gene expression in wild-type cells and in isogenic mutants lacking CcpA, HprK, or the HprK phosphorylatable serine in HPr. Binding site identification revealed which genes are likely to be primarily or secondarily regulated by CcpA. Most genes subject to CcpA-dependent regulation are regulated fully by HprK and partially by serine-phosphorylated HPr [HPr(Ser-P)]. A positive linear correlation was noted between the dependencies of catabolite-repressible gene expression on CcpA and HprK, but no such relationship was observed for catabolite-activated genes, suggesting that large numbers of the latter genes are not regulated by the CcpA-HPr(Ser-P) complex. Many genes that mediate nitrogen or phosphorus metabolism as well as those that function in stress responses proved to be subject to CcpA-dependent glucose control. While nitrogen-metabolic genes may be subject to either glucose repression or activation, depending on the gene, almost all glucose-responsive phosphorus-metabolic genes exhibit activation while almost all glucose-responsive stress genes show repression. These responses are discussed from physiological standpoints. These studies expand our appreciation of CcpA-mediated catabolite control and provide insight into potential interregulon control mechanisms in gram-positive bacteria.
PMCID: PMC1280314  PMID: 16267306

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