CcpA-mediated Carbon Catabolite Repression Research Articles

Elimination of carbon catabolite repression in Clostridium tyrobutyricum for enhanced butyric acid production from lignocellulosic hydrolysates

Clostridium tyrobutyricum, a gram-positive anaerobic bacterium, is recognized as the promising butyric acid producer. But, the existence of carbon catabolite repression (CCR) is the major drawback for C. tyrobutyricum to efficiently use the lignocellulosic biomass. In this study, the xylose pathway genes were first identified and verified. Then, the potential regulatory mechanisms of CCR in C. tyrobutyricum were proposed and the predicted engineering targets were experimental validated. Inactivation of hprK blocked the CcpA-mediated CCR and resulted in simultaneous conversion of glucose and xylose, although xylose consumption was severe lagging behind. Deletion of xylR further shortened the lag phase of xylose utilization. When hprK and xylR were inactivated together, the CCR in C. tyrobutyricum was completely eliminated. Consequently, ATCC 25755/ΔhprKΔxylR showed significant increase in butyrate productivity (1.8 times faster than the control) and excellent butyric acid fermentation performance using both mixed sugars (11.0–11.9 g/L) and undetoxified lignocellulosic hydrolysates (12.4–13.4 g/L).

Staphylococcus aureus Does Not Synthesize Arginine from Proline under Physiological Conditions.

The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or proC deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of proC, it inhibited the growth of the ccpA deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway. IMPORTANCE Staphylococcus aureus is a versatile Gram-positive human pathogen infecting various human organs. The bacterium's versatility is partly due to efficient metabolic regulation via the carbon catabolite repression system (CCR). S. aureus is known to interconvert proline and arginine, and CCR represses the synthesis of both amino acids. However, when CCR is released by a nonpreferred carbon source, S. aureus can synthesize proline but not arginine. In this study, we show that, in S. aureus, the intracellular concentration of pyrroline-5-carboxylate (P5C), the degradation product of proline and the substrate of proline synthesis, is too low to synthesize arginine from proline. These results call into question the notion that S. aureus synthesizes arginine from proline.

Transcriptome profile of carbon catabolite repression in an efficient l-(+)-lactic acid-producing bacterium Enterococcus mundtii QU25 grown in media with combinations of cellobiose, xylose, and glucose.

Enterococcus mundtii QU25, a non-dairy lactic acid bacterium of the phylum Firmicutes, is capable of simultaneously fermenting cellobiose and xylose, and is described as a promising strain for the industrial production of optically pure l-lactic acid (≥ 99.9%) via homo-fermentation of lignocellulosic hydrolysates. Generally, Firmicutes bacteria show preferential consumption of sugar (usually glucose), termed carbon catabolite repression (CCR), while hampering the catabolism of other sugars. In our previous study, QU25 exhibited apparent CCR in a glucose-xylose mixture phenotypically, and transcriptional repression of the xylose operon encoding initial xylose metabolism genes, likely occurred in a CcpA-dependent manner. QU25 did not exhibit CCR phenotypically in a cellobiose-xylose mixture. The aim of the current study is to elucidate the transcriptional change associated with the simultaneous utilization of cellobiose and xylose. To this end, we performed RNA-seq analysis in the exponential growth phase of E. mundtii QU25 cells grown in glucose, cellobiose, and/or xylose as either sole or co-carbon sources. Our transcriptomic data showed that the xylose operon was weakly repressed in cells grown in a cellobiose-xylose mixture compared with that in cells grown in a glucose-xylose mixture. Furthermore, the gene expression of talC, the sole gene encoding transaldolase, is expected to be repressed by CcpA-mediated CCR. QU25 metabolized xylose without using transaldolase, which is necessary for homolactic fermentation from pentoses using the pentose-phosphate pathway. Hence, the metabolism of xylose in the presence of cellobiose by QU25 may have been due to 1) sufficient amounts of proteins encoded by the xylose operon genes for xylose metabolism despite of the slight repression of the operon, and 2) bypassing of the pentose-phosphate pathway without the TalC activity. Accordingly, we have determined the targets of genetic modification in QU25 to metabolize cellobiose, xylose and glucose simultaneously for application of the lactic fermentation from lignocellulosic hydrolysates.

Surfactin: A Quorum-Sensing Signal Molecule to Relieve CCR in Bacillus amyloliquefaciens.

Bacillus utilize preferred sugars such as glucose over other carbon sources due to carbon catabolite repression (CCR). Surfactin is a small signal molecule to regulate the quorum-sensing (QS) response such as biofilm formation and sporulation in B. subtilis. Here, the srfA operon for synthesis of surfactin was mutated for disrupting the production of surfactin in B. amyloliquefaciens. The srfA-mutant strain showed a defective biofilm and sporulation but could be restored by addition with surfactin, indicating that surfactin is a QS signal molecule in B. amyloliquefaciens. Unexpectedly, mutation of srfA also led to the cells’ death although nutrients were still enough to support the bacterial growth during this period. Analysis of transcriptomes found that the srfA-mutant strain could not relieve CCR to use non-preferred carbon sources after glucose exhaustion due to deficiency of surfactin. This was further verified by the fact that addition with glucose could dramatically restore the growth, and addition with surfactin could improve the enzymes’ activity (e.g., glucanase and α-amylase) to use non-preferred carbon sources in the srfA-mutant strain. After glucose exhaustion, the cells produce surfactin to relieve CCR for utilizing non-preferred sugars. As a signal molecule to regulate QS, surfactin also directly or indirectly relieves the CcpA-mediated CCR to utilize non-preferred carbon sources countering nutrient limitation (e.g., glucose deprivation) in the environment. In conclusion, our findings provide the first evidence that the QS signal molecule of surfactin is also involved in relieving the CcpA-mediated CCR in B. amyloliquefaciens.

Expression of the Lactobacillus plantarum malE gene is regulated by CcpA and a MalR-like protein

Lactobacillus plantarum is commonly used in the food industry as a starter in various fermentations, especially in vegetable fermentations, in which starch is a common substrate. This polysaccharide, which is obtained from potatoes or corn and is hydrolysed mainly to maltose and glucose by acids or enzymes, is commercially used for the production of lactate by lactic acid fermentation. In this study, we describe the regulation of malE gene expression in L. plantarum. This gene, located in a 7-gene cluster, probably organized in an operon, encodes a putative maltose/maltodextrin-binding protein. We studied the expression of malE in L. plantarum LM3 (wild type) and in LM3-2 (ccpA1), which carries a null mutation in the ccpA gene, encoding the catabolite control protein A (CcpA). In the presence of glucose, expression of the MalE protein was higher in the mutant strain as compared to that in the wild-type strain. Transcription of the malE gene was induced by maltose and regulated by a CcpA-mediated carbon catabolite repression. Further, we isolated strains carrying mutations in 2 genes, lp_0172 and lp_0173, whose deduced amino acid sequences share significant identity with MalR, a regulator of the maltose operon in several gram-positive bacteria. A double mutant exhibited glucose-insensitive malE transcription, while absence of the functional Lp_0172 open reading frame had no effect on malE expression.

Comparative proteome analysis of alkaliphilic Bacillus sp. N16-5 grown on different carbon sources

To determine the impact of carbohydrates on the metabolic pathway in alkaliphiles, proteomes were obtained from cultures containing different carbohydrates and were resolved on two-dimensional gel electrophoresis (2-DE). The proteomes were compared to determine differentially expressed proteins. A novel alkaliphilic bacterium (alkaliphilic Bacillus sp. N16-5 isolated from Wudunur Soda Lake, China) was isolated in media with five different carbon sources (glucose, mannose, galactose, arabinose, and xylose). Comparative proteome analysis identified 61 differentially expressed proteins, which were mainly involved in carbohydrate metabolism, amino acid transport, and metabolism, as well as energy production and conversion. The comparison was based on the draft genome sequence of strain N16-5. The abundance of enzymes involved in central metabolism was significantly changed when exposed to various carbohydrates. Notably, catabolite control protein A (CcpA) was up-regulated under all carbon sources compared with glucose. In addition, pentose exhibited a stronger effect than hexose in CcpA-mediated carbon catabolite repression. These results provided a fundamental understanding of carbohydrate metabolism in alkaliphiles.

Carbon Catabolite Repression in Bacillus subtilis : Quantitative Analysis of Repression Exerted by Different Carbon Sources

In many bacteria glucose is the preferred carbon source and represses the utilization of secondary substrates. In Bacillus subtilis, this carbon catabolite repression (CCR) is achieved by the global transcription regulator CcpA, whose activity is triggered by the availability of its phosphorylated cofactors, HPr(Ser46-P) and Crh(Ser46-P). Phosphorylation of these proteins is catalyzed by the metabolite-controlled kinase HPrK/P. Recent studies have focused on glucose as a repressing substrate. Here, we show that many carbohydrates cause CCR. The substrates form a hierarchy in their ability to exert repression via the CcpA-mediated CCR pathway. Of the two cofactors, HPr is sufficient for complete CCR. In contrast, Crh cannot substitute for HPr on substrates that cause a strong repression. Determination of the phosphorylation state of HPr in vivo revealed a correlation between the strength of repression and the degree of phosphorylation of HPr at Ser46. Sugars transported by the phosphotransferase system (PTS) cause the strongest repression. However, the phosphorylation state of HPr at its His15 residue and PTS transport activity have no impact on the global CCR mechanism, which is a major difference compared to the mechanism operative in Escherichia coli. Our data suggest that the hierarchy in CCR exerted by the different substrates is exclusively determined by the activity of HPrK/P.

CcpA-Mediated Repression of Streptolysin S Expression and Virulence in the Group A Streptococcus

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 DeltaccpA 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 DeltaccpA 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.