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Emodin was extracted from Rheum officinale Baill by ultrasound-assisted extraction (UAE), and ethanol was chosen as the suitable solvent through SEM and molecular dynamic simulation. Under the optimum conditions (power 541 W, time 23 min, liquid to material ratio 13:1 mL/g, ethanol concentration 83 %) predicted by RSM, the yield of emodin was 2.18 ± 0.11 mg/g. Moreover, ultrasound power and time displayed the significant effects on the extraction process. Extracting dynamics analysis indicated that the extraction process of emodin by UAE conformed to Fick's second diffusion law. The results of antibacterial experiments suggested that emodin can damage cell membrane and inhibit the expression of cps2A, sao, mrp, epf, neu and the hemolytic activity of S. suis. Biolayer interferometry and FT-IR multi-peak fitting assays demonstrated that emodin induced a secondary conformational shift in CcpA. Molecular docking and molecular dynamics confirmed that emodin bound to CcpA through hydrogen bonding (ALA248, GLU249, GLY129 and ASN196) and π-π T-shaped interaction (TYR225 and TYR130), and the mutation of amino acid residues affected the affinity of CcpA to emodin. Therefore, emodin inhibited the sugar utilization of S. suis through binding to CcpA, and CcpA may be a potential target to inhibit the growth of S. suis.

Catabolite control protein A (CcpA), an important global regulatory protein, is extensively found in S. aureus. Many studies have reported that CcpA plays a pivotal role in regulating the tricarboxylic acid cycle and pathogenicity. Moreover, the CcpA-knockout Staphylococcus aureus (S. aureus) in diabetic mice, compared with the wild-type, showed a reduced colonization rate in the tissues and organs and decreased inflammatory factor expression. However, the effect of CcpA-knockout S. aureus on the host's energy metabolism in a high-glucose environment and its mechanism of action remain unclear. S. aureus, a common and major human pathogen, is increasingly found in patients with obesity and diabetes, as recent clinical data reveal. To address this issue, we generated CcpA-knockout S. aureus strains with different genetic backgrounds to conduct in-depth investigations. In vitro experiments with high-glucose-treated cells and an in vivo model study with type 1 diabetic mice were used to evaluate the unknown effect of CcpA-knockout strains on both the glucose and lipid metabolism phenotypes of the host. We found that the strains caused an abnormal metabolic phenotype in type 1 diabetic mice, particularly in reducing random and fasting blood glucose and increasing triglyceride and fatty acid contents in the serum. In a high-glucose environment, CcpA-knockout S. aureus may activate the hepatic STAT5/PDK4 pathway and affect pyruvate utilization. An abnormal metabolic phenotype was thus observed in diabetic mice. Our findings provide a better understanding of the molecular mechanism of glucose and lipid metabolism disorders in diabetic patients infected with S. aureus. [ABSTRACT FROM AUTHOR]

As a potent, pleiotropic regulatory protein in Gram-positive bacteria, catabolite control protein A (CcpA) mediates the transcriptional control of carbohydrate metabolism in Streptococcus bovis, a lactate-producing bacterium that plays an essential role in rumen acidosis in dairy cows. Although the rumen uptake of carbohydrates is multi-substrate, the focus of S. bovis research thus far has been on the glucose. With the aid of gene deletion, whole-genome sequencing, and transcriptomics, we have unraveled the role of CcpA in carbohydrate metabolism, on the one hand, and acidosis, on the other, and we show that the S. bovis strain S1 encodes "Carbohydrate-Active Enzymes" and that ccpA deletion slows the organism's growth rate and modulates the organic acid fermentation pathways toward lower lactate, higher formate, and acetate in the maltose and cellobiose. Furthermore, this study revealed the different regulatory functions of the CcpA protein in rumen metabolism and acidosis. [ABSTRACT FROM AUTHOR]

Catabolite control protein A (CcpA), an important global regulatory protein, is extensively found in S. aureus. Many studies have reported that CcpA plays a pivotal role in regulating the tricarboxylic acid cycle and pathogenicity. Moreover, the CcpA-knockout Staphylococcus aureus (S. aureus) in diabetic mice, compared with the wild-type, showed a reduced colonization rate in the tissues and organs and decreased inflammatory factor expression. However, the effect of CcpA-knockout S. aureus on the host’s energy metabolism in a high-glucose environment and its mechanism of action remain unclear. S. aureus, a common and major human pathogen, is increasingly found in patients with obesity and diabetes, as recent clinical data reveal. To address this issue, we generated CcpA-knockout S. aureus strains with different genetic backgrounds to conduct in-depth investigations. In vitro experiments with high-glucose-treated cells and an in vivo model study with type 1 diabetic mice were used to evaluate the unknown effect of CcpA-knockout strains on both the glucose and lipid metabolism phenotypes of the host. We found that the strains caused an abnormal metabolic phenotype in type 1 diabetic mice, particularly in reducing random and fasting blood glucose and increasing triglyceride and fatty acid contents in the serum. In a high-glucose environment, CcpA-knockout S. aureus may activate the hepatic STAT5/PDK4 pathway and affect pyruvate utilization. An abnormal metabolic phenotype was thus observed in diabetic mice. Our findings provide a better understanding of the molecular mechanism of glucose and lipid metabolism disorders in diabetic patients infected with S. aureus.

Abstract Background Aerobic growth provides benefits in biomass yield and stress tolerance of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). Catabolite control protein A (CcpA) is a master regulator involved in the aerobic and anaerobic growth, metabolic production and stress response in L. bulgaricus, but its potential molecular mechanisms remains unclear. The aim of this study is to elucidate the role of CcpA in L. bulgaricus in aerobic growth at the proteomic perspective. Results The differential proteomic analysis was performed on the L. bulgaricus ATCC11842 and its ccpA inactivated mutant strain using iTRAQ technology. A total of 132 differentially expressed proteins were obtained, among which 58 were up-regulated and 74 were down-regulated. These proteins were mainly involved in the cellular stress response, carbohydrate and energy metabolism, amino acid transport and protein synthesis, genetic information processing. Moreover, inactivation of ccpA negatively affected the expression of key enzymes involved in glycolysis pathway, while it enhanced the expression of proteins related to the pyruvate pathway, supporting the conclusion that CcpA mediated the shift from homolactic fermentation to mixed acid fermentation in L. bulgaricus. Conclusions Overall, these results showed that the role of CcpA in L. bulgaricus as a pleiotropic regulator in aerobic metabolism and stress response. This proteomic analysis also provide new insights into the CcpA-mediated regulatory network of L. bulgaricus and potential strategies to improve the production of starter and probiotic cultures based on the metabolic engineering of global regulators.

Ruminants may suffer from rumen acidosis when fed with high-concentrate diets due to the higher proliferation and overproduction of lactate by Streptococcus bovis. The catabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase (ldh) and pyruvate formate-lyase (pfl) in S. bovis , but its role in response to different carbon concentrations remains unclear. To characterize the regulatory mechanisms of CcpA in S. bovis S1 at different levels of carbon, herein, we analyzed the transcriptomic and physiological characteristics of S. bovis S1 and its ccpA mutant strain grown in glucose-excess and glucose-limited conditions. A reduced growth rate and a shift in fermentation pattern from homofermentation to heterofermentation were observed under glucose-limited condition as compared to glucose-excess condition, in S. bovis S1. Additionally, the inactivation of ccpA significantly affected the growth and end metabolites in both conditions. For the glycolytic intermediate, fructose 1,6-bisphosphate (FBP), the concentration significantly reduced at lower glucose conditions; its concentration decreased significantly in the ccpA mutant strain. Transcriptomic results showed that about 46% of the total genes were differentially transcribed between the wild-type strain and ccpA mutant strain grown in glucose-excess conditions; while only 12% genes were differentially transcribed in glucose-limited conditions. Different glucose concentrations led to the differential expression of 38% genes in the wild-type strain, while only half of these were differentially expressed in the ccpA -knockout strain. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the substrate glucose concentration significantly affected the gene expression in histidine metabolism, nitrogen metabolism, and some carbohydrate metabolism pathways. The deletion of ccpA affected several genes involved in carbohydrate metabolism, such as glycolysis, pyruvate metabolism, fructose and mannose metabolism, as well as in fatty acid biosynthesis pathways in bacteria grown in glucose-excess conditions; this effect was attenuated under glucose-limited conditions. Overall, these findings provide new information on gene transcription and metabolic mechanisms associated with substrate glucose concentration and validate the important role of CcpA in the regulation of carbon metabolism in S. bovis S1 at differential glucose availability. [ABSTRACT FROM AUTHOR]

Ruminants may suffer from rumen acidosis when fed with high-concentrate diets due to the higher proliferation and overproduction of lactate by Streptococcus bovis. The catabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase (ldh) and pyruvate formate-lyase (pfl) in S. bovis, but its role in response to different carbon concentrations remains unclear. To characterize the regulatory mechanisms of CcpA in S. bovis S1 at different levels of carbon, herein, we analyzed the transcriptomic and physiological characteristics of S. bovis S1 and its ccpA mutant strain grown in glucose-excess and glucose-limited conditions. A reduced growth rate and a shift in fermentation pattern from homofermentation to heterofermentation were observed under glucose-limited condition as compared to glucose-excess condition, in S. bovis S1. Additionally, the inactivation of ccpA significantly affected the growth and end metabolites in both conditions. For the glycolytic intermediate, fructose 1,6-bisphosphate (FBP), the concentration significantly reduced at lower glucose conditions; its concentration decreased significantly in the ccpA mutant strain. Transcriptomic results showed that about 46% of the total genes were differentially transcribed between the wild-type strain and ccpA mutant strain grown in glucose-excess conditions; while only 12% genes were differentially transcribed in glucose-limited conditions. Different glucose concentrations led to the differential expression of 38% genes in the wild-type strain, while only half of these were differentially expressed in the ccpA-knockout strain. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the substrate glucose concentration significantly affected the gene expression in histidine metabolism, nitrogen metabolism, and some carbohydrate metabolism pathways. The deletion of ccpA affected several genes involved in carbohydrate metabolism, such as glycolysis, pyruvate metabolism, fructose and mannose metabolism, as well as in fatty acid biosynthesis pathways in bacteria grown in glucose-excess conditions; this effect was attenuated under glucose-limited conditions. Overall, these findings provide new information on gene transcription and metabolic mechanisms associated with substrate glucose concentration and validate the important role of CcpA in the regulation of carbon metabolism in S. bovis S1 at differential glucose availability.

Abstract Background The catabolite control protein A (CcpA) is a master regulator of many important cellular processes in Gram-positive bacteria. In Lactobacillus plantarum, CcpA directly or indirectly controls the transcription of a large number of genes that are involved in carbohydrate metabolism, aerobic and anaerobic growth, stress response and metabolite production, but its role in response to different carbon sources remains unclear. Results Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth phase of wild-type and ccpA mutant strains of L. plantarum ST-III using fructooligosaccharides (FOS) or glucose as the sole carbon source. The inactivation of ccpA significantly affected the growth and production of metabolites under both carbon sources. About 15% of the total genes were significantly altered between wild-type and ccpA strains grown on glucose and the value is deceased to 12% when these two strains were compared on FOS, while only 7% were obviously changed due to the loss of CcpA when comparing strains grown on glucose and FOS. Although most of the differentially expressed genes mediated by CcpA are glucose dependent, FOS can also induce carbon catabolite repression (CCR) through the CcpA pathway. Moreover, the inactivation of ccpA led to a transformation from homolactic fermentation to mixed fermentation under aerobic conditions. CcpA can control genes directly by binding in the regulatory region of the target genes (mixed fermentation), indirectly through local regulators (fatty acid biosynthesis), or have a double effect via direct and indirect regulation (FOS metabolism). Conclusion Overall, our results show that CcpA plays a central role in response to carbon source and availability of L. plantarum and provide new insights into the complex and extended regulatory network of lactic acid bacteria.

Lactiplantibacillus plantarum is frequently exposed to salt stress during industrial applications. Catabolite control protein (CcpA) controls the transcription of many genes, but its role in the response to salt stress remains unclear. In this study, we used transcriptome analyses to investigate differences in the logarithmic growth phases of Lactiplantibacillus plantarum ST-III and its ccpA-knockout mutant when grown with or without salt and glycine betaine (GB). The deletion of ccpA significantly affected bacterial growth under different conditions. Among the comparisons, the highest proportion of differentially expressed genes (64%) was observed in the comparison between the wild-type and ccpA mutant grown with NaCl, whereas the lowest proportion (6%) was observed in the comparison between the ccpA mutant strain cultures grown with NaCl alone or with GB together. Transcriptomic analyses showed that CcpA could regulate GB uptake, activate iron uptake, produce acetyl-CoA, and affect fatty acid composition to maintain membrane lipid homeostasis in the adaptation of high-salinity conditions. Conclusively, these results demonstrate the importance of CcpA as a master regulator of these processes in response to salt stress, and provide new insights into the complex regulatory network of lactic acid bacteria. Key points: • The absence of CcpA significantly affected growth of L. plantarum and its response to salt stress. • CcpA regulates compatible solutes absorption and ions transport to resist salt stress. • CcpA alters fatty acids composition to maintain membrane lipid homeostasis towards salt stress. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

Background: Aerobic growth provides benefits in biomass yield and stress tolerance of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). Catabolite control protein A (CcpA) is a master regulator involved in the aerobic and anaerobic growth, metabolic production and stress response in L. bulgaricus, but its potential molecular mechanisms remains unclear. The aim of this study is to elucidate the role of CcpA in L. bulgaricus in aerobic growth at the proteomic perspective. Results: The differential proteomic analysis was performed on the L. bulgaricus ATCC11842 and its ccpA inactivated mutant strain using iTRAQ technology. A total of 132 differentially expressed proteins were obtained, among which 58 were up-regulated and 74 were down-regulated. These proteins were mainly involved in the cellular stress response, carbohydrate and energy metabolism, amino acid transport and protein synthesis, genetic information processing. Moreover, inactivation of ccpA negatively affected the expression of key enzymes involved in glycolysis pathway, while it enhanced the expression of proteins related to the pyruvate pathway, supporting the conclusion that CcpA mediated the shift from homolactic fermentation to mixed acid fermentation in L. bulgaricus. Conclusions: Overall, these results showed that the role of CcpA in L. bulgaricus as a pleiotropic regulator in aerobic metabolism and stress response. This proteomic analysis also provide new insights into the CcpA-mediated regulatory network of L. bulgaricus and potential strategies to improve the production of starter and probiotic cultures based on the metabolic engineering of global regulators. [ABSTRACT FROM AUTHOR]

Emodin was extracted from Rheum officinale Baill by ultrasound-assisted extraction (UAE), and ethanol was chosen as the suitable solvent through SEM and molecular dynamic simulation. Under the optimum conditions (power 541 W, time 23 min, liquid to material ratio 13:1 mL/g, ethanol concentration 83 %) predicted by RSM, the yield of emodin was 2.18 ± 0.11 mg/g. Moreover, ultrasound power and time displayed the significant effects on the extraction process. Extracting dynamics analysis indicated that the extraction process of emodin by UAE conformed to Fick's second diffusion law. The results of antibacterial experiments suggested that emodin can damage cell membrane and inhibit the expression of cps2A, sao, mrp, epf, neu and the hemolytic activity of S. suis. Biolayer interferometry and FT-IR multi-peak fitting assays demonstrated that emodin induced a secondary conformational shift in CcpA. Molecular docking and molecular dynamics confirmed that emodin bound to CcpA through hydrogen bonding (ALA248, GLU249, GLY129 and ASN196) and π-π T-shaped interaction (TYR225 and TYR130), and the mutation of amino acid residues affected the affinity of CcpA to emodin. Therefore, emodin inhibited the sugar utilization of S. suis through binding to CcpA, and CcpA may be a potential target to inhibit the growth of S. suis. [ABSTRACT FROM AUTHOR]

Galactooligosaccharides (GOS) are documented prebiotic compounds, but knowledge of the metabolic and regulatory mechanisms of GOS utilization by lactic acid bacteria is still limited. Here we used transcriptome and physiological analyses to investigate the differences in the logarithmic growth phase of Lactobacillus plantarum and L. plantarum ΔccpA metabolizing GOS or glucose as the sole source of carbohydrate. In total, 489 genes (16%) were differentially transcribed in the wild-type L. plantarum grown on glucose and GOS and the value is decreased to 7% due to the loss of ccpA. Only 6% genes were differentially expressed when the wild-type and the ccpA mutant were compared on GOS. Transcriptome data revealed that the carbon sources significantly affected the expression of several genes, and some of the genes were mediated by CcpA. In particular, lac and gal gene clusters resembled the corresponding clusters in L. acidophilus NCFM that are involved in GOS metabolism, indicating that these clusters may be participating in GOS utilization. Moreover, reverse transcription-PCR analysis showed that GOS-related gene clusters were organized in five independent polycistronic units. In addition, many commonalities were found between fructooligosaccharides and GOS metabolism in L. plantarum, including differentially expressed genes involved in oligosaccharide metabolism, conversion of metabolites, and changes in fatty acid biosynthesis. Overall, our findings provide new information on gene transcription and the metabolic mechanism associated with GOS utilization, and confirm that CcpA plays an important role in carbon metabolism regulation in L. plantarum.

Fructooligosaccharides (FOSs) metabolism in Lactobacillus plantarum is controlled by two gene clusters, and the global regulator catabolite control protein A (CcpA) may be involved in the regulation. To understand the mechanism, this study focused on the regulation relationships of CcpA toward target genes and the binding effects on the catabolite responsive element (cre). First, reverse transcription-PCR analysis of the transcriptional organization of the FOS-related gene clusters showed that they were organized in three independent polycistronic units. Diauxic growth, hierarchical utilization of carbohydrates and repression of FOS-related genes were observed in cultures containing FOS and glucose, suggesting carbon catabolite repression (CCR) control in FOS utilization. Knockout of ccpA gene eliminated these phenomena, indicating the principal role of this gene in CCR of FOS metabolism. Furthermore, six potential cre sites for CcpA binding were predicted in the regions of putative promoters of the two clusters. Direct binding was confirmed by electrophoretic mobility shift assays in vitro and chromatin immunoprecipitation in vivo. The results of the above studies suggest that CcpA is a vital regulator of FOS metabolism in L. plantarum and that CcpA-dependent CCR regulates FOS metabolism through the direct binding of CcpA toward the cre sites in the promoter regions of FOS-related clusters.

As a potent, pleiotropic regulatory protein in Gram-positive bacteria, catabolite control protein A (CcpA) mediates the transcriptional control of carbohydrate metabolism in Streptococcus bovis , a lactate-producing bacterium that plays an essential role in rumen acidosis in dairy cows. Although the rumen uptake of carbohydrates is multi-substrate, the focus of S. bovis research thus far has been on the glucose. With the aid of gene deletion, whole-genome sequencing, and transcriptomics, we have unraveled the role of CcpA in carbohydrate metabolism, on the one hand, and acidosis, on the other, and we show that the S. bovis strain S1 encodes "Carbohydrate-Active Enzymes" and that ccpA deletion slows the organism's growth rate and modulates the organic acid fermentation pathways toward lower lactate, higher formate, and acetate in the maltose and cellobiose. Furthermore, this study revealed the different regulatory functions of the CcpA protein in rumen metabolism and acidosis.IMPORTANCEThis study is important as it illustrates the varying regulatory role of the Streptococcus bovis catabolite control protein A protein in carbohydrate metabolism and the onset of acidosis in dairy cattle., Competing Interests: The authors declare no conflict of interest.

The catabolite control protein A (ccpA) regulates the carbon metabolism in Streptococcus suis type 2 and has pleiotropic regulatory functions in bacterial virulence and transcription. The present study systematically investigated ccpA activity in Streptococcus suis type 2 using isobaric tag for relative and absolute quantification (iTRAQ) liquid chromatography-tandem mass spectrometry-based proteomics. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses demonstrated that ccpA is an important protein for the regulation of metabolism, virulence and immune pathways in Streptococcus suis type 2. The present study therefore expanded the current understanding of the effects of ccpA on virulence, metabolic regulation and transcription in Streptococcus suis type 2 and other important pathogens. [ABSTRACT FROM AUTHOR]

Catabolite control protein A (CcpA) serves a key function in the catabolism of Streptococcus suis serotype 2 (S. suis 2) by affecting the biological function and metabolic regulatory mechanisms of this bacterium. The aim of the present study was to identify variations in CcpA expression in S. suis 2 using gene expression profile analysis. Using sequencing and functional analysis, CcpA was demonstrated to play a regulatory role in the expression and regulation of virulence genes, carbon metabolism and immunoregulation in S. suis 2. Gene Ontology and Kyto Encyclopedia of Genes and Genomes analyses indicated that CcpA in S. suis 2 is involved in the regulation of multiple metabolic processes. Furthermore, combined analysis of the transcriptome and metabolite data suggested that metabolites varied due to the modulation of gene expression levels under the influence of CcpA regulation. In addition, metabolic network analysis indicated that CcpA impacted carbon metabolism to a certain extent. Therefore, the present study has provided a more comprehensive analysis of the role of CcpA in the metabolic regulation of S. suis 2, which may facilitate future investigation into this mechanism. Furthermore, the results of the present study provide a foundation for further research into the regulatory function of CcpA and associated metabolic pathways in S. suis 2. [ABSTRACT FROM AUTHOR]

The aim of this study was to identify genes involved in biofilm development in the probiotic lactic acid bacterium Lactobacillus plantarum. The ability of L. plantarum LM3 and of some derivative mutant strains to form biofilm has been investigated. Biofilm microtitre plate assays showed that L. plantarum LM3-2, carrying a null mutation in the ccpA gene, coding the CcpA master regulator, was partially impaired in biofilm production compared to wild type (LM3). Moreover, we found three genes in the L. plantarum genome, hereby named flmA, flmB, and flmC, whose deduced amino acid sequences show significant identity with the Streptococcus mutans BrpA (biofilm regulatory protein A). We investigated the role of FlmA, FlmB, and FlmC in biofilm formation by isolating strains carrying null mutations in the corresponding genes. Our results suggest involvement of the Flm proteins in biofilm development. Moreover, transcriptional studies show that expression of flmA, flmB, and flmC is under the control of CcpA. These results, together with the reduced ability of LM3-2 ( ccpA1) to form biofilm, strongly suggest a positive role of the master regulator CcpA in biofilm development. [ABSTRACT FROM AUTHOR]

Catabolite control protein A (CcpA) is the major transcriptional regulator in carbon catabolite repression in several Gram-positive bacteria. We attempted to characterize the role of a CcpA homologue of Streptococcus suis type 2 in sugar metabolism and virulence. Addition of glucose or sucrose to the defined medium significantly reduced the activity of raffinose-inducible α-galactosidase, cellobiose-inducible β-glucosidase, and maltose-inducible α-glucosidase of the wild-type strain by about 9, 4, and 2-3 fold, respectively. Deletion of ccpA substantially derepressed the effects of repressing sugars on α-galactosidase or β-glucosidase activity. The ccpA deletion mutant showed reduced expression of virulence genes sly and eno ( P<0.05), decreased adhesion to and invasion into endothelial cells ( P<0.05), and attenuated virulence to mice with significant reduction of death rate and bacterial burden in organs, as compared to the wild-type strain. Both the in vitro and in vivo defect phenotypes were reversible by ccpA complementation. Thus, this study shows that CcpA of S. suis type 2 plays an important role in carbon catabolite repression and virulence. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: The catabolite control protein A (CcpA) from Bacillus megaterium is a member of the bacterial repressor protein family GalR–LacI. CcpA functions as master transcriptional regulator of carbon catabolite repression/regulation in firmicutes. Here we present the crystal structure of full-length apo CcpA at 2.5 Å resolution from B. megaterium. The structure reveals the location of the helix–turn–helix domain as well as the hinge region, which were not visible due to their high flexibility in earlier crystallographic studies on CcpA molecules. The structure of the apo CcpA homodimer in the present form is in contrast to other reported structures for CcpA. [Copyright &y& Elsevier]

Crystal structure determination of catabolite control protein A (CcpA) at 2.6 Å resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. In the crystal packing of apo-CcpA, the protein–protein contacts between the N-terminal three-helix bundle and the core domain consisted of interactions between the homodimers that were similar to those between the corepressor protein HPr and the CcpA N-subdomain in the ternary DNA complex. In contrast to the DNA complex, the apo-CcpA structure reveals large subdomain movements in the core, resulting in a complete loss of contacts between the N-subdomains of the homodimer. [ABSTRACT FROM AUTHOR]

Sucrose utilization in Staphylococcus xylosus is dependent on two genes, scrA and scrB; encoding a PTS permease and a sucrose phosphate hydrolase, respectively. The genes are encoded on separate loci and are transcribed from two promoters, PscrA and PscrB, both of which are controlled by the repressor ScrR by binding to the operator sequences OA and OB. In the scrA promoter region, a catabolite-responsive element (cre), operator for the global catabolite control protein CcpA, is also present, but its contribution to scrA regulation has not been determined. Using an integrative promoter probe plasmid, the activities of the promoters PscrA and PscrB were determined under different growth conditions. Both promoters are induced by sucrose and induction is prevented when glucose is also present. Without a functional CcpA, glucose-mediated prevention of induction is lost, clearly demonstrating that CcpA ensures hierarchical sugar utilization with glucose as preferred substrate. Measurements of promoter activities in the absence of a functional ScrR repressor indicated that CcpA also acts upon the operators OA and OB, albeit not as efficiently as on the genuine cre in PscrA. Besides determining the choice of the carbon source, CcpA has a second effect on sucrose gene expression. When sucrose is the sole carbon source, sucrose catabolism activates carbon catabolite repression and CcpA prevents full induction of the sucrose utilization genes by partially repressing the scrA promoter. Thus, CcpA-dependent regulation serves as a built-in autoregulatory device to restrict sucrose uptake. [ABSTRACT FROM AUTHOR]

CcpA is the master regulator for carbon catabolite regulation in Bacillus subtilis and regulates more than 300 genes by repression or activation. To reveal the effects of different functional domains of CcpA on various regulatory modes, we compared the activities of CcpA point mutants in activation (alsS, ackA) and repression (xynP, gntR) . CcpA variants mutated at residues in the HPrSerP-binding region without allosteric functions are inactive. On the other hand, CcpA variants mutated at residues that change their conformation upon HPrSerP or CrhP binding regulate only ackA . Another set of mutants with alterations in the corepressor-binding region show glucose-independent regulation of xynP. The data presented here demonstrate the involvement of HPrSerP and/or CrhP in activation of ackA and alsS by CcpA . Furthermore, these data also indicate that activation and repression mediated by CcpA may utilize different conformational changes of the protein. [ABSTRACT FROM AUTHOR]

The article presents a study that evaluates the cloning and characterization of the HPr kinase/phosphorylase (HPr-K/P) gene from Bacillus stearothermophilus 236. The key regulator of carbon catabolite repression/activation (CCR/CCA) in most gram-positive bacteria was cloned. The nucleotide sequence of the Complementation experiments showed that the cloned HPr-K/P gene product was functionally active in the Bacillus stearothermophilus cells.

Examines the cloning and characterization of the catabolite control protein A gene from Bacillus stearothermophilus No. 236. Assessment of the nucleotide sequence of the ccpA gene; Production of the CcpA protein belonging to the LacI/GaIR family of transcriptional regulators by a recombinant Escherichia coli strain; Deduction of the amino acid sequence.

Catabolite control protein A (CcpA) of the human pathogen Staphylococcus aureus is an essential DNA regulator for carbon catabolite repression and virulence, which facilitates bacterial survival and adaptation to a changing environment. Here, we report that copper (II) signaling mediates the DNA-binding capability of CcpA in vitro and in vivo. Copper (II) catalyzes the oxidation of two cysteine residues (Cys216 and Cys242) in CcpA to form intermolecular disulfide bonds between two CcpA dimers, which results in the formation and dissociation of a CcpA tetramer of CcpA from its cognate DNA promoter. We further demonstrate that the two cysteine residues on CcpA are important for S. aureus to resist host innate immunity, indicating that S. aureus CcpA senses the redox-active copper (II) ions as a natural signal to cope with environmental stress. Together, these findings reveal a novel regulatory mechanism for CcpA activity through copper (II)-mediated oxidation., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Catabolite control protein A (CcpA) serves a key function in the catabolism of Streptococcus suis serotype 2 ( S. suis 2 ) by affecting the biological function and metabolic regulatory mechanisms of this bacterium. The aim of the present study was to identify variations in CcpA expression in S. suis 2 using gene expression profile analysis. Using sequencing and functional analysis, CcpA was demonstrated to play a regulatory role in the expression and regulation of virulence genes, carbon metabolism and immunoregulation in S. suis 2 . Gene Ontology and Kyto Encyclopedia of Genes and Genomes analyses indicated that CcpA in S. suis 2 is involved in the regulation of multiple metabolic processes. Furthermore, combined analysis of the transcriptome and metabolite data suggested that metabolites varied due to the modulation of gene expression levels under the influence of CcpA regulation. In addition, metabolic network analysis indicated that CcpA impacted carbon metabolism to a certain extent. Therefore, the present study has provided a more comprehensive analysis of the role of CcpA in the metabolic regulation of S. suis 2 , which may facilitate future investigation into this mechanism. Furthermore, the results of the present study provide a foundation for further research into the regulatory function of CcpA and associated metabolic pathways in S. suis 2 .