Jessica B. Warner

CcpA-Dependent Carbon Catabolite Repression in Bacteria

SUMMARY Carbon catabolite repression (CCR) by transcriptional regulators follows different mechanisms in gram-positive and gram-negative bacteria. In gram-positive bacteria, CcpA-dependent CCR is mediated by phosphorylation of the phosphoenolpyruvate:sugar phosphotransferase system intermediate HPr at a serine residue at the expense of ATP. The reaction is catalyzed by HPr kinase, which is activated by glycolytic intermediates. In this review, the distribution of CcpA-dependent CCR among bacteria is investigated by searching the public databases for homologues of HPr kinase and HPr-like proteins throughout the bacterial kingdom and by analyzing their properties. Homologues of HPr kinase are commonly observed in the phylum Firmicutes but are also found in the phyla Proteobacteria, Fusobacteria, Spirochaetes, and Chlorobi, suggesting that CcpA-dependent CCR is not restricted to gram-positive bacteria. In the α and β subdivisions of the Proteobacteria, the presence of HPr kinase appears to be common, while in the γ subdivision it is more of an exception. The genes coding for the HPr kinase homologues of the Proteobacteria are in a gene cluster together with an HPr-like protein, termed XPr, suggesting a functional relationship. Moreover, the XPr proteins contain the serine phosphorylation sequence motif. Remarkably, the analysis suggests a possible relation between CcpA-dependent gene regulation and the nitrogen regulation system (Ntr) found in the γ subdivision of the Proteobacteria. The relation is suggested by the clustering of CCR and Ntr components on the genome of members of the Proteobacteria and by the close phylogenetic relationship between XPr and NPr, the HPr-like protein in the Ntr system. In bacteria in the phylum Proteobacteria that contain HPr kinase and XPr, the latter may be at the center of a complex regulatory network involving both CCR and the Ntr system.

Complementary Metal Ion Specificity of the Metal-Citrate Transporters CitM and CitH of Bacillus subtilis

Citrate uptake in Bacillus subtilis is stimulated by a wide range of divalent metal ions. The metal ions were separated into two groups based on the expression pattern of the uptake system. The two groups correlated with the metal ion specificity of two homologous B. subtilis secondary citrate transporters, CitM and CitH, upon expression in Escherichia coli. CitM transported citrate in complex with Mg(2+), Ni(2+), Mn(2+), Co(2+), and Zn(2+) but not in complex with Ca(2+), Ba(2+), and Sr(2+). CitH transported citrate in complex with Ca(2+), Ba(2+), and Sr(2+) but not in complex with Mg(2+), Ni(2+), Mn(2+), Co(2+), and Zn(2+). Both transporters did not transport free citrate. Nevertheless, free citrate uptake could be demonstrated in B. subtilis, indicating the expression of at least a third citrate transporter, whose identity is not known. For both the CitM and CitH transporters it was demonstrated that the metal ion promoted citrate uptake and, vice versa, that citrate promoted uptake of the metal ion, indicating that the complex is the transported species. The results indicate that CitM and CitH are secondary transporters that transport complexes of divalent metal ions and citrate but with a complementary metal ion specificity. The potential physiological function of the two transporters is discussed.

Catabolite repression and induction of the Mg2+-citrate transporter CitM of Bacillus subtilis

In Bacillus subtilis the citM gene encodes the Mg(2+)-citrate transporter. A target site for carbon catabolite repression (cre site) is located upstream of citM. Fusions of the citM promoter region, including the cre sequence, to the beta-galactosidase reporter gene were constructed and integrated into the amyE site of B. subtilis to study catabolic effects on citM expression. In parallel with beta-galactosidase activity, the uptake of Ni(2+)-citrate in whole cells was measured to correlate citM promoter activity with the enzymatic activity of the CitM protein. In minimal media, CitM was only expressed when citrate was present. The presence of glucose in the medium completely repressed citM expression; repression was also observed in media containing glycerol, inositol, or succinate-glutamate. Studies with B. subtilis mutants defective in the catabolite repression components HPr, Crh, and CcpA showed that the repression exerted by all these medium components was mediated via the carbon catabolite repression system. During growth on inositol and succinate, the presence of glutamate strongly potentiated the repression of citM expression by glucose. A reasonable correlation between citM promoter activity and CitM transport activity was observed in this study, indicating that the Mg(2+)-citrate uptake activity of B. subtilis is mainly regulated at the transcriptional level.

Growth of Bacillus subtilis on citrate and isocitrate is supported by the Mg2+-citrate transporter CitM.

Bacillus subtilis 168 was assayed for its growth on tricarboxylic acid (TCA) cycle intermediates and related compounds as the sole carbon sources. Growth of the organism was supported by citrate, D-isocitrate, succinate, fumarate and L-malate, whereas no growth was observed in the presence of cis-aconitate,2-oxoglutarate, D-malate, oxaloacetate and tricarballylate. Growth of the organism on the tricarboxylates citrate and D-isocitrate required the presence of functional CitM, an Mg(2+)-citrate transporter, whereas its growth on succinate, fumarate and L-malate appeared to be CitM-independent. Interestingly, the naturally occurring enantiomer D-isocitrate was favoured over L-isocitrate by the organism. Like citrate, D-isocitrate was shown to be an inducer of citM expression in B. subtilis. The addition of 1 mM Mg(2+) to the growth medium improved growth of the organism on both citrate and D-isocitrate, suggesting that D-isocitrate was taken up by CitM in complex with divalent metal ions. Subsequently, the ability of CitM to transport D-isocitrate was demonstrated by competition experiments and by heterologous exchange in right-side-out membrane vesicles prepared from E. coli cells expressing citM. None of the other TCA cycle intermediates and related compounds tested were recognized by CitM. Uptake experiments using radioactive (63)Ni(2+) provided direct evidence that D-isocitrate is transported in complex with divalent metal ions.

A Crh‐specific function in carbon catabolite repression in Bacillus subtilis

Carbon catabolite repression in Bacillus subtilis is mediated by phosphorylation of the phosphoenolpyruvate:carbohydrate phosphotransferase system intermediate HPr at a serine residue catalyzed by HPr kinase. The orthologous protein Crh functions in a similar way, but, unlike HPr, it is not functional in carbohydrate uptake. A specific function for Crh is not known. The role of HPr and Crh in repressing the citM gene encoding the Mg(2+)-citrate transporter was investigated during growth of B. subtilis on different carbon sources. In glucose minimal medium, full repression was supported by both HPr and Crh. Strains deficient in Crh or the regulatory function of HPr revealed the same repression as the wild-type strain. In contrast, in a medium containing succinate and glutamate, repression was specifically mediated via Crh. Repression was relieved in the Crh-deficient strain, but still present in the HPr mutant strain. The data are the first demonstration of a Crh-specific function in B. subtilis and suggest a role for Crh in regulation of expression during growth on substrates other than carbohydrates.

Impact of the Mg2+-citrate transporter CitM on heavy metal toxicity in Bacillus subtilis

Abstract.Bacillus subtilis possesses a secondary transporter, CitM, that is specific for the complex of citrate and Mg2+ but is also capable of transporting citrate in complex with the heavy metal ions Zn2+, Ni2+ and Co2+. We report on the impact of CitM activity on the toxicity of Zn2+, Ni2+ and Co2+ in B. subtilis. In a citM deletion mutant or under conditions in which CitM is not expressed, the toxic effects of the metals were reduced by the presence of citrate in the medium. In contrast, the presence of citrate dramatically enhanced toxicity when the Mg2+-citrate transporter was present in the membrane. It is demonstrated that the complex of Ni2+ and citrate is transported into the cell and that the uptake is responsible for the enhanced toxicity. At toxic concentrations of the metal ions, the cultures adapted by developing tolerance against these ions. Tolerant cells isolated by exposure to one of the metal ions remained tolerant after growth in the absence of toxic metal ions and were cross-tolerant against the other two toxic ions. Tolerant strains were shown to contain point mutations in the citM gene, which resulted in premature termination of translation.

Transporters involved in uptake of di- and tricarboxylates in Bacillus subtilis

Di- and tricarboxylates found as intermediates in the tricarboxylic acid cycle can be utilized by many bacteria and serve as carbon and energy source under aerobic and anaerobic conditions. A prerequisite for metabolism is that the carboxylates are transported into the cells across the cytoplasmic membrane. Bacillus subtilis is able to metabolize many di- and tricarboxylates and in this overview the available data on all known and putative di- and tricarboxylate transporters in B. subtilis is summarized. The B. subtilis transporters, that are of the secondary type, are discussed in the context of the protein families to which they belong. Available data on biochemical characterization, regulation of gene expression and the physiological function is summarized. It is concluded that in B. subtilis multiple transporters are present for tricarboxylic acid cycle intermediates.

CcpA-Independent Regulation of Expression of the Mg2+-Citrate Transporter Gene citM by Arginine Metabolism in Bacillus subtilis

Transcriptional regulation of the Mg(2+)-citrate transporter, CitM, the main citrate uptake system of Bacillus subtilis, was studied during growth in rich medium. Citrate in the growth medium was required for induction under all growth conditions. In Luria-Bertani medium containing citrate, citM expression was completely repressed during the exponential growth phase, marginally expressed in the transition phase, and highly expressed in the stationary growth phase. The repression was relieved when the cells were grown in spent Luria-Bertani medium. The addition of a mixture of 18 amino acids restored repression. L-Arginine in the mixture appeared to be solely responsible for the repression, and ornithine appeared to be an equally potent repressor of citM expression. Studies of mutant strains deficient in RocR and SigL, proteins required for the expression of the enzymes of the arginase pathway, confirmed that uptake into the cell and, most likely, conversion of arginine to ornithine were required for repression. Arginine-mediated repression was independent of a functional CcpA, the global regulator protein in carbon catabolite repression (CCR). Nevertheless, CCR-mediated repression was the major mechanism controlling the expression during exponential growth, while the newly described, CcpA-independent arginine-mediated repression was specifically apparent during the transition phase of growth.