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

The folding of an 85-residue protein, the histidine-containing phosphocarrier protein HPr, has been studied using a variety of techniques including DSC, CD, ANS fluorescence, and NMR spectroscopy. In both kinetic and equilibrium experiments the unfolding of HPr can be adequately described as a two-state process which does not involve the accumulation of intermediates. Thermodynamic characterization of the native and the transition states has been achieved from both equilibrium and kinetic experiments. The heat capacity change from the denatured state to the transition state (3. 2 kJ mol-1 K-1) is half of the heat capacity difference between the native and denatured states (6.3 kJ mol-1 K-1), while the solvent accessibility of the transition state (0.36) indicates that its compactness is closer to that of the native than that of the denatured state. The high value for the change in heat capacity upon unfolding results in the observation of cold denaturation at moderate denaturant concentrations. Refolding from high denaturant concentrations is, however, slow. The rate constant of folding in water, (14.9 s-1), is small compared to that reported for other proteins of similar size under similar conditions. This indicates that very fast refolding is not a universal character of small globular proteins which fold in the absence of detectable intermediates.

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. Citrate is abundant in nature and a natural constituent of all living cells. Most bacteria have transport systems in the cytoplasmic membrane that mediate the uptake of citrate. Internalized citrate can be utilized as a carbon and energy source under aerobic as well as under anaerobic conditions. Under aerobic conditions citrate dissimilation occurs via the tricarboxylic acid cycle, while under anaerobic conditions three different fermentative pathways have been described (reviewed in reference 7). All known bacterial citrate transporters are secondary transporters that use the energy stored in electrochemical gradients of protons or sodium ions to drive uptake. Mechanistically, the transporters couple the uptake of citrate to the uptake of one or more protons or sodium ions (25,26, 27). A special case are the citrate transporters found in lactic acid bacteria that catalyze heterologous exchange of citrate and lactate (precursor-product exchange) (4, 19). These transporters are involved in secondary proton motive force generation by translocation of net negative charge into the cell (20). A similar precursor-product exchange mechanism has been proposed for CitT, a citrate transporter of Escherichia coli that is induced under anaerobic conditions (23).Citrate forms stable complexes with divalent metal ions. Most citrate transporters are inhibited by the addition of divalent cations because they do not recognize the metal-citrate complex (18,25,28). However, in strains of the genera Pseudomonas, Klebsiella, Citrobacter, and Bacillus citrate transporters have evolved that specifically recognize citrate in complex with a divalent metal ion (5, 13, 17). It is believed that these organisms take up complexed citrate because it is available as such in their habitat.To date, the best-studied system for metal-citrate transport is the Mg 2ϩ -dependent citrate transporter CitM of Bacillus subtilis (6). CitM is a proton motive force-driven secondary citrate transporter that is strictly dependent on the presence of Mg 2ϩ . Regulation of expression of the transporter is under strict control of the medium composition. Expression requires the presence of citrate in the medium that activates a twocomponent signal transduction pathway (9, 32) and is under control of catabolite repression by rapidly metabolized carbon sources like glucose, inositol, and succinate (30a). CitM belongs to a novel family of secondary transporters that contains only six known members, three of which are found in B. subtilis. One of these, termed CitH, was also shown to be a citrate transporter. Since uptake of citrate catalyzed by CitH was inhibited by the presence of Mg 2ϩ in the assay buffer, it w...

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 ␤-galactosidase reporter gene were constructed and integrated into the amyE site of B. subtilis to study catabolic effects on citM expression. In parallel with ␤-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؉