Secondary transporters for citrate and the Mg(2+)-citrate complex in Bacillus subtilis are homologous proteins.

Citrate uptake in Bacillus subtilis is mediated by a secondary transporter that transports the complex of citrate and divalent metal ions. The gene coding for the transporter termed CitM was cloned, sequenced, and functionally expressed in Escherichia coli. Translation of the base sequence to the primary sequence revealed a transporter that is not homologous to any known secondary transporter. However, CitM shares 60% sequence identity with the gene product of open reading frame N15CR that is on the genome of B. subtilis and for which no function is known. The hydropathy profiles of the primary sequences of CitM and the unknown gene product are very similar, and secondary structure prediction algorithms predict 12 transmembrane-spanning segments for both proteins. Open reading frame N15CR was cloned and expressed in E. coli and was shown to be a citrate transporter as well. The transporter is termed CitH. A remarkable difference between the two transporters is that citrate uptake by CitM is stimulated by the presence of Mg2+ ions, while citrate uptake by CitH is inhibited by Mg2+. It is concluded that the substrate of CitM is the Mg(2+)-citrate complex and that CitH transports the free citrate anion. Uptake experiments in right-side-out membrane vesicles derived from E. coli cells expressing either CitM or CitH showed that both transporters catalyze electrogenic proton/substrate symport.     

Catabolite repression of the Bacillus subtilis gnt operon exerted by two catabolite-responsive elements

Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting catabolite-responsive element (cre) by a complex of CcpA, which is a member of the GalR-LacI family of bacterial regulatory proteins, and the seryl-phos-phorylated form of HPr (P-ser-HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the ere in the first gene (gntR) of the gnt operon (cre_down), this operon contains another ere located in the promoter region (cre_up). A translational gntR-lacZ fusion expressed under the control of various combinations of wild-type and mutant cre_down and cre_up was integrated into the chromosomal amyE locus, and then catabolite repression of p-galac-tosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by cre_up was probably independent of catabolite repression exerted by cre_down; both creup and cre_down catabolite repression involved CcpA. Catabolite repression exerted by cre_up was independent of P-ser-HPr, and catabolite repression exerted by cre_down was partially independent of P-ser-HPr. DNase I footprinting experiments indicated that a complex of CcpA and P-ser-HPr did not recognize cre_up, in contrast to its specific recognition of cre_down. However, CcpA complexed with glucose-6-phosphate specifically recognized cre_up as well as cre_down, but the physiological significance of this complexing is unknown.     

Catabolite repression of the Bacillus subtilis gnt operon mediated by the CcpA protein.

Inducer exclusion was not important in catabolite repression of the Bacillus subtilis gnt operon. The CcpA protein (also known as AlsA) was found to be necessary for catabolite repression of the gnt operon, and a mutation (crsA47, which is an allele of the sigA gene) partially affected this catabolite repression.     

Catabolite repression vs derepression, an approach to differentiation during sporulation in Bacillus subtilis

Glutamine, like glucose, repressed sporulation and the synthesis of mycobacillin and dipicolinic acid by Bacillus subtilis , and these syntheses were derepressed by dibutyryl cyclic GMP but not by dibutyryl cyclic AMP. Neither of these dibutyryl cyclic nucleotides affected sporulation or a number of spore-associated parameters in the strain under normal physiological conditions. Mutants insensitive to glutamine repression were indifferent to the addition of either of the dibutyryl cyclic nucleotides both in the presence and in the absence of glutamine. Sporulation resulted from the remission of repression obtained under the catabolically active state.     

Catabolite repression-resistant mutants of Bacillus subtilis.

Mutants of Bacillus subtilis that are able to sporulate under the condition of catabolite repression were isolated by a simple selection technique. The mutants used in the present study were able to grow normally on minimal medium with ammonium sulphate as the nitrogen source and glucose as the carbon source. Studies carried out with these mutants show that there is no close relation between catabolite repression of an inducible enzyme, acetoin dehydrogenase, and that of sporulation. Certain mutants are able to sporulate in the presence of all the carbon sources tested but some mutants are resistant only to the carbon source used in isolation. It is suggested that several metabolic steps may be affected in catabolite repression of sporulation.     

Catabolite repression vs derepression, an approach to differentiation during sporulation in Bacillus subtilis.

Glutamine, like glucose, repressed sporulation and the synthesis of mycobacillin and dipicolinic acid by Bacillus subtilis, and these syntheses were depressed by dibutyryl cyclic GMP but not by dibutyryl cyclic AMP. Neither of these dibutyryl cyclic nucleotides affected sporulation or a number of spore-associated parameters in the strain under normal physiological conditions. Mutants insensitive to glutamine repression were indifferent to the addition of either of the dibutyryl cyclic nucleotides both in the presence and in the absence of glutamine. Sporulation resulted from the remission of repression obtained under the catabolically active state.     

Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca(2+)-dipicolinate

Dormant Bacillus subtilis spores can be induced to germinate by nutrients, as well as by nonmetabolizable chemicals, such as a 1:1 chelate of Ca(2+) and dipicolinic acid (DPA). Nutrients bind receptors in the spore, and this binding triggers events in the spore core, including DPA excretion and rehydration, and also activates hydrolysis of the surrounding cortex through mechanisms that are largely unknown. As Ca(2+)-DPA does not require receptors to induce spore germination, we asked if this process utilizes other proteins, such as the putative cortex-lytic enzymes SleB and CwlJ, that are involved in nutrient-induced germination. We found that Ca(2+)-DPA triggers germination by first activating CwlJ-dependent cortex hydrolysis; this mechanism is different from nutrient-induced germination where cortex hydrolysis is not required for the early germination events in the spore core. Nevertheless, since nutrients can induce release of the spore's DPA before cortex hydrolysis, we examined if the DPA excreted from the core acts as a signal to activate CwlJ in the cortex. Indeed, endogenous DPA is required for nutrient-induced CwlJ activation and this requirement was partially remedied by exogenous Ca(2+)-DPA. Our findings thus define a mechanism for Ca(2+)-DPA-induced germination and also provide the first definitive evidence for a signaling pathway that activates cortex hydrolysis in response to nutrients.     

Catabolite repression during single and multiple induction inEscherichia coli

Intracellular concentration of cAMP regulates the synthesis of enzymes sensitive to catabolite repression. The relationship between the single and multiple induction of beta-galactosidase (EC 3.2.1.23), L-tryptophanase (EC 4.1.99.1), D-serine deaminase (EC 4.2.1.14), L-asparaginase (EC 3.5.1.1) and L-malate dehydrogenase (EC 1.1.1.37) was studied and the effect of cAMP level on the induction in Escherichia coli Crookes (ATCC 8739) was investigated. A varying degree of catabolite repression was observed during induction of individual enzymes induced separately on different energy sources. The synthesis of l-tryptophanase was most sensitive, whereas l-asparaginase was not influenced at all. Exogenous cAMP was found to overcome partially the catabolite repression of beta-galactosidase and D-serine deaminase, both during single induction. The synthesis of l-malate dehydrogenase was negatively influenced by the multiple induction even in the presence of cAMP; on the other hand, the synthesis of l-tryptophanase was stimulated, independently of the level of the exogenous cAMP. Similarly, the activity of L-asparaginase slightly but significantly increased during the multiple induction of all five enzymes; here too the activity increase did not depend on exogenous cAMP.     

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.