Catabolite repression and induction of the Mg(2+)-citrate transporter CitM of Bacillus subtilis

Abstract

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.

FIG. 1

Genetic organization of the citM gene on the B. subtilis chromosome. Arrows, direction of transcription; loops, transcription termination sites. citS and citT code for the two-component signal transduction system, citM codes for the secondary transporter of the Mg²⁺-citrate complex, and yflP and yflN code for unknown proteins. The genes are drawn to scale. Below is shown the alignment of the cre site located in the CitM promoter region and the consensus sequence as described previously (48). The citM cre site is centered at −24 bp relative to the start codon of the citM gene. Symbols for nucleotides in the consensus sequence: W, A or T; R, A or G; Y, C or T; N, A, G, C, or T.

FIG. 2

Ni²⁺-citrate uptake and citM promoter activity. Uptake of [1,5-¹⁴C]citrate in whole cells of B. subtilis 168 grown in CSE medium without further additions (□, CSE), with 10 mM citrate (○, CSEC), and with 10 mM citrate plus 10 mM glucose (▵, CSECG). (Inset) β-Galactosidase activity (in Miller units [MU]) of B. subtilis CM002 carrying the lacZ gene under the control of the citM promoter grown in the same media.

FIG. 3

Effect of different growth substrates on transport and promoter activities. B. subtilis strains 168 and CM002 were grown in C minimal medium with 10 mM citrate in the presence of no further additions (none), glucose (Gluc), glycerol (Gly), inositol (I), inositol and glutamate (IE), and succinate and glutamate (SE). Open bars, initial rates of uptake of [1,5-¹⁴C]citrate in the presence of 1 mM NiCl₂ by whole cells of B. subtilis 168; solid bars, β-galactosidase activity of B. subtilis CM002 grown in the different media in Miller units (MU).

FIG. 4

citM promoter activity of CCR mutants grown in CI medium. β-Galactosidase activities of B. subtilis strains CM002 (wt), CM004 (HPr), CM006 (Crh), CM008 (HPr/Crh), and CM010 (CcpA) grown in CI medium supplemented with citrate (black bars) and citrate plus glucose (gray bars) are shown. All strains carry the PcitM-lacZ fusion integrated in the amyE locus. (Inset) enlarged part of the graph.