Functional Characterization of CitM, the Mg2+-Citrate Transporter

Abstract

The CitM transporter from Bacillus subtilis transports citrate as a complex with Mg2+. In this study, CitM was functionally expressed and characterized in E. coli DH5a cells. In the presence of saturating Mg2+ concentrations, the Km for citrate in CitM was 274 mM, similar to previous studies using whole cells of B. subtilis. CitM has a high substrate specificity for citrate. Other di- and tricarboxylic acids including succinate, isocitrate, cis-aconitate and tricarballylic acid did not significantly inhibit the uptake of citrate in the presence of Mg2+. However, CitM accepts complexes of citrate with metal ions other than Mg2+. The highest rate of citrate transport was seen in the presence of Mg2+, followed in order of preference by Mn2+, Ba2+, Ni2+, Co2+ and Ca2+. Citrate transport by CitM appears to be proton coupled. The transport was inhibited in transport buffers more alkaline than pH 7.5 and not affected by pH at acidic values. Transport was also inhibited by ionophores that affect the transmembrane proton gradient, including FCCP, TCC and nigericin. Valinomycin did not affect the uptake by CitM, suggesting that transport is electroneutral. In conclusion, the cloned CitM transporter from B. subtilis expressed in E. coli has properties similar to the transporter in intact B. subtilis cells. The results support a transport model with a coupling stoichiometry of one proton coupled to the uptake of one complex of (Mg2+-citrate)1-.

Functional characterization and Me2+ ion specificity of a Ca2+–citrate transporter from Enterococcus faecalis

“…Citrate transport by the gene product of EfcitH located in the citrate fermentation operon of E. faecalis ATCC29212 was demonstrated by comparing the uptake of [1,[5][6][7][8][9][10][11][12][13][14] C]citrate in right-side-out (RSO) membrane vesicles prepared from cells of Escherichia coli BL21 containing either pET-EfcitH or the control vector pET28b, both induced with 0.25 mm isopropyl b-d-thiogalactopyranoside. The membranes were energized using the artificial electron donor system ascorbate ⁄ phenazine methosulfate (see Experimental procedures).…”

“…Cells were diluted to an D 660 of 1 in 50 mm Pipes, pH 6.1, in a total volume of 100 lL, and equilibrated at 30°C. [1,[5][6][7][8][9][10][11][12][13][14] C]Citrate (114 mCiAEmmol )1 ; Amersham BioSciences) was added at a final concentration of 4.4 lm. Uptake was stopped by the addition of 2 mL ice-cold 0.1 m LiCl, followed by immediate filtration over cellulose nitrate filters (0.45 lm, pore size).…”

Assessment of the Requirements for Magnesium Transporters in Bacillus subtilis

“…For example, the citrate transporter CitM is known to transport divalent ions with citrate (31,32). Therefore, to examine the functionally significant routes of magnesium transport, we investigated the effects of the mutational disruption of all known putative B. subtilis magnesium transport proteins, including MgtE, YqxL, YfjQ, YloB, and CitM.…”

Citrate Utilization byCorynebacterium glutamicumIs Controlled by the CitAB Two-Component System through Positive Regulation of the Citrate Transport GenescitHandtctCBA

“…Aerobic citrate utilization by bacteria possessing a complete tricarboxylic acid cycle usually requires only a citrate uptake system. Presently, at least five families of citrate transporters in bacteria have been characterized according to the classification system introduced by Saier (44) (6,30,31); (iii) the citrate:cation symporter family (TC no. 2.A.24), represented by CitS and CitW of K. pneumoniae (24,41); (iv) the divalent anion:Na ϩ symporter family (TC no.…”