Impact of the Mg 2+ -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 Mg 2+ but is also capable of transporting citrate in complex with the heavy metal ions Zn 2+ , Ni 2+ and Co 2+ . We report on the impact of CitM activity on the toxicity of Zn 2+ , Ni 2+ and Co 2+ 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 Mg 2+ -citrate transporter was present in the membrane. It is demonstrated that the complex of Ni 2+ 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.

Cobalt toxicity in anaerobic granular sludge: influence of chemical speciation

“…Although not only the free cobalt species can be transported into a microbial cell (e.g. vitamin B 12 [8], Co-citrate [23] and siderophores [45] are directly taken up by the methanogenic cell), similar IC 50 values for all three measurement sets (standard deviation of only 22%) were obtained when taking only the free cobalt species (Co 2? ) concentration into account (Table 5).…”

“…Although the free metal concentration strongly depends on precipitation [19], sorption [41] and speciation [43], the free metal ion is the most important chemical species for bacterial uptake [15] and therefore is also responsible for metal toxicity [13]. The presence of organic complexing ligands such as citrate (that decrease the free metal concentration) clearly influences the cobalt and nickel toxicity to bacteria, as is the case with Pseudomonas aeruginosa [6] or Bacillus subtilis [23]. These toxicity models were, however, established and verified for natural waters and higher organisms such as freshwater algae Chlorella kesslerii [15], urchin larvae [27] or mussels (Mytilus galloprovincialis) [2].…”

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).…”

Adaptive Gene Expression in Bacillus subtilis Strains Deleted for tetL

“…Previous studies failed to clarify the basis for the extreme Co 2ϩ sensitivity of JC112 (51). The microarray experiments revealed up-regulation in JC112 of citM, which is capable of taking up citrate in complex with a number of divalent cations, including toxic divalent cations such as Co 2ϩ (31,33). We hypothesized that up-regulation of citM in JC112 could facilitate use of citrate to offset the reduced use of malate caused by the down-regulation of maeN, since the Spiz-KM medium used routinely in growth experiments contained both malate and citrate.…”