Screening possible mechanisms mediating cadmium resistance in Rhizobium leguminosarum bv. viciae isolated from contaminated Portuguese soils

Environment heavy-metal contamination is now widespread. Soils may become contaminated from a variety of anthropogenic sources, such as smelters, mining, industry, and application of metal-containing pesticides and fertilizers. Soil microorganisms are very sensitive to moderate heavy-metal concentrations. Therefore, the present work was designed to screen possible mechanisms involved in Rhizobium's Cd resistance; with this purpose, we determined the tolerance levels of several isolates originated from sites with different heavy-metal contamination. Whole-cell-soluble proteins and plasmid profiles were analyzed. We also determined Cd cell concentrations and lipopolysaccharide (LPS) amounts. Results showed different tolerances among Rhizobium isolates; according to their maximum resistance level, isolates were divided in four groups: sensitive (0-125 μM CdCl₂), moderately tolerant (125-210 μM CdCl ₂), tolerant (250-500 μM CdCl₂), and extremely tolerant (≥750 μM CdCl₂). Intracellular Cd concentrations were lower when compared to wall-bound Cd. Unexpectedly, extremely tolerant isolates accumulated higher levels of metal, suggesting the presence of intracellular agents that prevent metal interfering with important metabolic pathways. The electrophoretic patterns of whole-cell-soluble proteins evidenced cadmium as an inducer of protein metabolism alterations, which were more evident in some polypeptides. Plasmid profiles also showed differences; most tolerant isolates presented two plasmids with molecular weights of 485 and 415 kb, indicating that extrachromosomal DNA may be involved in cadmium resistance. LPS showed to be a common mechanism of resistance. However, the degree of tolerance conferred by LPS is not enough to support tolerance to the higher levels of stress imposed. Presence of other resistance mechanisms is currently being investigated.