Characterization of metal-resistant soil eubacteria by polymerase chain reaction--denaturing gradient gel electrophoresis with isolation of resistant strains.

Contamination of soils with heavy metal ions is a major problem on industrial and defense-related sites worldwide. The bioavailability and mobility of these contaminants is partially determined by the microbial biomass present at these sites. In this study, we have assessed the effect of the addition of a mixture of toxic metal salts on the prokaryotic community of microcosms consisting of sandy-loam soil using direct molecular analysis of the recoverable eubacterial 16S rDNA molecules by polymerase chain reaction--denaturing gradient gel electrophoresis (PCR-DGGE) and limited phospholipid fatty acid analysis (PLFA). Addition of toxic metals (nonradioactive surrogates of Sr, Co, Cs, Cd) resulted in rapid (ca. 1 week) changes in the DGGE profile of the indigenous eubacterial community when compared with pristine controls. These changes were stable over the course of the experiment (8 weeks). No changes in the eubacterial population of control microcosms were detected. The major changes in community structure in metal-contaminated microcosms consisted of the appearance of four novel bands not detected in controls. Sequence analysis of these bands suggested that two organisms related to the genus Acinetobacter and two related to the genus Burkholderia carried a selective advantage over other indigenous eubacteria under heavy metal induced stress. The Burkholderia spp. were then cultured and further characterized using lipid analysis.