Screening for genetic diversity of isolates of anaerobic Fe(II)-oxidizing bacteria using DGGE and whole-cell hybridization

Nitrate-reducing bacteria, which grow anaerobically with Fe(II) as electron donor, were isolated from freshwater mud samples. Since extensive phylogenetic and physiological characterization of multiple strains is very time-consuming and labour-intensive, the isolates were first screened for genetic diversity by denaturing gradient gel electrophoresis (DGGE) and whole-cell hybridization. DGGE analysis of 16S rDNA fragments amplified from 12 strains indicated that three different types of bacteria had been independently isolated (type A-C). Whole-cell hybridization with domain- and group-specific oligonucleotide probes suggested that the type-A and -C isolates were members of the beta-subdivision of the Proteobacteria. The type-B isolates hybridized only with the bacterial probe but not with any of the probes specific for the alpha-, beta- or gamma-Proteobacteria. Based on these results representative strains of each type were chosen for further phylogenetic characterization using 16S rDNA sequencing. This analysis confirmed that the type-A and -C isolates were members of the beta-Proteobacteria. The type-B isolate was shown to be a member of the Xanthomonas group of the gamma-Proteobacteria. Our results demonstrate that probe GAM42a (specific for gamma-Proteobacteria; MANZ et al., 1992) does not hybridize to the 23S rRNA target sequence of this group of deep branching gamma-Proteobacteria: this was confirmed by hybridization experiments with Xanthomonas fragariae, which also failed to hybridize to this probe. 23S rDNA sequence analysis revealed that probe GAM42a has one mismatch with the target sequence of the type-B isolate and two mismatches with the target sequence of X. fragariae; Furthermore it was shown that double stranded DNA fragments of 626 bp length, which differed by as many as two to three nucleotides were not separated by DGGE. This suggested that rDNA fragments of closely related bacteria (99.8% sequence similarity or more) are not resolved by DGGE. We propose that the combined use of DGGE and whole-cell hybridization provides a rapid way to distinguish distantly related microbial isolates.