Microdiversity of phenol hydroxylase genes among phenol-degrading isolates of Alcaligenes sp. from an activated sludge system.

Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting classified 97 phenol-degrading isolates with identical amplified ribosomal DNA restriction analysis (ARDRA) patterns into six genotypic groups. The 16S rRNA gene of the representative isolate of each group had higher than 99.47% common identity with each other and higher than 98% identity with the type strain of Alcaligenes faecalis. PCR-TGGE (temperature gradient gel electrophoresis) analysis of the genes of the largest subunit of the multi-component phenol hydroxylase (LmPH) in each isolate followed with sequencing showed that isolates within each ERIC-PCR group had identical LmPH gene sequences. Among the six different ERIC-PCR groups, two were found to harbor two different LmPH genes encoding low- and high-Ks (affinity constants) phenol hydroxylases, and the low-Ks type LmPH was identical in sequence with one predominant LmPH of the parental activated sludge. Three ERIC-PCR groups had only the high-Ks type and one had no sequence similar to the known LmPHs. Our work suggests that there is no correlation between the phylogenetic groupings of phenol-degrading bacteria and their LmPH genotypes possibly due to extensive horizontal gene transfer of this functional gene.