Use of Polymerase Chain Reaction-amplified 16S rRNA Gene Sequences to Identify Pink-pigmented Bacteria Found in a Potable Water Treatment System.

Abstract

Two strains of pink-pigmented, chlorine-resistant bacteria, GK101 and GK118, previously isolated from pink-colored microbial biomass occurring in a potable water treatment system were studied phylogenetically by using 16S rRNA gene sequence information. 16S ribosomal DNA (rDNA) fragments of approximately 1.5kbp were amplified by the polymerase chain reaction (PCR) from crude cell lysates of the isolates and sequenced directly by a combined method consisting of linear PCR sequencing and automated fluorescence detection. Sequence comparisons and a phylogenetic tree analysis showed that the pink-pigmented isolates were closely related to Methylobacterium extorquens. Smaller fragments of 16S rDNA were also amplified from the bulk DNA extracted from the pink biomass, with a pair of PCR primers specific to the methylotrophs with the serine pathway or to proteobacteria of the alpha subclass. The PCR product was cloned by using the T-A cloning technique and sequenced as noted above. The results of this molecular approach suggested that bacteria phylogenetically the same as strain GK101 predominated in the pink-pigmented population in situ and that some other bacteria of the genus Methylobacterium might also be involved in formation of the pink-colored aggregates.