Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum cytophaga-flavobacter-bacteroides in the natural environment

TLDR

The probes were applied to analyze community composition in complex environmental samples. We designed and computationally optimized a panel of four 16S rRNA‑targeted oligonucleotide probes for the CFB phylum, then validated them by whole‑cell and non‑radioactive dot‑blot hybridization against reference strains. In activated sludge, flavobacteria‑cytophaga members were key constituents of flocs, while BAC303 revealed Bacteroides and Prevotella as major components of human faecal microflora.

Abstract

We designed a panel of four 16S rRNA-targeted oligonucleotide probes specific for bacteria of the phylum cytophaga-flavobacter-bacteroides (CFB). Probes CF319a and CF319b are targeted to members of the flavobacteria-cytophaga group and the genus Porphyromonas, whereas probe BAC303 has a target region characteristic for the genera Prevotella and Bacteroides within the bacteroides group. The probe FFE8b was developed for species-specific hybridizations with Flavobacterium ferrugineum. All probes were designed by computer-assisted sequence analysis and compared to all currently accessible 16S and 23S rRNA sequences. The oligonucleotides were further evaluated by whole-cell and non-radioactive dot-blot hybridization against reference strains of the CFB phylum and other major lineages of Bacteria. The newly developed probes were used together with other higher-order probes to analyse the structure and community composition in complex environments. In activated sludge samples, members of the flavobacteria-cytophaga group were revealed by in situ hybridization as important constituents of sludge flocs and characteristic colonizers of filamentous bacteria. By application of fluorescent probe BAC303, members of the genera Bacteroides and Prevotella could be visualized without prior cultivation as an important part of the human faecal microflora.