Identification of Oral Taxa from Rare Phyla/Candidate Divisions

The majority of taxa in the oral microbiome are members of six well known phyla: Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes, and Fusobacteria.  Less commonly encountered phyla include the Tenericutes, Chlamydiae and Synergistetes, and the uncultivated candidate division, TM7.  Objectives:  The primary objective of this study was to develop primers for selective PCR amplification and cloning of the 16S rRNA genes from five truly rare phyla/candidate divisions: Chloroflexi, Chlorobi, GN02, SR1, and WPS-2.  Methods:   16S rRNA clone sequences demonstrating the existence of these five phyla/divisions are known from our previous cloning studies (Dewhirst et al. 2010. J. Bacteriol 192:5002-17), the Human Microbiome Project, and unpublished studies of the oral microbiomes of dogs and cats.  16S rRNA sequences from these studies were aligned and used to retrieve additional related sequences from GenBank and Greengenes.  Based on aligned sequences from these phyla/divisions with reference sequences from the Human Oral Microbiome Database (HOMD), taxa selective forward and reverse primers were designed heuristically.  DNA pools were created from previous oral cloning studies.  Probe pairs, including “universal” 9-27F and 1492-1509R and 1525-1541R primers, were tested for amplification of DNA pools.  Primer combinations producing amplicons were cloned using TA cloning kits.  Results:   Nineteen primers were designed and synthesized.  Primer pairs for each of the five phyla/divisions produced an amplicon with at least one oral DNA pool.  Twenty-one 16S rRNA libraries were constructed and approximately 50 clones sequenced from each library.  Library analysis validated highly specific primer pairs for Chloroflexi, GN02 and SR1.  Primer pairs for WPS-2 and Chlorobi produced amplicons for Canine DNA pools but not the two tested human DNA pools. Conclusions:  We have designed highly selective primers for use in analyzing the presence and diversity of five rare oral phyla/divisions, and have identified six previously unrecognized oral taxa.  Supported by NIDRC grant DE016937.