rpoD gene pyrosequencing for the assessment of Pseudomonas diversity in a water sample from the Woluwe river

A water sample from a noncontaminated site at the source of the Woluwe River (Belgium) was analyzed by culture-dependent and -independent methods. Pseudomonas isolates were identified by sequencing and analysis of the rpoD gene. Cultureindependent methods consisted of cloning and pyrosequencing of a Pseudomonas rpoD amplicon from total DNA extracted from the same sample and amplified with selective rpoD gene primers. Among a total of 14,540 reads, 6,228 corresponded to Pseudomonas rpoD gene sequences by a BLAST analysis in the NCBI database. The selection criteria for the reads were sequences longer than 400 bp, an average Q40 value greater than 25, and >85% identity with a Pseudomonas species. Of the 6,228 Pseudomonas rpoD sequences, 5,345 sequences met the established criteria for selection. Sequences were clustered by phylogenetic analysis and by use of the QIIME software package. Representative sequences of each cluster were assigned by BLAST analysis to a known Pseudomonas species when the identity with the type strain was greater than or equal to 96%. Twenty-six species distributed among 12 phylogenetic groups or subgroups within the genus were detected by pyrosequencing. Pseudomonas stutzeri, P. moraviensis, and P. simiae were the only cultured species not detected by pyrosequencing. The predominant phylogenetic group within the Pseudomonas genus was the P. fluorescens group, as determined by culture-dependent and -independent analyses. In all analyses, a high number of putative novel phylospecies was found: 10 were identified in the cultured strains and 246 were detected by pyrosequencing, indicating that the diversity of Pseudomonas species has not been fully described. © 2014, American Society for Microbiology.Financial support was obtained from the Spanish MINECO through projects CGL2011-24318 and Consolider CSD2009-00006, as well as from funds for competitive research groups from the Government of the Balearic Islands (the last two funds with FEDER cofunding). D. Sánchez was the recipient of a predoctoral fellowship from the Conselleria d'Interior, Direcció General de Recerca, Desenvolupament Tecnològic i Innovació del Govern de les Illes Balears (FPI09), and the European Social Fund (ESF). M. Gomila is the recipient of a postdoctoral contract from the University of the Balearic Islands, funded by the Spanish Ministry of Education, Culture and Sports through the International Excellence Campus ProgramPeer Reviewe