Distribution and culturability of the uncultivated ‘AGG58 cluster’ of the Bacteroidetes phylum in aquatic environments

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Abstract
Members of the Bacteroidetes phylum are abundant in aquatic habitats when assessed by fluorescent in situ hybridisation and in some 16S rRNA gene libraries. In this study 16S rRNA gene clone libraries were constructed with bacterial primers that amplify Bacteroidetes sequences well (27F, 1492R) from coastal seawater near Plymouth (UK) during a phytoplankton bloom. Most of the clones (66%, 106/160) affiliated with the Bacteroidetes phylum, and of these 62% (66/106; or 41% 66/160 of the entire library) clustered with marine bacterioplankton clones env.agg58, Arctic97A-17, CF17, CF96 and CF101. This phylogenetic branch of Bacteroidetes was designated the ‘AGG58 cluster’, and its presence in various aquatic environments was investigated. Two pairs of AGG58-specific 16S rRNA-gene-targeted polymerase chain reaction (PCR) primers were designed and successfully used to detect the cluster in DNA extracts from three UK coastal seawater sites, and from freshwater River Taff epilithon. In addition, 600 putative Bacteroidetes strains were isolated from these sites on relatively high-nutrient agar media. AGG58 cluster specific probes were used to screen the amplified 16S rRNA gene products from the isolates, but no members of the AGG58 cluster were discovered. The least specific probe hybridised with one River Taff water isolate (RW262 NCIMB 13979) which formed a monophyletic group with the genera Crocinitomix, Brumimicrobium and Cryomorpha of the family Cryomorphaceae in the Bacteroidetes phylum. RW262 probably represents the first isolate of a new genus within this family. This study provides new evidence that the uncultivated AGG58 group is abundant, globally distributed, and can be rapidly detected with the new PCR primers described.