Identification of novel bacterial lineages as active members of microbial populations in a freshwater sediment using a rapid RNA extraction procedureand RT-PCR

A rapid method for the extraction of RNA from the indigenous bacterial comm unities in environmental samples was developed. The method was tested using anoxic sediment samples from a productive freshwater lake (Priest Pot, Cum bria, UK). The simple protocol yielded rRNA and mRNA of a purity suitable f or amplification by reverse transcriptase PCR (RT-PCR). The integrity of th e RT-PCR was demonstrated by amplifying 16S rRNA and mRNA for the mercury r esistance regulatory gene merR. The diversity of 16S rRNA sequences recover ed from RNA and DNA extracted from anoxic Priest Pot sediments was analysed . The 5' end of extracted 16S rRNA was amplified by RT-PCR and the 16S rRNA PCR products were cloned and sequenced to identify active constituents of the sediment bacterial community. Corresponding analyses were performed upo n DNA templates from the same sediment samples. Partial 16S rRNA sequences were obtained from a total of 147 clones (71 rRNA-derived and 76 rDNA-deriv ed). The clone libraries included sequences related to Pirellula staleyi, a n aerobic planktonic member of the Planctomycetales, and the recently descr ibed candidate bacterial division OP5. Sequences from these groups were rec overed in libraries generated from a DNA template but were also present in RNA-derived libraries. Previous studies of anoxic environments have identif ied sequences most closely related to Pirellula spp. This study, which util ized RT-PCR of 16S rRNA, has provided the first evidence that Pirellula-lik e bacteria are active in situ in an anoxic environment. Furthermore, member s of the recently described candidate division, OP5, were also identified a s active constituents of the bacterial community of anoxic Priest Pot sedim ents. This not only supports the widespread occurrence of OP5 members in di verse environments but suggests that they are active under anoxic condition s.