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

A rapid method for the extraction of RNA from the indigenous bacterial communities in environmental samples was developed. The method was tested using anoxic sediment samples from a productive freshwater lake (Priest Pot, Cumbria, UK). The simple protocol yielded rRNA and mRNA of a purity suitable for amplification by reverse transcriptase PCR (RT-PCR). The integrity of the RT-PCR was demonstrated by amplifying 16S rRNA and mRNA for the mercury resistance regulatory gene merR. The diversity of 16S rRNA sequences recovered 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 upon 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-derived). The clone libraries included sequences related to Pirellula staleyi, an aerobic planktonic member of the Planctomycetales, and the recently described candidate bacterial division OP5. Sequences from these groups were recovered in libraries generated from a DNA template but were also present in RNA-derived libraries. Previous studies of anoxic environments have identified sequences most closely related to Pirellula spp. This study, which utilized RT-PCR of 16S rRNA, has provided the first evidence that Pirellula-like bacteria are active in situ in an anoxic environment. Furthermore, members of the recently described candidate division, OP5, were also identified as active constituents of the bacterial community of anoxic Priest Pot sediments. This not only supports the widespread occurrence of OP5 members in diverse environments but suggests that they are active under anoxic conditions.