Structure of bacterial communities in aquatic systems as revealed by filter PCR

Collection of microbial biomass and extraction of DNA are the first steps o f many molecular approaches for examining uncultured microbes in aquatic ec osystems. Because of the difficulties of using large samples (up to 20 I) a nd the occasional ineffectiveness of DNA isolation procedures, we examined an alternative approach, 'filter PCR', which consists of filtering small vo lumes through polycarbonate filters and using sections of a filter directly in PCR. Positive amplification was achieved with as little as 25 mul of co astal seawater, corresponding to about 10000 bacterial cells, although larg er volumes (1 to 10 ml, depending on bacterial abundance) gave more consist ent results. Denaturing gradient gel electrophoresis (DGGE) revealed few di fferences in the 16S rRNA amplicons from filter PCR and from the standard a pproach using DNA isolated from several liters of coastal seawater. A clone library of 16S rRNA amplicons from filter PCR was slightly more diverse th an a clone library constructed by the standard approach. These results allo w us to explore variation in microbial community structure over a range of spatial scales and to examine the relative evenness of microbial communitie s in aquatic habitats. Our results indicate that filter PCR is as effective as the standard approach in retrieving bacterial genes from uncultured mic robes in aquatic environments.