Detection of abundant bacteria in the Weser estuary using culture-dependent and culture-independent approaches

To assess the composition and abundant members of the bacterial community in the Weser estuary, Germany, we applied a PCR-dependent approach simultaneously with a PCR-independent approach, the dilution culture technique. Dilution cultures growing in autoclaved ambient water without any nutrient addition from the limnetic, brackish and marine sections of the Weser estuary from May, August and November 1999 yielded growth to a dilution of 10(-6) to 10(-9), equivalent to a cultivation efficiency of 1.5 to 66%. Bacteria enriched in the highest dilution steps were analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. The results show that with increasing dilution steps, fewer (in 7 cases only 1) DGGE bands occurred that matched prominent bands in the DGGE patterns of the natural bacterial community. In the 7 cases with only 1 band, almost complete 16S rRNA gene sequences were retrieved, thus extending the limited sequence information provided by the DGGE approach. The dominant phylotypes included bacteria of clusters within alpha- and beta-Proteobacteria, the Flavobacteria/Sphingobacteria group of the Bacteroidetes phylum and Actinobacteria, comprising cultivated strains but also clusters of as yet uncultured phylotypes. Closely related bacteria and phylotypes in most of these clusters are well known from other limnetic and marine systems. We also obtained isolates from the high dilution steps. More than 30% of them, belonging to various clusters within alpha- and beta-Proteobacteria and the Flavobacteria/Sphingobacteria group, had a sequence similarity of < 96% to described species. alpha-Proteobacteria constituted the most abundant phylotypes of the DGGE bands in the high dilution steps and of isolates, whereas Flavobacteria/Sphingobacteria constituted only low proportions. There were pronounced differences in the composition of bacterial communities on solid- and liquid-phase media in enrichment cultures with algal extracts and a preferential growth of gamma-Proteobacteria that did not occur in the natural samples and the high dilution steps.