Community composition and activity state of estuarine bacterioplankton assessed using differential staining and metagenomic analysis of 16S rDNA and rRNA

Abstract

To better understand how resource availability controls estuarine microbial communities, we compared community composition at 2 sites in the tidal freshwater James River (Virginia, USA) which differed in nutrient concentrations and autochthonous production. In addition, we conducted laboratory microcosm experiments, wherein bacteria from the 2 sites received nutrient, light, and organic carbon amendments. Traditional, microscopy-based methods of staining and enumeration were used to determine the abundance of total, live, and active cells. DNA fingerprinting (terminal restriction fragment length polymorphism, TRFLP) and dual 16S rDNA-rRNA pyrosequencing were used to assess community composition and taxon-specific activity (from rRNA:rDNA). Enumeration revealed that total, live, and active (CTC+) bacteria were more abundant at the site where autochthonous production was higher, while DNA fingerprinting (TRFLP) and 16S rDNA pyrosequencing indicated high similarity in community composition at the 2 sites. In contrast, pyrosequencing results from rRNA revealed greater variation and suggest that the metabolically active fraction of the community differed between sites. Differences in rDNA- and rRNA-based libraries were also apparent in microcosm experiments, where resource amendments resulted in shifts in both the active and overall communities. In both environmental and microcosm samples, dual rDNA-rRNA pyrosequencing yielded higher estimates of the proportion of active cells (32 ± 3%) compared to differential staining and microscopy (13 ± 1 %). Though characterization of active taxa was sensitive to the presumed activity threshold (rRNA:rDNA), our findings suggest that variable resource conditions can give rise to unique assemblages of active taxa despite similarities in overall community composition. © Inter-Research 2013.