Application of Illumina next-generation sequencing to characterize the bacterial community of the Upper Mississippi River.

AIMS: A next-generation, Illumina-based sequencing approach was used to characterize the bacterial community at ten sites along the Upper Mississippi River to evaluate shifts in the community potentially resulting from upstream inputs and land use changes. Furthermore, methodological parameters including filter size, sample volume and sample reproducibility were evaluated to determine the best sampling practices for community characterization. METHODS AND RESULTS: Community structure and diversity in the river was determined using Illumina next-generation sequencing technology and the V6 hypervariable region of 16S rDNA. A total of 16,400 operational taxonomic units (OTUs) were observed (4594 ± 824 OTUs per sample). Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria and Verrucomicrobia accounted for 93.6 ± 1.3% of all sequence reads, and 90.5 ± 2.5% belonged to OTUs shared among all sites (n = 552). Among nonshared sequence reads at each site, 33-49% were associated with potentially anthropogenic impacts upstream of the second sampling site. Alpha diversity decreased with distance from the pristine headwaters, while rainfall and pH were positively correlated with diversity. Replication and smaller filter pore sizes minimally influenced the characterization of community structure. CONCLUSIONS: Shifts in community structure are related to changes in the relative abundance, rather than presence/absence of OTUs, suggesting a 'core bacterial community' is present throughout the Upper Mississippi River. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is among the first to characterize a large riverine bacterial community using a next-generation-sequencing approach and demonstrates that upstream influences and potentially anthropogenic impacts can influence the presence and relative abundance of OTUs downstream resulting in significant variation in community structure.

5'-nucleotidase activity in a eutrophic lake and an oligotrophic lake.

Differences in enzymatic hydrolysis of dissolved organic phosphorus and subsequent phosphorus uptake were compared by using dual-labeled (gamma-P and 2-H) ATP in oligotrophic Lake Michigan and a moderately eutrophic lake in southeastern Michigan. More than 50% of the phosphate that was hydrolyzed was immediately taken up into bacterium-sized particles in the eutrophic lake and at a near-shore site in Lake Michigan. Less than 50% of the hydrolyzed phosphate was taken up into bacterium-sized particles at an offshore site in Lake Michigan. It is hypothesized that differences in size-fractionated uptake were the result of greater phosphorus utilization capacity in bacteria in habitats where loading of organic carbon is greater. Substantial isotope dilution of labeled phosphate uptake by unlabeled phosphate occurred, which implied that the phosphate was hydrolyzed extracellularly in both systems. Comparable nucleotidase activities were measured in the eutrophic lake and Lake Michigan, but the significance of the phosphate regenerated relative to particulate phosphorus pools was an order of magnitude greater in Lake Michigan. Seventy percent of the nucleotidase activity was inhibited by 100 muM phosphate in the eutrophic lake, which suggests that most hydrolysis was by phosphatase. Therefore, nucleotidase activity may be more important to phosphorus regeneration in oligotrophic habitats than phosphatase activity.