Primer Design for an Accurate View of Picocyanobacterial Community Structure by Using High-Throughput Sequencing.

High-throughput sequencing (HTS) of the 16S rRNA gene has been used successfully to describe the structure and dynamics of microbial communities. Picocyanobacteria are important members of bacterioplankton communities, and, so far, they have predominantly been targeted using universal bacterial primers, providing a limited resolution of the picocyanobacterial community structure and dynamics. To increase such resolution, the study of a particular target group is best approached with the use of specific primers. Here, we aimed to design and evaluate specific primers for aquatic picocyanobacterial genera to be used with high-throughput sequencing. Since the various regions of the 16S rRNA gene have different degrees of conservation in different bacterial groups, we therefore first determined which hypervariable region of the 16S rRNA gene provides the highest taxonomic and phylogenetic resolution for the genera Synechococcus, Prochlorococcus, and Cyanobium An in silico analysis showed that the V5, V6, and V7 hypervariable regions appear to be the most informative for this group. We then designed primers flanking these hypervariable regions and tested them in natural marine and freshwater communities. We successfully detected that most (97%) of the obtained reads could be assigned to picocyanobacterial genera. We defined operational taxonomic units as exact sequence variants (zero-radius operational taxonomic units [zOTUs]), which allowed us to detect higher genetic diversity and infer ecologically relevant information about picocyanobacterial community composition and dynamics in different aquatic systems. Our results open the door to future studies investigating picocyanobacterial diversity in aquatic systems.IMPORTANCE The molecular diversity of the aquatic picocyanobacterial community cannot be accurately described using only the available universal 16S rRNA gene primers that target the whole bacterial and archaeal community. We show that the hypervariable regions V5, V6, and V7 of the 16S rRNA gene are better suited to study the diversity, community structure, and dynamics of picocyanobacterial communities at a fine scale using Illumina MiSeq sequencing. Due to its variability, it allows reconstructing phylogenies featuring topologies comparable to those generated when using the complete 16S rRNA gene sequence. Further, we successfully designed a new set of primers flanking the V5 to V7 region whose specificity for picocyanobacterial genera was tested in silico and validated in several freshwater and marine aquatic communities. This work represents a step forward for understanding the diversity and ecology of aquatic picocyanobacteria and sets the path for future studies on picocyanobacterial diversity.

Seasonality of freshwater bacterioplankton diversity in two tropical shallow lakes from the Brazilian Atlantic Forest.

Bacteria are highly important for the cycling of organic and inorganic matter in freshwater environments; however, little is known about the diversity of bacterioplankton in tropical systems. Studies on carbon and nutrient cycling in tropical lakes suggest a very different seasonality from that of temperate climates. Here, we used 16S rRNA gene next-generation sequencing (NGS) to investigate seasonal changes in bacterioplankton communities of two tropical lakes, which differed in trophic status and mixing regime. Our findings revealed seasonally and depth-wise highly dynamic bacterioplankton communities. Differences in richness and structure appeared strongly related to the physicochemical characteristics of the water column, especially phosphate, pH and oxygen. Bacterioplankton communities were dominated by common taxonomic groups, such as Synechococcus and Actinobacteria acI, as well as rare and poorly characterized taxa such as 'Candidatus Methylacidiphilum' (Verrucomicrobia). Stratification and oxygen depletion during the rainy season promoted the occurrence of anoxygenic phototrophic and methanotrophic bacteria important for carbon and nutrient cycling. Differences in lake mixing regime were associated with seasonal beta diversity. Our study is the first attempt to use NGS for cataloging the diversity of bacterioplankton communities in Brazilian lakes and thus contributes to the ongoing worldwide endeavor to characterize freshwater lake bacterioplankton signatures.

Marine bacterioplankton community turnover within seasonally hypoxic waters of a subtropical sound: Devil's Hole, Bermuda.

Understanding bacterioplankton community dynamics in coastal hypoxic environments is relevant to global biogeochemistry because coastal hypoxia is increasing worldwide. The temporal dynamics of bacterioplankton communities were analysed throughout the illuminated water column of Devil's Hole, Bermuda during the 6-week annual transition from a strongly stratified water column with suboxic and high-pCO2 bottom waters to a fully mixed and ventilated state during 2008. A suite of culture-independent methods provided a quantitative spatiotemporal characterization of bacterioplankton community changes, including both direct counts and rRNA gene sequencing. During stratification, the surface waters were dominated by the SAR11 clade of Alphaproteobacteria and the cyanobacterium Synechococcus. In the suboxic bottom waters, cells from the order Chlorobiales prevailed, with gene sequences indicating members of the genera Chlorobium and Prosthecochloris--anoxygenic photoautotrophs that utilize sulfide as a source of electrons for photosynthesis. Transitional zones of hypoxia also exhibited elevated levels of methane- and sulfur-oxidizing bacteria relative to the overlying waters. The abundance of both Thaumarcheota and Euryarcheota were elevated in the suboxic bottom waters (> 10(9) cells l(-1)). Following convective mixing, the entire water column returned to a community typical of oxygenated waters, with Euryarcheota only averaging 5% of cells, and Chlorobiales and Thaumarcheota absent.

Phylogenetic diversity of nonmarine picocyanobacteria.

We studied the phylogenetic diversity of nonmarine picocyanobacteria broadening the sequence data set with 43 new sequences of the 16S rRNA gene. The sequences were derived from monoclonal strains isolated from four volcanic high-altitude athalassohaline lakes in Mexico, five glacial ultraoligotrophic North Patagonian lakes and six Italian lakes of glacial, volcanic and morenic origin. The new sequences fall into a number of both novel and previously described clades within the phylogenetic tree of 16S rRNA gene. The new cluster of Lake Nahuel Huapi (North Patagonia) forms a sister clade to the subalpine cluster II and the marine Synechococcus subcluster 5.2. Our finding of the novel clade of 'halotolerants' close to the marine subcluster 5.3 (Synechococcus RCC307) constitutes an important demonstration that euryhaline and marine strains affiliate closely. The intriguing results obtained shed new light on the importance of the nonmarine halotolerants in the phylogenesis of picocyanobacteria.