Genome Streamlining, Proteorhodopsin, and Organic Nitrogen Metabolism in Freshwater Nitrifiers.

Microbial nitrification is a critical process governing nitrogen availability in aquatic systems. Freshwater nitrifiers have received little attention, leaving many unanswered questions about their taxonomic distribution, functional potential, and ecological interactions. Here, we reconstructed genomes to infer the metabolism and ecology of free-living picoplanktonic nitrifiers across the Laurentian Great Lakes, a connected series of five of Earth's largest lakes. Surprisingly, ammonia-oxidizing bacteria (AOB) related to Nitrosospira dominated over ammonia-oxidizing archaea (AOA) at nearly all stations, with distinct ecotypes prevailing in the transparent, oligotrophic upper lakes compared to Lakes Erie and Ontario. Unexpectedly, one ecotype of Nitrosospira encodes proteorhodopsin, which could enhance survival under conditions where ammonia oxidation is inhibited or substrate limited. Nitrite-oxidizing bacteria (NOB) "Candidatus Nitrotoga" and Nitrospira fluctuated in dominance, with the latter prevailing in deeper, less-productive basins. Genome reconstructions reveal highly reduced genomes and features consistent with genome streamlining, along with diverse adaptations to sunlight and oxidative stress and widespread capacity for organic nitrogen use. Our findings expand the known functional diversity of nitrifiers and establish their ecological genomics in large lake ecosystems. By elucidating links between microbial biodiversity and biogeochemical cycling, our work also informs ecosystem models of the Laurentian Great Lakes, a critical freshwater resource experiencing rapid environmental change. IMPORTANCE Microorganisms play critical roles in Earth's nitrogen cycle. In lakes, microorganisms called nitrifiers derive energy from reduced nitrogen compounds. In doing so, they transform nitrogen into a form that can ultimately be lost to the atmosphere by a process called denitrification, which helps mitigate nitrogen pollution from fertilizer runoff and sewage. Despite their importance, freshwater nitrifiers are virtually unexplored. To understand their diversity and function, we reconstructed genomes of freshwater nitrifiers across some of Earth's largest freshwater lakes, the Laurentian Great Lakes. We discovered several new species of nitrifiers specialized for clear low-nutrient waters and distinct species in comparatively turbid Lake Erie. Surprisingly, one species may be able to harness light energy by using a protein called proteorhodopsin, despite the fact that nitrifiers typically live in deep dark water. Our work reveals the unique biodiversity of the Great Lakes and fills key gaps in our knowledge of an important microbial group, the nitrifiers.

Microbial communities of the Laurentian Great Lakes reflect connectivity and local biogeochemistry.

The Laurentian Great Lakes are a vast, interconnected freshwater system spanning strong physicochemical gradients, thus constituting a powerful natural laboratory for addressing fundamental questions about microbial ecology and evolution. We present a comparative analysis of pelagic microbial communities across all five Laurentian Great Lakes, focusing on Bacterial and Archaeal picoplankton characterized via 16S rRNA amplicon sequencing. We collected samples throughout the water column from the major basins of each lake in spring and summer over 2 years. Two oligotypes, classified as LD12 (Alphaproteobacteria) and acI-B1 (Actinobacteria), were among the most abundant in every sample. At the same time, microbial communities showed distinct patterns with depth during summer stratification. Deep hypolimnion samples were frequently dominated by a Chloroflexi oligotype that reached up to 19% relative abundance. Stratified surface communities differed between the colder, less productive upper lakes (Superior, Michigan, Huron) and warmer, more productive lower lakes (Erie, Ontario), in part due to an Actinobacteria oligotype (acI-C2) that averaged 7.7% of sequences in the lower lakes but <0.2% in the upper lakes. Together, our findings suggest that both hydrologic connectivity and local selective pressures shape microbial communities in the Great Lakes and establish a framework for future investigations.

Reevaluating the Salty Divide: Phylogenetic Specificity of Transitions between Marine and Freshwater Systems.

Marine and freshwater microbial communities are phylogenetically distinct, and transitions between habitat types are thought to be infrequent. We compared the phylogenetic diversity of marine and freshwater microorganisms and identified specific lineages exhibiting notably high or low similarity between marine and freshwater ecosystems using a meta-analysis of 16S rRNA gene tag-sequencing data sets. As expected, marine and freshwater microbial communities differed in the relative abundance of major phyla and contained habitat-specific lineages. At the same time, and contrary to expectations, many shared taxa were observed in both habitats. Based on several metrics, we found that Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, and Betaproteobacteria contained the highest number of closely related marine and freshwater sequences, suggesting comparatively recent habitat transitions in these groups. Using the abundant alphaproteobacterial group SAR11 as an example, we found evidence that new lineages, beyond the recognized LD12 clade, are detected in freshwater at low but reproducible abundances; this evidence extends beyond the 16S rRNA locus to core genes throughout the genome. Our results suggest that shared taxa are numerous, but tend to occur sporadically and at low relative abundance in one habitat type, leading to an underestimation of transition frequency between marine and freshwater habitats. Rare taxa with abundances near or below detection, including lineages that appear to have crossed the salty divide relatively recently, may possess adaptations enabling them to exploit opportunities for niche expansion when environments are disturbed or conditions change. IMPORTANCE The distribution of microbial diversity across environments yields insight into processes that create and maintain this diversity as well as potential to infer how communities will respond to future environmental changes. We integrated data sets from dozens of freshwater lake and marine samples to compare diversity across open water habitats differing in salinity. Our novel combination of sequence-based approaches revealed lineages that likely experienced a recent transition across habitat types. These taxa are promising targets for studying physiological constraints on salinity tolerance. Our findings contribute to understanding the ecological and evolutionary controls on microbial distributions, and open up new questions regarding the plasticity and adaptability of particular lineages.

Phytoplankton succession affects the composition of Polynucleobacter subtypes in humic lakes.

Phytoplankton influence the composition of bacterial communities, but the taxonomic specificity of algal-bacterial interactions is unclear due to the aggregation of ecologically distinct bacterial populations by community characterization methods. Here we examine whether phytoplankton seasonal succession affects the composition of subtypes within the cosmopolitan freshwater bacterial genus Polynucleobacter. Changes in the composition of Polynucleobacter subtypes were characterized in samples collected weekly from May to August in 2003 and 2008 from three humic lakes using terminal restriction fragment length polymorphism fingerprinting of the protein-encoding cytochrome c oxidase ccoN gene. Changes in phytoplankton population abundances explained, on average, 30% of temporal variation in the composition of Polynucleobacter subtypes and the interaction between phytoplankton and the environment explained an additional 18% of temporal variation. The effect of phytoplankton on specific Polynucleobacter subtypes was experimentally confirmed by changes in Polynucleobacter subtype composition following incubation with different phytoplankton assemblages or a no-phytoplankton control. Phytoplankton-associated subtypes and differentiation in substrate use among subtypes likely contribute to the effects of phytoplankton on Polynucleobacter subtype composition. Interactions between unique Polynucleobacter populations and phytoplankton highlight the ecological significance and specificity of species interactions in freshwater communities.

Interactions between specific phytoplankton and bacteria affect lake bacterial community succession.

Time-series observations and a phytoplankton manipulation experiment were combined to test the hypothesis that phytoplankton succession effects changes in bacterial community composition. Three humic lakes were sampled weekly May-August and correlations between relative abundances of specific phytoplankton and bacterial operational taxonomic units (OTUs) in each time series were determined. To experimentally characterize the influence of phytoplankton, bacteria from each lake were incubated with phytoplankton from one of the three lakes or no phytoplankton. Following incubation, variation in bacterial community composition explained by phytoplankton treatment increased 65%, while the variation explained by bacterial source decreased 64%. Free-living bacteria explained, on average, over 60% of the difference between phytoplankton and corresponding no-phytoplankton control treatments. Fourteen out of the 101 bacterial OTUs that exhibited positively correlated patterns of abundance with specific algal populations in time-series observations were enriched in mesocosms following incubation with phytoplankton, and one out of 59 negatively correlated bacterial OTUs was depleted in phytoplankton treatments. Bacterial genera enriched in mesocosms containing specific phytoplankton assemblages included Limnohabitans (clade betI-A), Bdellovibrio and Mitsuaria. These results suggest that effects of phytoplankton on certain bacterial populations, including bacteria tracking seasonal changes in algal-derived organic matter, result in correlations between algal and bacterial community dynamics.

Temporal succession of putative glycolate-utilizing bacterioplankton tracks changes in dissolved organic matter in a high-elevation lake.

Shifts in the composition of dissolved organic matter (DOM) may be a principal factor effecting changes in bacterial community composition. Emerald Lake, a high-elevation lake with DOM dominated by terrestrial sources in the spring with increasing phytoplankton-derived inputs throughout the ice-free season, provided a natural experiment with which to investigate the importance of resource-mediated drivers. Glycolate-utilizing bacteria, a subset of the bacterial community able to use algal exudates, were characterized using DNA analysis of glycolate oxidase subunit D (glcD) genes from Emerald Lake samples collected approximately biweekly from ice-cover (June) through fall turnover (September). glcD genes were not detected in early-season samples when the lake was ice-covered and phytoplankton-derived resources were scarce. Following this period, glcD gene composition exhibited significant changes through time, which were strongly correlated with the combination of fluorescence index, an indicator of the proportion of lake vs. terrestrially derived DOM, and dissolved inorganic nitrate + nitrite. These results suggest that seasonal shifts in DOM source and environmental variables that affect the composition of algal-derived dissolved resources drive changes in the relative abundance and composition of putative glycolate-utilizing bacteria. These findings strengthen the evidence for shifts in DOM structuring bacterial communities.

Temporal patterns in glycolate-utilizing bacterial community composition correlate with phytoplankton population dynamics in humic lakes.

Previous observations of correlated community dynamics between phytoplankton and bacteria in lakes indicate that phytoplankton populations may influence bacterial community structure. To investigate the possibility that bacterial use of phytoplankton exudates contributes to observed patterns of community change, we characterized the diversity and dynamics of heterotrophic bacterioplankton with genetic potential to use glycolate, a photorespiration-specific exudate, in five lakes over a 15-week period. Culture-independent approaches were used to track different bacterial phylotypes represented by DNA sequence variation in the functional gene glycolate oxidase subunit D (glcD). glcD gene sequences from freshwater bacteria exhibited broad phylogenetic diversity, including sequences representing the Alpha-, Beta-, and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Verrucomicrobia. The majority of glcD gene sequences were betaproteobacterial, with 48% of the sequences clustering with the glcD gene from the cosmopolitan freshwater species Polynucleobacter necessarius. Terminal restriction fragment length polymorphism fingerprinting of the glcD gene revealed changes in glycolate-utilizing assemblages over time. An average of 39% of within-lake temporal variation in glycolate-utilizing assemblages across five lakes was explained by phytoplankton community composition and dynamics. The interaction between phytoplankton populations and the environment explained an additional 17% of variation on average. These observations offer new insight into the diversity and temporal dynamics of freshwater bacteria with genetic potential to use glycolate and support the hypothesis that algal exudates influence the structure of bacterial communities.