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Epigenetics is a rapidly developing field focused on deciphering chemical fingerprints that accumulate on human genomes over time. As the nascent idea of precision medicine expands to encompass epigenetic signatures of diagnostic and prognostic relevance, there is a need for methodologies that provide high-throughput DNA methylation profiling measurements. Here we report a novel quantification methodology for computationally reconstructing site-specific CpG methylation status from next generation sequencing (NGS) data using methyl-sensitive restriction endonucleases (MSRE). An integrated pipeline efficiently incorporates raw NGS metrics into a statistical discrimination platform to identify functional linkages between shifts in epigenetic DNA methylation and disease phenotypes in samples being analyzed. In this pilot proof-of-concept study we quantify and compare DNA methylation in blood serum of individuals with Parkinson's Disease relative to matched healthy blood profiles. Even with a small study of only six samples, a high degree of statistical discrimination was achieved based on CpG methylation profiles between groups, with 1008 statistically different CpG sites ( p < 0.0025, after false discovery rate correction). A methylation load calculation was used to assess higher order impacts of methylation shifts on genes and pathways and most notably identified FGF3, FGF8, HTT, KMTA5, MIR8073, and YWHAG as differentially methylated genes with high relevance to Parkinson's Disease and neurodegeneration (based on PubMed literature citations). Of these, KMTA5 is a histone methyl-transferase gene and HTT is Huntington Disease Protein or Huntingtin, for which there are well established neurodegenerative impacts. The future need for precision diagnostics now requires more tools for exploring epigenetic processes that may be linked to cellular dysfunction and subsequent disease progression.

Unlabelled: Bacteria often respond to environmental stimuli using transcriptional control, but this may not be the case for marine bacteria such as "Candidatus Pelagibacter ubique," a cultivated representative of the SAR11 clade, the most abundant organism in the ocean. This bacterium has a small, streamlined genome and an unusually low number of transcriptional regulators, suggesting that transcriptional control is low in Pelagibacter and limits its response to environmental conditions. Transcriptome sequencing during batch culture growth revealed that only 0.1% of protein-encoding genes appear to be under transcriptional control in Pelagibacter and in another oligotroph (SAR92) whereas >10% of genes were under transcriptional control in the copiotrophs Polaribacter sp. strain MED152 and Ruegeria pomeroyi When growth levels changed, transcript levels remained steady in Pelagibacter and SAR92 but shifted in MED152 and R. pomeroyi Transcript abundances per cell, determined using an internal RNA sequencing standard, were low (<1 transcript per cell) for all but a few of the most highly transcribed genes in all four taxa, and there was no correlation between transcript abundances per cell and shifts in the levels of transcription. These results suggest that low transcriptional control contributes to the success of Pelagibacter and possibly other oligotrophic microbes that dominate microbial communities in the oceans., Importance: Diverse heterotrophic bacteria drive biogeochemical cycling in the ocean. The most abundant types of marine bacteria are oligotrophs with small, streamlined genomes. The metabolic controls that regulate the response of oligotrophic bacteria to environmental conditions remain unclear. Our results reveal that transcriptional control is lower in marine oligotrophic bacteria than in marine copiotrophic bacteria. Although responses of bacteria to environmental conditions are commonly regulated at the level of transcription, metabolism in the most abundant bacteria in the ocean appears to be regulated by other mechanisms., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Aerobic anoxygenic phototrophic (AAP) bacteria are photoheterotrophs that despite their low abundances have been hypothesized to play an ecologically and biogeochemically important role in aquatic systems. Characterizing this role requires a better understanding of the in situ dynamics and activity of AAP bacteria. Here we provide the first assessment of the single-cell activity of freshwater AAP bacteria and their contribution to total bacterial production across lakes spanning a wide trophic gradient, and explore the role of light in regulating AAP activity. The proportion of cells that were active in leucine incorporation and the level of activity per cell were consistently higher for AAP than for bulk bacteria across lakes. As a result, AAP bacteria contributed disproportionately more to total bacterial production than to total bacterial abundance. Interestingly, although environmentally driven patterns in activity did not seem to differ largely between AAP and bulk bacteria, their response to light did, and exposure to light resulted in increases in the proportion of active AAP bacteria with no clear effect on their cell-specific activity. This suggests that light may play a role in the activation of AAP bacteria, enabling these photoheterotrophs to contribute more to the carbon cycle than suggested by their abundance.

Interpretation of 16S ribosomal RNA (rRNA) to 16S rRNA gene ratios (rRNA:rDNA) is based on a limited number of studies with rapidly growing copiotrophic bacteria. The most abundant bacteria in the ocean are oligotrophs, which probably grow more slowly than those bacteria whose rRNA:rDNA versus growth rate relationships are known. To examine whether rRNA:rDNA varies differently in oligotrophic marine bacteria than in copiotrophic bacteria, we used quantitative PCR and reverse transcriptase quantitative PCR to measure rRNA:rDNA in two marine copiotrophs and in two marine oligotrophs, including Candidatus Pelagibacter ubique HTCC1062, a coastal isolate of SAR11, the most abundant bacterial clade in the ocean. The rRNA:rDNA ratios for the two copiotrophs were similar to those expected on the basis of an analysis of previously studied copiotrophic bacteria, while the ratios for the two oligotrophs were substantially lower than predicted even given their slow growth rates. The rRNA:rDNA ratios determined along a transect in the Delaware estuary suggested that SAR11 bacteria grow at rates close to the growth rate in culture, while rates of the two copiotrophs were far below those observed in laboratory cultures. Our results have implications for interpreting rRNA:rDNA from natural communities, understanding growth strategies and comparing regulatory mechanisms in copiotrophs and oligotrophs.

There is now evidence that aerobic anoxygenic phototrophic (AAP) bacteria are widespread across aquatic systems, yet the factors that determine their abundance and activity are still not well understood, particularly in freshwaters. Here we describe the patterns in AAP abundance, cell size and pigment content across wide environmental gradients in 43 temperate and boreal lakes of Québec. AAP bacterial abundance varied from 1.51 to 5.49 x 105 cells mL-1, representing <1 to 37% of total bacterial abundance. AAP bacteria were present year-round, including the ice-cover period, but their abundance relative to total bacterial abundance was significantly lower in winter than in summer (2.6% and 7.7%, respectively). AAP bacterial cells were on average two-fold larger than the average bacterial cell size, thus AAP cells made a greater relative contribution to biomass than to abundance. Bacteriochlorophyll a (BChla) concentration varied widely across lakes, and was not related to AAP bacterial abundance, suggesting a large intrinsic variability in the cellular pigment content. Absolute and relative AAP bacterial abundance increased with dissolved organic carbon (DOC), whereas cell-specific BChla content was negatively related to chlorophyll a (Chla). As a result, both the contribution of AAP bacteria to total prokaryotic abundance, and the cell-specific BChla pigment content were positively correlated with the DOC:Chla ratio, both peaking in highly colored, low-chlorophyll lakes. Our results suggest that photoheterotrophy might represent a significant ecological advantage in highly colored, low-chlorophyll lakes, where DOC pool is chemically and structurally more complex.

Aerobic anoxygenic phototrophic (AAP) bacteria are well known to be abundant in estuaries, coastal regions and in the open ocean, but little is known about their activity in any aquatic ecosystem. To explore the activity of AAP bacteria in the Delaware estuary and coastal waters, single-cell (3)H-leucine incorporation by these bacteria was examined with a new approach that combines infrared epifluorescence microscopy and microautoradiography. The approach was used on samples from the Delaware coast from August through December and on transects through the Delaware estuary in August and November 2011. The percent of active AAP bacteria was up to twofold higher than the percentage of active cells in the rest of the bacterial community in the estuary. Likewise, the silver grain area around active AAP bacteria in microautoradiography preparations was larger than the area around cells in the rest of the bacterial community, indicating higher rates of leucine consumption by AAP bacteria. The cell size of AAP bacteria was 50% bigger than the size of other bacteria, about the same difference on average as measured for activity. The abundance of AAP bacteria was negatively correlated and their activity positively correlated with light availability in the water column, although light did not affect (3)H-leucine incorporation in light-dark experiments. Our results suggest that AAP bacteria are bigger and more active than other bacteria, and likely contribute more to organic carbon fluxes than indicated by their abundance.

Heterotrophic bacteria are well known to be key players in the turnover of dissolved organic material (DOM) in the oceans, but the relationship between DOM uptake and bacterial clades is still not well understood. Here we explore the turnover and single-cell use of glucose, an amino acid mixture, N-acetylglucosamine (NAG), and protein by gammaproteobacterial clades in coastal waters of the West Antarctic Peninsula in summer and fall. More than 60% of the cells within two closely related gammaproteobacterial clades, Ant4D3 and Arctic96B-16, were active in using the amino acid mixture, protein, and NAG. In contrast, an average of only 7% of all SAR86 cells used amino acids and protein even in summer when DOM use was high. In addition to DOM uptake within a group, we explored the contribution of the three gammaproteobacterial groups to total community uptake of a compound. SAR86 contributed 5- to 10-fold less than the other gammaproteobacterial subgroups to the uptake of all compounds. We found that the overall contribution of the Ant4D3 clade to DOM uptake was highest, whereas the SAR86 clade contributed the least to DOM turnover in West Antarctic Peninsula waters. Our results suggest that the low growth activity of a bacterial clade leads to low abundance, fewer active cells and a low contribution to the turnover of DOM components.

Characterizing both growth and abundance is important in understanding the role of bacterial communities in biogeochemical cycling of global oceans. However, these two quantities are seldom measured together for specific bacterial clades. Our goal was to examine growth and abundance of three gammaproteobacterial subgroups, including SAR86, at the single-cell level by microautoradiography combined with fluorescence in situ hybridization (FISH) in coastal waters of the west Antarctic Peninsula region during two austral summers and one austral fall. We found that the SAR86 clade was less abundant and grew more slowly than two related gammaproteobacterial clades, Ant4D3 and Arctic96B-16. Over 60% of Ant4D3 and Arctic96B-16 cells incorporated leucine, while only 25% of SAR86 cells were active in both summer and fall. We also explored using the size of the FISH image as another measure of single-cell activity. There was a linear relationship between FISH cell size and incorporation of leucine for all bacteria, Ant4D3 and Arctic96B-16, but not for SAR86. FISH sizes of SAR86 cells were at least threefold smaller than cells in the other clades. Our results suggest slow growth of SAR86 in the perennially cold waters of the west Antarctic Peninsula., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Dissolved organic matter (DOM) and heterotrophic bacteria are highly diverse components of the ocean system, and their interactions are key in regulating the biogeochemical cycles of major elements. How chemical and phylogenetic diversity are linked remains largely unexplored to date. To investigate interactions between bacterial diversity and DOM, we followed the response of natural bacterial communities to two sources of phytoplankton-derived DOM over six bacterial generation times in continuous cultures. Analyses of total hydrolysable neutral sugars and amino acids, and ultrahigh resolution mass spectrometry revealed large differences in the chemical composition of the two DOM sources. According to 454 pyrosequences of 16S ribosomal ribonucleic acid genes, diatom-derived DOM sustained higher levels of bacterial richness, evenness and phylogenetic diversity than cyanobacteria-derived DOM. These distinct community structures were, however, not associated with specific taxa. Grazing pressure affected bacterial community composition without changing the overall pattern of bacterial diversity levels set by DOM. Our results demonstrate that resource composition can shape several facets of bacterial diversity without influencing the phylogenetic composition of bacterial communities, suggesting functional redundancy at different taxonomic levels for the degradation of phytoplankton-derived DOM., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Aerobic anoxygenic phototrophic (AAP) bacteria are found in a range of aquatic and terrestrial environments, potentially playing unique roles in biogeochemical cycles. Although known to occur in the Arctic Ocean, their ecology and the factors that govern their community structure and distribution in this extreme environment are poorly understood. Here, we examined summer AAP abundance and diversity in the North East Pacific and the Arctic Ocean with emphasis on the southern Beaufort Sea. AAP bacteria comprised up to 10 and 14% of the prokaryotic community in the bottom nepheloid layer and surface waters of the Mackenzie plume, respectively. However, relative AAP abundances were low in offshore waters. Environmental pufM clone libraries revealed that AAP bacteria in the Alphaproteobacteria and Betaproteobacteria classes dominated in offshore and in river-influenced surface waters, respectively. The most frequent AAP group was a new uncultivated betaproteobacterial clade whose abundance decreased along the salinity gradient of the Mackenzie plume even though its photosynthetic genes were actively expressed in offshore waters. Our data indicate that AAP bacterial assemblages represented a mixture of freshwater and marine taxa mostly restricted to the Arctic Ocean and highlight the substantial influence of riverine inputs on their distribution in coastal environments., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

The influence of arsenite [As(III)] on natural microbial communities and the capacity of exposed communities to oxidize As(III) has not been well explored. In this study, we conducted soil column experiments with a natural microbial community exposed to different carbon conditions and a continuous flow of As(III). We measured the oxidation rates of As(III) to As(V), and the composition of the bacterial community was monitored by 454 pyrosequencing of 16S rRNA genes. The diversity of As(III)-oxidizing bacteria was examined with the aox gene, which encodes the enzyme involved in As(III) oxidation. Arsenite oxidation was high in the live soil regardless of the carbon source and below detection in sterilized soil. In columns amended with 200 μmol kg(-1) of As (III), As(V) concentrations reached 158 μmol kg(-1) in the column effluent, while As(III) decreased to unmeasurable levels. Although the number of bacterial taxa decreased by as much as twofold in treatments amended with As(III), some As(III)-oxidizing bacterial groups increased up to 20-fold. Collectively, the data show the large effect of As(III) on bacterial diversity, and the capacity of natural communities from a soil with low initial As contamination to oxidize large inputs of As(III)., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Environmental conditions in the western Arctic Ocean range from constant light and nutrient depletion in summer to complete darkness and sea ice cover in winter. This seasonal environmental variation is likely to have an effect on the use of dissolved organic matter (DOM) by heterotrophic bacteria in surface water. However, this effect is not well studied and we know little about the activity of specific bacterial clades in the surface oceans. The use of DOM by three bacterial subgroups in both winter and summer was examined by microautoradiography combined with fluorescence in situ hybridization. We found selective use of substrates by these groups, although the abundances of Ant4D3 (Antarctic Gammaproteobacteria), Polaribacter (Bacteroidetes), and SAR11 (Alphaproteobacteria) were not different between summer and winter in the Beaufort and Chukchi Seas. The number of cells taking up glucose within all three bacterial groups decreased significantly from summer to winter, while the percentage of cells using leucine did not show a clear pattern between seasons. The uptake of the amino acid mix increased substantially from summer to winter by the Ant4D3 group, although such a large increase in uptake was not seen for the other two groups. Use of glucose by bacteria, but not use of leucine or the amino acid mix, related strongly to inorganic nutrients, chlorophyll a, and other environmental factors. Our results suggest a switch in use of dissolved organic substrates from summer to winter and that the three phylogenetic subgroups examined fill different niches in DOM use in the two seasons.

This study examined total uptake of extracellular polymeric substances (EPS) and glucose and the percentage of prokaryotic cells (Bacteria, Crenarchaea and Euryarchaea) consuming these compounds in the major water masses at the DYFAMED site (NW Mediterranean Sea). The potential assimilation rates of EPS at 10 m depth were higher but on the same order of magnitude as those at 2000 m depth (from 43.4 to 29.0 pmol l(-1)  h(-1) ). In contrast, glucose assimilation rates decreased with depth from 49.4 to 0.07 pmol l(-1)  h(-1) at 10 and 2000 m depth respectively. Microautoradiography analyses indicated similar percentages of active cells assimilating EPS at 10 and 2000 m depth (13% and 10% of the total-cells). The combination of microautoradiography and catalysed reporter deposition fluorescence in situ hybridization (MICRO-CARD-FISH) analyses revealed that the percentages of Bacteria assimilating (3) H-carbohydrates decreased with depth by twofold for EPS. In contrast, the contribution by Euryarchaea to EPS consumption increased with depth by sixfold. At 2000 m, 50% of active cells consuming (3) H-carbohydrates were Euryarchaea. These data highlight potential differences in the roles of Bacteria and Archaea in the deep sea biogeochemical cycles and shed light on the importance of deep-sea Euryarchaea in the degradation of dissolved organic matter., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Ammonia oxidation, the first step in nitrification, is performed by certain Beta- and Gammaproteobacteria and Crenarchaea to generate metabolic energy. Ammonia monooxygenase (amoA) genes from both Bacteria and Crenarchaea have been found in a variety of marine ecosystems, but the relative importance of Bacteria versus Crenarchaea in ammonia oxidation is unresolved, and seasonal comparisons are rare. In this study, we compared the abundance of betaproteobacterial and crenarchaeal amoA genes in the coastal Arctic Ocean during summer and winter over 2 years. Summer and winter betaproteobacterial amoA clone libraries were significantly different, although the gene sequences were similar to those found in temperate and polar environments. Betaproteobacterial and crenarchaeal amoA genes were 30- to 115-fold more abundant during the winter than during the summer in both years of the study. Archaeal amoA genes were more abundant than betaproteobacterial amoA genes in the first year, but betaproteobacterial amoA was more abundant than archaeal amoA the following year. The ratio of archaeal amoA gene copies to marine group I crenarchaeal 16S rRNA genes averaged 2.9 over both seasons and years, suggesting that ammonia oxidation was common in Crenarchaea at this location. Potential nitrification rates, as well as the total amoA gene abundance, were highest in the winter when competition with phytoplankton was minimal and ammonium concentrations were the highest. These results suggest that ammonium concentrations were important in determining the rates of ammonia oxidation and the abundance of ammonia-oxidizing Betaproteobacteria and Crenarchaea.

Anaerobic anoxygenic phototrophic bacteria can be found in the suboxic waters of shallow stratified coastal systems, and may play important roles in the total primary production of subtropical stratified coastal lagoons. We investigated the spatiotemporal variability of light CO(2) fixation and net oxygen production in the stratified Conceição Lagoon (Brazil) in summer and fall of 2007, as well as the contribution of bacteriochlorophyll a (BChl a)-containing bacteria to photosynthetically driven electron transfer. Both chlorophyll a (Chl a) and BChl a varied in space, while only BChl a varied in time (three-fold increase from summer to fall). In summer, net oxygen production and light CO(2) fixation were correlated, with both having higher rates with higher Chl a concentrations in the enclosed region of the lagoon. In fall, CO(2) fixation was decoupled from oxygen production. Denaturing gradient gel electrophoresis revealed that bacterial communities of oxic site 12 and suboxic site 33 formed one cluster, different from other oxic samples within the lagoon. In addition, BChl a/Chl a ratios at these sites were high, 40% and 45%, respectively. Light acted as the main factor controlling the BChl a concentration and CO(2) fixation rates. High turbidity within the enclosed area of the lagoon explained high BChl a and decoupling between CO(2) fixation and oxygen production in oxygenated waters. Contribution of purple sulfur bacteria to total bacterial density in suboxic waters was 1.2%, and their biomass contributed to a much higher percentage (12.2%) due to their large biovolume. Our results indicate a significant contribution of anaerobic anoxygenic bacteria to the primary production of the "dead zone" of Conceição Lagoon.

Aerobic anoxygenic phototrophic bacteria (AAP) represent an important fraction of bacterioplankton assemblages in various oceanic regimes. Although their abundance and distribution have been explored recently in diverse oceanic regions, the environmental factors controlling the population structure and diversity of these photoheterotrophic bacteria remain poorly understood. Here, we investigate the horizontal and vertical distributions and the genetic diversity of AAP populations collected in late summer throughout the Mediterranean Sea using pufM-temporal temperature gel gradient electrophoresis (TTGE) and clone library analyses. The TTGE profiles and clone libraries analyzed using multivariate statistical methods demonstrated a horizontal and vertical zonation of AAP assemblages. Physicochemical parameters such as pH, inorganic nitrogen compounds, photosynthetically active radiation, total organic carbon and to a lesser extent particulate organic nitrogen and phosphorus, and biogenic activities (e.g. bacterial production, cell densities), acted in synergy to explain the population changes with depth. About half of the pufM sequences were <94% identical to known sequences. The AAP populations were predominantly (~80%) composed of Gammaproteobacteria, unlike most previously explored marine systems. Our results suggest that genetically distinct ecotypes inhabiting different niches may exist in natural AAP populations of the Mediterranean Sea whose genetic diversity is typical of oligotrophic environments., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Photoheterotrophic microbes use organic substrates and light energy to satisfy their demand for carbon and energy and seem to be well adapted to eutrophic estuarine and oligotrophic oceanic environments. One type of photoheterotroph, aerobic anoxygenic phototrophic (AAP) bacteria, is especially abundant in particle-rich, turbid estuaries. To explore questions regarding the controls of these photoheterotrophic bacteria, we examined their abundance by epifluorescence microscopy, concentrations of the light-harvesting pigment, bacteriochlorophyll a (BChl a) and the diversity of pufM and 16S ribosomal RNA (rRNA) genes in the Chesapeake Bay. Concentrations of BChl a varied substantially, much more so than AAP bacterial abundance, along the estuarine salinity gradient. The BChl a concentration was correlated with turbidity only when oceanic and estuarine waters were considered together. Concentrations of BChl a and BChl a quotas were higher in particle-associated than in free-living AAP bacterial communities and appear to reflect physiological adaptation, not different AAP bacterial communities; pufM genes did not differ between particle-associated and free-living communities. In contrast, particle-associated and free-living bacterial communities were significantly different, on the basis of the analysis of 16S rRNA genes. The BChl a quota of AAP bacteria was not correlated with turbidity, suggesting that pigment synthesis varies in direct response to particles, not light attenuation. The AAP bacteria seem to synthesize more BChl a when dissolved and particulate substrates are available than when only dissolved materials are accessible, which has implications for understanding the impact of substrates on the level of photoheterotrophy compared with heterotrophy in AAP bacteria.

Bacterial communities in the surface layer of the oceans consist of a few abundant phylotypes and many rare ones, most with unknown ecological functions and unclear roles in biogeochemical processes. To test hypotheses about relationships between abundant and rare phylotypes, we examined bacterial communities in the western Arctic Ocean using pyrosequence data of the V6 region of the 16S rRNA gene. Samples were collected from various locations in the Chukchi Sea, the Beaufort Sea and Franklin Bay in summer and winter. We found that bacterial communities differed between summer and winter at a few locations, but overall there was no significant difference between the two seasons in spite of large differences in biogeochemical properties. The sequence data suggested that abundant phylotypes remained abundant while rare phylotypes remained rare between the two seasons and among the Arctic regions examined here, arguing against the 'seed bank' hypothesis. Phylotype richness was calculated for various bacterial groups defined by sequence similarity or by phylogeny (phyla and proteobacterial classes). Abundant bacterial groups had higher within-group diversity than rare groups, suggesting that the ecological success of a bacterial lineage depends on diversity rather than on the dominance of a few phylotypes. In these Arctic waters, in spite of dramatic variation in several biogeochemical properties, bacterial community structure was remarkably stable over time and among regions, and any variation was due to the abundant phylotypes rather than rare ones.

Proteorhodopsin (PR)-containing bacteria are hypothesized to use both light and organic compounds as energy sources. Recent studies have found that PR is common in marine microorganisms, but the impact of light on the growth of PR-containing organisms and PR transcription in the environment remains unclear. We examined the diversity of PR genes and transcripts by PCR amplification and sequencing in Delaware coastal waters. Clone libraries of PR DNA and cDNA (from mRNA) revealed large differences between bacterial groups in expression of PR genes. We then evaluated by quantitative PCR the impact of light on growth and PR expression in PR-containing SAR11 bacteria (SAR11-PR) and a population of Flavobacteria (Flavobacteria-PR). This experiment was conducted in 30 l microcosms exposed to continuous light, continuous dark, and 12 h-12 h dark-light cycles for 5 days. We found a strong upregulation of PR expression by light in Flavobacteria-PR and SAR11-PR. The abundance of PR transcripts per PR cell was enhanced up to 120-fold under continuous light and up to 20-fold under dark-light cycles while continuous darkness led to very low levels of PR mRNA. This upregulation of PR expression was correlated with the abundance of PR genes, indicating net growth of SAR11-PR cells and Flavobacteria-PR under dark-light cycles. SAR11-PR and Flavobacteria-PR abundance decreased under continuous light despite upregulation of PR expression, and continuous darkness led to low abundances of both populations. Collectively, these data suggest that light affects growth of PR-containing bacteria and regulation of PR mRNA synthesis in natural communities.

Metagenomic studies characterize both the composition and diversity of uncultured viral and microbial communities. BLAST-based comparisons have typically been used for such analyses; however, sampling biases, high percentages of unknown sequences, and the use of arbitrary thresholds to find significant similarities can decrease the accuracy and validity of estimates. Here, we present Genome relative Abundance and Average Size (GAAS), a complete software package that provides improved estimates of community composition and average genome length for metagenomes in both textual and graphical formats. GAAS implements a novel methodology to control for sampling bias via length normalization, to adjust for multiple BLAST similarities by similarity weighting, and to select significant similarities using relative alignment lengths. In benchmark tests, the GAAS method was robust to both high percentages of unknown sequences and to variations in metagenomic sequence read lengths. Re-analysis of the Sargasso Sea virome using GAAS indicated that standard methodologies for metagenomic analysis may dramatically underestimate the abundance and importance of organisms with small genomes in environmental systems. Using GAAS, we conducted a meta-analysis of microbial and viral average genome lengths in over 150 metagenomes from four biomes to determine whether genome lengths vary consistently between and within biomes, and between microbial and viral communities from the same environment. Significant differences between biomes and within aquatic sub-biomes (oceans, hypersaline systems, freshwater, and microbialites) suggested that average genome length is a fundamental property of environments driven by factors at the sub-biome level. The behavior of paired viral and microbial metagenomes from the same environment indicated that microbial and viral average genome sizes are independent of each other, but indicative of community responses to stressors and environmental conditions.

Photoheterotrophic microbes, which are capable of utilizing dissolved organic materials and harvesting light energy, include coccoid cyanobacteria (Synechococcus and Prochlorococcus), aerobic anoxygenic phototrophic (AAP) bacteria, and proteorhodopsin (PR)-containing bacteria. Our knowledge of photoheterotrophic microbes is largely incomplete, especially for high-latitude waters such as the Arctic Ocean, where photoheterotrophs may have special ecological relationships and distinct biogeochemical impacts due to extremes in day length and seasonal ice cover. These microbes were examined by epifluorescence microscopy, flow cytometry, and quantitative PCR (QPCR) assays for PR and a gene diagnostic of AAP bacteria (pufM). The abundance of AAP bacteria and PR-containing bacteria decreased from summer to winter, in parallel with a threefold decrease in the total prokaryotic community. In contrast, the abundance of Synechococcus organisms did not decrease in winter, suggesting that their growth was supported by organic substrates. Results from QPCR assays revealed no substantial shifts in the community structure of AAP bacteria and PR-containing bacteria. However, Arctic PR genes were different from those found at lower latitudes, and surprisingly, they were not similar to those in Antarctic coastal waters. Photoheterotrophic microbes appear to compete successfully with strict heterotrophs during winter darkness below the ice, but AAP bacteria and PR-containing bacteria do not behave as superior competitors during the summer.

Biovolume is an important characteristic of cells that shapes the contribution of microbes to total biomass and biogeochemical cycling. Most studies of bacterial cell volumes use DAPI (4',6'-diamidino-2-phenylindole), which stains nucleic acids and therefore only a portion of the cell. We used SYPRO Ruby protein stain combined with fluorescence in situ hybridization to examine biovolumes of bacteria in the total community, as well in phylogenetic subgroups. Protein-based volumes varied more and were consistently larger than DNA-based volumes by 3.3-fold on average. Bacterial cells were ca. 30% larger in the Arctic Ocean and Antarctic coastal waters than in temperate regimes. We hypothesized that geographic differences in the abundance of specific bacterial groups drove the observed patterns in biovolume. In support of this hypothesis, we found that Gammaproteobacteria and members of the Sphingobacteria-Flavobacteria group were larger in higher-latitude waters and that the mean volumes of both groups were larger than the mean bacterial volume in all environments tested. The mean cell size of SAR11 bacteria was larger than the mean cell size of the total bacterial community on average, although this varied. Protein staining increases the accuracy of biovolume measurements and gives insights into how the biomass of marine microbial communities varies over time and space.

The diversity and abundance of glycosyl hydrolase family 5 (GH5) were studied in the North Atlantic Ocean. This family was chosen because of the large number of available sequences from cultured bacteria, the variety of substrates it targets, and the high number of similar sequences in the Sargasso Sea environmental genome database. Three clone libraries of a GH5 subcluster were constructed from the Mid-Atlantic Bight and the eastern and western North Atlantic Ocean. The two North Atlantic Ocean libraries did not differ from each other but both were significantly less diverse than the Mid-Atlantic Bight library. The abundance of GH5 genes estimated by quantitative PCR was positively correlated with chlorophyll concentrations in the eastern part of a transect from Fort Pierce, Florida, to the Azores and in a depth profile, suggesting that the supply of labile organic material selects for GH5-bearing bacteria in these waters. However, the data suggest that only <1% of all bacteria harbor the GH5 subcluster. These and other data suggest that the hydrolysis of polysaccharides requires complicated multi-enzyme systems.

Proteorhodopsin (PR) is a light-driven proton pump that has been found in a variety of marine bacteria, including Pelagibacter ubique, a member of the ubiquitous SAR11 clade. The goals of this study were to explore the diversity of PR genes and to estimate their abundance in the North Atlantic Ocean using quantitative polymerase chain reaction (QPCR). We found that PR genes in the western portion of the Sargasso Sea could be grouped into 27 clusters, but five clades had the most sequences. Sets of specific QPCR primers were designed to examine the abundance of PR genes in the following four of the five clades: SAR11 (P. ubique and other SAR11 Alphaproteobacteria), BACRED17H8 (Alphaproteobacteria), HOT2C01 (Alphaproteobacteria) and an uncultured subgroup of the Flavobacteria. Two groups (SAR11 and HOT2C01) dominated PR gene abundance in oligotrophic waters, but were significantly less abundant in nutrient- and chlorophyll-rich waters. The other two groups (BACRED17H8 and Flavobacteria subgroup NASB) were less abundant in all waters. Together, these four PR gene types were found in 50% of all bacteria in the Sargasso Sea. We found a significant negative correlation between total PR gene abundance and nutrients and chlorophyll but no significant correlation with light intensity for three of the four PR types in the depth profiles north of the Sargasso Sea. Our data suggest that PR is common in the North Atlantic Ocean, especially in SAR11 bacteria and another marine alphaproteobacterial group (HOT2C01), and that these PR-bearing bacteria are most abundant in oligotrophic waters.

We examined the contribution of photoheterotrophic microbes--those capable of light-mediated assimilation of organic compounds--to bacterial production and amino acid assimilation along a transect from Florida to Iceland from 28 May to 9 July 2005. Bacterial production (leucine incorporation at a 20 nM final concentration) was on average 30% higher in light than in dark-incubated samples, but the effect varied greatly (3% to 60%). To further characterize this light effect, we examined the abundance of potential photoheterotrophs and measured their contribution to bacterial production and amino acid assimilation (0.5 nM addition) using flow cytometry. Prochlorococcus and Synechococcus were abundant in surface waters where light-dependent leucine incorporation was observed, whereas aerobic anoxygenic phototrophic bacteria were abundant but did not correlate with the light effect. The per-cell assimilation rates of Prochlorococcus and Synechococcus were comparable to or higher than those of other prokaryotes, especially in the light. Picoeukaryotes also took up leucine (20 nM) and other amino acids (0.5 nM), but rates normalized to biovolume were much lower than those of prokaryotes. Prochlorococcus was responsible for 80% of light-stimulated bacterial production and amino acid assimilation in surface waters south of the Azores, while Synechococcus accounted for on average 12% of total assimilation. However, nearly 40% of the light-stimulated leucine assimilation was not accounted for by these groups, suggesting that assimilation by other microbes is also affected by light. Our results clarify the contribution of cyanobacteria to photoheterotrophy and highlight the potential role of other photoheterotrophs in biomass production and dissolved-organic-matter assimilation.

Little is known about the abundance, distribution, and ecology of aerobic anoxygenic phototrophic (AAP) bacteria, particularly in oligotrophic environments, which represent 60% of the ocean. We investigated the abundance of AAP bacteria across the South Pacific Ocean, including the center of the gyre, the most oligotrophic water body of the world ocean. AAP bacteria, Prochlorococcus, and total prokaryotic abundances, as well as bacteriochlorophyll a (BChl a) and divinyl-chlorophyll a concentrations, were measured at several depths in the photic zone along a gradient of oligotrophic conditions. The abundances of AAP bacteria and Prochlorococcus were high, together accounting for up to 58% of the total prokaryotic community. The abundance of AAP bacteria alone was up to 1.94 x 10(5) cells ml(-1) and as high as 24% of the overall community. These measurements were consistent with the high BChl a concentrations (up to 3.32 x 10(-3) microg liter(-1)) found at all stations. However, the BChl a content per AAP bacterial cell was low, suggesting that AAP bacteria are mostly heterotrophic organisms. Interestingly, the biovolume and therefore biomass of AAP bacteria was on average twofold higher than that of other prokaryotic cells. This study demonstrates that AAP bacteria can be abundant in various oligotrophic conditions, including the most oligotrophic regime of the world ocean, and can account for a large part of the bacterioplanktonic carbon stock.

This study examined bacterial community structure of biofilms on stainless steel and polycarbonate in seawater from the Delaware Bay. Free-living bacteria in the surrounding seawater were compared to the attached bacteria during the first few weeks of biofilm growth. Surfaces exposed to seawater were analyzed by using 16S rDNA libraries, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE). Community structure of the free-living bacterial community was different from that of the attached bacteria according to FISH and DGGE. In particular, alpha-proteobacteria dominated the attached communities. Libraries of 16S rRNA genes revealed that representatives of the Rhodobacterales clade were the most abundant members of biofilm communities. Changes in community structure during biofilm growth were also examined by DGGE analysis. We hypothesized that bacterial communities on dissimilar surfaces would initially differ and become more similar over time. In contrast, the compositions of stainless steel and polycarbonate biofilms were initially the same, but differed after about 1 week of biofilm growth. These data suggest that the relationship between surface properties and biofilm community structure changes as biofilms grow on surfaces such as stainless steel and polycarbonate in estuarine water.

The abundance of aerobic anoxygenic phototrophic (AAP) bacteria, cyanobacteria, and heterotrophs was examined in the Mid-Atlantic Bight and the central North Pacific Gyre using infrared fluorescence microscopy coupled with image analysis and flow cytometry. AAP bacteria comprised 5% to 16% of total prokaryotes in the Atlantic Ocean but only 5% or less in the Pacific Ocean. In the Atlantic, AAP bacterial abundance was as much as 2-fold higher than that of Prochlorococcus spp. and 10-fold higher than that of Synechococcus spp. In contrast, Prochlorococcus spp. outnumbered AAP bacteria 5- to 50-fold in the Pacific. In both oceans, subsurface abundance maxima occurred within the photic zone, and AAP bacteria were least abundant below the 1% light depth. The abundance of AAP bacteria rivaled some groups of strictly heterotrophic bacteria and was often higher than the abundance of known AAP bacterial genera (Erythrobacter and Roseobacter spp.). Concentrations of bacteriochlorophyll a (BChl a) were low ( approximately 1%) compared to those of chlorophyll a in the North Atlantic. Although the BChl a content of AAP bacteria per cell was typically 20- to 250-fold lower than the divinyl-chlorophyll a content of Prochlorococcus, the pigment content of AAP bacteria approached that of Prochlorococcus in shelf break water. Our results suggest that AAP bacteria can be quite abundant in some oceanic regimes and that their distribution in the water column is consistent with phototrophy.

To determine whether metagenomic libraries sample adequately the dominant bacteria in aquatic environments, we examined the phylogenetic make-up of a large insert metagenomic library constructed with bacterial DNA from the Delaware River, a polymerase chain reaction (PCR) library of 16S rRNA genes, and community structure determined by fluorescence in situ hybridization (FISH). The composition of the libraries and community structure determined by FISH differed for the major bacterial groups in the river, which included Actinobacteria, beta-proteobacteria and Cytophaga-like bacteria. Beta-proteobacteria were underrepresented in the metagenomic library compared with the PCR library and FISH, while Cytophaga-like bacteria were more abundant in the metagenomic library than in the PCR library and in the actual community according to FISH. The Delaware River libraries contained bacteria belonging to several widespread freshwater clusters, including clusters of Polynucleobacter necessarius, Rhodoferax sp. Bal47 and LD28 beta-proteobacteria, the ACK-m1 and STA2-30 clusters of Actinobacteria, and the PRD01a001B Cytophaga-like bacteria cluster. Coverage of bacteria with > 97% sequence identity was 65% and 50% for the metagenomic and PCR libraries respectively. Rarefaction analysis of replicate PCR libraries and of a library constructed with re-conditioned amplicons indicated that heteroduplex formation did not substantially impact the composition of the PCR library. This study suggests that although it may miss some bacterial groups, the metagenomic approach can sample other groups (e.g. Cytophaga-like bacteria) that are potentially underrepresented by other culture-independent approaches.

Sequence analysis of environmental DNA promises to provide new insights into the ecology and biogeochemistry of uncultured marine microbes. In this study we used the Sargasso Sea Whole Genome Sequence (WGS) data set to search for hydrolases used by Cytophaga-like bacteria to degrade biopolymers such as polysaccharides and proteins. Analysis of the Sargasso WGS data for contigs bearing both the 16S rRNA genes of Cytophaga-like bacteria and hydrolase genes revealed a cellulase gene (celM) most similar to the gene found in Cytophaga hutchinsonii. A BLAST search of the entire Sargasso Sea WGS data set indicated that celM was the most abundant cellulase-like gene in the Sargasso Sea. However, the similarity between CelM-like cellulases and peptidases belonging to metalloprotease family M42 led us to question whether CelM is involved in the degradation of polysaccharides or proteins. PCR primers were designed for the celM genes in the Sargasso Sea WGS data set and used to identify celM in a fosmid library constructed with prokaryotic DNA from the western Arctic Ocean. Expression analysis of the Cytophaga-like Arctic CelM, which is 63% identical and 77% similar to CelM in C. hutchinsonii, indicated that there was peptidase activity, whereas cellulase activity was not detected. Our analysis suggests that the celM gene plays a role in the degradation of protein by Cytophaga-like bacteria. The abundance of peptidase genes in the Cytophaga-like fosmid clone provides further evidence for the importance of Cytophaga-like bacteria in the degradation of protein in high-molecular-weight dissolved organic matter.

The contribution of major bacterial groups to the assimilation of extracellular polymeric substances (EPS) and glucose in the Delaware Estuary was assessed using microautoradiography and fluorescence in situ hybridization. Bacterial groups contributed to EPS and glucose assimilation in part according to their distribution in the estuary. Abundance of the phylogenetic groups explained 35% and 55% of the variation in EPS and glucose assimilation, respectively. Actinobacteria contributed 70% to glucose assimilation in freshwater, while Alphaproteobacteria assimilated 60% of this compound in saline water. In contrast, various bacterial groups dominated the assimilation of EPS. Actinobacteria and Betaproteobacteria contributed the most in the freshwater section, whereas Cytophaga-like bacteria and Alpha- and Gammaproteobacteria participated in EPS assimilation in the lower part of the estuary. In addition, we examined the fraction of bacteria in each group that assimilated glucose or EPS. Overall, the fraction of bacteria in all groups that assimilated glucose was higher than the fraction that assimilated EPS (15 to 30% versus 5 to 20%, respectively). We found no correlation between the relative abundance of a group in the estuary and the fraction of bacteria actively assimilating glucose or EPS; the more active groups were often less abundant. Our results imply that the bacterial community in the Delaware Estuary is not controlled solely by "bottom-up" factors such as dissolved organic matter.

Members of the SAR11 clade often dominate the composition of marine microbial communities, yet their contribution to biomass production and the flux of dissolved organic matter (DOM) is unclear. In addition, little is known about the specific components of the DOM pool utilized by SAR11 bacteria. To better understand the role of SAR11 bacteria in the flux of DOM, we examined the assimilation of leucine (a measure of biomass production), as well as free amino acids, protein, and glucose, by SAR11 bacteria in the Northwest Atlantic Ocean. We found that when SAR11 bacteria were >25% of total prokaryotes, they accounted for about 30 to 50% of leucine incorporation, suggesting that SAR11 bacteria were major contributors to bacterial biomass production and the DOM flux. Specific growth rates of SAR11 bacteria either equaled or exceeded growth rates for the total prokaryotic community. In addition, SAR11 bacteria were typically responsible for a greater portion of amino acid assimilation (34 to 61%) and glucose assimilation (45 to 57%) than of protein assimilation (< or = 34%). These data suggest that SAR11 bacteria do not utilize various components of the DOM pool equally and may be more important to the flux of low-molecular-weight monomers than to that of high-molecular-weight polymers.