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In oligotrophic oceans, the smallest eukaryotic phytoplankton are both significant primary producers and predators of abundant bacteria such as Prochlorococcus. However, the drivers and consequences of community dynamics among these diverse protists are not well understood. Here, we investigated how trophic strategies along the autotrophy-mixotrophy spectrum vary in importance over time and across depths at Station ALOHA in the North Pacific Subtropical Gyre. We combined picoeukaryote community composition from a 28-month time-series with traits of diverse phytoplankton isolates from the same location, to examine trophic strategies across 13 operational taxonomic units and 8 taxonomic classes. We found that autotrophs and slower-grazing mixotrophs tended to prevail deeper in the photic zone, while the most voracious mixotrophs were relatively abundant near the surface. Within the mixed layer, there was greater phagotrophy when conditions were most stratified and when Chl a concentrations were lowest, although the greatest temporal variation in trophic strategy occurred at intermediate depths (45-100 m). Dynamics at this site are consistent with previously described spatial patterns of trophic strategies. The success of relatively phagotrophic phytoplankton at shallower depths in the most stratified waters suggests that phagotrophy is a competitive strategy for acquiring nutrients when energy from light is plentiful., (© 2024 John Wiley & Sons Ltd.)

Marine microbes are important in biogeochemical cycling, but the nature and magnitude of their contributions are influenced by their associated viruses. In the presence of a lytic virus, cells that have evolved resistance to infection have an obvious fitness advantage over relatives that remain susceptible. However, susceptible cells remain extant in the wild, implying that the evolution of a fitness advantage in one dimension (virus resistance) must be accompanied by a fitness cost in another dimension. Identifying costs of resistance is challenging because fitness is context-dependent. We examined the context dependence of fitness costs in isolates of the picophytoplankton genus Micromonas and their co-occurring dsDNA viruses using experimental evolution. After generating 88 resistant lineages from two ancestral Micromonas strains, each challenged with one of four distinct viral strains, we found resistance led to a 46% decrease in mean growth rate under high irradiance and a 19% decrease under low. After a year in culture, the experimentally selected lines remained resistant, but fitness costs had attenuated. Our results suggest that the cost of resistance in Micromonas is dependent on environmental conditions and the duration of population adaptation, illustrating the dynamic nature of fitness costs of viral resistance among marine protists., (© 2024 John Wiley & Sons Ltd.)

We present the genome assembly of the pennate diatom Epithemia pelagica strain UHM3201 (Ochrophyta; Bacillariophyceae; Rhopalodiales; Rhopalodiaceae) and that of its cyanobacterial endosymbiont (Chroococcales: Aphanothecaceae). The genome sequence of the diatom is 60.3 megabases in span, and the cyanobacterial genome has a length of 2.48 megabases. Most of the diatom nuclear genome assembly is scaffolded into 15 chromosomal pseudomolecules. The organelle genomes have also been assembled, with the mitochondrial genome 40.08 kilobases and the plastid genome 130.75 kilobases in length. A number of other prokaryote MAGs were also assembled., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Schvarcz CR et al.)

Viruses in the phylum Nucleocytoviricota are large, complex and have an exceptionally diverse metabolic repertoire. Some encode hundreds of products involved in the translation of mRNA into protein, but none was known to encode any of the proteins in ribosomes, the central engines of translation. With the discovery of the eL40 gene in FloV-SA2, we report the first example of a eukaryotic virus encoding a ribosomal protein and show that this gene is also present and expressed in other uncultivated marine giant viruses. FloV-SA2 also encodes a "group II" viral rhodopsin, a viral light-activated protein of unknown function previously only reported in metagenomes. FloV-SA2 is thus a valuable model system for investigating new mechanisms by which viruses manipulate eukaryotic cell metabolism., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2024.)

A large fraction of marine primary production is performed by diverse small protists, and many of these phytoplankton are phagotrophic mixotrophs that vary widely in their capacity to consume bacterial prey. Prior analyses suggest that mixotrophic protists as a group vary in importance across ocean environments, but the mechanisms leading to broad functional diversity among mixotrophs, and the biogeochemical consequences of this, are less clear. Here we use isolates from seven major taxa to demonstrate a tradeoff between phototrophic performance (growth in the absence of prey) and phagotrophic performance (clearance rate when consuming Prochlorococcus ). We then show that trophic strategy along the autotrophy-mixotrophy spectrum correlates strongly with global niche differences, across depths and across gradients of stratification and chlorophyll a . A model of competition shows that community shifts can be explained by greater fitness of faster-grazing mixotrophs when nutrients are scarce and light is plentiful. Our results illustrate how basic physiological constraints and principles of resource competition can organize complexity in the surface ocean ecosystem.

The south shore of O'ahu, Hawai'i is one of the most visited coastal tourism areas in the United States with some of the highest instances of recreational waterborne disease. A population of the pathogenic bacterium Vibrio vulnificus lives in the estuarine Ala Wai Canal in Honolulu which surrounds the heavily populated tourism center of Waikīkī. We developed a statistical model to predict V. vulnificus dynamics in this system using environmental measurements from moored oceanographic and atmospheric sensors in real time. During a year-long investigation, we analyzed water from 9 sampling events at 3 depths and 8 sites along the canal (n = 213) for 36 biogeochemical variables and V. vulnificus concentration using quantitative polymerase chain reaction (qPCR) of the hemolysin A gene (vvhA). The best multiple linear regression model of V. vulnificus concentration, explaining 80% of variation, included only six predictors: 5-day average rainfall preceding water sampling, daily maximum air temperature, water temperature, nitrate plus nitrite, and two metrics of humic dissolved organic matter (DOM). We show how real-time predictions of V. vulnificus concentration can be made using these models applied to the time series of water quality measurements from the Pacific Islands Ocean Observing System (PacIOOS) as well as the PacIOOS plume model based on the Waikīkī Regional Ocean Modeling System (ROMS) products. These applications highlight the importance of including DOM variables in predictive modeling of V. vulnificus and the influence of rain events in elevating nearshore concentrations of V. vulnificus. Long-term climate model projections of locally downscaled monthly rainfall and air temperature were used to predict an overall increase in V. vulnificus concentration of approximately 2- to 3-fold by 2100. Improving these predictive models of microbial populations is critical for management of waterborne pathogen risk exposure, particularly in the wake of a changing global climate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Small eukaryotic phytoplankton are major contributors to global primary production and marine biogeochemical cycles. Many taxa are thought to be mixotrophic, but quantitative studies of phagotrophy exist for very few. In addition, little is known about consumers of Prochlorococcus, the abundant cyanobacterium at the base of oligotrophic ocean food webs. Here we describe thirty-nine new phytoplankton isolates from the North Pacific Subtropical Gyre (Station ALOHA), all flagellates ~2-5 µm diameter, and we quantify their ability to graze Prochlorococcus. The mixotrophs are from diverse classes (dictyochophytes, haptophytes, chrysophytes, bolidophytes, a dinoflagellate, and a chlorarachniophyte), many from previously uncultured clades. Grazing ability varied substantially, with specific clearance rate (volume cleared per body volume) varying over ten-fold across isolates and six-fold across genera. Slower grazers tended to create more biovolume per prey biovolume consumed. Using qPCR we found that the haptophyte Chrysochromulina was most abundant among the isolated mixotrophs at Station ALOHA, with 76-250 cells mL -1 across depths in the upper euphotic zone (5-100 m). Our results show that within a single ecosystem the phototrophs that ingest bacteria come from many branches of the eukaryotic tree, and are functionally diverse, indicating a broad range of strategies along the spectrum from phototrophy to phagotrophy., (© 2022. The Author(s).)

To better understand the controls on the opportunistic human pathogen Vibrio vulnificus in warm tropical waters, we conducted a year-long investigation in the Ala Wai Canal, a channelized estuary in Honolulu, HI. The abundance of V. vulnificus, as determined by quantitative PCR (qPCR) of the hemolysin gene ( vvhA ), varied spatially and temporally by nearly 4 orders of magnitude (≤3 to 14,000 mL -1 ). Unlike in temperate and subtropical systems, temperatures were persistently warm (19 to 31°C) and explained little of the variability in V. vulnificus abundance. Salinity (1 to 36 ppt) had a significant, but nonlinear, relationship with V. vulnificus abundance with the highest vvhA concentrations (>2,500 mL -1 ) observed only at salinities from 7 to 22 ppt. V. vulnificus abundances were lower on average during the summer dry season, when waters were warmer but more saline. The highest canal-wide average abundances were observed during a time of modest rainfall, when moderate salinities and elevated concentrations of reduced nitrogen species and silica suggested a groundwater influence. Parallel quantification of the vcgC gene suggested that C-type strains, which are responsible for most human infections, comprised 25% of the total V. vulnificus on average, but their relative contribution was greater at higher salinities, suggesting a broader salinity tolerance. Generalized regression models suggested that up to 67% of sample-to-sample variation ( n  = 202) in log-transformed V. vulnificus abundance was explained using the measured environmental variables, and up to 97% of the monthly variation in canal-wide average concentrations ( n  = 13) was explained with the best subset of four variables. IMPORTANCE Our data illustrate that, in the absence of strong seasonal variation in water temperature in the tropics, variation in salinity driven by rainfall becomes a primary controlling variable on V. vulnificus abundance. There is thus a tendency for a rainfall-driven seasonal cycle in V. vulnificus abundance which is inverted from the temperature-driven seasonal cycle at higher latitudes. However, stochasticity in rainfall and its nonlinear, indirect effects on V. vulnificus concentration means that high abundances can occur at any location in the canal at any time of year, making it challenging to predict concentrations of this pathogen at a high temporal or spatial resolution. Much of the variability in canal-wide average concentrations, on the other hand, was explained by a few variables that reflect the magnitude of freshwater input to the system, suggesting that relative risk of exposure to this pathogen could be predicted as an average for the system.

Persistent nitrogen depletion in sunlit open ocean waters provides a favorable ecological niche for nitrogen-fixing (diazotrophic) cyanobacteria, some of which associate symbiotically with eukaryotic algae. All known marine examples of these symbioses have involved either centric diatom or haptophyte hosts. We report here the discovery and characterization of two distinct marine pennate diatom-diazotroph symbioses, which until now had only been observed in freshwater environments. Rhopalodiaceae diatoms Epithemia pelagica sp. nov. and Epithemia catenata sp. nov. were isolated repeatedly from the subtropical North Pacific Ocean, and analysis of sequence libraries reveals a global distribution. These symbioses likely escaped attention because the endosymbionts lack fluorescent photopigments, have nifH gene sequences similar to those of free-living unicellular cyanobacteria, and are lost in nitrogen-replete medium. Marine Rhopalodiaceae-diazotroph symbioses are a previously overlooked but widespread source of bioavailable nitrogen in marine habitats and provide new, easily cultured model organisms for the study of organelle evolution., (© 2022. The Author(s).)

Understanding how phenotypes emerge from genotypes is a foundational goal in biology. As challenging as this task is when considering cellular life, it is further complicated in the case of viruses. During replication, a virus as a discrete entity (the virion) disappears and manifests itself as a metabolic amalgam between the virus and the host (the virocell). Identifying traits that unambiguously constitute a virus's phenotype is straightforward for the virion, less so for the virocell. Here, we present a framework for categorizing virus phenotypes that encompasses both virion and virocell stages and considers functional and performance traits of viruses in the context of fitness. Such an integrated view of virus phenotype is necessary for comprehensive interpretation of viral genome sequences and will advance our understanding of viral evolution and ecology., (© 2021. Springer Nature Limited.)

Motivation: Metagenomic approaches hold the potential to characterize microbial communities and unravel the intricate link between the microbiome and biological processes. Assembly is one of the most critical steps in metagenomics experiments. It consists of transforming overlapping DNA sequencing reads into sufficiently accurate representations of the community's genomes. This process is computationally difficult and commonly results in genomes fragmented across many contigs. Computational binning methods are used to mitigate fragmentation by partitioning contigs based on their sequence composition, abundance or chromosome organization into bins representing the community's genomes. Existing binning methods have been principally tuned for bacterial genomes and do not perform favorably on viral metagenomes., Results: We propose Composition and Coverage Network (CoCoNet), a new binning method for viral metagenomes that leverages the flexibility and the effectiveness of deep learning to model the co-occurrence of contigs belonging to the same viral genome and provide a rigorous framework for binning viral contigs. Our results show that CoCoNet substantially outperforms existing binning methods on viral datasets., Availability and Implementation: CoCoNet was implemented in Python and is available for download on PyPi (https://pypi.org/). The source code is hosted on GitHub at https://github.com/Puumanamana/CoCoNet and the documentation is available at https://coconet.readthedocs.io/en/latest/index.html. CoCoNet does not require extensive resources to run. For example, binning 100k contigs took about 4 h on 10 Intel CPU Cores (2.4 GHz), with a memory peak at 27 GB (see Supplementary Fig. S9). To process a large dataset, CoCoNet may need to be run on a high RAM capacity server. Such servers are typically available in high-performance or cloud computing settings., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Viruses span an impressive size range, with genome length varying a thousandfold and virion volume nearly a millionfold. For cellular organisms the scaling of traits with size is a pervasive influence on ecological processes, but whether size plays a central role in viral ecology is unknown. Here, we focus on viruses of aquatic unicellular organisms, which exhibit the greatest known range of virus size. We outline hypotheses within a quantitative framework, and analyse data where available, to consider how size affects the primary components of viral fitness. We argue that larger viruses have fewer offspring per infection and slower contact rates with host cells, but a larger genome tends to increase infection efficiency, broaden host range, and potentially increase attachment success and decrease decay rate. These countervailing selective pressures may explain why a breadth of sizes exist and even coexist when infecting the same host populations. Oligotrophic ecosystems may be enriched in "giant" viruses, because environments with resource-limited phagotrophs at low concentrations may select for broader host range, better control of host metabolism, lower decay rate and a physical size that mimics bacterial prey. Finally, we describe where further research is needed to understand the ecology and evolution of viral size diversity., (© 2020 John Wiley & Sons Ltd.)

Potassium ion (K + ) channels have been observed in diverse viruses that infect eukaryotic marine and freshwater algae. However, experimental evidence for functional K + channels among these alga-infecting viruses has thus far been restricted to members of the family Phycodnaviridae, which are large, double-stranded DNA viruses within the phylum Nucleocytoviricota . Recent sequencing projects revealed that alga-infecting members of Mimiviridae , another family within this phylum, may also contain genes encoding K + channels. Here we examine the structural features and the functional properties of putative K + channels from four cultivated members of Mimiviridae . While all four proteins contain variations of the conserved selectivity filter sequence of K + channels, structural prediction algorithms suggest that only two of them have the required number and position of two transmembrane domains that are present in all K + channels. After in vitro translation and reconstitution of the four proteins in planar lipid bilayers, we confirmed that one of them, a 79 amino acid protein from the virus Tetraselmis virus 1 (TetV-1), forms a functional ion channel with a distinct selectivity for K + over Na + and a sensitivity to Ba 2+ . Thus, virus-encoded K + channels are not limited to Phycodnaviridae but also occur in the members of Mimiviridae . The large sequence diversity among the viral K + channels implies multiple events of lateral gene transfer.

We present an extension of the Minimum Information about any (x) Sequence (MIxS) standard for reporting sequences of uncultivated virus genomes. Minimum Information about an Uncultivated Virus Genome (MIUViG) standards were developed within the Genomic Standards Consortium framework and include virus origin, genome quality, genome annotation, taxonomic classification, biogeographic distribution and in silico host prediction. Community-wide adoption of MIUViG standards, which complement the Minimum Information about a Single Amplified Genome (MISAG) and Metagenome-Assembled Genome (MIMAG) standards for uncultivated bacteria and archaea, will improve the reporting of uncultivated virus genomes in public databases. In turn, this should enable more robust comparative studies and a systematic exploration of the global virosphere.

Viruses are integral to ecological and evolutionary processes, but we have a poor understanding of what drives variation in key traits across diverse viruses. For lytic viruses, burst size, latent period, and genome size are primary characteristics controlling host-virus dynamics. Here we synthesize data on these traits for 75 strains of phytoplankton viruses, which play an important role in global biogeochemistry. We find that primary traits of the host (genome size, growth rate) explain 40%-50% of variation in burst size and latent period. Specifically, burst size and latent period both exhibit saturating relationships versus the host∶virus genome size ratio, with both traits increasing at low genome size ratios while showing no relationship at high size ratios. In addition, latent period declines as host growth rate increases. We analyze a model of latent period evolution to explore mechanisms that could cause these patterns. The model predicts that burst size may often be set by the host genomic resources available for viral construction, while latent period evolves to permit this maximal burst size, modulated by host metabolic rate. These results suggest that general mechanisms may underlie the evolution of diverse viruses. Future extensions of this work could help explain viral regulation of host populations, viral influence on community structure and diversity, and viral roles in biogeochemical cycles.

The family Mimiviridae contains uncommonly large viruses, many of which were isolated using a free-living amoeba as a host. Although the genomes of these and other mimivirids that infect marine heterokont and haptophyte protists have now been sequenced, there has yet to be a genomic investigation of a mimivirid that infects a member of the Viridiplantae lineage (green algae and land plants). Here we characterize the 668-kilobase complete genome of TetV-1, a mimivirid that infects the cosmopolitan green alga Tetraselmis (Chlorodendrophyceae). The analysis revealed genes not previously seen in viruses, such as the mannitol metabolism enzyme mannitol 1-phosphate dehydrogenase, the saccharide degradation enzyme alpha-galactosidase, and the key fermentation genes pyruvate formate-lyase and pyruvate formate-lyase activating enzyme. The TetV genome is the largest sequenced to date for a virus that infects a photosynthetic organism, and its genes reveal unprecedented mechanisms by which viruses manipulate their host's metabolism., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Microbial life has been detected well into the igneous crust of the seafloor (i.e., the oceanic basement), but there have been no reports confirming the presence of viruses in this habitat. To detect and characterize an ocean basement virome, geothermally heated fluid samples (ca. 60 to 65°C) were collected from 117 to 292 m deep into the ocean basement using seafloor observatories installed in two boreholes (Integrated Ocean Drilling Program [IODP] U1362A and U1362B) drilled in the eastern sediment-covered flank of the Juan de Fuca Ridge. Concentrations of virus-like particles in the fluid samples were on the order of 0.2 × 10 5 to 2 × 10 5  ml -1 ( n = 8), higher than prokaryote-like cells in the same samples by a factor of 9 on average (range, 1.5 to 27). Electron microscopy revealed diverse viral morphotypes similar to those of viruses known to infect bacteria and thermophilic archaea. An analysis of virus-like sequences in basement microbial metagenomes suggests that those from archaeon-infecting viruses were the most common (63 to 80%). Complete genomes of a putative archaeon-infecting virus and a prophage within an archaeal scaffold were identified among the assembled sequences, and sequence analysis suggests that they represent lineages divergent from known thermophilic viruses. Of the clustered regularly interspaced short palindromic repeat (CRISPR)-containing scaffolds in the metagenomes for which a taxonomy could be inferred (163 out of 737), 51 to 55% appeared to be archaeal and 45 to 49% appeared to be bacterial. These results imply that the warmed, highly altered fluids in deeply buried ocean basement harbor a distinct assemblage of novel viruses, including many that infect archaea, and that these viruses are active participants in the ecology of the basement microbiome. IMPORTANCE The hydrothermally active ocean basement is voluminous and likely provided conditions critical to the origins of life, but the microbiology of this vast habitat is not well understood. Viruses in particular, although integral to the origins, evolution, and ecology of all life on earth, have never been documented in basement fluids. This report provides the first estimate of free virus particles (virions) within fluids circulating through the extrusive basalt of the seafloor and describes the morphological and genetic signatures of basement viruses. These data push the known geographical limits of the virosphere deep into the ocean basement and point to a wealth of novel viral diversity, exploration of which could shed light on the early evolution of viruses., (Copyright © 2017 Nigro et al.)

Reverse transcription, quantitative PCR (RT-qPCR) is a sensitive method for quantification of specific RNA targets, but the first step of the assay, reverse transcription, is notoriously variable and sensitive to reaction conditions. In this study, we used purified Bacteriophage MS2 genomic RNA as a model virus target to test two different RT enzymes (SuperScript II and SuperScript III), two RT-priming strategies (gene-specific primers and random hexamers), and varying background RNA concentrations (0-50ngμl -1 ) to determine how these variables influence the efficiency of reverse transcription over a range of target concentrations (10 1 -10 7 copies μl -1 ). The efficiency of the RT reaction was greatly improved by increasing both background RNA and primer concentrations, but the benefit provided by background RNA was source dependent. At a given target concentration, similar RT efficiencies were achieved with gene-specific primers and random hexamers, but the latter required much higher concentrations. With random hexamers, we observed a systematic variation in RT reaction efficiency as a function of target concentration. Using an RNA standard curve that was also subject to RT effectively normalized for this systematic variability, but the assay accuracy depended critically on the length of the standard RNA extending to the 3' end of the qPCR target site. Our results shed some light on previous contradictory conclusions in the literature, and provide insights that may aid in the design of RT-qPCR assays and the design of synthetic RNA standards when full-length material is not available., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Early work on marine algal viruses focused exclusively on those having DNA genomes, but recent studies suggest that RNA viruses, especially those with positive-sense, single-stranded RNA (+ssRNA) genomes, are abundant in tropical and temperate coastal seawater. To test whether this was also true of polar waters, we estimated the relative abundances of RNA and DNA viruses using a mass ratio approach and conducted shotgun metagenomics on purified viral samples collected from a coastal site near Palmer Station, Antarctica on six occasions throughout a summer phytoplankton bloom (November-March). Our data suggest that RNA viruses contributed up to 65% of the total virioplankton (8-65%), and that, as observed previously in warmer waters, the majority of RNA viruses in these Antarctic RNA virus metagenomes had +ssRNA genomes most closely related to viruses in the order Picornavirales. Assembly of the metagenomic reads resulted in five novel, nearly complete genomes, three of which had features similar to diatom-infecting viruses. Our data are consistent with the hypothesis that RNA viruses influence diatom bloom dynamics in Antarctic waters., (© 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Plating environmental samples on vibrio-selective chromogenic media is a commonly used technique that allows one to quickly estimate concentrations of putative vibrio pathogens or to isolate them for further study. Although this approach is convenient, its usefulness depends directly on how well the procedure selects against false positives. We tested whether a chromogenic medium, CHROMagar Vibrio (CaV), used alone (single-plating) or in combination (double-plating) with a traditional medium thiosulfate-citrate-bile-salts (TCBS), could improve the discrimination among three pathogenic vibrio species (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus) and thereby decrease the number of false-positive colonies that must be screened by molecular methods. Assays were conducted on water samples from two estuarine environments (one subtropical, one tropical) in a variety of seasonal conditions. The results of the double-plating method were confirmed by PCR and 16S rRNA sequencing. Our data indicate that there is no significant difference in the false-positive rate between CaV and TCBS when using a single-plating technique, but determining color changes on the two media sequentially (double-plating) reduced the rate of false positive identification in most cases. The improvement achieved was about two-fold on average, but varied greatly (from 0- to 5-fold) and depended on the sampling time and location. The double-plating method was most effective for V. vulnificus in warm months, when overall V. vulnificus abundance is high (false positive rates as low as 2%, n=178). Similar results were obtained for V. cholerae (minimum false positive rate of 16%, n=146). In contrast, the false positive rate for V. parahaemolyticus was always high (minimum of 59%, n=109). Sequence analysis of false-positive isolates indicated that the majority of confounding isolates are from the Vibrionaceae family, however, members of distantly related bacterial groups were also able to grow on vibrio-selective media, even when using the double-plating method. In conclusion, the double-plating assay is a simple means to increase the efficiency of identifying pathogenic vibrios in aquatic environments and to reduce the number of molecular assays required for identity confirmation. However, the high spatial and temporal variability in the performance of the media mean that molecular approaches are still essential to obtain the most accurate vibrio abundance estimates from environmental samples., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Anodic aluminum oxide (AAO) filters have high porosity and can be manufactured with a pore size that is small enough to quantitatively capture viruses. These properties make the filters potentially useful for harvesting total microbial communities from water samples for molecular analyses, but their performance for nucleic acid extraction has not been systematically or quantitatively evaluated. In this study, we characterized the flux of water through commercially produced nanoporous (0.02 μm) AAO filters (Anotop; Whatman) and used isolates (a virus, a bacterium, and a protist) and natural seawater samples to test variables that we expected would influence the efficiency with which nucleic acids are recovered from the filters. Extraction chemistry had a significant effect on DNA yield, and back flushing the filters during extraction was found to improve yields of high-molecular-weight DNA. Using the back-flush protocol, the mass of DNA recovered from microorganisms collected on AAO filters was ≥ 100% of that extracted from pellets of cells and viruses and 94% ± 9% of that obtained by direct extraction of a liquid bacterial culture. The latter is a minimum estimate of the relative recovery of microbial DNA, since liquid cultures include dissolved nucleic acids that are retained inefficiently by the filter. In conclusion, we demonstrate that nucleic acids can be extracted from microorganisms on AAO filters with an efficiency similar to that achievable by direct extraction of microbes in suspension or in pellets. These filters are therefore a convenient means by which to harvest total microbial communities from multiple aqueous samples in parallel for subsequent molecular analyses., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Viruses have a profound influence on the ecology and evolution of plankton, but our understanding of the composition of the aquatic viral communities is still rudimentary. This is especially true of those viruses having RNA genomes. The limited data that have been published suggest that the RNA virioplankton is dominated by viruses with positive-sense, single-stranded (+ss) genomes that have features in common with those of eukaryote-infecting viruses in the order Picornavirales (picornavirads). In this study, we investigated the diversity of the RNA virus assemblages in tropical coastal seawater samples using targeted PCR and metagenomics. Amplification of RNA-dependent RNA polymerase (RdRp) genes from fractions of a buoyant density gradient suggested that the distribution of two major subclades of the marine picornavirads was largely congruent with the distribution of total virus-like RNA, a finding consistent with their proposed dominance. Analyses of the RdRp sequences in the library revealed the presence of many diverse phylotypes, most of which were related only distantly to those of cultivated viruses. Phylogenetic analysis suggests that there were hundreds of unique picornavirad-like phylotypes in one 35-liter sample that differed from one another by at least as much as the differences among currently recognized species. Assembly of the sequences in the metagenome resulted in the reconstruction of six essentially complete viral genomes that had features similar to viruses in the families Bacillarna-, Dicistro-, and Marnaviridae. Comparison of the tropical seawater metagenomes with those from other habitats suggests that +ssRNA viruses are generally the most common types of RNA viruses in aquatic environments, but biases in library preparation remain a possible explanation for this observation., Importance: Marine plankton account for much of the photosynthesis and respiration on our planet, and they influence the cycling of carbon and the distribution of nutrients on a global scale. Despite the fundamental importance of viruses to plankton ecology and evolution, most of the viruses in the sea, and the identities of their hosts, are unknown. This report is one of very few that delves into the genetic diversity within RNA-containing viruses in the ocean. The data expand the known range of viral diversity and shed new light on the physical properties and genetic composition of RNA viruses in the ocean., (Copyright © 2014 Culley et al.)

Strain HIMB11 is a planktonic marine bacterium isolated from coastal seawater in Kaneohe Bay, Oahu, Hawaii belonging to the ubiquitous and versatile Roseobacter clade of the alphaproteobacterial family Rhodobacteraceae. Here we describe the preliminary characteristics of strain HIMB11, including annotation of the draft genome sequence and comparative genomic analysis with other members of the Roseobacter lineage. The 3,098,747 bp draft genome is arranged in 34 contigs and contains 3,183 protein-coding genes and 54 RNA genes. Phylogenomic and 16S rRNA gene analyses indicate that HIMB11 represents a unique sublineage within the Roseobacter clade. Comparison with other publicly available genome sequences from members of the Roseobacter lineage reveals that strain HIMB11 has the genomic potential to utilize a wide variety of energy sources (e.g. organic matter, reduced inorganic sulfur, light, carbon monoxide), while possessing a reduced number of substrate transporters.

Metagenomic analyses of marine viruses generate an overview of viral genes present in a sample, but the percentage of the resulting sequence fragments that can be reassembled is low and the phenotype of the virus from which a given sequence derives is usually unknown. In this study, we employed physical fractionation to characterize the morphological and genomic traits of a subset of uncultivated viruses from a natural marine assemblage. Viruses from Kāne'ohe Bay, Hawai'i were fractionated by equilibrium buoyant density centrifugation in a cesium chloride (CsCl) gradient, and one fraction from the CsCl gradient was then further fractionated by strong anion-exchange chromatography. One of the fractions resulting from this two-dimensional separation appeared to be dominated by only a few virus types based on genome sizes and morphology. Sequences generated from a shotgun clone library of the viruses in this fraction were assembled into significantly more numerous contigs than have been generated with previous metagenomic investigations of whole DNA viral assemblages with comparable sequencing effort. Analysis of the longer contigs (up to 6.5 kb) assembled from our metagenome allowed us to assess gene arrangement in this subset of marine viruses. Our results demonstrate the potential for physical fractionation to facilitate sequence assembly from viral metagenomes and permit linking of morphological and genomic data for uncultivated viruses.

Viruses are abundant in the ocean and a major driving force in plankton ecology and evolution. It has been assumed that most of the viruses in seawater contain DNA and infect bacteria, but RNA-containing viruses in the ocean, which almost exclusively infect eukaryotes, have never been quantified. We compared the total mass of RNA and DNA in the viral fraction harvested from seawater and using data on the mass of nucleic acid per RNA- or DNA-containing virion, estimated the abundances of each. Our data suggest that the abundance of RNA viruses rivaled or exceeded that of DNA viruses in samples of coastal seawater. The dominant RNA viruses in the samples were marine picorna-like viruses, which have small genomes and are at or below the detection limit of common fluorescence-based counting methods. If our results are typical, this means that counts of viruses and the rate measurements that depend on them, such as viral production, are significantly underestimated by current practices. As these RNA viruses infect eukaryotes, our data imply that protists contribute more to marine viral dynamics than one might expect based on their relatively low abundance. This conclusion is a departure from the prevailing view of viruses in the ocean, but is consistent with earlier theoretical predictions.

Investigating the bacteriophages of vibrios has led to significant insights into the evolution and pathogenicity of their host strains. This report presents the first complete genome sequence of a bacteriophage that infects the deadly human pathogen Vibrio vulnificus. The phage was isolated from the surface waters of the Ala Wai Canal, which is part of an urban watershed in eastern Honolulu, Hawaii, USA. The phage particle is icosahedral, with a diameter of 35-40 nm, and a small non-contractile tail. The genome was sequenced in its entirety, rendering a 38 kb sequence located on a single, linear, circularly permuted chromosome. Here, we present the annotation and genomic features of the bacteriophage, VvAW1.

Strain HIMB100 is a planktonic marine bacterium in the class Alphaproteobacteria. This strain is of interest because it is one of the first known isolates from a globally ubiquitous clade of marine bacteria known as SAR116 within the family Rhodospirillaceae. Here we describe preliminary features of the organism, together with the draft genome sequence and annotation. This is the second genome sequence of a member of the SAR116 clade. The 2,458,945 bp genome contains 2,334 protein-coding and 42 RNA genes.

We investigated the abundance, distribution, and virulence gene content of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus in the waters of southern Lake Pontchartrain in Louisiana on four occasions from October 2005 to September 2006, using selective cultivation and molecular assays. The three targeted pathogenic vibrios were generally below the detection level in January 2006, when the water was cold (13°C), and most abundant in September 2006, when the lake water was warmest (30°C). The maximum values for these species were higher than reported previously for the lake by severalfold to orders of magnitude. The only variable consistently correlated with total vibrio abundance within a single sampling was distance from shore (P = 0.000). Multiple linear regression of the entire data set revealed that distance from shore, temperature, and turbidity together explained 82.1% of the variability in total vibrio CFU. The log-transformed mean abundance of V. vulnificus CFU in the lake was significantly correlated with temperature (P = 0.014), but not salinity (P = 0.625). Virulence-associated genes of V. cholerae (ctx) and V. parahaemolyticus (trh and tdh) were not detected in any isolates of these species (n = 128 and n = 20, respectively). In contrast, 16S rRNA typing of V. vulnificus (n = 298) revealed the presence of both environmental (type A) and clinical (type B) strains. The percentage of the B-type V. vulnificus was significantly higher in the lake in October 2005 (35.8% of the total) than at other sampling times (P ≤ 0.004), consistent with the view that these strains represent distinct ecotypes.

Background: Viruses have a profound influence on both the ecology and evolution of marine plankton, but the genetic diversity of viral assemblages, particularly those in deeper ocean waters, remains poorly described. Here we report on the construction and analysis of a viral metagenome prepared from below the euphotic zone in a temperate, eutrophic bay of coastal California., Methods: We purified viruses from approximately one cubic meter of seawater collected from 200 m depth in Monterey Bay, CA. DNA was extracted from the virus fraction, sheared, and cloned with no prior amplification into a plasmid vector and propagated in E. coli to produce the MBv200m library. Random clones were sequenced by the Sanger method. Sequences were assembled then compared to sequences in GenBank and to other viral metagenomic libraries using BLAST analyses., Results: Only 26% of the 881 sequences remaining after assembly had significant (E≤0.001) BLAST hits to sequences in the GenBank nr database, with most being matches to bacteria (15%) and viruses (8%). When BLAST analysis included environmental sequences, 74% of sequences in the MBv200m library had a significant match. Most of these hits (70%) were to microbial metagenome sequences and only 0.7% were to sequences from viral metagenomes. Of the 121 sequences with a significant hit to a known virus, 94% matched bacteriophages (Families Podo-, Sipho-, and Myoviridae) and 6% matched viruses of eukaryotes in the Family Phycodnaviridae (5 sequences) or the Mimivirus (2 sequences). The largest percentages of hits to viral genes of known function were to those involved in DNA modification (25%) or structural genes (17%). Based on reciprocal BLAST analyses, the MBv200m library appeared to be most similar to viral metagenomes from two other bays and least similar to a viral metagenome from the Arctic Ocean., Conclusions: Direct cloning of DNA from diverse marine viruses was feasible and resulted in a distribution of virus types and functional genes at depth that differed in detail, but were broadly similar to those found in surface marine waters. Targeted viral analyses are useful for identifying those components of the greater marine metagenome that circulate in the subcellular size fraction.

Cyanobacteria are thought to be the main N(2)-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N(2) fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.

Concentrations of viruses and prokaryotes in the alkaline, moderately hypersaline, seasonally stratified Mono Lake are among the highest reported for a natural aquatic environment. We used electron microscopy to test whether viral morphological characteristics differed among the epilimnion, metalimnion, and the anoxic hypolimnion of the lake and to determine how the properties of viruses in Mono Lake compare to other aquatic environments. Viral capsid size distributions were more similar in the metalimnion and hypolimnion of Mono Lake, while viral tail lengths were more similar in the epilimnion and metalimnion. The percentage of tailed viruses decreased with depth and the relative percentages of tailed phage families changed with depth. The presence of large (>125 nm capsid), untailed viruses in the metalimnion and hypolimnion suggests that eukaryotic viruses are produced in these suboxic and anoxic, hypersaline environments. Capsid diameters of viruses were larger on average in Mono Lake compared to other aquatic environments, and no lemon-shaped or filamentous viruses were found, in contrast to other high-salinity or high-altitude lakes and seas. Our data suggest that the physically and chemically distinct layers of Mono Lake harbor different viral assemblages, and that these assemblages are distinct from other aquatic environments that have been studied. Furthermore, we found that filtration of a sample through a 0.22-µm pore-size filter significantly altered the distribution of viral capsid diameters and tail lengths, resulting in a relative depletion of viruses having larger capsids and longer tails. This observation highlights the potential for bias in molecular surveys of viral diversity, which typically rely on filtration through 0.2- or 0.22-µm pore-size membrane filters to remove bacteria during sample preparation.

Viruses in the family Phycodnaviridae infect autotrophic protists in aquatic environments. Application of a PCR assay targeting the DNA polymerase of viruses in this family has revealed that phycodnaviruses are quite diverse and appear to be widespread, but a limited number of environments have been examined so far. In this study, we examined the sequence diversity among viral DNA pol genes amplified by PCR from subtropical coastal waters of O'ahu, Hawai'i. A total of 18 novel prasinovirus-like sequences were detected along with two other divergent sequences that differ at the genus-level relative to other sequences in the family. Of the 20 new sequence types reported here, three were serendipitously found to contain protein introns, or inteins. Sequence analysis of the inteins suggested that all three have self-splicing domains and are apparently capable of removing themselves from the translated polymerase protein. Two of the three also appear to be 'active', meaning they encode all the motifs necessary for a complete dodecapeptide homing endonuclease, and are therefore capable of horizontal transfer. A subsequent PCR survey of our samples with intein-specific primers suggested that intein-containing phycodnaviruses are common in this environment. A search for similar sequences in metagenomic data sets from other oceans indicated that viral inteins are also widespread, but how these genetic parasites might be influencing the ecology and evolution of phycodnaviruses remains unclear.

Viruses are ubiquitous in the sea and appear to outnumber all other forms of marine life by at least an order of magnitude. Through selective infection, viruses influence nutrient cycling, community structure, and evolution in the ocean. Over the past 20 years we have learned a great deal about the diversity and ecology of the viruses that constitute the marine virioplankton, but until recently the emphasis has been on DNA viruses. Along with expanding knowledge about RNA viruses that infect important marine animals, recent isolations of RNA viruses that infect single-celled eukaryotes and molecular analyses of the RNA virioplankton have revealed that marine RNA viruses are novel, widespread, and genetically diverse. Discoveries in marine RNA virology are broadening our understanding of the biology, ecology, and evolution of viruses, and the epidemiology of viral diseases, but there is still much that we need to learn about the ecology and diversity of RNA viruses before we can fully appreciate their contributions to the dynamics of marine ecosystems. As a step toward making sense of how RNA viruses contribute to the extraordinary viral diversity in the sea, we summarize in this review what is currently known about RNA viruses that infect marine organisms.

Background: In environmental sequencing projects, a mix of DNA from a whole microbial community is fragmented and sequenced, with one of the possible goals being to reconstruct partial or complete genomes of members of the community. In communities with high diversity of species, a significant proportion of the sequences do not overlap any other fragment in the sample. This problem will arise not only in situations with a relatively even distribution of many species, but also when the community in a particular environment is routinely dominated by the same few species. In the former case, no genomes may be assembled at all, while in the latter case a few dominant species in an environment will always be sequenced at high coverage to the detriment of coverage of the greater number of sparse species., Methods and Results: Here we show that, with the same global sequencing effort, separating the species into two or more sub-communities prior to sequencing can yield a much higher proportion of sequences that can be assembled. We first use the Lander-Waterman model to show that, if the expected percentage of singleton sequences is higher than 25%, then, under the uniform distribution hypothesis, splitting the community is always a wise choice. We then construct simulated microbial communities to show that the results hold for highly non-uniform distributions. We also show that, for the distributions considered in the experiments, it is possible to estimate quite accurately the relative diversity of the two sub-communities., Conclusion: Given the fact that several methods exist to split microbial communities based on physical properties such as size, density, surface biochemistry, or optical properties, we strongly suggest that groups involved in environmental sequencing, and expecting high diversity, consider splitting their communities in order to maximize the information content of their sequencing effort.

Viruses are an integral component of the marine food web, contributing to the disease and mortality of essentially every type of marine life, yet the diversity of viruses in the sea, especially those with RNA genomes, remains very poorly characterized. Isolates of RNA-containing viruses that infect marine plankton are still rare, and the only cultivation-independent surveys of RNA viral diversity reported so far were conducted for temperate coastal waters of British Columbia. Here, we report on our improvements to a previously used protocol to investigate the diversity of marine picorna-like viruses and our results from applying this protocol in subtropical waters. The original protocol was simplified by using direct filtration, rather than tangential flow filtration, to harvest viruses from seawater, and new degenerate primers were designed to amplify a fragment of the RNA-dependent RNA polymerase gene by reverse transcription-PCR from RNA extracted from the filters. Whereas the original protocol was unsuccessful in a preliminary test, the new protocol resulted in amplification of picorna-like virus sequences in every sample of subtropical and temperate coastal seawater assayed. These polymerase sequences formed a diverse, but monophyletic cluster along with other sequences amplified previously from seawater and sequences from isolates infecting marine protists. Phylogenetic analysis suggested that our sequences represent at least five new genera and 24 new species of RNA viruses. These results contribute to our understanding of RNA virus diversity and suggest that picorna-like viruses are a source of mortality for a wide variety of marine protists.

Floodwaters in New Orleans from Hurricanes Katrina and Rita were observed to contain high levels of fecal indicator bacteria and microbial pathogens, generating concern about long-term impacts of these floodwaters on the sediment and water quality of the New Orleans area and Lake Pontchartrain. We show here that fecal indicator microbe concentrations in offshore waters from Lake Pontchartrain returned to prehurricane concentrations within 2 months of the flooding induced by these hurricanes. Vibrio and Legionella species within the lake were more abundant in samples collected shortly after the floodwaters had receded compared with samples taken within the subsequent 3 months; no evidence of a long-term hurricane-induced algal bloom was observed. Giardia and Cryptosporidium were detected in canal waters. Elevated levels of fecal indicator bacteria observed in sediment could not be solely attributed to impacts from floodwaters, as both flooded and nonflooded areas exhibited elevated levels of fecal indicator bacteria. Evidence from measurements of Bifidobacterium and bacterial diversity analysis suggest that the fecal indicator bacteria observed in the sediment were from human fecal sources. Epidemiologic studies are highly recommended to evaluate the human health effects of the sediments deposited by the floodwaters.

Diazotrophic community structure in microbial mats from Guerrero Negro (GN), Baja California, Mexico, was studied using polymerase chain reaction amplification of the nifH gene and a newly developed nifH oligonucleotide microarray. Ninety-six oligonucleotide probes designed for nifH sequences from cultivated isolates and the environment were printed on glass microarrays. Phylogenetic analysis showed that the probes represented all of the main nifH clusters. Specificity was tested by (i) evaluation of cross hybridization using individual targets, and (ii) comparison of the observed hybridization signals and those predicted from the sequences cloned from microbial mats. Signal intensity had a positive relationship with target concentration and the percentage identity between probe and target. Under moderate stringency and high target concentration, specificity of the probes varied from 77% to 100% with the individual targets tested. At the end of a 7-month long nutrient manipulation experiment in GN microbial mats, no expression of nitrogen fixation under nitrogen loading was detected, although a diverse community of diazotrophs was detected. The diversity in diazotrophic population present was higher than in the population expressing the nifH gene, and there were taxa specific differences in response to nutrients. The nifH microarray is a powerful tool for diazotroph community analysis in the marine environment.

Marine picoplankton are central mediators of many oceanic biogeochemical processes, but much of their biology and ecology remains ill defined. One approach to better defining these environmentally significant microbes involves the acquisition of genomic data that can provide information about genome content, metabolic capabilities, and population variability in picoplankton assemblages. Previously, we constructed and phylogenetically screened a Bacterial Artificial Chromosome (BAC) library from surface water picoplankton of Monterey Bay. To further describe niche partitioning, metabolic variability, and population structure in coastal picoplankton populations, we constructed and compared several picoplankton BAC libraries recovered from different depths in Monterey Bay. To facilitate library screening, a rapid technique was developed (ITS-LH-PCR) to identify and quantify ribosomal RNA (rRNA) gene-containing BAC clones in BAC libraries. The approach exploited natural length variations in the internal transcribed spacer (ITS) located between SSU and LSU rRNA genes, as well as the presence and location of tRNA-alanine coding genes within the ITS. The correspondence between ITS-LH-PCR fragment sizes and 16S rRNA gene phylogenies facilitated rapid identification of rRNA genes in BAC clones without requiring direct DNA sequencing. Using this approach, 35 phylogenetic groups (previously identified by cultivation or PCR-based rRNA gene surveys) were detected and quantified among the BAC clones. Since the probability of recovering chimeric rRNA gene sequences in large insert BAC clones was low, we used these sequences to identify potentially chimeric sequences from previous PCR amplified clones deposited in public databases. Full-length SSU rRNA gene sequences from picoplankton BAC libraries, cultivated bacterioplankton, and nonchimeric RNA genes were then used to refine phylogenetic analyses of planktonic marine gamma Proteobacteria, Roseobacter, and Rhodospirillales species.

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.

Investigations of the distribution and diversity of nitrogen-fixing microorganisms in natural environments have often relied on PCR amplification and sequence analysis of a portion of one of the key enzymes in nitrogen fixation, dinitrogenase reductase, encoded by nifH. Recent work has suggested that DNA macroarrays provide semiquantitative fingerprints of diversity within mixtures of nifH amplicons (G. F. Steward, B. D. Jenkins, B. B. Ward, and J. P. Zehr, Appl. Environ. Microbiol. 70:1455-1465, 2004). Here we report the application of macroarrays for a study in the Chesapeake Bay. Samples from different locations in the bay yielded distinct fingerprints. Analysis of replicates and samples from different locations by cluster analysis showed that replicates clustered together, whereas different samples formed distinct clusters. There was a correspondence between the hybridization pattern observed and that predicted from the distribution of sequence types in a corresponding clone library. Some discrepancies between the methods were observed which are likely a result of the high nifH sequence diversity in the Chesapeake Bay and the limited number of sequences represented on this version of the array. Analyses of sequences in the clone library indicate that the Chesapeake Bay harbors unique, phylogenetically diverse diazotrophs. The macroarray hybridization patterns suggest that there are spatially variable communities of diazotrophs, which have been confirmed by quantitative PCR methods (S. M. Short, B. D. Jenkins, and J. P. Zehr, Appl. Environ. Microbiol., in press). The results show that DNA macroarrays have great potential for mapping the spatial and temporal variability of functional gene diversity in the environment.

We investigated the diversity of nitrogenase genes in the alkaline, moderately hypersaline Mono Lake, California to determine (1) whether nitrogen-fixing (diazotrophic) populations were similar to those in other aquatic environments and (2) if there was a pattern of distribution of phylotypes that reflected redox conditions, as well as (3) to identify populations that could be important in N dynamics in this nitrogen-limited lake. Mono Lake has been meromictic for almost a decade and has steep gradients in oxygen and reduced compounds that provide a wide range of aerobic and anaerobic habitats. We amplified a fragment of the nitrogenase gene (nifH) from planktonic DNA samples collected at three depths representing oxygenated surface waters, the oxycline, and anoxic, ammonium-rich deep waters. Forty-three percent of the 90 sequences grouped in nifH Cluster I. The majority of clones (57%) grouped in Cluster III, which contains many known anaerobic bacteria. Cluster I and Cluster III sequences were retrieved at every depth indicating little vertical zonation in sequence types related to the prominent gradients in oxygen and ammonia. One group in Cluster I was found most often at every depth and accounted for 29% of all the clones. These sequences formed a subcluster that contained other environmental clones, but no cultivated representatives. No significant nitrogen fixation was detected by the 15N2 method after 48 h of incubation of surface, oxycline, or deep waters, suggesting that pelagic diazotrophs were contributing little to nitrogen fluxes in the lake. The failure to measure any significant nitrogen fixation, despite the detection of diverse and novel nitrogenase genes throughout the water column, raises interesting questions about the ecological controls on diazotrophy in Mono Lake and the distribution of functional genes in the environment.