Metagenomic Characterization of Chesapeake Bay Virioplankton

Viruses are ubiquitous and abundant throughout the biosphere. In marine systems, virus-mediated processes can have significant impacts on microbial diversity and on global biogeocehmical cycling. However, viral genetic diversity remains poorly characterized. To address this shortcoming, a metagenomic library was constructed from Chesapeake Bay virioplankton. The resulting sequences constitute the largest collection of long-read double-stranded DNA (dsDNA) viral metagenome data reported to date. BLAST homology comparisons showed that Chesapeake Bay virioplankton contained a high proportion of unknown (homologous only to environmental sequences) and novel (no significant homolog) sequences. This analysis suggests that dsDNA viruses are likely one of the largest reservoirs of unknown genetic diversity in the biosphere. The taxonomic origin of BLAST homologs to viral library sequences agreed well with reported abundances of cooccurring bacterial subphyla within the estuary and indicated that cyanophages were abundant. However, the low proportion of Siphophage homologs contradicts a previous assertion that this family comprises most bacteriophage diversity. Identification and analyses of cyanobacterial homologs of the psbA gene illustrated the value of metagenomic studies of virioplankton. The phylogeny of inferred PsbA protein sequences suggested that Chesapeake Bay cyanophage strains are endemic in that environment. The ratio of psbA homologous sequences to total cyanophage sequences in the metagenome indicated that the psbA gene may be nearly universal in Chesapeake Bay cyanophage genomes. Furthermore, the low frequency of psbD homologs in the library supports the prediction that Chesapeake Bay cyanophage populations are dominated by Podoviridae.     

Comparative Genomic and Phylogenomic Analyses Reveal a Conserved Core Genome Shared by Estuarine and Oceanic Cyanopodoviruses

Podoviruses are among the major viral groups that infect marine picocyanobacteria Prochlorococcus and Synechococcus. Here, we reported the genome sequences of five Synechococcus podoviruses isolated from the estuarine environment, and performed comparative genomic and phylogenomic analyses based on a total of 20 cyanopodovirus genomes. The genomes of all the known marine cyanopodoviruses are highly syntenic. A pan-genome of 349 clustered orthologous groups was determined, among which 15 were core genes. These core genes make up nearly half of each genome in length, reflecting the high level of genome conservation among this cyanophage type. The whole genome phylogenies based on concatenated core genes and gene content were highly consistent and confirmed the separation of two discrete marine cyanopodovirus clusters MPP-A and MPP-B. The genomes within cluster MPP-B grouped into subclusters mainly corresponding to Prochlorococcus or Synechococcus host types. Auxiliary metabolic genes tend to occur in a specific phylogenetic group of these cyanopodoviruses. All the MPP-B phages analyzed here encode the photosynthesis gene psbA, which are absent in all the MPP-A genomes thus far. Interestingly, all the MPP-B and two MPP-A Synechococcus podoviruses encode the thymidylate synthase gene thyX, while at the same genome locus all the MPP-B Prochlorococcus podoviruses encode the transaldolase gene talC. Both genes are hypothesized to have the potential to facilitate the biosynthesis of deoxynucleotide for phage replication. Inheritance of specific functional genes could be important to the evolution and ecological fitness of certain cyanophage genotypes. Our analyses demonstrate that cyanopodoviruses of estuarine and oceanic origins share a conserved core genome and suggest that accessory genes may be related to environmental adaptation.     

Marine Cyanophages Demonstrate Biogeographic Patterns throughout the Global Ocean

Myoviruses and podoviruses that infect cyanobacteria are the two major groups of marine cyanophages, but little is known of how their phylogenetic lineages are distributed in different habitats. In this study, we analyzed the phylogenetic relationships of cyanopodoviruses and cyanomyoviruses based on the existing genomes. The 28 cyanomyoviruses were classified into four clusters (I to IV), and 19 of the 20 cyanopodoviruses were classified into two clusters, MPP-A and MPP-B, with four subclusters within cluster MPP-B. These genomes were used to recruit cyanophage-like fragments from microbial and viral metagenomes to estimate the relative abundances of these cyanophage lineages. Our results showed that cyanopodoviruses and cyanomyoviruses are both abundant in various marine environments and that clusters MPP-B, II and III appear to be the most dominant lineages. Cyanopodoviruses and cluster I and IV cyanomyoviruses exhibited habitat-related variability in their relative levels of abundance, while cluster II and III cyanomyoviruses appeared to be consistently dominant in various habitats. Multivariate analyses showed that reads that mapped to Synechococcus phages and Prochlorococcus phages had distinct distribution patterns that were significantly correlated to those of Synechococcus and Prochlorococcus, respectively. The Mantel test also revealed a strong correlation between the community compositions of cyanophages and picocyanobacteria. Given that cyanomyoviruses tend to have a broad host range and some can cross-infect Synechococcus and Prochlorococcus, while cyanopodoviruses are commonly host specific, the observation that their community compositions both correlated significantly with that of picocyanobacteria was unexpected. Although cyanomyoviruses and cyanopodoviruses differ in host specificity, their biogeographic distributions are likely both constrained by the picocyanobacterial community.     

Population Dynamics of Chesapeake Bay Virioplankton: Total-Community Analysis by Pulsed-Field Gel Electrophoresis

Recognition of viruses as the most abundant component of aquatic microbial communities has stimulated investigations of the impact of viruses on bacterio- and phytoplankton host communities. From results of field studies to date, it is concluded that in most aquatic environments, a reduction in the number of bacteria on a daily basis is caused by viral infection. However, the modest amount of in situ virus-mediated mortality may be less significant than viral infection serving to maintain clonal diversity in the host communities directly, through gene transmission (i.e., transduction), and indirectly, by elimination of numerically dominant host species. If the latter mechanism for controlling community diversity prevails, then the overall structure of aquatic viral communities would be expected to change as well over short seasonal and spatial scales. To determine whether this occurs, pulsed-field gel electrophoresis (PFGE) was used to monitor the population dynamics of Chesapeake Bay virioplankton for an annual cycle (1 year). Virioplankton in water samples collected at six stations along a transect running the length of the bay were concentrated 100-fold by ultrafiltration. Viruses were further concentrated by ultracentrifugation, and the concentrated samples were embedded in agarose. PFGE analysis of virus DNA in the agarose plugs yielded several distinct bands, ranging from 50 to 300 kb. Principal-component and cluster analyses of the virus PFGE fingerprints indicated that changes in virioplankton community structure were correlated with time, geographical location, and extent of water column stratification. From the results of this study, it is concluded that, based on the dynamic nature of the Chesapeake Bay virioplankton community structure, the clonal diversity of bacterio- and phytoplankton host communities is an important component of the virus community.     

Prevalence of highly host-specific cyanophages in the estuarine environment

Cyanophages that infect coastal and oceanic Synechococcus have been studied extensively. However, no cyanophages infecting estuarine Synechococcus have been reported. In this study, seven cyanophages (three podoviruses, three siphoviruses and one myovirus) isolated from four estuarine Synechococcus strains were characterized in terms of their morphology, host range, growth and genetic features. All the podoviruses and siphoviruses were highly host specific. For the first time, the photosynthesis gene ( psbA ) was found in two podoviruses infecting estuarine Synechococcus . However, the psbA gene was not detected in the three siphoviruses. The psbA sequences from the two Synechococcus podoviruses clustered with some environmental psbA sequences, forming a unique cluster distantly related to previous known psbA clusters. Our results suggest that the psbA among Synechococcus podoviruses may evolve independently from the psbA of Synechococcus myoviruses. All three estuarine Synechococcus podoviruses contained the DNA polymerase ( pol ) gene, and clustered with other podoviruses that infect oceanic Synechococcus and Prochlorococcus , suggesting that the DNA pol is conserved among marine picocyanobacterial podoviruses. Prevalence of host-specific cyanophages in the estuary suggests that Synechococcus and their phages in the estuarine ecosystem may develop a host–phage relationship different from what have been found in the open ocean.     

Randomly amplified polymorphic DNA PCR as a tool for assessment of marine viral richness

Recent discoveries have uncovered considerable genetic diversity among aquatic viruses and raised questions about the variability of this diversity within and between environments. Studies of the temporal and spatial dynamics of aquatic viral assemblages have been hindered by the lack of a common genetic marker among viruses for rapid diversity assessments. Randomly amplified polymorphic DNA (RAPD) PCR bypasses this obstacle by sampling at the genetic level without requiring viral isolation or previous sequence knowledge. In this study, the utility of RAPD-PCR for assessing DNA viral richness within Chesapeake Bay water samples was evaluated. RAPD-PCR using single 10-mer oligonucleotide primers successfully produced amplicons from a variety of viral samples, and banding patterns were highly reproducible, indicating that each band likely represents a single amplicon originating from viral template DNA. In agreement with observations from other community profiling techniques, resulting RAPD-PCR banding patterns revealed more temporal than spatial variability in Chesapeake Bay virioplankton assemblages. High-quality hybridization probes and sequence information were also easily generated from single RAPD-PCR products or whole reactions. Thus, the RAPD-PCR technique appears to be practical and efficient for routine use in high-resolution viral diversity studies by providing assemblage comparisons through fingerprinting, probing, or sequence information.     

Amplification of DNA polymerase gene fragments from viruses infecting microalgae.

Nested PCR with three highly degenerate primers was used for amplification and identification of DNA polymerase (pol) genes from viruses which infect three genera of microalgae. Group-specific primers (AVS1 and AVS2) were designed on the basis of inferred amino acid sequences unique to the DNA pol genes of viruses (PBCV-1 and NY-2A) that infect an endosymbiotic Chlorella-like alga (Chlorophyceae) and a virus (MpV-SP1) which infects the photosynthetic flagellate Micromonas pusilla (Prasinophyceae). In addition, a nested primer (POL) was designed on the basis of the highly conserved amino acid sequence YGDTDS found in most B-family (alpha-like) DNA pol genes. These primers were used to amplify DNA from the three viruses, PBCV-1, NY-2A, and MpV-SP1, for which the primers were designed, as well as eight clonal isolates of genetically distinct viruses which infect M. pusilla and others which infect Chrysochromulina spp. (Prymnesiophyceae), suggesting that these are a group of related viruses. In contrast, no product resulted from using DNA from viruses which infect the marine brown algae Ectocarpus siliculosis and Feldmannia sp. (Phaeophyceae), suggesting that these viruses may not be closely related to those that infect microalgae. These primers were also used to amplify DNA from natural virus communities. Our results indicate that nested PCR, even under low-stringency conditions, can be used as a rapid method to verify the presence in seawater of a group of related viruses which infect microalgae. Sequence analysis of these fragments should provide information on the genetic diversity and potentially the phyletic relationships among these viruses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic diversity and temporal dynamics of phytoplankton viruses in East Lake,China

Phytoplankton viruses are important components of aquatic ecosystems. However, their prevalence and genetic diversity in marine and freshwater systems are largely under estimated owing to the immense size of water bodies and limitations in virus discovery techniques. In this study, we conducted a 1-year survey of phytoplankton virus communities by collecting surface water monthly from an inland lake(East Lake) in China between May 2012 and April 2013. We examined four phytoplankton viruses, i.e., myoviruses, podoviruses, siphoviruses, and phycodnaviruses, and seven sets of primers were used to target conserved genes within these four species. In this year-long investigation, a total of 358 different virus-related sequences from four virus families were obtained. All virus families were detected in all months, except for cyanopodoviruses, which were only identified during eight of the 12 months surveyed. Moreover, virus abundance and diversity changed dynamically over time. Phylogenetic analysis revealed that the majority of viral sequences from East Lake, China displayed distinct clustering patterns compared with published sequences. These results supported the existence of a highly diverse and unique phytoplankton virus community in East Lake, China.     

Fingerprinting Diazotroph Communities in the Chesapeake Bay by Using a DNA Macroarray

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.     

Genome sequences of siphoviruses infecting marine Synechococcus unveil a diverse cyanophage group and extensive phage-host genetic exchanges

Investigating the interactions between marine cyanobacteria and their viruses (phages) is important towards understanding the dynamic of ocean's primary productivity. Genome sequencing of marine cyanophages has greatly advanced our understanding about their ecology and evolution. Among 24 reported genomes of cyanophages that infect marine picocyanobacteria, 17 are from cyanomyoviruses and six from cyanopodoviruses, and only one from cyanosiphovirus (Prochlorococcus phage P-SS2). Here we present four complete genome sequences of siphoviruses (S-CBS1, S-CBS2, S-CBS3 and S-CBS4) that infect four different marine Synechococcus strains. Three distinct subtypes were recognized among the five known marine siphoviruses (including P-SS2) in terms of morphology, genome architecture, gene content and sequence similarity. Our study revealed that cyanosiphoviruses are genetically diverse with polyphyletic origin. No core genes were found across these five cyanosiphovirus genomes, and this is in contrast to the fact that many core genes have been found in cyanomyovirus or cyanopodovirus genomes. Interestingly, genes encoding three structural proteins and a lysozyme of S-CBS1 and S-CBS3 showed homology to a prophage-like genetic element in two freshwater Synechococcus elongatus genomes. Re-annotation of the prophage-like genomic region suggests that S.?elongatus may contain an intact prophage. Cyanosiphovirus genes involved in DNA metabolism and replication share high sequence homology with those in cyanobacteria, and further phylogenetic analysis based on these genes suggests that ancient and selective genetic exchanges occurred, possibly due to past prophage integration. Metagenomic analysis based on the Global Ocean Sampling database showed that cyanosiphoviruses are present in relatively low abundance in the ocean surface water compared to cyanomyoviruses and cyanopodoviruses.     

RNA viruses in the sea

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.     

Shotgun metagenomics indicates novel family A DNA polymerases predominate within marine virioplankton

Virioplankton have a significant role in marine ecosystems, yet we know little of the predominant biological characteristics of aquatic viruses that influence the flow of nutrients and energy through microbial communities. Family A DNA polymerases, critical to DNA replication and repair in prokaryotes, are found in many tailed bacteriophages. The essential role of DNA polymerase in viral replication makes it a useful target for connecting viral diversity with an important biological feature of viruses. Capturing the full diversity of this polymorphic gene by targeted approaches has been difficult; thus, full-length DNA polymerase genes were assembled out of virioplankton shotgun metagenomic sequence libraries (viromes). Within the viromes novel DNA polymerases were common and found in both double-stranded (ds) DNA and single-stranded (ss) DNA libraries. Finding DNA polymerase genes in ssDNA viral libraries was unexpected, as no such genes have been previously reported from ssDNA phage. Surprisingly, the most common virioplankton DNA polymerases were related to a siphovirus infecting an α-proteobacterial symbiont of a marine sponge and not the podoviral T7-like polymerases seen in many other studies. Amino acids predictive of catalytic efficiency and fidelity linked perfectly to the environmental clades, indicating that most DNA polymerase-carrying virioplankton utilize a lower efficiency, higher fidelity enzyme. Comparisons with previously reported, PCR-amplified DNA polymerase sequences indicated that the most common virioplankton metagenomic DNA polymerases formed a new group that included siphoviruses. These data indicate that slower-replicating, lytic or lysogenic phage populations rather than fast-replicating, highly lytic phages may predominate within the virioplankton.     

Genetic diversity in marine algal virus communities as revealed by sequence analysis of DNA polymerase genes.

Algal-virus-specific PCR primers were used to amplify DNA polymerase gene (pol) fragments (683 to 689 bp) from the virus-sized fraction (0.02 to 0.2 microns) concentrated from inshore and offshore water samples collected from the Gulf of Mexico. Algal-virus-like DNA pol genes were detected in five samples collected from the surface and deep chlorophyll maximum. PCR products from an offshore station were cloned, and the genetic diversity of 33 fragments was examined by restriction fragment length polymorphism and sequence analysis. The five different genotypes or operational taxonomic units (OTUs) that were identified on the basis of restriction fragment length polymorphism banding patterns were present in different relative abundances (9 to 34%). One clone from each OTU was sequenced, and phylogenetic analysis showed that all of the OTUs fell within the family Phycodnaviridae. Four of the OTUs fell within a group of viruses (MpV) which infect the photosynthetic picoplankter Micromonas pusilla. The genetic diversity among these genotypes was as large as that previously found for MpV isolates from different oceans. The remaining genotype formed its own clade between viruses which infect M. pusilla and Chrysochromulina brevifilum. These results imply that marine virus communities contain a diverse assemblage of MpV-like viruses, as well as other unknown members of the Phycodnaviridae.     

Metagenomics: Read Length Matters

Obtaining an unbiased view of the phylogenetic composition and functional diversity within a microbial community is one central objective of metagenomic analysis. New technologies, such as 454 pyrosequencing, have dramatically reduced sequencing costs, to a level where metagenomic analysis may become a viable alternative to more-focused assessments of the phylogenetic (e.g., 16S rRNA genes) and functional diversity of microbial communities. To determine whether the short (~100 to 200 bp) sequence reads obtained from pyrosequencing are appropriate for the phylogenetic and functional characterization of microbial communities, the results of BLAST and COG analyses were compared for long (~750 bp) and randomly derived short reads from each of two microbial and one virioplankton metagenome libraries. Overall, BLASTX searches against the GenBank nr database found far fewer homologs within the short-sequence libraries. This was especially pronounced for a Chesapeake Bay virioplankton metagenome library. Increasing the short-read sampling depth or the length of derived short reads (up to 400 bp) did not completely resolve the discrepancy in BLASTX homolog detection. Only in cases where the long-read sequence had a close homolog (low BLAST E-score) did the derived short-read sequence also find a significant homolog. Thus, more-distant homologs of microbial and viral genes are not detected by short-read sequences. Among COG hits, derived short reads sampled at a depth of two short reads per long read missed up to 72% of the COG hits found using long reads. Noting the current limitation in computational approaches for the analysis of short sequences, the use of short-read-length libraries does not appear to be an appropriate tool for the metagenomic characterization of microbial communities.     

Sediment denitrifier community composition and nirS gene expression investigated with functional gene microarrays

A functional gene microarray was used to investigate denitrifier community composition and nitrite reductase (nirS) gene expression in sediments along the estuarine gradient in Chesapeake Bay, USA. The nirS oligonucleotide probe set was designed to represent a sequence database containing 539 Chesapeake Bay clones, as well as sequences from many other environments. Greatest nirS diversity was detected at the freshwater station at the head of the bay and least diversity at the higher salinity station near the mouth of the Bay. The most common OTUs from the sequence database were detected on the array with high signal strength in most samples. One of the most abundant OTUs, CB2-S-138, was identified as dominant at the mid-bay site by both microarray and quantitative PCR assays, but it comprised a much smaller fraction of the assemblage in the north and south bay samples. cDNA (transcribed from total RNA extracts) targets were hybridized to the same array to compare the profiles of community composition at the DNA (relative abundance) and mRNA (gene expression) levels. Only the three dominant denitrifying groups (in terms of relative strength of DNA hybridization signal) were detected at the mRNA level. These results suggest that the most actively denitrifying groups are responsible for most nirS expression as well.     

Global survey of diversity among environmental saltwater Bacteriovoracaceae

Halophilic Bacteriovorax (Bx), formerly known as the marine Bdellovibrio, are Gram-negative, predatory bacteria found in saltwater systems. To assess their genetic diversity and geographical occurrence, the small subunit rRNA (ssu-rRNA) gene sequences were analysed from 111 marine, salt lake and estuarine isolates recovered from 27 locations around the world. Phylogenetic analysis of these isolates using Geobacter as the outgroup revealed eight distinct ribotype clusters each with at least two isolates. Each cluster was composed of isolates with >or= 96.5% similarity in ssu-rRNA sequences. Three single isolate outliers were observed. Many of the Bx ribotypes were widely dispersed among different types of ecosystems (e.g. cluster III was recovered from the Great Salt Lake, the Atlantic Ocean, Pacific Ocean, Chesapeake Bay and gills of aquarium fish). However, cluster V was only recovered from a single ecosystem, estuaries. Cluster V was originally detected in the Chesapeake Bay and subsequently in the Pamlico Sound/Neuse River system. Principal coordinate analysis revealed that the sequences of the isolates from different environments were distinct from each other. The results of this study reveal the saltwater Bx to be phylogenetically and environmentally more diverse than was previously known.     

Endospores of halophilic bacteria of the family Bacillaceae isolated from non-saline Japanese soil may be transported by Kosa event (Asian dust storm)

Background

Natural microbial communities are extremely complex and dynamic systems in terms of their population structure and functions. However, little is known about the in situ functions of the microbial communities.

Results

This study describes the application of proteomic approaches (metaproteomics) to observe expressed protein profiles of natural microbial communities (metaproteomes). The technique was validated using a constructed community and subsequently used to analyze Chesapeake Bay microbial community (0.2 to 3.0 μm) metaproteomes. Chesapeake Bay metaproteomes contained proteins from pI 4–8 with apparent molecular masses between 10–80 kDa. Replicated middle Bay metaproteomes shared ~92% of all detected spots, but only shared 30% and 70% of common protein spots with upper and lower Bay metaproteomes. MALDI-TOF analysis of highly expressed proteins produced no significant matches to known proteins. Three Chesapeake Bay proteins were tentatively identified by LC-MS/MS sequencing coupled with MS-BLAST searching. The proteins identified were of marine microbial origin and correlated with abundant Chesapeake Bay microbial lineages, Bacteroides and α-proteobacteria.

Conclusion

Our results represent the first metaproteomic study of aquatic microbial assemblages and demonstrate the potential of metaproteomic approaches to link metagenomic data, taxonomic diversity, functional diversity and biological processes in natural environments.     

Phylogenetic Diversity of Sequences of Cyanophage Photosynthetic Gene psbA in Marine and Freshwaters

Many cyanophage isolates which infect the marine cyanobacteria Synechococcus spp. and Prochlorococcus spp. contain a gene homologous to psbA, which codes for the D1 protein involved in photosynthesis. In the present study, cyanophage psbA gene fragments were readily amplified from freshwater and marine samples, confirming their widespread occurrence in aquatic communities. Phylogenetic analyses demonstrated that sequences from freshwaters have an evolutionary history that is distinct from that of their marine counterparts. Similarly, sequences from cyanophages infecting Prochlorococcus and Synechococcus spp. were readily discriminated, as were sequences from podoviruses and myoviruses. Viral psbA sequences from the same geographic origins clustered within different clades. For example, cyanophage psbA sequences from the Arctic Ocean fell within the Synechococcus as well as Prochlorococcus phage groups. Moreover, as psbA sequences are not confined to a single family of phages, they provide an additional genetic marker that can be used to explore the diversity and evolutionary history of cyanophages in aquatic environments.     

Diversity of ammonia monooxygenase (amoA) genes across environmental gradients in Chesapeake Bay sediments

Chesapeake Bay, the largest estuary in North America, encompasses a wide range of nutrient loading and trophic levels from the rivers and upper Bay to the sea, providing an ideal natural environment in which to explore relationships between functional diversity, physical/chemical complexity and ecosystem function (e.g. nitrification). In this study, amoA gene fragments (encoding subunit A of the key nitrification enzyme, ammonia monooxygenase) were PCR‐amplified from DNA extracted from sediment cores collected at five stations spanning gradients of salinity, ammonium, nitrate, oxygen and organic carbon along the Bay and Choptank River, a subestuary of the Bay. Phylogenetic analysis of ～30 amoA clones from each station revealed extensive diversity within the β‐Proteobacteria group of ammonia‐oxidizing bacteria (AOB), with the vast majority of sequences falling into coherent phylogenetic clusters distinct from sequences of cultivated AOB. Over 70% of the clones fell into two major phylogenetic clusters that appear to represent novel groups of Nitrosomonas‐like and Nitrosospira‐like amoA sequences that may be specific to estuarine and marine environments. Rarefaction analysis, estimators of genetic variation and dissimilarity indices all revealed differences in the relative amoA‐based diversity and/or richness among most of the stations, with the highest diversity at the North Bay station and the lowest at the mesohaline stations. Although salinity appears to play a role, no single physical or chemical parameter entirely explains the pattern of diversity along the estuary, suggesting that a complex combination of environmental factors may shape the overall level of AOB diversity in this dynamic environment.