Analysis of a viral metagenomic library from 200 m depth in Monterey Bay, California constructed by direct shotgun cloning

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>We purified viruses from approximately one cubic meter of seawater collected from 200m 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 <it>E. coli </it>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.</p> <p>Results</p> <p>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 <it>Podo</it>-, <it>Sipho</it>-, and <it>Myoviridae</it>) and 6% matched viruses of eukaryotes in the Family <it>Phycodnaviridae </it>(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.</p> <p>Conclusions</p> <p>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.</p

The Sorcerer II Global Ocean Sampling Expedition: Metagenomic Characterization of Viruses within Aquatic Microbial Samples

Viruses are the most abundant biological entities on our planet. Interactions between viruses and their hosts impact several important biological processes in the world's oceans such as horizontal gene transfer, microbial diversity and biogeochemical cycling. Interrogation of microbial metagenomic sequence data collected as part of the Sorcerer II Global Ocean Expedition (GOS) revealed a high abundance of viral sequences, representing approximately 3% of the total predicted proteins. Cluster analyses of the viral sequences revealed hundreds to thousands of viral genes encoding various metabolic and cellular functions. Quantitative analyses of viral genes of host origin performed on the viral fraction of aquatic samples confirmed the viral nature of these sequences and suggested that significant portions of aquatic viral communities behave as reservoirs of such genetic material. Distributional and phylogenetic analyses of these host-derived viral sequences also suggested that viral acquisition of environmentally relevant genes of host origin is a more abundant and widespread phenomenon than previously appreciated. The predominant viral sequences identified within microbial fractions originated from tailed bacteriophages and exhibited varying global distributions according to viral family. Recruitment of GOS viral sequence fragments against 27 complete aquatic viral genomes revealed that only one reference bacteriophage genome was highly abundant and was closely related, but not identical, to the cyanomyovirus P-SSM4. The co-distribution across all sampling sites of P-SSM4-like sequences with the dominant ecotype of its host, Prochlorococcus supports the classification of the viral sequences as P-SSM4-like and suggests that this virus may influence the abundance, distribution and diversity of one of the most dominant components of picophytoplankton in oligotrophic oceans. In summary, the abundance and broad geographical distribution of viral sequences within microbial fractions, the prevalence of genes among viral sequences that encode microbial physiological function and their distinct phylogenetic distribution lend strong support to the notion that viral-mediated gene acquisition is a common and ongoing mechanism for generating microbial diversity in the marine environment

Going Deeper: Metagenome of a Hadopelagic Microbial Community

The paucity of sequence data from pelagic deep-ocean microbial assemblages has severely restricted molecular exploration of the largest biome on Earth. In this study, an analysis is presented of a large-scale 454-pyrosequencing metagenomic dataset from a hadopelagic environment from 6,000 m depth within the Puerto Rico Trench (PRT). A total of 145 Mbp of assembled sequence data was generated and compared to two pelagic deep ocean metagenomes and two representative surface seawater datasets from the Sargasso Sea. In a number of instances, all three deep metagenomes displayed similar trends, but were most magnified in the PRT, including enrichment in functions for two-component signal transduction mechanisms and transcriptional regulation. Overrepresented transporters in the PRT metagenome included outer membrane porins, diverse cation transporters, and di- and tri-carboxylate transporters that matched well with the prevailing catabolic processes such as butanoate, glyoxylate and dicarboxylate metabolism. A surprisingly high abundance of sulfatases for the degradation of sulfated polysaccharides were also present in the PRT. The most dramatic adaptational feature of the PRT microbes appears to be heavy metal resistance, as reflected in the large numbers of transporters present for their removal. As a complement to the metagenome approach, single-cell genomic techniques were utilized to generate partial whole-genome sequence data from four uncultivated cells from members of the dominant phyla within the PRT, Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Planctomycetes. The single-cell sequence data provided genomic context for many of the highly abundant functional attributes identified from the PRT metagenome, as well as recruiting heavily the PRT metagenomic sequence data compared to 172 available reference marine genomes. Through these multifaceted sequence approaches, new insights have been provided into the unique functional attributes present in microbes residing in a deeper layer of the ocean far removed from the more productive sun-drenched zones above

Cell-free cloning using φ29 DNA polymerase

We describe conditions for rolling-circle amplification (RCA) of individual DNA molecules 5–7 kb in size by >10(9)-fold, using φ29 DNA polymerase. The principal difficulty with amplification of small amounts of template by RCA using φ29 DNA polymerase is “background” DNA synthesis that usually occurs when template is omitted, or at low template concentrations. Reducing the reaction volume while keeping the amount of template fixed increases the template concentration, resulting in a suppression of background synthesis. Cell-free cloning of single circular molecules by using φ29 DNA polymerase was achieved by carrying out the amplification reactions in very small volumes, typically 600 nl. This procedure allows cell-free cloning of individual synthetic DNA molecules that cannot be cloned in Escherichia coli, for example synthetic phage genomes carrying lethal mutations. It also allows cell-free cloning of genomic DNA isolated from bacteria. This DNA can be sequenced directly from the φ29 DNA polymerase reaction without further amplification. In contrast to PCR amplification, RCA using φ29 DNA polymerase does not produce mutant jackpots, and the high processivity of the enzyme eliminates stuttering at homopolymer tracts. Cell-free cloning has many potential applications to both natural and synthetic DNA. These include environmental DNA samples that have proven difficult to clone and synthetic genes encoding toxic products. The method may also speed genome sequencing by eliminating the need for biological cloning

Biogeographic partitioning of Southern Ocean microorganisms revealed by metagenomics

We performed a metagenomic survey (6.6Gbp of 454 sequence data) of Southern Ocean (SO) microorganisms during the austral summer of 2007-2008, examining the genomic signatures of communities across a latitudinal transect from Hobart (44°S) to the Mertz Glacier, Antarctica (67°S). Operational taxonomic units (OTUs) of the SAR11 and SAR116 clades and the cyanobacterial genera Prochlorococcus and Synechococcus were strongly overrepresented north of the Polar Front (PF). Conversely, OTUs of the Gammaproteobacterial Sulfur Oxidizer-EOSA-1 (GSO-EOSA-1) complex, the phyla Bacteroidetes and Verrucomicrobia and order Rhodobacterales were characteristic of waters south of the PF. Functions enriched south of the PF included a range of transporters, sulfur reduction and histidine degradation to glutamate, while branched-chain amino acid transport, nucleic acid biosynthesis and methionine salvage were overrepresented north of the PF. The taxonomic and functional characteristics suggested a shift of primary production from cyanobacteria in the north to eukaryotic phytoplankton in the south, and reflected the different trophic statuses of the two regions. The study provides a new level of understanding about SO microbial communities, describing the contrasting taxonomic and functional characteristics of microbial assemblages either side of the PF. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd

Virophage control of antarctic algal host–virus dynamics

Viruses are abundant ubiquitous members of microbial communities and in the marine environment affect population structure and nutrient cycling by infecting and lysing primary producers. Antarctic lakes are microbially dominated ecosystems supporting truncated food webs in which viruses exert a major influence on the microbial loop. Here we report the discovery of a virophage (relative of the recently described Sputnik virophage) that preys on phycodnaviruses that infect prasinophytes (phototrophic algae). By performing metaproteogenomic analysis on samples from Organic Lake, a hypersaline meromictic lake in Antarctica, complete virophage and near-complete phycodnavirus genomes were obtained. By introducing the virophage as an additional predator of a predator–prey dynamic model we determined that the virophage stimulates secondary production through the microbial loop by reducing overall mortality of the host and increasing the frequency of blooms during polar summer light periods. Virophages remained abundant in the lake 2 y later and were represented by populations with a high level of major capsid protein sequence variation (25–100% identity). Virophage signatures were also found in neighboring Ace Lake (in abundance) and in two tropical lakes (hypersaline and fresh), an estuary, and an ocean upwelling site. These findings indicate that virophages regulate host–virus interactions, influence overall carbon flux in Organic Lake, and play previously unrecognized roles in diverse aquatic ecosystems