Metagenomic characterization of Chesapeake Bay virioplankton

Abstract

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.

FIG. 1.

Distribution of translated BLAST (tBLASTx against nucleotide databases and BLASTx against protein databases) matches between all database “types.” The upper, largest circle represents matches to the env-nt and/or env-nr database comprised mostly of Sargasso Sea bacterial metagenomic data. The leftmost circle represents matches to either of the traditional GenBank (nt and nr) databases. The bottom right circle represents matches to any of a series of small viral metagenomes from terrestrial and marine environments (see Materials and Methods for details). Intersections of circles represent sequences that had BLAST homology to more than one database type, and the center area represents sequences with homology to all three types.

FIG. 2.

Distribution of translated BLAST sequence matches across taxonomic domains sorted by match quality. Sequences were placed in nonredundant bins according to quality (i.e., E value), and relative domain percentages were calculated for each bin. The smallest E values represent the highest-quality matches on the left. The least confident matches are on the right, with a maximum E value of 10⁻³. The numbers of sequences in the bins are indicated above the bars.

FIG. 3.

Distribution of translated BLAST prokaryotic homolog sequences. The data are organized according to prokaryotic phyla. Data for completed prokaryote genomes in GenBank (at the time of metagenome sequence comparison) are shown to illustrate groups that are overrepresented (e.g., cyanobacteria and Proteobacteria) or underrepresented (e.g., Firmicutes) in the metagenome relative to the subject database. CB, Chesapeake Bay.

FIG. 4.

Viral metagenome translated BLAST homologs sorted according to annotated functional gene category. Each sequence was assigned to a presumptive functional category based on the highest-quality sequence homolog. The most likely phylogenetic affiliations (virus, bacteria, prophage, and mobile element) for each sequence category are indicated. Asterisks indicate categories that could not contain prokaryote sequences because they are purely viral functions.

FIG. 5.

Positions of Chesapeake Bay virioplankton BLAST homologs on the Prochlorococcus phage P-SSM2 genome. Regions with high levels of coverage are indicated by brackets. Only translated BLAST homologs with E values below 10⁻⁶ are shown. psbA, core photosystem II reaction center protein; nrdA&B, alpha and beta subunits of ribonucleoside reductase.

FIG. 6.

Phylogenetic tree of PsbA amino acid sequences deduced by a comparison of viral metagenome sequences with PsbA amino acid sequences derived from public databases. The tree is based on alignment of 187 homologous positions. Scale bar = 0.05 substitution per position.

FIG. 7.

PFGE gel of virioplankton concentrates used to construct the Chesapeake Bay metagenome library. The numbers above the lanes indicate the stations in the bay (see Materials and Methods for the location of each station). For station 858, surface and bottom samples are indicated by the suffixes “s” and “b,” respectively. The numbers on the left indicate marker band sizes (in kilobases). Marker lanes M contained concatemers of phage λ genomes (with resolvable bands at positions ranging from 291 to 48.5 kb) mixed with a HindIII digest of λ genomic DNA (23.1 and 9.4 kb). The viral concentrate from the bottom water sample at station 858 was not used in construction of the metagenome library in this study.