Phylogenetic diversity of marine cyanophage isolates and natural virus communities as revealed by sequences of viral capsid assembly protein gene g20

Abstract

In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different Synechococcus groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity.

FIG. 1.

Phylogenetic affiliation of cyanophage isolates based on 552-bp nucleotide sequences between primers CPS1 and CPS8. Coliphage T4 was used as an outgroup. Values at tree branches are bootstrap values with 100 replicates. The scale bar is equivalent to 0.1 replacement per site. Only bootstrap values larger than 50 are shown.

FIG. 2.

Phylogenetic relationships among the clones amplified with primers CPS1 and CPS8 from concentrated natural virus communities at Skidaway Estuarine surface SE1 (a), Gulf Stream surface GS26 (b), Gulf Stream DCM GS27 (c), Sargasso Sea surface SS48 (d), Sargasso Sea DCM SS47 (e), and Sargasso Sea DCM SS40 (f). The neighbor-joining tree was constructed based on 552-bp nucleotide sequence alignment with T4 as the outgroup. Only bootstrap values of greater than 50 are shown. Each value in parentheses is the number of clones bearing identical nucleotide sequences in a community. The clusters were assigned on the basis of Fig. 3.

FIG. 2.

Phylogenetic relationships among the clones amplified with primers CPS1 and CPS8 from concentrated natural virus communities at Skidaway Estuarine surface SE1 (a), Gulf Stream surface GS26 (b), Gulf Stream DCM GS27 (c), Sargasso Sea surface SS48 (d), Sargasso Sea DCM SS47 (e), and Sargasso Sea DCM SS40 (f). The neighbor-joining tree was constructed based on 552-bp nucleotide sequence alignment with T4 as the outgroup. Only bootstrap values of greater than 50 are shown. Each value in parentheses is the number of clones bearing identical nucleotide sequences in a community. The clusters were assigned on the basis of Fig. 3.

FIG. 3.

Neighbor-joining tree showing the phylogenetic affiliation of cyanophage isolates (boldface type) and representative clones from all six of the natural virus communities studied. The tree was constructed on the basis of a 176-amino-acid sequence alignment with T4 as the outgroup. Each value in parentheses is the number of different nucleotide sequences in the same cluster and same community as the representative clone. Clusters A through F and I through III were assigned on the basis of phylogenetic relatedness. Bootstrap values of less than 50 were not shown. The scale bar indicates 0.1 substitution per site.