Nearly identical bacteriophage structural gene sequences are widely distributed in both marine and freshwater environments

Abstract

Primers were designed to amplify a 592-bp region within a conserved structural gene (g20) found in some cyanophages. The goal was to use this gene as a proxy to infer genetic richness in natural cyanophage communities and to determine if sequences were more similar in similar environments. Gene products were amplified from samples from the Gulf of Mexico, the Arctic, Southern, and Northeast and Southeast Pacific Oceans, an Arctic cyanobacterial mat, a catfish production pond, lakes in Canada and Germany, and a depth of ca. 3,246 m in the Chuckchi Sea. Amplicons were separated by denaturing gradient gel electrophoresis, and selected bands were sequenced. Phylogenetic analysis revealed four previously unknown groups of g20 clusters, two of which were entirely found in freshwater. Also, sequences with >99% identities were recovered from environments that differed greatly in temperature and salinity. For example, nearly identical sequences were recovered from the Gulf of Mexico, the Southern Pacific Ocean, an Arctic freshwater cyanobacterial mat, and Lake Constance, Germany. These results imply that closely related hosts and the viruses infecting them are distributed widely across environments or that horizontal gene exchange occurs among phage communities from very different environments. Moreover, the amplification of g20 products from deep in the cyanobacterium-sparse Chuckchi Sea suggests that this primer set targets bacteriophages other than those infecting cyanobacteria.

FIG. 1.

Negative images of denaturing gradient gels of PCR-amplified g20 gene fragments. Labeled bands were excised from the gels, cloned, and then sequenced. Bands in the same lane with the same letter beside them (a, b, or c) had >99% identical nucleotides. From within each lane, only one sequence with >99% identical nucleotides was included in further analyses. Bands with labels that have an asterisk and are shown in bold were included in the phylogenetic analysis.

FIG. 2.

Neighbor-joining tree of g20 fragments. Bootstrap values are shown as percentages to the lower left of the appropriate nodes. Sequences of cyanophage isolates do not have a bar next to them, and sequences with a black, white, or gray bar to the right are environmental g20 sequences that were obtained from GenBank. Environmental sequences obtained during the present study are indicated by colored bars, and the sequences of the cyanophage isolates obtained during the present study are named S-PWM3 and S-PWM4. The three letters in the sequence name refer to the location and the two numbers refer to the year of collection. Clusters A to F and I to III were assigned by Zhong et al. (41), clusters N1 to N4 were assigned by Wang et al. (35), and clusters 1 to 6 were assigned by Dorigo et al. (5). We assigned clusters G, H, J, K, and CSP. All clusters were assigned on the basis of phylogenetic relatedness. The scale bar represents the number of amino acid substitutions per residue.