Portal protein diversity and phage ecology

Abstract

Oceanic phages are critical components of the global ecosystem, where they play a role in microbial mortality and evolution. Our understanding of phage diversity is greatly limited by the lack of useful genetic diversity measures. Previous studies, focusing on myophages that infect the marine cyanobacterium Synechococcus, have used the coliphage T4 portal-protein-encoding homologue, gene 20 (g20), as a diversity marker. These studies revealed 10 sequence clusters, 9 oceanic and 1 freshwater, where only 3 contained cultured representatives. We sequenced g20 from 38 marine myophages isolated using a diversity of Synechococcus and Prochlorococcus hosts to see if any would fall into the clusters that lacked cultured representatives. On the contrary, all fell into the three clusters that already contained sequences from cultured phages. Further, there was no obvious relationship between host of isolation, or host range, and g20 sequence similarity. We next expanded our analyses to all available g20 sequences (769 sequences), which include PCR amplicons from wild uncultured phages, non-PCR amplified sequences identified in the Global Ocean Survey (GOS) metagenomic database, as well as sequences from cultured phages, to evaluate the relationship between g20 sequence clusters and habitat features from which the phage sequences were isolated. Even in this meta-data set, very few sequences fell into the sequence clusters without cultured representatives, suggesting that the latter are very rare, or sequencing artefacts. In contrast, sequences most similar to the culture-containing clusters, the freshwater cluster and two novel clusters, were more highly represented, with one particular culture-containing cluster representing the dominant g20 genotype in the unamplified GOS sequence data. Finally, while some g20 sequences were non-randomly distributed with respect to habitat, there were always numerous exceptions to general patterns, indicating that phage portal proteins are not good predictors of a phage's host or the habitat in which a particular phage may thrive.

Fig. 1

Evolutionary relationships determined using 183 amino acids of the portal protein gene (g20) amplified from cultured phage isolates (names begin with ‘S-’ or ‘P-’ and are coloured orange or green for Synechococcus or Prochlorococcus phages respectively) from this study (italicized), as well as previous studies (non-italicized), and environmental g20 sequences (names in black) (Zhong et al., 2002; Marston and Sallee, 2003). Clusters defined by Zhong and colleagues (2002) are as follows: clusters I–III contain g20 sequences from cultured phage isolates, while clusters A–F represent only environmental g20 sequences. Clusters containing identical g20 protein sequences are numbered with alphanumeric numbers (1–13). For cultured phages, the phage isolate names are followed by black lettering that indicates the original host strain used for isolation, while the phage host range is indicated as high light-adapted Prochlorococcus (green circle or dash), low light-adapted Prochlorococcus (blue circle or dash) or Synechococcus (orange circle or dash). The circles represent cross-infection was observed within this group of hosts tested, whereas a dash indicates that no cross-infection was observed. Isolates not available for host range testing have no indication of their host range. The tree shown was inferred by neighbour-joining as described in the Experimental procedures. Support values shown at the nodes are neighbour-joining bootstrap/maximum parsimony bootstrap/maximum likelihood quartet puzzling support (only values > 50 are shown). Well-supported nodes (as defined in Experimental procedures) are designated by italicized support values, including six nodes that represent subclusters within the culture-containing clusters I–III. The g20 sequence from the non-cyanomyophage isolate T4 was used as an outgroup to root this tree.

Fig. 2

Evolutionary relationships determined using 554 base pairs of the portal protein gene (g20) from 769 available g20 sequences. Clusters defined by Zhong and colleagues (2002) are identified as culture-based clusters I–III and environmental-sequence-only clusters A–F. New clusters defined since Zhong and colleagues (2002) are indicated with the preface ‘new cluster’, a number and a brief description. The tree shown is the consensus (majority rules) tree from 11 GARLI iterations inferred using the maximum likelihood criterion (see Experimental procedures), with the Aeromonas phage Aeh1 g20 sequence used as an outgroup to root the tree. Three colour rings reflect the habitat type from which the g20 sequence originated. For most of these sequences (GOS sequences), there is ribotype dot-blot and metagenomic information about the microbial community structure at the site, while for non-GOS sequences such information was assumed where reasonable to do so (see Table 3 legend). The inner ring is the microbial community structure information listed as Rusch and colleagues (2007)-defined environmental categories, while the other two rings reflect the temperature and salinity of the original sampling site.