Isolation and analysis of Gordonia terrae phages reveals novel aspects of lysis, virion morphology, and host range

Matthew Benczkowski, Melanie Umbaugh, Pitt SEA-PHAGES 2015-16 students, Rebecca Bortz, Emily Furbee, Sarah Grubb, Deborah Jacobs-Sera, Matthew Montgomery, Marcie Warner, Welkin H Pope, Graham F Hatfull

Analysis of more than 1,000 Mycobacterium smegmatis bacteriophage (mycobacteriophage) genomes provides significant insights into phage diversity and evolution. To gain a broader perspective on phage biology, University of Pittsburgh students utilized an alternate host, Gordonia terrae 3612, for phage isolation. G. terrae is a member of the same taxonomic order (Corynebacteriales) as M. smegmatis; this relatedness facilitates analysis of the evolutionary pressures affecting different classes of phage genes. Isolation of Gordonia phages may also have medical and industrial applications: Gordonia species are implicated in catheter-related infections in humans and as contributors to wastewater sludge. Pitt students isolated 182 Gordonia phages; 30 of which have been sequenced and annotated. These Gordonia phages have been categorized into 8 clusters and 12 singletons, and their diversity appears to be at least as great if not greater than the mycobacteriophages. The Gordonia phage genomes have an average length of 54,897 bp (ranging from 41,857 bp [Nyceirae] to 98,136 bp [Yvonnetastic]), average G+C content of 65.4% (ranging from 58.9% [Hotoboro] to 68.4% [Phinally]; the host G+C content is 67.8%), and 88 genes (ranging from 66 [Emalyn] to 198 [Yvonnetastic]). Gordonia phage genomes share similar architectures with mycobacteriophages: the left arms contain the virion structure and assembly genes, genes involved in lysogeny (if present) are centrally located, and the right arms contain DNA metabolism genes, orphams, and genes of no known function. Despite genome architecture similarities to mycobacteriophages, Gordonia phages display a number of distinct attributes. The enzymatic functions of mycobacteriophage lysin A’s are commonly separated into two polypeptides in Gordonia phages and lysin B genes are found in unexpected locations. Numerous phages isolated on G. terrae were also able to infect one or more of the following hosts: G. lacunae, G. westfalica, G. rubripertincta, and G. neofelifaecis. This promiscuity in host range is rarely observed in the mycobacteriophages. Gordonia phages also exhibit distinct morphological features. Gordonia phages frequently possess exceptionally long, flexible tails; tape measure genes in Hotorobo, Benczkowski14, and Katyusha range in size from 9,084 bp to 9,984 bp and are among the longest such genes discovered. To date, Pupper is the sole contractile tailed Gordonia phage to be identified. Furthermore, Cluster CZ (composed entirely of Gordonia phages) is the first cluster to contain phages with both prolate and isometric capsids; this can likely be attributed to the discovery of variability in the major capsid genes present in individual members of this cluster. These distinctive aspects of Gordonia phages, combined with the discovery of a large collection of new orphams in these phages, support the notion that the phage population is vast and continually expanding.