Enterococcal Bacteriophages and Genome Defense

Bacteriophages (phages) are viruses that infect bacteria. Similar to the viruses of plants and animals, phages are inert and are unable to propagate themselves in the absence of a host. Phages depend on host metabolism to provide the organic material and machinery necessary for their replication and for the subsequent packaging of the viral genetic material during phage particle biosynthesis. Phages are associated with nearly all known bacterial taxa and, as a result, are found in diverse environments that range from soil to oceans and even in deserts (Prestel, Salamitou, & DuBow, 2008; Prigent, Leroy, Confalonieri, Dutertre, & DuBow, 2005; Srinivasiah, Bhavsar, Thapar, Liles, Schoenfeld, & Wommack, 2008; Wommack & Colwell, 2000). Phages are found either directly associated with their bacterial hosts or in large numbers as free virions in the environment. Since there is a vast distribution of phages across the globe, it is possible to theorize that phages constitute the most abundant biological entities on earth. Their numbers have been estimated to reach as high as 10 particles with the potential for 10 phage infections occurring every second (Pedulla, et al., 2003; Wommack & Colwell, 2000). As many more phage genome sequences have become available in recent years, it is obvious that phages are extremely incongruent at the genomic level. This diversity in genetic makeup is proposed to result from the fastidious replication of phage particles during the infection of highly permissive hosts. During these infections, phages are able to exchange DNA within host genomes through recombination, and continually generate diversity as a result (Hendrix, Smith, Burns, Ford, & Hatfull, 1999). The vast majority of phages belong to the order of , which are tailed phages that have dsDNA and an isometric capsid. is comprised of three phylogenetically-related families that are discriminated by tail morphology: (long contractile tails), (long non-contractile tails), and (short tails) (Ackermann, 2007; Krupovic, Prangishvili, Hendrix, & Bamford, 2011). The most well-studied tailed phages are the coliphages ʎ (), T4 (), and T7 () which infect and which have served as workhorses for elucidating the mechanisms of modern molecular genetics and biochemistry (Johnson, Poteete, Lauer, Sauer, Ackers, & Ptashne, 1981; Miller, Kutter, Mosiq, Arisaka, Kunisawa, & Rüger, 2003; Ptashne, et al., 1980; Tabor & Richardson, 1985). Far less abundant are the non-tailed phages, which encompass numerous families with great morphological distinction; these include phages that are filamentous (long filaments to short rods), polyhedral (vesicular and envelope-like), and pleomorphic (including those that are lemon, droplet, and ampule shaped) (Ackermann, 2007). The nucleic acid content of phage genomes is either DNA or RNA and both double and single stranded DNA and RNA phages have been identified. In addition, the size of the phage genome can range from under ten kilobases to several hundred kilobases. Phages have evolved replication strategies that can be lytic, lysogenic (temperate), or chronic. Chronic replication results in the continual, non-lethal shedding of virions by protrusion through the membrane. All phages have common life-cycle stages of adsorption, DNA injection and replication, virion production, and release. Tailed phages mediate host cell lysis through the combined action of a holin, which perforates the membrane, and an endolysin (lysin), which hydrolyses cell wall peptidoglycan. Lytic phages are restricted to a life-cycle that results in the lysis of their host. Temperate phages have two possible life-cycles: lysis, or the recombination of their genome at a chromosomal attachment site using a phage-encoded integrase. Temperate phages are maintained within the host chromosome by transcriptional repressors that determine when the phage undergoes an infectious or lytic switch. The lytic switch occurs when conditions within their host promote excision. Excision usually proceeds during times of hardship when host health is threatened, either by physical stress or by chemical stress, such as antibiotics, ultraviolet (UV) light, or reactive oxygen species (Allen, et al., 2011; DeMarini & Lawrence, 1992; Little & Mount, 1982). Temperate phages provide key insights into the evolution of bacterial pathogenesis, since many temperate phages encode virulence factors used by pathogenic bacteria during both human and animal infections (Bensing, Siboo, & Sullam, 2001; Brüssow, Canchaya, & Hardt, 2004; Novick, Christie, & Penadés, 2010).