Novel Bacteriophages Containing a Genome of Another Bacteriophage within Their Genomes Maud M. Swanson 1. , Brian Reavy 1. *, Kira S. Makarova 2 , Peter J. Cock 1 , David W. Hopkins 1¤ , Lesley Torrance 1 , Eugene V. Koonin 2 , Michael Taliansky 1 1 The James Hutton Institute, Invergowrie, Dundee, United Kingdom, 2 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, United States of America Abstract A novel bacteriophage infecting Staphylococus pasteuri was isolated during a screen for phages in Antarctic soils. The phage named SpaA1 is morphologically similar to phages of the family Siphoviridae. The 42,784 bp genome of SpaA1 is a linear, double-stranded DNA molecule with 39 protruding cohesive ends. The SpaA1 genome encompasses 63 predicted protein- coding genes which cluster within three regions of the genome, each of apparently different origin, in a mosaic pattern. In two of these regions, the gene sets resemble those in prophages of Bacillus thuringiensis kurstaki str. T03a001 (genes involved in DNA replication/transcription, cell entry and exit) and B. cereus AH676 (additional regulatory and recombination genes), respectively. The third region represents an almost complete genome (except for the short terminal segments) of a distinct bacteriophage, MZTP02. Nearly the same gene module was identified in prophages of B. thuringiensis serovar monterrey BGSC 4AJ1 and B. cereus Rock4-2. These findings suggest that MZTP02 can be shuttled between genomes of other bacteriophages and prophages, leading to the formation of chimeric genomes. The presence of a complete phage genome in the genome of other phages apparently has not been described previously and might represent a ‘fast track’ route of virus evolution and horizontal gene transfer. Another phage (BceA1) nearly identical in sequence to SpaA1, and also including the almost complete MZTP02 genome within its own genome, was isolated from a bacterium of the B. cereus/B. thuringiensis group. Remarkably, both SpaA1 and BceA1 phages can infect B. cereus and B. thuringiensis, but only one of them, SpaA1, can infect S. pasteuri. This finding is best compatible with a scenario in which MZTP02 was originally contained in BceA1 infecting Bacillus spp, the common hosts for these two phages, followed by emergence of SpaA1 infecting S. pasteuri. Citation: Swanson MM, Reavy B, Makarova KS, Cock PJ, Hopkins DW, et al. (2012) Novel Bacteriophages Containing a Genome of Another Bacteriophage within Their Genomes. PLoS ONE 7(7): e40683. doi:10.1371/journal.pone.0040683 Editor: Ramy K. Aziz, Cairo University, Egypt Received March 1, 2012; Accepted June 14, 2012; Published July 17, 2012 Copyright: ß 2012 Swanson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work of authors MMS, BR, PJC, DH, LT and MT was funded by the Scottish Government’s Rural and Environment Science and Analytical Services (RESAS) Division. KSM and EVK are supported by the intramural funds of the United States Department of Health and Human Services (National Library of Medicine, National Institutes of Health). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Brian.Reavy@hutton.ac.uk . These authors contributed equally to this work. ¤ Current address: School of Life Sciences, Heriot-Watt University, Edinburgh, United Kingdom Introduction Viruses are the most abundant entities in the biosphere. In marine and soil habitats, the number of virus particles exceeds the number of cells by at least an order of magnitude [1–3]. Numerous viruses infect organisms from all branches of cellular life. However, virus research has traditionally focused on viruses that infect humans, other vertebrates and plants due to the obvious medical and agricultural importance of these viruses. In addition, viruses infecting several model bacteria (bacteriophages) have been studied in detail thanks primarily to their utility as tools of molecular biology. Viruses from diverse environments are incomparably less thoroughly characterized but recently environ- mental genomics and metagenomics of viruses have become rapidly growing research areas [4–7]. A total of about 2300 viruses are recognized by the Interna- tional Committee on Taxonomy of Viruses [8] but this is likely to be a gross underestimate because of the enormous diversity of viruses in unsampled or poorly investigated habitats (see for example, [9], [10]. Virus particles are abundant in air, water and soils [1], [5], [11–15]. Recent metagenomic analyses have revealed hitherto unknown diverse assemblages of viruses in these environments [6], [9], [10], [16], [17]. For example, Fierer et al. [10] reported that the majority of the 4577 virus-related nucleotide sequences found in soils from different ecosystems showed no similarity to previously described sequences. Analysis of metage- nomic data suggests novel patterns of virus evolution and reveals new groups of viruses providing unprecedented insights into the composition and dynamics of the virus world [7]. Viruses, in particular transducing bacteriophages, have been long known to make major contributions to gene exchange between bacteria [18]. Recently, a distinct class of defective bacteriophages, the Gene Transfer Agents (GTAs) [19], have been characterized as apparent dedicated vehicles for horizontal gene transfer that might account for extensive gene flow in bacterial and archaeal PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40683