All blastx results with an E-value of < 0.001 were analysed using the megan software to identify the different taxa present in reclaimed and potable water viral communities. Since viral particles were purified extensively prior to isolating DNA and RNA, contigs with their best matches to proteins from cellular organisms (i.e. bacteria, archaea and eukaryotes) were further compared against the ACLAME database to identify proteins related to mobile genetic elements (i.e. plasmids and phages). For all metagenomes, more than 60% of the sequences that were classified as bacteria were re-classified as plasmids or phages after performing a blastx analysis against the ACLAME database. Although sequences similar to eukaryotes and archaea were not as abundant as the bacterial sequences, 15–56% of eukaryotic and 40–70% of archaeal sequences were also re-classified in the same manner. The discrepancy between the results obtained from searching the GenBank versus ACLAME databases may be due to an abundance of unidentified prophage-like sequences within microbial genomes in GenBank (Fouts, 2006). Similarities to mobile genetic elements are common in previously sequenced DNA viral metagenomes from other sources (Breitbart et al., 2002; 2003; 2004; Bench et al., 2007; Kim et al., 2008).
Hits to mobile genetic elements dominated all the viral metagenomes (Fig. 2B). For reclaimed water DNA libraries, 51% of the known sequences were similar to viral proteins, whereas the majority of the sequences in the reclaimed water RNA libraries and potable water DNA library were similar to proteins found in plasmids. More than 99% of plasmid-like protein sequences were identified after re-analysing sequences with hits to cellular organisms through the ACLAME database. Although most of the sequences identified were similar to hypothetical proteins, numerous integrases, transposases, recombinases and replication-associated proteins, among others, were identified. Since viral particles were purified by CsCl gradients and DNase treatment to remove contaminating cells and free DNA, plasmids should have been eliminated before viral DNA and RNA isolation. However, phages and plasmids share a number of characteristics as both contain machinery for gene transfer and replication. Moreover, there may be genetic exchange between plasmids, phages and other mobile genetic elements within a bacterial host (Boltner et al., 2002; Mark Osborn and Böltner, 2002), and this exchange may lead to gene organization and protein similarities between plasmids and phages (Hazen et al., 2007). In addition, some prophages (e.g. pKO2, PY4, P1, N15, LE1, φ20 and φBB-1) replicate in their hosts as low-copy-number plasmids instead of integrating into the host genomes (Ikeda and Tomizawa, 1968; Inal and Karunakaran, 1996; Eggers et al., 2000; Girons et al., 2000; Ravin et al., 2000; Briani et al., 2001; Casjens et al., 2004). It is possible that there is an abundance of previously undescribed phages containing plasmid-like proteins in potable water as 52% of the identified sequences had similarities to plasmid proteins (Fig. 2B). The abundance of plasmid-like sequences in reclaimed water RNA libraries may reflect the abundance of novel RNA viruses with plasmid-like properties such as the endornaviruses. Currently, four species of dsRNA viruses with plasmid-like properties found in some rice and bean species have been classified by the International Committee on Taxonomy of Viruses as members of the Endornavirus genus (Gibbs et al., 2005). In addition, double-stranded RNA viruses with plasmid-like properties have been found in plants, algae, fungi, protozoa and insects and, thus, endorna-like viruses may be widely distributed among eukaryotes (Horiuchi and Fukuhara, 2004; Fukuhara et al., 2006; Osaki et al., 2006).