Replications of Two Closely Related Groups of Jumbo Phages Show Different Level of Dependence on Host-encoded RNA Polymerase

Abstract

Ralstonia solanacearum phages ΦRP12 and ΦRP31 are jumbo phages isolated in Thailand. Here we show that they exhibit similar virion morphology, genome organization and host range. Genome comparisons as well as phylogenetic and proteomic tree analyses support that they belong to the group of ΦKZ-related phages, with their closest relatives being R. solanacearum phages ΦRSL2 and ΦRSF1. Compared with ΦRSL2 and ΦRSF1, ΦRP12 and ΦRP31 possess larger genomes (ca. 280 kbp, 25% larger). The replication of ΦRP12 and ΦRP31 was not affected by rifampicin treatment (20 μg/ml), suggesting that phage-encoded RNAPs function to start and complete the infection cycle of these phages without the need of host-encoded RNAPs. In contrast, ΦRSL2 and ΦRSF1, encoding the same set of RNAPs, did not produce progeny phages in the presence of rifampicin (5 μg/ml). This observation opens the possibility that some ΦRP12/ΦRP31 factors that are absent in ΦRSL2 and ΦRSF1 are involved in their host-independent transcription.

Keywords: Ralstonia solanacearum; genomic analysis; jumbo phages; virion-associated-RNA polymerase; ΦKZ-like phages.

Figure 1

Genome comparison among five ΦKZ-related phage genomes. (A) Linear genome alignment of five ΦKZ-related phages. Red and blue lines between genomes represent sequence similarities (≥50% identity) detected by TBLASTX in the same and reverse orientations, respectively. (B) Dot-plot comparison among five ΦKZ-related phages. Red and blue lines represent sequence similarities detected by TBLASTX in the same and reverse orientations, respectively.

Figure 2

Proteomic and phylogenetic relationships between ΦRP12/ΦRP31 and other phages. (A) A proteomic tree produced by the BIONJ program (Gascuel, 1997) based on TBLASTX genomic sequence comparisons of 61 phage genomes. Branch lengths from the root were scaled logarithmically. In this logarithmic representation, nodes that were at distances smaller than 0.001 from the root were agglomerated into the root point. (B,C) Maximum likelihood phylogenetic trees of the tail sheath and terminase large subunit proteins, respectively. Statistical support at node is given as bootstrap values. Number at scale bar indicates the number of substitutions per site.

Figure 3

Phylogenetic relationships of virion-associated and early-expressed RNAP homologs. Maximum likelihood phylogenetic trees of RNA polymerase β subunits (A) and β′ subunits (B). ORFs corresponding to each subunit were concatenated before building sequence alignments. Black rectangles correspond to proposed gene duplications. Bootstrap values are given along the branches. Number at scale bar indicates the number of substitutions per site.

Figure 4

Proteomic analysis of virion proteins of ΦRP31. Proteins from purified ΦRP31 particles were separated by SDS-PAGE and stained with Coomassie blue. The protein bands excised from the SDS-PAGE gel were subjected to trypsin digestion and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS, LTQ Orbitrap XL). Tandem mass spectrometry data were assigned to tryptic peptides encoded by phage open reading frames using an established procedure (Ahmad et al., 2014). Asterisks indicate the fragmented β and β′ subunits of virion-associated-RNAP. VSP: virion structural protein.