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.

Xanthomonas citri jumbo phage XacN1 exhibits a wide host range and high complement of tRNA genes

“…Phage structural proteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (4–15% polyacrylamide) were stained with Coomassie Brilliant Blue, excised from the in-gel and digested with trypsin. The tryptic peptides were trapped with a short ODS column (PepMap 100; 5 × 0.3 mm ID, Thermo Fisher Scientific Inc. as described before 4 , Waltham, MA, USA, 30 µL/min) and separated with a long ODS column (Capillary Column; 120 × 0.075 mm ID, Nikkyo Technos, Tokyo, Japan, 0.2 µL/min) using nano-liquid chromatography (Ultimate 3000 RSLC, Thermo Fisher Scientific Inc.). The eluate was continuously introduced into a nanoESI source and analyzed by mass spectrometry (MS) and MS/MS (LTQ Orbitrap XL, Thermo Fisher Scientific Inc.).…”

“…The MS and MS/MS spectra were generated in the positive ion mode using Orbitrap ( m/z 300–1500) and Iontrap (data-dependent scan of top five peaks using CID), respectively, with the capillary source voltage at 1.5 kV and the transfer capillary temperature at 200 °C. The MS/MS data was assigned to tryptic peptides encoded by ORFs as described before 4 with the Xcalibur program ver. 2.0 (Thermo Fisher Scientific Inc.).…”

“…They usually show large virions, being capable of enclosing a larger genome than those of other smaller phages 2 . Jumbo phages are classified into several evolutionarily unrelated clades 3 , 4 . The largest reported phage is Bacillus phage G with a 160 nm capsid and a 453 nm tail.…”

Phage-Antibiotic Combination Treatments: Antagonistic Impacts of Antibiotics on the Pharmacodynamics of Phage Therapy?

“…By way of example, regarding antibiotic interference with phage infection activity, Matsui et al [68] describe four phages of Ralstonia solanacearum-ΦRSF1, ΦRSL1, ΦRSL2, and ΦRSB1-for which phage virion production is fully blocked given bacterial exposure to 5 µg/mL rifampin (20 min incubation prior to phage infection). Here the calculated MIC for this host was 3 µg/mL, and thus phage virion-production was inhibited given exposure to~2× MIC.…”

“…Matsui et al [68] tested the ability of phages ΦRP12 and ΦRP31 of R. solanacearum to replicate in the presence of up to 20 µg/mL of rifampin (MIC = 5 µg/mL). At 1× or 2× MIC, resulting titers were reduced by about one log, whereas at 4× MIC, i.e., 20 µg/mL, they were reduced by roughly 2 logs.…”