Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum

Abstract

A filamentous bacteriophage, designated ϕRs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that ϕRs551 is a member of the family Inoviridae, and is about 1200 nm long and 7 nm wide. ϕRs551 has a genome of 7929 nucleotides containing 14 open reading frames, and is the first isolated virion that contains a resolvase (ORF13) and putative type-2 phage repressor (ORF14). Unlike other R. solanacearum phages isolated from soil, the genome sequence of ϕRs551 is not only 100% identical to its prophage sequence in the deposited genome of R. solanacearum strain UW551 from which the phage was isolated, but is also surprisingly found with 100% identity in the deposited genomes of 10 other phylotype II sequevar 1 strains of R. solanacearum. Furthermore, it is homologous to genome RS-09-161, resulting in the identification of a new prophage, designated RSM10, in a R. solanacearum strain from India. When ORF13 and a core attP site of ϕRs551 were either deleted individually or in combination, phage integration was not observed, suggesting that similar to other filamentous R. solanacearum ϕRSM phages, ϕRs551 relies on its resolvase and the core att sequence for site-directed integration into its susceptible R. solanacearum strain. The integration occurred four hours after phage infection. Infection of a susceptible R. solanacearum strain RUN302 by ϕRs551 resulted in less fluidal colonies and EPS production, and reduced motilities of the bacterium. Interestingly, infection of RUN302 by ϕRs551 also resulted in reduced virulence, rather than enhanced or loss of virulence caused by other ϕRSM phages. Study of bacteriophages of R. solanacearum would contribute to a better understanding of the phage-bacterium-environment interactions in order to develop integrated management strategies to combat R. solanacearum.

Filamentous phages: masters of a microbial sharing economy

“…The RSM1‐like phages (including RSM3, and Rs551) have been implicated in cooperatively trading away virulence to acquire drug resistance in a plant disease model. This the phage does by reducing Ralstonia 's production of extracellular polysaccharide, reducing twitching motility, increasing cell aggregation and reducing the expression of key virulence genes . Given the phage modifies the host to ameliorate virulence, they have been proposed as biocontrol agents .…”

Prophage Rs551 and Its Repressor Gene orf14 Reduce Virulence and Increase Competitive Fitness of Its Ralstonia solanacearum Carrier Strain UW551

“…For the past 10 years, a wide range of lysogenic and lytic bacteriophages specifically infecting R. solanacearum have been isolated from soil of crop fields, and their use as potential biocontrol agents has been explored ( Yamada et al, 2007 ; Fujiwara et al, 2011 ; Bhunchoth et al, 2015 ). We recently isolated a filamentous lysogenic bacteriophage ϕRs551 directly from the race 3 biovar 2 strain UW551 of R. solanacearum grown under normal culture conditions ( Ahmad et al, 2017 ). The phage has a particle size of about 1,200 nm in length and 7 nm in width, and has a genome size of 7,929 nucleotide with 14 open reading frames ( Ahmad et al, 2017 ).…”

“…We recently isolated a filamentous lysogenic bacteriophage ϕRs551 directly from the race 3 biovar 2 strain UW551 of R. solanacearum grown under normal culture conditions ( Ahmad et al, 2017 ). The phage has a particle size of about 1,200 nm in length and 7 nm in width, and has a genome size of 7,929 nucleotide with 14 open reading frames ( Ahmad et al, 2017 ). In contrast with other R. solanacearum phages isolated from soil, ϕRs551 is the first isolated phage that contains a resolvase (ORF13) and a putative type-2 phage repressor (ORF14), although how this repressor maintains a prophage state and phage immunity in ϕRs551 had not been studied.…”

“…In contrast with other R. solanacearum phages isolated from soil, ϕRs551 is the first isolated phage that contains a resolvase (ORF13) and a putative type-2 phage repressor (ORF14), although how this repressor maintains a prophage state and phage immunity in ϕRs551 had not been studied. In addition, the genome sequence of ϕRs551 is surprisingly found with 100% identity in the deposited genomes of 11 race 3 biovar 2 phylotype IIB sequevar 1 strains of R. solanacearum , indicating evolutionary importance ( Ahmad et al, 2017 ). Infection of a susceptible R. solanacearum strain RUN302 by ϕRs551 resulted in colonies with less fluidal appearance and reduced EPS production, motility, and virulence ( Ahmad et al, 2017 ).…”

“…In addition, the genome sequence of ϕRs551 is surprisingly found with 100% identity in the deposited genomes of 11 race 3 biovar 2 phylotype IIB sequevar 1 strains of R. solanacearum , indicating evolutionary importance ( Ahmad et al, 2017 ). Infection of a susceptible R. solanacearum strain RUN302 by ϕRs551 resulted in colonies with less fluidal appearance and reduced EPS production, motility, and virulence ( Ahmad et al, 2017 ). It is unclear, however, what effects the phage or the putative phage repressor has on its host strain R. solanacearum UW551.…”