Characterization of Φ2954, a newly isolated bacteriophage containing three dsRNA genomic segments

Abstract

BackgroundBacteriophage Φ12 is a member of the Cystoviridae and is distinct from Φ6, the first member of that family. We have recently isolated a number of related phages and five showed high similarity to Φ12 in the amino acid sequences of several proteins. Bacteriophage Φ2954 is a member of this group.ResultsΦ2954 was isolated from radish leaves and was found to have a genome of three segments of double-stranded RNA (dsRNA), placing it in the Cystoviridae. The base sequences for many of the genes and for the segment termini were similar but not identical to those of bacteriophage Φ12. However, the host specificity was for the type IV pili of Pseudomonas syringae HB10Y rather than for the rough LPS to which Φ12 attaches. Reverse genetics techniques enabled the production of infectious phage from cDNA copies of the genome. Phage were constructed with one, two or three genomic segments. Phage were also produced with altered transcriptional regulation. Although the pac sequences of Φ2954 show no similarity to those of Φ12, segment M of Φ2954 could be acquired by Φ12 resulting in a change of host specificity.ConclusionsWe have isolated a new member of the bacteriophage family Cystoviridae and find that although it shows similarity to other members of the family, it has unique properties that help to elucidate viral strategies for genomic packaging and gene expression.

New enveloped dsRNA phage from freshwater habitat

“…The genetic organization of wNN resembles that of w6 and other identified cystoviruses (Gottlieb et al, , 2002Hoogstraten et al, 2000;McGraw et al, 1986;Mindich et al, 1988;Qiao et al, 2000Qiao et al, , 2010, and putative functions could be assigned to the ORFs based on chromosomal position, size and sequence homology with w6 ( Fig. 2, Table 1).…”

“…Similarly to other cystoviruses, all the ORFs of the wNN genome are oriented in the same direction (Gottlieb et al, , 2002Hoogstraten et al, 2000;McGraw et al, 1986;Mindich et al, 1988;Qiao et al, 2000Qiao et al, , 2010. The ORFs contain a translational initiation codon AUG, except for ORFs 5 and 10, which begin with the codon GUG.…”

“…All isolates were obtained from plant debris in the USA, and they infect Gram-negative bacteria, mainly phytopathogenic Pseudomonas syringae strains. Complete genome sequences are available for four cystoviral isolates in addition to w6 (Gottlieb et al, , 2002Hoogstraten et al, 2000;McGraw et al, 1986;Mindich et al, 1988;Qiao et al, 2000Qiao et al, , 2010. Some cystoviruses are genetically remarkably similar to w6, whereas others are only distantly related.…”

“…In addition to Pseudomonas phage w6 (Vidaver et al, 1973), the type virus of the Cystoviridae, several putative cystoviruses have been isolated (Mindich et al, 1999;O'Keefe et al, 2010;Silander et al, 2005;Qiao et al, 2010), but have not been formally classified by the International Committee on Taxonomy of Viruses. All isolates were obtained from plant debris in the USA, and they infect Gram-negative bacteria, mainly phytopathogenic Pseudomonas syringae strains.…”

“…Some cystoviruses are genetically remarkably similar to w6, whereas others are only distantly related. Phage w6 and its distant relative w2954 (Mindich et al, 1999;Qiao et al, 2010) attach to a specific type IV pilus on the host bacterium, whereas the other distant relatives of w6 (w8, w12 and w13) bind directly to rough lipopolysaccharide on the host outer membrane (Mindich et al, 1999).…”