Characterization of Phi2954, a newly isolated bacteriophage containing three dsRNA genomic segments

Abstract

Background: Bacteriophage Phi12 is a member of the Cystoviridae and is distinct from Phi6, the first member of that family. We have recently isolated a number of related phages and five showed high similarity to Phi12 in the amino acid sequences of several proteins. Bacteriophage Phi2954 is a member of this group.

Results: Phi2954 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 Phi12. However, the host specificity was for the type IV pili of Pseudomonas syringae HB10Y rather than for the rough LPS to which Phi12 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 Phi2954 show no similarity to those of Phi12, segment M of Phi2954 could be acquired by Phi12 resulting in a change of host specificity.

Conclusions: We 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.

Figure 1

Bacteriophage Φ2954 was purified by zone and equilibrium centrifugation in sucrose gradients and applied to an 18% polyacrylamide gel for electrophoresis. The gel was stained with Coomasi blue. Purified Φ6 virions were displayed for comparison.

Figure 2

Genetic maps of the genomic segments of Φ2954. Restriction sites utilized in the construction of phage variants are shown. PstI and XbaI sites are present in the plasmid vectors for the cDNA copies.

Figure 3

Sequence comparisons at the 5' termini of transcripts of Φ2954, Φ12 and Φ6. Note that in each case the sequence of L is different from those of S and M.

Figure 4

Stem loop structures at the 3' termini of the Φ2954 transcripts.

Figure 5

In vitro transcription by nucleocapsids of Φ2954 having the normal 5' L sequence of ACAAA and a mutant, Φ3528, with the sequence GCAAA.

Figure 6

Agarose gel electrophoresis of genomic segments of Φ12, Φ2954 and a Φ12 that has acquired segment M of Φ2954.