Genome sequences of two closely related Vibrio parahaemolyticus phages, VP16T and VP16C - PubMed (original) (raw)

Comparative Study

Victor Seguritan et al. J Bacteriol. 2003 Nov.

Abstract

Two bacteriophages of an environmental isolate of Vibrio parahaemolyticus were isolated and sequenced. The VP16T and VP16C phages were separated from a mixed lysate based on plaque morphology and exhibit 73 to 88% sequence identity over about 80% of their genomes. Only about 25% of their predicted open reading frames are similar to genes with known functions in the GenBank database. Both phages have cos sites and open reading frames encoding proteins closely related to coliphage lambda's terminase protein (the large subunit). Like in coliphage lambda and other siphophages, a large operon in each phage appears to encode proteins involved in DNA packaging and capsid assembly and presumably in host lysis; we refer to this as the structural operon. In addition, both phages have open reading frames closely related to genes encoding DNA polymerase and helicase proteins. Both phages also encode several putative transcription regulators, an apparent polypeptide deformylase, and a protein related to a virulence-associated protein, VapE, of Dichelobacter nodosus. Despite the similarity of the proteins and genome organization, each of the phages also encodes a few proteins not encoded by the other. We did not identify genes closely related to genes encoding integrase proteins belonging to either the tyrosine or serine recombinase family, and we have no evidence so far that these phages can lysogenize the V. parahaemolyticus strain 16 host. Surprisingly for active lytic viruses, the two phages have a codon usage that is very different than that of the host, suggesting the possibility that they may be relative newcomers to growth in V. parahaemolyticus. The DNA sequences should allow us to characterize the lifestyles of VP16T and VP16C and the interactions between these phages and their host at the molecular level, as well as their relationships to other marine and nonmarine phages.

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Figures

FIG. 1.

FIG. 1.

Restriction analysis of phage genomic DNA isolated from the VP16 lysates. Phage DNA was purified from the original lysate (mixed) and from lysates made from clear and turbid plaques from the original lysate after three successive plaque purifications on LB medium containing 2.5% NaCl. The phage DNA was restricted with _Hpa_I or _Xmn_I. After restriction digestion at 37°C, the digests were either kept at 37°C before they were loaded on the agarose gel or incubated for 20 min at 65°C and then loaded on the agarose gel.

FIG. 2.

FIG. 2.

Electron micrographs of VP16C and VP16T particles. The micrographs show phage particles from several independent lysates. Although a few of the micrographs showed phage particles like those shown in the VP16C-a panel, the vast majority of micrographs showed phage particles like those shown in the rest of the panels. Note that many of the particles show a disruption in the middle of the tail collar, while the collars are compressed together in a significant fraction of the remaining particles.

FIG. 3.

FIG. 3.

Genomic maps of VP16T and VP16C. ORFs are color coded based on the BLAST E-values and are labeled largely based on the BLAST hits shown in Table 2. BLAST hits returning hypothetical proteins or weak E-values (≤1e−2) are shown as unlabeled color-coded ORFs. Gray ORFs do not exhibit homology to GenBank entries. An ORF labeled with a solid diamond is a VP16 ORF that was identified by the neural network as an ORF that encodes a possible capsid protein. The amino acid sequences of VP16T proteins were found in VP16T ORF sequences labeled TS1 (ANELATGWVQ), TS2 (LIKLT), and TS3 (ADRHIL). Similarly, amino acid sequences of VP16C proteins were encoded in the VP16C ORF sequences labeled CS1 (ANELATGWVQ) and CS2 (LVKLS). ORFs encoding sequences shorter than 70 amino acids are not included in the map for ease of display, but all ORFs are listed in Table 2.

FIG. 4.

FIG. 4.

SDS-polyacrylamide gel electrophoresis analysis of VP16C and VP16T phage capsid proteins. TS and CS are structural proteins from the turbid- and clear-plaque phages, respectively. Twenty or 30 μl of phage lysate was mixed with Laemmli buffer containing SDS, boiled for 10 min, and loaded on a 4 to 20% polyacrylamide gradient gel that was electrophoresed with Tris-glycine runing buffer. Color-coded molecular weight markers were electrophoresed in parallel.

FIG. 5.

FIG. 5.

Comparison of genome maps of several siphophages. Sfi21 and Sfi11 are S. thermophilus phages. L5, M. tuberculosis phage L5; C31, Streptococcus coelicolor φC31; λ, coliphage lambda.

FIG. 6.

FIG. 6.

Plot of frequency of occurrence versus length of identical sequences in the two phages. Only sequences consisting of at least 20 contiguous identical nucleotides were considered.

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