Complete genomic sequence of the virulent Salmonella bacteriophage SP6

Abstract

We report the complete genome sequence of enterobacteriophage SP6, which infects Salmonella enterica serovar Typhimurium. The genome contains 43,769 bp, including a 174-bp direct terminal repeat. The gene content and organization clearly place SP6 in the coliphage T7 group of phages, but there is approximately 5 kb at the right end of the genome that is not present in other members of the group, and the homologues of T7 genes 1.3 through 3 appear to have undergone an unusual reorganization. Sequence analysis identified 10 putative promoters for the SP6-encoded RNA polymerase and seven putative rho-independent terminators. The terminator following the gene encoding the major capsid subunit has a termination efficiency of about 50% with the SP6-encoded RNA polymerase. Phylogenetic analysis of phages related to SP6 provided clear evidence for horizontal exchange of sequences in the ancestry of these phages and clearly demarcated exchange boundaries; one of the recombination joints lies within the coding region for a phage exonuclease. Bioinformatic analysis of the SP6 sequence strongly suggested that DNA replication occurs in large part through a bidirectional mechanism, possibly with circular intermediates.

FIG. 1.

Electron micrographs of two SP6 virions, negatively stained with uranyl acetate. Scale bar = 100 nm.

FIG. 2.

Map of the SP6 genome. Rectangles represent genes, and the different colors indicate different classes of genes, as indicated in the key below the map. All transcription is from left to right. The gene numbering scheme is described in Materials and Methods; the designations in parentheses are the names of the homologous T7 genes. Promoters and terminators are indicated above the line by designations that begin with P and t, respectively, and below the line by symbols. Gene functions, either determined experimentally or inferred by homology, are indicated where they are known. The scale is in kilobase pairs.

FIG. 3.

Putative phage-specific promoters of SP6. Ten potential SP6 promoters were identified by searching for sequences closely related to three previously identified promoters (7), shown here as P6, P8, and P9. Each promoter designation is indicated on the left and is followed by the nucleotide position at +1. The letters at the top indicate the effects of mutations at positions on promoter activity in vivo, as reported by Shin et al. (53). The four letters indicate how many of the three possible substitutions lead to a reduction in activity to a level that is less than one-third the consensus level (i.e., P8); A indicates that all three substitutions reduced activity to this extent, B indicates that two substitutions reduced activity to this extent, C indicates that one substitution reduced activity to this extent, and D indicates that no substitution reduced activity to this extent (53). We do not know how these putative promoters are related to those described previously (34).

FIG. 4.

Transcription termination: denaturing polyacrylamide gel analysis of in vitro transcription products synthesized from linearized and circular pCSMl0l templates with SP6RP. The numbers on the left indicate the lengths of the transcripts (in nucleotides). Lanes 1, 2, and 3, transcription products of closed circular, HindIII-digested, and PvuII-digested pCSM101 templates, respectively; lanes 4, 5, and 6, transcription products of closed circular, HindIII-digested, and PvuII-digested pSP6/T7-19 (parent plasmid) templates, respectively.

FIG. 5.

Comparison of five T7-like phages. All genes in SP6 with identifiable homologues in the other taxa are colored; in addition, gene 49 (similar to P22 tail spike protein gene 9) is shown in green. Genes in black or in blue show phages T3 and φYeO3-12 as most closely related, while those that are in red show phages T3 and T7 as most closely related. The genes indicated in blue appear to have been inverted in the genome (by displacement and inversion of gene order) but have individually reinverted so that they are transcribed in the same direction as all other genes. There appear to have been at least two large recombination events involving large portions of the genome; these domains may involve genes with cooperating functions (RNA transactions, DNA replication, head assembly, and tail assembly). At the bottom, phylogenies are constructed for selected genes using neighbor joining (47). Bootstrap values for each dendrogram are shown. In addition, the results of Felsenstein tests of phylogenetically informative sites (16) are shown below each phylogeny, indicating the numbers of sites supporting (i) T3,φYeo-T7**, (ii) T3,T7-φYeo**, and (iii) T7,φYeo-T3** phylogenies, respectively, where the double asterisk indicates the homologous genes from either phage SP6 (line 1) or gh-1 (line 2).

FIG. 6.

Analysis of phylogenetically informative sites showing support for two distinct phylogenies for the N terminus and C terminus of the gp20 (T7-gp6) protein. This pattern was not seen for any other gene (see Fig. 5). (A) Phylogeny of the gene as a whole. (B) Phylogenetically informative sites extracted from an alignment of the homologues of the protein from the four phages indicated. The numbers indicate positions in the alignment. (C) Phylogeny of the N terminus of the protein. The sequences were divided at aligned residue 180 (C-terminal residue of the SCDKDFKTIP sequence in T7), which was between the two sites defining domains with different phylogenetic histories. This phylogeny is supported robustly (bootstrap value for both nodes, 100%). (D) Distinctly different phylogeny inferred from the C-terminal portion of the gp6 exonuclease.

FIG. 7.

Mapping the origin of replication by mutational bias. The genome was analyzed by circular permutation to find breakpoints where changes in strand-specific degenerate-octomeric skew were the strongest. Two sites were identified; positions were refined after fine-scale analysis of the distribution of several hundred octomers showing at least 80% strand bias, as shown in the center. The graph shows the cumulative distribution of the sequences on the Watson-and-Crick strands of DNA for 3% intervals of the genome.