The genome of Bacillus subtilis bacteriophage SPO1

Abstract

We report the genome sequence of Bacillus subtilis phage SPO1. The unique genome sequence is 132,562 bp long, and DNA packaged in the virion (the chromosome) has a 13,185-bp terminal redundancy, giving a total of 145,747 bp. We predict 204 protein-coding genes and 5 tRNA genes, and we correlate these findings with the extensive body of investigations of SPO1, including studies of the functions of the 61 previously defined genes and studies of the virion structure. Sixty-nine percent of the encoded proteins show no similarity to any previously known protein. We identify 107 probable transcription promoters; most are members of the promoter classes identified in earlier studies, but we also see a new class that has the same sequence as the host sigma K promoters. We find three genes encoding potential new transcription factors, one of which is a distant homologue of the host sigma factor K. We also identify 75 probable transcription terminator structures. Promoters and terminators are generally located between genes and together with earlier data give what appears to be a rather complete picture of how phage transcription is regulated. There are complete genome sequences available for five additional phages of Gram-positive hosts that are similar to SPO1 in genome size and in composition and organization of genes. Comparative analysis of SPO1 in the context of these other phages yields insights about SPO1 and the other phages that would not be apparent from the analysis of any one phage alone. These include assigning identities as well as probable functions for several specific genes and inferring evolutionary events in the phages' histories. The comparative analysis also allows us to put SPO1 into a phylogenetic context. We see a pattern similar to what has been noted in phage T4 and its relatives, in which there is minimal successful horizontal exchange of genes among a "core" set of genes that includes most of the virion structural genes and some genes of DNA metabolism, but there is extensive horizontal transfer of genes over the remainder of the genome. There is a correlation between genes in rapid evolutionary flux through these genomes and genes that are small.

Figure 1. Gene and transcription unit map of the SPO1 genome

The sequence of the SPO1 genome is represented with the two copies of its terminal direct repeats highlighted with yellow boxes. Predicted genes are indicated by rectangular boxes; rightward-transcribed (green) are denoted above the kbp scale and leftward-transcribed (red) genes are below. Black and blue arrows denote rightward and leftward transcription units (defined in the text), vertical green and red lines mark predicted promoters and terminators, respectively. E, M, L, and K represent early, middle, late, and sigma K promoters, respectively. Selected gene names are shown on the gene rectangles, and transcript numbers are shown above each transcript. Established or predicted functions of the encoded proteins are indicated below the gene rectangles. Those predicted only from sequence comparisons are underlined.

Figure 2. SPO1 virion proteins

The protein components of SPO1 virions (purified by CsCl density step gradient centrifugation129) are displayed by SDS-12.5% polyacrylamide gel electrophoresis and stained with Coomaissie brilliant blue. A molecular weight scale is shown on the left, and the five bands subjected to N-terminal amino acid sequence analysis are marked on the right with the SPO1 gene that is thought to encode them and their N-terminal amino acid sequence in parentheses.

Figure 3. Similar gene organization in the morphogenetic gene clusters of the SPO1-like phages

The head and tail gene regions are shown for each of the SPO1-like phages and phage LE1. Genes are represented by rectangles; each phage’s genes are indicated by a different color except that white boxes indicate genes that have no matches; red genes are inside introns, and terminase, coat and tail sheath genes are yellow for orientation. Blue lines connect homologous genes. Beneath each gene cluster is a scale in kbp. Note that all the homologous genes have similar locations except for LP65 genes 129–132 and 134 which are located far from this cluster, and SPO1 gene 2.11 which is separated by a large insertion (see text). Above the SPO1 map the putative functions are shown (“tail” refers to genes of unknown function with homologues in tail gene clusters of phages outside the SPO1-like phage group).

Figure 4. Sequence relationship between SPO1 head and protease proteins

A. The shaded rectangle shows the location and extent of sequence similarity between the major capsid protein and the head maturation protease of SPO1. Sequence matching other phage proteases, corresponding to the catalytic domain, and the site of maturation cleavage, are indicated. B. Similar relationship between the sequences of the phage T4 major capsid protein and a small, separately encoded T4 protein gp61.1.

Figure 5. Tail gene comparisons

Alignment of a portion of the tail gene regions of the SPO1-like group of phages analyzed here. Orthologous genes, represented as rectangles filled with the same color, have been classified into the same Phamily by the computer program Phamerator, and they are connected in the figure by red lines. SPO1 gene 10.1 and its homologues in the other phages are proposed to encode the tail tube protein. SPO1 genes 10.2 and 11.1 are proposed as the genes encoding the tail assembly chaperones related by a programmed translational frameshift. This figure has been extracted from the figure showing the alignment of the complete genomes of these six phages, shown as Figure S3 in SUPPLEMENTARY MATERIAL. Gene names are shown inside the gene rectangle; Phamily number is shown above each gene; the three lavender rectangles in Twort gene 106 represent introns. For more details, see SUPPLEMENTARY MATERIAL.

Figure 6. Tail gene frameshifting

The mRNA sequences shown are from the overlap region of the two ORFs in the phage tail genes that are proposed to sponsor a +1 programmed translational frameshift (see text). Bases to the left are grouped to indicate the codons in the incoming frame and bases to the right indicate the outgoing frame. A tRNA in the P site of the ribosome (tRNA-Lys for SPO1 and tRNA-Phe for the other phages) is proposed to shift one base forward at the frameshift site. Numbers in the “Position” column indicate the position of the frameshift site relative to the termination codon of the upstream gene, in codons.

Figure 7. Size distributions of SPO1 genes

Bars indicate the number of SPO1 genes in each size category; black, genes that have no detected homologues in the other phages of the SPO1 superfamily; gray, genes with at least one homologue in this group of phages.

Figure 8. Comparative organization of SPO1 and closely related genomes

The genomes of the six phages named at the left are shown above each of their published kbp scales. The orange boxes on the SPO1 genome mark its long terminal repeats (the possible terminal redundancies of the other five phages are not indicated). The genes with the following functions are found in the regions indicated by colored boxes above the scales as follows: virion assembly (yellow); DNA metabolism (red); lysis (green); and tRNAs (blue with number of tRNA genes indicated). Red lines connect genes that lie in parallel locations, present in most members of the group, and that map outside the virion assembly gene cluster (see Figure 3 for such relationships within the latter cluster); blue lines indicate some examples of genes present in most of the genomes that are in different locations. Green shading between genomes denotes recognizable overall nucleotide sequence similarity. Note that phages K and G1 are nearly identical except for three insertions (noted by black triangles) in G1 relative to K. Black arrows below the kbp scales denote the general direction of transcription in the various regions of each genome.

Figure 9. Neighbor-joining trees of four SPO1 core gene products

Clustal W was used to construct neighbor-joining trees of the indicated six proteins. The scales of the trees are approximately equal, with 5% difference indicated by a bar near each tree. Non-SPO1-like phage LE1 is included in the four virion structural protein trees (coat, portal, sheath and baseplate proteins); phage A511 (a recently sequenced close relative of phage P10047) genes are included where they were available; orthologues from T4 and T4-like subgroups pseudo-T4 (phage RB49), Schizo-T4 (phage Aeh1), and Exo-T4 (phages PSSM4 and Syn957) are included for comparison in the two lower trees; if the T4-like orthologues are included in the top four trees they also fall outside the SPO1-like branches (not shown). Bootstrap values from 1000 repetitions are shown for each branch. Branching orders are very similar for the six SPO1-like phage proteins in all six trees with only minor differences in branch lengths. The SPO1 group and T4 group orthologues (including the virion proteins in the upper four trees, not shown) formed separate branches with very high bootstrap support.