Comparative Genomics of Prophages Sato and Sole Expands the Genetic Diversity Found in the Genus Betatectivirus

Abstract

Tectiviruses infecting the Bacillus cereus group represent part of the bacterial "plasmid repertoire" as they behave as linear plasmids during their lysogenic cycle. Several novel tectiviruses have been recently found infecting diverse strains belonging the B. cereus lineage. Here, we report and analyze the complete genome sequences of phages Sato and Sole. The linear dsDNA genome of Sato spans 14,852 bp with 32 coding DNA sequences (CDSs), whereas the one of Sole has 14,444 bp comprising 30 CDSs. Both phage genomes contain inverted terminal repeats and no tRNAs. Genomic comparisons and phylogenetic analyses placed these two phages within the genus Betatectivirus in the family Tectiviridae. Additional comparative genomic analyses indicated that the "gene regulation-genome replication" module of phages Sato and Sole is more diverse than previously observed among other fully sequenced betatectiviruses, displaying very low sequence similarities and containing some ORFans. Interestingly, the ssDNA binding protein encoded in this genomic module in phages Sato and Sole has very little amino acid similarity with those of reference betatectiviruses. Phylogenetic analyses showed that both Sato and Sole represent novel tectivirus species, thus we propose to include them as two novel species in the genus Betatectivirus.

Keywords: Bacillus phages; betatectiviruses; lysogeny; plasmidial prophages; tectivirus; temperate phages.

Figure 4

Phylogenetic relationships of phages Sato and Sole with other phages within the family Tectiviridae. (a–c) Phylogenetic trees resulting from maximum likelihood inference on multiple amino acid sequence alignments of the three canonical tectiviral proteins: the DNA polymerase (a), packaging ATPase (b), and major capsid protein (c). The general reverse transcriptase model was used to compute the maximum likelihood trees. Bootstrap values (1000 iterations) above 60% are indicated for each node. Scale bars in (a–c) represent the numbers of substitutions per site. Protein GenBank accession numbers are indicated in parentheses. (d) Virus Intergenomic Distance Calculator (VIRIDIC) [42] generated heatmap incorporating intergenomic similarity values (right half) and alignment indicators (left half and top annotation). Phage genome GenBank accession numbers are indicated. Color boxes (a–d) highlight phages belonging to genera Alphatectivirus (rose), Betatectivirus (blue), Gammatectivirus (grey), Deltatectivirus (green) and Epsilontectivirus (yellow). Note that Forthebois, WheeHeim and Toil are actinophages, isolated from the Gram-positive bacteria Streptomyces scabiei for the two formers [24] and from Rhodococcus opacus for the latter [58].

Figure 1

Genetic maps of tectiviruses Sato (top) and Sole (bottom). Predicted genes and their direction of transcription are represented as block arrows. Inverted terminal repeats (ITR) are shown as red arrows at both ends of the genomes. The color key at the bottom-right indicates known and postulated gene functions [4,10,22,23,44,45,46]. Three genetic modules based of functional grouping, together with the highly variable region (HVR), are indicated above the genomes. The rulers represent base pairs in the phage genomes. MerR-type, a putative transcriptional regulator; SSB, ssDNA binding protein; TP, terminal protein.

Figure 2

Plasmid(s) electrophoresis profiles of strains (1) B. thuringiensis GBJ002/GIL16; (2) emetic B. cereus AND1284; and (3) B. cereus s.l. VD166, harboring plasmidial tectiviruses GIL16, Sato and Sole, respectively. The tectiviral prophages in a linear plasmid state (~15 kb) are indicated. chr, linearized chromosomal DNA.

Figure 3

Genome comparisons of phages Sato and Sole with other betatectiviruses and the tectivirus-like element pBClin15. Predicted genes and direction of transcription are represented as block arrows. CDSs numbers are indicated inside the block arrows. Canonical tectiviral proteins and well conserved proteins among betatectiviruses are color-coded: light purple, terminal protein (TP); dark purple, B-family DNA polymerase (DNAPolB); rose, LexA transcriptional regulator; yellow, packaging ATPase (ATPase); orange, major capsid protein (MCP); light green, muramidase; dark green, transglycosylase. The other CDSs are in blue. ITR, inverted terminal repeat; HVR, highly variable region. Conserved regions are grey-shaded, with the color intensity indicating the percentage of amino acid identity. The comparisons were done by tBLASTx, and similarities with E values lower than 0.001 were plotted using EasyFig (v.2.1) [33]. Scale and percentage of amino acid identity are indicated at the bottom-right. Genome lengths in base pairs (bp) are indicated for each molecule. GenBank accession numbers are listed in Table 3.