Analyses of four new Caulobacter Phicbkviruses indicate independent lineages

Abstract

Bacteriophages with genomes larger than 200 kbp are considered giant phages, and the giant Phicbkviruses are the most frequently isolated Caulobacter crescentus phages. In this study, we compare six bacteriophage genomes that differ from the genomes of the majority of Phicbkviruses. Four of these genomes are much larger than those of the rest of the Phicbkviruses, with genome sizes that are more than 250 kbp. A comparison of 16 Phicbkvirus genomes identified a 'core genome' of 69 genes that is present in all of these Phicbkvirus genomes, as well as shared accessory genes and genes that are unique for each phage. Most of the core genes are clustered into the regions coding for structural proteins or those involved in DNA replication. A phylogenetic analysis indicated that these 16 CaulobacterPhicbkvirus genomes are related, but they represent four distinct branches of the Phicbkvirus genomic tree with distantly related branches sharing little nucleotide homology. In contrast, pairwise comparisons within each branch of the phylogenetic tree showed that more than 80 % of the entire genome is shared among phages within a group. This conservation of the genomes within each branch indicates that horizontal gene transfer events between the groups are rare. Therefore, the Phicbkvirus genus consists of at least four different phylogenetic branches that are evolving independently from one another. One of these branches contains a 27-gene inversion relative to the other three branches. Also, an analysis of the tRNA genes showed that they are relatively mobile within the Phicbkvirus genus.

Keywords: Bacteriophage; Caulobacter; DNA virus; genomics; pan-genome.

Fig. 1.

Transmission electron micrographs of Phicbkviruses CcrSC, CcrPW, CcrBL9 and CcrBL10. Scale bars are 200 nm.

Fig. 2.

Genome organization of four new phages. The DNA plot image represents the genome organization of CcrBL9, CcrSC, CcrPW and CcrBL10. The outside track is the genome of CcrBL9, next going inwards is CcrSC, then CcrPW and CcrBL10. Located in the centre of the plot is the GC content of the CcrBL9 genome. The marks facing outwards represent greater than average GC content, while the marks facing inward represent lower than average GC content. The red boxes indicate structural genes, the green boxes represent DNA replication genes, the purple boxes represent genes exclusive to single phages, the dark blue boxes represent genes that are exclusive to phages in the same branch, and the light blue boxes represent genes located in other genomes within in NCBI GenBank database.

Fig. 3.

Comparison of eight Phicbkviruses with CbK as the reference. The BRIG image [29] illustrates the variability of amino acid conservation within different Phicbkvirus genomes. The brightness of the hue determines the overall percentage identity match to the reference genome ranging from 35–100 % identity. Percentage identities less than 35 % are represented by no coloration.

Fig. 4.

Maximum-likelihood phylogenetic analysis of ten Phicbkvirus genomes. This tree is based upon the core genome SNP matrix generated during the PanX pipeline [27]. Maximum likelihood was determined using the FastTree and RaxML programs that utilize the Jukes–Cantor model. Tree viewer was used for visualization of the .json file [28]. The tree is drawn to scale based upon single nucleotide differences. Each cluster is located on its own branch.

Fig. 5.

Large inversion within the CcrSC/CcrBL9 phage genomes. The consensus organization of all CbK-like phage protein-coding genes within this region is located at the bottom. The CcrSC/CcrBL9 phage genome organization of the same region is shown at the top. The red boxes are genes annotated as hypothetical, the turquoise boxes are DNA replication genes, the royal blue boxes are genes associated with RNA metabolism, and the green boxes structural proteins.