Xanthomonas citri jumbo phage XacN1 exhibits a wide host range and high complement of tRNA genes

Abstract

Xanthomonas virus (phage) XacN1 is a novel jumbo myovirus infecting Xanthomonas citri, the causative agent of Asian citrus canker. Its linear 384,670 bp double-stranded DNA genome encodes 592 proteins and presents the longest (66 kbp) direct terminal repeats (DTRs) among sequenced viral genomes. The DTRs harbor 56 tRNA genes, which correspond to all 20 amino acids and represent the largest number of tRNA genes reported in a viral genome. Codon usage analysis revealed a propensity for the phage encoded tRNAs to target codons that are highly used by the phage but less frequently by its host. The existence of these tRNA genes and seven additional translation-related genes as well as a chaperonin gene found in the XacN1 genome suggests a relative independence of phage replication on host molecular machinery, leading to a prediction of a wide host range for this jumbo phage. We confirmed the prediction by showing a wider host range of XacN1 than other X. citri phages in an infection test against a panel of host strains. Phylogenetic analyses revealed a clade of phages composed of XacN1 and ten other jumbo phages, indicating an evolutionary stable large genome size for this group of phages.

Figure 1

Electron micrograph of XacN1. Phage particles were stained with 1% ammonium molybdate. Bar = 100 nm.

Figure 2

Comparison of codon usages between XacN1 and X. citri. Red dots represent codons for which corresponding tRNAs are encoded in both XacN1 and X. citri. Blue dots represent codons for which corresponding tRNAs are encoded only in X. citri. In gray area, relative codon frequencies are similar between XacN1 and X. citri (i.e. ratio of relative codon frequencies is between 0.9 and 1.1).

Figure 3

Proteomic analysis of virion proteins of XacN1. Virion proteins separated by SDS-PAGE were visualized with Coomassie Brilliant Blue. The protein bands were excised from the gel, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry (LTQ Orbitrap XL). Assignment of tandem mass spectrometry data to tryptic peptides encoded by phage open reading frames was completed using an established procedure. ND: Not determined exactly.

Figure 4

Phylogenetic relationships between XacN1 and other phages. Maximum likelihood phylogenetic trees of terminase large subunit proteins. The length of the bars outside the tree is proportional to the genome size of each phage. Red and blue bars represent genomes greater than 300 kbp and 200 kbp, respectively. Black bars represent genomes smaller than 200 kbp.

Figure 5

Genome comparison among eleven XacN1 related phages. Red and blue lines of the dot-plot represent sequence similarities detected by TBLASTX in the same and reverse orientations, respectively.

Figure 6

Distribution of translation-associated genes in jumbo phages and other smaller phages. Box plots represented the number of translation-associated genes including tRNA genes.