Comparative Analyses of Bacteriophage Genomes.

Bacterial viruses (bacteriophages or phages) are the most abundant and diverse biological entities on Earth. There is a renewed worldwide interest in phage-centered research motivated by their enormous potential as antimicrobials to cope with multidrug-resistant pathogens. An ever-growing number of complete phage genomes are becoming available, derived either from newly isolated phages (cultivated phages) or recovered from metagenomic sequencing data (uncultivated phages). Robust comparative analysis is crucial for a comprehensive understanding of genotypic variations of phages and their related evolutionary processes, and to investigate the interaction mechanisms between phages and their hosts. In this chapter, we present a protocol for phage comparative genomics employing tools selected out of the many currently available, focusing on complete genomes of phages classified in the class Caudoviricetes. This protocol provides accurate identification of similarities, differences, and patterns among new and previously known complete phage genomes as well as phage clustering and taxonomic classification.

Isolation and characterization of vB_XciM_LucasX, a new jumbo phage that infects Xanthomonas citri and Xanthomonas fuscans.

Citrus canker is one of the main bacterial diseases that affect citrus crops and is caused by Xanthomonas citri which affects all citrus species worldwide. New strategies to control citrus canker are necessary and the use of bacteriophages as biocontrol agent could be an alternative. Phages that infect Xanthomonas species have been studied, such as XacN1, a myovirus that infects X. citri. Here we report the isolation and characterization of a new jumbo phage, vb_XciM_LucasX, which infects X. citri and X. fuscans. Transmission electron microscopy allowed classification of LucasX in the Myoviridae family, which was corroborated by its genomic sequencing, annotation, and proteome clustering. LucasX has a 305,651 bp-long dsDNA genome. ORF prediction and annotation revealed 157 genes encoding putative structural proteins such as capsid and tail related proteins and phage assembly associated proteins, however, for most of the structural proteins it was not possible assign specific functions. Its genome encodes several proteins related to DNA replication and nucleotide metabolism, five putative RNA polymerases, at least one homing endonuclease mobile element, a terminase large subunit (TerL), an endolysin and many proteins classified as beneficial to the host. Proteome clustering and phylogeny analyses showed that LucasX is a new jumbo phage having as its closest neighbor the Xanthomonas jumbo phage Xoo-sp14. LucasX presented a burst size of 40 PFU/infected cell of X. citri 306, was completely inactivated at temperatures above 50°C, presented survival lower than 25% after 80 s of exposition to artificial UV light and had practically no tolerance to concentrations above 2.5 g/L NaCl or 40% ethanol. LucasX presented optimum pH at 7 and a broad range of Xanthomonas hosts, infecting twenty-one of the twenty-three strains tested. Finally, the LucasX yield was dependent on the host strain utilized, resulting one order of magnitude higher in X. fuscans C 752 than in X. citri 306, which points out to the possibility of phage yield improvement, an usual challenge for biocontrol purposes.