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

PLoS One. 2022 Apr 14;17(4):e0266891. doi: 10.1371/journal.pone.0266891. eCollection 2022.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriophages*
  • Citrus* / microbiology
  • Myoviridae
  • Plant Diseases / microbiology
  • Plant Diseases / prevention & control
  • Proteome
  • Xanthomonas* / genetics

Substances

  • Proteome

Supplementary concepts

  • Xanthomonas citri
  • Xanthomonas fuscans

Grants and funding

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001 (VM) and by Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP (Programa Jovem Pesquisador - Proc. 2007/50910-2). FPNR was supported by a fellowship from CAPES. AMDS and JCS received a Research Fellowship Award from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). EM facility utilized at LME/IQSC was supported by FAPESP (grant 2009/54216-9). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.