Genomic characterization of JG068, a novel virulent podovirus active against Burkholderia cenocepacia

Abstract

BackgroundAs is true for many other antibiotic-resistant Gram-negative pathogens, members of the Burkholderia cepacia complex (BCC) are currently being assessed for their susceptibility to phage therapy as an antimicrobial treatment. The objective of this study was to perform genomic and limited functional characterization of the novel BCC phage JG068 (vB_BceP_JG068).ResultsJG068 is a podovirus that forms large, clear plaques on Burkholderia cenocepacia K56-2. Host range analysis indicates that this phage can infect environmental, clinical, and epidemic isolates of Burkholderia multivorans, B. cenocepacia, Burkholderia stabilis, and Burkholderia dolosa, likely through interaction with the host lipopolysaccharide as a receptor. The JG068 chromosome is 41,604 base pairs (bp) in length and is flanked by 216 bp short direct terminal repeats. Gene expression originates from both host and phage promoters and is in the forward direction for all 49 open reading frames. The genome sequence shows similarity to Ralstonia phage ϕRSB1, Caulobacter phage Cd1, and uncharacterized genetic loci of blood disease bacterium R229 and Burkholderia pseudomallei 1710b. CoreGenesUniqueGenes analysis indicates that JG068 belongs to the Autographivirinae subfamily and ϕKMV-like phages genus. Modules within the genome encode proteins involved in DNA-binding, morphogenesis, and lysis, but none associated with pathogenicity or lysogeny. Similar to the signal-arrest-release (SAR) endolysin of ϕKMV, inducible expression of the JG068 SAR endolysin causes lysis of Escherichia coli that is dependent on the presence of an N-terminal signal sequence. In an in vivo assay using the Galleria mellonella infection model, treatment of B. cenocepacia K56-2-infected larvae with JG068 results in a significant increase in larval survival.ConclusionsAs JG068 has a broad host range, does not encode virulence factors, is obligately lytic, and has activity against an epidemic B. cenocepacia strain in vivo, this phage is a highly promising candidate for BCC phage therapy development.

Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas

“…5). This configuration, with lysis and terminase genes intermingled, was previously observed for phage SP6 and the phiKMVlike phage JG068 (48,49).…”

Complete Genomic and Lysis-Cassette Characterization of the Novel Phage, KBNP1315, which Infects Avian Pathogenic Escherichia coli (APEC)

“…Our observation that ORF 51 could trigger host cell lysis independent of holin function confirms that KBNP1315 uses a SAR endolysin in its lysis system. Although the catalytic triad (E-8aa-D/C-5aa-T) found in the endolysin sequences from P1, 21, phiKMV, Era103 and T4 is not conserved in ORF 51 ( S2 Fig ), the enzymatic activity of the latter is comparable with those of SAR endolysins containing these catalytic residues [ 23 , 24 , 25 ]. Most SAR endolysins (roughly 85%) contain the lysozyme-type catalytic domain; however, the lack of conserved catalytic residues in ORF 51 may indicate that it has a different type of catalytic domain.…”

The isolation and characterization of two Stenotrophomonas maltophilia bacteriophages capable of cross-taxonomic order infectivity

“…DLP1 and DLP2 genomic DNA was isolated from bacteriophage lysate using the Wizard Lambda DNA purification system (Promega Corp., Madison, WI) with a modified protocol [ 60 , 61 ]. A 10 ml aliquot of filter-sterilized phage lysate (propagated on D1585 with agarose medium) was treated with 10 μl DNase I (Thermo Scientific, Waltham, MA), 100 μl 100x DNase I buffer (1 M Tris–HCl, 0.25 M MgCl 2 , 10 mM CaCl 2 ), and 6 μl RNase (Thermo Scientific) and incubated 1 h at 37 °C to degrade the bacterial nucleic acids.…”