Genomes and Characterization of Phages Bcep22 and BcepIL02, Founders of a Novel Phage Type in Burkholderia cenocepacia

Abstract

Within the Burkholderia cepacia complex, B. cenocepacia is the most common species associated with aggressive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeutic B. cenocepacia podophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages' coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates determination of a phage lifestyle, a key consideration in the selection of therapeutic phages.

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

“…PCR using phage-specific primers did not detect the presence of phage lysogens in resistant isolates, indicating that resistance was not due to lysogeny. Additionally, we examined the potential for abortive lysogeny using infection at a high MOI and measuring survival (27). As shown in Table 6, following infection with Sano or Prado, there was no significant difference between predicted and actual survivors, indicating that phage infection at a high MOI did not lead to the establishment of repression.…”

“…To test whether evidence for abortive lysogeny (i.e., the establishment of repression) could be found, we followed a procedure described previously by Gill et al (27), except that reversibly bound phages were removed by three successive washes. Liquid cultures of logarithmically growing Xanthomonas strain EC-12 were cultured to an OD 600 of ϳ0.3 to 0.5.…”

Characterization of DC1, a Broad-Host-Range Bcep22-Like Podovirus

“…BcepIL02 was recently identified as a member of a new phage type, the Bcep22-like phages. To date, this phage type contains only two viruses: the 62,714-bp BcepIL02 (FJ937737) and the 63,882-bp Bcep22 (AY349011) (5). However, we have recently characterized a third member of this podovirus group, a broadhost-range Bcep22-like phage named DC1 (vB_BceP_DC1) (10).…”

“…While the originally described Bcep22-like phages were reported to specifically infect B. cenocepacia (3,5), the relatively broader host range of DC1 is a significant advantage with respect to clinical use. In contrast to Bcep22 and BcepIL02, which infect B. cenocepacia PC184 (BcepIL02), AU0728 (BcepIL02), and AU1054 (both phages) (3,5), the DC1 host range includes B. cepacia LMG 18821, B. cenocepacia C6433, PC184, and CEP511, and Burkholderia stabilis LMG 18870 (22). The B. cepacia and B. stabilis strains are CF isolates, while the B. cenocepacia strains are CF epidemic isolates (19).…”

“…Although DC1 has certain drawbacks with respect to its potential for use in phage therapy (i.e., genes for lysogeny and a putative lipid A palmitoyltransferase [5]), it also has three key advantages. First, the DC1 host range is relatively broad, infecting clinical strains of multiple BCC species.…”