Duarte, Iris (2012-08). Characterization of a Broad Host Range Tailocin from Burkholderia. Doctoral Dissertation.
Members of the Burkholderia cepacia complex (Bcc) are plant and human opportunistic pathogens. Essentially all Bcc isolates demonstrate in vitro broad-spectrum antibiotic resistance. In fact, many clinical isolates are resistant to all currently available antibiotics, rendering therapy ineffective. There is a substantial need to develop new antimicrobial therapies. The potential use of phage-tail-like high molecular weight bacteriocins, or "tailocins", as alternative anti-bacterial agents against Bcc was investigated. A tailocin, designated Bcep0425, produced by B.cenocepacia strain BC0425 was determined to have broad host range activity against members of the Bcc. Targeted mutagenesis of genes involved in the biosynthesis of the bacterial lipopolysaccharide (LPS) was conducted to determine the receptor site and it was determined that L-rhamnose and alpha-glucose associated with the LPS core were the receptors. Genetic analysis and targeted mutagenesis of the tailocin encoding genes was conducted in the host strain, B. cenocepacia BC0425, to determine the genetic organization of the tailocin Bcep0425 gene cluster and to confirm gene functions. We report for the first time genes involved in replication and integration that are associated with a pyocin/tailocin gene cluster. Additionally, a new class (IV) of holin was identified as part of the lysis cassette. Genetic analysis of the tailocin encoding genes revealed a high degree of similarity to defective phages identified in sequenced Burkholderia genomes. Two novel transcriptional regulators, bctN and bctR, along with recA were found to be involved in the induction of Bcep0425. Numerous studies have focused on the characterization of pyocins from Pseudomonas, but there have been no molecular investigations of tailocins from Burkholderia. This constitutes the first molecular characterization of a phage tail-like bacteriocin from Burkholderia.