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Identification and characterization of ?H111-1: A novel myovirus with broad activity against clinical isolates of Burkholderia cenocepacia.

ABSTRACT: Characterization of prophages in sequenced bacterial genomes is important for virulence assessment, evolutionary analysis, and phage application development. The objective of this study was to identify complete, inducible prophages in the cystic fibrosis (CF) clinical isolate Burkholderia cenocepacia H111. Using the prophage-finding program PHAge Search Tool (PHAST), we identified three putative intact prophages in the H111 sequence. Virions were readily isolated from H111 culture supernatants following extended incubation. Using shotgun cloning and sequencing, one of these virions (designated ?H111-1 [vB_BceM_?H111-1]) was identified as the infective particle of a PHAST-detected intact prophage. ?H111-1 has an extremely broad host range with respect to B. cenocepacia strains and is predicted to use lipopolysaccharide (LPS) as a receptor. Bioinformatics analysis indicates that the prophage is 42,972 base pairs in length, encodes 54 proteins, and shows relatedness to the virion morphogenesis modules of AcaML1 and "Vhmllikevirus" myoviruses. As ?H111-1 is active against a broad panel of clinical strains and encodes no putative virulence factors, it may be therapeutically effective for Burkholderia infections.

SUBMITTER: Lynch KH

PROVIDER: S-EPMC3829948 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Identification and characterization of ϕH111-1: A novel myovirus with broad activity against clinical isolates of <i>Burkholderia cenocepacia.</i>

Lynch Karlene H KH Liang Yongjie Y Eberl Leo L Wishart David S DS Dennis Jonathan J JJ

Bacteriophage 20131001 4

Characterization of prophages in sequenced bacterial genomes is important for virulence assessment, evolutionary analysis, and phage application development. The objective of this study was to identify complete, inducible prophages in the cystic fibrosis (CF) clinical isolate <i>Burkholderia cenocepacia</i> H111. Using the prophage-finding program PHAge Search Tool (PHAST), we identified three putative intact prophages in the H111 sequence. Virions were readily isolated from H111 culture superna ...[more]

PMID: 24265978

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Project description:The study of the minimum set of genes required to sustain life is a fundamental question in biological research. Recent studies on bacterial essential genes suggested that between 350 and 700 genes are essential to support autonomous bacterial cell growth. Essential genes are of interest as potential new antimicrobial drug targets; hence, our aim was to identify the essential genome of the cystic fibrosis (CF) isolate Burkholderia cenocepacia H111. Using a transposon sequencing (Tn-Seq) approach, we identified essential genes required for growth in rich medium under aerobic and microoxic conditions as well as in a defined minimal medium with citrate as a sole carbon source. Our analysis suggests that 398 genes are required for autonomous growth in rich medium, a number that represents only around 5% of the predicted genes of this bacterium. Five hundred twenty-six genes were required to support growth in minimal medium, and 434 genes were essential under microoxic conditions (0.5% O2). A comparison of these data sets identified 339 genes that represent the minimal set of essential genes required for growth under all conditions tested and can be considered the core essential genome of B. cenocepacia H111. The majority of essential genes were found to be located on chromosome 1, and few such genes were located on chromosome 2, where most of them were clustered in one region. This gene cluster is fully conserved in all Burkholderia species but is present on chromosome 1 in members of the closely related genus Ralstonia, suggesting that the transfer of these essential genes to chromosome 2 in a common ancestor contributed toward the separation of the two genera.IMPORTANCE Transposon sequencing (Tn-Seq) is a powerful method used to identify genes that are essential for autonomous growth under various conditions. In this study, we have identified a set of "core essential genes" that are required for growth under multiple conditions, and these genes represent potential antimicrobial targets. We also identified genes specifically required for growth under low-oxygen and nutrient-limited environments. We generated conditional mutants to verify the results of our Tn-Seq analysis and demonstrate that one of the identified genes was not essential per se but was an artifact of the construction of the mutant library. We also present verified examples of genes that were not truly essential but, when inactivated, showed a growth defect. These examples have identified so-far-underestimated shortcomings of this powerful method.

Dataset Information

Identification and characterization of ?H111-1: A novel myovirus with broad activity against clinical isolates of Burkholderia cenocepacia.

Publications

Identification and characterization of ϕH111-1: A novel myovirus with broad activity against clinical isolates of <i>Burkholderia cenocepacia.</i>

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