Emergence of a Competence-Reducing Filamentous Phage from the Genome of Acinetobacter baylyi ADP1

Abstract

Bacterial genomes commonly contain prophage sequences as a result of past infections with lysogenic phages. Many of these integrated viral sequences are believed to be cryptic, but prophage genes are sometimes coopted by the host, and some prophages may be reactivated to form infectious particles when cells are stressed or mutate. We found that a previously uncharacterized filamentous phage emerged from the genome of Acinetobacter baylyi ADP1 during a laboratory evolution experiment. This phage has a genetic organization similar to that of the Vibrio cholerae CTX phage. The emergence of the ADP1 phage was associated with the evolution of reduced transformability in our experimental populations, so we named it the competence-reducing acinetobacter phage (CRA). Knocking out ADP1 genes required for competence leads to resistance to CRA infection. Although filamentous bacteriophages are known to target type IV pili, this is the first report of a phage that apparently uses a competence pilus as a receptor. A. baylyi may be especially susceptible to this route of infection because every cell is competent during normal growth, whereas competence is induced only under certain environmental conditions or in a subpopulation of cells in other bacterial species. It is possible that CRA-like phages restrict horizontal gene transfer in nature by inhibiting the growth of naturally transformable strains. We also found that prophages with homology to CRA exist in several strains of Acinetobacter baumannii. These CRA-like A. baumannii prophages encode toxins similar to CTX that might contribute to the virulence of this opportunistic multidrug-resistant pathogen. IMPORTANCEWe observed the emergence of a novel filamentous phage (CRA) from the genome of Acinetobacter baylyi ADP1 during a longterm laboratory evolution experiment. CRA is the first bacteriophage reported to require the molecular machinery involved in the uptake of environmental DNA for infection. Reactivation and evolution of CRA reduced the potential for horizontal transfer of genes via natural transformation in our experiment. Risk of infection by similar phages may limit the expression and maintenance of bacterial competence in nature. The closest studied relative of CRA is the Vibrio cholerae CTX phage. Variants of CRA are found in the genomes of Acinetobacter baumannii strains, and it is possible that phage-encoded toxins contribute to the virulence of this opportunistic multidrug-resistant pathogen.

New Shuttle Vectors for Gene Cloning and Expression in Multidrug-Resistant Acinetobacter Species

“…After plasmid extraction, the naturally competent A. baylyi BD413 trpE27 and the transposon insertion derivative of A. baumannii ATCC 19606 T trpE19 were transformed with each plasmid. Transformation of A. baylyi was performed as previously described (43). Briefly, 70 l of an A. baylyi BD413 trpE27 bacterial culture grown 18 h in TSB was combined with 1 ml of fresh TSB and 130 ng of each plasmid DNA.…”

New Shuttle Vectors for Real-Time Gene Expression Analysis in Multidrug-Resistant Acinetobacter Species: In Vitro and In Vivo Responses to Environmental Stressors

“…Transformation efficiency (TE) was determined by transformation of 75 ng and 500 ng of plasmid DNA in calcium-competent E. coli and electrocompetent Acinetobacter species cells, respectively. Naturally competent A. baylyi BD413 was transformed with 150 ng of plasmid, as reported previously (64). Transformants were plated on LB agar supplemented with the appropriate concentration of Gm.…”

“…electrocompetent cells were transformed by electroporation(74); A. baylyi naturally competent cells were transformed as previously reported(64). Data are the means Ϯ standard deviations from three independent experiments.…”

One-Day Construction Of Multiplex Arrays to Harness Natural CRISPR Systems

“…Next, we attempted to delete two genes at once, creating a 6x array targeting both bap and the CRAΦ prophage, which binds the competence machinery when activated, complicating horizontal gene transfer experiments 48 . The pBAV1spec-CRISPR 6xCRA-BAP construct had the correct array length in 6 of 8 E. coli clones ( Figure 4A, right half), but we were unable to use it to obtain a double genomic deletion in A. baylyi, likely due to the relative inefficiency of circular plasmids in natural transformation.…”