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. 2016 Nov 4;198(23):3209-3219.
doi: 10.1128/JB.00424-16. Print 2016 Dec 1.

Emergence of a Competence-Reducing Filamentous Phage From the Genome of Acinetobacter Baylyi ADP1

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Emergence of a Competence-Reducing Filamentous Phage From the Genome of Acinetobacter Baylyi ADP1

Brian A Renda et al. J Bacteriol. .
Free PMC article

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 CTXϕ 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 (CRAϕ). Knocking out ADP1 genes required for competence leads to resistance to CRAϕ 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 CRAϕ-like phages restrict horizontal gene transfer in nature by inhibiting the growth of naturally transformable strains. We also found that prophages with homology to CRAϕ exist in several strains of Acinetobacter baumannii These CRAϕ-like A. baumannii prophages encode toxins similar to CTXϕ that might contribute to the virulence of this opportunistic multidrug-resistant pathogen.

Importance: We observed the emergence of a novel filamentous phage (CRAϕ) from the genome of Acinetobacter baylyi ADP1 during a long-term laboratory evolution experiment. CRAϕ is the first bacteriophage reported to require the molecular machinery involved in the uptake of environmental DNA for infection. Reactivation and evolution of CRAϕ 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 CRAϕ is the Vibrio cholerae CTXϕ phage. Variants of CRAϕ 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.

Figures

FIG 1
FIG 1
Laboratory-evolved A. baylyi ADP1 strain P5-C produces a factor that inhibits natural transformation and growth of wild-type ADP1 cells. (A) Profiling the transformability of two evolved ADP1 populations (designated P5 and P3) at 1,000 generations unearthed an anomaly showing that transformation of the mixed population was significantly less efficient than expected from averaging measurements of 15 randomly selected clones tested independently. Error bars show estimated 95% confidence intervals. For the ancestral strain (Anc) and mixed-population samples (Pop), averages and confidence limits were calculated from log-transformed replicate measurements (n = 3). For the mixed-population values (Clone Avg), confidence limits on the averages of the clone transformation frequencies were estimated from 1,000 bootstrap resamplings of these 15 measurements. Each of the 15 clone transformation frequencies used in these calculations was itself the average value of log-transformed replicate measurements (n = 3). (B) Adding the cell-free supernatant from one of these 15 clones (P5-C) inhibited transformation of wild-type ADP1, whereas cell-free supernatant from this ancestral ADP1 strain (Anc) had no effect on transformation. Boiling the P5-C supernatant ameliorated this inhibition. Error bars are 95% confidence limits estimated from log-transformed transformation frequency measurements (n = 3). (C) Cell-free supernatant (Sup) from clone P5-C slows the growth rate of wild-type ADP1 cells. Again, cell-free supernatant from ancestral ADP1 had no effect on its own growth, and the inhibitory effect of P5-C supernatant was lost upon boiling.
FIG 2
FIG 2
P5-C contains mutations in a putative filamentous prophage that is integrated into the A. baylyi ADP1 genome. (A) Illumina sequencing of DNA isolated from P5-C cells showed increased read-depth coverage of the prophage region relative to the remainder of the genome. Individual reads also spanned a new sequence junction that is consistent with the replication of circular single-stranded DNA (ssDNA) phage genomes by this strain as a result of reactivating the phage. (B) Genes in the ADP1 prophage region are arranged in a manner that is consistent with it encoding a filamentous phage, which we have named the competence-reducing acinetobacter phage (CRAϕ). Putative functions of CRAϕ genes (shown above) were assigned based on homology to V. cholerae CTXϕ (shown below) (6, 24) and the general structure of filamentous phages (23). The CRAϕ prophage genes include putative homologs of genes (cep, ace, and zot) encoding three bacteriophage proteins that act as virulence factors in Vibrio cholerae, but CRAϕ does not encode a homolog of the cholera toxin that is present in CTXϕ (ctxAB). Flanking copies of the repetitive sequence (RS) region containing the rst genes (ACIAD1861 to ACIAD1857 and ACIAD1850 to ACIAD1846) have identical sequences in the wild-type ADP1 genome (indicated by shading in the same color). Mutations in this region in clone P5-C were localized to the RS1 copy. Circularization produces molecules consistent with the connection illustrated with a dashed line.
FIG 3
FIG 3
Visualization and biochemical analysis of CRAϕ particles. (A) A transmission electron micrograph of purified CRAϕ shows elongated structures that resemble those of other filamentous phages. (B) SDS-PAGE gel of CRAϕ proteins. Three bands were excised and identified as either the putative pVIII major coat protein encoded by ACIAD1855 (dark bands labeled 1 and 2) or the putative pV DNA binding protein encoded by ACIAD1857 (faint band labeled 3) from their trypsin fragments by LC-MS/MS (see Table S2 in the supplemental material).
FIG 4
FIG 4
Knockout of ADP1 genes affecting competence confers resistance to CRAϕ. Growth curves show the effect of sterile-filtered supernatant from the ancestral ADP1 strain (Anc) versus that from the CRAϕ-producing clone (P5-C) on the growth of various single-gene-knockout mutants of wild-type ADP1. Loss-of-function mutations in pilB, comC, barA, pgi, and ACIAD3148 were observed during the original evolution experiment in which reactivated CRAϕ emerged from the genome. The comP knockout was tested because this gene is known to be required for competence. Each strain-supernatant combination was tested in triplicate. Error bars show 95% confidence intervals for OD measurements, but some are obscured by the symbols.
FIG 5
FIG 5
CRAϕ activation correlates with reduced population transformability and the evolution of reduced competence. For two independent populations, P3 (A) and P5 (B), from a long-term evolution experiment (15), we assayed the overall transformability of cells in whole-population samples archived at 100-generation intervals (graphs). Error bars are 95% confidence intervals estimated from log-transformed measurements (n = 3). We further tested these samples for the presence of circularized CRAϕ DNA (phage detected) using a qualitative PCR assay (see Fig. S2 in the supplemental material), except in the cases of two samples for which it was not determined (n.d.). For P3, data on the population structure of competence (representing the percentage of fully competent clones) at 200-generation intervals are from a prior study (15). The value for P5 at 1,000 generations is from the current study. In each case, fully competent clones were defined as those having a transformation frequency of >0.0002, and ≥10 clones selected randomly from the whole-population sample were tested at each time point. −, no PCR band for circularized phage episome; +, weak band; ++, moderate band; +++, strong band.

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