Method for host-independent detection of generalized transducing bacteriophages in natural habitats

Abstract

Despite an increasing interest in horizontal gene transfer in bacteria, the role of generalized transduction in this process has not been well investigated yet. Certainly one of the reasons is that only a small fraction of general transducing bacteriophages have been characterized, because many bacterial hosts needed for propagation and identification are not culturable or are simply unknown. A method for host-independent detection of transducing bacteriophages was developed. Phage-encapsulated DNA was used as a template for PCR amplification of 16S ribosomal DNA using primers specific for the 16S rRNA genes of most eubacteria. Sequencing of the cloned amplification products permits the identification of the host bacteria. The Salmonella phage P22 was used as an example. Applying this method to a sample of the supernatant of the mixed liquor in the aeration tank of an activated sludge treatment works revealed the presence of transducing phages infecting several bacterial species for which such phages have not yet been described. This method is suitable for estimating the contribution of generalized transduction to horizontal gene transfer in different habitats.

FIG. 1

Protocol for preparation and amplification of phage-encapsulated DNA.

FIG. 2

Agarose gel analysis of PCR amplification products. We performed 16S rDNA amplification with primers 28F and 1492R and control insert amplification with standard primers M13 reverse and M13 (−20) forward. Lane M, 1-kb ladder (New England Biolabs); lanes 1 and 5, amplification product of the control insert of plasmid pHpaC; lanes 2 to 4, test for the presence of the control insert of plasmid pHpaC or 16S rDNA, respectively, in phage lysates. All lysates were spiked with pHpaC, free DNA was degraded, and encapsulated DNA subsequently was extracted. Lane 2, control insert amplification of a P22 lysate; lane 3, 16S rDNA amplification of a P22 lysate; lane 4, 16S rDNA amplification of a lambda lysate. The 900-bp pHpaC amplification product is invisible at the applicated DNA amounts in lanes 1 and 5.