A broad-host-range, generalized transducing phage (SN-T) acquires 16S rRNA genes from different genera of bacteria

Abstract

Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera: Sphaerotilus natans, the host from which SN-T was originally isolated, and Pseudomonas aeruginosa. In sequential infections, SN-T harbored only 16S rRNA gene sequences of the final host as determined by restriction fragment length polymorphism analysis. The frequency of 16S rRNA gene sequences in SN-T populations was determined to be 1 x 10(-9) transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria.

FIG. 1.

Protocol for phage infection (where SN-ΤφPP denotes sequential infection of P. aeruginosa by SN-T, SN-ΤφSP denotes sequential infection of S. natans followed by P. aeruginosa, etc.) and subsequent DNA preparation, amplification, and analysis by RFLP.

FIG. 2.

Agarose gel electrophoresis of 16S rRNA gene PCR-amplified region (8 to 1,525 bp). Lanes: 1, 1-kb ladder; 2, E. coli; 3, D3112; 4, UT1; 5, D3; 6, no DNA; 7, 1-kb ladder.

FIG. 3.

Agarose gel electrophoresis of 16S rRNA gene PCR-amplified region (8 to 1,525 bp). Lanes: 1, P. aeruginosa; 2, S. natans; 3, SN-ΤφPP; 4, SN-ΤφSS; 5, SN-ΤφSP; 6, SN-ΤφPS; 7, 1-kb ladder.

FIG. 4.

Agarose gel electrophoresis of SN-T uninfected cell-only controls. Lanes: 1, 1-kb ladder; 2, P. aeruginosa genomic positive control; 3, SN-ΤφPP; 4, SN-ΤφSS; 5, SN-ΤφSP; 6, SN-ΤφPS.

FIG. 5.

Restriction fragments of the PCR-amplified region (8 to 1,525 bp) of the 16S rRNA gene from bacterial and phage genomic DNA. Lanes: 1, uncut; 2, cut with AgeI; 3, cut with BamHI.

FIG. 6.

Relative frequencies of each bacterial 16S rRNA gene sequence in SN-T. Frequencies were determined by dilution series of SN-T followed by DNA extraction and PCR amplification. Replicates were run for each dilution.