Gene transfer by transduction in the marine environment

Abstract

To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we established transduction systems by using marine phage host isolates. Plasmid pQSR50, which contains transposon Tn5 and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays. Both marine bacterial isolates and concentrated natural bacterial communities were used as recipients in transduction studies. Transductants were detected by a gene probe complementary to the neomycin phosphotransferase (nptII) gene in Tn5. The transduction frequencies ranged from 1.33 x 10(-7) to 5.13 x 10(-9) transductants/PFU in studies performed with the bacterial isolates. With the mixed bacterial communities, putative transductants were detected in two of the six experiments performed. These putative transductants were confirmed and separated from indigenous antibiotic-resistant bacteria by colony hybridization probed with the nptII probe and by PCR amplification performed with two sets of primers specific for pQSR50. The frequencies of plasmid transduction in the mixed bacterial communities ranged from 1.58 x 10(-8) to 3.7 x 10(-8) transductants/PFU. Estimates of the transduction rate obtained by using a numerical model suggested that up to 1.3 x 10(14) transduction events per year could occur in the Tampa Bay Estuary. The results of this study suggest that transduction could be an important mechanism for horizontal gene transfer in the marine environment.

FIG. 1

Map of plasmid pQSR50, including the locations of PCR primers. Arrows indicate primer directions.

FIG. 2

Dot blot hybridization of plasmid DNAs from E. coli RM1259, donor strain HOPE-1, recipient strain HSIC, and transductants with the nptII probe.

FIG. 3

Southern hybridization of undigested and HindIII-digested plasmid DNAs from transductants, E. coli RM1259, donor strain HOPE-1, and recipient strain HSIC, probed with the nptII probe. Lanes 1 through 20, plasmid DNAs from transductants; lanes 21 and 22, plasmid DNA from E. coli RM1259; lanes 23 and 24, plasmid DNA from donor strain HOPE-1; lanes 25 and 26, plasmid DNA from recipient strain HSIC. Odd-numbered lanes contained undigested plasmid DNA, whereas even-numbered lanes contained HindIII-digested DNA. Molecular weights (M) (in kilobases) are indicated on the left.

FIG. 4

Southern hybridization of undigested (odd-numbered lanes) and HindIII-digested (even-numbered lanes) plasmid DNAs from transductants, E. coli RM1259, and donor and recipient strains probed with the T-ΦHSIC riboprobe. For the contents of the lanes see the legend to Figure 3.

FIG. 5

Colony hybridization after a transduction assay performed with indigenous bacterial communities from Tampa Bay as recipients. (A) Control (no lysate added). (B) Transduction with HOPE-2–T-φD1b lysate. (C) Transduction with HOPE-1–T-φHSIC lysate. (D) Transduction with UV-treated HOPE-1– T-φHSIC lysate.

FIG. 6

Autoradiograph after Southern transfer of an agarose gel containing PCR products obtained with primers JP44 and JP52. The template DNAs were plasmid DNAs of transductants (lanes 1 through 7), plasmid DNAs of indigenous antibiotic-resistant bacteria (lanes 8 through 12), and pQSR50 DNA (lane 13). Lane 14 was a negative control (no template DNA). The preparations were hybridized with the nptII probe. Lane M contained a molecular weight marker; sizes (in base pairs) are indicated on the left.

FIG. 7

PCR amplification performed with primers JP64 and JP65 and EcoRI-digested amplification products. The template DNAs were DNAs from plasmids of transductants (lanes 1 through 10), indigenous antibiotic-resistant bacteria (lanes 11 and 12), and pQSR50 (lanes 13 and 14). Odd-numbered lanes contained uncut DNA, and even-numbered lanes contained EcoRI-cut DNA. Lane M contained a molecular weight ladder; sizes (in base pairs) are indicated on the right.