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Applied and Environmental Microbiology, May 2007, p. 3291-3299, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.02890-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level{triangledown}

Takehiko Kenzaka,1 Katsuji Tani,1,2 Akiko Sakotani,1,2 Nobuyasu Yamaguchi,1 and Masao Nasu1*

Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Osaka 565-0871, Japan,1 Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi 584-8540, Japan2

Received 14 December 2006/ Accepted 14 March 2007

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 x 10–8 to 2 x 10–6, 1 x 10–8 to 4 x 10–8, and <4 x 10–9 to 4 x 10–8 per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 x 10–4 to 1 x 10–3, 9 x 10–4 to 3 x 10–3, and 5 x 10–4 to 4 x 10–3 for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.

* Corresponding author. Mailing address: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Osaka 565-0871, Japan. Phone: 81-6-6879-8170. Fax: 81-6-6879-8174. E-mail: nasu{at}phs.osaka-u.ac.jp

{triangledown} Published ahead of print on 23 March 2007.

Applied and Environmental Microbiology, May 2007, p. 3291-3299, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.02890-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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