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

• Published in: Applied and Environmental Microbiology Vol. 73; no. 10; pp. 3291 - 3299
• Main Authors: Kenzaka, Takehiko, Tani, Katsuji, Sakotani, Akiko, Yamaguchi, Nobuyasu, Nasu, Masao
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.05.2007
• Subjects: Ampicillin Resistance - genetics; Bacteriology; Bacteriophage P1 - genetics; Bacteriophage P1 - physiology; Bacteriophage T4 - genetics; Bacteriophage T4 - physiology; Base Sequence; Biological and medical sciences; Coliphages - genetics; Coliphages - physiology; Colony Count, Microbial; DNA, Viral - chemistry; DNA, Viral - genetics; E coli; Environmental Microbiology; Escherichia coli; Escherichia coli - genetics; Escherichia coli - physiology; Escherichia coli - virology; Fluorescence in situ hybridization; Fundamental and applied biological sciences. Psychology; Gene Transfer, Horizontal; Genes; Genetic Vectors; In Situ Hybridization, Fluorescence - methods; Microbial Viability; Microbiology; Molecular Sequence Data; Phage P1; Protozoa; Sequence Analysis; Transduction, Genetic; Viral Plaque Assay; Virus; Escherichia coli; Isolated cell; Bacteria; Enterobacteriaceae; Phage; Genetic transfer
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.02890-06

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.