High-frequency phage-mediated gene transfer among Escherichia coli cells, determined at the single-cell level.
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 3x10(-8) to 2x10(-6), 1x10(-8) to 4x10(-8), and <4x10(-9) to 4x10(-8) per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7x10(-4) to 1x10(-3), 9x10(-4) to 3x10(-3), and 5x10(-4) to 4x10(-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.
Kenzaka
Takehiko
T
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Osaka 565-0871, Japan.
Tani
Katsuji
K
Sakotani
Akiko
A
Yamaguchi
Nobuyasu
N
Nasu
Masao
M
D000668:Ampicillin Resistance / Q000235:genetics; D017101:Bacteriophage P1 / Q000235:genetics / Q000502:physiology; D017122:Bacteriophage T4 / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D003090:Coliphages / Q000235:genetics* / Q000502:physiology; D015169:Colony Count, Microbial; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology / Q000821:virology*; D022761:Gene Transfer, Horizontal; D005822:Genetic Vectors; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D050296:Microbial Viability; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D014161:Transduction, Genetic; D010948:Viral Plaque Assay
D004279:DNA, Viral