title: Quantitative determination of free-DNA uptake in river bacteria at the single-cell level by in situ rolling-circle amplification.
abstract: Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.
authors.affiliation: Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamada-oka, Suita, Osaka 565-0871, Japan.
authors.forename: Fumito
authors.initials: F
authors.lastname: Maruyama
authors.forename: Katsuji
authors.initials: K
authors.lastname: Tani
authors.forename: Takehiko
authors.initials: T
authors.lastname: Kenzaka
authors.forename: Nobuyasu
authors.initials: N
authors.lastname: Yamaguchi
authors.forename: Masao
authors.initials: M
authors.lastname: Nasu
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001693:Biological Transport, Active; D002463:Cell Membrane Permeability; D004274:DNA, Recombinant / Q000235:genetics* / Q000378:metabolism*; D005618:Fresh Water / Q000382:microbiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007564:Japan; D007700:Kinetics; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D004274:DNA, Recombinant; D049452:Green Fluorescent Proteins