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Applied and Environmental Microbiology, September 2006, p. 6248-6256, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.03035-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Quantitative Determination of Free-DNA Uptake in River Bacteria at the Single-Cell Level by In Situ Rolling-Circle Amplification

Fumito Maruyama, Katsuji Tani, Takehiko Kenzaka, Nobuyasu Yamaguchi, and Masao Nasu*

Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan

Received 23 December 2005/ Accepted 22 June 2006

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.

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

Applied and Environmental Microbiology, September 2006, p. 6248-6256, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.03035-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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