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.

Methods for Whole Cell Detection of Microorganisms

“…Over the past decade, several distinct methods for generating sequence-specific fluorescence signals within intact bacterial cells have been developed, primarily for environmental microbiology applications. These include in situ PCR (25)(26)(27)(28)(29), in situ reverse transcription-PCR (30)(31)(32), in situ reverse transcription (33), chromosomal painting (34,35), in situ rolling circle amplification (36,37), in situ loop-mediated signal amplification (38) and fluorescence in situ hybridization (FISH) (39)(40)(41)(42)(43). Descriptions of each method are given below, followed by a discussion of their potential benefits and drawbacks for use in routine diagnostics in environmental, food and clinical microbiology applications.…”

“…In the RCA reaction, a circulizable probe is hybridized to its target on a plasmid or on the chromosome, then the circle is closed via ligation. A primer then directs DNA polymerase to extend the circular sequence progressively around the circle, leading to the formation of the long linear product of tandem repeats (36,37,46). The population of products formed is generally distributed over a wide range of lengths, typically appearing as a continuous smear of high molecular weight DNA when viewed on an electrophoretic gel (46).…”

“…The population of products formed is generally distributed over a wide range of lengths, typically appearing as a continuous smear of high molecular weight DNA when viewed on an electrophoretic gel (46). Detection of the tandemly repeated sequences in the amplicon is then facilitated using fluorescently-labeled detector or "decorator" probes (36,37,47). For in situ RCA, this entire process is carried out within the context of whole bacterial cells.…”

“…Rolling Circle Amplification (RCA) is an isothermal method for nucleic acid amplification that targets short sequences (~ 40 or fewer bases) and generates long, single-stranded amplicons comprised of tandemly repeated sequences that can subsequently be detected via hybridization (36,37,46). In standard RCA techniques, amplicon production proceeds in a linear fashion; however, modifications of this technique variously termed "ramification", "cascade" or "hyperbranched" RCA have been developed to yield geometric amplicon production (46).…”

Application of Real-Time Long and Short Polymerase Chain Reaction for Sensitive Monitoring of the Fate of Extracellular Plasmid DNA Introduced into River Waters

“…Then, both real-time long and short PCRs were used to quantify the plasmid encoding a green fluorescent protein gene (gfp) introduced in river water after separating the water sample into extracellular and cellular fractions to analyze the dynamics of the target plasmid in detail. In an assessment of the fate of the plasmid DNA, DNA uptake frequencies in bacterial communities were also determined by detecting transformants expressing the gfp gene on the plasmid and taking up gfp at the single cell level using in situ rolling circle amplification (in situ RCA) 27,28) .…”