Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples

Abstract

The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 106 Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations.

Use of a chiA probe for detection of chitinase genes in bacteria from the Chesapeake Bay

“…However, there have been few attempts to apply molecular approaches to ecological studies of chitinolytic bacteria in the marine environment, using, for example, nucleic acid probes for detection of bacteria possessing genes encoding for chitinase. Detection and quantitative determination of the presence (or absence) of a gene encoding a particular characteristic can be achieved with great speed and accuracy by nucleic acid hybridization, thus circumventing many limitations associated with conventional bacteriological methods [11,12].…”

Nucleic‐acid‐based methods for monitoring the performance of in situ bioremediation

“…Studies using colony hybridization and hybridization to enrichment cultures have shown a correlation between a catabolic activity measurement and/or a most-probable number (MPN) enumeration of a specific catabolic type and an estimate of the microbial population density possessing the trait using a nucleic acid probe. The utility of DNA-targeted methods for monitoring bioremediation was first widely recognized in a report of pond sediment microcosms spiked with synthetic oil and analysed by colony hybridization (Sayler et al 1985). Exposure to synthetic oil increased the percentage of the total CFU that hybridized to the catabolic plasmids TOL and NAH7 up to 10-fold and > 3%fold, respectively, compared to nonspiked control microcosms.…”

Molecular ecology of microbes: A review of promises, pitfalls and true progress

“…Since the introduced bacteria often survive at a low population density, close to, or even below the detection level, our insight in the ecology of rhizobia is still incomplete. The same holds for plant pathogens [18][19][20], human and animal pathogens [21][22][23][24][25][26], and microbes involved in the degradation of xenobiotics [27][28][29]. 187…”

“…DNA probes used in environmental studies may consist of relatively long cloned fragments of plasmids (e.g. [20,22,54,55]) or genes, such as cloned copper resistance genes [19,56], enterotoxin genes [57,58], and genes involved in the degradation of xenobiotics [29,[59][60][61][62]. Alternatively, the probes may consist of short (usual 20-30) nucleic acid sequences defined as oligonucleotide probes.…”