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Ecosystems, including engineered ones, are complex systems in which microorganisms occur in heterogenous communities. Their behaviour in the environment is often unknown due to the lack of proper detection and identification techniques. Molecular ecology is a new field in which microbes can be recognized and their function can be understood at the DNA/RNA level without unreliable steps of cultivation of microbes. During the last few years genetically modified microbes have been constructed by recombinant DNA techniques for putative use in the environment. The slow progress in this field is due to the lack of integration of microbial ecology and molecular biology. In the present review, examples will be given of the use of DNA probes and marker genes in our study on the ecology of genetically modified microbes and wild-type recalcitrant microorganisms that are difficult to cultivate or even 'non-culturable'. Emphasis is given to the development and use of oligonucleotide probes directed towards 16S rRNA, to detect microbes in various engineered ecosystems: (i) Frankia in root nodules, and (ii) propionate-oxidizing sulfate-reducing bacteria in anaerobic granular sludge. Expression of genes is demonstrated by studies on the localization of nifH transcripts in root nodules of Coriaria and Alnus. In addition we will describe examples of the use of marker genes (gusA gene and aphV gene) to study competition and genetic stability of released engineered Rhizobium and Streptomyces strains.

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