In situ detection of genes in bacterial cells using flourescent dye-labeled polynucleotide probes (RING-FISH): Improvements of specificity and permeabilization as well as its application for environmental studies

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Recently, we could for the first time successfully visualize bacterial genes at cellular level by RING-FISH (Recognition of Individual Genes by Fluorescent In Situ Hybridization). The method is based upon a modified in situ cell hybridization procedure using gene specific polynucleotide transcript probes. The presence of the respective genes is indicated by probe conferred cell fluorescence. The typically halo-like cell fluorescence is usually visualized by laser scanning microscopy. Whereas conventional FISH applying rRNA-targeted oligonucleotide probes benefits from natural target amplification, the detection of single or oligocopy genes essentially depends upon intensive signal amplification. As shown recently, the latter is conferred by inter-molecular probe network formation. Although the general applicability of this new, powerful and attractive technique has been shown, further essential improvements are necessary before the method can be used for environmental studies. Given that the approach has been applied for a small selection of genes and few gram-negative species thus far, it has to be adapted for a broader spectrum of genes (especially nifH) and bacteria (especially gram-positives). Concerning probe specificity limitations result from the need for polynucleotide probes. Synthetic constructs comprising multiple units of gene specific oligonucleotides have to be developed to ensure higher specificity. In this context, major efforts are needed to solve the problem of network formation by synthetic probe constructs. Moreover, the permeabilization of gram-positive bacteria has to be improved for a better access of polynucleotide probes. Environmental studies will be performed concerning the assignment of nifH genes classified by RING-FISH to microbial cells identified by conventional rRNA FISH.