In situ probing of gram-positive bacteria with high DNA G+C content using 23S rRNA-targeted oligonucleotides.

Lehrstuhl fur Mikrobiologie, Tec hn isc he U n ive rsitat Munchen, ArcisstraBe 16, D80290 Mu nchen, Germany 235-rRNA-targeted oligonucleotide probes were designed for the phylogenetic group Gram-positive bacteria with high G + C content of DNA ' (GPBHGC). A sequence idiosyncrasy in two adjacent base pairs in the stem of helix 69 in domain IV of the 235 rRNA is present in all hitherto analysed strains of GPBHGC. An oligonucleotide probe targeted to this region hybridized only with strains of GPBHGC and was successfully used for in situ monitoring of these cells in activated sludge. Another unique feature of the 235 rRNA molecules of GPBHGC is a large insertion in domain 111. Fluorescent oligonucleotides targeted to the highly variable regions of the rRNA within the insertions of Corynebacterium glutamicum DSM 2O30OT, Aureobacterium testaceum DSM 20166 and Brevibacterium sp. DSM 20165 hybridized specifically to their target strains, whereas probing with oligonucleotides complementary to the rRNA-coding strand of the 235 rDNA and to the spacer between 165 and 235 rRNA of C. glutamicum did not result in detectable fluorescence. This confirmed that the large 235 insertions are indeed present in 235 rRNAs of GPBHGC and provide potential target sites for highly specific nucleic acid probes.

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