All members of the ‘Polynucleobacter subcluster D’ possessed the oligonucleotide sequence 59-AA(T/G)CCCT(A/T)AGGGGGAAA-39 within the 16S rRNA gene (E. coli positions 181–197). In almost all cases, the presence of this sequence could be determined by fluorescent in situ hybridization of whole cells by using a mixture of the fluorescently labelled oligonucleotide probes PnecD1-181 and PnecD2-181 (Hahn et al., 2005). The sequences of these two probes differ in the second variable position (E. coli position 188) of the diagnostic sequence, but not in the first variable position (E. coli position 183). Analysis of 104 sequences forming the phylogenetic cluster representing the proposed new taxon, Polynucleobacter
cosmopolitanus sp. nov., demonstrated that only one of the novel strains differed from the probe sequences in the first variable position of the diagnostic sequence. Besides the FISH probe, a reverse line blot hybridization probe for the detection of the proposed new taxon in environmental samples has been developed and applied successfully.