Several candidate division TM7 filamentous bacteria have been previously described from soils and activated sludge plants, and several FISH (Fluorescence In Situ Hybridization) probes have been designed to target members of this candidate division. Two of these probes, TM7-305 and TM7-905, when used in a survey of activated sludge samples in both Japan and Australia, invariably hybridized to other filamentous organisms recognized as the Eikelboom morphotype 1851 and 0041/0675. Unexpectedly, the type 1851 filament morphotype also responded to the CHL1851 FISH probe designed to target type 1851 Chloroflexi, and both morphotypes responded to the CFX1223 and GNSB941 FISH probes designed to target most members of the Chloroflexi phylum. These observations suggested either the existence of novel Eikelboom morphotype 1851 and 0041/0675 filaments possessing both the Chloroflexi and TM7 target sites, or that hybridization by either probe was not specific for its intended target group.
16S rRNA clone libraries from samples containing type 1851 TM7-305 positive filaments in both countries yielded Chloroflexi clones with high sequence similarity to Kouleothrix aurantiaca. These contained the TM7-305 probe target site, except that they possessed a single base deletion. This probe appears to hybridize with it by forming a ‘loop out’ over this missing base. Furthermore, the TM7-905 FISH probe, designed to target members of the entire candidate division TM7 also hybridized with Eikelboom morphotype 0041/0675 filaments responding to the same phylum level Chloroflexi probes. In silico analysis revealed many Chloroflexi 16S rRNA sequences possessed only a single mismatch to the TM7-905 probe target. When competitor probes for both the TM7-305 and TM7-905 Chloroflexi non-target sites were applied, no fluorescent signal was seen in any filamentous organisms also hybridizing with the Chloroflexi probes. This study highlights the importance of regular FISH probe assessment, and the subsequent design and use of appropriate competitor probes.