Desulfotomaculum genus- and subgenus-specific 16S rRNA hybridization probes for environmental studies

Based on comparative analysis of 16S rRNA sequences and the recently establ ished phylogeny of the genus Desulfotomaculum, a set of phylogenetically ne sted hybridization probes was developed and characterized. A genus-specific probe targets all known Desulfotomaculum species (with the exception of De sulfotomaculum acetoxidans), and five specific probes target subclusters wi thin the Desulfotomaculum genus. The dissociation temperature of each probe was determined experimentally. Probe specificities were verified through h ybridizations with pure culture rRNA isolated from a wide variety of target and non-target organisms and through an evaluation of probe 'nesting' usin g samples obtained from four different environments. Fixation and hybridiza tion conditions for fluorescence in situ hybridizations were also optimized . The probes were used in quantitative membrane hybridizations to determine the abundance of Desulfotomaculum species in thermophilic anaerobic digest ers, in soil, in human faeces and in pig colon samples. Desulfotomaculum rR NA accounted for 0.3-2.1% of the total rRNA in the digesters, 2.6-6.6% in s oil, 1.5-3.3% in human faeces and 2.5-6.2% in pig colon samples.