Bacterial strain BF36(T), isolated from the biofilm of a tufa deposit in a hard water rivulet, was characterized by a polyphasic taxonomic approach. Cells of these organisms were Gram-negative, motile, nonpigmented, rod-shaped, non-endospore-forming, and facultatively anaerobic. Cells, organized in loose consortia, were coated by a massive slime layer. Phylogenetic analyses using 16S rRNA gene sequences showed that strain BF36T was a member of the family Enterobacteriaceae, class Gammaproteobacteria, displaying a moderate degree of relationship (96.5% sequence similarity) to Sodalis glossinidius and "Sodalis pallipedes," intracellular symbionts of the tsetse fly Glossinis morsitans morsitans. Dendrograms of relationship generated by different algorithms consistently grouped isolate BF36(T) with Sodalis glossinidius, Pragia fontium, Budvicia aquatica, Serratia rubideae, and Brenneria spp (94.7-95.8% similarity) which also share many common metabolic properties. Differences between strain BF36(T) and Sodalis glossinidius DSM 13495(T) are seen in motility and in the pattern of substrates utilized. Membership to the family was also confirmed by a fatty acid profile consisting of major amounts of C-16:0 and C(16:1 omega)7, by the presence of isoprenoids of the ubiquinone Q8 and menaquinone MK8 types and a DNA G + C content of 54.2 mol%. The decision to classify strain BF36(T) into a new genus Biostraticola gen. nov. is based on its distant phylogenetic position as compared to any other representative of the family and the significant phenotypic differences to its nearest phylogenetic neighbor, Sodalis glossinidius. BF36(T) represents the type species, for which the name Biostraticola tofi sp. nov. is proposed. The type strain is BF36(T) (DSM 19580(T); CIP109699(T)).