From a local hot spring of Myagdi, in the western geothermal area of Nepal, Two aerobic, Gram-negative, non-motile, non-sporulating, yellow-pigmented thermophilic bacteria, strains TH-1 and TH-2, were isolated. They exhibited circular, convex, non-transparent and yellow pigment colonies. The isolates grew at 55–75°C (optimally at 72°C) and at pH 6.5–8.5 (optimally at pH 7.5–8.0). Both culture dependent and culture independent strategies were implemented for the identification of the isolates. Phylogenetic analysis of 16S rRNA gene sequences and analysis of FAME as well as levels of DNA–DNA relatedness together indicated that the new isolates represented a novel species of the genus Thermus with closest affinity to Thermus aquaticus, Thermus igniterrae, Thermus thermophilus and Thermus arciformis. The genomic DNA G+C contents of strains TH-1 and TH-2 were 62.3 mol% and 64 mol% Compared with their closest relatives, strains TH-1 and TH-2 were able to assimilate a wider range of carbohydrates, amino acids and organic acids as sole carbon sources for growth. The isolates were found to be very effective in production of various Extracellular enzymes like amylase, pectinase, protease, caseinase, gelatinase and lipase. The new isolates have very high potential for industrial upragades for production of thermostable enzymes. The new isolates had higher combined levels of 15:0 iso and 15:0 anteiso compared with their closest relatives. Based on the results of physiological and biochemical tests, polyphasic taxonomic characterization and phylogenetic analyses, the isolates strains could not be classified as representing any species of the genus Thermus with a validly published name. Thus the two strains TH-1 and TH-2 are considered to represent a novel species of the genus Thermus, for which the name Thermus nepalensis sp. nov. is proposed.
