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Taxonomic studies were performed on four strains (D-1/D-an/IIT, C/C-an/B1T, A-1/C-an/C1T and A-1/C-an/IT) of anaerobic, gram-positive, spore-forming bacteria originally isolated from a mat sample retrieved from a shallow, moated area around Lake Fryxell, an Antarctic freshwater lake. Phylogenetic analyses based on 16S rRNA gene sequence data indicated that these strains are affiliated with cluster I clostridia and form a coherent group with Clostridium estertheticum and Clostridium laramiense. Similarity values among 16S rRNA gene sequences within this assemblage ranged between 96.7 and 99.8%. Despite the close phylogenetic relationship, several distinguishing phenotypic traits were found among the novel strains using a polyphasic approach. All strains were psychrophilic, but the temperature optimum for growth differed markedly, ranging from 4 to 16 degrees C. In addition, substrate utilization patterns, fermentation end products, cellular fatty acid profiles and morphological traits enabled a clear differentiation between the strains. DNA-DNA hybridization experiments revealed that each of the four novel strains represents a distinct species, with DNA-DNA similarity values to related strains in the range 16-62%. In contrast, the type strains of C. estertheticum and C. laramiense shared 79% DNA-DNA similarity, indicating a close relationship at the species level. On the basis of genetic and phenotypic properties, it is proposed to designate four novel species of the genus Clostridium to harbour the newly isolated strains: Clostridium frigoris sp. nov. (type strain D-1/D-an/IIT=DSM 14204T=ATCC BMAA-579T), Clostridium lacusfryxellense sp. nov. (type strain C/C-an/B1T=DSM 14205T=ATCC BAA-580T), Clostridium bowmaniisp. nov. (type strain A-1/C-an/C1T=DSM 14206T=ATCC BAA-581T) and Clostridium psychrophilum sp. nov. (type strain A-1/C-an/IT=DSM 14207T=ATCC BAA-582T). It is also proposed to unite C. laramiense and C. estertheticum under C. estertheticum. The subspecies C. estertheticum subsp. laramiense subsp. nov. is established, represented by strain ATCC 51254T (=DSM 14884T). The type strain of C. estertheticum subsp. estertheticum remains NCIMB 12511T (=DSM 8809T).

Twenty-seven of 37 non-toxigenic, urease-negative strains originally identified as Clostridium bifermentans that were isolated in the Antarctic are reidentified as C. sordellii by the tests for DNA-DNA homology, by the absence of mannose in the cell wall, and by growth inhibition of mannose. The test for cell wall sugar components of urease-negative and -positive strains of C. sordellii revealed that glucose, mannose, and rhamnose could not be detected in any of eight urease-negative strains used by galactose was detectable in seven of the eight strains and that glucose or galactose or both of the two sugars were present in the urease-positive strains tested.