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Four strains of an obligately anaerobic spore-forming bacterium were isolated from soil samples from a donkey pasture in Pakistan. Comparative 16s rRNA sequence analysis demonstrated that the strains are members of phylogenetic cluster I of the genus Clostridium (Collins et al. 1994). The strains are mesophilic, nonsaccharolytic, and nonproteolytic, utilize glutamate and histidine, and produce indole. Acetate, butyrate, ethanol, hydrogen, and carbon dioxide are the products of fermentation. Although the strains phenotypically resemble the classical glutamate-fermenting clostridia, such as Clostridium cochlearium, Clostridium tetanomorphum, Clostridium tetani, and especially Clostridium malenominatum, they differ from these organisms in sugar utilization, cellular fatty acid composition, and cellular protein pattern and by a 16s rRNA sequence divergence value of approximately 4 to 8%. Phylogenetically, the strains are more closely related to Clostridium estertheticum (sequence divergence, approximately 5%) and Clostridium subterminale (sequence divergence, approximately 5%) but are phenotypically readily distinguished from these species. On the basis of phenotypic and genotypic criteria, we conclude that the four strains are members of a new species of the genus Clostridium, for which the name Clostridium pascui is proposed. The type strain is strain Cm19 (= DSM 10365).In the course of previous studies aimed at isolation and characterization of glutamate-fermenting bacteria, which led to the revival of the species Clostridium tetanomoiphum (26), we recovered four clostridial strains which could not be unambiguously identified as strains of any of the classical glutamate-fermenting species, such as Clostridium cochlearium, Clostridium tetanomorphum, Clostridium tetani, Clostridium malenominatum, and Clostridium limosum. When the identification key of Cat0 et al. (3) was used, the strains closely resembled C. malenominatum. However, differences in electrophoretic cellular protein patterns did not support this classification.In an attempt to clarify the taxonomic position of these unknown isolates and to determine their relationships with other glutamate-fermenting species, we conducted further phenotypic and genotypic investigations. On the basis of the findings of these studies, we concluded that the four glutamatefermenting soil isolates represent a new species of the genus Clostridium, for which the name Clostridium pascui is proposed. MATERIALS AND METHODSBacteria. Strains Cm17, Cm19T (T = type strain), Cm20, and Cm21 were isolated from soil samples taken from a donkey pasture in Pakistan. The isolation procedure described by Hippe et al. (13) was used with glutamate as the substrate. The isolates were tested for toxigenicity, and none was positive. Strains of C. mulenominatum (DSM 1127T), C. cochlearium (DSM 666, DSM 667, DSM 2153, DSM 3588, and DSM 1285T), C. tetanomorphum (DSM 528, DSM 665, and DSM 4474T), C. limosum (DSM 1400T), Clostridium symbiosum (DSM 934T), Clostridium subterminale (DSM 758, DSM 2636, ...

Growth of various bacteria, especially aerobic hydrogen-oxidizing bacteria, in the presence of 2 to 100% (v/v) oxygen in the gas atmosphere was evaluated. The bacterial strains included Alcaligenes eutrophus, A. paradoxus, Aquaspirillum autotrophicum, Arthrobacter spec. strain 11 X, Escherichia coli, Arthrobacter globiformis, Nocardia opaca, N. autotrophica, Paracoccus denitrificans, Pseudomonas facilis, P. putida, and Xanthobacter autotrophicus. Under heterotrophic conditions with fructose or gluconate as substrates neither colony formation on solid medium nor the growth rates in liquid media were drastically impaired by up to 100% oxygen. In contrast, autotrophic growth--with hydrogen, carbon dioxide and up to 80% oxygen in the gas atmosphere--was strongly depressed by high oxygen concentrations. However, only the growth rate, not the viability of the cells, was decreased. Growth retardation was accompanied by a decrease of hydrogenase activity.

A total of 46 glutamate-degrading strains of the genus Clostridium, including 36 new isolates, were examined for deoxyribonucleic acid (DNA)-DNA homology, protein patterns produced by polyacrylamide gel electrophoresis, and physiological characteristics in order to determine the differences between Clostridium cochleurium and "Clostridium tetunomorphum." Thirty-four newly isolated strains could be assigned to the "C.tetunomorphum" group; two strains were classified as nontoxigenic Clostridium tetuni strains. The levels of DNA-DNA homology between C. cochleurium and "C. tetunomorphum" were only 10 to 19%, and the protein patterns showed that there were great differences between both groups and other glutamate-degrading species.Biochemical tests supported these results. In contrast to C. cochleurium, all "C. tetunomorphum" strains fermented carbohydrates (at least glucose and maltose); they also produced lipase and degraded ethanolamine. Moreover, C. cochleurium, like other glutamate-degrading species, needed a higher concentration of yeast extract than "C. tetunomorphum" when it was grown on glutamate. On the basis of our results we propose C. tetunomorphum sp. nov., nom. rev. The type strain is strain NCTC 543 (= DSM 4474).Formerly, the classical glutamate-fermenting clostridial species included Clostridium cochlearium (Douglas, Fleming, and Colebrook 1919) Bergey, Harrison, Breed, Hammer, and Huntoon 1923; Clostridium tetanomorphum" (Bulloch,

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