Proinflammatory and prothrombotic effects on human vascular endothelial cells of immune-cell-derived LIGHT.

OBJECTIVE: LIGHT (TNFSF 14) belongs to the tumor necrosis factor superfamily and is expressed by activated T cells as well as various types of antigen presenting cells. LIGHT binds to its cellular receptors TR2 and LTbetaR and has a co-stimulatory role in T cell activation. Here, we compared the relative expression of LIGHT in different immune cells and the biological activity of immune cell-derived LIGHT on endothelial cells. METHODS AND RESULTS: Surface expression of LIGHT and mRNA production by PBMC and isolated T cells (CD4+ or CD8+) significantly increased after stimulation with PMA (Phorbolester-12- Myristat-13-Acetat)+ionomycin. No LIGHT expression on PMA stimulated monocytes or monocytic-like THP-1 cells could be detected; differentiation of monocytes and THP-1 cells into macrophages, however, resulted in up-regulation of LIGHT. Supernatants of stimulated T cells contained higher concentrations of soluble LIGHT than macrophage supernatants normalized to cell numbers; release of soluble LIGHT was found to be dependent on metalloproteinase activity. Size determination of released soluble LIGHT by size exclusion chromatography revealed a molecular mass of approximately 60 kDa, suggesting a trimeric form. Released soluble LIGHT induced expression of proinflammatory antigens ICAM-1, tissue factor and IL-8 in human endothelial cells and caused apoptosis of IFN-g pretreated endothelial cells. Soluble LIGHT was detected at low levels in sera of healthy controls and was significantly enhanced in sera of patients with chronic hepatitis C and rheumatoid arthritis (24.93+/-9.41 vs. 129.53+/-49.14 and 172.13+/-77.64; p<0.0005). CONCLUSION: These findings suggest that among immune cells activated T lymphocytes are the main source of soluble LIGHT with released amounts of soluble LIGHT markedly higher compared to platelets. Immune cell-derived membrane-bound and soluble trimeric LIGHT is biologically active, inducing proinflammatory changes in endothelial cells. Enhanced plasma levels of soluble LIGHT in patients with chronic infections suggest a role of LIGHT in systemic inflammatory activation.

Petrotoga olearia sp. nov. and Petrotoga sibirica sp. nov., two thermophilic bacteria isolated from a continental petroleum reservoir in Western Siberia.

Strictly anaerobic, thermophilic bacteria (strains SL24T, SL25T, SL27, SL29 and SL32) were isolated from a deep, continental oil reservoir in Western Siberia (Russia). These motile, rod-shaped organisms were surrounded by a sheath-like structure, a feature characteristic of the Thermotogales. On the basis of partial 16S rDNA sequences (500 nucleotides), strains SL25T, SL27, SL29 and SL32 were identical. Therefore, only strains SL24T and SL25T were studied in detail. The optimum temperature for growth of both strains was 55 degrees C. Their optimum pH for growth was 7.5 and their optimum NaCl concentration was between 20 and 30 g l(-1). The novel isolates reduced elemental sulfur and cystine, but not thiosulfate or sulfate, to hydrogen sulfide. The G+C contents of the genomic DNA of strains SL24T and SL25T were respectively 35 and 33 mol%. Phylogenetically, both strains are most closely related to Petrotoga miotherma, there being 98.9-99.4% similarity between their 16S rDNA sequences. Phenotypic properties and DNA-DNA hybridization experiments indicate that the strains belong to two novel species, for which the names Petrotoga olearia (type strain SL24T = DSM 13574T = JCM 11234T) and Petrotoga sibirica (type strain SL25T= DSM 13575T = JCM 11235T) are proposed.

Nautilia lithotrophica gen. nov., sp. nov., a thermophilic sulfur-reducing epsilon-proteobacterium isolated from a deep-sea hydrothermal vent.

A novel, strictly anaerobic, thermophilic sulfur-reducing bacterium, strain 525T, was isolated from tubes of the deep-sea hydrothermal vent polychaete Alvinella pompejana, collected on the East Pacific Rise (13 degrees N). This organism grew in the temperature range 37-68 degrees C, the optimum being 53 degrees C, and in the pH range 6.4-7.4, the optimum being 6.8-7.0. The NaCl range for growth was 0.8-5.0%, the optimum being 3.0%. Strain 525T grew lithoautotrophically with H2 as energy source, S0 as electron acceptor and CO2 as carbon source. Alternatively, strain 525T was able to use formate as an energy source. The G+C content of the genomic DNA was 34.7 mol%. Phylogenetic analysis of the 16S rDNA gene sequence placed strain 525T in the epsilon-subclass of the Proteobacteria, where it forms a deep cluster with recently isolated relatives. On the basis of phenotypic and phylogenetic differences between strain 525T and its closest phylogenetic relatives, it is proposed that the new isolate should be described as a member of a new genus, Nautilia, for which the name Nautilia lithotrophica gen. nov., sp. nov. is proposed. The type strain is strain 525T (= DSM 13520T).

Thermosipho geolei sp. nov., a thermophilic bacterium isolated from a continental petroleum reservoir in Western Siberia.

Three strictly anaerobic, thermophilic bacteria (SL31T, SL30 and MLM39636) were isolated from a deep continental oil reservoir in Western Siberia (Russia). Following the mid-exponential phase of growth, the non-motile rod-shaped organisms were surrounded by a sheath-like structure. As DNA-DNA hybridizations showed that these strains were highly related genomically, only strain SL31T was studied in detail. The temperature range for growth of strain SL31T was between 45 and 75 degrees C, with optimum growth at 70 degrees C. Its optimum pH and NaCl concentration for growth were pH 7.5 and 20-30 g l(-1), respectively. The novel isolate reduced elemental sulfur and cystine, but not thiosulfate or sulfate, to hydrogen sulfide. The G+C content of the genomic DNA was 30.0 mol %. As determined by 16S rDNA sequence analysis, this organism belonged to the genus Thermosipho. DNA-DNA hybridization levels between strain SL31T and type strains of the previously described species of Thermosipho were less than 10%. On the basis of physiological and molecular properties, it is proposed that this organism should be placed in a new species, Thermosipho geolei sp. nov. The novel organism represents the first species of the genus Thermosipho that has been isolated from a petroleum reservoir. The type strain is SL31T ( = DSM 13256T = JCM 10986T).

Isolation and characterization of Thermococcus sibiricus sp. nov. from a Western Siberia high-temperature oil reservoir.

Anaerobic organotrophic hyperthermophilic Archaea were isolated from five of eight samples from oil wells of the Samotlor oil reservoir (depth, 1,799-2,287 m; temperature, 60 degrees-84 degrees C). Three strains were isolated in pure cultures and characterized phylogenetically on the basis of comparison of the 16S rRNA gene sequences. All strains belonged to a new species of the genus Thermococcus, with Thermococcus litoralis, Thermococcus aggregans, Thermococcus fumicolans, and Thermococcus alcaliphilus being the nearest relatives (range of sequence similarity, 97.2%-98.8%). Strain MM 739 was studied in detail. The new isolate grew on peptides but not on carbohydrates. Elemental sulfur had a stimulatory effect on growth. The temperature range for growth was between 40 degrees and 88 degrees C, with the optimum at 78 degrees C; the pH range was 5.8 to 9.0, with the optimum around 7.3; and the salinity range was 0.5% to 7.0%, with the optimum at 1.8%-2.0%. The doubling time at optimal growth conditions was about 43 min. The G+C content of the DNA was 38.4 mol%. The DNA-DNA relatedness between strain MM 739 and T. litoralis was 27%; between strain MM 739 and T. aggregans, it was 22%. Based on the phenotypic and genomic differences with known Thermococcus species, the new species Thermococcus sibiricus is proposed. The isolation of a hyperthermophilic archaeum from a deep subsurface environment, significantly remote from shallow or abyssal marine hot vents, indicates the existence of a subterranean biosphere inhabited by indigenous hyperthermophilic biota.

16S rDNA diversity of cultured and uncultured prokaryotes of a mat sample from Lake Fryxell, McMurdo Dry Valleys, Antarctica.

The prokaryotic diversity of aerobic and anaerobic bacterial isolates and of bacterial and archaeal 16S rDNA clones was determined for a microbial mat sample from the moated region of Lake Fryxell, McMurdo Dry Valleys, Antarctica. Among the anaerobic bacteria, members of Clostridium estertheticum and some other psychrotolerant strains dominated whereas methanogens and other Archaea were lacking. Isolates highly related to Flavobacterium hibernum, Janthiniobacterium lividum, and Arthrobacter flavus were among the aerobic bacteria most frequently isolated. Assessment of more than 350 partial 16S rDNA clone sequences of libraries generated by Bacteria- and Archaea-specific PCR primers revealed a rich spectrum of bacterial diversity but only two different archaeal clone sequences. Among the Bacteria, representative sequences belonged to the class Proteobacteria, order Verrucomicrobiales, class Actinobacteria, Clostridium/Bacillus subphylum of Gram-positives, and the Cytophaga-Flavobacterium-Bacteroides phylum. The clones formed about 70 higher taxonomy groups (<98% sequence similarity) and 133 potential species, i.e., groups of clones sharing greater than 98% similarity. Only rarely were clone sequences found to be highly related to Lake Fryxell isolates and to strains of described species. Subsequent analysis of ten sequencing batches of 36 individual clones indicated that the diversity might be still higher than had been assessed.

Clostridium hathewayi sp. nov., from human faeces.

A strictly anoxic, Gram-positive, sporeforming, rod-shaped bacterium was isolated from a chemostat inoculated with human faeces. The bacterium used carbohydrate as fermentable substrates, producing acetate, ethanol, carbon dioxide and hydrogen as the major products of glucose metabolism, and possessed a G + C content of 50.7 to 50.9 mol%. Comparative 16S rRNA gene sequencing showed that the unidentified bacterium represents a previously unrecognised sub-line within the Clostridium coccoides rRNA group of organisms. The nearest relatives of the unknown bacterium corresponded to Clostridium algidixylanolyticum, C. aerotolerans, C. celerecrescens, C. indolis, C. sphenoides, C. methoxybenzovorans and C. xylanolyticum but 16S rRNA sequence divergence values of >4% demonstrated that it represents a novel species. Based on the presented findings a new species, Clostridium hathewayi, is described. The type strain of Clostridium hathewayi is DSM = 13479T (= CCUG 43506 T).

Reclassification of Clostridium quercicolum as Dendrosporobacter quercicolus gen. nov., comb. nov.

Morphological features, genomic DNA base composition and 16S rDNA sequence similarities, as well as a distinct phospholipid pattern, whole-cell fatty acid distribution and the occurrence of the lipoquinone 'lipid F', indicate that Clostridium quercicolum belongs to the Sporomusa-Pectinatus-Selenomonas phyletic group and possesses only a remote relationship to members of the genus Clostridium sensu stricto. On the basis of these results, the new genus and combination Dendrosporobacter quercicolus gen. nov., comb. nov. are proposed.

Leptospirillum gen. nov. (ex Markosyan 1972), nom. rev., including Leptospirillum ferrooxidans sp. nov. (ex Markosyan 1972), nom. rev. and Leptospirillum thermoferrooxidans sp. nov. (Golovacheva et al. 1992).

The name Leptospirillum ferrooxidans is not in the Approved Lists of Bacterial Names (1980), nor has it been subsequently validly published. In accordance with the rules of the International Code of Nomenclature of Bacteria, the name Leptospirillum for the genus (gen. nov., nom. rev.) and Leptospirillum ferrooxidans for the species (sp. nov., nom. rev.) is revived here. The type species is Leptospirillum ferrooxidans strain L15T (= DSM 2705T). The second species in the genus is Leptospirillum thermoferrooxidans (Golovacheva et al. 1992) (type strain L-88T; Institute of Microbiology, INMI, Moscow, Russia).

Anoxybacillus pushchinensis gen. nov., sp. nov., a novel anaerobic, alkaliphilic, moderately thermophilic bacterium from manure, and description of Anoxybacillus flavitherms comb. nov.

A new strictly anaerobic, alkaliphilic, moderately thermophilic, fermentative, spore-forming bacterium, strain K1T, was isolated from manure samples (pH 6-8). Cells were Gram-positive, straight, non-motile rods that grew at temperatures of 37-66 degrees C (optimum at 62 degrees C) and in a pH range of 8.0-10.5 (optimum at 9.5-9.7). The bacterium fermented D-glucose, sucrose, D-fructose, D-trehalose and starch as carbon and energy sources. It required vitamins and its growth is stimulated by yeast extract. The major metabolic products were H2 and acetate. Cells were catalase-negative and could reduce nitrate to nitrite. The G+C content of the DNA was 42.2 mol%. Based on the phenotypic properties and 16S rDNA sequencing and DNA-DNA hybridization data, strain K1T (= DSM 12423T = ATCC 700785T = VKM B-2193T) was assigned to the new genus Anoxybacillus gen. nov., as a representative of a new species, Anoxybacillus pushchinensis sp. nov. 'Bacillus flavothermus' strain d.y., which was found to be closely related to strain K1T, is described as Anoxybacillus flavithermus comb. nov. (type strain = d.y.T = DSM 2641T).

Description of Desulfotomaculum sp. Groll as Desulfotomaculum gibsoniae sp. nov.

Strain GrollT, isolated from fresh water, is a mesophilic, spore-forming, sulfate-reducing bacterium that uses a large variety of substrates as electron donors ranging from simple organic compounds to long-chain fatty acids and several aromatic compounds. Sulfate, thiosulfate and sulfite are used as electron acceptors. Homoacetogenic growth occurs under sulfate-free conditions. Substrate oxidation is usually complete, leading to CO2, but acetate or other fatty acids can accumulate at high substrate concentrations. The G + C content of the DNA is 54.8 mol%. Strain GrollT was found to be phenotypically and phylogenetically different from known members of the genus Desulfotomaculum. 16S rRNA gene sequence analyses show that this organism falls within the radiation of the genus Desulfotomaculum cluster and has < 96% sequence similarity to previously described species. The name Desulfotomaculum gibsoniae sp. nov. is proposed for this strain; the type strain is GrollT (= DSM 7213T).

A re-evaluation of the taxonomy of the genus Anaerovibrio, with the reclassification of Anaerovibrio glycerini as Anaerosinus glycerini gen. nov., comb. nov., and Anaerovibrio burkinabensis as Anaeroarcus burkinensis [corrig.] gen. nov., comb. nov.

Chemotaxonomic, electron microscopic and 16S rRNA gene sequence analyses of the three described species of the genus Anaerovibrio demonstrated only remote similarities to each other. The 16S rRNA gene sequence similarities between Anaerovibrio lipolytica, Anaerovibrio glycerini and Anaerovibrio burkinabensis and the derived phylogenetic relationships of the three species studied fell below genus level. All three species clustered within the Sporomusa-Pectinatus-Selenomonas phyletic group. Each species showed a distinct phospholipid pattern and whole-cell fatty acid distribution. Several isoprenologues of the lipoquinone 'lipid F' were found to differ in their quantitative distribution in the Anaerovibrio species. On the basis of these results, the new genera Anaerosinus gen. nov. and Anaeroarcus gen. nov. are proposed. The type species of Anaerosinus is Anaerosinus glycerini comb. nov., and the type species of Anaeroarcus is Anaeroarcus burkinensis [corrig.] comb. nov. The genus Anaerovibrio is consequently restricted to a single species, namely Anaerovibrio lipolyticus [corrig.]

Phylogenetic basis for a taxonomic dissection of the genus Clostridium.

The 16S rDNA-based phylogenetic analysis of the genus Clostridium has been completed by determination of the phylogenetic position of the type strains of 15 species and two non-validated species. These strains are members of phylogenetic clusters I, III, IV, V, IX, XIVa and XVIII as defined previously by Collins et al. [Int. J. Syst. Bacteriol. 44 (1994) 812-826]. Members of the genus Clostridium span a large evolutionary distance and the genus is not a phylogenetically coherent taxon but is intermixed with members of different genera, exhibiting a combination of Clostridium- and non-Clostridium-type properties. Anaerobacter polyendosporus, Syntrophococcus sucromutans and Acetivibrio multivorans also cluster within the radiation of Clostridium species. Although several taxa have been described for former Clostridium species with distinct phenotypic properties, the majority of Clostridium species, which are not members of the core cluster I, can at present not be reclassified as long as taxon-specific, phenotypic properties are not available.

Phylogenetic analysis of Formivibrio citricus, Propionivibrio dicarboxylicus, Anaerobiospirillum thomasii, Succinimonas amylolytica and Succinivibrio dextrinosolvens and proposal of Succinivibrionaceae fam. nov.

The phylogenetic position of Gram-negative, strictly anaerobic, non-spore-forming bacteria, representing four different genera, was determined by analysis of their 16S rDNA sequences. Formivibrio citricus and Propionivibrio dicarboxylicus are members of the beta-subclass of the class Proteobacteria. While Formivibrio citricus stands phylogenetically isolated, Propionivibrio dicarboxylicus is moderately related to members of the genus Rhodocyclus. Succinimonas amylolytica and Succinivibrio dextrinosolvens are members of the gamma-subclass of the class Proteobacteria in which they, together with members of the genus Anaerobiospirillum and Ruminobacter amylophilus, form a separate line of descent. This phylogenetic group is described as Succinivibrionaceae fam. nov.

Desulfurella kamchatkensis sp. nov. and desulfurella propionica sp. nov., new sulfur-respiring thermophilic bacteria from Kamchatka thermal environments.

Two strains of moderately thermophilic bacteria, which reduce elemental sulfur to hydrogen sulfide, were isolated from volcanic sources in Kamchatka. Strain K-119T was obtained from a thermophilic microbial community associated with Thermothrix thiopara, and strain U-8T was isolated from a cyanobacterial mat inhabiting a sulfide-rich hot spring. Cells of both strains are short Gram-negative rods, motile with one polar flagellum (strain K-119T) or non-motile (strain U-8T). Both strains are obligate anaerobes, have temperature otima of 54-55 degrees C and pH optima of 6.9-7.2. Molecular hydrogen, acetate, fumarate, malate, pyruvate, lactate and long-chain saturated fatty acids served as growth substrates for both species; strain U-8T was also able to grow on propionate. All substrates were oxidized completely, H2S and CO2 being the only metabolic products. Elemental sulfur was obligately required for growth of strain K-119T, whereas strain U-8T was able to grow also with thiosulfate as electron acceptor and on pyruvate without an external electron acceptor. The DNA G + C contents of strains K-119T and U-8T were 31.6 and 32.2 mol%, respectively. Phenotypic features and the results of 16S rRNA sequencing indicate the affiliation of the new isolates to the genus Desulfurella. The DNA-DNA hybridization with Desulfurella acetivorans was 40% for strain K-119T and 55% for strain U-8T; the DNA-DNA hybridization between the new isolates was 32%. Based on the results of morphological, physiological and phylogenetic studies the following two new species are proposed: Desulfurella kamchatkensis sp. nov. with the type strain K-119T (= DSM 10409T) and Desulfurella propionica sp. nov. with the type strain U-8T (= DSM 10410T).

Macrorestriction analysis of Desulfurella acetivorans and Desulfurella multipotens.

The genomes of the phylogenetically and physiologically unique bacteria Desulfurella acetivorans DSM 5264T and D. multipotens DSM 8415T were characterized and compared by pulsed field gel electrophoresis (PFGE). Macrorestriction patterns made of large PFGE separated DNA fragments were generated by digesting the genomic DNAs of both strains with the rare cutting restriction endonucleases ApaI, AscI, EagI, RsrII, SacII, SalI as well as with the intron encoded endonuclease I-CeuI. The sum of calculated fragment sizes from digests of the first six enzymes yielded estimates for the chromosome sizes of D. acetivorans with a mean of 1939.0 +/- 26.0 kb and for D. multipotens with a mean of 1864.0 +/- 23.0 kb. Within the patterns obtained from EagI and RsrII cleavages the apparent differences could be attributed to DNA insertion or deletion and to point mutation. The single, circular chromosomes of the two strains contain two copies of 23S rRNA genes each. Different extrachromosomal elements were detected in both strains.

Phylogenetic analysis of the genus Desulfotomaculum: evidence for the misclassification of Desulfotomaculum guttoideum and description of Desulfotomaculum orientis as Desulfosporosinus orientis gen. nov., comb. nov.

Almost complete 16S ribosomal DNA (rDNA) sequences were determined for the type strains of nine species belonging to the genus Desulfotomaculum and for seven strains described as strains of this genus. The sequences were compared with previously published 16S rDNA and rRNA sequences of the type strains of the other species of the genus. The majority of the species form a phylogenetically coherent cluster within the Clostridium-Bacillus subphylum of gram-positive bacteria. The cluster consists of phylogenetically well-separated lineages containing (i) Desulfotomaculum nigrificans, Desulfotomaculum aeronauticum, and Desulfotomaculum ruminis, (ii) Desulfotomaculum geothermicum, Desulfotomaculum thermosapovorans, and Desulfotomaculum sapomandens, (iii) Desulfotomaculum kuznetsovii, Desulfotomaculum australicum, and Desulfotomaculum thermocisternum, (iv) Desulfotomaculum thermobenzoicum and Desulfotomaculum thermoacetoxidans, and (v) Desulfotomaculum acetoxidans. Some as-yet-undescribed Desulfotomaculum strains are phylogenetically well-separated from strains of the described species. Desulfotomaculum guttoideum shares extremely high 16S rDNA similarity with certain Clostridium species (e.g., Clostridium sphenoides and Clostridium celerecrescens) and is most likely a misidentified species. Desulfotomaculum orientis represents a new genus which branches most closely to the genus Desulfitobacterium. The name Desulfosporosinus orientis gen. nov., comb. nov., is proposed for this taxon.

Purification and Properties of a Thermostable Pullulanase from a Newly Isolated Thermophilic Anaerobic Bacterium, Fervidobacterium pennavorans Ven5.

Extremely thermophilic anaerobic fermentative bacteria growing at temperatures between 50 and 80(deg)C (optimum, 65 to 70(deg)C) were isolated from mud samples collected at Abano Terme spa (Italy). The cells were gram-negative motile rods, about 1.8 (mu)m in length and 0.6 (mu)m in width, occurring singly and in pairs. Cells commonly formed spheroids at one end similar to Fervidobacterium islandicum and Fervidobacterium nodosum. The new isolate differs from F. nodosum by the 7% higher G+C content of its DNA (40.6 mol%) but is similar to Fervidobacterium pennavorans and F. islandicum in its G+C content and phenotypic properties. The phylogenetic dendrogram indicates that strain Ven5 belongs to the order Thermotogales and shows the highest 16S ribosomal DNA sequence similarity to F. pennavorans, F. islandicum, and F. nodosum, with similarities of 99.0, 98.6, and 96.0%, respectively. During growth on starch the strain produced a thermostable pullulanase of type I which preferentially hydrolyzed (alpha)-1,6 glucosidic linkages. The enzyme was purified 65-fold by anion-exchange, gel permeation, and hydrophobic chromatography. The native pullulanase has a molecular mass of 240,000 Da and is composed of three subunits, each with a molecular mass of 77,600 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimal conditions for the activity and stability of the purified pullulanase were pH 6.0 and 85(deg)C. At pH 6.0, the half-life of the enzyme was over 2 h at 80(deg)C and 5 min at 90(deg)C. This is the first report on the presence of pullulanase type I in an anaerobic bacterium.

Schwartzia succinivorans gen. nov., sp. nov., another ruminal bacterium utilizing succinate as the sole energy source.

Four strains of gram-negative, anaerobic, non-spore-forming bacteria that were curved rods which were motile by means of flagella originating from the concave side of the cells and which fermented succinate quantitatively to propionate were isolated from high dilutions of rumen ingesta obtained from cows on pasture. The bacteria were asaccharolytic and not proteolytic and did not ferment amino acids or peptides. Succinate was the only substrate fermented. Rumen fluid together with yeast extract was required for good growth on succinate. Growth on succinate was enhanced in the presence of fumarate. The strains did not grow at 22 degrees C, and growth at 45 degrees C was in all cases less than growth at 39 degrees C. The cellular fatty acid compositions of all four strains were determined. The DNA base composition was about 46 mol% G + C. The complete 16S ribosomal DNA sequence of the type strain (strain S1-1) was determined, and the phylogenetic relationships were analyzed. The most closely related genera were the genera Selenomonas, Zymophilus, and Pectinatus, whereas the recently described succinate-fermenting organism Succiniclasticum ruminis was distantly related. The name proposed for these strains is Schwartzia succinivorans gen. nov., sp. nov.; the type strain is strain S1-1 (= DSM 10502). These organisms are common inhabitants of the rumina of cows on pasture.

Clostridium pascui sp. nov., a new glutamate-fermenting sporeformer from a pasture in Pakistan.

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).