Purification and characterization of an extracellular protease produced by psychrotolerant Clostridium sp. LP3 from lake sediment of Leh, India

An anaerobic, proteolytic bacterium isolated from lake sediments of Leh, India, was characterized with respect to morphology, biochemical characteristics, and 16S rRNA sequence and was identified as Clostridium species, with closest similarity to Clostridium subterminale. Isolate LP3 was psychrophilic, forming maximum cell mass between 10 and 20 degrees C, and produced extracellular protease. Growth was observed in the pH range of 7.0-8.5, with optimum at pH 7.5. Protease was purified 62.4-fold with a total yield of 17.5%. The effects of temperature, pH, and salt concentration on enzyme activity were studied. Protease was found to be a serine-type metallo-enzyme, active in a broad range of pHs. It was thermolabile and resistant to sodium dodecyl sulfate. Enzyme kinetics showed a tendency to increase Km with an increase in temperature for casein substrate.     

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).     

Clostridium asparagiforme sp. nov., isolated from a human faecal sample

An obligatory anaerobic, Gram-positive, rod-shaped organism was isolated from faeces of a healthy human donor. It was characterized using biochemical, phenotypic and molecular taxonomic methods. The organism produced acetate, lactate, and ethanol as the major products of glucose fermentation. The G + C content was 53 mol%. Based on comparative 16S rRNA gene sequencing, the unidentified bacterium is a member of the Clostridium subphylum of the Gram-positive bacteria, and most closely related to species of the Clostridium coccoides cluster (rRNA cluster XIVa) [M.D. Collins et al., The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations, Int. J. Syst. Bacteriol. 44 (1994) 812-826]. Clostridium bolteae and Clostridium clostridioforme were identified as the most closely related described species. A 16S rRNA sequence divergence value of > 3% suggested that the isolate represents a new species. This was also supported by the gyrase-encoding gyrB gene sequences. Based on these findings, we propose the novel bacterium from human faeces to be classified as a new species, Clostridium asparagiforme. The type strain of C. asparagiforme is N6 (DSM 15981 and CCUG 48471).     

Clostridium bolteae sp. nov., isolated from human sources

Seven obligately anaerobic, gram-positive, rod-shaped, spore-forming organisms isolated from human sources were characterized using phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing showed that the strains were genetically highly related to each other (displaying >99% sequence similarity) and represent a previously unknown sub-line within the Clostridium coccoides rRNA group of organisms. Strains of the unidentified bacterium used carbohydrate as fermentable substrates, producing acetic acid and lactic acid as the major products of glucose metabolism. The closest described species to the novel bacterium corresponded to Clostridium clostridioforme, although a 16S rRNA sequence divergence of 3% demonstrated they represent different species. Genomic DNA-DNA pairing studies confirmed the separateness of the unknown species and Clostridium clostridioforme. Based on phenotypic and phylogenetic evidence, it is therefore proposed that the unknown bacterium, be classified as Clostridium bolteae sp. nov. The type strain of Clostridium bolteae is WAL 16351T (= ATCC(T) = BAA-613T, CCUG(T) = 46953T).     

Characterization of Clostridium sp. RKD producing botulinum-like neurotoxin

A Gram positive, motile, rod-shaped, strictly anaerobic bacterium isolated from intestine of decaying fish was identified as Clostridium sp. RKD and produced a botulinum type B-like neurotoxin as suggested by mouse bioassay and protection with anti botulinum antibodies. The neurotoxicity was functionally characterized by the phrenic nerve hemi-diaphragm assay. Phylogenetic analysis based on 16S rDNA sequence, placed it at a different position from the reported strains of Clostridium botulinum. The strain exhibited differences from both Clostridium botulinum and Clostridium tetani with respect to morphological, biochemical and chemotaxonomic characteristics. Botulinum group specific and serotype specific primers amplified the DNA fragments of 260 and 727 bp, respectively, indicating presence of botulinum type 'B' toxin gene. Sequence of nearly 700 bp amplified using primers specific for botulinum neurotoxin type B gene, did not show any significant match in the database when subjected to BLAST search.     

Clostridium bartlettii sp. nov., isolated from human faeces

Seven obligately anaerobic, Gram-positive, rod-shaped, spore-forming organisms isolated from human faecal specimens were characterized using phenotypic and molecular taxonomic methods. Strains of the unidentified bacterium used carbohydrates as fermentable substrates, producing acetic acid, isovaleric acid and phenylacetic acid (PAA) as the major products of glucose metabolism, and possessed a G +C content of approximately 29.8 mol%. Comparative 16S rRNA gene sequencing showed that the 7 strains were genetically highly related to each other (displaying >99.5% sequence similarity) and represent a previously unknown sub-line within the Clostridium Cluster XI. The closest described species to the novel bacterium is Clostridium glycolicum, although a 16S rRNA sequence divergence of 4% demonstrates that they represent different species. Genomic DNA-DNA pairing studies confirmed the separateness of the unknown species and C. glycolicum (30.6% similarity between the proposed type strain of the novel species, WAL 16138, and C. glycolicum ATCC 14880(T)). Based on morphologic, phenotypic and phylogenetic evidence, it is therefore proposed that the unknown bacterium be classified as C. bartlettii sp. nov. The type strain of C. bartlettii is WAL 16138(T) (= ATCCBAA-827(T)=CCUG48940(T)).     

A new nitrogen-fixing Clostridium species from a high Arctic ecosystem.

A hitherto undescribed species of yellow-pigmented, Gram-negative Clostridium sp., possessing nitrogenase activity, has been isolated from a number of sampling sites on the Truelove Lowland of Devon Island in the Canadian high Arctic. This bacterium, tentatively designated Clostridium arcticum sp. nov., accounted for 19% of all isolates recovered which were capable of anaerobic nitrogen fixation.     

Characterization of a new xylanolytic bacterium, Clostridium xylanovorans sp. nov.

A new xylanolytic bacterium designated strain HESP1T (T = type strain) was isolated from a methanogenic digester. Strain HESP1T was a motile, rod shaped, spore-forming bacterium, which possessed a Gram-positive type cell wall. Glucose, fructose, lactose, trehalose, maltose, raffinose, sucrose, xylan, mannitol, cellobiose, galactose, mannose, melibiose, ribose were fermented to produce, acetate, butyrate, H2, CO2, formate, isobutyrate, and ethanol. Fumarate was fermented to acetate. Glycerol and methanol were also utilized. Sulfate, thiosulfate, nitrate, sulfur and fumarate were not used as electron acceptors. Strain HESP1T had a G + C content of 40 mol% and grew optimally at 37 degrees C and pH 7 on a fructose containing medium. Phylogenetically, strain HESP1T was most related to Clostridium aminovalericum (similarity of 94%) than to C. populeti, C. herbivorans and Eubacterium xylanophilum (average similarity of 92%), all members of subcluster XIVa of the low G + C containing Gram-positive branch. However, strain HESP1T shared little phenotypic and genotypic traits with C. aminovalericum and on the basis of this and phylogenetic evidence, we propose to tentatively designate strain HESP1T as a new species of the genus Clostridium, Clostridium xylanovorans sp. nov. The type strain is HESP1T (= DSM 12503).     

The growth-promoting effect of Beijerinckia and Clostridium sp. cultures on some agricultural crops

New strains of Beijerinckia mobilis and Clostridium sp. isolated from the pea rhizosphere were studied with respect to their promoting effect on the growth and development of some agricultural crops. Seed soaking in bacterial suspensions followed by the soil application of the suspensions or their application by means of foliar spraying was found to be the most efficient method of bacterization. The application of B. mobilis and Clostridium sp. cultures in combination with mineral fertilizers increased the crop production by 1.5-2.5 times. The study of the population dynamics of B. mobilis by the method of genetic marking showed that this bacterium quickly colonized the rhizoplane of plants and, therefore, had characteristics of an r-strategist. At the same time, Clostridium sp. was closer to K-strategists, since this bacterium slowly colonized the econiches studied. The introduction of the bacteria into soil did not affect the indigenous soil bacterial complex. The presence of Clostridium sp. slowed down the colonization of roots by the fungal mycelium. The possible mechanisms of the plant growth-promoting activity of B. mobilis and Clostridium sp. are discussed.     

A novel keratinase from Clostridium sporogenes bv. pennavorans bv. nov., a thermotolerant organism isolated from solfataric muds

A bacterium which can grow on chicken feathers and which exhibits keratinolytic activity was isolated from solfataric muds. It was classified as belonging to the genus Clostridium and closely related to C. sporogenes. Based on its unique capability to degrade chicken feathers, it was designated as Clostridium sporogenes bv. pennavorans bv. nov. The keratinase purified from the culture supernatant is a monomer of 28.7kDa molecular mass. The enzyme is relatively thermostable and is active over a broad range of temperature and pH. Specific enzymatic assays demonstrate that keratinase can act on a large variety of soluble and insoluble protein substrates.     

丙酮丁醇梭菌磷酸化蛋白质组分析

近年的研究揭示,细菌细胞中蛋白质的磷酸化状态可能调节信号或代谢通路的生物活性。丙酮丁醇梭菌是一个重要的工业菌株,在酸性条件下能够生成大量的有机溶剂。然而,调节丙酮丁醇梭菌有机溶剂生成的分子机制尚未完全阐明。采用双向电泳和质谱联用的技术,比较了该菌在产酸期与产有机溶剂期间的差异蛋白质谱图。特别关注了那些分子量接近但具有不同等电点的蛋白质。在高有机溶剂生成速率的丙酮丁醇梭菌中,发现了8个电泳斑点簇呈现明显的酸移而且伴随光密度强度的变化。质谱分析数据表明,这些蛋白质均含有磷酸化修饰的肽段。生物信息学分析预示,这些蛋白质参与了有机溶剂的生成过程。但究竟它们的磷酸化状态如何调控有机溶剂生成仍需更为深入地研究。     

Comparison of the chitinolytic properties of Clostridium sp. strain 9.1 and a chitin-degrading bacterium from the intestinal tract of the plaice, Pleuronectes platessa (L.)

The chitinolytic properties of a facultatively anaerobic bacterium isolated from the hindgut of plaice were compared with those of Clostridium sp. strain 9.1, a bacterium isolated from anoxic estuarine sediment. The chitinolytic enzyme systems of the gut isolate and strain 9.1 both released N,N'-diacetylchitobiose (NAG2) as the major hydrolysis end-product. During the hydrolysis of chitin, there was transient accumulation of a non-sedimentary chitin fraction which was not detectable by high-performance liquid chromatography. Growth on NAG2 repressed chitinase synthesis in the gut isolate but not in the Clostridium species. Thiol reagents were strongly inhibitory to the chitinase of the strict anaerobe but did not affect the hydrolytic enzymes of the gut isolate. When the two bacteria were cocultured with chitin as the sole carbon and energy source, Clostridium sp. strain 9.1 was always outcompeted. Experiments with batch and phauxostat cultures showed that the competitiveness of strain 9.1 could be improved dramatically by the inclusion in the cocultures of a non-chitinolytic bacterium capable of fermenting chitin oligomers. The cooperation between the oligomer-fermenting species and the Clostridium sp. is discussed in relation to the regulation of chitinolytic activity in the latter organism.     

The F- or V-type Na(+)-ATPase of the thermophilic bacterium Clostridium fervidus.

Clostridium fervidus is a thermophilic, anaerobic bacterium which uses solely Na+ as a coupling ion for energy transduction. Important features of the primary Na+ pump (ATPase) that generates the sodium motive force are presented. The advantage of using a sodium rather than a proton motive force at high temperatures becomes apparent from the effect of temperature on H+ and Na+ permeation in liposomes.     

Physiological interactions between a mesophilic cellulolytic Clostridium and a non-cellulolytic bacterium

Abstract A mesophilic cellulolytic bacterium ( Clostridium strain C7) capable of N₂ fixation and a non-cellulolytic bacterium ( Klebsiella strain W1), both isolated from freshwater environments rich in decaying plant material, were co-cultured in a chemically defined, vitamin-deficient medium containing cellulose as the carbon and energy source. In the co-culture, an extracellular cellulase complex produced by the Clostridium hydrolyzed cellulose to soluble sugars that served as fermentable substrates for the Klebsiella . In turn, the Klebsiella excreted growth factors, identified as biotin and p -aminobenzoic acid, which were required by the Clostridium . Furthermore, demonstration of NH₄⁺-repressible acetylene reduction by co-cultures growing in medium lacking combined nitrogen showed that the Clostridium fixed N₂, thus allowing growth of the Klebsiella , which was not a nitrogen fixer. The mutualistic relationships observed in the co-cultures may be representative of interactions that take place in natural environments in which cellulose-containing plant materials are biodegraded.     

Enzymes Produced by a Pseudomonas Species Which Inactivate Inhibitors of Certain Viral Hemagglutinins. I. Identification and Purification of a Proteinase and Phospholipase C

Filtrates from cultures of a psychrophilic Pseudomonas species, which inactivate serum inhibitors of certain viral hemagglutinins, were shown to contain both lecithinase (phospholipase C) and a proteolytic enzyme with elastase activity. The bacterium was cultivated under conditions favoring production of the respective enzymes, and the enzymes were purified by ammonium sulfate precipitation followed by column chromatography or by gel filtration. The elastase was obtained in crystalline form and was recrystallized. It has properties similar to those of a number of other bacterial elastases but is more heat-labile than most. Although a high degree of purification was achieved for the lecithinase, as evidenced by an increase in specific activity, it was not obtained in crystalline form. Partially purified preparations of the lecithinase had extremely high activity compared to that of commercial preparations of phospholipase C from Clostridium welchii.     

Clostridium viride sp. nov., a strictly anaerobic bacterium using 5-aminovalerate as growth substrate,previously assigned to Clostridium aminovalericum

Strain T2–7, a 5-aminovalerate-fermenting bacterium previously classified as Clostridium aminovalericum, was further characterized, both physiologically and phylogenetically. Comparative sequencing analysis of the almost complete 16S rDNA revealed that strain T2–7 forms a distinct lineage within a phylogenetically coherent cluster of gram-positive bacteria currently assigned to the genus Clostridium. Strain T2–7 grew with 5-aminovalerate, 5-hydroxyvalerate, 4-hydroxybutyrate, vinylacetate, and crotonate, and required yeast extract and l-cysteine for growth. Other substrates were not utilized. The fermentation products, depending on the growth substrate, were ammonia, acetate, propionate, butyrate, and valerate. Sulphur was reduced by a mechanism not linked to energy conservation. Other acceptors were not utilized. Cells were gram-positive pointed-ended ovals, motile by means of two subpolar flagella, and possessed a gram-positive cell wall structure with an S-layer of hexagonally arranged subunits of 18.5 nm diameter. The DNA mol% G+C was 41.5. Strain T2–7 (DSM 6836) is proposed as the type strain of a new species, Clostridium viride sp. nov. Dedicated to H. A. Barker on the occasion of his 87th birthday     

Isolation of bacteriophage-like particles from uninduced Clostridium tetani cultures.

The isolation of hexagonal-headed, tailless, bacteriophage-like particles from uninduced cultures of Clostridium tetani is described. Clear, round, 1--3 mm diameter plaques were noted on Clostrisel agar plates, which were overlaid with soft agar inoculated with 7--14 day broth cultures. Particles were detected by transmission electron microscopy from broth cultures seeded with scrapings from the plaques. Both electron dense and electron lucent heads were noted. An electron dense head was observed attached to the surface of a dividing bacterium.     

A lithotrophic Clostridium strain with extremely thermoresistant spores isolated from a pectin-limited continuous culture of Clostridium thermosaccharolyticum strain Haren

Abstract A thermophilic Clostridium sp. with extremely thermoresistant spores was isolated from a pectin-limited continuous culture of Clostridium thermosaccharolyticum . The decimal reduction time of the spores was 70 min at 121°C. Because of the ability of the bacterium to grow both heterotrophically and lithotrophically, it has a potential for infecting a wide range of thermophilic anaerobic cultures.     

The abattoir source of culturable psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled venison

AIMS: To identify the abattoir source(s) of culturable psychrophilic clostridia causing 'blown pack' spoilage of vacuum-packed chilled meats. METHODS AND RESULTS: Psychrophilic and psychrotolerant clostridia were isolated from hides, faeces and tonsils of deer slaughter stock, and from a meat plant environment. The isolates were differentiated using restriction fragment length polymorphism analysis of the 16S rDNA gene (PCR-RFLP) and 16S-23S rDNA internal transcribed spacer (ITS) analysis. PCR-RFLP group I clostridia were found to have restriction patterns indistinguishable from the patterns of 'blown pack'-causing Clostridium gasigenes DB1A(T) and R26. Gas production in packs inoculated with vegetative cells of PCR-RFLP group I clostridia was first evident after 14 days at 2 degrees C. The prevalence of these clostridia was similar in hide and faecal samples from slaughter animals, but these micro-organisms were absent from tonsils and the meat plant environment. Banding patterns of PCR-RFLP group II clostridia showed some cross-similarity with patterns of the 'blown pack'-causing micro-organism Cl. estertheticum DSM 8809(T) and Cl. estertheticum-like meat strains. The majority of clostridia in PCR-RFLP group II were found in the faeces of slaughter animals. Isolates representing PCR-RFLP group II did not, however, produce gas in vacuum packs stored at 2 degrees C for 84 days. CONCLUSIONS: The data suggest that soil particles attached to hide or present in faeces are the most probable primary reservoir from which 'blown pack' clostridia are introduced onto carcasses. Therefore, dressing procedure hygiene remains paramount in order to control the spread of psychrophilic Clostridium spp. in a meat plant. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper provides information critical for controlling 'blown pack' spoilage in meat processing plants. It reports on the use of molecular techniques for determination of abattoir sources of 'blown pack'-causing clostridia.     

Novel psychrophilic anaerobic spore-forming bacterium from the overcooled water brine in permafrost: description Clostridium algoriphilum sp. nov.

A gram-positive, motile, strict anaerobic spore-forming bacterium was isolated from the over-cooled brine in the permafrost. The optimal temperature for isolate growth was 5-6 degrees C at pH 6.8-7.2. The bacterium was growing on the medium rich in saccharides and disaccharides. Out of polysaccharides tested, only xylan sustained the growth. Fermentation of the hexoses led to the formation of acetate, butyrate, lactate, H2,CO2 and some formate and ethanol. Cell wall peptidoglycan contained meso-diaminopimelic acid. The major fatty acids of the cell wall were C(14:0) and C(16:1c9). The content of G-C pairs in DNA was 31.4 mol%. As phylogenetic analysis has shown, it is closely linked to the members of cluster 1 of Clostridium. It differs from the other species of the genus by the substrates necessary for the growth, products forming as a result of the fermentation and content of the fatty acids in the cell wall. Thus, it was suggested to describe this strain as a new species named Clostridium algoriphilum. Type strain 14D1 was deposited into the Russian Collection of the Microorganisms VKM B-2271T and German Collection of the Microorganisms DSM 16153T .