Romboutsia sedimentorum sp. nov., isolated from an alkaline-saline lake sediment and emended description of the genus Romboutsia.

A Gram-stain-positive, spore-forming, obligately anaerobic bacterium, designated LAM201(T), was isolated from sediment samples from an alkaline-saline lake located in Daqing oilfield, Daqing City, PR China. Cells of strain LAM201(T) were non-motile and straight or spiral rod-shapes. Strain LAM201(T) was able to utilize glucose, fructose, maltose, trehalose and sorbitol as the sole carbon source. Acetic acid, ethanol, iso-butanoic acid and iso-valeric acid were the main products of glucose fermentation. The major fatty acids of LAM201(T) were C(16 : 0) (26.7%) and C(18 : 0) (11.2%). The main polar lipids were four unknown glycolipids and five unknown phospholipids. The predominant cell-wall sugars were ribose and galactose. The cell-wall peptidoglycan of strain LAM201(T) contained alanine, glycine, glutamic acid and aspartic acid. Sodium sulfite was used as the electron acceptor. The G+C content of the genomic DNA was 32±0.8 mol%, as determined by the T(m) method. Analysis of the 16S rRNA gene sequence indicated that the isolate belonged to the genus Romboutsia and was most closely related to Romboutsia lituseburensis DSM 797(T) and Romboutsia ilealis CRIB(T) with 97.3% and 97.2% similarities, respectively. The DNA-DNA hybridization values between strain LAM201(T) and the two reference strains were 37% and 31%, respectively. On the basis of its phenotypic, phylogenetic and chemotaxonomic characteristics, strain LAM201(T) is suggested to represent a novel species within the genus Romboutsia , for which the name Romboutsia sedimentorum sp. nov. is proposed. The type strain is LAM201(T) ( = ACCC 00717(T) = JCM 19607(T)).

Comparison of environmental and isolate Sulfobacillus genomes reveals diverse carbon, sulfur, nitrogen, and hydrogen metabolisms.

BACKGROUND: Bacteria of the genus Sulfobacillus are found worldwide as members of microbial communities that accelerate sulfide mineral dissolution in acid mine drainage environments (AMD), acid-rock drainage environments (ARD), as well as in industrial bioleaching operations. Despite their frequent identification in these environments, their role in biogeochemical cycling is poorly understood. RESULTS: Here we report draft genomes of five species of the Sulfobacillus genus (AMDSBA1-5) reconstructed by cultivation-independent sequencing of biofilms sampled from the Richmond Mine (Iron Mountain, CA). Three of these species (AMDSBA2, AMDSBA3, and AMDSBA4) have no cultured representatives while AMDSBA1 is a strain of S. benefaciens, and AMDSBA5 a strain of S. thermosulfidooxidans. We analyzed the diversity of energy conservation and central carbon metabolisms for these genomes and previously published Sulfobacillus genomes. Pathways of sulfur oxidation vary considerably across the genus, including the number and type of subunits of putative heterodisulfide reductase complexes likely involved in sulfur oxidation. The number and type of nickel-iron hydrogenase proteins varied across the genus, as does the presence of different central carbon pathways. Only the AMDSBA3 genome encodes a dissimilatory nitrate reducatase and only the AMDSBA5 and S. thermosulfidooxidans genomes encode assimilatory nitrate reductases. Within the genus, AMDSBA4 is unusual in that its electron transport chain includes a cytochrome bc type complex, a unique cytochrome c oxidase, and two distinct succinate dehydrogenase complexes. CONCLUSIONS: Overall, the results significantly expand our understanding of carbon, sulfur, nitrogen, and hydrogen metabolism within the Sulfobacillus genus.

Response of the methanogenic microbial communities in Amazonian oxbow lake sediments to desiccation stress.

Methanogenic microbial communities in soil and sediment function only when the environment is inundated and anoxic. In contrast to submerged soils, desiccation of lake sediments happens only rarely. However, some predictions suggest that extreme events of drying will become more common in the Amazon region, and this will promote an increase in sediments drying and exposure. We asked whether and how such methanogenic communities can withstand desiccation stress. Therefore, we determined the rates and pathways of CH(4) production (analysis of CH(4) and δ(13) C of CH(4), CO(2) and acetate), the copy numbers of bacterial and archaeal 16S rRNA genes and mcrA genes (quantitative PCR), and the community composition of Archaea and Bacteria (T-RFLP and pyrosequencing) in oxbow lake sediments of rivers in the Brazilian Amazon region. The rivers were of white water, black water and clear water type. The measurements were done with sediment in fresh state and after drying and rewetting. After desiccation and rewetting the composition of both, the archaeal and bacterial community changed. Since lake sediments from white water rivers exhibited only negligible methanogenic activity, probably because of relatively high iron and low organic matter content, they were not further analysed. The other sediments produced CH(4), with hydrogenotrophic methanogenesis usually accounting for > 50% of total activity. After desiccation and rewetting, archaeal and bacterial gene copy numbers decreased. The bacterial community showed a remarkable increase of Clostridiales from about 10% to > 30% of all Bacteria, partially caused by proliferation of specific taxa as the numbers of OTU shared with fresh sediment decreased from about 9% to 3%. Among the Archaea, desiccation specifically enhanced the relative abundance of either Methanocellales (black water) and/or Methanosarcinaceae (clear water). Despite the changes in gene copy numbers and composition of the microbial community, rates of CH(4) production even increased after desiccation-rewetting, demonstrating that the function of the methanogenic microbial community had not been impaired. This result indicates that the increase in extreme events of drying may increase methane production in flooded sediments.

Hungatella effluvii gen. nov., sp. nov., an obligately anaerobic bacterium isolated from an effluent treatment plant, and reclassification of Clostridium hathewayi as Hungatella hathewayi gen. nov., comb. nov.

A Gram-stain-positive, rod-shaped, spore-forming and strictly anaerobic bacterium, designated UB-B.2(T), was isolated from an industrial effluent anaerobic digester sample. It grew optimally at 30 °C and pH 7.0. Comparative analysis of the 16S rRNA gene sequence confirmed that strain UB-B.2(T) was closely related to Clostridium hathewayi DSM 13479(T) (97.84% similarity), a member of rRNA gene cluster XIVa of the genus Clostridium, and formed a coherent cluster with other related members of the Blautia (Clostridium) coccoides rRNA group in phylogenetic analyses. The end products of glucose fermentation by strain UB-B.2(T) were acetate and propionate. The G+C content of the DNA was 51.4 mol%. Although strain UB-B.2(T) showed 97.8% 16S rRNA gene sequence identity to the type strain of C. hathewayi, it exhibited only 38.4% relatedness at the whole-genome level. It also showed differences from its closest phylogenetic relative, C. hathewayi DSM 13479(T), in phenotypic characteristics such as hydrolysis of aesculin, starch and urea and fermentation end products. Both strains showed phenotypic differences from the members of rRNA gene cluster XIVa of the genus Clostridium. Based on these differences, C. hathewayi DSM 13479(T) and strain UB-B.2(T) were identified as representatives of a new genus of the family Clostridiaceae. Thus, we propose the reclassification of Clostridium hathewayi as Hungatella hathewayi gen. nov., comb. nov., the type species of the new genus (type strain DSM 13479(T) = CCUG 43506(T) = MTCC 10951(T)). Strain UB-B.2(T) ( = MTCC 11101(T) = DSM 24995(T)) is assigned to the novel species Hungatella effluvii gen. nov., sp. nov as the type strain.

Anaerostipes rhamnosivorans sp. nov., a human intestinal, butyrate-forming bacterium.

A novel butyrate-producing bacterium, strain 1y-2(T), was isolated from a stool sample of a 1-year-old, healthy Dutch infant. The isolate was obtained by using lactate and acetate as sources of carbon and energy. The strain was Gram-variable, strictly anaerobic and spore-forming and formed curly rod-shaped cells that fermented glucose into butyrate, lactate, formate and acetate as main products. The DNA G+C content of the strain was 44.5 mol% and its major cellular fatty acids were C12:0, iso-C19:1 I and C16:0. Strain 1y-2(T) was related to Anaerostipes caccae DSM 14662(T) based on 16S rRNA gene sequence analysis, with 3% divergence, but hybridization studies of their genomic DNA revealed only 33% relatedness. Moreover, strain 1y-2(T) showed marked physiological and biochemical differences from known species of the genus Anaerostipes. Based on phylogenetic, chemotypic and phenotypic criteria, we propose that strain 1y-2(T) should be classified in the genus Anaerostipes within a novel species, Anaerostipes rhamnosivorans sp. nov. The type strain is 1y-2(T) ( = DSM 26241(T) = KCTC 15316(T)).

Description of Symbiobacterium ostreiconchae sp. nov., Symbiobacterium turbinis sp. nov. and Symbiobacterium terraclitae sp. nov., isolated from shellfish, emended description of the genus Symbiobacterium and proposal of Symbiobacteriaceae fam. nov.

Three novel moderately anaerobic, thermophilic, rod-shaped bacterial strains, KY38(T), KY46(T) and KA13(T), were isolated from shellfish collected on the Pacific coastline of Enoshima, Japan. Phylogenetic analysis of the 16S rRNA gene sequences indicated that these bacteria belong to the genus Symbiobacterium, sharing sequence similarities of 97.8% (KY38(T)), 96.4% (KY46(T)) and 93.3% (KA13(T)) with the type strain of Symbiobacterium thermophilum, the only species of the genus with a validly published name. These isolates reduced nitrate and grew optimally at 55-60 °C. Strains KY38(T) and KA13(T) formed endospore-like structures in the terminal or subterminal part of their cells at low frequencies. Genomic DNA G+C contents were 68.8 (KY38(T)), 67.2 (KY46(T)) and 67.1 (KA13(T)) mol%. The isolates all presented the predominant menaquinone MK-6, major fatty acids iso-C15:0, C16:0 and iso-C17:0 and the major polar lipids phosphatidylglycerol, phosphatidylethanolamine and unknown glycol-containing phospholipids. On the basis of their morphological, physiological and phylogenetic properties, strains KY38(T), KY46(T) and KA13(T) represent three novel species, for which the names Symbiobacterium ostreiconchae sp. nov. (type strain KY38(T) = DSM 27624(T) = KCTC 4567(T) = JCM 15048(T)), Symbiobacterium turbinis sp. nov. (type strain KY46(T) = DSM 27625(T) = KCTC 4568(T) = JCM 15996(T)) and Symbiobacterium terraclitae sp. nov. (type strain KA13(T) = DSM 27138(T) = KCTC 4569(T) = JCM 15997(T)) are proposed. An emended description of the genus Symbiobacterium is also presented. The phylogenetic distinctiveness of the genus Symbiobacterium indicates its affiliation with a novel family, for which the name Symbiobacteriaceae fam. nov. is proposed.

Aneurinibacillus soli sp. nov., isolated from mountain soil.

A novel bacterial strain designated CB4(T) was isolated from soil from the Hallasan, Jeju, Korea. Strain CB4(T) was found to be strictly aerobic, Gram-stain-positive, rod-shaped, motile and formed creamy greyish colonies on nutrient agar. The major fatty acids were identified as iso-C(15:0) and iso-C(16:0), and the predominant isoprenoid quinone as MK-7. The cell-wall peptidoglycan contained glycine and alanine as the diagnostic amino acids and phosphatidyl-N-methylethanolamine, phosphatidylethanolamine, diphosphatidylglycerol and an unidentified aminophospholipid as the polar lipids. The genomic DNA G+C content of strain CB4(T) was 46.5 mol%. Phylogenetic analysis, based on 16S rRNA gene sequence similarities, showed that strain CB4(T) forms a deep branch within the genus Aneurinibacillus, sharing the highest level of sequence homology with Aneurinibacillus aneurinilyticus DSM 5562(T) (96.5%). On the basis of the phenotypic, chemotaxonomic and phylogenetic characteristics, strain CB4(T) is considered to represent a novel species within the genus Aneurinibacillus, for which the name Aneurinibacillus soli sp. nov. is proposed. The type strain is CB4(T) ( =KCTC 33505(T) =CECT 8566(T)). An emended description of the genus Aneurinibacillus is also proposed.

Mobilitalea sibirica gen. nov., sp. nov., a halotolerant polysaccharide-degrading bacterium.

A novel strictly anaerobic, halotolerant, organotrophic bacterium, strain P3M-3(T), was isolated from a microbial mat formed under the flow of hot water emerging from a 2775 m-deep well in Tomsk region (western Siberia, Russia). Cells of strain P3M-3(T) were straight and curved rods, 0.2-0.4 µm in width and 1.5-20 µm in length. Strain P3M-3(T) grew optimally at 37 °C, pH 7.0-7.5 and in a NaCl concentration of 15 g l(-1). Under optimum growth conditions, the doubling time was 1 h. The isolate was able to ferment a variety of mono-, di- and polysaccharides, including microcrystalline cellulose. Acetate, ethanol, H2 and CO2 were the main products of glucose fermentation. The DNA G+C content was 33.4 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain P3M-3(T) was a member of family Lachnospiraceae, whose representatives are also found in Clostridium cluster XIVa. 16S rRNA gene sequence similarity with Clostridium jejuense HY-35-12(T), the closest relative, was 93.9%. A novel genus and species, Mobilitalea sibirica gen. nov., sp. nov., are proposed based on phylogenetic analysis and physiological properties of the novel isolate. The type strain of the type species is P3M-3(T) ( = DSM 26468(T) = VKM B-2804(T)).

Characterization of Romboutsia ilealis gen. nov., sp. nov., isolated from the gastro-intestinal tract of a rat, and proposal for the reclassification of five closely related members of the genus Clostridium into the genera Romboutsia gen. nov., Intestinibacter gen. nov., Terrisporobacter gen. nov. and Asaccharospora gen. nov.

A Gram-positive staining, rod-shaped, non-motile, spore-forming obligately anaerobic bacterium, designated CRIBT, was isolated from the gastro-intestinal tract of a rat and characterized. The major cellular fatty acids of strain CRIBT were saturated and unsaturated straight-chain C12-C19 fatty acids, with C16:0 being the predominant fatty acid. The polar lipid profile comprised six glycolipids, four phospholipids and one lipid that did not stain with any of the specific spray reagents used. The only quinone was MK-6. The predominating cell-wall sugars were glucose and galactose. The peptidoglycan type of strain CRIBT was A1σ lanthionine-direct. The genomic DNA G+C content of strain CRIBT was 28.1 mol%. On the basis of 16S rRNA gene sequence similarity, strain CRIBT was most closely related to a number of species of the genus Clostridium, including Clostridium lituseburense (97.2%), Clostridium glycolicum (96.2%), Clostridium mayombei (96.2%), Clostridium bartlettii (96.0%) and Clostridium irregulare (95.5%). All these species show very low 16S rRNA gene sequence similarity (<85%) to the type strain of Clostridium butyricum, the type species of the genus Clostridium. DNA-DNA hybridization with closely related reference strains indicated reassociation values below 32%. On the basis of phenotypic and genetic studies, a novel genus, Romboutsia gen. nov., is proposed. The novel isolate CRIBT (=DSM 25109T=NIZO 4048T) is proposed as the type strain of the type species, Romboutsia ilealis gen. nov., sp. nov., of the proposed novel genus. It is proposed that C. lituseburense is transferred to this genus as Romboutsia lituseburensis comb. nov. Furthermore, the reclassification into novel genera is proposed for C. bartlettii, as Intestinibacter bartlettii gen. nov., comb. nov. (type species of the genus), C. glycolicum, as Terrisporobacter glycolicus gen. nov., comb. nov. (type species of the genus), C. mayombei, as Terrisporobacter mayombei gen. nov., comb. nov., and C. irregulare, as Asaccharospora irregularis gen. nov., comb. nov. (type species of the genus), on the basis of additional data collected in this study. In addition, an emendation of the species Peptostreptococcus anaerobius and the order Eubacteriales is provided.

Substrate-driven gene expression in Roseburia inulinivorans: importance of inducible enzymes in the utilization of inulin and starch.

Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene-expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide/inulin utilization genes induced during growth on inulin included one encoding a ß-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, whereas a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis showed that the ß-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked sugar phosphotransferase transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the use of inulin. The R. inulinivorans ß-fructofuranosidase was overexpressed in Escherichia coli and shown to hydrolyze fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosaccharides. Genes induced on starch included the major extracellular α-amylase and two distinct α-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles.

Cloning, expression and characterization of a novel thermophilic polygalacturonase from Caldicellulosiruptor bescii DSM 6725.

We cloned the gene ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3). After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA). The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polygalacturonic acid (PGA), and released monogalacturonic acid as its sole product. The Vmax and Km of CbPelA were 384.6 U·mg⁻¹ and 0.31 mg·mL⁻¹, respectively. CbPelA was also able to hydrolyze methylated pectin (48% and 10% relative activity on 20%-34% and 85% methylated pectin, respectively). The high thermo-activity and methylated pectin hydrolization activity of CbPelA suggest that it has potential applications in the food and textile industry.

Butyricicoccus pullicaecorum in inflammatory bowel disease.

OBJECTIVE: Many species within the phylum Firmicutes are thought to exert anti-inflammatory effects. We quantified bacteria belonging to the genus Butyricicoccus in stools of patients with ulcerative colitis (UC) and Crohn's disease (CD). We evaluated the effect of Butyricicoccus pullicaecorum in a rat colitis model and analysed the ability to prevent cytokine-induced increases in epithelial permeability. DESIGN: A genus-specific quantitative PCR was used for quantification of Butyricicoccus in stools from patients with UC or CD and healthy subjects. The effect of B pullicaecorum on trinitrobenzenesulfonic (TNBS)-induced colitis was assessed and the effect of B pullicaecorum culture supernatant on epithelial barrier function was investigated in vitro. RESULTS: The average number of Butyricicoccus in stools from patients with UC and CD in active (UC: 8.61 log10/g stool; CD: 6.58 log10/g stool) and remission phase (UC: 8.69 log10/g stool; CD: 8.38 log10/g stool) was significantly lower compared with healthy subjects (9.32 log10/g stool) and correlated with disease activity in CD. Oral administration of B pullicaecorum resulted in a significant protective effect based on macroscopic and histological criteria and decreased intestinal myeloperoxidase (MPO), tumour necrosis factor α (TNFα) and interleukin (IL)-12 levels. Supernatant of B pullicaecorum prevented the loss of transepithelial resistance (TER) and the increase in IL-8 secretion induced by TNFα and interferon γ (IFN gamma) in a Caco-2 cell model. CONCLUSIONS: Patients with inflammatory bowel disease have lower numbers of Butyricicoccus bacteria in their stools. Administration of B pullicaecorum attenuates TNBS-induced colitis in rats and supernatant of B pullicaecorum cultures strengthens the epithelial barrier function by increasing the TER.

Biochemical and structural insights into xylan utilization by the thermophilic bacterium Caldanaerobius polysaccharolyticus.

Hemicellulose is the next most abundant plant cell wall component after cellulose. The abundance of hemicellulose such as xylan suggests that their hydrolysis and conversion to biofuels can improve the economics of bioenergy production. In an effort to understand xylan hydrolysis at high temperatures, we sequenced the genome of the thermophilic bacterium Caldanaerobius polysaccharolyticus. Analysis of the partial genome sequence revealed a gene cluster that contained both hydrolytic enzymes and also enzymes key to the pentose-phosphate pathway. The hydrolytic enzymes in the gene cluster were demonstrated to convert products from a large endoxylanase (Xyn10A) predicted to anchor to the surface of the bacterium. We further use structural and calorimetric studies to demonstrate that the end products of Xyn10A hydrolysis of xylan are recognized and bound by XBP1, a putative solute-binding protein, likely for transport into the cell. The XBP1 protein showed preference for xylo-oligosaccharides as follows: xylotriose > xylobiose > xylotetraose. To elucidate the structural basis for the oligosaccharide preference, we solved the co-crystal structure of XBP1 complexed with xylotriose to a 1.8-Å resolution. Analysis of the biochemical data in the context of the co-crystal structure reveals the molecular underpinnings of oligosaccharide length specificity.

Pullulanibacillus uraniitolerans sp. nov., an acidophilic, U(VI)-resistant species isolated from an acid uranium mill tailing effluent and emended description of the genus Pullulanibacillus.

Two Gram-positive-staining, rod-shaped, endospore-forming isolates (UG-2(T) and UG-3), with an optimum growth temperature of around 37 °C and an optimum pH for growth of about 4, were recovered from an acidic effluent of the uranium mill tailing at Urgeiriça in Central Portugal. On the basis of 16S rRNA gene sequence similarity, the strains belonged to the family Sporolactobacillaceae and were closely related to Pullulanibacillus naganoensis ATCC 53909(T) (97.9 %). Unlike P. naganoensis, strains UG-2(T) and UG-3 grew in medium containing up to 5000 p.p.m. U(VI) but did not hydrolyse pullulan. Chemotaxonomic data also supported the affiliation of strains UG-2(T) and UG-3 to the genus Pullulanibacillus. Physiological and biochemical tests along with fatty acid composition allowed differentiation of strains UG-2(T) and UG-3 from P. naganoensis. It is suggested that strains UG-2(T) and UG-3 represent a novel species, for which the name Pullulanibacillus uraniitolerans is proposed; the type strain is UG-2(T) (=DSM 19429(T) = LMG 24205(T)). An emended description of the genus Pullulanibacillus is also proposed.

Natranaerobaculum magadiense gen. nov., sp. nov., an anaerobic, alkalithermophilic bacterium from soda lake sediment.

An obligately alkaliphilic, anaerobic, thermo- and halotolerant, spore-forming bacterium was isolated from sediments of soda lake Magadi (Kenya) and designated strain Z-1001(T). Cells of strain Z-1001(T) were straight, Gram-positive rods, slowly motile. Strain Z-1001(T) was found to be an obligate anaerobe. It grew within a pH range from 7.5 to 10.7 with an optimum at 9.25-9.5 (at 40 °C), a temperature range from 20 to 57 °C with an optimum at 45-50 °C, and a NaCl concentration range from 0 to 1.55 M with an optimum at 1.2-1.4 M. Peptides, such as meat and yeast extracts, peptone and tryptone, were fermented by Z-1001(T). Carbohydrates did not support growth. With yeast extract as an electron donor, strain Z-1001(T) reduced S(2)O(3)(2-), NO(-)(3), AsO(3-)(4), Fe(III) citrate and anthraquinone-2,6-disulfonate (AQDS) as electron acceptors. The isolate was able to grow oligotrophically with a very small amount of yeast extract: 0.03 g l(-1). The main fatty acids were C16 : 0, C16 : 1ω7c, C18 : 0 and C18 : 1ω9. The DNA G+C content of the isolate was 35.6 mol%. 16S rRNA gene sequence analysis showed that strain Z-1001(T) is a member of family Natranaerobiaceae, clustering with the type strain of Natranaerobius thermophilus (95.8-96.0 % sequence similarity). On the basis of physiological and phylogenetic data it is proposed that strain Z-1001(T) ( = DSM 24923(T) = VKM B-2666(T)) represents a novel genus and species, Natranaerobaculum magadiense gen. nov., sp. nov.

Intestinimonas butyriciproducens gen. nov., sp. nov., a butyrate-producing bacterium from the mouse intestine.

A Gram-positive, spore-forming, non-motile, strictly anaerobic rod-shaped bacterium was isolated from the caecal content of a TNF(deltaARE) mouse. The isolate, referred to as strain SRB-521-5-I(T), was originally cultured on a reduced agar medium containing yeast extract, rumen fluid and lactic acid as main energy and carbon sources. Phylogenetic analysis of partial 16S rRNA genes revealed that the species most closely related to strain SRB-521-5-I(T) were Flavonifractor plautii and Pseudoflavonifractor capillosus (<95 % sequence similarity; 1436 bp). In contrast to F. plautii and P. capillosus, strain SRB-521-5-I(T) contained a substantial amount of C18 : 0 dimethylacetal. Additional major fatty acids were C14 : 0 methyl ester, C16 : 0 dimethylacetal and C18 : 0 aldehyde. Strain SRB-521-5-I(T) differed in its enzyme profile from F. plautii and P. capillosus by being positive for dextrin, maltotriose, turanose, dl-lactic acid and d-lactic acid methyl ester but negative for d-fructose. In reduced Wilkins-Chalgren-Anaerobe broth, strain SRB-521-5-I(T) produced approximately 8 mM butyrate and 4 mM acetate. In contrast to F. plautii, the strain did not metabolize flavonoids. It showed intermediate resistance towards the antibiotics ciprofloxacin, colistin and tetracycline. Based on genotypic and phenotypic characteristics, we propose the name Intestinimonas butyriciproducens gen. nov., sp. nov. to accommodate strain SRB-521-5-I(T) ( = DSM 26588(T) = CCUG 63529(T)) as the type strain.

Oceanirhabdus sediminicola gen. nov., sp. nov., an anaerobic bacterium isolated from sea sediment.

A novel anaerobic bacterium, designated NH-JN4(T) was isolated from a sediment sample collected in the South China Sea. Cells were Gram-stain-positive, spore-forming, peritrichous and rod-shaped (0.5-1.2×2.2-7 µm). The temperature and pH ranges for growth were 22-42 °C and pH 6.0-8.5. Optimal growth occurred at 34-38 °C and pH 6.5-7.0. The NaCl concentration range for growth was 0.5-6 % (w/v) with an optimum of 2.5 %. Catalase and oxidase were not produced. Substrates which could be utilized were peptone, tryptone, yeast extract, beef extract and glycine. Main fermentation products from PYG medium were formate, acetate, butyrate and ethanol. Strain NH-JN4(T) could utilize sodium sulfite as an electron acceptor. No respiratory quinone was detected. The predominant fatty acids were anteiso-C15 : 0, C16 : 0, iso-C15 : 0, anteiso-C17 : 0 and C16 : 0 DMA. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and glycolipids. The DNA G+C content was 35.8 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain NH-JN4(T) was a member of family Clostridiaceae, and was most closely related to Clostridium limosum ATCC 25620(T), Clostridium proteolyticum DSM 3090(T), Clostridium histolyticum ATCC 19401(T) and Clostridium tepidiprofundi SG 508(T), showing 94.0, 93.0, 92.9 and 92.3 % sequence similarity, respectively. On the basis of phenotypic, genotypic and chemotaxonomic properties, strain NH-JN4(T) represents a novel species of a new genus in the family Clostridiaceae, for which the name Oceanirhabdus sediminicola gen. nov., sp. nov. is proposed. The type strain of the type species is NH-JN4(T) ( = JCM 18501(T) = CCTCC AB 2013103(T) = KCTC 15322(T)).

Reconstructing a chloride-binding site in a bacterial neurotransmitter transporter homologue.

In ion-coupled transport proteins, occupation of selective ion-binding sites is required to trigger conformational changes that lead to substrate translocation. Neurotransmitter transporters, targets of abused and therapeutic drugs, require Na(+) and Cl(-) for function. We recently proposed a chloride-binding site in these proteins not present in Cl(-)-independent prokaryotic homologues. Here we describe conversion of the Cl(-)-independent prokaryotic tryptophan transporter TnaT to a fully functional Cl(-)-dependent form by a single point mutation, D268S. Mutations in TnaT-D268S, in wild type TnaT and in serotonin transporter provide direct evidence for the involvement of each of the proposed residues in Cl(-) coordination. In both SERT and TnaT-D268S, Cl(-) and Na(+) mutually increased each other's potency, consistent with electrostatic interaction through adjacent binding sites. These studies establish the site where Cl(-) binds to trigger conformational change during neurotransmitter transport.

Suppression of Clostridium difficile in the gastrointestinal tracts of germfree mice inoculated with a murine isolate from the family Lachnospiraceae.

The indigenous microbial community of the gastrointestinal (GI) tract determines susceptibility to Clostridium difficile colonization and disease. Previous studies have demonstrated that antibiotic-treated mice challenged with C. difficile either developed rapidly lethal C. difficile infection or were stably colonized with mild disease. The GI microbial community of animals with mild disease was dominated by members of the bacterial family Lachnospiraceae, while the gut community in moribund animals had a predominance of Escherichia coli. We investigated the roles of murine Lachnospiraceae and E. coli strains in colonization resistance against C. difficile in germfree mice. Murine Lachnospiraceae and E. coli isolates were cultured from wild-type mice. The ability of each of these isolates to interfere with C. difficile colonization was tested by precolonizing germfree mice with these bacteria 4 days prior to experimental C. difficile challenge. Mice precolonized with a murine Lachnospiraceae isolate, but not those colonized with E. coli, had significantly decreased C. difficile colonization, lower intestinal cytotoxin levels and exhibited less severe clinical signs and colonic histopathology. Infection of germfree mice or mice precolonized with E. coli with C. difficile strain VPI 10463 was uniformly fatal by 48 h, but only 20% mortality was seen at 2 days in mice precolonized with the Lachnospiraceae isolate prior to challenge with VPI 10463. These findings confirm that a single component of the GI microbiota, a murine Lachnospiraceae isolate, could partially restore colonization resistance against C. difficile. Further study of the members within the Lachnospiraceae family could lead to a better understanding of mechanisms of colonization resistance against C. difficile and novel therapeutic approaches for the treatment and prevention of C. difficile infection.

Genome-guided analysis of physiological and morphological traits of the fermentative acetate oxidizer Thermacetogenium phaeum.

BACKGROUND: Thermacetogenium phaeum is a thermophilic strictly anaerobic bacterium oxidizing acetate to CO(2) in syntrophic association with a methanogenic partner. It can also grow in pure culture, e.g., by fermentation of methanol to acetate. The key enzymes of homoacetate fermentation (Wood-Ljungdahl pathway) are used both in acetate oxidation and acetate formation. The obvious reversibility of this pathway in this organism is of specific interest since syntrophic acetate oxidation operates close to the energetic limitations of microbial life. RESULTS: The genome of Th. phaeum is organized on a single circular chromosome and has a total size of 2,939,057 bp. It comprises 3.215 open reading frames of which 75% could be assigned to a gene function. The G+C content is 53.88 mol%. Many CRISPR sequences were found, indicating heavy phage attack in the past. A complete gene set for a phage was found in the genome, and indications of phage action could also be observed in culture. The genome contained all genes required for CO(2) reduction through the Wood-Ljungdahl pathway, including two formyl tetrahydrofolate ligases, three carbon monoxide dehydrogenases, one formate hydrogenlyase complex, three further formate dehydrogenases, and three further hydrogenases. The bacterium contains a menaquinone MQ-7. No indications of cytochromes or Rnf complexes could be found in the genome. CONCLUSIONS: The information obtained from the genome sequence indicates that Th. phaeum differs basically from the three homoacetogenic bacteria sequenced so far, i.e., the sodium ion-dependent Acetobacterium woodii, the ethanol-producing Clostridium ljungdahlii, and the cytochrome-containing Moorella thermoacetica. The specific enzyme outfit of Th. phaeum obviously allows ATP formation both in acetate formation and acetate oxidation.