Methanomethylophilus alvi gen. nov., sp. nov., a Novel Hydrogenotrophic Methyl-Reducing Methanogenic Archaea of the Order Methanomassiliicoccales Isolated from the Human Gut and Proposal of the Novel Family Methanomethylophilaceae fam. nov.

The methanogenic strain Mx-05T was isolated from the human fecal microbiome. A phylogenetic analysis based on the 16S rRNA gene and protein marker genes indicated that the strain is affiliated with the order Methanomassiliicoccales. It shares 86.9% 16S rRNA gene sequence identity with Methanomassiliicoccus luminyensis, the only member of this order previously isolated. The cells of Mx-05T were non-motile cocci, with a diameter range of 0.4-0.7 µm. They grew anaerobically and reduced methanol, monomethylamine, dimethylamine, and trimethylamine into methane, using H2 as an electron donor. H2/CO2, formate, ethanol, and acetate were not used as energy sources. The growth of Mx-05T required an unknown medium factor(s) provided by Eggerthella lenta and present in rumen fluid. Mx-05T grew between 30 °C and 40 °C (optimum 37 °C), over a pH range of 6.9-8.3 (optimum pH 7.5), and between 0.02 and 0.34 mol.L-1 NaCl (optimum 0.12 mol.L-1 NaCl). The genome is 1.67 Mbp with a G+C content of 55.5 mol%. Genome sequence annotation confirmed the absence of the methyl branch of the H4MPT Wood-Ljungdahl pathway, as described for other Methanomassiliicoccales members. Based on an average nucleotide identity analysis, we propose strain Mx-05T as being a novel representative of the order Methanomassiliicoccales, within the novel family Methanomethylophilaceae, for which the name Methanomethylophilus alvi gen. nov, sp. nov. is proposed. The type strain is Mx-05T (JCM 31474T).

Growth temperature and chromatinization in archaea.

DNA in cells is associated with proteins that constrain its structure and affect DNA-templated processes including transcription and replication. HU and histones are the main constituents of chromatin in bacteria and eukaryotes, respectively, with few exceptions. Archaea, in contrast, have diverse repertoires of nucleoid-associated proteins (NAPs). To analyse the evolutionary and ecological drivers of this diversity, we combined a phylogenomic survey of known and predicted NAPs with quantitative proteomic data. We identify the Diaforarchaea as a hotbed of NAP gain and loss, and experimentally validate candidate NAPs in two members of this clade, Thermoplasma volcanium and Methanomassiliicoccus luminyensis. Proteomic analysis across a diverse sample of 19 archaea revealed that NAP investment varies from <0.03% to >5% of total protein. This variation is predicted by growth temperature. We propose that high levels of chromatinization have evolved as a mechanism to prevent uncontrolled helix denaturation at higher temperatures, with implications for the origin of chromatin in both archaea and eukaryotes.

Lentils and Yeast Fibers: A New Strategy to Mitigate Enterotoxigenic Escherichia coli (ETEC) Strain H10407 Virulence?

Dietary fibers exhibit well-known beneficial effects on human health, but their anti-infectious properties against enteric pathogens have been poorly investigated. Enterotoxigenic Escherichia coli (ETEC) is a major food-borne pathogen that causes acute traveler's diarrhea. Its virulence traits mainly rely on adhesion to an epithelial surface, mucus degradation, and the secretion of two enterotoxins associated with intestinal inflammation. With the increasing burden of antibiotic resistance worldwide, there is an imperious need to develop novel alternative strategies to control ETEC infections. This study aimed to investigate, using complementary in vitro approaches, the inhibitory potential of two dietary-fiber-containing products (a lentil extract and yeast cell walls) against the human ETEC reference strain H10407. We showed that the lentil extract decreased toxin production in a dose-dependent manner, reduced pro-inflammatory interleukin-8 production, and modulated mucus-related gene induction in ETEC-infected mucus-secreting intestinal cells. We also report that the yeast product reduced ETEC adhesion to mucin and Caco-2/HT29-MTX cells. Both fiber-containing products strengthened intestinal barrier function and modulated toxin-related gene expression. In a complex human gut microbial background, both products did not elicit a significant effect on ETEC colonization. These pioneering data demonstrate the promising role of dietary fibers in controlling different stages of the ETEC infection process.

Archaea, specific genetic traits, and development of improved bacterial live biotherapeutic products: another face of next-generation probiotics.

Trimethylamine (TMA) and its oxide TMAO are important biomolecules involved in disease-associated processes in humans (e.g., trimethylaminuria and cardiovascular diseases). TMAO in plasma (pTMAO) stems from intestinal TMA, which is formed from various components of the diet in a complex interplay between diet, gut microbiota, and the human host. Most approaches to prevent the occurrence of such deleterious molecules focus on actions to interfere with gut microbiota metabolism to limit the synthesis of TMA. Some human gut archaea however use TMA as terminal electron acceptor for producing methane, thus indicating that intestinal TMA does not accumulate in some human subjects. Therefore, a rational alternative approach is to eliminate neo-synthesized intestinal TMA. This can be achieved through bioremediation of TMA by these peculiar methanogenic archaea, either by stimulating or providing them, leading to a novel kind of next-generation probiotics referred to as archaebiotics. Finally, specific components which are involved in this archaeal metabolism could also be used as intestinal TMA sequesters, facilitating TMA excretion along with stool. Referring to a standard pharmacological approach, these TMA traps could be synthesized ex vivo and then delivered into the human gut. Another approach is the engineering of known probiotic strain in order to metabolize TMA, i.e., live engineered biotherapeutic products. These alternatives would require, however, to take into account the necessity of synthesizing the 22nd amino acid pyrrolysine, i.e., some specificities of the genetics of TMA-consuming archaea. Here, we present an overview of these different strategies and recent advances in the field that will sustain such biotechnological developments. KEY POINTS: • Some autochthonous human archaea can use TMA for their essential metabolism, a methyl-dependent hydrogenotrophic methanogenesis. • They could therefore be used as next-generation probiotics for preventing some human diseases, especially cardiovascular diseases and trimethylaminuria. • Their genetic capacities can also be used to design live recombinant biotherapeutic products. • Encoding of the 22nd amino acid pyrrolysine is necessary for such alternative developments.

Obligate sugar oxidation in Mesotoga spp., phylum Thermotogae, in the presence of either elemental sulfur or hydrogenotrophic sulfate-reducers as electron acceptor.

Mesotoga prima strain PhosAc3 is a mesophilic representative of the phylum Thermotogae comprising only fermentative bacteria so far. We show that while unable to ferment glucose, this bacterium is able to couple its oxidation to reduction of elemental sulfur. We demonstrate furthermore that M. prima strain PhosAc3 as well as M. prima strain MesG1 and Mesotoga infera are able to grow in syntrophic association with sulfate-reducing bacteria (SRB) acting as hydrogen scavengers through interspecies hydrogen transfer. Hydrogen production was higher in M. prima strain PhosAc3 cells co-cultured with SRB than in cells cultured alone in the presence of elemental sulfur. We propose that the efficient sugar-oxidizing metabolism by M. prima strain PhosAc3 in syntrophic association with a hydrogenotrophic sulfate-reducing bacterium can be extrapolated to all members of the Mesotoga genus. Genome comparison of Thermotogae members suggests that the metabolic difference between Mesotoga and Thermotoga species (sugar oxidation versus fermentation) is mainly due to the absence of the bifurcating [FeFe]-hydrogenase in the former. Such an obligate oxidative process for using sugars, unusual within prokaryotes, is the first reported within the Thermotogae. It is hypothesized to be of primary ecological importance for growth of Mesotoga spp. in the environments that they inhabit.

Balneicella halophila gen. nov., sp. nov., an anaerobic bacterium, isolated from a thermal spring and description of Balneicellaceae fam. nov.

A mesophilic anaerobic bacterium, designated KHALHBd91T was isolated from the moderately hot spring of Hammam Biadha, Tunisia. The strain was Gram-staining-negative, non-sporulating, non-motile and rod-shaped, appearing singly (0.5-2.0×0.5-1 µm). It grew anaerobically at temperatures between 20 and 50 °C (optimum 37 °C) and at pH values between 5.5 and 7.8 (optimum 7.0). It required NaCl for growth, with growth observed at up 8.5 % and an optimum at 2.5 %. KHALHBd91T used glucose, galactose, maltose, pyruvate, lactate, fumarate and yeast extract as electron donors. The end-products from glucose fermentation were acetate, propionate, succinate and CO2. Nitrate, nitrite, thiosulfate, elemental sulfur, sulfate and sulfite were not used as terminal electron acceptors. The predominant cellular fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The respiratory quinone was MK-6. The main polar lipids consisted of lipids, phospholipids, glycolipids, aminolipids, phosphoaminoglycolipids and phosphatidylethanolamine. The DNA G+C content was 35.0 mol%. Phylogenetic analysis of the small-subunit ribosomal 16S rRNA gene sequence indicated that KHALHBd91T had Marinifilum fragile and Marinifilum flexuosum (phylum Bacteroidetes, class Bacteroidia, order Bacteroidales) as its closest relatives (similarity of 86.7 and 87.8 % respectively). The phylogenetic and physiological data fro the present study strongly indicate that the isolate represents a novel genus and species of a novel family, Balneicella halophila gen. nov., sp. nov., in the family Balneicellaceaefam. nov. The type strain is KHALHBd91T (=DSM28579T=JCM19909T).

Draft genome sequence of Mesotoga strain PhosAC3, a mesophilic member of the bacterial order Thermotogales, isolated from a digestor treating phosphogypsum in Tunisia.

Mesotoga strain PhosAc3 was the first mesophilic cultivated member of the order Thermotogales. This genus currently contain two described species, M. prima and M. infera. Strain PhosAc3, isolated from a Tunisian digestor treating phosphogypsum, is phylogenetically closely related to M. prima strain MesG1.Ag.4.2(T). Strain PhosAc3 has a genome of 3.1 Mb with a G+C content of 45.2%. It contains 3,051 protein-coding genes of which 74.6% have their best reciprocal BLAST hit in the genome of the type species, strain MesG1.Ag.4.2(T). For this reason we propose to assign strain PhosAc3 as a novel ecotype of the Mesotoga prima species. However, in contrast with the M. prima type strain, (i) it does not ferment sugars but uses them only in the presence of elemental sulfur as terminal electron acceptor, (ii) it produces only acetate and CO2 from sugars, whereas strain MesG1.Ag.4.2(T) produces acetate, butyrate, isobutyrate, isovalerate, 2-methyl-butyrate and (iii) sulfides are also end products of the elemental sulfur reduction in theses growth conditions.

Fusibacter fontis sp. nov., a sulfur-reducing, anaerobic bacterium isolated from a mesothermic Tunisian spring.

Strain KhalAKB1T, a mesophilic, anaerobic, rod-shaped bacterium, was isolated from water collected from a mesothermic Tunisian spring. Cells were Gram-staining-positive rods, occurring singly or in pairs and motile by one lateral flagellum. Strain KhalAKB1T grew at 15-45 °C (optimum 30 °C), at pH 5.5-8.5 (optimum pH 7.0) and in the presence of 0-35 g NaCl l- 1 (optimum 1 g NaCl l- 1). It fermented yeast extract and a wide range of carbohydrates including cellobiose, d-glucose, d-ribose, sucrose, d-xylose, maltose, d-galactose and starch as electron donors. Acetate, ethanol, CO2 and H2 were end products of glucose metabolism. It reduced elemental sulfur, but not sulfate, thiosulfate or sulfite, into sulfide. The DNA G+C content was 37.6 mol%. The predominant cellular fatty acids were C14 : 0 and C16 : 0. Phylogenetic analysis of the 16S rRNA gene sequence suggested Fusibacter bizertensis as the closest relative of this isolate (identity of 97.2 % to the type strain). Based on phenotypic, phylogenetic and genotypic taxonomic characteristics, strain KhalAKB1T is proposed to be assigned to a novel species within the genus Fusibacter, order Clostridiales, Fusibacter fontis sp. nov. The type strain is KhalAKB1T ( = DSM 28450T = JCM 19912T).

Characterization of Desulfovibrio biadhensis sp. nov., isolated from a thermal spring.

A novel anaerobic, mesophilic, slightly halophilic sulfate-reducing bacterium, designated strain Khaled BD4(T), was isolated from waters of a Tunisian thermal spring. Cells were vibrio-shaped or sigmoids (5-7×1-1.5 µm) and occurred singly or in pairs. Strain Khaled BD4(T) was Gram-stain-negative, motile and non-sporulated. It grew at 25-45 °C (optimum 37 °C), at pH 5.5-8.3 (optimum pH 7.0) and with 0.5-8% NaCl (optimum 3%). It required vitamins or yeast extract for growth. Sulfate, thiosulfate, sulfite and elemental sulfur served as terminal electron acceptors, but not fumarate, nitrate or nitrite. Strain Khaled BD4(T) utilized H2 in the presence of 2 mM acetate (carbon source), but also lactate, formate, pyruvate and fumarate in the presence of sulfate. Lactate was incompletely oxidized to acetate. Amongst substrates used, only pyruvate was fermented. Desulfoviridin and c-type cytochrome were present. The G+C content of the DNA was 54.6 mol%. The main fatty acids were anteiso -C(15 : 0), iso-C(18 : 0), iso-C(17 : 0) and iso-C(14 : 0). Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain Khaled BD4(T) had Desulfovibrio giganteus DSM 4123(T) (96.7% similarity) as its closest phylogenetic relative. On the basis of 16S rRNA gene sequence comparisons together with genetic and physiological characteristics, strain Khaled BD4(T) is assigned to a novel bacterial species, for which the name Desulfovibrio biadhensis sp. nov. is proposed. The type strain is Khaled BD4(T) ( = DSM 28904(T) = JCM 30146(T)).

Fusibacter tunisiensis sp. nov., isolated from an anaerobic reactor used to treat olive-mill wastewater.

Strain BELH1(T), a novel mesophilic, anaerobic, halotolerant, rod-shaped bacterium, was isolated from a Tunisian wastewater digester. The cells of the strain are motile, measure 0.5×2-5 µm, and occur singly or in pairs. The strain reduced thiosulfate and elemental sulfur (but not sulfate or sulfite) into sulfide. It grew at 15-40 °C (optimum 30 °C), pH 5.8-8.4 (optimum 7) and with 0-10 % (w/v) NaCl (optimum 3.0 %). The genomic DNA G+C content of strain BELH1(T) was 38.2 mol% and the strain's predominant cellular fatty acids were C(14:0), a summed feature that contained iso-C(17:1) and/or anteiso-C(17:1) B, and C(16:0). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel strain was most closely related to Fusibacter paucivorans (94.8 % sequence similarity). Based on phenotypic, phylogenetic and taxonomic characteristics, strain BELH1(T) represents a novel species of the genus Fusibacter, for which the name Fusibacter tunisiensis sp. nov. is proposed. The type strain is BELH1(T) ( = DSM 24436(T) = JCM 17481(T)).