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Mucus production is initiated before birth and provides mucin glycans to the infant gut microbiota. Bifidobacteria are the major bacterial group in the feces of vaginally delivered and breast milk-fed infants. Among the bifidobacteria, only Bifidobacterium bifidum is able to degrade mucin and to release monosaccharides which can be used by other gut microbes colonizing the infant gut. Eubacterium hallii is an early occurring commensal that produces butyrate and propionate from fermentation metabolites but that cannot degrade complex oligo-and polysaccharides. We aimed to demonstrate that mucin crossfeeding initiated by B. bifidum enables growth and metabolite formation of E. hallii leading to short-chain fatty acid (SCFA) formation. Growth and metabolite formation of co-cultures of B. bifidum, of Bifidobacterium breve or Bifidobacterium infantis, which use mucin-derived hexoses and fucose, and of E. hallii were determined. Growth of E. hallii in the presence of lactose and mucin monosaccharides was tested. In co-culture fermentations, the presence of B. bifidum enabled growth of the other strains. B. bifidum/B. infantis co-cultures yielded acetate, formate, and lactate while co-cultures of B. bifidum and E. hallii formed acetate, formate, and butyrate. In three-strain co-cultures, B. bifidum, E. hallii, and B. breve or B. infantis produced up to 16 mM acetate, 5 mM formate, and 4 mM butyrate. The formation of propionate (approximately 1 mM) indicated cross-feeding on fucose. Lactose, galactose, and GlcNAc were identified as substrates of E. hallii. This study shows that trophic interactions of bifidobacteria and E. hallii lead to the formation of acetate, butyrate, propionate, and formate, potentially contributing to intestinal SCFA formation with potential benefits for the host and for microbial colonization of the infant gut. The ratios of SCFA formed differed depending on the microbial species involved in mucin cross-feeding.

Tungsten metabolism was found to be prevalent in the human gut microbiome, which is involved in the detoxification of food and antimicrobial aldehydes, as well as in the production of beneficial SCFAs. In this study, we characterized a protein in the human gut microbe, Eubacterium limosum , that stores tungstate in preparation for its use in enzymes involved in SCFA generation. This revealed several families of tungstate binding proteins that are also involved in tungstate transport and tungstate-dependent regulation and are widely distributed in the human gut microbiome. Elucidating how tungsten is stored and transported in the human gut microbes contributes to our understanding of the human gut microbiome and its impact on human health.

Abstract Eubacterium limosum is an acetogenic bacterium of potential industrial relevance for its ability to efficiently metabolize a range of single carbon compounds. However, extracellular polymeric substance (EPS) produced by the type strain ATCC 8486 is a serious impediment to bioprocessing and genetic engineering. To remove these barriers, here we bioinformatically identified genes involved in EPS biosynthesis, and targeted several of the most promising candidates for inactivation, using a homologous recombination-based approach. Deletion of a single genomic region encoding homologues for epsABC, ptkA, and tmkA resulted in a strain incapable of producing EPS. This strain is significantly easier to handle by pipetting and centrifugation, and retains important wild-type phenotypes including the ability to grow on methanol and carbon dioxide and limited oxygen tolerance. Additionally, this strain is also more genetically tractable with a 2-fold increase in transformation efficiency compared to the highest previous reports. This work advances a simple, rapid protocol for gene knockouts in E. limosum using only the native homologous recombination machinery. These results will hasten the development of this organism as a workhorse for valorization of single carbon substrates, as well as facilitate exploration of its role in the human gut microbiota. We developed a rapid, simple protocol for gene deletion in the gas-fermenting microbe Eubacterium limosum, and used this to abolish biofilm formation to improve handling and genetic engineering.

Background: The global alteration of the gut microbial community (dysbiosis) plays an important role in the pathogenesis of inflammatory bowel diseases (IBDs). However, bacterial species that characterize dysbiosis in IBD remain unclear. In this study, we assessed the alteration of the fecal microbiota profile in patients with Crohn's disease (CD) using 16S rRNA sequencing. Summary: Fecal samples from 10 inactive CD patients and 10 healthy individuals were subjected to 16S rRNA sequencing. The V3-V4 hypervariable regions of 16S rRNA were sequenced by the Illumina MiSeq™II system. The average of 62,201 reads per CD sample was significantly lower than the average of 73,716 reads per control sample. The genera Bacteroides, Eubacterium, Faecalibacterium and Ruminococcus significantly decreased in CD patients as compared to healthy controls. In contrast, the genera Actinomyces and Bifidobacterium significantly increased in CD patients. At the species level, butyrate-producing bacterial species, such as Blautia faecis, Roseburia inulinivorans, Ruminococcus torques, Clostridium lavalense, Bacteroides uniformis and Faecalibacterium prausnitzii were significantly reduced in CD patients as compared to healthy individuals (p < 0.05). These results of 16S rRNA sequencing were confirmed in additional CD patients (n = 68) and in healthy controls (n = 46) using quantitative PCR. The abundance of Roseburia inulinivorans and Ruminococcus torques was significantly lower in C-reactive protein (CRP)-positive CD patients as compared to CRP-negative CD patients (p < 0.05). Key Message: The dysbiosis of CD patients is characterized by reduced abundance of multiple butyrate-producing bacteria species.

Ruminal microorganisms hydrogenate polyunsaturated fatty acids (PUFA) present in forages and thereby restrict the availability of health-promoting PUFA in meat and milk. The aim of this study was to investigate PUFA metabolism and the influence of PUFA on members of the ruminal microflora. Eleven of 26 predominant species of ruminal bacteria metabolised linoleic acid (LA; cis-9,cis-12-18:2) substantially. The most common product was vaccenic acid (trans-11-18:1), produced by species related to Butyrivibrio fibrisolvens. α-Linolenic acid (LNA; cis-9,cis-12,cis-15-18:3) was metabolised mostly by the same species. The fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n - 3)) and docosahexaenoic acid (DHA; 22:6(n - 3)) were not metabolised. Cellulolytic bacteria did not grow in the presence of any PUFA at 50 μg ml-¹, nor did some butyrate-producing bacteria, including the stearate producer Clostridium proteoclasticum, Butyrivibrio hungatei and Eubacterium ruminantium. Toxicity to growth was ranked EPA > DHA > LNA > LA. Cell integrity, as measured using propidium iodide, was damaged by LA in all 26 bacteria, but to different extents. Correlations between its effects on growth and apparent effects on cell integrity in different bacteria were low. Combined effects of LA and sodium lactate in E. ruminantium and C. proteoclasticum indicated that LA toxicity is linked to metabolism in butyrate-producing bacteria. PUFA also inhibited the growth of the cellulolytic ruminal fungi, with Neocallimastix frontalis producing small amounts of cis-9,trans-11-18:2 (CLA) from LA. Thus, while dietary PUFA might be useful in suppressing the numbers of biohydrogenating ruminal bacteria, particularly C. proteoclasticum, care should be taken to avoid unwanted effects in suppressing cellulolysis.

Eubacterium limosum ZL-II was described to convert secoisolariciresinol (SECO) to its demethylating product 4,4′-dihydroxyenterodiol (DHEND) under anoxic conditions. However, the reaction cascade remains unclear. Here, the O -demethylase being responsible for the conversion was identified and characterized. Nine genes encoding two methyltransferase-Is (MT-I), two corrinoid proteins (CP), two methyltransferase-IIs (MT-II), and three activating enzymes (AE) were screened, cloned, and expressed in Escherichia coli . Four of the nine predicted enzymes, including ELI_2003 (MT-I), ELI_2004 (CP), ELI_2005 (MT-II), and ELI_0370 (AE), were confirmed to constitute the O -demethylase in E. limosum ZL-II. The complete O -demethylase (combining the four components) reaction system was reconstructed in vitro. As expected, the demethylating products 3-demethyl-SECO and DHEND were both produced. During the reaction process, ELI_2003 (MT-I) initially catalyzed the transfer of methyl group from SECO to the corrinoid of ELI_2004 ([Co I ]-CP), yielding demethylating products and [CH 3 -Co III ]-CP; then ELI_2005 (MT-II) mediated the transfer of methyl group from [CH 3 -Co III ]-CP to tetrahydrofolate, forming methyltetrahydrofolate and [Co I ]-CP. Due to the low redox potential of [Co II ]/[Co I ], [Co I ]-CP was oxidized to [Co II ]-CP immediately in vitro, and ELI_0370 (AE) was responsible for catalyzing the reduction of [Co II ]-CP to its active form [Co I ]-CP. The active-site residues in ELI_2003, ELI_2005, and ELI_0370 were subsequently determined using molecular modeling combined with site-directed mutagenesis. To our knowledge, this is the first study on the identification and characterization of a four-component O -demethylase from E. limosum ZL-II, which will facilitate the development of method to artificial synthesis of related bioactive chemicals.

Inflammatory bowel disease (IBD) is considered to result from interplay between host and intestinal microbiota. While IBD in adults has shown to be associated with marked changes in the intestinal microbiota, there are only a few studies in children, and particularly studies focusing on therapeutic responses are lacking. Hence, this prospective study addressed the intestinal microbiota in pediatric IBD especially related to the level of inflammation. In total, 68 pediatric patients with IBD and 26 controls provided stool and blood samples in a tertiary care hospital and 32 received anti-tumor necrosis factor-α (anti-TNF-α). Blood inflammatory markers and fecal calprotectin levels were determined. The intestinal microbiota was characterized by phylogenetic microarray and qPCR analysis. The microbiota varied along a gradient of increasing intestinal inflammation (indicated by calprotectin levels), which was associated with reduced microbial richness, abundance of butyrate producers, and relative abundance of Gram-positive bacteria (especially Clostridium clusters IV and XIVa). A significant association between microbiota composition and inflammation was indicated by a set of bacterial groups predicting the calprotectin levels (area under curve (AUC) of 0.85). During the induction of anti-TNF-α, the microbial diversity and similarity to the microbiota of controls increased in the responder group by week 6, but not in the non-responders (P<0.01; response related to calprotectin levels). The abundance of six groups of bacteria including those related to Eubacterium rectale and Bifidobacterium spp. predicted the response to anti-TNF-α medication. Intestinal microbiota represents a potential biomarker for correlating the level of inflammation and therapeutic responses to be further validated.

Background Acetogenic bacteria constitute promising biocatalysts for the conversion of CO 2 /H 2 or synthesis gas (H 2 /CO/CO 2 ) into biofuels and value-added biochemicals. These microorganisms are naturally capable of autotrophic growth via unique acetogenesis metabolism. Despite their biosynthetic potential for commercial applications, a systemic understanding of the transcriptional and translational regulation of the acetogenesis metabolism remains unclear. Results By integrating genome-scale transcriptomic and translatomic data, we explored the regulatory logic of the acetogenesis to convert CO 2 into biomass and metabolites in Eubacterium limosum . The results indicate that majority of genes associated with autotrophic growth including the Wood-Ljungdahl pathway, the reduction of electron carriers, the energy conservation system, and gluconeogenesis were transcriptionally upregulated. The translation efficiency of genes in cellular respiration and electron bifurcation was also highly enhanced. In contrast, the transcriptionally abundant genes involved in the carbonyl branch of the Wood-Ljungdahl pathway, as well as the ion-translocating complex and ATP synthase complex in the energy conservation system, showed decreased translation efficiency. The translation efficiencies of genes were regulated by 5′UTR secondary structure under the autotrophic growth condition. Conclusions The results illustrated that the acetogenic bacteria reallocate protein synthesis, focusing more on the translation of genes for the generation of reduced electron carriers via electron bifurcation, rather than on those for carbon metabolism under autotrophic growth.

Background: Several observational studies and clinical trials have shown that the gut microbiota is associated with urological cancers. However, the causal relationship between gut microbiota and urological cancers remains to be elucidated due to many confounding factors. Methods: In this study, we used two thresholds to identify gut microbiota GWAS from the MiBioGen consortium and obtained data for five urological cancers from the UK biobank and Finngen consortium, respectively. We then performed a two-sample Mendelian randomization (MR) analysis with Wald ratio or inverse variance weighted as the main method. We also performed comprehensive sensitivity analyses to verify the robustness of the results. In addition, we performed a reverse MR analysis to examine the direction of causality. Results: Our study found that family Rikenellaceae, genus Allisonella, genus Lachnospiraceae UCG001, genus Oscillibacter, genus Eubacterium coprostanoligenes group, genus Eubacterium ruminantium group, genus Ruminococcaceae UCG013, and genus Senegalimassilia were related to bladder cancer; genus Ruminococcus torques group, genus Oscillibacter, genus Barnesiella, genus Butyricicoccus, and genus Ruminococcaceae UCG005 were related to prostate cancer; class Alphaproteobacteria, class Bacilli, family Family XI, genus Coprococcus2, genus Intestinimonas, genus Lachnoclostridium, genus Lactococcus, genus Ruminococcus torques group, and genus Eubacterium brachy group were related to renal cell cancer; family Clostridiaceae 1, family Christensenellaceae, genus Eubacterium coprostanoligenes group, genus Clostridium sensu stricto 1, and genus Eubacterium eligens group were related to renal pelvis cancer; family Peptostreptococcaceae, genus Romboutsia, and genus Subdoligranulum were related to testicular cancer. Comprehensive sensitivity analyses proved that our results were reliable. Conclusions: Our study confirms the role of specific gut microbial taxa on urological cancers, explores the mechanism of gut microbiota on urological cancers from a macroscopic level, provides potential targets for the screening and treatment of urological cancers, and is dedicated to providing new ideas for clinical research.

Rheumatoid arthritis (RA) is a chronic autoimmune disease with a complex and diverse etiology. The onset of RA is closely associated with intestinal flora, which is essential for immune regulation. Fecal samples of 22 healthy controls and 38 patients with newly diagnosed RA were used for performing 16S rRNA sequencing, microbiota diversity assessment, and functional enrichment analysis. Through integrative analysis of random forest feature selection and bidirectional Mendelian randomization (MR), was prioritized as a key bacterial candidate associated with RA. Furthermore, was used to treat the arthritis model mice by gavage treatment, and we evaluated joint inflammation and immune cell profile in mice. Finally, untargeted metabolomics was used to evaluate the changes in serum and fecal metabolites in the arthritis mouse model before and after intervention. The beta diversity of the intestinal flora exhibited significant differences between RA patients and healthy controls (HC). Functional enrichment analysis revealed that RA patients' intestinal microbiota functions were enriched in pathways like genetic information processing and material metabolism. Further random forest model revealed , etc., and twelve genera with characteristic significance in RA patients. According to further MR analysis, and had a protective effect on RA, and reverse MR analysis showed no evidence of a causal relationship between these groups and RA. experiments showed that after the administration of , the joint inflammation of the mice was relatively slight, the bone destruction and bone density of the joints improved, the proportion of Treg and follicular regulatory T cells (Tfr) cells increased, and the proportion of follicular helper T cells (Tfh) cells decreased. Metabolomic analysis revealed significant changes in both serum and fecal metabolites in mice with collagen-induced arthritis (CIA) compared with healthy controls. The changes in metabolites such as butyric acid were reversed after treatment with . The study demonstrates that has a protective effect on RA. significantly attenuates joint inflammation in mouse models by may regulating the expression level of butyrate, ameliorating the Treg and Tfr/Tfh immune imbalance status, and re-establishing the immune tolerance. These findings serve as valuable references for future studies on the pathogenesis of RA and the development of new therapeutic approaches.