A Reverse Ecology Approach Based on a Biological Definition of Microbial Populations.

Delineating ecologically meaningful populations among microbes is important for identifying their roles in environmental and host-associated microbiomes. Here, we introduce a metric of recent gene flow, which when applied to co-existing microbes, identifies congruent genetic and ecological units separated by strong gene flow discontinuities from their next of kin. We then develop a pipeline to identify genome regions within these units that show differential adaptation and allow mapping of populations onto environmental variables or host associations. Using this reverse ecology approach, we show that the human commensal bacterium Ruminococcus gnavus breaks up into sharply delineated populations that show different associations with health and disease. Defining populations by recent gene flow in this way will facilitate the analysis of bacterial and archaeal genomes using ecological and evolutionary theory developed for plants and animals, thus allowing for testing unifying principles across all biology.

Paneth Cell-Derived Lysozyme Defines the Composition of Mucolytic Microbiota and the Inflammatory Tone of the Intestine.

Paneth cells are the primary source of C-type lysozyme, a ß-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1-/- hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.

The arginine and nitric oxide metabolic pathway regulate the gut colonization and expansion of Ruminococcous gnavus.

Ruminococcus gnavus is a mucolytic commensal bacterium whose increased gut colonization has been associated with chronic inflammatory and metabolic diseases in humans. Whether R. gnavus metabolites can modulate host intestinal physiology remains largely understudied. We performed untargeted metabolomic and bulk RNA-seq analyses using R. gnavus monocolonization in germ-free mice. Based on transcriptome-metabolome correlations, we tested the impact of specific arginine metabolites on intestinal epithelial production of nitric oxide (NO) and examined the effect of NO on the growth of various strains of R. gnavus in vitro and in nitric oxide synthase 2 (Nos2)-deficient mice. R. gnavus produces specific arginine, tryptophan, and tyrosine metabolites, some of which are regulated by the environmental richness of sialic acid and mucin. R. gnavus colonization promotes expression of amino acid transporters and enzymes involved in metabolic flux of arginine and associated metabolites into NO. R. gnavus induced elevated levels of NOS2, while Nos2 ablation resulted in R. gnavus expansion in vivo. The growth of various R. gnavus strains can be inhibited by NO. Specific R. gnavus metabolites modulate intestinal epithelial cell NOS2 abundance and reduce epithelial barrier function at higher concentrations. Intestinal colonization and interaction with R. gnavus are partially regulated by an arginine-NO metabolic pathway, whereby a balanced control by the gut epithelium may restrain R. gnavus growth in healthy individuals. Disruption in this arginine metabolic regulation will contribute to the expansion and blooming of R. gnavus.

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1.

The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA containing either TTTA, TCTA, TCCA, or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent, crRNA-guided DNA cleavage by the Cpf1 family nucleases.

Microbiota-induced inflammatory responses in bladder tumors promote epithelial-mesenchymal transition and enhanced immune infiltration.

The intratumoral microbiota can modulate the tumor immune microenvironment (TIME); however, the underlying mechanism by which intratumoral microbiota influences the TIME in urothelial carcinoma of the bladder (UCB) remains unclear. To address this, we collected samples from 402 patients with UCB, including paired host transcriptome and tumor microbiome data, from The Cancer Genome Atlas (TCGA). We found that the intratumoral microbiome profiles were significantly correlated with the expression pattern of epithelial-mesenchymal transition (EMT)-related genes. Furthermore, we detected that the genera Lachnoclostridium and Sutterella in tumors could indirectly promote the EMT program by inducing an inflammatory response. Moreover, the inflammatory response induced by these two intratumoral bacteria further enhanced intratumoral immune infiltration, affecting patient survival and response to immunotherapy. In addition, an independent immunotherapy cohort of 348 patients with bladder cancer was used to validate our results. Collectively, our study elucidates the potential mechanism by which the intratumoral microbiota influences the TIME of UCB and provides a new guiding strategy for the targeted therapy of UCB.NEW & NOTEWORTHY The intratumoral microbiota may mediate the bladder tumor inflammatory response, thereby promoting the epithelial-mesenchymal transition program and influencing tumor immune infiltration.

3-Hydroxypropionate production from myo-inositol by the gut acetogen Blautia schinkii.

Species of the genus Blautia are not only abundant in the human gut but also contribute to human well-being. Our study demonstrates that the gut acetogen Blautia schinkii can grow on myo-inositol. We identified the pathway of myo-inositol degradation through a combination of physiological and biochemical studies, genome-wide expression profiling and homology searches. Initially, myo-inositol is oxidized to 2-keto-myo-inositol. This compound is then metabolized by a series of enzymes - a dehydratase, hydrolase, isomerase and kinase - to form 2-deoxy-5-keto-d-gluconic acid 6-phosphate. This intermediate is split by an aldolase into malonate semialdehyde and dihydroxyacetone phosphate, which is an intermediate of the Embden-Meyerhof-Parnas pathway. This pathway leads to the production of pyruvate and, subsequently, acetate. Concurrently, malonate semialdehyde is reduced to 3-hydroxypropionate (3-HP). The genes responsible for myo-inositol degradation are clustered on the genome, except for the gene encoding the aldolase. We identified the putative aldolase Fba_3 and 3-HP dehydrogenase Adh1 encoding genes bioinformatically and verified them biochemically using enzyme assays with heterologously produced and purified protein. The major fermentation end products were 3-HP and acetate, produced in similar amounts. The production of the unusual fermentation end product 3-HP is significant not only for human health but also for the potential bioindustrial production of this highly desired compound.

The utilization of N-acetylgalactosamine and its effect on the metabolism of amino acids in Erysipelotrichaceae strain.

BACKGROUND: The metabolism of gut microbiota produces bioactive metabolites that modulate host physiology and promote self-growth. Erysipelotrichaceae is one of the most common anaerobic microorganism families in the gut, which has been discovered to play a vital role in host metabolic disorders and inflammatory diseases. Our previous study found that N-acetylgalactosamine (GalNAc) in caecal content of pigs significantly affected the abundance of Erysipelotrichaceae strains. However, it remains unknown how GalNAc feeding in vitro culture affects the expression levels of genes in the GalNAc metabolic pathway and the concentrations of intermediate metabolites in the Erysipelotrichaceae strain. Whether GalNAc feeding should influence the metabolism of other nutrients, such as amino acids, remains unrevealed. RESULTS: In this study, whole-genome sequence, transcriptome, and metabolome data were analyzed to assess the utilization of a Erysipelotrichaceae strain on GalNAc. The results showed the presence of a complete GalNAc catabolism pathway in the genome of this Erysipelotrichaceae strain. GalNAc feeding to this Erysipelotrichaceae strain significantly changed the expression levels of genes involved in glycolysis and tricarboxylic acid (TCA) cycle. Meanwhile, the concentrations of lactate, pyruvate, citrate, succinate and malate from the glycolysis and TCA cycle were significantly increased. In addition, transcriptome analysis indicated that the genes involved in the metabolism of amino acids were affected by GalNAc, including lysA (a gene involved in lysine biosynthesis) that was significantly down-regulated. The intracellular concentrations of 14 amino acids in the Erysipelotrichaceae strain were significantly increased after feeding GalNAc. CONCLUSIONS: Our findings comfirmed and extended our previous works that demonstrated the utilization of GalNAc by Erysipelotrichaceae strain, and explained the possible mechanism of GalNAc affecting the abundance of Erysipelotrichaceae strain in vitro.

Sequence similarity network analysis of drug- and dye-modifying azoreductase enzymes found in the human gut microbiome.

Drug metabolism by human gut microbes is often exemplified by azo bond reduction in the anticolitic prodrug sulfasalazine. Azoreductase activity is often found in incubations with cell cultures or ex vivo gut microbiome samples and contributes to the xenobiotic metabolism of drugs and food additives. Applying metagenomic studies to personalized medicine requires knowledge of the genes responsible for sulfasalazine and other drug metabolism, and candidate genes and proteins for drug modifications are understudied. A representative gut-abundant azoreductase from Anaerotignum lactatifermentan DSM 14214 efficiently reduces sulfasalazine and another drug, phenazopyridine, but could not reduce all azo-bonded drugs in this class. We used enzyme kinetics to characterize this enzyme for its NADH-dependent reduction of these drugs and food additives and performed computational docking to provide the groundwork for understanding substrate specificity in this family. We performed an analysis of the Flavodoxin-like fold InterPro family (IPR003680) by computing a sequence similarity network to classify distinct subgroups of the family and then performed chemically-guided functional profiling to identify proteins that are abundant in the NIH Human Microbiome Project dataset. This strategy aims to reduce the number of unique azoreductases needed to characterize one protein family in the diverse set of potential drug- and dye-modifying activities found in the human gut microbiome.

Semiautomated design and soluble expression of a chimeric antigen TbpAB01 from Glaesserella parasuis.

Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi-epitope chimeric antigen (ChA) and screening two classes of protein-assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin-binding protein A of Glaesserella parasuis. Our engineered strain, which co-expresses P17 tagged-ChA and endogenous chaperones groEL-ES, yields a 0.346 g/L highly soluble ChA with the property of HPS-positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5-L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications.

The human gut symbiont Ruminococcus gnavus shows specificity to blood group A antigen during mucin glycan foraging: Implication for niche colonisation in the gastrointestinal tract.

The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-ß-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.

Cloning, characterization of ß-glucosidase from Furfurilactobacillus rossiae in bioconversion and its efficacy.

Minor ginsenosides produced by ß-glucosidase are interesting biologically and pharmacologically. In this study, new ginsenoside-hydrolyzing glycosidase from Furfurilactobacillus rossiae DCYL3 was cloned and expressed in Escherichia coli strain BL21. The enzyme converted Rb1 and Gyp XVII into Rd and compound K following the pathways: Rb1→Rd and Gyp XVII→F2→CK, respectively at optimal condition: 40 °C, 15 min, and pH 6.0. Furthermore, we examined the cytotoxicity, NO production, ROS generation, and gene expression of Gynostemma extract (GE) and bioconverted Gynostemma extract (BGE) in vitro against A549 cell lines for human lung cancer and macrophage RAW 264.7 cells for antiinflammation, respectively. As a result, BGE demonstrated significantly greater toxicity than GE against lung cancer at a dose of 500 µg/mL but in normal cells showed lower toxicity. Then, we indicated an enhanced generation of ROS, which may be boosting cancer cell toxicity. By blocking the intrinsic way, BGE increased p53, Bax, Caspase 3, 9, and while Bcl2 is decreased. At 500 µg/mL, the BGE sample was less toxic in normal cells and decreased the LPS-treated NO and ROS level to reduce inflammation. In addition, BGE inhibited the expression of pro-inflammatory genes COX-2, iNOS, IL-6, and IL-8 in RAW 264.7 cells than the sample of GE. In conclusion, FrBGL3 has considerable downstream applications for high-yield, low-cost, effective manufacture of minor ginsenosides. Moreover, the study's findings imply that BGE would be potential materials for anti-cancer and anti-inflammatory agent after consideration of future studies.

•The first time ß-glucosidase (FrBGL3) from Furfurilactobacillus rossiae was identified and characterized.•FrBGL3 activity in ginsenoside and gypenoside bioconversion were found and confirmed.•Application in Gynostemma extract bioconversion by FrBGL3 boosts anti-inflammatory and anti-cancer activities.

Proposal for reclassification of the species Hungatella xylanolytica as Lacrimispora xylanisolvens nom. nov. and transfer of the genus Hungatella to the family Lachnospiraceae.

Hungatella xylanolytica X5-1T is an anaerobic, xylan-fermenting bacterium first isolated from methane-producing cattle manure. Initially identified as Bacteroides xylanolyticus, this species was later reclassified as H. xylanolytica in 2019. Although this reclassification found support through Genome blast Distance Phylogeny analysis which placed H. xylanolytica X5-1T into the same clade as Hungatella effluvii DSM 24995T, it was contradicted by 16S rRNA gene phylogenetic analysis, which associated it with a set of misnamed Clostridium species later reassigned into the genus Lacrimispora. To ascertain its taxonomic position, comparative analyses were performed to re-examine the relationship between H. xylanolytica X5-1T and all species of the genera Hungatella and Lacrimispora. The ranges of 16S rRNA gene sequence similarity, average amino acid identity, and percentage of conserved protein prediction values were higher between H. xylanolytica X5-1T and species of the genus Lacrimispora than Hungatella. In addition, H. xylanolytica X5-1T was found to harbour genes and pathways conserved and exclusive to species within the genus Lacrimispora but not Hungatella. Essentially, in both the 16S rRNA gene phylogenetic tree and the core-genome phylogenomic tree, H. xylanolytica X5-1T clustered into the same clade as species of the genus Lacrimispora, distinct from species of the genus Hungatella. It is thus clear that H. xylanolytica X5-1T represents a species within the genus Lacrimispora, which we propose to reclassify as Lacrimispora xylanisolvens nom. nov. Finally, based on the results from the phylogenetic and comparative analyses, the genus Hungatella was transferred to the family Lachnospiraceae.

Reclassification of Butyrivibrio crossotus Moore et al. 1976 (Approved Lists 1980) into a novel genus as Eshraghiella crossota gen. nov., comb. nov.

The reclassification of Butyrivibrio crossotus Moore et al. 1976 (Approved Lists 1980) as Eshraghiella crossota gen. nov., comb. nov. is proposed within the family Lachnospiraceae. This reclassification is based on differences revealed through the analysis of 16S rRNA, groEL, recA, and rpoB genes, as well as genome sequences, distinguishing it from other Butyrivibrio species. Comparative analysis showed that B. crossotus exhibited digital DNA-DNA hybridization (dDDH) values of 19.40-27.20% and average nucleotide identities based on blast (ANIb) values of 67.06-67.64% with other Butyrivibrio species. These values are significantly below the species delineation thresholds (dDDH, 70%; ANIb, 95-96%), justifying the proposed reclassification. Additionally, the results of the average amino acid identity (AAI) analysis indicated that this species shares 59.22-60.17% AAI with the other species of the genus Butyrivibrio, which is below the AAI threshold (65%) for a genus boundary. In addition, biochemical and morphological characteristics also support the proposal that this species is different from other species of the genus Butyrivibrio. The type strain is ATCC 29175T (DSM 2876T=T9-40AT).

Lacrimispora brassicae sp. nov. isolated from fermented cabbage, and proposal of Clostridium indicum Gundawar et al. 2019 and Clostridium methoxybenzovorans Mechichi et al. 1999 as heterotypic synonyms of Lacrimispora amygdalina (Parshina et al. 2003) Haas and Blanchard 2020 and Lacrimispora indolis (McClung and McCoy 1957) Haas and Blanchard 2020, respectively.

A Gram-stain-negative, endospore-forming, rod-shaped, indole-producing bacterial strain, designated YZC6T, was isolated from fermented cabbage. Strain YZC6T grew at 10-37  °C, pH 5.5-8.5, and with up to 2  % (w/v) NaCl. The major cellular fatty acids were C16 : 0 and C18 : 1 cis 11 dimethyl acetal. Phylogenetic analysis of the 16S rRNA gene revealed that strain YZC6T belonged to the genus Lacrimispora and was closely related to Lacrimispora aerotolerans DSM 5434T (98.3  % sequence similarity), Lacrimispora saccharolytica WM1T (98.1  %), and Lacrimispora algidixylanolytica SPL73T (98.1  %). The average nucleotide identity based on blast (below 87.8  %) and digital DNA-DNA hybridization (below 36.1 %) values between the novel isolate and its corresponding relatives showed that strain YZC6T could be readily distinguished from its closely related species. Based on genotypic, phenotypic, and chemotaxonomic data, a novel Lacrimispora species, Lacrimispora brassicae sp. nov., was proposed, with YZC6T as the type strain (=MAFF 212518T=JCM 32810T=DSM 112100T). This study also proposed Clostridium indicum Gundawar et al. 2019 as a later heterotypic synonym of Lacrimispora amygdalina (Parshina et al. 2003) Haas and Blanchard 2020 and Clostridium methoxybenzovorans Mechichi et al. 1999 as a later heterotypic synonym of Lacrimispora indolis (McClung and McCpy 1957) Haas and Blanchard 2020.

Transcriptional recording by CRISPR spacer acquisition from RNA.

The ability to record transcriptional events within a cell over time would help to elucidate how molecular events give rise to complex cellular behaviours and states. However, current molecular recording technologies capture only a small set of defined stimuli. Here we use CRISPR spacer acquisition to capture and convert intracellular RNAs into DNA, enabling DNA-based storage of transcriptional information. In Escherichia coli, we show that defined stimuli, such as an RNA virus or arbitrary sequences, as well as complex stimuli, such as oxidative stress, result in quantifiable transcriptional records that are stored within a population of cells. We demonstrate that the transcriptional records enable us to classify and describe complex cellular behaviours and to identify the precise genes that orchestrate differential cellular responses. In the future, CRISPR spacer acquisition-mediated recording of RNA followed by deep sequencing (Record-seq) could be used to reconstruct transcriptional histories that describe complex cell behaviours or pathological states.

Characterization of a novel GH30 non-specific endoxylanase AcXyn30B from Acetivibrio clariflavus.

The xylanolytic enzymes Clocl_1795 and Clocl_2746 from glycoside hydrolase (GH) family 30 are highly abundant in the hemicellulolytic system of Acetivibrio clariflavus (Hungateiclostridium, Clostridium clariflavum). Clocl_1795 has been shown to be a xylobiohydrolase AcXbh30A releasing xylobiose from the non-reducing end of xylan and xylooligosaccharides. In this work, biochemical characterization of Clocl_2746 is presented. The protein, designated AcXyn30B, shows low sequence similarity to other GH30 members and phylogenetic analysis revealed that AcXyn30B and related proteins form a separate clade that is proposed to be a new subfamily GH30_12. AcXyn30B exhibits similar specific activity on glucuronoxylan, arabinoxylan, and aryl glycosides of linear xylooligosaccharides suggesting that it is a non-specific xylanase. From polymeric substrates, it releases the fragments of degrees of polymerization (DP) 2-6. Hydrolysis of different xylooligosaccharides indicates that AcXyn30B requires at least four occupied catalytic subsites for effective cleavage. The ability of the enzyme to hydrolyze a wide range of substrates is interesting for biotechnological applications. In addition to subfamilies GH30_7, GH30_8, and GH30_10, the newly proposed subfamily GH30_12 further widens the spectrum of GH30 subfamilies containing xylanolytic enzymes. KEY POINTS: Bacterial GH30 endoxylanase from A. clariflavus (AcXyn30B) has been characterized AcXyn30B is non-specific xylanase hydrolyzing various xylans and xylooligosaccharides Phylogenetic analysis placed AcXyn30B in a new GH30_12 subfamily.

Gut microbiota predict retinopathy in patients with diabetes: A longitudinal cohort study.

The gut microbiota has emerged as an independent risk factor for diabetes and its complications. This research aimed to delve into the intricate relationship between the gut microbiome and diabetic retinopathy (DR) through a dual approach of cross-sectional and prospective cohort studies. In our cross-sectional study cross-sectional investigation involving ninety-nine individuals with diabetes, distinct microbial signatures associated with DR were identified. Specifically, gut microbiome profiling revealed decreased levels of Butyricicoccus and Ruminococcus torques group, alongside upregulated methanogenesis pathways among DR patients. These individuals concurrently exhibited lower concentrations of short-chain fatty acids in their plasma. Leveraging machine learning models, including random forest classifiers, we constructed a panel of microbial genera and genes that robustly differentiated DR cases. Importantly, these genera also demonstrated significant correlations with dietary patterns and the molecular profiles of peripheral blood mononuclear cells. Building upon these findings, our prospective cohort study followed 62 diabetes patients over a 2-year period to assess the predictive value of these microbial markers. The results underlined the panel's efficacy in predicting DR incidence. By stratifying patients based on the predictive genera and metabolites identified in the cross-sectional phase, we established significant associations between reduced levels of Butyricicoccus, plasma acetate, and increased susceptibility to DR. This investigation not only deepens our understanding of how gut microbiota influences DR but also underscores the potential of microbial markers as early indicators of disease risk. These insights hold promise for developing targeted interventions aimed at mitigating the impact of diabetic complications. KEY POINTS: • Microbial signatures are differed in diabetic patients with and without retinopathy • DR-related taxa are linked to dietary habits and transcriptomic profiles • Lower abundances of Butyricicoccus and acetate were prospectively associated with DR.

Reclassification of Aestuariicella albida as Pseudomaricurvus albidus comb. nov. and Aestuariicella hydrocarbonica as Pseudomaricurvus hydrocarbonicus comb. nov. Based on Comparative Genomics and Molecular Synapomorphies.

The genus Aestuariicella has been recently reclassified as a member of the family Cellvibrionaceae. However, the taxonomic position of the genus as a distinct member of the family has not been clarified. In the present study, we performed multilayered analyses anchored on genome sequences to clarify the relationship between the genera Aestuariicella and Pseudomaricurvus within the family Cellvibrionaceae. Phylogenetic analyses based on 16S rRNA gene, RNA polymerase beta subunit (RpoB) protein, and core gene sequences showed a well-supported tight cluster formed by the members of the two genera. Moreover, the analysis of the average amino acid identity (AAI) revealed that the members of the two genera shared 68.16-79.48% AAI, values which were within the range of observed AAI (≥ 67.23%) among the members of the same genus within the family Cellvibrionaceae. Members of the two genera also shared several common characteristics. Furthermore, molecular synapomorphies in a form of conserved signature indels were identified in six protein sequences that were exclusively shared by the members of the two genera. Based on the phylogenetic and molecular evidence presented here, we propose the reclassification of the species Aestuariicella albida and Aestuariicella hydrocarbonica as Pseudomaricurvus albidus comb. nov. and Pseudomaricurvus hydrocarbonicus comb. nov., respectively.

Elucidation of an anaerobic pathway for metabolism of l-carnitine-derived γ-butyrobetaine to trimethylamine in human gut bacteria.

Trimethylamine (TMA) is an important gut microbial metabolite strongly associated with human disease. There are prominent gaps in our understanding of how TMA is produced from the essential dietary nutrient l-carnitine, particularly in the anoxic environment of the human gut where oxygen-dependent l-carnitine-metabolizing enzymes are likely inactive. Here, we elucidate the chemical and genetic basis for anaerobic TMA generation from the l-carnitine-derived metabolite γ-butyrobetaine (γbb) by the human gut bacterium Emergencia timonensis We identify a set of genes up-regulated by γbb and demonstrate that the enzymes encoded by the induced γbb utilization (bbu) gene cluster convert γbb to TMA. The key TMA-generating step is catalyzed by a previously unknown type of TMA-lyase enzyme that utilizes a putative flavin cofactor to catalyze a redox-neutral transformation. We identify additional cultured and uncultured host-associated bacteria that possess the bbu gene cluster, providing insights into the distribution of anaerobic γbb metabolism. Lastly, we present genetic, transcriptional, and metabolomic evidence that confirms the relevance of this metabolic pathway in the human gut microbiota. These analyses indicate that the anaerobic pathway is a more substantial contributor to TMA generation from l-carnitine in the human gut than the previously proposed aerobic pathway. The discovery and characterization of the bbu pathway provides the critical missing link in anaerobic metabolism of l-carnitine to TMA, enabling investigation into the connection between this microbial function and human disease.

Capsular polysaccharide correlates with immune response to the human gut microbe Ruminococcus gnavus.

Active inflammatory bowel disease (IBD) often coincides with increases of Ruminococcus gnavus, a gut microbe found in nearly everyone. It was not known how, or if, this correlation contributed to disease. We investigated clinical isolates of R. gnavus to identify molecular mechanisms that would link R. gnavus to inflammation. Here, we show that only some isolates of R. gnavus produce a capsular polysaccharide that promotes a tolerogenic immune response, whereas isolates lacking functional capsule biosynthetic genes elicit robust proinflammatory responses in vitro. Germ-free mice colonized with an isolate of R. gnavus lacking a capsule show increased measures of gut inflammation compared to those colonized with an encapsulated isolate in vivo. These observations in the context of our earlier identification of an inflammatory cell-wall polysaccharide reveal how some strains of R. gnavus could drive the inflammatory responses that characterize IBD.