CoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing.

The expansion of the CRISPR-Cas toolbox is highly needed to accelerate the development of therapies for genetic diseases. Here, through the interrogation of a massively expanded repository of metagenome-assembled genomes, mostly from human microbiomes, we uncover a large variety (n = 17,173) of type II CRISPR-Cas loci. Among these we identify CoCas9, a strongly active and high-fidelity nuclease with reduced molecular size (1004 amino acids) isolated from an uncultivated Collinsella species. CoCas9 is efficiently co-delivered with its sgRNA through adeno associated viral (AAV) vectors, obtaining efficient in vivo editing in the mouse retina. With this study we uncover a collection of previously uncharacterized Cas9 nucleases, including CoCas9, which enriches the genome editing toolbox.

A potential marker of radiation based on 16S rDNA in the rat model: Intestinal flora.

The gastrointestinal microbiota plays an important role in the function of the host intestine. However, little is currently known about the effects of irradiation on the microorganisms colonizing the mucosal surfaces of the gastrointestinal tract. The aim of this study was to investigate the effects of X-ray irradiation on the compositions of the large intestinal Microbiotas of the rat. The gut microbiotas in control mice and mice receiving irradiation with different dose treatment were characterized by high-throughput sequencing of the bacterial 16S rDNA gene and their metabolites were detected by gas chromatography-mass spectrometry. Unexpectedly, the diversity was increased mildly at 2Gy irradiation, and dose dependent decreased at 4Gy, 6Gy, 8Gy irradiation. The phyla with large changes in phylum level are Firmicutes, Bacteroides and Proteobacteria; the abundance ratio of Firmicutes/Bacteroides is inverted; and when 8Gy is irradiated, the phylum abundance level was significantly increased. At the genus level, the abundance levels of Phascolarctobacterium, Ruminococcaceae and Lachnospiraceae increased at 2Gy irradiation, and significantly decreased at 4Gy, 6Gy, and 8Gy irradiation; the abundance level of Prevotellaceae diminished at 2Gy irradiation, and enhanced at 4Gy, 6Gy, 8Gy irradiation; The abundance level of Violet bacteria (Christenellaceae) and Lactobacillus attenuated in a dose-dependent manner; Lachnoclostridium enhanced in a dose-dependent manner; Bacteroides was in 4Gy, 6Gy, 8Gy The abundance level increased significantly during irradiation; the abundance level of Shigella (Escherichia-Shigella) only increased significantly during 8Gy irradiation. Lefse predicts that the biomarker at 0Gy group is Veillonellaceae, the biomarker at 2Gy group is Firmicutes, the biomarkers at 4Gy group are Dehalobacterium and Dehalobacteriaceae, the biomarkers at 6Gy group are Odoribacter, and the biomarkers at 8Gy group are Anaerotruncus, Holdemania, Proteus, Bilophila, Desufovibrionales and Deltaproteobacteria. Overall, the data presented here reveal that X-ray irradiation can cause imbalance of the intestinal flora in rats; different doses of irradiation can cause different types of bacteria change. Representative bacteria can be selected as biomarkers for radiation damage and repair.This may contribute to the development of radiation resistance in the future.

Gut Microbiome Dysbiosis in Patients with Endometrial Cancer vs. Healthy Controls Based on 16S rRNA Gene Sequencing.

Metabolic diseases like obesity, diabetes, and hypertension are considered major risk factors associated with endometrial cancer. Considering that an imbalance in the gut microbiome may lead to metabolic alterations, we hypothesized that alteration in the gut microbioma might be an indirect factor in the development of endometrial cancer. Our aim was to profile the gut microbiota of patients with endometrial cancer compared with healthy controls in this study. Thus, we used 16S rRNA high-throughput gene sequencing on the Illumina NovaSeq platform to profile microbial communities. Fecal samples were collected from 33 endometrial cancer patients (EC group) and 32 healthy controls (N group) between February 2021 and July 2021. The total numbers of operational taxonomic units (OTUs) in the N and EC groups were 28,537 and 18,465, respectively, while the number of OTUs shared by the two groups was 4771. This study was the first to report that the alpha diversity of the gut microbiota was significantly reduced in endometrial cancer patients vs. healthy controls. Also, there was a significant difference in the distribution of microbiome between the two groups: the abundance of Firmicutes, Clostridia, Clostridiales, Ruminococcaceae, Faecalibacterium, and Gemmiger_formicis decreased, while that of Proteobacteria, Gammaproteobacteria, Enterobacteriales, Enterobacteriaceae and Shigella increased significantly in the EC group vs. healthy controls (all p < 0.05). The predominant intestinal microbiota of the endometrial cancer patients was Proteobacteria, Gammaproteobacteria, Enterobacteriales, Enterobacteriaceae, and Shigella. These results imply that adjusting the composition of the gut microbiota and maintaining microbiota homeostasis may be an effective strategy for preventing and treating endometrial cancer.

Crohn's disease recurrence updates: first surgery vs. surgical relapse patients display different profiles of ileal microbiota and systemic microbial-associated inflammatory factors.

Background and aims: Crohn's disease (CD) pathogenesis is still unclear. Remodeling in mucosal microbiota and systemic immunoregulation may represent an important component in tissue injury. Here, we aim to characterize the ileal microbiota in both pathological and healthy settings and to evaluate the correlated systemic microbial-associated inflammatory markers comparing first-time surgery and relapse clinical conditions. Methods: We enrolled 28 CD patients at surgery; we collected inflamed and non-inflamed mucosa tissues and blood samples from each patient. Bacterial wall adherence was observed histologically, while its composition was assessed through amplicon sequencing of the 16S rRNA gene. In addition, we evaluated the systemic microRNA (miRNA) using quantitative real-time PCR amplification and free fatty acids (FFAs) using gas chromatography-mass spectroscopy. Results: The total number of mucosal adherent microbiota was enriched in healthy compared to inflamed mucosa. In contrast, the phylum Tenericutes, the family Ruminococcaceae, and the genera Mesoplasma and Mycoplasma were significantly enriched in the pathological setting. Significant microbiota differences were observed between the relapse and first surgery patients regarding the families Bacillaceae 2 and Brucellaceae and the genera Escherichia/Shigella, Finegoldia, Antrobacter, Gemmatimonas, Moraxella, Anoxibacillus, and Proteus. At the systemic level, we observed a significant downregulation of circulating miR-155 and miR-223, as well as 2-methyl butyric, isobutyric, and hexanoic (caproic) acids in recurrence compared to the first surgery patients. In addition, the level of hexanoic acid seems to act as a predictor of recurrence risk in CD patients (OR 18; 95% confidence interval 1.24-261.81; p = 0.006). Conclusions: We describe a dissimilarity of ileal microbiota composition comparing CD and healthy settings, as well as systemic microbial-associated inflammatory factors between first surgery and surgical relapse. We suggest that patterns of microbiota, associated with healthy ileal tissue, could be involved in triggering CD recurrence. Our findings may provide insight into the dynamics of the gut microbiota-immunity axis in CD surgical recurrence, paving the way for new diagnostics and therapeutics aimed not only at reducing inflammation but also at maintaining a general state of eubiosis in healthy tissue.

Analysis of Urinary Flora Characteristics in Urinary Tumor Based on 16S rRNA Sequence.

The relationship between urinary system tumors and urothelial microorganisms remains unexplored. This study is aimed at exploring the relationship between urinary flora and urinary tumors and identifying potential biomarkers for urinary tumors and new targets for prevention. We included four healthy adults (control group) and six patients diagnosed with urinary tract tumors (tumor group). In both groups, 10 and 50 ml clean middle urine samples were reserved. The 10 ml samples were analyzed (including pH, specific gravity, and leukocytes) using an automatic urine analyzer, and the 50 ml samples were analyzed by DNA extraction, 16S rRNA gene amplification, and high-throughput sequencing. The correlation between routine urine analysis and sequencing results was also analyzed. Testing using the DESeq2 method showed that, at the order level, there were significant differences in the abundance of Caulobacterales between the urinary flora of the two groups (P < 0.05); family level, Bacteroidaceae, Actinomycetaceae, and Tsukamurellaceae (P < 0.05); genus level, Finegoldia, Varibaculum, Actinobaculum, Propionimicrobium, Bacteroides, Brevundimonas, and Tsukamurella (P < 0.05). LEfSe analysis found specific bacteria at the genus level in the urinary flora of the tumor group, namely, Finegoldia (genus Digestiflora) (P < 0.001) and Varibaculum (P < 0.001). Further correlation analysis showed that both species were positively correlated with the urine pH (P < 0.05). PICRUSt analysis showed significant differences in the two functional pathways of cell transformation and metabolism (P < 0.05). Combined with the results of bioinformatics analysis, some differential bacteria may be new biomarkers for urologic tumors, and there may be a correlation between urine pH and tumor occurrence. However, large-scale prospective studies and in vitro and in vivo experiments are required to further test and verify these findings.

First reported case of Lachnoanaerobaculum gingivalis bacteremia in an acute myeloid leukemia patient with oral mucositis during high dose chemotherapy.

CASE DESCRIPTION: Lachnoanaerobaculum gingivalis is an obligate anaerobe identified in a human dental plaque in 2019. Here, we report the first case of L. gingivalis bacteremia in a patient with oral mucositis during chemotherapy. L. gingivalis was confirmed by 16S rRNA gene analysis but not by MALDI-TOF-MS. CONCLUSION: During chemotherapy in patients with oral mucositis, we should consider the possibility of L. gingivalis bacteremia.

Profiling gut microbiota and bile acid metabolism in critically ill children.

Broad-spectrum antimicrobial use during the treatment of critical illness influences gastrointestinal fermentation endpoints, host immune response and metabolic activity including the conversion of primary to secondary bile acids. We previously observed reduced fermentation capacity in the faecal microbiota of critically ill children upon hospital admission. Here, we further explore the timecourse of the relationship between the microbiome and bile acid profile in faecal samples collected from critically ill children. The microbiome was assayed by sequencing of the 16S rRNA gene, and faecal water bile acids were measured by liquid chromatography mass spectrometry. In comparison to admission faecal samples, members of the Lachnospiraceae recovered during the late-acute phase (days 8-10) of hospitalisation. Patients with infections had a lower proportion of Lachnospiraceae in their gut microbiota than controls and patients with primary admitting diagnoses. Keystone species linked to ecological recovery were observed to decline with the length of PICU admission. These species were further suppressed in patients with systemic infection, respiratory failure, and undergoing surgery. Bile acid composition recovers quickly after intervention for critical illness which may be aided by the compositional shift in Lachnospiraceae. Our findings suggest gut microbiota recovery can be readily assessed via measurement of faecal bile acids.

The Prebiotic Effect of Australian Seaweeds on Commensal Bacteria and Short Chain Fatty Acid Production in a Simulated Gut Model.

Diet is known to affect the composition and metabolite production of the human gut microbial community, which in turn is linked with the health and immune status of the host. Whole seaweeds (WH) and their extracts contain prebiotic components such as polysaccharides (PS) and polyphenols (PP). In this study, the Australian seaweeds, Phyllospora comosa, Ecklonia radiata, Ulva ohnoi, and their PS and PP extracts were assessed for potential prebiotic activities using an in vitro gut model that included fresh human faecal inoculum. 16S rRNA sequencing post gut simulation treatment revealed that the abundance of several taxa of commensal bacteria within the phylum Firmicutes linked with short chain fatty acid (SCFA) production, and gut and immune function, including the lactic acid producing order Lactobacillales and the chief butyrate-producing genera Faecalibacteria, Roseburia, Blautia, and Butyricicoccus were significantly enhanced by the inclusion of WH, PS and PP extracts. After 24 h fermentation, the abundance of total Firmicutes ranged from 57.35−81.55% in the WH, PS and PP samples, which was significantly greater (p ≤ 0.01) than the inulin (INU) polysaccharide control (32.50%) and the epigallocatechingallate (EGCG) polyphenol control (67.13%); with the exception of P. comosa PP (57.35%), which was significantly greater than INU only. However, all WH, PS and PP samples also increased the abundance of the phylum Proteobacteria; while the abundance of the phylum Actinobacteria was decreased by WH and PS samples. After 24 h incubation, the total and individual SCFAs present, including butyric, acetic and propionic acids produced by bacteria fermented with E. radiata and U. ohnoi, were significantly greater than the SCFAs identified in the INU and EGCG controls. Most notably, total SCFAs in the E. radiata PS and U. ohnoi WH samples were 227.53 and 208.68 µmol/mL, respectively, compared to only 71.05 µmol/mL in INU and 7.76 µmol/mL in the EGCG samples. This study demonstrates that whole seaweeds and their extracts have potential as functional food ingredients to support normal gut and immune function.

Gut Microbiome and Metabolome Changes in Mice With Acute Vestibular Deficit.

Vestibular deficit is a very common disorder in clinical practice and is characterized by vertigo, spontaneous nystagmus, and autonomic nervous symptoms, including nausea, vomiting, and sweating. In addition, the comorbidity of vestibular deficit and anxiety has long been an integral component of the medical literature. Previous studies have suggested that the mechanisms underlying this comorbidity involved overlap of vestibular and cerebellar networks. Emerging evidence has shown that the microbiota-gut-brain axis plays a key role in the regulation of affective disorders. Thus, we hypothesized that the gut microbiota may be involved in the comorbidity of vestibular deficit and anxiety. To verify this, we constructed a unilateral labyrinthectomy mouse model to simulate vestibular deficit. Then, 16S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC-MS) were used to analyze the microbiome and metabolome of the cecal samples collected from mice in the unilateral labyrinthectomy, sham surgery, and control groups. Notably, unilateral labyrinthectomy shaped the composition of the mouse gut microbiome, resulting in increased abundance of Lachnospiraceae NK4A136 group, Odoribacter and Roseburia and decreased abundance of Prevotella and Parasutterella at the genus level. Tax4Fun functional prediction indicated a decrease in tryptophan metabolism in mice in the unilateral labyrinthectomy group. Moreover, functional correlation of changes in gut microbes and metabolites between different groups showed that the oleamide level was negatively correlated with Odoribacter abundance (r = -0.89, p = 0.0002). The butyric acid level was positively correlated with Parasutterella abundance (r = 0.85, p = 0.0010). The propanoate level was negatively correlated with Prevotella abundance (r = -0.81, p = 0.0020). The 20-HETE level was positively correlated with Parasutterella abundance (r = 0.84, p = 0.0013). The altered microbes and metabolites were closely related to the pathogenesis of affective disorders. Our results not only offer novel insights into the vestibular deficit comorbid with anxiety but also build an important basis for future research on this etiology.

Firmicutes and Blautia in gut microbiota lessened in chronic liver diseases and hepatocellular carcinoma patients: a pilot study.

The gut microbiota system plays a vital role in liver diseases. This study aimed to address the diversity of gut microbiota and its correlations with clinical parameters in healthy individuals, chronic liver disease (CLD), and hepatocellular carcinoma (HCC) patients. Fecal specimens of nine healthy individuals, 11 CLD, and 21 HCC were collected. The diversity of gut microbiota was examined by PCR and Illumina MiSeq sequencing and analyzed using 16S rRNA gene sequencing database. The correlations between gut microbiota and the clinical parameters of participants were also addressed. Compared to healthy individuals, Firmicutes at a phylum level decreased in CLD and HCC patients and Proteobacteria increased (p < 0.05). The composition of Blautia on a genus level in CLD and HCC patients significantly decreased compared to healthy controls (p < 0.05). Firmicutes composition was negatively associated with age and number of males (p < 0.05) and was positively associated with monocytes, high-density lipoprotein cholesterol (HDL-C), and estimated glomerular filtration rate (eGFR) levels (p < 0.05). At a genus level, Blautia composition was negatively associated with cirrhosis, age, and number of males (p < 0.01), while it was positively associated with red blood cells (RBCs), triglycerides, HDL-C, and lymphocyte levels (p < 0.05). Conclusively, there was a significant compositional difference in gut microbiota in CLD and HCC patients compared with healthy subjects. Firmicutes and Blautia in gut microbiota system lessened in CLD and HCC patients. Clinical biochemical parameters have an impact on the diversity of gut microbiota in liver diseases.

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.

Screening of Human Gut Bacterial Culture Collection Identifies Species That Biotransform Quercetin into Metabolites with Anticancer Properties.

We previously demonstrated that flavonoid metabolites inhibit cancer cell proliferation through both CDK-dependent and -independent mechanisms. The existing evidence suggests that gut microbiota is capable of flavonoid biotransformation to generate bioactive metabolites including 2,4,6-trihydroxybenzoic acid (2,4,6-THBA), 3,4-dihydroxybenzoic acid (3,4-DHBA), 3,4,5-trihyroxybenzoic acid (3,4,5-THBA) and 3,4-dihydroxyphenylacetic acid (DOPAC). In this study, we screened 94 human gut bacterial species for their ability to biotransform flavonoid quercetin into different metabolites. We demonstrated that five of these species were able to degrade quercetin including Bacillus glycinifermentans, Flavonifractor plautii, Bacteroides eggerthii, Olsenella scatoligenes and Eubacterium eligens. Additional studies showed that B. glycinifermentans could generate 2,4,6-THBA and 3,4-DHBA from quercetin while F. plautii generates DOPAC. In addition to the differences in the metabolites produced, we also observed that the kinetics of quercetin degradation was different between B. glycinifermentans and F. plautii, suggesting that the pathways of degradation are likely different between these strains. Similar to the antiproliferative effects of 2,4,6-THBA and 3,4-DHBA demonstrated previously, DOPAC also inhibited colony formation ex vivo in the HCT-116 colon cancer cell line. Consistent with this, the bacterial culture supernatant of F. plautii also inhibited colony formation in this cell line. Thus, as F. plautii and B. glycinifermentans generate metabolites possessing antiproliferative activity, we suggest that these strains have the potential to be developed into probiotics to improve human gut health.

Mucosa-associated gut microbiota reflects clinical course of ulcerative colitis.

This longitudinal study was designed to elucidate whether gut microbiota is associated with relapse and treatment response in ulcerative colitis (UC) patients. Fifty-one patients with UC were enrolled between 2012 and 2017, and followed up through 2020. Colon mucosal biopsy were obtained at enrollment, and 16S ribosomal RNA sequencing was performed using extracted RNA. Of the 51 patients, 24 were in remission and 27 had active UC at enrollment. Of the 24 patients in remission, 17 maintained remission and 7 developed relapse during follow-up. The 7 patients with relapse showed lower diversity, with a lower proportion of Clostridiales (p = 0.0043), and a higher proportion of Bacteroides (p = 0.047) at enrollment than those without relapse. The 27 patients with active UC were classified into response (n = 6), refractory (n = 13), and non-response (n = 8) groups according to their treatment response in 6 months. The refractory and non-response groups showed lower diversity with a lower proportion of Prevotella (p = 0.048 and 0.043) at enrollment than the response group. This study is the first demonstration that reduced diversity and particular microbes are associated with the later clinical course of relapse events and treatment response in UC.

Aminipila terrae sp. nov., a strictly anaerobic bacterium isolated from river sediment.

In this study, aimed at investigating and characterizing river sediment bacteria, we isolated a Gram-stain-positive, rod-shaped, obligate anaerobic bacterium, strain CBA3637T, from the sediment of the Geum River. This strain grew at 10-40 °C (optimum, 30 °C), 0-1% NaCl (optimum, 0%), and pH 7-8 (optimum, pH 7). The 16S rRNA gene sequence comparison revealed Aminipila butyrica DSM 103574T to be the closest relative of strain CBA3637T (96.6-96.7% similarity); and both strains clustered together in phylogenetic analysis. The genome of strain CBA3637T was found to consist of a single chromosome (3.51 Mbp; 36.98% G + C content). Comparative genomic analysis of the strain CBA3637T with A. butyrica DSM 103574T revealed that strain CBA3637T possessed five unique pathways related to polyamine biosynthesis, lipopolysaccharide metabolism, pyrimidine metabolism, and cofactor and vitamin metabolism. Strain CBA3637T contained C14:0, C16:0, and C18:1 ω9c as the major fatty acids, and diphosphatidylglycerol as the major polar lipid. No respiratory quinone was observed. Biochemical, chemotaxonomic, and genotypic data revealed that the strain CBA3637T is a representative of a novel species within the genus Aminipila, for which the name Aminipila terrae is proposed. The type strain is CBA3637T (= KACC 21651T = DSM 110662T).

Affective disorders impact prevalence of Flavonifractor and abundance of Christensenellaceae in gut microbiota.

Background Affective disorders (AD) have been associated with a higher prevalence of the gut Flavonifractor genus and a lower abundance of the gut Christensenellaceae family. Objective and methods By pooling two independent study samples of patients with AD (n = 176), their unaffected first-degree relatives (n = 70) and healthy controls (n = 101) we aimed to replicate and extend our prior findings of differential Flavonifractor prevalence and Christensenellaceae abundance when comparing patients with AD and healthy controls. The gut microbiota was profiled using 16S rRNA gene amplicon sequencing. Results The pattern of higher prevalence of Flavonifractor and lower Centered Log-Ratio (CLR) abundance of Christensenellaceae was associated with AD. In generalized linear models the CLR abundance of Christensenellaceae was lower in patients with AD (p = 0.024), and in smokers (p = 1.9*10-4), and inversely associated with increasing waist circumference (p = 0.031). The prevalence of Flavonifractor was higher in patients with AD (p = 0.033) and in smokers (p = 0.036). No impact of psychotropic medication was found. The CLR abundance of Christensenellaceae (p = 0.041), but not the prevalence of Flavonifractor (p = 0.20) could distinguish non-smoking patients with AD from non-smoking healthy controls, whereas no such associations were found in smokers. Unaffected relatives neither differed from patients with AD nor from healthy controls. Conclusion Compared with findings in healthy controls, AD was associated with a significantly lower CLR abundance of the health-linked Christensenellaceae and a significantly higher prevalence of Flavonifractor; findings that are associated with enhanced oxidative stress and systemic low-grade inflammation. If our observations are validated in future independent studies, they support the notion that parts of aberrant gut microbiota are shared by AD and states of dysmetabolism.

Challenges Faced by Highly Polyploid Bacteria with Limits on DNA Inheritance.

Most studies of bacterial reproduction have centered on organisms that undergo binary fission. In these models, complete chromosome copies are segregated with great fidelity into two equivalent offspring cells. All genetic material is passed on to offspring, including new mutations and horizontally acquired sequences. However, some bacterial lineages employ diverse reproductive patterns that require management and segregation of more than two chromosome copies. Epulopiscium spp., and their close relatives within the Firmicutes phylum, are intestinal symbionts of surgeonfish (family Acanthuridae). Each of these giant (up to 0.6 mm long), cigar-shaped bacteria contains tens of thousands of chromosome copies. Epulopiscium spp. do not use binary fission but instead produce multiple intracellular offspring. Only ∼1% of the genetic material in an Epulopiscium sp. type B mother cell is directly inherited by its offspring cells. And yet, even in late stages of offspring development, mother-cell chromosome copies continue to replicate. Consequently, chromosomes take on a somatic or germline role. Epulopiscium sp. type B is a strict anaerobe and while it is an obligate symbiont, its host has a facultative association with this intestinal microorganism. Therefore, Epulopiscium sp. type B populations face several bottlenecks that could endanger their diversity and resilience. Multilocus sequence analyses revealed that recombination is important to diversification in populations of Epulopiscium sp. type B. By employing mechanisms common to others in the Firmicutes, the coordinated timing of mother-cell lysis, offspring development and congression may facilitate the substantial recombination observed in Epulopiscium sp. type B populations.

Discovery of an ene-reductase for initiating flavone and flavonol catabolism in gut bacteria.

Gut microbial transformations of flavonoids, an enormous class of polyphenolic compounds abundant in plant-based diets, are closely associated with human health. However, the enzymes that initiate the gut microbial metabolism of flavones and flavonols, the two most abundant groups of flavonoids, as well as their underlying molecular mechanisms of action remain unclear. Here, we discovered a flavone reductase (FLR) from the gut bacterium, Flavonifractor plautii ATCC 49531 (originally assigned as Clostridium orbiscindens DSM 6740), which specifically catalyses the hydrogenation of the C2-C3 double bond of flavones/flavonols and initiates their metabolism as a key step. Crystal structure analysis revealed the molecular basis for the distinct catalytic property of FLR. Notably, FLR and its widespread homologues represent a class of ene-reductases that has not been previously identified. Genetic and biochemical analyses further indicated the importance of FLR in gut microbial consumption of dietary and medicinal flavonoids, providing broader insight into gut microbial xenobiotic transformations and possible guidance for personalized nutrition and medicine.

Perturbation of the primary acceptor chlorophyll site in the heliobacterial reaction center by coordinating amino acid substitution.

All known Type I photochemical reaction center protein complexes contain a form of the pigment chlorophyll a in their primary electron acceptor site (termed ec3). In the reaction center from the primitive heliobacteria (HbRC), all of the pigment cofactors are bacteriochlorophyll g except in the ec3 sites, which contain 81-hydroxychlorophyll a. To explore the energetic flexibility of this site, we performed site-directed mutagenesis on two of the amino acids of the PshA core polypeptide responsible for coordinating the 81-hydroxychlorophyll a. These two amino acids are serine-545, which coordinates the central Mg(II) through an intermediary water molecule, and serine-553, which participates in a hydrogen bond with the 131-keto O atom. Mutagenesis of serine-545 to histidine (S545H) changes how the chlorophyll's central Mg(II) is coordinated, with the result of decreasing the chlorophyll's site energy. Mutagenesis of serine-545 to methionine (S545M), which was made to mimic the ec3 site of Photosystem I, abolishes chlorophyll binding and charge separation altogether. Mutagenesis of serine-553 to alanine (S553A) removes the aforementioned hydrogen bond, increasing the site energy of the chlorophyll. In the S545H and S553A mutants, the forward and reverse electron transfer rates from ec3 are both faster. This coincides with a decrease in both the quantum yield of initial charge separation and the overall photochemical quantum yield. Taken together, these data indicate that wild-type HbRC is optimized for overall photochemical efficiency, rather than just for maximizing the forward electron transfer rate. The necessity for a chlorophyll a derivative at the ec3 site is also discussed.

Classification of Changes in the Fecal Microbiota Associated with Colonic Adenomatous Polyps Using a Long-Read Sequencing Platform.

The microbiota is the community of microorganisms that colonizes the oral cavity, respiratory tract, and gut of multicellular organisms. The microbiota exerts manifold physiological and pathological impacts on the organism it inhabits. A growing body of attention is being paid to host-microbiota interplay, which is highly relevant to the development of carcinogenesis. Adenomatous polyps are considered a common hallmark of colorectal cancer, the second leading cause of carcinogenesis-mediated death worldwide. In this study, we examined the relevance between targeted operational taxonomic units and colonic polyps using short- and long-read sequencing platforms. The gut microbiota was assessed in 132 clinical subjects, including 53 healthy participants, 36 patients with occult blood in the gut, and 43 cases with adenomatous polyps. An elevation in the relative abundance of Klebsiella pneumonia, Fusobacterium varium, and Fusobacterium mortiferum was identified in patients with adenomatous polyps compared with the other groups using long-read sequencing workflow. In contrast, the relatively high abundances of Blautia luti, Bacteroides plebeius, and Prevotella copri were characterized in the healthy groups. The diversities in gut microbiota communities were similar in all recruited samples. These results indicated that alterations in gut microbiota were characteristic of participants with adenomatous polyps, which might be relevant to the further development of CRC. These findings provide a potential contribution to the early prediction and interception of CRC occurrence.

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.