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Members of the conserved Argonaute protein family use small RNA guides to locate their mRNA targets and regulate gene expression and suppress mobile genetic elements in eukaryotes 1 , 2 . Argonautes are also present in many bacterial and archaeal species 3 – 5 . Unlike eukaryotic proteins, several prokaryotic Argonaute proteins use small DNA guides to cleave DNA, a process known as DNA interference 6 – 10 . However, the natural functions and targets of DNA interference are poorly understood, and the mechanisms of DNA guide generation and target discrimination remain unknown. Here we analyse the activity of a bacterial Argonaute nuclease from Clostridium butyricum ( Cb Ago) in vivo. We show that Cb Ago targets multicopy genetic elements and suppresses the propagation of plasmids and infection by phages. Cb Ago induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. The loading of Cb Ago with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interaction was reported for the acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes that encode Ago nucleases are enriched in CRISPR–Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest that the recognition of foreign nucleic acids by prokaryotic defence systems involves common principles. Argonaute protein from the bacterium C. butyricum targets multicopy genetic elements and functions in the suppression of plasmid and phage propagation, and there appears to be a DNA-mediated immunity pathway in prokaryotes.

Clostridium butyricum inhabits various anoxic environments, including soil and the human gut. Here, this common bacterium comes into contact with abundant plant-derived flavonoids. Metabolization of these bioactive polyphenols has been studied in recent years, particularly focusing on gut bacteria due to the proposed health-promoting properties of these dietary constituents. Based on an initial report in 1997 on eriodictyol degradation (Miyake et al. 1997, J Agric Food Chem, 45:3738–3742), the present study systematically investigated C. butyricum for its ability to convert a set of structurally diverse flavonoids. Incubation experiments revealed that C. butyricum deglycosylated flavonoid O -glucosides but only when glucose was absent. Moreover, aglycone members of flavone, flavanone, dihydrochalcone, and flavanonol subclasses were degraded. The C-ring cleavage of the flavanones, naringenin and eriodictyol, was stereospecific and finally resulted in formation of the corresponding hydroxyphenylpropionic acids. Stereospecific C-ring cleavage of the flavanonol taxifolin led to taxifolin dihydrochalcone. C. butyricum did neither cleave flavonols and isoflavones nor catalyze de-rhamnosylation, demethylation, or dehydroxylation of flavonoids. Genes encoding potential flavonoid-metabolizing enzymes were detected in the C. butyricum genome. Overall, these findings indicate that C. butyricum utilizes flavonoids as alternative substrates and, as observed for the dihydrochalcone phloretin, can eliminate growth-inhibiting flavonoids through degradation. Key points • Clostridium butyricum deglycosylated flavonoid O-glucosides. • Clostridium butyricum converted members of several flavonoid subclasses. • Potential flavonoid-metabolizing enzymes are encoded in the C. butyricum genome.

Background The extracellular vesicles (EVs) traffic constitutes an essential pathway of cellular communication. And the molecules in EVs produced by procaryotes help in maintaining homeostasis, addressing microbial imbalance and infections, and regulating the immune system. Despite the fact that Clostridium butyricum ( C. butyricum ) is commonly used for treating ulcerative colitis (UC), the potential role of C. butyricum -secreted EVs in commensals-host crosstalk remains unclear. Results Here, we performed flow cytometry, western blot, immunohistochemistry and 16S rRNA analysis to explore the role of C. butyricum -derived EVs on macrophage polarization and gut microbiota composition in a dextran sulfate sodium (DSS)-induced UC mouse model. The antibiotic cocktail-induced microbiome depletion and faecal transplantations were used to further investigate the mechanisms by which EVs regulate macrophage balance. Our findings showed that C. butyricum -derived EVs improved the remission of murine colitis and polarized the transformation of macrophages to the M2 type. Furthermore, C. butyricum -derived EVs restored gut dysbiosis and altered the relative abundance of Helicobacter, Escherichia-Shigella, Lactobacillus , Akkermansia and Bacteroides, which, in turn, faecal transplantations from EVs-treated mice relieved the symptoms of UC and improved the impact of EVs on the reprogramming of the M2 macrophages. Conclusion C. butyricum -derived EVs could protect against DSS-induced colitis by regulating the repolarization of M2 macrophages and remodelling the composition of gut microbiota, suggesting the potential efficacy of EVs from commensal and probiotic Clostridium species against UC.

Clostridium butyricum, a probiotic commonly prescribed in Asia, most notably as MIYA-BM (Miyarisan Pharmaceutical Co., Ltd.; https://www.miyarisan.com), occasionally leads to bacteremia. The prevalence and characteristics of C. butyricum bacteremia and its bacteriologic and genetic underpinnings remain unknown. We retrospectively investigated patients admitted to Osaka University Hospital during September 2011-February 2023. Whole-genome sequencing revealed 5 (0.08%) cases of C. butyricum bacteremia among 6,576 case-patients who had blood cultures positive for any bacteria. Four patients consumed MIYA-BM, and 1 patient consumed a different C. butyricum-containing probiotic. Most patients had compromised immune systems, and common symptoms included fever and abdominal distress. One patient died of nonocclusive mesenteric ischemia. Sequencing results confirmed that all identified C. butyricum bacteremia strains were probiotic derivatives. Our findings underscore the risk for bacteremia resulting from probiotic use, especially in hospitalized patients, necessitating judicious prescription practices.

Lactate-utilizing bacteria (LUB) are intestinal bacteria that produce butyrate from lactate and acetate, key metabolites in the gut. As LUB help maintain lactate and butyrate concentrations in the intestinal tract, they are promising probiotic candidates. Clostridium butyricum TO-A (CBTOA) has reportedly been effective in treating various gastrointestinal issues in humans and animals. Although CBTOA is known to increase intestinal butyrate levels, it is unclear how it utilizes lactate and acetate, similar to LUB, to produce butyrate. We investigated lactate utilization-related genes in CBTOA and examined the relationship between lactate and acetate utilization and butyrate production using peptone–yeast medium supplemented with D-lactate, L-lactate, and/or acetate. This study demonstrates for the first time that the probiotic strain CBTOA harbors lactate utilization-related genes and efficiently produces butyrate only in the presence of exogenous lactate and acetate instead of sugars. Furthermore, CBTOA expresses a lactate racemase that enables the bacterium to utilize both lactate enantiomers while regulating the ratio of D-lactate to L-lactate in the intestinal microenvironment via racemization. In conclusion, CBTOA efficiently produces butyrate utilizing lactate and acetate, similar to LUB; therefore, CBTOA could be an efficient butyrate supplier as a probiotic strain in the intestinal tract.

Background. Necrotizing enterocolitis (NEC) is the most common and serious gastrointestinal disorder among preterm neonates. We aimed to assess a specific gut microbiota profile associated with NEC. Methods. Stool samples and clinical data were collected from 4 geographically independent neonatal intensive care units, over a 48-month period. Thirty stool samples from preterm neonates with NEC (n = 15) and controls (n = 15) were analyzed by 16S ribosomal RNA pyrosequencing and culture-based methods. The results led us to develop a specific quantitative polymerase chain reaction (qPCR) assay for Clostridium butyricum, and we tested stool samples from preterm neonates with NEC (n = 93) and controls (n = 270). We sequenced the whole genome of 16 C. butyricum strains, analyzed their phylogenetic relatedness, tested their culture supernatants for cytotoxic activity, and searched for secreted toxins. Results. Clostridium butyricum was specifically associated with NEC using molecular and culture-based methods (15/15 vs 2/15; P < .0001) or qPCR (odds ratio, 45.4 [95% confidence interval, 26.2–78.6]; P < .0001). Culture supernatants of C. butyricum strains from preterm neonates with NEC (n = 14) exhibited significant cytotoxic activity (P = .008), and we identified in all a homologue of the β-hemolysin toxin gene shared by Brachyspira hyodysenteriae, the etiologic agent of swine dysentery. The corresponding protein was secreted by a NEC-associated C. butyricum strain. Conclusions. NEC was associated with C. butyricum strains and dysbiosis with an oxidized, acid, and poorly diversified gut microbiota. Our findings highlight the plausible toxigenic mechanism involved in the pathogenesis of NEC.

Background Clostridium spp. has demonstrated therapeutic potential in cancer treatment through intravenous or intratumoral administration. This approach has expanded to include non-pathogenic clostridia for the treatment of various diseases, underscoring the innovative concept of oral-spore vaccination using clostridia. Recent advancements in the field of synthetic biology have significantly enhanced the development of Clostridium -based bio-therapeutics. These advancements are particularly notable in the areas of efficient protein overexpression and secretion, which are crucial for the feasibility of oral vaccination strategies. Here, we present two examples of genetically engineered Clostridium candidates: one as an oral cancer vaccine and the other as an antiviral oral vaccine against SARS-CoV-2. Results Using five validated promoters and a signal peptide derived from Clostridium sporogenes , a series of full-length NY-ESO-1/CTAG1, a promising cancer vaccine candidate, expression vectors were constructed and transformed into C. sporogenes and Clostridium butyricum . Western blotting analysis confirmed efficient expression and secretion of NY-ESO-1 in clostridia, with specific promoters leading to enhanced detection signals. Additionally, the fusion of a reported bacterial adjuvant to NY-ESO-1 for improved immune recognition led to the cloning difficulties in E. coli . The use of an AUU start codon successfully mitigated potential toxicity issues in E. coli , enabling the secretion of recombinant proteins in C. sporogenes and C. butyricum . We further demonstrate the successful replacement of PyrE loci with high-expression cassettes carrying NY-ESO-1 and adjuvant-fused NY-ESO-1, achieving plasmid-free clostridia capable of secreting the antigens. Lastly, the study successfully extends its multiplex genetic manipulations to engineer clostridia for the secretion of SARS-CoV-2-related Spike_S1 antigens. Conclusions This study successfully demonstrated that C. butyricum and C. sporogenes can produce the two recombinant antigen proteins (NY-ESO-1 and SARS-CoV-2-related Spike_S1 antigens) through genetic manipulations, utilizing the AUU start codon. This approach overcomes challenges in cloning difficult proteins in E. coli . These findings underscore the feasibility of harnessing commensal clostridia for antigen protein secretion, emphasizing the applicability of non-canonical translation initiation across diverse species with broad implications for medical or industrial biotechnology.

In this study, Clostridium butyricum TO-A culture supernatant (CBCS) or butyric acid was added to a culture medium of human cervical carcinoma HeLa S3 cells, and changes in DNA-repair-related gene promoter activities were investigated. The HeLa S3 cells were transfected with a luciferase (Luc) expression vector containing approximately 500 bp of the 5′-upstream region of several human DNA-repair-related genes and cultured with a medium containing the CBCS (10%) or butyric acid (2.5 mM). The cells were harvested after 19 to 42 h of incubation. A Luc assay revealed that the human ATM, PARG, PARP1, and RB1 gene promoter activities were significantly increased. A Western blot analysis showed that the amounts of the proteins encoded by these genes markedly increased. Furthermore, 8, 24, and 48 h after the addition of the CBCS (10%), total RNA was extracted and subjected to RNAseq analysis. The results showed that the expression of several inflammation- and DNA-replication/repair-related genes, including NFKB and the MCM gene groups, decreased markedly after 8 h. However, the expression of the histone genes increased after 24 h. Elucidation of the mechanism by which the CBCS and butyrate affect the expression of genes that encode DNA-repair-associated proteins may contribute to the prevention of carcinogenesis, the risk of which rises in accordance with aging.

Previously, a whole-genome comparison of three Clostridium butyricum type E strains from Italy and the United States with different C. botulinum type E strains indicated that the bont/e gene might be transferred between the two clostridia species through transposition. However, transposable elements (TEs) have never been identified close to the bont/e gene. Herein, we report the whole genome sequences for four neurotoxigenic C. butyricum type E strains that originated in China. An analysis of the obtained genome sequences revealed the presence of a novel putative TE upstream of the bont/e gene in the genome of all four strains. Two strains of environmental origin possessed an additional copy of the putative TE in their megaplasmid. Similar putative TEs were found in the megaplasmids and, less frequently, in the chromosomes of several C. butyricum strains, of which two were neurotoxigenic C. butyricum type E strains, and in the chromosome of a single C. botulinum type E strain. We speculate that the putative TE might potentially transpose the bont/e gene at the intracellular and inter-cellular levels. However, the occasional TE occurrence in the clostridia genomes might reflect rare transposition events.

Background Biohydrogen production from agro-industrial wastes through dark fermentation offers several advantages including eco-friendliness, sustainability, and the simplicity of the process. This study aimed to produce biohydrogen from fruit and vegetable peel wastes (FVPWs) by anaerobic fermentative bacteria isolated from domestic wastewater. Kinetic analysis of the produced biohydrogen by five isolates on a glucose medium was analyzed using a modified Gompertz model (MGM). Besides, the feasibility of hydrogen production by Clostridium butyricum NE95 using FVPWs as substrates was investigated. Results The bacterial isolate NE95 was selected as the highest biohydrogen producer with maximum biohydrogen production (H max ) of 1617.67 ± 3.84 mL/L, R max (MGM) of 870.77 mL/L/h and lag phase (λ) of 28.37 h. NE95 was phenotypically and genetically identified as C. butyricum and its 16 S rRNA gene sequence was deposited in the GenBank under the accession number PP581833. The genetic screening of hydrogenase gene clusters indicated the presence of Fe-Fe hydrogenase gene in C. butyricum NE95. C. butyricum NE95 showed the ability to produce biohydrogen from different FVPWs, with watermelon and melon peels being the most promising feedstocks for fermentation. It was revealed that using a mixture (1:1, w/w) of watermelon and melon peels as a substrate for C. butyricum NE95 significantly increased biohydrogen yield with H max of 991.00 ± 10.54 mL/L, R max of 236.31 mL/L/h, λ of 33.92 h and a high accuracy of R 2 (0.997). Conclusions The study highlights the effectiveness of C. butyricum NE95 on the valorization of FVPWs and generates a sustainable source of biohydrogen production.