Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.

A metabolic pathway for bile acid dehydroxylation by the gut microbiome.

The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 µM and are known to block the growth of Clostridium difficile1, promote hepatocellular carcinoma2 and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. 3). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids4; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool.

Tuning strand displacement kinetics enables programmable ZTP riboswitch dynamic range in vivo.

A large body of work has shown that transcriptional riboswitches function through internal strand displacement mechanisms that guide the formation of alternative structures which drive regulatory outcomes. Here, we sought to investigate this phenomenon using the Clostridium beijerinckii pfl ZTP riboswitch as a model system. Using functional mutagenesis with Escherichia coli gene expression assays, we show that mutations designed to slow strand displacement of the expression platform enable precise tuning of riboswitch dynamic range (2.4-34-fold), depending on the type of kinetic barrier introduced, and the position of the barrier relative to the strand displacement nucleation site. We also show that expression platforms from a range of different Clostridium ZTP riboswitches contain sequences that impose these barriers to affect dynamic range in these different contexts. Finally, we use sequence design to flip the regulatory logic of the riboswitch to create a transcriptional OFF-switch, and show that the same barriers to strand displacement tune dynamic range in this synthetic context. Together, our findings further elucidate how strand displacement can be manipulated to alter the riboswitch decision landscape, suggesting that this could be a mechanism by which evolution tunes riboswitch sequence, and providing an approach to optimize synthetic riboswitches for biotechnology applications.

Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR.

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.

Genomic characterization of prophage elements in Clostridium clostridioforme: an understudied component of the intestinal microbiome.

Genome sequencing of Clostridium clostridioforme strain LM41 revealed the presence of an atypically high proportion of mobile genetic elements for this species, with a particularly high abundance of prophages. Bioinformatic analysis of prophage sequences sought to characterize these elements and identify prophage-linked genes contributing to enhanced fitness of the host bacteria in the dysbiotic gut. Using PHASTER, PhageScope and manual curation, this work has identified 15 prophages: 4 predicted to be intact, 2 predicted to be defective and 9 which are unclassified. Quantitative PCR (qPCR) analysis revealed spontaneous release of four of the LM41 prophages (φ1, φ2, φ4 and φ10) into the culture supernatant, with virion-like particles visualized using transmission electron microscopy. The majority (12/14) of these particles had morphology akin to podoviruses, which is consistent with morphology predictions for φ1 and φ4. We observed diversity in the lysogeny mechanisms utilized by the prophages, with examples of the classical λ-like CI/Cro system, the ICEBs1 ImmR/ImmA-like system and the Mu-like C/Ner system. Classical morons, such as toxins or immune evasion factors, were not observed. We did, however, identify a variety of genes with roles in mediating restriction modification and genetic diversity, as well as some candidate genes with potential roles in host adaptation. Despite being the most abundant entities in the intestine, there is a dearth of information about phages associated with members of the microbiome. This work begins to shed light on the contribution of these elements to the lifestyle of C. clostridioforme LM41.

Clostridium neonatale antimicrobial susceptibility, genetic resistance determinants, and genotyping: a multicentre spatiotemporal retrospective analysis.

BACKGROUND: Clostridium neonatale was isolated during an outbreak of neonatal necrotizing enterocolitis (NEC) in 2002. C. neonatale was validated as a new species within the genus Clostridium sensu stricto in 2018. In the present study, we evaluated the antimicrobial susceptibility, genetic determinants of resistance, and phylogenetic relationships of a collection of clinical isolates of C. neonatale. METHODS: C. neonatale strains (n = 68) were isolated from the stools of preterm neonates who either developed NEC or were asymptomatic carriers of C. neonatale in different periods and in different hospitals. Antimicrobial susceptibility was determined by the disc diffusion method. The MICs of clindamycin, cefotaxime and tetracycline were determined. Genetic determinants of resistance were screened by PCR (n = 68) and WGS (n = 35). Genotyping of the isolates was performed by MLST. RESULTS: Antimicrobial resistance was found to clindamycin (n = 24; 35%), cefotaxime (n = 7; 10%) and tetracycline (n = 1; 1%). One clindamycin-resistant isolate carried erm(B) by PCR. In addition, one isolate carrying tet(M) was tetracycline resistant (MIC = 16 mg/L) and 44 isolates carrying either tet(O), tet(32) or tet(M) were tetracycline susceptible (MICs < 16 mg/L). MLST showed that ST2 and ST15 were significantly associated with tet(32) (P < 0.0001) and tet(O) (P < 0.0001), respectively. From WGS, we identified aph(3')-IIa and blaTEM-116 genes and a blaCBP-1-like gene. CONCLUSIONS: C. neonatale is susceptible to anti-anaerobic molecules but resistant to clindamycin, cefotaxime and tetracycline. Genes encoding tetracycline ribosomal protection, macrolide-lincosamide-streptogramin B rRNA methyltransferase, aminoglycoside 3'-phosphotransferase and ß-lactamases have been identified in genomic regions flanked by mobile genetic elements.

A novel glycoside hydrolase 43-like enzyme from Clostridium boliviensis is an endo-xylanase and a candidate for xylooligosaccharide production from different xylan substrates.

An uncharacterized gene encoding a glycoside hydrolase family 43-like enzyme from Clostridium boliviensis strain E-1 was identified from genomic sequence data, and the encoded enzyme, CbE1Xyn43-l, was produced in Escherichia coli. CbE1Xyn43-l (52.9 kDa) is a two-domain endo-ß-xylanase consisting of a C-terminal CBM6 and a GH43-like catalytic domain. The positions of the catalytic dyad conserved in GH43, the catalytic base (Asp74), and proton donor (Glu240) were identified in alignments including GH43-enzymes of known 3D-structure from different subfamilies. CbE1Xyn43-l is active at pH 7.0-9.0, with optimum temperature at 65°C, and a more than 7 days' half-life in irreversible deactivation studies at this temperature. The enzyme hydrolyzed birchwood xylan, quinoa stalks glucuronoarabinoxylan, and wheat arabinoxylan with xylotriose and xylotetraose as major hydrolysis products. CbE1Xyn43-l also released xylobiose from pNPX2 with low turnover (kcat of 0.044 s-1) but was inactive on pNPX, showing that a degree of polymerization of three (DP3) was the smallest hydrolyzable substrate. Divalent ions affected the specific activity on xylan substrates, which dependent on the ion could be increased or decreased. In conclusion, CbE1Xyn43-l from C. boliviensis strain E-1 is the first characterized member of a large group of homologous hypothetical proteins annotated as GH43-like and is a thermostable endo-xylanase, producing xylooligosaccharides of high DP (xylotriose and xylotetraose) producer. IMPORTANCE: The genome of Clostridium boliviensis strain E-1 encodes a number of hypothetical enzymes, annotated as glycoside hydrolase-like but not classified in the Carbohydrate Active Enzyme Database (CAZy). A novel thermostable GH43-like enzyme is here characterized as an endo-ß-xylanase of interest in the production of prebiotic xylooligosaccharides (XOs) from different xylan sources. CbE1Xyn43-l is a two-domain enzyme composed of a catalytic GH43-l domain and a CBM6 domain, producing xylotriose as main XO product. The enzyme has homologs in many related Clostridium strains which may indicate a similar function and be a previously unknown type of endo-xylanase in this evolutionary lineage of microorganisms.

Transcriptomic characterization of recombinant Clostridium beijerinckii NCIMB 8052 expressing methylglyoxal synthase and glyoxal reductase from Clostridium pasteurianum ATCC 6013.

Bioconversion of abundant lactose-replete whey permeate to value-added chemicals holds promise for valorization of this expanding food processing waste. Efficient conversion of whey permeate-borne lactose requires adroit microbial engineering to direct carbon to the desired chemical. An engineered strain of Clostridium beijerinckii NCIMB 8052 (C. beijerinckii_mgsA+mgR) that produces 87% more butanol on lactose than the control strain was assessed for global transcriptomic changes. The results revealed broadly contrasting gene expression patterns in C. beijerinckii_mgsA+mgR relative to the control strain. These were characterized by widespread decreases in the abundance of mRNAs of Fe-S proteins in C. beijerinckii_mgsA+mgR, coupled with increased differential expression of lactose uptake and catabolic genes, iron uptake genes, two-component signal transduction and motility genes, and genes involved in the biosynthesis of vitamins B5 and B12, aromatic amino acids (particularly tryptophan), arginine, and pyrimidines. Conversely, the mRNA patterns suggest that the L-aspartate-dependent de novo biosynthesis of NAD as well as biosynthesis of lysine and asparagine and metabolism of glycine and threonine were likely down-regulated. Furthermore, genes involved in cysteine and methionine biosynthesis and metabolism, including cysteine desulfurase-a central player in Fe-S cluster biosynthesis-equally showed reductions in mRNA abundance. Genes involved in biosynthesis of capsular polysaccharides and stress response also showed reduced mRNA abundance in C. beijerinckii_mgsA+mgR. The results suggest that remodeling of cellular and metabolic networks in C. beijerinckii_mgsA+mgR to counter anticipated effects of methylglyoxal production from heterologous expression of methylglyoxal synthase led to enhanced growth and butanol production in C. beijerinckii_mgsA+mgR. IMPORTANCE: Biological production of commodity chemicals from abundant waste streams such as whey permeate represents an opportunity for decarbonizing chemical production. Whey permeate remains a vastly underutilized feedstock for bioproduction purposes. Thus, enhanced understanding of the cellular and metabolic repertoires of lactose-mediated production of chemicals such as butanol promises to identify new targets that can be fine tuned in recombinant and native microbial strains to engender stronger coupling of whey permeate-borne lactose to value-added chemicals. Our results highlight new genetic targets for future engineering of C. beijerinckii for improved butanol production on lactose and ultimately in whey permeate.

Strain-dependent induction of primary bile acid 7-dehydroxylation by cholic acid.

BACKGROUND: Bile acids (BAs) are steroid-derived molecules with important roles in digestion, the maintenance of host metabolism, and immunomodulation. Primary BAs are synthesized by the host, while secondary BAs are produced by the gut microbiome through transformation of the former. The regulation of microbial production of secondary BAs is not well understood, particularly the production of 7-dehydroxylated BAs, which are the most potent agonists for host BA receptors. The 7-dehydroxylation of cholic acid (CA) is well established and is linked to the expression of a bile acid-inducible (bai) operon responsible for this process. However, little to no 7-dehydroxylation has been reported for other host-derived BAs (e.g., chenodeoxycholic acid, CDCA or ursodeoxycholic acid, UDCA). RESULTS: Here, we demonstrate that the 7-dehydroxylation of CDCA and UDCA by the human isolate Clostridium scindens is induced when CA is present, suggesting that CA-dependent transcriptional regulation is required for substantial 7-dehydroxylation of these primary BAs. This is supported by the finding that UDCA alone does not promote expression of bai genes. CDCA upregulates expression of the bai genes but the expression is greater when CA is present. In contrast, the murine isolate Extibacter muris exhibits a distinct response; CA did not induce significant 7-dehydroxylation of primary BAs, whereas BA 7-dehydroxylation was promoted upon addition of germ-free mouse cecal content in vitro. However, E. muris was found to 7-dehydroxylate in vivo. CONCLUSIONS: The distinct expression responses amongst strains indicate that bai genes are regulated differently. CA promoted bai operon gene expression and the 7-dehydroxylating activity in C. scindens strains. Conversely, the in vitro activity of E. muris was promoted only after the addition of cecal content and the isolate did not alter bai gene expression in response to CA. The accessory gene baiJ was only upregulated in the C. scindens ATCC 35704 strain, implying mechanistic differences amongst isolates. Interestingly, the human-derived C. scindens strains were also capable of 7-dehydroxylating murine bile acids (muricholic acids) to a limited extent. This study shows novel 7-dehydroxylation activity in vitro resulting from the presence of CA and suggests distinct bai gene expression across bacterial species.

Genetic diversity, biofilm formation, and Vancomycin resistance of clinical Clostridium innocuum isolates.

BACKGROUND: Clostridium innocuum, previously considered a commensal microbe, is a spore-forming anaerobic bacterium. C. innocuum displays inherent resistance to vancomycin and is associated with extra-intestinal infections, antibiotic-associated diarrhea, and inflammatory bowel disease. This study seeks to establish a multilocus sequence typing (MLST) scheme to explore the correlation between C. innocuum genotyping and its potential pathogenic phenotypes. METHODS: Fifty-two C. innocuum isolates from Linkou Chang Gung Memorial Hospital (CGMH) in Taiwan and 60 sequence-available C. innocuum isolates from the National Center for Biotechnolgy Information Genome Database were included. The concentrated sequence of housekeeping genes in C. innocuum was determined by amplicon sequencing and used for MLST and phylogenetic analyses. The biofilm production activity of the C. innocuum isolates was determined by crystal violet staining. RESULTS: Of the 112 C. innocuum isolates, 58 sequence types were identified. Maximum likelihood estimation categorized 52 CGMH isolates into two phylogenetic clades. These isolates were found to be biofilm producers, with isolates in clade I exhibiting significantly higher biofilm production than isolates in clade II. The sub-inhibitory concentration of vancomycin seemed to minimally influence biofilm production by C. innocuum isolates. Nevertheless, C. innocuum embedded in the biofilm structure demonstrated resistance to vancomycin treatments at a concentration greater than 256 µg/mL. CONCLUSIONS: This study suggests that a specific genetic clade of C. innocuum produces a substantial amount of biofilm. Furthermore, this phenotype assists C. innocuum in resisting high concentrations of vancomycin, which may potentially play undefined roles in C. innocuum pathogenesis.

An efficient cre-based workflow for genomic integration and expression of large biosynthetic pathways in Eubacterium limosum.

Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis-independent recombinase-assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum. We then integrated a non-ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum, which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.

Clostridium omnivorum sp. nov., isolated from anoxic soil under the treatment of reductive soil disinfestation.

Reductive soil disinfestation (RSD), also known as biological soil disinfestation, is a bioremediation method used to suppress soil-borne plant pathogens by stimulating the activity of indigenous anaerobic bacteria in the soil. An anaerobic bacterial strain (E14T) was isolated from an anoxic soil sample subjected to RSD treatment and then comprehensively characterized. Cells of the strain were Gram-stain-positive, curved to sigmoid, and spore-forming rods. Cells were motile with a polar flagellum. Strain E14T grew in peptone-yeast extract broth, indicating that it utilized proteinous compounds. Strain E14T was also saccharolytic and produced acetate, isobutyrate, butyrate, isovalerate and gases (H2 and CO2) as fermentation products. The strain did not decompose any of examined polysaccharides except for starch. The major cellular fatty acids of strain E14T were iso-C15:0 and iso-C15:0 DMA. The closest relative to strain E14T, based on 16S rRNA gene sequences, was Clostridium thermarum SYSU GA15002T (96.2 %) in the Clostridiaceae. Whole-genome analysis of strain E14T showed that its genome was 4.66 Mb long with a genomic DNA G+C content of 32.5 mol%. The average nucleotide identity (ANIb) between strain E14T and C. thermarum SYSU GA15002T was 69.0 %. The presence of the genes encoding glycolysis and butyrate production via the acetyl-CoA pathway was confirmed through genome analysis. Based on the obtained phylogenetic, genomic and phenotypic data, we propose that strain E14T should be assigned to the genus Clostridium in the family Clostridiaceae as Clostridium omnivorum sp. nov. The type strain is E14T (=NBRC 115133T=DSM 114974T).

Clostridium aquiflavi sp. nov., isolated from yellow water of Nongxiangxing baijiu in the Yibin region of China.

A Gram-stain-positive, strictly anaerobic, endospore-forming and rod-shaped (0.6-0.8×2.7-13.1 µm) bacterium, designated as 5 N-1T, was isolated from a yellow water sample collected from the manufacturing process of Nongxiangxing baijiu in the Yibin region of Sichuan, PR China. Growth occurred at 15-40 °C (optimum growth at 37 °C), at pH 6.0-9.0 (optimum growth at pH 7.0) and in NaCl concentrations of 0-1 % (w/v) and ethanol concentrations of 0-2 % (v/v). The major fatty acids in strain 5 N-1T were C16 : 0, C18 : 0 and C14 : 0. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unidentified aminophospholipids and one unidentified lipid. Phylogenetic analysis of its 16S rRNA gene sequence indicated that strain 5 N-1T was most closely related to Clostridium weizhouense YB-6T (97.70 %) and Clostridium uliginosum DSM 12992T (97.56 %). The average nucleotide identity and digital DNA‒DNA hybridization values between strain 5 N-1T and the above two type strains were 80.89 and 80.05 % and 25.80 and 25.30 %, respectively, which were all below the species thresholds. The genome size of strain 5 N-1T was 3.5 Mbp and the DNA G+C content was 27.5 mol%. Based on the results of phenotypic and genotypic analyses, strain 5 N-1T represents a novel species of the genus Clostridium, for which the name Clostridium aquiflavi sp. nov. is proposed. The type strain is Clostridium aquiflavi 5 N-1T (=CICC 24886T=JCM 35355T).

Clostridium tanneri sp. nov., isolated from the faecal material of an alpaca.

A strictly anaerobic, Gram-stain-negative rod-shaped bacterium, designated A1-XYC3T, was isolated from the faeces of an alpaca (Lama pacos). On the basis of the results of a comparative 16S rRNA gene sequence analysis, the isolate was assigned to the genus Clostridium with the highest sequence similarities to Clostridium magnum DSM 2767T (96.8 %), Clostridium carboxidivorans P7T (96.3 %) and Clostridium aciditolerans JW/YJL-B3T (96.1 %). The average nucleotide identity between A1-XYC3T, C. magnum, C. carboxidivorans and C. aciditolerans was 77.4, 76.1 and 76.6  %, respectively. The predominant components of the cellular fatty acids of A1-XYC3T were C14 : 0, C16 : 0 and summed feature 10, containing C18:0/C17:0 cyclo. The DNA G+C content was 32.4 mol%. On the basis of biochemical, phylogenetic, genotypic and chemotaxonomic criteria, this isolate represents a novel species within Clostridium sensu stricto for which the name Clostridium tanneri sp. nov. is proposed. The type strain of this species is strain A1-XYC3T (=CCM 9376T=NRRL B-65691T).

Development of a genetic engineering toolbox for syngas-utilizing acetogen Clostridium sp. AWRP.

BACKGROUND: Clostridium sp. AWRP (AWRP) is a novel acetogenic bacterium isolated under high partial pressure of carbon monoxide (CO) and can be one of promising candidates for alcohol production from carbon oxides. Compared to model strains such as C. ljungdahlii and C. autoethanogenum, however, genetic manipulation of AWRP has not been established, preventing studies on its physiological characteristics and metabolic engineering. RESULTS: We were able to demonstrate the genetic domestication of AWRP, including transformation of shuttle plasmids, promoter characterization, and genome editing. From the conjugation experiment with E. coli S17-1, among the four replicons tested (pCB102, pAMß1, pIP404, and pIM13), three replicated in AWRP but pCB102 was the only one that could be transferred by electroporation. DNA methylation in E. coli significantly influenced transformation efficiencies in AWRP: the highest transformation efficiencies (102-103 CFU/µg) were achieved with unmethylated plasmid DNA. Determination of strengths of several clostridial promoters enabled the establishment of a CRISPR/Cas12a genome editing system based on Acidaminococcus sp. BV3L6 cas12a gene; interestingly, the commonly used CRISPR/Cas9 system did not work in AWRP, although it expressed the weakest promoter (C. acetobutylicum Pptb) tested. This system was successfully employed for the single gene deletion (xylB and pyrE) and double deletion of two prophage gene clusters. CONCLUSIONS: The presented genome editing system allowed us to achieve several genome manipulations, including double deletion of two large prophage groups. The genetic toolbox developed in this study will offer a chance for deeper studies on Clostridium sp. AWRP for syngas fermentation and carbon dioxide (CO2) sequestration.

Lactate-mediated mixotrophic co-cultivation of Clostridium drakei and recombinant Acetobacterium woodii for autotrophic production of volatile fatty acids.

BACKGROUND: Acetogens, a diverse group of anaerobic autotrophic bacteria, are promising whole-cell biocatalysts that fix CO2 during their growth. However, because of energetic constraints, acetogens exhibit slow growth and the product spectrum is often limited to acetate. Enabling acetogens to form more valuable products such as volatile fatty acids during autotrophic growth is imperative for cementing their place in the future carbon neutral industry. Co-cultivation of strains with different capabilities has the potential to ease the limiting energetic constraints. The lactate-mediated co-culture of an Acetobacterium woodii mutant strain, capable of lactate production, with the Clostridium drakei SL1 type strain can produce butyrate and hexanoate. In this study, the preceding co-culture is characterized by comparison of monocultures and different co-culture approaches. RESULTS: C. drakei grew with H2 + CO2 as main carbon and energy source and thrived when further supplemented with D-lactate. Gas phase components and lactate were consumed in a mixotrophic manner with acetate and butyrate as main products and slight accumulation of hexanoate. Formate was periodically produced and eventually consumed by C. drakei. A lactate-mediated co-culture of the A. woodii [PbgaL_ldhD_NFP] strain, engineered for autotrophic lactate production, and C. drakei produced up to 4 ± 1.7 mM hexanoate and 18.5 ± 5.8 mM butyrate, quadrupling and doubling the respective titers compared to a non-lactate-mediated co-culture. Further co-cultivation experiments revealed the possible advantage of sequential co-culture over concurrent approaches, where both strains are inoculated simultaneously. Scanning electron microscopy of the strains revealed cell-to-cell contact between the co-culture partners. Finally, a combined pathway of A. woodii [PbgaL_ldhD_NFP] and C. drakei for chain-elongation with positive ATP yield is proposed. CONCLUSION: Lactate was proven to be a well-suited intermediate to combine the high gas uptake capabilities of A. woodii with the chain-elongation potential of C. drakei. The cell-to-cell contact observed here remains to be further characterized in its nature but hints towards diffusive processes being involved in the co-culture. Furthermore, the metabolic pathways involved are still speculatory for C. drakei and do not fully explain the consumption of formate while H2 + CO2 is available. This study exemplifies the potential of combining metabolically engineered and native bacterial strains in a synthetic co-culture.

Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination.

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.

Helicovermis profundi gen. nov., sp. nov., a novel mesophilic, asporogenous bacterium within the Clostridia isolated from a deep-sea hydrothermal vent chimney.

A novel mesophilic bacterial strain, designated S502T, was isolated from a deep-sea hydrothermal vent at Suiyo Seamount, Japan. Cells were Gram-positive, asporogenous, motile, and curved rods, measuring 1.6-5.6 µm in length. The strain was an obligate anaerobe that grew fermentatively on complex substrates such as yeast extract and Bacto peptone. Elemental sulfur stimulated the growth of the strain, and was reduced to hydrogen sulfide. The strain grew within a temperature range of 10-23 °C (optimum at 20 °C), pH range of 4.8-8.3 (optimum at 7.4), and a NaCl concentration range of 1.0-4.0% (w/v) (optimum at 3.0%, w/v). Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolate was a member of the class Clostridia, with Fusibacter paucivorans strain SEBR 4211T (91.1% sequence identity) being its closest relative. The total size of the genome of the strain was 3.12 Mbp, and a G + C content was 28.2 mol%. The highest values for average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) value of strain S502T with relatives were 67.5% (with Marinisporobacter balticus strain 59.4MT), 51.5% (with M. balticus strain 59.4MT), and 40.9% (with Alkaliphilus serpentinus strain LacTT), respectively. Based on a combination of phylogenetic, genomic, and phenotypic characteristics, we propose strain S502T to represent a novel genus and species, Helicovermis profundi gen. nov., sp. nov., with the type strain S502T (= DSM 112048T = JCM 39167T).

Exploiting the Thermotolerance of Clostridium Strain M1NH for Efficient Caproic Acid Fermentation from Ethanol and Acetic Acid.

A novel thermotolerant caproic acid-producing bacterial strain, Clostridium M1NH, was successfully isolated from sewage sludge. Ethanol and acetic acid at a molar ratio of 4:1 proved to be the optimal substrates, yielding a maximum caproic acid production of 3.5 g/L. Clostridium M1NH exhibited remarkable tolerance to high concentrations of ethanol (up to 5% v/v), acetic acid (up to 5% w/v), and caproic acid (up to 2% w/v). The strain also demonstrated a wide pH tolerance range (pH 5.5-7.5) and an elevated temperature optimum between 35 and 40 °C. Phylogenetic analysis based on 16S rRNA gene sequences revealed that Clostridium M1NH shares a 98% similarity with Clostridium luticellarii DSM 29923 T. The robustness of strain M1NH and its efficient caproic acid production from low-cost substrates highlight its potential for sustainable bio-based chemical production. The maximum caproic acid yield achieved by Clostridium M1NH was 1.6-fold higher than that reported for C. kluyveri under similar fermentation conditions. This study opens new avenues for valorizing waste streams and advancing a circular economy model in the chemical industry.

Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences.

RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a 'Quick' method to generate a single PAM prediction or in a 'Comprehensive' method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.