Characterisation of acetogen formatotrophic potential using Eubacterium limosum.

Formate is a promising energy carrier that could be used to transport renewable electricity. Some acetogenic bacteria, such as Eubacterium limosum, have the native ability to utilise formate as a sole substrate for growth, which has sparked interest in the biotechnology industry. However, formatotrophic metabolism in E. limosum is poorly understood, and a system-level characterisation in continuous cultures is yet to be reported. Here, we present the first steady-state dataset for E. limosum formatotrophic growth. At a defined dilution rate of 0.4 d-1, there was a high specific uptake rate of formate (280 ± 56 mmol/gDCW/d; gDCW = gramme dry cell weight); however, most carbon went to CO2 (150 ± 11 mmol/gDCW/d). Compared to methylotrophic growth, protein differential expression data and intracellular metabolomics revealed several key features of formate metabolism. Upregulation of phosphotransacetylase (Pta) appears to be a futile attempt of cells to produce acetate as the major product. Instead, a cellular energy limitation resulted in the accumulation of intracellular pyruvate and upregulation of pyruvate formate ligase (Pfl) to convert formate to pyruvate. Therefore, metabolism is controlled, at least partially, at the protein expression level, an unusual feature for an acetogen. We anticipate that formate could be an important one-carbon substrate for acetogens to produce chemicals rich in pyruvate, a metabolite generally in low abundance during syngas growth. KEY POINTS: First Eubacterium limosum steady-state formatotrophic growth omics dataset High formate specific uptake rate, however carbon dioxide was the major product Formate may be the cause of intracellular stress and biofilm formation.

Association of gut microbiome and metabolites with onset and treatment response of patients with pemphigus vulgaris.

Background: Gut dysbiosis and gut microbiome-derived metabolites have been implicated in both disease onset and treatment response, but this has been rarely demonstrated in pemphigus vulgaris (PV). Here, we aim to systematically characterize the gut microbiome to assess the specific microbial species and metabolites associated with PV. Methods: We enrolled 60 PV patients and 19 matched healthy family members, and collected 100 fecal samples (60 treatment-naïve, 21 matched post-treatment, and 19 controls). Metagenomic shotgun sequencing and subsequent quality control/alignment/annotation were performed to assess the composition and microbial species, in order to establish the association between gut microbiome with PV onset and treatment response. In addition, we evaluated short-chain fatty acids (SCFAs) in PV patients through targeted metabolomics analysis. Results: The diversity of the gut microbiome in PV patients deviates from the healthy family members but not between responder and non-responder, or before and after glucocorticoid treatment. However, the relative abundance of several microbial species, including the pathogenic bacteria (e.g., Escherichia coli) and some SCFA-producing probiotics (e.g., Eubacterium ventriosum), consistently differed between the two groups in each comparison. Escherichia coli was enriched in PV patients and significantly decreased after treatment in responders. In contrast, Eubacterium ventriosum was enriched in healthy family members and significantly increased particularly in responders after treatment. Consistently, several gut microbiome-derived SCFAs were enriched in healthy family members and significantly increased after treatment (e.g., butyric acid and valeric acid). Conclusions: This study supports the association between the gut microbiome and PV onset, possibly through disrupting the balance of gut pathogenic bacteria and probiotics and influencing the level of gut microbiome-derived SCFAs. Furthermore, we revealed the potential relationship between specific microbial species and glucocorticoid treatment.

Genome-Based Reclassification of Strain KIST612, Previously Classified as Eubacterium limosum, into a New Strain of Eubacterium callanderi.

The strain KIST612, initially identified as E. limosum, was a suspected member of E. callanderi due to differences in phenotype, genotype, and average nucleotide identity (ANI). Here, we found that E. limosum ATCC 8486T and KIST612 are genetically different in their central metabolic pathways, such as that of carbon metabolism. Although 16S rDNA sequencing of KIST612 revealed high identity with E. limosum ATCC 8486T (99.2%) and E. callanderi DSM 3662T (99.8%), phylogenetic analysis of housekeeping genes and genome metrics clearly indicated that KIST612 belongs to E. callanderi. The phylogenies showed that KIST612 is closer to E. callanderi DSM 3662T than to E. limosum ATCC 8486T. The ANI between KIST612 and E. callanderi DSM 3662T was 99.8%, which was above the species cut-off of 96%, Meanwhile, the ANI value with E. limosum ATCC 8486T was not significant, showing only 94.6%. The digital DNA-DNA hybridization (dDDH) results also supported the ANI values. The dDDH between KIST612 and E. callanderi DSM 3662T was 98.4%, whereas between KIST612 and E. limosum ATCC 8486T, it was 57.8%, which is lower than the species cut-off of 70%. Based on these findings, we propose the reclassification of E. limosum KIST612 as E. callanderi KIST612.

Deletion of biofilm synthesis in Eubacterium limosum ATCC 8486 improves handling and transformation efficiency.

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

[Research progress of Eubacterium and its metabolite short-chain fatty acids in regulating type 2 diabetes mellitus].

Intestinal flora and its metabolites are closely related to the progression of type 2 diabetes mellitus(T2DM). Eubacterium is one of the dominant intestinal flora, and its metabolites short-chain fatty acids (SCFAs) play a leading role in regulating intestinal metabolic balance. It has been reported that SCFAs can regulate the secretion of glucagon-like peptide-1, improve the function of pancreatic ß cells, participate in bile acids metabolism and regulate the production of inflammatory factors in T2DM. Based on the above research background, this article mainly reviews the relationship between Eubacterium and its metabolite SCFAs and T2DM and its regulatory mechanism.

Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum.

Acetogenic bacteria are a unique biocatalyst that highly promises to develop the sustainable bioconversion of carbon oxides (e.g., CO and CO2) into multicarbon biochemicals. Genotype-phenotype relationships are important for engineering their metabolic capability to enhance their biocatalytic performance; however, systemic investigation on the fitness contribution of individual gene has been limited. Here, we report genome-scale CRISPR interference screening using 41,939 guide RNAs designed from the E. limosum genome, one of the model acetogenic species, where all genes were targeted for transcriptional suppression. We investigated the fitness contributions of 96% of the total genes identified, revealing the gene fitness and essentiality for heterotrophic and autotrophic metabolisms. Our data show that the Wood-Ljungdahl pathway, membrane regeneration, membrane protein biosynthesis, and butyrate synthesis are essential for autotrophic acetogenesis in E. limosum. Furthermore, we discovered genes that are repression targets that unbiasedly increased autotrophic growth rates fourfold and acetoin production 1.5-fold compared to the wild-type strain under CO2-H2 conditions. These results provide insight for understanding acetogenic metabolism and genome engineering in acetogenic bacteria.

Research progress of Eubacterium and its metabolite short-chain fatty acids in regulating type 2 diabetes mellitus / 中华预防医学杂志

Intestinal flora and its metabolites are closely related to the progression of type 2 diabetes mellitus(T2DM). Eubacterium is one of the dominant intestinal flora, and its metabolites short-chain fatty acids (SCFAs) play a leading role in regulating intestinal metabolic balance. It has been reported that SCFAs can regulate the secretion of glucagon-like peptide-1, improve the function of pancreatic β cells, participate in bile acids metabolism and regulate the production of inflammatory factors in T2DM. Based on the above research background, this article mainly reviews the relationship between Eubacterium and its metabolite SCFAs and T2DM and its regulatory mechanism.

Genetic manipulation of the human gut bacterium Eggerthella lenta reveals a widespread family of transcriptional regulators.

Eggerthella lenta is a prevalent human gut Actinobacterium implicated in drug, dietary phytochemical, and bile acid metabolism and associated with multiple human diseases. No genetic tools are currently available for the direct manipulation of E. lenta. Here, we construct shuttle vectors and develop methods to transform E. lenta and other Coriobacteriia. With these tools, we characterize endogenous E. lenta constitutive and inducible promoters using a reporter system and construct inducible expression systems, enabling tunable gene regulation. We also achieve genome editing by harnessing an endogenous type I-C CRISPR-Cas system. Using these tools to perform genetic knockout and complementation, we dissect the functions of regulatory proteins and enzymes involved in catechol metabolism, revealing a previously unappreciated family of membrane-spanning LuxR-type transcriptional regulators. Finally, we employ our genetic toolbox to study the effects of E. lenta genes on mammalian host biology. By greatly expanding our ability to study and engineer gut Coriobacteriia, these tools will reveal mechanistic details of host-microbe interactions and provide a roadmap for genetic manipulation of other understudied human gut bacteria.

Butyrate-producing Eubacterium rectale suppresses lymphomagenesis by alleviating the TNF-induced TLR4/MyD88/NF-κB axis.

Microbiota-induced tumorigenesis is well established in solid tumors of the gastrointestinal tract but rarely explored in hematologic malignancies. To determine the role of gut microbiota in lymphoma progression, we performed metagenomic sequencing on human primary gastrointestinal B cell lymphomas. We identified a distinct microbiota profile of intestinal lymphoma, with significantly decreased symbiotic microbes, particularly the genus Eubacterium and notably butyrate-producing Eubacterium rectale. Transfer of E. rectale-deficit microbiota of intestinal lymphoma patients to mice caused inflammation and tumor necrosis factor (TNF) production. Conversely, E. rectale treatment reduced TNF levels and the incidence of lymphoma in sensitized Eµ-Myc mice. Moreover, lipopolysaccharide from the resident microbiota of lymphoma patients and mice synergizes with TNF signaling and reinforces the NF-κB pathway via the MyD88-dependent TLR4 signaling, amalgamating in enhanced intestinal B cell survival and proliferation. These findings reveal a mechanism of inflammation-associated lymphomagenesis and a potential clinical rationale for therapeutic targeting of gut microbiota.

Development of highly characterized genetic bioparts for efficient gene expression in CO2-fixing Eubacterium limosum.

Acetogenic bacteria demonstrate industrial potential for utilizing carbon dioxide (CO2) for biochemical production using the Wood-Ljungdahl pathway. However, the metabolic engineering of acetogenic bacteria has been hampered by the limited number of available genetic bioparts for gene expression. Here, we integrated RNA sequencing, ribosome profiling, differential RNA sequencing, and RNA 3'-end sequencing results of Eubacterium limosum to establish genetic bioparts, such as promoters, 5' untranslated regions, and transcript terminators, to regulate transcriptional and translational expression of genes composing of biosynthetic pathways. In addition, a transformation method for the strain was developed to efficiently deliver the obtained genetic bioparts into cells, resulting in a transformation efficiency of 2.5 × 105 CFU/µg DNA. Using this method, the genetic bioparts were efficiently introduced, and their strengths were measured, which were then applied to optimize the heterologous expression of acetolactate synthase and acetolactate decarboxylase for non-native biochemical acetoin production. The strategy developed in this study is the first report on integrating multi-omics data for biopart development of CO2 or syngas utilizing acetogenic bacteria, which lays a foundation for the efficient production of biochemicals from CO2 or syngas as a carbon feedstock under autotrophic growth conditions.

Eubacterium coprostanoligenes and Methanoculleus identified as potential producers of metabolites that contribute to swine manure foaming.

AIM: Swine manure foaming is a major problem, causing damage to property, livestock, and people. Here, we identified the main chemicals and microbes that contribute to foaming. METHODS AND RESULTS: Foaming and non-foaming swine manure were sampled from farms in Iowa and Illinois. Targeted and untargeted metabolomics analyses identified chemical markers that differed between foaming and non-foaming manure and between manure layers. Microbial community analysis and metagenomics were performed on a subset of samples. Foam contained significantly higher levels of total bile acids and long chain fatty acids like palmitic, stearic and oleic acid than the other manure layers. Foam layers also had significantly higher levels of ubiquinone 9 and ubiquinone 10. The slurry layer of foaming samples contained more alanine, isoleucine/leucine, diacylglycerols (DG), phosphtatidylethanolamines, and vitamin K2, while ceramide was significantly increased in the slurry layer of non-foaming samples. Eubacterium coprostanoligenes and Methanoculleus were more abundant in foaming samples, and E. coprostanoligenes was significantly correlated with levels of DG. Genes involved in diacylglycerol biosynthesis and in the biosynthesis of branched-chain hydrophobic amino acids were overrepresented in foaming samples. CONCLUSIONS: A mechanism for manure foaming is hypothesized in which proliferation of Methanoculleus leads to excessive production of methane, while production of DG by E. coprostanoligenes and hydrophobic proteins by Methanosphaera stadtmanae facilitates bubble formation and stabilization. SIGNIFICANCE AND IMPACT OF STUDY: While some chemical and biological treatments have been developed to treat swine manure foaming, its causes remain unknown. We identified key microbes and metabolites that correlate with foaming and point to possible roles of other factors like animal feed.

High methanol-to-formate ratios induce butanol production in Eubacterium limosum.

Unlike gaseous C1 feedstocks for acetogenic bacteria, there has been less attention on liquid C1 feedstocks, despite benefits in terms of energy efficiency, mass transfer and integration within existing fermentation infrastructure. Here, we present growth of Eubacterium limosum ATCC8486 using methanol and formate as substrates, finding evidence for the first time of native butanol production. We varied ratios of methanol-to-formate in batch serum bottle fermentations, showing butyrate is the major product (maximum specific rate 220 ± 23 mmol-C gDCW-1 day-1 ). Increasing this ratio showed methanol is the key feedstock driving the product spectrum towards more reduced products, such as butanol (maximum titre 2.0 ± 1.1 mM-C). However, both substrates are required for a high growth rate (maximum 0.19 ± 0.011 h-1 ) and cell density (maximum 1.2 ± 0.043 gDCW l-1 ), with formate being the preferred substrate. In fact, formate and methanol are consumed in two distinct growth phases - growth phase 1, on predominately formate and growth phase 2 on methanol, which must balance. Because the second growth varied according to the first growth on formate, this suggests butanol production is due to overflow metabolism, similar to 2,3-butanediol production in other acetogens. However, further research is required to confirm the butanol production pathway in E. limosum, particularly given, unlike other substrates, methanol likely results in mostly NADH generation, not reduced ferredoxin.

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.

Characterization of Protein-Nucleic Acid Complexes by Size-Exclusion Chromatography Coupled with Light Scattering, Absorbance, and Refractive Index Detectors.

Size-exclusion chromatography (SEC) coupled with multiangle light scattering detection (SEC/MALS) enables determination of the molecular weight, oligomeric state, and stoichiometry of protein-nucleic acid complexes in solution. Often such complexes show anomalous behavior on SEC, thus presenting a challenge in determination of molecular weight and stoichiometry based solely on the elution position from SEC. In contrast to analytical ultracentrifugation, the SEC/MALS analysis is not affected by the shape of the complex. Here we describe the use of SEC/MALS for characterization of the stoichiometry of the complex between the reverse transcriptase (RT) domain from group II intron-maturase from Eubacterium rectale and intron RNA, and for monitoring protein dimerization that is driven by interaction between single-stranded DNA upstream of the P1 promoter, known as FUSE and FUSE binding protein-interacting repressor (FIR).

Fecal g. Streptococcus and g. Eubacterium_coprostanoligenes_group combined with sphingosine to modulate the serum dyslipidemia in high-fat diet mice.

BACKGROUND & AIMS: Although high-fat diet (HFD) could impact the composition of fecal microbiome and their metabolites, it is still largely unknown which fecal bacteria and metabolites are relatively important in responding to the HFD. This study aimed to identify the crucial fecal bacteria and metabolites in the HFD mice using a microbial-metabolite network, and to investigate the synergistic mediation effect of the crucial fecal bacteria and metabolites on serum dyslipidemia induced by the HFD. METHODS: The 16srDNA sequencing and the ultra-performance liquid chromatography (UPLC/TOF MSMS) platform were performed to characterize the composition and function of fecal microbiome, and metabolites in the HFD. The microbial-metabolite network, correlation and mediation analyses were performed to examine the relationships among fecal microbiome, metabolites, and serum dyslipidemia indicators. Mice models were conducted to evaluate the effect of fecal metabolite on dyslipidemia. RESULTS: Compared to the control, 32 genera were altered in the HFD, including 26 up-regulated and 6 down-regulated. A total of 42 altered pathways were observed between the control and HFD, and the "Glycosphingolipid biosynthesis" was identified as the most significant pathway (fold change = 0.64; p < 0.001). Meanwhile, 49 fecal metabolites were altered in the HFD, and the fecal microbiome was associated with the fecal metabolism (M2 = 0.776, p = 0.008). Based on the microbial-metabolite network, two major hub genera were screened (HUB1: g. Streptococcus, HUB2: g. Eubacterium_coprostanoligenes_group), and one bacterial metabolite, sphingosine, was found in this study. Further, the HUB2 was positively associated with fecal sphingosine (r = 0.646, p = 0.001), and its downstream metabolic pathway, "Glycosphingolipid biosynthesis" pathway (r = 0.544, p = 0.009). The regulatory relationship between the HUB2 and sphingosine synergistically mediated the effect of HFD on TCHO (33.7%), HDL-C (37.3%), and bodyweight (36.7%). Besides, compared to the HFD, the HFD with sphingosine supplementation had lower bodyweight (35.12 ± 1.23 vs. 39.42 ± 1.25, p < 0.001), TG (0.44 ± 0.08 vs. 0.52 ± 0.05, p = 0.002), TCHO (3.81 ± 0.34 vs. 4.51 ± 0.38, p = 0.002), and LDL-c (0.82 ± 0.09 vs. 0.97 ± 0.15, p = 0.016). CONCLUSIONS: The g. Streptococcus and g. Eubacterium_coprostanoligenes are two hub genera in the fecal micro-ecosystem of the HFD, and the g. Eubacterium_coprostanoligenes mediates the effect of HFD on dyslipidemia through sphingosine. Sphingosine supplementation can improve dyslipidemia induced by HFD.

Butyrate Levels in the Transition from an Infant- to an Adult-Like Gut Microbiota Correlate with Bacterial Networks Associated with Eubacterium Rectale and Ruminococcus Gnavus.

Relatively little is known about the ecological forces shaping the gut microbiota composition during infancy. Therefore, the objective of the present study was to identify the nutrient utilization- and short-chain fatty acid (SCFA) production potential of gut microbes in infants during the first year of life. Stool samples were obtained from mothers at 18 weeks of pregnancy and from infants at birth (first stool) at 3, 6, and 12-months of age from the general population-based PreventADALL cohort. We identified the taxonomic and SCFA composition in 100 mother-child pairs. The SCFA production and substrate utilization potential of gut microbes were observed by multiomics (shotgun sequencing and proteomics) on six infants. We found a four-fold increase in relative butyrate levels from 6 to 12 months of infant age. The increase was correlated to Eubacterium rectale and its bacterial network, and Faecalibacterium prausnitzii relative abundance, while low butyrate at 12 months was correlated to Ruminococcus gnavus and its associated network of bacteria. Both E. rectale and F. prausnitzii expressed enzymes needed for butyrate production and enzymes related to dietary fiber degradation, while R. gnavus expressed mucus-, fucose, and human milk oligosaccharides (HMO)-related degradation enzymes. Therefore, we believe that the presence of E. rectale, its network, and F. prausnitzii are key bacteria in the transition from an infant- to an adult-like gut microbiota with respect to butyrate production. Our results indicate that the transition from an infant- to an adult-like gut microbiota with respect to butyrate producing bacteria, occurs between 6 and 12 months of infant age. The bacteria associated with the increased butyrate ratio/levels were E. rectale and F. prausnitzii, which potentially utilize a variety of dietary fibers based on the glycoside hydrolases (GHs) expressed. R. gnavus with a negative association to butyrate potentially utilizes mucin, fucose, and HMO components. This knowledge could have future importance in understanding how microbial metabolites can impact infant health and development.

Butyrate producing colonic Clostridiales metabolise human milk oligosaccharides and cross feed on mucin via conserved pathways.

The early life human gut microbiota exerts life-long health effects on the host, but the mechanisms underpinning its assembly remain elusive. Particularly, the early colonization of Clostridiales from the Roseburia-Eubacterium group, associated with protection from colorectal cancer, immune- and metabolic disorders is enigmatic. Here, we describe catabolic pathways that support the growth of Roseburia and Eubacterium members on distinct human milk oligosaccharides (HMOs). The HMO pathways, which include enzymes with a previously unknown structural fold and specificity, were upregulated together with additional glycan-utilization loci during growth on selected HMOs and in co-cultures with Akkermansia muciniphila on mucin, suggesting an additional role in enabling cross-feeding and access to mucin O-glycans. Analyses of 4599 Roseburia genomes underscored the preponderance and diversity of the HMO utilization loci within the genus. The catabolism of HMOs by butyrate-producing Clostridiales may contribute to the competitiveness of this group during the weaning-triggered maturation of the microbiota.

ErCas12a CRISPR-MAD7 for Model Generation in Human Cells, Mice, and Rats.

MAD7 is an engineered class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) system isolated from Eubacterium rectale. Analogous to Cas9, it is an RNA-guided nuclease with demonstrated gene editing activity in Escherichia coli and yeast cells. Here, we report that MAD7 is capable of generating indels and fluorescent gene tagging of endogenous genes in human HCT116 and U2OS cancer cell lines, respectively. In addition, MAD7 is highly proficient in generating indels, small DNA insertions (23 bases), and larger integrations ranging from 1 to 14 kb in size in mouse and rat embryos, resulting in live-born transgenic animals. Due to the different protospacer adjacent motif requirement, small-guide RNA, and highly efficient targeted gene disruption and insertions, MAD7 can expand the CRISPR toolbox for genome enginnering across different systems and model organisms.

Open microbiome dominated by Clostridium and Eubacterium converts methanol into i-butyrate and n-butyrate.

Isobutyrate (i-butyrate) is a versatile platform chemical, whose acid form is used as a precursor of plastic and emulsifier. It can be produced microbially either using genetically engineered organisms or via microbiomes, in the latter case starting from methanol and short-chain carboxylates. This opens the opportunity to produce i-butyrate from non-sterile feedstocks. Little is known on the ecology and process conditions leading to i-butyrate production. In this study, we steered i-butyrate production in a bioreactor fed with methanol and acetate under various conditions, achieving maximum i-butyrate productivity of 5.0 mM day-1, with a concurrent production of n-butyrate of 7.9 mM day-1. The production of i-butyrate was reversibly inhibited by methanogenic inhibitor 2-bromoethanesulfonate. The microbial community data revealed the co-dominance of two major OTUs during co-production of i-butyrate and n-butyrate in two distinctive phases throughout a period of 54 days and 28 days, respectively. The cross-comparison of product profile with microbial community composition suggests that the relative abundance of Clostridium sp. over Eubacterium sp. is correlated with i-butyrate productivity over n-butyrate productivity.

Insights into the Phylogeny and Evolution of Cold Shock Proteins: From Enteropathogenic Yersinia and Escherichia coli to Eubacteria.

Psychrotrophic foodborne pathogens, such as enteropathogenic Yersinia, which are able to survive and multiply at low temperatures, require cold shock proteins (Csps). The Csp superfamily consists of a diverse group of homologous proteins, which have been found throughout the eubacteria. They are related to cold shock tolerance and other cellular processes. Csps are mainly named following the convention of those in Escherichia coli. However, the nomenclature of certain Csps reflects neither their sequences nor functions, which can be confusing. Here, we performed phylogenetic analyses on Csp sequences in psychrotrophic enteropathogenic Yersinia and E. coli. We found that representative Csps in enteropathogenic Yersinia and E. coli can be clustered into six phylogenetic groups. When we extended the analysis to cover Enterobacteriales, the same major groups formed. Moreover, we investigated the evolutionary and structural relationships and the origin time of Csp superfamily members in eubacteria using nucleotide-level comparisons. Csps in eubacteria were classified into five clades and 12 subclades. The most recent common ancestor of Csp genes was estimated to have existed 3585 million years ago, indicating that Csps have been important since the beginning of evolution and have enabled bacterial growth in unfavorable conditions.