Characterisation of Agr quorum sensing in Clostridium autoethanogenum

The Gram-positive, anaerobic, acetogenic bacterium Clostridium autoethanogenum is regarded as an important biocatalyst in the current advancement of industrial gas fermentation. The ever-widening diversity of industrially-relevant acetogenic bacteria has inspired a rational approach into utilising industrial waste gases as a viable feedstock, with goals of mitigating greenhouse gas emissions, and supporting an alternative means of fuel and high-value chemical production. Combined with this effort, is the expanding repertoire of gene editing tools that have allowed for the improvement of gas fermentation processes and increased spectrum of fermentative products. Despite these advances, there remain many pertinent questions, which addressed, can further the understanding of metabolism and physiology in acetogenic bacteria. This includes cell-to-cell communication and signalling, Quorum Sensing. In this project, these questions are addressed through the study of the Agr QS system in C. autoethanogenum. Signalling peptide genes, agrD1 and agrD2 were disabled separately and more importantly, in tandem, which effectively abolished Agr signalling. Phenotypic characterisation of the double agrD mutants revealed a significant increase in ethanol at the expense of acetate output. Further observations exhibited a complete utilisation of the fructose carbon source, and the inability to fully re-assimilate CO2. These findings markedly contrasted with the wild type, and both single knock-out agrD mutants. Proteomics and enzyme activity analysis of the double AgrD mutant revealed a marked down-regulation of Wood-Ljungdahl pathway genes that included the CO2-assimilating, carbon monoxide dehydrogenase / acetyl-CoA synthase complex subunits and hydrogenases. An up-regulation of alcohol dehydrogenases was observed explaining ethanol increases, alongside an unexpected upregulation of bacterial micro-compartment clusters. These findings led to the hypothesis that the C. autoethanogenum Agr system influences the ancient Wood-Ljungdahl pathway, primarily as a means of survival by managing carbon-source utilisation and regulation

Adaptation during propagation improves Clostridium autoethanogenum tolerance towards benzene, toluene and xylenes during gas fermentation

Benzene, toluene and xylenes (BTX) are a group of compounds detected in many crude syngas mixtures. However, BTX have been identified to negatively affect microorganisms, including acetogenic species that are capable of fermenting syngas into valuable biocommodities. In order to overcome BTX inhibitory effects, we describe stepwise adaptation in Clostridium autoethanogenum that leads to tolerance to up to 0.5 mM benzene, 0.21 mM toluene and 0.07 mM xylenes. This is equivalent to eightfold of that which is found in a wood gasification plant syngas stream. Fully adapted cultures matched growth, acetate and ethanol product concentrations, and CO consumption compared to the control. The results demonstrate an efficient route towards producing a highly tolerant, industrially relevant acetogenic strain

A genome-scale model of Clostridium autoethanogenum reveals optimal bioprocess conditions for high-value chemical production from carbon monoxide

Clostridium autoethanogenum is an industrial microbe used for the commercial-scale production of ethanol from carbon monoxide. While significant progress has been made in the attempted diversification of this bioprocess, further improvements are desirable, particularly in the formation of the high-value platform chemicals, such as 2,3-butanediol. A new, experimentally parameterised genome scale model of C. autoethanogenum predicts dramatically increased 2,3-butanediol production under non-carbon-limited conditions when thermodynamic constraints on hydrogen production are considered

Whole genome sequence and manual annotation of Clostridium autoethanogenum, an industrially relevant bacterium

Clostridium autoethanogenum is an acetogenic bacterium capable of producing high value commodity chemicals and biofuels from the C1 gases present in synthesis gas. This common industrial waste gas can act as the sole energy and carbon source for the bacterium that converts the low value gaseous components into cellular building blocks and industrially relevant products via the action of the reductive acetyl-CoA (Wood-Ljungdahl) pathway. Current research efforts are focused on the enhancement and extension of product formation in this organism via synthetic biology approaches. However, crucial to metabolic modelling and directed pathway engineering is a reliable and comprehensively annotated genome sequence

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Characterisation of Agr quorum sensing in Clostridium autoethanogenum

The Gram-positive, anaerobic, acetogenic bacterium Clostridium autoethanogenum is regarded as an important biocatalyst in the current advancement of industrial gas fermentation. The ever-widening diversity of industrially-relevant acetogenic bacteria has inspired a rational approach into utilising industrial waste gases as a viable feedstock, with goals of mitigating greenhouse gas emissions, and supporting an alternative means of fuel and high-value chemical production. Combined with this effort, is the expanding repertoire of gene editing tools that have allowed for the improvement of gas fermentation processes and increased spectrum of fermentative products. Despite these advances, there remain many pertinent questions, which addressed, can further the understanding of metabolism and physiology in acetogenic bacteria. This includes cell-to-cell communication and signalling, Quorum Sensing. In this project, these questions are addressed through the study of the Agr QS system in C. autoethanogenum. Signalling peptide genes, agrD1 and agrD2 were disabled separately and more importantly, in tandem, which effectively abolished Agr signalling. Phenotypic characterisation of the double agrD mutants revealed a significant increase in ethanol at the expense of acetate output. Further observations exhibited a complete utilisation of the fructose carbon source, and the inability to fully re-assimilate CO2. These findings markedly contrasted with the wild type, and both single knock-out agrD mutants. Proteomics and enzyme activity analysis of the double AgrD mutant revealed a marked down-regulation of Wood-Ljungdahl pathway genes that included the CO2-assimilating, carbon monoxide dehydrogenase / acetyl-CoA synthase complex subunits and hydrogenases. An up-regulation of alcohol dehydrogenases was observed explaining ethanol increases, alongside an unexpected upregulation of bacterial micro-compartment clusters. These findings led to the hypothesis that the C. autoethanogenum Agr system influences the ancient Wood-Ljungdahl pathway, primarily as a means of survival by managing carbon-source utilisation and regulation

Additional file 1: of Whole genome sequence and manual annotation of Clostridium autoethanogenum, an industrially relevant bacterium

Discrepancies occurring between the current and Brown et al. finished genome sequence of C. autoethanogenum. This table shows all of the discrepancies that occur when our finished genome sequence (CLAU) is mapped against the Brown et al. finished genome sequence (BRO). Mutation column describes the mutation occurring in the CLAU genome compared to the BRO genome. Gene / region gives the gene name where the discrepancy occurs, ← / ← or similar denotes that the discrepancy occurred in a non-coding region between the named genes. Homopolymer length indicates the number of the same base occurring consecutively at the site of the discrepancy. Amino acid length gives the annotated protein length of the gene in which the discrepancy occurs, *indicates protein codes for multiple stop codons and ^indicates that no stop codon was found in the annotation. The sequence identity is relative to the CLAU C. autoethanogenum genome sequence when protein BLAST searched on the NCBI database. CLAU, C. autoethanogenum finished genome sequence in present study; CLJU, C. ljungdahlii DSM 13528 finished genome sequence (GCA_000143685.1); BRO, Brown et al. C. autoethanogenum finished genome sequence (GCA_000484505.1); CAUT, Bruno-Barcena et al. C. autoethanogenum draft genome sequence (GCA_000427255.1); NF, not found. (DOCX 73 kb

Cardiac myosin activation with omecamtiv mecarbil in systolic heart failure

BACKGROUND The selective cardiac myosin activator omecamtiv mecarbil has been shown to improve cardiac function in patients with heart failure with a reduced ejection fraction. Its effect on cardiovascular outcomes is unknown. METHODS We randomly assigned 8256 patients (inpatients and outpatients) with symptomatic chronic heart failure and an ejection fraction of 35% or less to receive omecamtiv mecarbil (using pharmacokinetic-guided doses of 25 mg, 37.5 mg, or 50 mg twice daily) or placebo, in addition to standard heart-failure therapy. The primary outcome was a composite of a first heart-failure event (hospitalization or urgent visit for heart failure) or death from cardiovascular causes. RESULTS During a median of 21.8 months, a primary-outcome event occurred in 1523 of 4120 patients (37.0%) in the omecamtiv mecarbil group and in 1607 of 4112 patients (39.1%) in the placebo group (hazard ratio, 0.92; 95% confidence interval [CI], 0.86 to 0.99; P = 0.03). A total of 808 patients (19.6%) and 798 patients (19.4%), respectively, died from cardiovascular causes (hazard ratio, 1.01; 95% CI, 0.92 to 1.11). There was no significant difference between groups in the change from baseline on the Kansas City Cardiomyopathy Questionnaire total symptom score. At week 24, the change from baseline for the median N-terminal pro-B-type natriuretic peptide level was 10% lower in the omecamtiv mecarbil group than in the placebo group; the median cardiac troponin I level was 4 ng per liter higher. The frequency of cardiac ischemic and ventricular arrhythmia events was similar in the two groups. CONCLUSIONS Among patients with heart failure and a reduced ejection, those who received omecamtiv mecarbil had a lower incidence of a composite of a heart-failure event or death from cardiovascular causes than those who received placebo. (Funded by Amgen and others; GALACTIC-HF ClinicalTrials.gov number, NCT02929329; EudraCT number, 2016 -002299-28.)