Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum.

The majority of the genes present in bacterial genomes remain poorly characterized, with up to one-third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to those species in which high rates of DNA transfer can be achieved. Here, we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum by using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as the sole carbon and energy source. IMPORTANCE Although microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterized, leaving the function of many as either hypothetical or entirely unknown. High-throughput transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here, we developed approaches to facilitate transposon insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases CO and CO2 into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth, providing insights into the utilization of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon insertion sequencing to other microbes where DNA transfer has until now represented a barrier to progress.

Clinical Trial-Ready Patient Cohorts for Multiple System Atrophy: Coupling Biospecimen and iPSC Banking to Longitudinal Deep-Phenotyping.

Multiple system atrophy (MSA) is a fatal neurodegenerative disease of unknown etiology characterized by widespread aggregation of the protein alpha-synuclein in neurons and glia. Its orphan status, biological relationship to Parkinson's disease (PD), and rapid progression have sparked interest in drug development. One significant obstacle to therapeutics is disease heterogeneity. Here, we share our process of developing a clinical trial-ready cohort of MSA patients (69 patients in 2 years) within an outpatient clinical setting, and recruiting 20 of these patients into a longitudinal "n-of-few" clinical trial paradigm. First, we deeply phenotype our patients with clinical scales (UMSARS, BARS, MoCA, NMSS, and UPSIT) and tests designed to establish early differential diagnosis (including volumetric MRI, FDG-PET, MIBG scan, polysomnography, genetic testing, autonomic function tests, skin biopsy) or disease activity (PBR06-TSPO). Second, we longitudinally collect biospecimens (blood, CSF, stool) and clinical, biometric, and imaging data to generate antecedent disease-progression scores. Third, in our Mass General Brigham SCiN study (stem cells in neurodegeneration), we generate induced pluripotent stem cell (iPSC) models from our patients, matched to biospecimens, including postmortem brain. We present 38 iPSC lines derived from MSA patients and relevant disease controls (spinocerebellar ataxia and PD, including alpha-synuclein triplication cases), 22 matched to whole-genome sequenced postmortem brain. iPSC models may facilitate matching patients to appropriate therapies, particularly in heterogeneous diseases for which patient-specific biology may elude animal models. We anticipate that deeply phenotyped and genotyped patient cohorts matched to cellular models will increase the likelihood of success in clinical trials for MSA.

Bronchoalveolar lavage fluid lymphocytosis in chronic hypersensitivity pneumonitis: a systematic review and meta-analysis.

BACKGROUND: The role of bronchoalveolar lavage fluid (BALF) lymphocyte percentage in diagnosing chronic hypersensitivity pneumonitis (CHP) is unclear. We conducted a systematic review and meta-analysis of bronchoalveolar lavage (BAL) lymphocyte percentage in the diagnosis of CHP. METHODS: We searched Medline, Embase and the Cochrane Library from inception to August 2019. Individual patient data were obtained to test performance characteristics of BAL lymphocyte percentage at different thresholds. Random-effects models were used for pooled estimates, with comparisons made between CHP and non-CHP interstitial lung diseases (ILDs). RESULTS: Fifty-three studies were included in the systematic review and 42 in the meta-analysis. The pooled estimate for BAL lymphocyte percentage was 42.8% (95% CI 37.7-47.8, I2=95.3%) in CHP, 10.0% (95% CI 6.9-13.1, I2=91.2%) in idiopathic pulmonary fibrosis (IPF), 23.1% (95% CI 3.0-43.2, I2=85.2%) in non-IPF idiopathic interstitial pneumonia (IIP), 23.4% (95% CI 11.0-35.9, I2=45.7%) in connective-tissue disease associated ILD (CTD-ILD) and 31.2% (95% CI 17.6-44.8, I2=95.2%) in sarcoidosis. Results differed between CHP and IPF (p<0.0001), non-IPF IIP (p=0.0309) or CTD-ILD (p=0.0824), but not between CHP and sarcoidosis (p=0.0966). Using individual patient data from eight studies, a lymphocyte percentage threshold of >20% provided a sensitivity of 68.1% and a specificity of 64.8% for CHP. Higher thresholds provided lower sensitivity with higher specificity. Older age and ever having smoked were associated with lower lymphocyte percentage in CHP. CONCLUSIONS: BAL lymphocyte percentage is higher in CHP compared to IPF and other IIPs, with higher thresholds providing improved specificity at the cost of sensitivity. However, the parent studies are at risk of incorporation bias and prospective studies should evaluate the additive discriminate value of BAL lymphocyte percentage to accurately diagnose CHP.

Oxygen in patients with fibrotic interstitial lung disease: an international Delphi survey.

RATIONALE: Patients with fibrotic interstitial lung disease (ILD) frequently develop resting or exertional hypoxaemia. There is heterogeneity in clinical practice and a paucity of evidence guiding supplemental oxygen use in this patient population. The objectives of this study were to build international expert-based consensus on the indications and goals of supplemental oxygen from the perspective of healthcare providers, and identify potential barriers to its access. METHODS: Semistructured interviews and a comprehensive literature search informed items for the Delphi survey, with items not meeting consensus included in round 2. Round 3 contained survey questions regarding regional funding coverage for oxygen therapy. A priori definitions of consensus were median scores of 4 (agree) to 5 (strongly agree) for "agreement", 1 (strongly disagree) to 2 (disagree) for "disagreement" or 3 (unsure) with an interquartile range of 0-1. RESULTS: 42 out of 45 (93%) experts completed all three survey rounds, representing 17 countries. 20 out of 36 items met consensus for agreement or disagreement, 10 items met consensus for unsure and four items did not meet consensus. Experts agreed that oxygen should be recommended for patients with severe resting hypoxaemia and in cases of exertional desaturation to <85-89%, particularly with attributable symptoms or exercise limitation. There are regional differences in funding coverage for oxygen, based on desaturation thresholds, clinical symptoms and testing requirements. CONCLUSIONS: Experts achieved consensus on 20 items guiding supplemental oxygen use in fibrotic ILD. These findings may inform research, clinical recommendations and funding policy.

Engineering of vitamin prototrophy in Clostridium ljungdahlii and Clostridium autoethanogenum.

Clostridium autoethanogenum and Clostridium ljungdahlii are physiologically and genetically very similar strict anaerobic acetogens capable of growth on carbon monoxide as sole carbon source. While exact nutritional requirements have not been reported, we observed that for growth, the addition of vitamins to media already containing yeast extract was required, an indication that these are fastidious microorganisms. Elimination of complex components and individual vitamins from the medium revealed that the only organic compounds required for growth were pantothenate, biotin and thiamine. Analysis of the genome sequences revealed that three genes were missing from pantothenate and thiamine biosynthetic pathways, and five genes were absent from the pathway for biotin biosynthesis. Prototrophy in C. autoethanogenum and C. ljungdahlii for pantothenate was obtained by the introduction of plasmids carrying the heterologous gene clusters panBCD from Clostridium acetobutylicum, and for thiamine by the introduction of the thiC-purF operon from Clostridium ragsdalei. Integration of panBCD into the chromosome through allele-coupled exchange also conveyed prototrophy. C. autoethanogenum was converted to biotin prototrophy with gene sets bioBDF and bioHCA from Desulfotomaculum nigrificans strain CO-1-SRB, on plasmid and integrated in the chromosome. The genes could be used as auxotrophic selection markers in recombinant DNA technology. Additionally, transformation with a subset of the genes for pantothenate biosynthesis extended selection options with the pantothenate precursors pantolactone and/or beta-alanine. Similarly, growth was obtained with the biotin precursor pimelate combined with genes bioYDA from C. acetobutylicum. The work raises questions whether alternative steps exist in biotin and thiamine biosynthesis pathways in these acetogens.

A novel conjugal donor strain for improved DNA transfer into Clostridium spp.

Clostridium encompasses species which are relevant to human and animal disease as well as species which have industrial potential, for instance, as producers of chemicals and fuels or as tumour delivery vehicles. Genetic manipulation of these target organisms is critical for advances in these fields. DNA transfer efficiencies, however, vary between species. Low efficiencies can impede the progress of research efforts. A novel conjugal donor strain of Escherichia coli has been created which exhibits a greater than 10-fold increases in conjugation efficiency compared to the traditionally used CA434 strain in the three species tested; C. autoethanogenum DSM 10061, C. sporogenes NCIMB 10696 and C. difficile R20291. The novel strain, designated 'sExpress', does not methylate DNA at Dcm sites (CCWGG) which allows circumvention of cytosine-specific Type IV restriction systems. A robust protocol for conjugation is presented which routinely produces in the order of 105 transconjugants per millilitre of donor cells for C. autoethanogenum, 106 for C. sporogenes and 102 for C. difficile R20291. The novel strain created is predicted to be a superior conjugal donor in a wide range of species which possess Type IV restriction systems.

A roadmap for gene system development in Clostridium.

Clostridium species are both heroes and villains. Some cause serious human and animal diseases, those present in the gut microbiota generally contribute to health and wellbeing, while others represent useful industrial chassis for the production of chemicals and fuels. To understand, counter or exploit, there is a fundamental requirement for effective systems that may be used for directed or random genome modifications. We have formulated a simple roadmap whereby the necessary gene systems maybe developed and deployed. At its heart is the use of 'pseudo-suicide' vectors and the creation of a pyrE mutant (a uracil auxotroph), initially aided by ClosTron technology, but ultimately made using a special form of allelic exchange termed ACE (Allele-Coupled Exchange). All mutants, regardless of the mutagen employed, are made in this host. This is because through the use of ACE vectors, mutants can be rapidly complemented concomitant with correction of the pyrE allele and restoration of uracil prototrophy. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Once available, the pyrE host may be used to stably insert all manner of application specific modules. Examples include, a sigma factor to allow deployment of a mariner transposon, hydrolases involved in biomass deconstruction and therapeutic genes in cancer delivery vehicles. To date, provided DNA transfer is obtained, we have not encountered any clostridial species where this technology cannot be applied. These include, Clostridium difficile, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium perfringens, Clostridium sporogenes, Clostridium pasteurianum, Clostridium ljungdahlii, Clostridium autoethanogenum and even Geobacillus thermoglucosidasius.

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

BACKGROUND: 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. RESULTS: We performed next generation sequencing using Illumina MiSeq technology on the DSM10061 strain of Clostridium autoethanogenum and observed 243 single nucleotide discrepancies when compared to the published finished sequence (NCBI: GCA_000484505.1), with 59.1 % present in coding regions. These variations were confirmed by Sanger sequencing and subsequent analysis suggested that the discrepancies were sequencing errors in the published genome not true single nucleotide polymorphisms. This was corroborated by the observation that over 90 % occurred within homopolymer regions of greater than 4 nucleotides in length. It was also observed that many genes containing these sequencing errors were annotated in the published closed genome as encoding proteins containing frameshift mutations (18 instances) or were annotated despite the coding frame containing stop codons, which if genuine, would severely hinder the organism's ability to survive. Furthermore, we have completed a comprehensive manual curation to reduce errors in the annotation that occur through serial use of automated annotation pipelines in related species. As a result, different functions were assigned to gene products or previous functional annotations rejected because of missing evidence in various occasions. CONCLUSIONS: We present a revised manually curated full genome sequence for Clostridium autoethanogenum DSM10061, which provides reliable information for genome-scale models that rely heavily on the accuracy of annotation, and represents an important step towards the manipulation and metabolic modelling of this industrially relevant acetogen.

Pathogenicity and virulence of Clostridium botulinum.

Clostridium botulinum, a polyphyletic Gram-positive taxon of bacteria, is classified purely by their ability to produce botulinum neurotoxin (BoNT). BoNT is the primary virulence factor and the causative agent of botulism. A potentially fatal disease, botulism is classically characterized by a symmetrical descending flaccid paralysis, which is left untreated can lead to respiratory failure and death. Botulism cases are classified into three main forms dependent on the nature of intoxication; foodborne, wound and infant. The BoNT, regarded as the most potent biological substance known, is a zinc metalloprotease that specifically cleaves SNARE proteins at neuromuscular junctions, preventing exocytosis of neurotransmitters, leading to muscle paralysis. The BoNT is now used to treat numerous medical conditions caused by overactive or spastic muscles and is extensively used in the cosmetic industry due to its high specificity and the exceedingly small doses needed to exert long-lasting pharmacological effects. Additionally, the ability to form endospores is critical to the pathogenicity of the bacteria. Disease transmission is often facilitated via the metabolically dormant spores that are highly resistant to environment stresses, allowing persistence in the environment in unfavourable conditions. Infant and wound botulism infections are initiated upon germination of the spores into neurotoxin producing vegetative cells, whereas foodborne botulism is attributed to ingestion of preformed BoNT. C. botulinum is a saprophytic bacterium, thought to have evolved its potent neurotoxin to establish a source of nutrients by killing its host.

Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations.

Base editors are recent multiplex gene editing tools derived from the Cas9 nuclease of Streptomyces pyogenes. They can target and modify a single nucleotide in the genome without inducing double-strand breaks (DSB) of the DNA helix. As such, they hold great potential for the engineering of microbes that lack effective DSB repair pathways such as homologous recombination (HR) or non-homologous end-joining (NHEJ). However, few applications of base editors have been reported in prokaryotes to date, and their advantages and drawbacks have not been systematically reported. Here, we used the base editors Target-AID and Target-AID-NG to introduce nonsense mutations into four different coding sequences of the industrially relevant Gram-positive bacterium Clostridium autoethanogenum. While up to two loci could be edited simultaneously using a variety of multiplexing strategies, most colonies exhibited mixed genotypes and most available protospacers led to undesired mutations within the targeted editing window. Additionally, fifteen off-target mutations were detected by sequencing the genome of the resulting strain, among them seven single-nucleotide polymorphisms (SNP) in or near loci bearing some similarity with the targeted protospacers, one 15 nt duplication, and one 12 kb deletion which removed uracil DNA glycosylase (UDG), a key DNA repair enzyme thought to be an obstacle to base editing mutagenesis. A strategy to process prokaryotic single-guide RNA arrays by exploiting tRNA maturation mechanisms is also illustrated.

Design, Analysis, and Implementation of a Novel Biochemical Pathway for Ethylene Glycol Production in Clostridium autoethanogenum.

The platform chemical ethylene glycol (EG) is used to manufacture various commodity chemicals of industrial importance, but largely remains synthesized from fossil fuels. Although several novel metabolic pathways have been reported for its bioproduction in model organisms, none has been reported for gas-fermenting, non-model acetogenic chassis organisms. Here, we describe a novel, synthetic biochemical pathway to convert acetate into EG in the industrially important gas-fermenting acetogen,Clostridium autoethanogenum. We not only developed a computational workflow to design and analyze hundreds of novel biochemical pathways for EG production but also demonstrated a successful pathway construction in the chosen host. The EG production was achieved using a two-plasmid system to bypass unfeasible expression levels and potential toxic enzymatic interactions. Although only a yield of 0.029 g EG/g fructose was achieved and therefore requiring further strain engineering efforts to optimize the designed strain, this work demonstrates an important proof-of-concept approach to computationally design and experimentally implement fully synthetic metabolic pathways in a metabolically highly specific, non-model host organism.

Agr Quorum Sensing influences the Wood-Ljungdahl pathway in Clostridium autoethanogenum.

Acetogenic bacteria are capable of fermenting CO2 and carbon monoxide containing waste-gases into a range of platform chemicals and fuels. Despite major advances in genetic engineering and improving these biocatalysts, several important physiological functions remain elusive. Among these is quorum sensing, a bacterial communication mechanism known to coordinate gene expression in response to cell population density. Two putative agr systems have been identified in the genome of Clostridium autoethanogenum suggesting bacterial communication via autoinducing signal molecules. Signal molecule-encoding agrD1 and agrD2 genes were targeted for in-frame deletion. During heterotrophic growth on fructose as a carbon and energy source, single deletions of either gene did not produce an observable phenotype. However, when both genes were simultaneously inactivated, final product concentrations in the double mutant shifted to a 1.5:1 ratio of ethanol:acetate, compared to a 0.2:1 ratio observed in the wild type control, making ethanol the dominant fermentation product. Moreover, CO2 re-assimilation was also notably reduced in both hetero- and autotrophic growth conditions. These findings were supported through comparative proteomics, which showed lower expression of carbon monoxide dehydrogenase, formate dehydrogenase A and hydrogenases in the ∆agrD1∆agrD2 double mutant, but higher levels of putative alcohol and aldehyde dehydrogenases and bacterial micro-compartment proteins. These findings suggest that Agr quorum sensing, and by inference, cell density play a role in carbon resource management and use of the Wood-Ljungdahl pathway as an electron sink.

Diaphragmatic paralysis in COVID-19: a rare cause of postacute sequelae of COVID-19 dyspnoea.

We describe a 56-year-old female patient hospitalised with COVID-19 in April 2020 who had persistent respiratory symptoms after radiographic and microbiologic recovery. X-ray of the chest demonstrated an elevated right hemidiaphragm while fluoroscopy confirmed unilateral diaphragmatic paralysis. Symptoms resolved gradually, concurrent with restoration of right hemidiaphragm function. Thus, we describe a rare cause of postacute sequelae of COVID-19 dyspnoea.

Rapid prediction of adverse outcomes for acute normotensive pulmonary embolism: derivation of the Calgary Acute Pulmonary Embolism score.

BACKGROUND: Acute pulmonary embolism (PE) has a wide spectrum of outcomes, but the best method to risk-stratify normotensive patients for adverse outcomes remains unclear. METHODS: A multicentre retrospective cohort study of acute PE patients admitted from emergency departments in Calgary, Canada, between 2012 and 2017 was used to develop a refined acute PE risk score. The composite primary outcome of in-hospital PE-related death or haemodynamic decompensation. The model was internally validated using bootstrapping and the prognostic value of the derived risk score was compared to the Bova score. RESULTS: Of 2067 patients with normotensive acute PE, the primary outcome (haemodynamic decompensation or PE-related death) occurred in 32 (1.5%) patients. In simplified Pulmonary Embolism Severity Index high-risk patients (n=1498, 78%), a multivariable model used to predict the primary outcome retained computed tomography (CT) right-left ventricular diameter ratio ≥1.5, systolic blood pressure 90-100 mmHg, central pulmonary artery clot and heart rate ≥100 beats·min-1 with a C-statistic of 0.89 (95% CI 0.82-0.93). Three risk groups were derived using a weighted score (score, prevalence, primary outcome event rate): group 1 (0-3, 73.8%, 0.34%), group 2 (4-6, 17.6%, 5.8%), group 3 (7-9, 8.7%, 12.8%) with a C-statistic 0.85 (95% CI 0.78-0.91). In comparison the prevalence (primary outcome) by Bova risk stages (n=1179) were stage I 49.8% (0.2%); stage II 31.9% (2.7%); and stage III 18.4% (7.8%) with a C-statistic 0.80 (95% CI 0.74-0.86). CONCLUSIONS: A simple four-variable risk score using clinical data immediately available after CT diagnosis of acute PE predicts in-hospital adverse outcomes. External validation of the Calgary Acute Pulmonary Embolism score is required.

A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences.

Phenotypic complementation of gene knockouts is an essential step in establishing function. Here, we describe a simple strategy for 'gold standard' complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. The method relies on the prior incorporation of a unique 24 nucleotide (nt) 'bookmark' sequence into the mutant allele to act as a guide RNA target during its Cas9-mediated replacement with the WT allele. The bookmark comprises a 23 nt Cas9 target sequence plus an additional nt to ensure the deletion is in-frame. Here, bookmarks are tailored to Streptococcus pyogenes CRISPR/Cas9 but could be designed for any CRISPR/Cas system. For proof of concept, nine bookmarks were tested in Clostridium autoethanogenum. Complementation efficiencies reached 91%. As complemented strains are indistinguishable from their progenitors, concerns over contamination may be satisfied by the incorporation of 'watermark' sequences into the complementing genes.