Metabolic Response of Clostridium ljungdahlii to Oxygen Exposure

ABSTRACTClostridium ljungdahliiis an important synthesis gas-fermenting bacterium used in the biofuels industry, and a preliminary investigation showed that it has some tolerance to oxygen when cultured in rich mixotrophic medium. Batch cultures not only continue to grow and consume H2, CO, and fructose after 8% O2exposure, but fermentation product analysis revealed an increase in ethanol concentration and decreased acetate concentration compared to non-oxygen-exposed cultures. In this study, the mechanisms for higher ethanol production and oxygen/reactive oxygen species (ROS) detoxification were identified using a combination of fermentation, transcriptome sequencing (RNA-seq) differential expression, and enzyme activity analyses. The results indicate that the higher ethanol and lower acetate concentrations were due to the carboxylic acid reductase activity of a more highly expressed predicted aldehyde oxidoreductase (CLJU_c24130) and thatC. ljungdahlii's primary defense upon oxygen exposure is a predicted rubrerythrin (CLJU_c39340). The metabolic responses of higher ethanol production and oxygen/ROS detoxification were found to be linked by cofactor management and substrate and energy metabolism. This study contributes new insights into the physiology and metabolism ofC. ljungdahliiand provides new genetic targets to generateC. ljungdahliistrains that produce more ethanol and are more tolerant to syngas contaminants.

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Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

Applied and Environmental Microbiology ◽

10.1128/aem.03666-13 ◽

2014 ◽

Vol 80 (8) ◽

pp. 2410-2416 ◽

Cited By ~ 65

Author(s):

Areen Banerjee ◽

Ching Leang ◽

Toshiyuki Ueki ◽

Kelly P. Nevin ◽

Derek R. Lovley

Keyword(s):

Ethanol Production ◽

Genetic Manipulation ◽

Electron Flow ◽

Model Organism ◽

Inducible Expression ◽

Carbon Flow ◽

Wild Type ◽

Content Type ◽

Microbial Electrosynthesis ◽

Clostridium Ljungdahlii

ABSTRACTThe development of tools for genetic manipulation ofClostridium ljungdahliihas increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox forC. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported forClostridium perfringens, was investigated. The plasmid pAH2, originally developed forC. perfringenswith agusAreporter gene, functioned as an effective lactose-inducible system inC. ljungdahlii. Lactose induction ofC. ljungdahliicontaining pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression ofadhE130-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression ofadhE1in a strain in whichadhE1and theadhE1homologadhE2had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression ofadhE2similarly failed to restore ethanol production, suggesting thatadhE1is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

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A Genetic System for Clostridium ljungdahlii: a Chassis for Autotrophic Production of Biocommodities and a Model Homoacetogen

Applied and Environmental Microbiology ◽

10.1128/aem.02891-12 ◽

2012 ◽

Vol 79 (4) ◽

pp. 1102-1109 ◽

Cited By ~ 118

Author(s):

Ching Leang ◽

Toshiyuki Ueki ◽

Kelly P. Nevin ◽

Derek R. Lovley

Keyword(s):

Ethanol Production ◽

Gene Deletion ◽

Genetic Manipulation ◽

Electron Flow ◽

Genetic System ◽

Content Type ◽

Suicide Vector ◽

Double Deletion ◽

Clostridium Ljungdahlii ◽

Genomic Studies

ABSTRACTMethods for genetic manipulation ofClostridium ljungdahliiare of interest because of the potential for production of fuels and other biocommodities from carbon dioxide via microbial electrosynthesis or more traditional modes of autotrophy with hydrogen or carbon monoxide as the electron donor. Furthermore, acetogenesis plays an important role in the global carbon cycle. Gene deletion strategies required for physiological studies ofC. ljungdahliihave not previously been demonstrated. An electroporation procedure for introducing plasmids was optimized, and four different replicative origins for plasmid propagation inC. ljungdahliiwere identified. Chromosomal gene deletion via double-crossover homologous recombination with a suicide vector was demonstrated initially with deletion of the gene for FliA, a putative sigma factor involved in flagellar biogenesis and motility inC. ljungdahlii. Deletion offliAyielded a strain that lacked flagella and was not motile. To evaluate the potential utility of gene deletions for functional genomic studies and to redirect carbon and electron flow, the genes for the putative bifunctional aldehyde/alcohol dehydrogenases,adhE1andadhE2, were deleted individually or together. Deletion ofadhE1, but notadhE2, diminished ethanol production with a corresponding carbon recovery in acetate. The double deletion mutant had a phenotype similar to that of theadhE1-deficient strain. Expression ofadhE1intranspartially restored the capacity for ethanol production. These results demonstrate the feasibility of genetic investigations of acetogen physiology and the potential for genetic manipulation ofC. ljungdahliito optimize autotrophic biocommodity production.

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Whole-Transcriptome Shotgun Sequencing (RNA-seq) Screen Reveals Upregulation of Cellobiose and Motility Operons of Lactobacillus ruminis L5 during Growth on Tetrasaccharides Derived from Barley β-Glucan

Applied and Environmental Microbiology ◽

10.1128/aem.01887-13 ◽

2013 ◽

Vol 79 (18) ◽

pp. 5661-5669 ◽

Cited By ~ 21

Author(s):

Blair Lawley ◽

Ian M. Sims ◽

Gerald W. Tannock

Keyword(s):

Growth Phase ◽

Shotgun Sequencing ◽

Rna Seq ◽

Content Type ◽

Batch Cultures ◽

Reverse Transcription Quantitative Pcr ◽

Altered Gene ◽

Lactobacillus Ruminis ◽

Early Growth Phase ◽

Whole Transcriptome

ABSTRACTLactobacillus ruminisis an inhabitant of human bowels and bovine rumens. None of 10 isolates (three from bovine rumen, seven from human feces) ofL. ruministhat were tested could utilize barley β-glucan for growth. Seven of the strains ofL. ruminiswere, however, able to utilize tetrasaccharides (3-O-β-cellotriosyl-d-glucose [LDP4] or 4-O-β-laminaribiosyl-d-cellobiose [CDP4]) present in β-glucan hydrolysates for growth. The tetrasaccharides were generated by the use of lichenase or cellulase, respectively. To learn more about the utilization of tetrasaccharides byL. ruminis, whole-transcriptome shotgun sequencing (RNA-seq) was tested as a transcriptional screen to detect altered gene expression when an autochthonous human strain (L5) was grown in medium containing CDP4. RNA-seq results were confirmed and extended by reverse transcription-quantitative PCR assays of selected genes in two upregulated operons when cells were grown as batch cultures in medium containing either CDP4 or LDP4. The cellobiose utilization operon had increased transcription, particularly in early growth phase, whereas the chemotaxis/motility operon was upregulated in late growth phase. Phenotypic changes were seen in relation to upregulation of chemotaxis/flagellar operons: flagella were rarely seen by electron microscopy on glucose-grown cells but cells cultured in tetrasaccharide medium were commonly flagellated. Chemotactic movement toward tetrasaccharides was demonstrated in capillary cultures.L. ruminisutilized 3-O-β-cellotriosyl-d-glucose released by β-glucan hydrolysis due to bowel commensalCoprococcussp., indicating that cross feeding of tetrasaccharide between bacteria could occur. Therefore, the RNA-seq screen and subsequent experiments had utility in revealing foraging attributes of gut commensalLactobacillus ruminis.

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Unravelling the Reduction Pathway as an Alternative Metabolic Route to Hydroxycinnamate Decarboxylation in Lactobacillus plantarum

Applied and Environmental Microbiology ◽

10.1128/aem.01123-18 ◽

2018 ◽

Vol 84 (15) ◽

Cited By ~ 10

Author(s):

Laura Santamaría ◽

Inés Reverón ◽

Félix López de Felipe ◽

Blanca de las Rivas ◽

Rosario Muñoz

Keyword(s):

Lactic Acid ◽

Lactobacillus Plantarum ◽

Metabolic Response ◽

Reductase Activity ◽

Bacterial Species ◽

Hydroxycinnamic Acids ◽

Hydroxyl Group ◽

Transcriptomic Response ◽

Content Type

ABSTRACT Lactobacillus plantarum is the lactic acid bacterial species most frequently found in plant-food fermentations where hydroxycinnamic acids are abundant. L. plantarum efficiently decarboxylates these compounds and also reduces them, yielding substituted phenylpropionic acids. Although the reduction step is known to be induced by a hydroxycinnamic acid, the enzymatic machinery responsible for this reduction pathway has not been yet identified and characterized. A previous study on the transcriptomic response of L. plantarum to p-coumaric acid revealed a marked induction of two contiguous genes, lp_1424 and lp_1425, encoding putative reductases. In this work, the disruption of these genes abolished the hydroxycinnamate reductase activity of L. plantarum, supporting their involvement in such chemical activity. Functional in vitro studies revealed that Lp_1425 (HcrB) exhibits hydroxycinnamate reductase activity but was unstable in solution. In contrast, Lp_1424 (HcrA) was inactive but showed high stability. When the hcrAB genes were co-overexpressed, the formation of an active heterodimer (HcrAB) was observed. Since L. plantarum reductase activity was only observed on hydroxycinnamic acids (o-coumaric, m-coumaric, p-coumaric, caffeic, ferulic, and sinapic acids), the presence of a hydroxyl group substituent on the benzene ring appears to be required for activity. In addition, hydroxycinnamate reductase activity was not widely present among lactic acid bacteria, and it was associated with the presence of hcrAB genes. This study revealed that L. plantarum hydroxycinnamate reductase is a heterodimeric NADH-dependent coumarate reductase acting on a carbon-carbon double bond. IMPORTANCE Lactobacillus plantarum is a bacterial species frequently found in the fermentation of vegetables where hydroxycinnamic acids are present. The bacterial metabolism on these compounds during fermentation plays a fundamental role in the biological activity of hydroxycinnamates. L. plantarum strains exhibit an as yet unknown reducing activity, transforming hydroxycinnamates to substituted phenylpropionic acids, which possess higher antioxidant activity than their precursors. The protein machinery involved in hydroxycinnamate reduction, HcrAB, was genetically identified and characterized. The heterodimeric NADH-dependent coumarate reductase HcrAB described in this work provides new insights on the L. plantarum metabolic response to counteract the stressful conditions generated by food phenolics.

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Ethanol Metabolism Dynamics in Clostridium ljungdahlii Grown on Carbon Monoxide

Applied and Environmental Microbiology ◽

10.1128/aem.00730-20 ◽

2020 ◽

Vol 86 (14) ◽

Cited By ~ 2

Author(s):

Zi-Yong Liu ◽

De-Chen Jia ◽

Kun-Di Zhang ◽

Hai-Feng Zhu ◽

Quan Zhang ◽

...

Keyword(s):

Carbon Monoxide ◽

Ethanol Production ◽

Biomass Production ◽

Ethanol Oxidation ◽

Ethanol Yield ◽

Ethanol Metabolism ◽

Content Type ◽

Atp Production ◽

Clostridium Ljungdahlii ◽

Gas Fermentation

ABSTRACT Bioethanol production from syngas using acetogenic bacteria has attracted considerable attention in recent years. However, low ethanol yield is the biggest challenge that prevents the commercialization of syngas fermentation into biofuels using microbial catalysts. The present study demonstrated that ethanol metabolism plays an important role in recycling NADH/NAD+ during autotrophic growth. Deletion of bifunctional aldehyde/alcohol dehydrogenase (adhE) genes leads to significant growth deficiencies in gas fermentation. Using specific fermentation technology in which the gas pressure and pH were constantly controlled at 0.1 MPa and 6.0, respectively, we revealed that ethanol was formed during the exponential phase, closely accompanied by biomass production. Then, ethanol was oxidized to acetate via the aldehyde ferredoxin oxidoreductase pathway in Clostridium ljungdahlii. A metabolic experiment using 13C-labeled ethanol and acetate, redox balance analysis, and comparative transcriptomic analysis demonstrated that ethanol production and reuse shared the metabolic pathway but occurred at different growth phases. IMPORTANCE Ethanol production from carbon monoxide (CO) as a carbon and energy source by Clostridium ljungdahlii and “Clostridium autoethanogenum” is currently being commercialized. During gas fermentation, ethanol synthesis is NADH-dependent. However, ethanol oxidation and its regulatory mechanism remain incompletely understood. Energy metabolism analysis demonstrated that reduced ferredoxin is the sole source of NADH formation by the Rnf-ATPase system, which provides ATP for cell growth during CO fermentation. Therefore, ethanol production is tightly linked to biomass production (ATP production). Clarification of the mechanism of ethanol oxidation and biosynthesis can provide an important reference for generating high-ethanol-yield strains of C. ljungdahlii in the future.

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The influence of the initial concentrations of glucose and yeast extract on the ethanolic fermentation by Pachysolen tannophilus

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19900854 ◽

1990 ◽

Vol 55 (3) ◽

pp. 854-866 ◽

Cited By ~ 5

Author(s):

Rodríguez V. Bravo ◽

Rubio F. Camacho ◽

Villasclaras S. Sánchez ◽

Vico M. Castro

Keyword(s):

Cell Growth ◽

Ethanol Production ◽

Yeast Extract ◽

Culture Medium ◽

Ethanolic Fermentation ◽

Pachysolen Tannophilus ◽

Significant Component ◽

Batch Cultures

The ethanolic fermentation in batch cultures of Pachysolen tannophilus was studied experimentally varying the initial concentrations of two of the components in the culture medium: glucose between 0 and 200 g l-1 and yeast extract between 0 and 8 g l-1. The yeast extract appears to be a significant component both in cell growth and for ethanol production.

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Mitochondrial Mistranslation in Brain Provokes a Metabolic Response Which Mitigates the Age-Associated Decline in Mitochondrial Gene Expression

International Journal of Molecular Sciences ◽

10.3390/ijms22052746 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2746

Author(s):

Dimitri Shcherbakov ◽

Reda Juskeviciene ◽

Adrián Cortés Sanchón ◽

Margarita Brilkova ◽

Hubert Rehrauer ◽

...

Keyword(s):

Gene Expression ◽

Metabolic Response ◽

Mitochondrial Gene ◽

Tca Cycle ◽

Brain Mitochondria ◽

Mitochondrial Gene Expression ◽

Rna Seq ◽

The Tca Cycle ◽

Neurological Phenotype

Mitochondrial misreading, conferred by mutation V338Y in mitoribosomal protein Mrps5, in-vivo is associated with a subtle neurological phenotype. Brain mitochondria of homozygous knock-in mutant Mrps5V338Y/V338Y mice show decreased oxygen consumption and reduced ATP levels. Using a combination of unbiased RNA-Seq with untargeted metabolomics, we here demonstrate a concerted response, which alleviates the impaired functionality of OXPHOS complexes in Mrps5 mutant mice. This concerted response mitigates the age-associated decline in mitochondrial gene expression and compensates for impaired respiration by transcriptional upregulation of OXPHOS components together with anaplerotic replenishment of the TCA cycle (pyruvate, 2-ketoglutarate).

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Relationship of patient shame to working alliance and satisfaction: a preliminary investigation

The Journal of Mental Health Training Education and Practice ◽

10.1108/jmhtep-10-2017-0059 ◽

2019 ◽

Vol 14 (4) ◽

pp. 251-263

Author(s):

Daniel J. Carabellese ◽

Michael J. Proeve ◽

Rachel M. Roberts

Keyword(s):

Patient Satisfaction ◽

Working Alliance ◽

Preliminary Investigation ◽

Community Members ◽

Content Type ◽

Mediation Analyses ◽

External Shame ◽

Relationship Of ◽

The Relationship

Purpose The purpose of this paper is to explore the relationship of two distinct variants of dispositional shame (internal and external shame) with collaborative, purpose-driven aspects of the patient–provider relationship (working alliance) and patient satisfaction. The aim of this research was to conduct a preliminary investigation into the relevance of dispositional shame in a general healthcare population. Design/methodology/approach In total, 127 community members (mean age 25.9 years) who reported that they had regularly seen a GP over the past year were recruited at an Australian university. Participants were asked to reflect on their relationship with their GP, and completed instruments assessing various domains of shame, as well as working alliance and patient satisfaction. Findings Non-parametric correlations were examined to determine the direction and strength of relationships, as well as conducting mediation analyses where applicable. Small, negative correlations were evident between external shame and working alliance. Both external and internal shame measures were also negatively correlated with patient satisfaction. Finally, the relationship of external shame to patient satisfaction was partially mediated by working alliance. Practical implications Both the reported quality of patient–provider working alliance, and level of patient satisfaction are related to levels of dispositional shame in patients, and working alliance may act as a mediator for this relationship. Originality/value The findings from this preliminary study suggest that internal and external shame are important factors to consider in the provision of medical care to maximise the quality of patient experience and working alliance.

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Application of the 1-µsec pulsed-dye laser to the treatment of experimental cerebral vasospasm

Journal of Neurosurgery ◽

10.3171/jns.1991.75.2.0271 ◽

1991 ◽

Vol 75 (2) ◽

pp. 271-276 ◽

Cited By ~ 12

Author(s):

Atsushi Teramura ◽

Robert Macfarlane ◽

Christopher J. Owen ◽

Ralph de la Torre ◽

Kenton W. Gregory ◽

...

Keyword(s):

Cerebral Vasospasm ◽

Basilar Artery ◽

Laser Energy ◽

Autologous Blood ◽

Preliminary Investigation ◽

Dye Laser ◽

Pulsed Dye Laser ◽

Content Type

✓ Laser energy of 480 nm was applied in 1-µsec pulses varying between 2.2 and 10 mJ to in vitro and in vivo models of cerebral vasospasm. First, the pulsed-dye laser was applied intravascularly via a 320-µm fiber to basilar artery segments from six dogs. The segments were mounted in a vessel-perfusion apparatus and constricted to, on average, 70% of resting diameter by superfusion with dog hemolysate. Immediate increase in basilar artery diameter occurred to a mean of 83% of control. In a second model, the basilar artery was exposed transclivally in the rabbit. In three normal animals, superfusion of the artery with rabbit hemolysate resulted in a reduction of mean vessel diameter to 81% of control. Following extravascular application of the laser, vessels returned to an average of 106% of the resting state. In six rabbits, the basilar artery was constricted by two intracisternal injections of autologous blood, 3 days apart. Two to 4 days after the second injection, the basilar artery was exposed. Extravascular laser treatment from a quartz fiber placed perpendicular to the vessel adventitia resulted in an immediate 53% average increase in caliber to an estimated 107% of control. No reconstriction was observed over a period of up to 5 hours. Morphologically, damage to the arterial wall was slight. This preliminary investigation suggests that the 1-µsec pulsed-dye laser may be of benefit in the treatment of cerebral vasospasm.

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