scholarly journals Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

2014 ◽  
Vol 80 (8) ◽  
pp. 2410-2416 ◽  
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.

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

2012 ◽  
Vol 79 (4) ◽  
pp. 1102-1109 ◽  
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.

scholarly journals Converting Carbon Dioxide to Butyrate with an Engineered Strain of Clostridium ljungdahlii

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Trevor L. Woodard ◽  
Derek R. Lovley
Keyword(s):  
Carbon Dioxide ◽  
Electron Flow ◽  
Microbial Conversion ◽  
Gene Encoding ◽  
Content Type ◽  
Microbial Electrosynthesis ◽  
Clostridium Ljungdahlii ◽  
Coa Transferase ◽  
Organic Products ◽  
Key Enzyme

ABSTRACTMicrobial conversion of carbon dioxide to organic commodities via syngas metabolism or microbial electrosynthesis is an attractive option for production of renewable biocommodities. The recent development of an initial genetic toolbox for the acetogenClostridium ljungdahliihas suggested thatC. ljungdahliimay be an effective chassis for such conversions. This possibility was evaluated by engineering a strain to produce butyrate, a valuable commodity that is not a natural product ofC. ljungdahliimetabolism. Heterologous genes required for butyrate production from acetyl-coenzyme A (CoA) were identified and introduced initially on plasmids and in subsequent strain designs integrated into theC. ljungdahliichromosome. Iterative strain designs involved increasing translation of a key enzyme by modifying a ribosome binding site, inactivating the gene encoding the first step in the conversion of acetyl-CoA to acetate, disrupting the gene which encodes the primary bifunctional aldehyde/alcohol dehydrogenase for ethanol production, and interrupting the gene for a CoA transferase that potentially represented an alternative route for the production of acetate. These modifications yielded a strain in which ca. 50 or 70% of the carbon and electron flow was diverted to the production of butyrate with H2or CO as the electron donor, respectively. These results demonstrate the possibility of producing high-value commodities from carbon dioxide withC. ljungdahliias the catalyst.IMPORTANCEThe development of a microbial chassis for efficient conversion of carbon dioxide directly to desired organic products would greatly advance the environmentally sustainable production of biofuels and other commodities.Clostridium ljungdahliiis an effective catalyst for microbial electrosynthesis, a technology in which electricity generated with renewable technologies, such as solar or wind, powers the conversion of carbon dioxide and water to organic products. Other electron donors forC. ljungdahliiinclude carbon monoxide, which can be derived from industrial waste gases or the conversion of recalcitrant biomass to syngas, as well as hydrogen, another syngas component. The finding that carbon and electron flow inC. ljungdahliican be diverted from the production of acetate to butyrate synthesis is an important step toward the goal of renewable commodity production from carbon dioxide with this organism.

scholarly journals Substrate Inhibition of VanA by d-Alanine Reduces Vancomycin Resistance in a VanX-Dependent Manner

2016 ◽  
Vol 60 (8) ◽  
pp. 4930-4939 ◽  
Author(s):  
Lizah T. van der Aart ◽  
Nicole Lemmens ◽  
Willem J. van Wamel ◽  
Gilles P. van Wezel
Keyword(s):  
Cell Wall ◽  
Streptomyces Coelicolor ◽  
Substrate Inhibition ◽  
Model Organism ◽  
Vancomycin Resistance ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
Lipid Ii ◽  
Vancomycin Resistant

ABSTRACTThe increasing resistance of clinical pathogens against the glycopeptide antibiotic vancomycin, a last-resort drug against infections with Gram-positive pathogens, is a major problem in the nosocomial environment. Vancomycin inhibits peptidoglycan synthesis by binding to thed-Ala–d-Ala terminal dipeptide moiety of the cell wall precursor lipid II. Plasmid-transferable resistance is conferred by modification of the terminal dipeptide into the vancomycin-insensitive variantd-Ala–d-Lac, which is produced by VanA. Here we show that exogenousd-Ala competes withd-Lac as a substrate for VanA, increasing the ratio of wild-type to mutant dipeptide, an effect that was augmented by several orders of magnitude in the absence of thed-Ala–d-Ala peptidase VanX. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that high concentrations ofd-Ala led to the production of a significant amount of wild-type cell wall precursors, whilevanX-null mutants produced primarily wild-type precursors. This enhanced the efficacy of vancomycin in the vancomycin-resistant model organismStreptomyces coelicolor, and the susceptibility of vancomycin-resistant clinical isolates ofEnterococcus faecium(VRE) increased by up to 100-fold. The enhanced vancomycin sensitivity ofS. coelicolorcells correlated directly to increased binding of the antibiotic to the cell wall. Our work offers new perspectives for the treatment of diseases associated with vancomycin-resistant pathogens and for the development of drugs that target vancomycin resistance.

scholarly journals Metabolic Response of Clostridium ljungdahlii to Oxygen Exposure

2015 ◽  
Vol 81 (24) ◽  
pp. 8379-8391 ◽  
Author(s):  
Jason M. Whitham ◽  
Oscar Tirado-Acevedo ◽  
Mari S. Chinn ◽  
Joel J. Pawlak ◽  
Amy M. Grunden
Keyword(s):  
Ethanol Production ◽  
Metabolic Response ◽  
Reductase Activity ◽  
Preliminary Investigation ◽  
Rna Seq ◽  
Product Analysis ◽  
Content Type ◽  
Batch Cultures ◽  
Oxygen Exposure ◽  
Clostridium Ljungdahlii

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.

scholarly journals Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

2015 ◽  
Vol 81 (21) ◽  
pp. 7394-7402 ◽  
Author(s):  
Nan Li ◽  
Ting Qin ◽  
Xiao Lin Zhang ◽  
Bei Huang ◽  
Zhi Xin Liu ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Bacterial Pathogen ◽  
Infected Fish ◽  
Economic Losses ◽  
Flavobacterium Columnare ◽  
Wild Type ◽  
Single Strain ◽  
Content Type

ABSTRACTFlavobacterium columnareis an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. TheF. johnsoniaeompApromoter (PompA) was fused tosacBto construct a counterselectable marker forF. columnare.F. columnarecarrying PompA-sacBfailed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacBwas constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes,cslAandcslB, were deleted. The ΔcslAand ΔcslBmutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslAΔcslB) was completely deficient in chondroitin lyase activity. Cells ofF. columnarewild-type strain G4and of the chondroitin lyase-deficient ΔcslAΔcslBmutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors ofF. columnarebut may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylumBacteroidetes.

scholarly journals Respiratory Chain Analysis of Zymomonas mobilis Mutants Producing High Levels of Ethanol

2012 ◽  
Vol 78 (16) ◽  
pp. 5622-5629 ◽  
Author(s):  
Takeshi Hayashi ◽  
Tsuyoshi Kato ◽  
Kensuke Furukawa
Keyword(s):  
Ethanol Production ◽  
Dehydrogenase Activity ◽  
Respiratory Chain ◽  
Zymomonas Mobilis ◽  
Nadh Dehydrogenase ◽  
Nucleotide Sequence Analysis ◽  
Wild Type ◽  
Aerobic Growth ◽  
Content Type ◽  
Respiratory Chains

ABSTRACTWe previously isolated respiratory-deficient mutant (RDM) strains ofZymomonas mobilis, which exhibited greater growth and enhanced ethanol production under aerobic conditions. These RDM strains also acquired thermotolerance. Morphologically, the cells of all RDM strains were shorter compared to the wild-type strain. We investigated the respiratory chains of these RDM strains and found that some RDM strains lost NADH dehydrogenase activity, whereas others exhibited reduced cytochromebd-type ubiquinol oxidase or ubiquinol peroxidase activities. Complementation experiments restored the wild-type phenotype. Some RDM strains seem to have certain mutations other than the corresponding respiratory chain components. RDM strains with deficient NADH dehydrogenase activity displayed the greatest amount of aerobic growth, enhanced ethanol production, and thermotolerance. Nucleotide sequence analysis revealed that all NADH dehydrogenase-deficient strains were mutated within thendhgene, which includes insertion, deletion, or frameshift. These results suggested that the loss of NADH dehydrogenase activity permits the acquisition of higher aerobic growth, enhanced ethanol production, and thermotolerance in this industrially important strain.

scholarly journals GFPuv-Expressing Recombinant Rickettsia typhi: a Useful Tool for the Study of Pathogenesis and CD8+ T Cell Immunology in R. typhi Infection

2017 ◽  
Vol 85 (6) ◽  
Author(s):  
Matthias Hauptmann ◽  
Nicole Burkhardt ◽  
Ulrike Munderloh ◽  
Svenja Kuehl ◽  
Ulricke Richardt ◽  
...  
Keyword(s):  
T Cell ◽  
Genetic Manipulation ◽  
Interleukin 2 ◽  
Scid Mice ◽  
Wild Type ◽  
Rickettsia Typhi ◽  
Content Type ◽  
First Time

ABSTRACT Rickettsia typhi is the causative agent of endemic typhus, a disease with increasing incidence worldwide that can be fatal. Because of its obligate intracellular life style, genetic manipulation of the pathogen is difficult. Nonetheless, in recent years, genetic manipulation tools have been successfully applied to rickettsiae. We describe here for the first time the transformation of R. typhi with the pRAM18dRGA plasmid that originally derives from Rickettsia amblyommatis and encodes the expression of GFPuv (green fluorescent protein with maximal fluorescence when excited by UV light). Transformed R. typhi (R. typhi GFPuv) bacteria are viable, replicate with kinetics similar to those of wild-type R. typhi in cell culture, and stably maintain the plasmid and GFPuv expression under antibiotic treatment in vitro and in vivo during infection of mice. CB17 SCID mice infected with R. typhi GFPuv succumb to the infection with kinetics similar to those for animals infected with wild-type R. typhi and develop comparable pathology and bacterial loads in the organs, demonstrating that the plasmid does not influence pathogenicity. In the spleen and liver of infected CB17 SCID mice, the bacteria are detectable by immunofluorescence microscopy in neutrophils and macrophages by histological staining. Finally, we show for the first time that transformed rickettsiae can be used for the detection of CD8+ T cell responses. GFP-specific restimulation of spleen cells from R. typhi GFPuv-infected BALB/c mice elicits gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 2 (IL-2) secretion by CD8+ T cells. Thus, R. typhi GFPuv bacteria are a novel, potent tool to study infection with the pathogen in vitro and in vivo and the immune response to these bacteria.

scholarly journals Growth Dynamics and Survival of Liberibacter crescens BT-1, an Important Model Organism for the Citrus Huanglongbing Pathogen “Candidatus Liberibacter asiaticus”

2019 ◽  
Vol 85 (21) ◽  
Author(s):  
Marta Sena-Vélez ◽  
Sean D. Holland ◽  
Manu Aggarwal ◽  
Nick G. Cogan ◽  
Mukesh Jain ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Genetic Manipulation ◽  
Model Organism ◽  
Growth Dynamics ◽  
Culture Conditions ◽  
Citrus Greening ◽  
Close Relative ◽  
Logarithmic Phase ◽  
Content Type ◽  
Liberibacter Asiaticus

ABSTRACT Liberibacter crescens is the only cultured member of its genus, which includes the devastating plant pathogen “Candidatus Liberibacter asiaticus,” associated with citrus greening/Huanglongbing (HLB). L. crescens has a larger genome and greater metabolic flexibility than “Ca. Liberibacter asiaticus” and the other uncultured plant-pathogenic Liberibacter species, and it is currently the best model organism available for these pathogens. L. crescens grows slowly and dies rapidly under current culture protocols and this extreme fastidiousness makes it challenging to study. We have determined that a major cause of rapid death of L. crescens in batch culture is its alkalinization of the medium (to pH 8.5 by the end of logarithmic phase). The majority of this alkalinization is due to consumption of alpha-ketoglutaric acid as its primary carbon source, with a smaller proportion of the pH rise due to NH3 production. Controlling the pH rise with higher buffering capacity and lower starting pH improved recoverability of cells from 10-day cultures by >1,000-fold. We have also performed a detailed analysis of L. crescens growth with total cell numbers calibrated to the optical density and the percentage of live and recoverable bacteria determined over 10-day time courses. We modified L. crescens culture conditions to greatly enhance survival and increase maximum culture density. The similarities between L. crescens and the pathogenic liberibacters make this work relevant to efforts to culture the latter organisms. Our results also suggest that growth-dependent pH alteration that overcomes medium buffering should always be considered when growing fastidious bacteria. IMPORTANCE Liberibacter crescens is a bacterium that is closely related to plant pathogens that have caused billions of dollars in crop losses in recent years. Particularly devastating are citrus losses due to citrus greening disease, also known as Huanglongbing, which is caused by “Candidatus Liberibacter asiaticus” and carried by the Asian citrus psyllid. L. crescens is the only close relative of “Ca. Liberibacter asiaticus” that can currently be grown in culture, and it therefore serves as an important model organism for the growth, genetic manipulation, and biological control of the pathogenic species. Here, we show that one of the greatest limitations to L. crescens growth is the sharp increase in alkaline conditions it produces as a consequence of consumption of its preferred nutrient source. In addition to new information about L. crescens growth and metabolism, we provide new guidelines for culture conditions that improve the survival and yield of L. crescens.

scholarly journals Ethanol Metabolism Dynamics in Clostridium ljungdahlii Grown on Carbon Monoxide

2020 ◽  
Vol 86 (14) ◽  
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.

scholarly journals A Replicative Plasmid Vector Allows Efficient Complementation of Pathogenic Leptospira Strains

2015 ◽  
Vol 81 (9) ◽  
pp. 3176-3181 ◽  
Author(s):  
Christopher J. Pappas ◽  
Nadia Benaroudj ◽  
Mathieu Picardeau
Keyword(s):  
Genetic Manipulation ◽  
Shuttle Vector ◽  
Plasmid Vector ◽  
Wild Type ◽  
Pathogenic Leptospira ◽  
Content Type ◽  
Large Panel ◽  
Stable Plasmid ◽  
The Stability ◽  
Functional Copy

ABSTRACTLeptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including theparA,parB, andrepgenes into pMAT, a plasmid that cannot replicate inLeptospiraspp. and contains a backbone consisting of anaadAcassette,oriR6K, andoriTRK2/RP4. The inserted DNA segment was isolated from a 52-kb region withinLeptospiramayottensisstrain 200901116 that is not found in the closely related strainL. mayottensis200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use oftranscomplementation in the pathogenLeptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of theperRgene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel ofLeptospirastrains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation ofLeptospiraspp.

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