@article {3086, title = {Transcriptomic profiles of Clostridium ljungdahlii during lithotrophic growth with syngas or H and CO compared to organotrophic growth with fructose.}, journal = {Sci Rep}, volume = {7}, year = {2017}, month = {2017 Oct 13}, pages = {13135}, abstract = {

Clostridium ljungdahlii derives energy by lithotrophic and organotrophic acetogenesis. C. ljungdahlii was grown organotrophically with fructose and also lithotrophically, either with syngas - a gas mixture containing hydrogen (H), carbon dioxide (CO), and carbon monoxide (CO), or with H and CO. Gene expression was compared quantitatively by microarrays using RNA extracted from all three conditions. Gene expression with fructose and with H/CO was compared by RNA-Seq. Upregulated genes with both syngas and H/CO (compared to fructose) point to the urea cycle, uptake and degradation of peptides and amino acids, response to sulfur starvation, potentially NADPH-producing pathways involving (S)-malate and ornithine, quorum sensing, sporulation, and cell wall remodeling, suggesting a global and multicellular response to lithotrophic conditions. With syngas, the upregulated (R)-lactate dehydrogenase gene represents a route of electron transfer from ferredoxin to NAD. With H/CO, flavodoxin and histidine biosynthesis genes were upregulated. Downregulated genes corresponded to an intracytoplasmic microcompartment for disposal of methylglyoxal, a toxic byproduct of glycolysis, as 1-propanol. Several cytoplasmic and membrane-associated redox-active protein genes were differentially regulated. The transcriptomic profiles of C. ljungdahlii in lithotrophic and organotrophic growth modes indicate large-scale physiological and metabolic differences, observations that may guide biofuel and commodity chemical production with this species.

}, keywords = {Carbon Dioxide, Carbon Monoxide, Clostridium, Fructose, Hydrogen, NADP, Transcriptome}, issn = {2045-2322}, doi = {10.1038/s41598-017-12712-w}, author = {Aklujkar, Muktak and Leang, Ching and Shrestha, Pravin M and Shrestha, Minita and Lovley, Derek R} } @article {3130, title = {Lactose-inducible system for metabolic engineering of Clostridium ljungdahlii.}, journal = {Appl Environ Microbiol}, volume = {80}, year = {2014}, month = {2014 Apr}, pages = {2410-6}, abstract = {

The development of tools for genetic manipulation of Clostridium ljungdahlii has 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 for C. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported for Clostridium perfringens, was investigated. The plasmid pAH2, originally developed for C. perfringens with a gusA reporter gene, functioned as an effective lactose-inducible system in C. ljungdahlii. Lactose induction of C. ljungdahlii containing pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression of adhE1 30-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression of adhE1 in a strain in which adhE1 and the adhE1 homolog adhE2 had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression of adhE2 similarly failed to restore ethanol production, suggesting that adhE1 is 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.

}, keywords = {Acetic Acid, Acetone, Acetyl Coenzyme A, Alcohol Dehydrogenase, Carbon, Clostridium, Ethanol, Fructose, Gene Expression, Gene Expression Regulation, Bacterial, Lactose, Metabolic Engineering, Metabolic Flux Analysis, Transcriptional Activation}, issn = {1098-5336}, doi = {10.1128/AEM.03666-13}, author = {Banerjee, Areen and Leang, Ching and Ueki, Toshiyuki and Nevin, Kelly P and Lovley, Derek R} } @article {3148, title = {The Rnf complex of Clostridium ljungdahlii is a proton-translocating ferredoxin:NAD+ oxidoreductase essential for autotrophic growth.}, journal = {mBio}, volume = {4}, year = {2012}, month = {2012 Dec 26}, pages = {e00406-12}, abstract = {

UNLABELLED: It has been predicted that the Rnf complex of Clostridium ljungdahlii is a proton-translocating ferredoxin:NAD(+) oxidoreductase which contributes to ATP synthesis by an H(+)-translocating ATPase under both autotrophic and heterotrophic growth conditions. The recent development of methods for genetic manipulation of C. ljungdahlii made it possible to evaluate the possible role of the Rnf complex in energy conservation. Disruption of the C. ljungdahlii rnf operon inhibited autotrophic growth. ATP synthesis, proton gradient, membrane potential, and proton motive force collapsed in the Rnf-deficient mutant with H(2) as the electron source and CO(2) as the electron acceptor. Heterotrophic growth was hindered in the absence of a functional Rnf complex, as ATP synthesis, proton gradient, and proton motive force were significantly reduced with fructose as the electron donor. Growth of the Rnf-deficient mutant was also inhibited when no source of fixed nitrogen was provided. These results demonstrate that the Rnf complex of C. ljungdahlii is responsible for translocation of protons across the membrane to elicit energy conservation during acetogenesis and is a multifunctional device also implicated in nitrogen fixation.

IMPORTANCE: Mechanisms for energy conservation in the acetogen Clostridium ljungdahlii are of interest because of its potential value as a chassis for the production of biocommodities with novel electron donors such as carbon monoxide, syngas, and electrons derived from electrodes. Characterizing the components implicated in the chemiosmotic ATP synthesis during acetogenesis by C. ljungdahlii is a prerequisite for the development of highly productive strains. The Rnf complex has been considered the prime candidate to be the pump responsible for the formation of an ion gradient coupled with ATP synthesis in multiple acetogens. However, experimental evidence for a proton-pumping Rnf complex has been lacking. This study establishes the C. ljungdahlii Rnf complex as a proton-translocating ferredoxin:NAD(+) oxidoreductase and demonstrates that C. ljungdahlii has the potential of becoming a model organism to study proton translocation, electron transport, and other functions of the Rnf complex in energy conservation or other processes.

}, keywords = {Adenosine Triphosphate, Autotrophic Processes, Clostridium, Energy Metabolism, Fructose, Gene Knockout Techniques, Genes, Essential, Nitrogen, Operon, Oxidoreductases, Proton-Motive Force}, issn = {2150-7511}, doi = {10.1128/mBio.00406-12}, author = {Tremblay, Pier-Luc and Zhang, Tian and Dar, Shabir A and Leang, Ching and Lovley, Derek R} }