title: LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313.
abstract: Organisms regulate gene expression in response to the environment to coordinate metabolic reactions.Clostridium thermocellumexpresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 inC. thermocellumATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a β-1,3 disaccharide). To better understand the threeC. thermocellumLacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313lacIgenes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5- to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ΔglyR1strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2strain, and α-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3strain, upregulation of the two genes adjacent toglyR3in thecelC-glyR3-licAoperon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters.IMPORTANCEUnderstandingC. thermocellumgene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specificC. thermocellumLacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation inC. thermocellum.
authors.affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.|Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Charlotte M
authors.initials: CM
authors.lastname: Wilson
authors.affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.|Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Dawn M
authors.initials: DM
authors.lastname: Klingeman
authors.affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.|Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Caleb
authors.initials: C
authors.lastname: Schlachter
authors.affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.|Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Mustafa H
authors.initials: MH
authors.lastname: Syed
authors.affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.|Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Chia-Wei
authors.initials: CW
authors.lastname: Wu
authors.affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.|Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Guss
authors.affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA brownsd@ornl.gov.|Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Brown
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002475:Cellobiose / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D004187:Disaccharides / Q000378:metabolism; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D053263:Gene Regulatory Networks; D016680:Genome, Bacterial; D006026:Glycoside Hydrolases / Q000187:drug effects / Q000235:genetics; D008074:Lipoproteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D009876:Operon / Q000235:genetics; D008897:Panicum / Q000378:metabolism; D011134:Polysaccharides / Q000235:genetics; D018085:Regulon / Q000235:genetics*; D017423:Sequence Analysis, RNA; D017384:Sequence Deletion; D014157:Transcription Factors; D059467:Transcriptome; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D004187:Disaccharides; D008074:Lipoproteins; D011134:Polysaccharides; D014157:Transcription Factors; C051928:lipoprotein-associated coagulation inhibitor; C014637:laminaribiose; D002475:Cellobiose; C007916:hemicellulose; D002482:Cellulose; D006026:Glycoside Hydrolases; C023305:hemicellulase; C028020:licheninase
keywords: EMSA; LacI; RNA-seq; Ruminiclostridium; consolidated bioprocessing; gene regulation; transcriptomics