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Title: LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

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. In one LacI regulator termed GlyR3 inC. thermocellumATCC 27405 we identified 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. Furthermore, 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-licAoperonmore » 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. Finally, 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.« less
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center and Biosciences Division
Publication Date:
Grant/Contract Number:
AC05-00OR22725
Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 83; Journal Issue: 5; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Research Org:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; EMSA; Lacl; RNA-seq; ruminiclostridium; transcriptomics; consolidated bioprocessing; gene regulation
OSTI Identifier:
1344258

Wilson, Charlotte M., Klingeman, Dawn M., Schlachter, Caleb, Syed, Mustafa H., Wu, Chia-wei, Guss, Adam M., Brown, Steven D., and Parales, Rebecca E.. LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313. United States: N. p., Web. doi:10.1128/AEM.02751-16.
Wilson, Charlotte M., Klingeman, Dawn M., Schlachter, Caleb, Syed, Mustafa H., Wu, Chia-wei, Guss, Adam M., Brown, Steven D., & Parales, Rebecca E.. LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313. United States. doi:10.1128/AEM.02751-16.
Wilson, Charlotte M., Klingeman, Dawn M., Schlachter, Caleb, Syed, Mustafa H., Wu, Chia-wei, Guss, Adam M., Brown, Steven D., and Parales, Rebecca E.. 2016. "LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313". United States. doi:10.1128/AEM.02751-16. https://www.osti.gov/servlets/purl/1344258.
@article{osti_1344258,
title = {LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313},
author = {Wilson, Charlotte M. and Klingeman, Dawn M. and Schlachter, Caleb and Syed, Mustafa H. and Wu, Chia-wei and Guss, Adam M. and Brown, Steven D. and Parales, Rebecca E.},
abstractNote = {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. In one LacI regulator termed GlyR3 inC. thermocellumATCC 27405 we identified 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. Furthermore, 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. Finally, 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.},
doi = {10.1128/AEM.02751-16},
journal = {Applied and Environmental Microbiology},
number = 5,
volume = 83,
place = {United States},
year = {2016},
month = {12}
}