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LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313.

ABSTRACT: Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a ?-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes 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 ?glyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ?glyR2 strain, and ?-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ?glyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters.IMPORTANCE Understanding C. thermocellum gene 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 specific C. thermocellum LacI 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 in C. thermocellum.

SUBMITTER: Wilson CM

PROVIDER: S-EPMC5311418 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313.

Wilson Charlotte M CM Klingeman Dawn M DM Schlachter Caleb C Syed Mustafa H MH Wu Chia-Wei CW Guss Adam M AM Brown Steven D SD

Applied and environmental microbiology 20170215 5

Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a β-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, del ...[more]

PMID: 28003194

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Project description:Clostridium thermocellum is a candidate for cellulosic ethanol production. It expresses enzymes for both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. Understanding how this organism regulates gene expression is of importance for developing a better fundamental understanding of this industrial relevant bacterium. We are primarily interested in gene regulation by three predicted LacI regulators. A previous report of one LacI regulator in C. thermocellum ATCC27405 (Cthe_2808) indicated this was a transcriptional repressor of neighboring genes with repressor activity relieved in the presence of laminaribiose (a β-1,3 disaccharide). The current work is aimed at understanding if a homolog of this LacI transcriptional regulator in C. thermocellum DSM1313 and two others putatively characterized as LacI regulators are in fact global regulatory proteins with extensive regulons or, if like the majority of LacI transcription factors, the regulation is limited to local genomic regions. Each of the three LacI regulators was deleted in C. thermocellum DSM1313. Genome re-sequencing was used to confirm the deletion and ensure nonspecific recombination events were avoided during the gene deletion strategy. Growth of each strain on cellobiose was unaffected by any of the gene deletions under pH controlled fermentations. Global gene expression patterns taken at mid-log phase for each strain identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly up-regulated (up to 9 fold) in the absence of each LacI regulator. Thus suggesting these were repressed under wild type conditions. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters with putative motifs ranging from 16-18 bp. Work is ongoing to confirm the specific binding motif recognized by the two previously un-characterized transcription factors and assess the occurrence of this motif in the C. thermocellum DSM1313 genome. The identification of LacI repressor activity on hemicellulose 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 in C. thermocellum.

Project description:Clostridium thermocellum is a candidate for cellulosic ethanol production. It expresses enzymes for both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. Understanding how this organism regulates gene expression is of importance for developing a better fundamental understanding of this industrial relevant bacterium. We are primarily interested in gene regulation by three predicted LacI regulators. A previous report of one LacI regulator in C. thermocellum ATCC27405 (Cthe_2808) indicated this was a transcriptional repressor of neighboring genes with repressor activity relieved in the presence of laminaribiose (a Î²-1,3 disaccharide). The current work is aimed at understanding if a homolog of this LacI transcriptional regulator in C. thermocellum DSM1313 and two others putatively characterized as LacI regulators are in fact global regulatory proteins with extensive regulons or, if like the majority of LacI transcription factors, the regulation is limited to local genomic regions. Each of the three LacI regulators was deleted in C. thermocellum DSM1313. Genome re-sequencing was used to confirm the deletion and ensure nonspecific recombination events were avoided during the gene deletion strategy. Growth of each strain on cellobiose was unaffected by any of the gene deletions under pH controlled fermentations. Global gene expression patterns taken at mid-log phase for each strain identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly up-regulated (up to 9 fold) in the absence of each LacI regulator. Thus suggesting these were repressed under wild type conditions. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters with putative motifs ranging from 16-18 bp. Work is ongoing to confirm the specific binding motif recognized by the two previously un-characterized transcription factors and assess the occurrence of this motif in the C. thermocellum DSM1313 genome. The identification of LacI repressor activity on hemicellulose 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 in C. thermocellum. Four strains of C. thermocellum DSM1313 including a parental strain (Îhpt) and three others with deletion in lacI transcription factors were characterised by RNA-seq. The strains were grown in 1L bioreactors, cells (from 50 ml samples) were harvested at mid exponential, late exponential and 30 min after acid production halted during the transition to stationary phase. The growth studies were performed in triplicate and thirty six samples were analyzed by RNA-sequencing.

Project description:Clostridium thermocellum is a promising consolidated bioprocessing candidate organism capable of directly converting lignocellulosic biomass to ethanol. Current ethanol yields, productivities, and growth inhibitions are industrial deployment impediments for commodity fuel production by this bacterium. Redox imbalance under certain conditions and in engineered strains may contribute to incomplete substrate utilization and may direct fermentation products to undesirable overflow metabolites. Towards a better understanding of redox metabolism in C. thermocellum, we established continuous growth conditions and analyzed global gene expression during addition of two stress chemicals (methyl viologen and hydrogen peroxide) which changed the fermentation redox potential.The addition of methyl viologen to C. thermocellum DSM 1313 chemostat cultures caused an increase in ethanol and lactate yields. A lower fermenter redox potential was observed in response to methyl viologen exposure, which correlated with a decrease in cell yield and significant differential expression of 123 genes (log2 > 1.5 or log2 < -1.5, with a 5 % false discovery rate). Expression levels decreased in four main redox-active systems during methyl viologen exposure; the [NiFe] hydrogenase, sulfate transport and metabolism, ammonia assimilation (GS-GOGAT), and porphyrin/siroheme biosynthesis. Genes encoding sulfate transport and reduction and porphyrin/siroheme biosynthesis are co-located immediately downstream of a putative iscR regulatory gene, which may be a cis-regulatory element controlling expression of these genes. Other genes showing differential expression during methyl viologen exposure included transporters and transposases.The differential expression results from this study support a role for C. thermocellum genes for sulfate transport/reduction, glutamate synthase-glutamine synthetase (the GS-GOGAT system), and porphyrin biosynthesis being involved in redox metabolism and homeostasis. This global profiling study provides gene targets for future studies to elucidate the relative contributions of prospective pathways for co-factor pool re-oxidation and C. thermocellum redox homeostasis.

Dataset Information

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313.

Publications

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313.

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