GSE68423
Understanding the Regulatory Networks of Three LacI Transcriptional Repressors in Clostridium thermocellum DSM1313
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 ß-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. Overall design: Four strains of C. thermocellum DSM1313 including a parental strain (?hpt) 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.