The unique set of putative membrane-associated anti-σ factors in Clostridium thermocellum suggests a novel extracellular carbohydrate-sensing mechanism involved in gene regulation

Spirochaeta thermophila is a thermophilic, free-living, and cellulolytic anaerobe. The genome sequence data for this organism have revealed a high density of genes encoding enzymes from more than 30 glycoside hydrolase (GH) families and a noncellulosomal enzyme system for (hemi)cellulose degradation. Functional screening of a fosmid library whose inserts were mapped on the S. thermophila genome sequence allowed the functional annotation of numerous GH open reading frames (ORFs). Seven different GH ORFs from the S. thermophila DSM 6192 genome, all putative ␤-glycanase ORFs according to sequence similarity analysis, contained a highly conserved novel GH-associated module of unknown function at their C terminus. Four of these GH enzymes were experimentally verified as xylanase, ␤-glucanase, ␤-glucanase/carboxymethylcellulase (CMCase), and CMCase. Binding experiments performed with the recombinantly expressed and purified GH-associated module showed that it represents a new carbohydrate-binding module (CBM) that binds to microcrystalline cellulose and is highly specific for this substrate. In the course of this work, the new CBM type was only detected in Spirochaeta, but recently we found sequences with detectable similarity to the module in the draft genomes of Cytophaga fermentans and Mahella australiensis, both of which are phylogenetically very distant from S. thermophila and noncellulolytic, yet inhabit similar environments. This suggests a possibly broad distribution of the module in nature.

Section: Resultsmentioning

confidence: 99%

Novel Family of Carbohydrate-Binding Modules Revealed by the Genome Sequence of Spirochaeta thermophila DSM 6192

Angelov

Loderer

Pompei

et al. 2011

“…Previous studies have demonstrated negative regulation of noncellulosomal enzymes by GlyR3 transcription factor (11,19). Positive regulation of cellulosome enzymes through cellulose and xylan interactions with anti-factors has been reported (17,20,21). Further studies are required to refine knowledge of C. thermocellum gene-regulatory networks contributing to the control and composition of the cellulosome and the lignocellulolytic abilities of the organism, regulation of important physiological responses, and cross talk between networks.…”

Section: Discussionmentioning

confidence: 98%

“…However, only a small proportion of C. thermocellum transcriptional regulators have been studied. Previous work has demonstrated the inducible expression of the celC-glyR3-licA operon (11,19), the involvement of alternative sigma factors with their cognate anti-sigma factors in the regulation of cellulolytic genes, including celS (17,20,21), and histidine kinase genes involved in the regulation of sporulation (22).…”

mentioning

confidence: 99%

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Wilson

Klingeman

Schlachter

et al. 2017

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 ΔglyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2 strain, and ␣-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3 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.

“…Previously, Stevenson and Weimer conducted a real-time PCR survey of the expression of 17 C. thermocellum genes involved in cellulose hydrolysis, intracellular phosphorylation, and catabolite repression and correlated their expression profiles with the formation of fermentation products (31). Most recently, sigma factor signaling in C. thermocellum was established by an elegant combination of bioinformatics and biochemical approaches (17). Collectively, these studies point to the ability of C. thermocellum to exquisitely control expression of certain known genes in response to growth rate and the presence of insoluble cellulose or soluble compounds such as cellobiose.…”

mentioning

confidence: 99%

“…The most highly expressed genes from this set encode glycoside hydrolases, scaffoldins, and other proteins known to participate in cellulose utilization. A large number of transcriptional regulators, signal transduction proteins (34), and sigma factors (17) were also identified, thus expanding our understanding of potential mechanisms of control for clostridial gene expression and protein translation. The expression data also revealed that certain genes among multiple copies from the glycolytic, ethanol fermentation, and H 2 evolution pathways were highly expressed under conditions that led to ethanol production.…”

mentioning

confidence: 99%

Global Gene Expression Patterns in Clostridium thermocellum as Determined by Microarray Analysis of Chemostat Cultures on Cellulose or Cellobiose

Riederer

Takasuka

Makino³

et al. 2011

A microarray study of chemostat growth on insoluble cellulose or soluble cellobiose has provided substantial new information on Clostridium thermocellum gene expression. This is the first comprehensive examination of gene expression in C. thermocellum under defined growth conditions. Expression was detected from 2,846 of 3,189 genes, and regression analysis revealed 348 genes whose changes in expression patterns were growth rate and/or substrate dependent. Successfully modeled genes included those for scaffoldin and cellulosomal enzymes, intracellular metabolic enzymes, transcriptional regulators, sigma factors, signal transducers, transporters, and hypothetical proteins. Unique genes encoding glycolytic pathway and ethanol fermentation enzymes expressed at high levels simultaneously with previously established maximal ethanol production were also identified. Ranking of normalized expression intensities revealed significant changes in transcriptional levels of these genes. The pattern of expression of transcriptional regulators, sigma factors, and signal transducers indicates that response to growth rate is the dominant global mechanism used for control of gene expression in C. thermocellum.