Three cellulosomal xylanase genes inClostridium thermocellum are regulated by both vegetative SigA (σA) and alternative SigI6 (σI6) factors

Sand, Andy; Holwerda, Evert K.; Ruppertsberger, Natalie M.; Maloney, Marybeth; Olson, Daniel G.; Nataf, Yakir; Borovok, Ilya; Sonenshein, Abraham L.; Bayer, Edward A.; Lamed, Raphael; Lynd, Lee R.; Shoham, Yuval

doi:10.1016/j.febslet.2015.08.026

Cited by 21 publications

(17 citation statements)

References 62 publications

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“…Interestingly, the C. thermocellum genome encodes eight alternative RNAP sigma I factors ( I1 to I8 ), of which six, I1 to I6 , were shown to be involved in regulating cellulosomal components (8)(9)(10)(11). The cognate membrane-associated anti-I factors (RsgI1 to RsgI6) contain C-terminal CBMs that are displayed on the cell surface, suggesting that they serve as putative sensors for extracellular polysaccharide substrates (8)(9)(10)(11). Recently, using a heterologous B. subtilis host system, we showed that the C. thermocellum alternative RNAP factors I3 and I6 recognized a predicted I -dependent promoter of the primary scaffoldin gene cipA, located approximately 850 bp from the start codon (10).…”

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confidence: 99%

Revisiting the Regulation of the Primary Scaffoldin Gene in Clostridium thermocellum

Ora

Muñoz-Gutiérrez

Bayer

et al. 2017

Appl Environ Microbiol

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Cellulosomes are considered to be one of the most efficient systems for the degradation of plant cell wall polysaccharides. The central cellulosome component comprises a large, noncatalytic protein subunit called scaffoldin. Multiple saccharolytic enzymes are incorporated into the scaffoldins via specific high-affinity cohesin-dockerin interactions. Recently, the regulation of genes encoding certain cellulosomal components by multiple RNA polymerase alternative σ factors has been demonstrated in () In the present report, we provide experimental evidence demonstrating that the gene, which encodes the primary cellulosomal scaffoldin, is regulated by several alternative σ factors and by the vegetative σ factor. Furthermore, we show that previously suggested transcriptional start sites (TSSs) of are actually posttranscriptional processed sites. By using comparative bioinformatic analysis, we have also identified highly conserved σ- and σ-dependent promoters upstream of the primary scaffoldin-encoding genes of other clostridia, namely, ,, , and sp. strain Bc-iso-3. Interestingly, a previously identified TSS of the primary scaffoldin CbpA gene of matches the predicted σ-dependent promoter identified in the present work rather than the previously proposed σ promoter. With the exception of , both σ and σ promoters of primary scaffoldin genes are located more than 600 nucleotides upstream of the start codon, yielding long 5'-untranslated regions (5'-UTRs). Furthermore, these 5'-UTRs have highly conserved stem-loop structures located near the start codon. We propose that these large 5'-UTRs may be involved in the regulation of both the primary scaffoldin and other cellulosomal components. Cellulosome-producing bacteria are among the most effective cellulolytic microorganisms known. This group of bacteria has biotechnological potential for the production of second-generation biofuels and other biocommodities from cellulosic wastes. The efficiency of cellulose hydrolysis is due to their cellulosomes, which arrange enzymes in close proximity on the cellulosic substrate, thereby increasing synergism among the catalytic domains. The backbone of these multienzyme nanomachines is the scaffoldin subunit, which has been the subject of study for many years. However, its genetic regulation is poorly understood. Hence, from basic and applied points of view, it is imperative to unravel the regulatory mechanisms of the scaffoldin genes. The understanding of these regulatory mechanisms can help to improve the performance of the industrially relevant strains of and related cellulosome-producing bacteria to the consolidated bioprocessing of biomass.

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confidence: 99%

Revisiting the Regulation of the Primary Scaffoldin Gene in Clostridium thermocellum

Ora

Muñoz-Gutiérrez

Bayer

et al. 2017

Appl Environ Microbiol

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“…, Table S1), indicating that the glucose‐derived cellulosome was steadily produced at low level, while the cellulosome synthesis with cellobiose and Avicel was greatly stimulated compared to glucose. According to previous reports, the expression of cellulosomal genes is regulated by both the primary sigma factor SigA and the alternative sigma factor SigI (Nataf et al ., ; Sand et al ., ; Munoz‐Gutierrez et al ., ). The housekeeping SigA is for constitutive cellulosome synthesis at low level, while the environment‐sensitive SigIs induce the high‐level production of the cellulosome.…”

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confidence: 98%

Inducing effects of cellulosic hydrolysate components of lignocellulose on cellulosome synthesis in Clostridium thermocellum

Feng

Liu

et al. 2018

Microbial Biotechnology

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SummaryCellulosome is a highly efficient supramolecular machine for lignocellulose degradation, and its substrate‐coupled regulation requires soluble transmembrane signals. However, the inducers for cellulosome synthesis and the inducing effect have not been clarified quantitatively. Values of cellulosome production capacity (CPC) and estimated specific activity (eSA) were calculated based on the primary scaffoldin ScaA to define the stimulating effects on the cellulosome synthesis in terms of quantity and quality respectively. The estimated cellulosome production of Clostridium thermocellum on glucose was at a low housekeeping level. Both Avicel and cellobiose increased CPCs of the cells instead of the eSAs of the cellulosome. The CPC of Avicel‐grown cells was over 20‐fold of that of glucose‐grown cells, while both Avicel‐ and glucose‐derived cellulosomes showed similar eSA. The CPC of cellobiose‐grown cells was also over three times higher than glucose‐grown cells, but the eSA of cellobiose‐derived cellulosome was 16% lower than that of the glucose‐derived cellulosome. Our results indicated that cello‐oligosaccharides played the key roles in inducing the synthesis of the cellulosome, but non‐cellulosic polysaccharides showed no inducing effects.

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“…Until now, the knowledge we have regarding the regulation of cellulosomal genes by C . thermocellum σ I -like factors is a recent report of Sand and co-workers [ 27 ] which showed that the xylanase genes xyn10Z (or Clo1313_2635 according to the DSM 1313 genome annotation), xyn11B (Clo1313_0522) and xyn10D (Clo1313_0177) were under the control of σ I6 . Previously, Nataf and co-workers [ 19 ] showed that the cellulase gene celS ( cel48S ) was likely under the control of σ I1 .…”

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confidence: 99%

Decoding Biomass-Sensing Regulons of Clostridium thermocellum Alternative Sigma-I Factors in a Heterologous Bacillus subtilis Host System

et al. 2016

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The Gram-positive, anaerobic, cellulolytic, thermophile Clostridium (Ruminiclostridium) thermocellum secretes a multi-enzyme system called the cellulosome to solubilize plant cell wall polysaccharides. During the saccharolytic process, the enzymatic composition of the cellulosome is modulated according to the type of polysaccharide(s) present in the environment. C. thermocellum has a set of eight alternative RNA polymerase sigma (σ) factors that are activated in response to extracellular polysaccharides and share sequence similarity to the Bacillus subtilis σI factor. The aim of the present work was to demonstrate whether individual C. thermocellum σI-like factors regulate specific cellulosomal genes, focusing on C. thermocellum σI6 and σI3 factors. To search for putative σI6- and σI3-dependent promoters, bioinformatic analysis of the upstream regions of the cellulosomal genes was performed. Because of the limited genetic tools available for C. thermocellum, the functionality of the predicted σI6- and σI3-dependent promoters was studied in B. subtilis as a heterologous host. This system enabled observation of the activation of 10 predicted σI6-dependent promoters associated with the C. thermocellum genes: sigI6 (itself, Clo1313_2778), xyn11B (Clo1313_0522), xyn10D (Clo1313_0177), xyn10Z (Clo1313_2635), xyn10Y (Clo1313_1305), cel9V (Clo1313_0349), cseP (Clo1313_2188), sigI1 (Clo1313_2174), cipA (Clo1313_0627), and rsgI5 (Clo1313_0985). Additionally, we observed the activation of 4 predicted σI3-dependent promoters associated with the C. thermocellum genes: sigI3 (itself, Clo1313_1911), pl11 (Clo1313_1983), ce12 (Clo1313_0693) and cipA. Our results suggest possible regulons of σI6 and σI3 in C. thermocellum, as well as the σI6 and σI3 promoter consensus sequences. The proposed -35 and -10 promoter consensus elements of σI6 are CNNAAA and CGAA, respectively. Additionally, a less conserved CGA sequence next to the C in the -35 element and a highly conserved AT sequence three bases downstream of the -10 element were also identified as important nucleotides for promoter recognition. Regarding σI3, the proposed -35 and -10 promoter consensus elements are CCCYYAAA and CGWA, respectively. The present study provides new clues for understanding these recently discovered alternative σI factors.

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Three cellulosomal xylanase genes inClostridium thermocellum are regulated by both vegetative SigA (σ^A) and alternative SigI6 (σ^I6) factors

Cited by 21 publications

References 62 publications

Revisiting the Regulation of the Primary Scaffoldin Gene in Clostridium thermocellum

Revisiting the Regulation of the Primary Scaffoldin Gene in Clostridium thermocellum

Inducing effects of cellulosic hydrolysate components of lignocellulose on cellulosome synthesis in Clostridium thermocellum

Decoding Biomass-Sensing Regulons of Clostridium thermocellum Alternative Sigma-I Factors in a Heterologous Bacillus subtilis Host System

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