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Clostridium Thermocellum Cellulosomal Genes Are Regulated by Extracytoplasmic Polysaccharides Via Alternative Sigma Factors

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Clostridium Thermocellum Cellulosomal Genes Are Regulated by Extracytoplasmic Polysaccharides Via Alternative Sigma Factors Clostridium thermocellum cellulosomal genes are regulated by extracytoplasmic polysaccharides via alternative sigma factors Yakir Natafa, Liat Baharib, Hamutal Kahel-Raiferc, Ilya Borovokc, Raphael Lamedc, Edward A. Bayerb, Abraham L. Sonensheind, and Yuval Shohama,1 aDepartment of Biotechnology and Food Engineering, Technion–Israel Institute of Technology, Haifa 32000, Israel; bDepartment of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel; cDepartment of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv 69978, Israel; and dDepartment of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111 Edited* by Arnold L. Demain, Drew University, Madison, NJ, and approved September 21, 2010 (received for review August 17, 2010) Clostridium thermocellum produces a highly efficient cellulolytic The known number of dockerin-bearing enzymes in C. ther- extracellular complex, termed the cellulosome, for hydrolyzing mocellum is approximately eight times more than the number of plant cell wall biomass. The composition of the cellulosome is af- cohesins in the scaffoldin subunit. Consequently, the composi- fected by the presence of extracellular polysaccharides; however, tion of the cellulosome is governed by the relative amounts of the the regulatory mechanism is unknown. Recently, we have identi- available dockerin-containing polypeptides that presumably are fied in C. thermocellum a set of putative σ and anti-σ factors that incorporated randomly into the complex (2). Individual cellulo- include extracellular polysaccharide-sensing components [Kahel- some complexes would therefore differ in their exact content and Raifer et al. (2010) FEMS Microbiol Lett 308:84–93]. These factor- distribution of subunits (11). The various cellulosomal genes in encoding genes are homologous to the Bacillus subtilis bicistronic C. thermocellum, for the most part, are monocistronic, scattered operon sigI-rsgI, which encodes for an alternative σI factor and its throughout the chromosome (12), and their expression was I cognate anti-σ regulator RsgI that is functionally regulated by an shown to be affected by the carbon source and the growth rate extracytoplasmic signal. In this study, the binding of C. thermocel- (13–23). Several general regulatory mechanisms were proposed I lum putative anti-σ factors to their corresponding σ factors was to be involved, including carbon catabolite repression (2, 21) and measured, demonstrating binding specificity and dissociation con- alternative σ factors (14). Surprisingly, the only regulator that stants in the range of 0.02 to 1 μM. Quantitative real-time RT-PCR has been characterized so far is GlyR3, which negatively regu- measurements revealed three- to 30-fold up-expression of the al- lates celC, a noncellulosomal cellulase gene (24). Although C. σ ternative factor genes in the presence of cellulose and xylan, thus thermocellum can utilize mainly cellodextrins and possesses connecting their expression to direct detection of their extracellular specific ABC sugar transporters for their selective uptake (25, polysaccharide substrates. Cellulosomal genes that are putatively 26), the bacterium encodes and differentially expresses numer- σ σI1 σI6 fi regulated by two of these factors, or , were identi ed based ous cellulosomal glycoside hydrolases that act on hemicellulose σI1 on the sequence similarity of their promoters. The ability of to and other cellulose-associated polysaccharides (23). These direct transcription from the sigI1 promoter and from the promoter enzymes are required for unmasking the cellulose fibers from the of celS (encodes the family 48 cellulase) was demonstrated in vitro surrounding hemicellulose fibers. Thus, the bacterium must by runoff transcription assays. Taken together, the results reveal σ possess a regulatory system that allows it to sense and react to a regulatory mechanism in which alternative factors are involved the presence of high molecular weight polysaccharides in the in regulating the cellulosomal genes via an external carbohydrate- extracellular surroundings without importing their low molecular sensing mechanism. weight soluble components intracellularly. Recently, we have identified in C. thermocellum a set of six biomass | carbohydrate binding modules | Clostridium regulation | putative operons encoding alternative σ factors and their cognate glycoside hydrolases | anti-sigma factors membrane-associated anti-σ factors that may play a role in reg- ulating cellulosomal genes (Table 1) (27). Deduced amino acid ram-positive thermophilic Clostridium thermocellum is an sequences of these σ factors share homology to the well char- Ganaerobic bacterium with a highly efficient cellulolytic sys- acterized Bacillus subtilis alternative σ factor, σI (28–30). The tem. The hallmark of the system is an extracellular multienzyme second gene in these operons encodes for a multimodular pro- complex, termed the cellulosome (1–4). As the bacterium is also tein that contains one strongly predicted transmembrane helix. capable of producing ethanol, it potentially could be integrated The approximate 165-residue N-termini of these transmembrane into a consolidated bioprocessing system for the production of proteins are homologous to the N-terminal segment of the B. cellulosic ethanol as a renewable source of energy (5–7). The subtilis anti-σI factor RsgI. The extracellular modules of these cellulosome complex consists of a noncatalytic polypeptide, the RsgI-like proteins appear to have polysaccharide-related func- scaffoldin, that mediates the attachment of nine catalytic sub- tions, and include carbohydrate-binding modules (e.g., CBM3, units and the binding to cellulose via an internal family 3 cellu- CBM42), sugar-binding elements (e.g., PA14), and a glycoside lose-binding module (CBM3). The cellulosomal enzymes possess hydrolase family 10 (GH10) module. In fact, two CBM3s from a dockerin module that binds tenaciously to the nine scaffoldin- borne cohesin modules, thus forming the complex (7–9). The scaffoldin subunit also includes a special type of dockerin mod- Author contributions: Y.N., I.B., R.L., E.A.B., A.L.S., and Y.S. designed research; Y.N., L.B., and H.K.-R. performed research; Y.N., L.B., I.B., R.L., E.A.B., A.L.S., and Y.S. analyzed data; ule (type II dockerin) for the attachment of the cellulosome to Y.N., L.B., I.B., R.L., E.A.B., A.L.S., and Y.S. wrote the paper. a complementary cohesin (type II) that is positioned on the cell The authors declare no conflict of interest. surface via cell surface anchoring proteins (10). Thus, the scaf- *This Direct Submission article had a prearranged editor. foldin mediates the attachment of the catalytic units, as well as 1To whom correspondence should be addressed. E-mail: [email protected]. the binding of the complex and the entire cell to insoluble This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. crystalline cellulose. 1073/pnas.1012175107/-/DCSupplemental. 18646–18651 | PNAS | October 26, 2010 | vol. 107 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1012175107 Downloaded by guest on September 29, 2021 Table 1. σ/anti-σ pairs proposed to participate in regulating cellulosomal genes σ/anti-σ genes, N-terminal anti-σ C-terminal Target σ/anti-σ locus tag domain, aa residues* sensing domain polysaccharides † σI1-RsgI1 Cthe_0058-9 52 CBM3 Cellulose σI2-RsgI2 Cthe_0268-7 67 CBM3 Cellulose σI3-RsgI3 Cthe_0315-6 57 PA14 dyad Pectin† σI4-RsgI4 Cthe_0403-4 52 CBM3 Cellulose† σI5-RsgI5 Cthe_1272-3 50 CBM42 Arabinoxylan † σI6-RsgI6 Cthe_2120-19 54 GH10 Xylans, cellulose σ24C-Rsi24C Cthe_1470-1 90 GH5 Cellulose† *Not including the transmembrane domain. † Confirmed experimentally (27, 31). RsgI1 (Cthe_0059) and RsgI4 (Cthe_0404) were found to bind operon in B. subtilis and various operons encoding ECF σ factors. cellulose, the PA14 dyad domains of RsgI3 (Cthe_0315) interact This arrangement suggests an extracellular sensing mechanism strongly with pectin (27) and the xylanase GH10 module of that regulates the activity of the σI-like σ factor via its interactions RsgI6 (Cthe_2119) interacts with xylans and cellulose (31). In with a cognate anti-σ peptide. To demonstrate the binding spec- addition, C. thermocellum encodes another transmembrane ificity of the putative σI-like factors for their corresponding anti-σ protein (Rsi24C; Table 1) with a carbohydrate-related function factors we have cloned, expressed, and purified representative (glycoside hydrolase family 5) (31); in this case, the σ factor gene recombinant σIs and anti-σ domains of their cognate RsgIs, which (sig24C) located upstream is weakly homologous to the B. subtilis are predicted to be on the N terminus (termed RsgIN; Table 1). extracytoplasmic function (ECF) σ factor σW (32). These findings The entire sigI-like structural genes were cloned fused to His-tags, strongly suggest that alternative σ factors are involved in regu- whereas the anti-σ domains were designed to contain segments of lating the cellulosomal genes via an external carbohydrate- only 51 to 90 aa residues of the N-terminal RsgI-like (27) or sensing mechanism. Rsi24C domain, again fused to His-tags at their N terminus. Of In this study, we demonstrate the binding specificity of rep- the seven protein pairs (6 σI-RsgIN pairs and σ24C-Rsi24CN) resentative anti-σI factors to their corresponding σ factors, reveal tested, three σI-RsgIN pairs (pairs 1, 2, and 6) were efficiently the expression profiles of the σ factors
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