Revisiting the Regulation of the Primary Scaffoldin Gene in Clostridium thermocellum.
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 σIfactors has been demonstrated inClostridium(Ruminiclostridium)thermocellumIn the present report, we provide experimental evidence demonstrating that theC. thermocellum cipAgene, which encodes the primary cellulosomal scaffoldin, is regulated by several alternative σIfactors and by the vegetative σAfactor. Furthermore, we show that previously suggested transcriptional start sites (TSSs) ofC. thermocellum cipAare actually posttranscriptional processed sites. By using comparative bioinformatic analysis, we have also identified highly conserved σI- and σA-dependent promoters upstream of the primary scaffoldin-encoding genes of other clostridia, namely,Clostridium straminisolvens,Clostridium clariflavum,Acetivibrio cellulolyticus, andClostridiumsp. strain Bc-iso-3. Interestingly, a previously identified TSS of the primary scaffoldin CbpA gene ofClostridium cellulovoransmatches the predicted σI-dependent promoter identified in the present work rather than the previously proposed σApromoter. With the exception ofC. cellulovorans, both σIand σApromoters 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.IMPORTANCECellulosome-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 ofC. thermocellumand related cellulosome-producing bacteriaen routeto the consolidated bioprocessing of biomass.
Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv, Israel.
Lizett
L
Ortiz de Ora
Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv, Israel.
Iván
I
Muñoz-Gutiérrez
Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel.
Edward A
EA
Bayer
Department of Biotechnology and Food Engineering, Technion-IIT, Haifa, Israel.
Yuval
Y
Shoham
Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv, Israel lamedr@post.tau.ac.il IlyaBo@tauex.tau.ac.il.
Raphael
R
Lamed
Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv, Israel lamedr@post.tau.ac.il IlyaBo@tauex.tau.ac.il.
Ilya
I
Borovok
D020121:5' Untranslated Regions; D001426:Bacterial Proteins / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D002482:Cellulose / Q000378:metabolism*; D044603:Cellulosomes / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000378:metabolism; D024363:Transcription Initiation Site
D020121:5' Untranslated Regions; D001426:Bacterial Proteins; D002352:Carrier Proteins; D012808:Sigma Factor; C074074:cellulose binding protein A, Clostridium cellulovorans; D002482:Cellulose
5′-UTR; Clostridium thermocellum; cellulosome; cipA; gene regulation; promoters; scaffoldin; sigma factors