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Regulation of biomass degradation by alternative ? factors in cellulolytic clostridia.

ABSTRACT: Bacteria can adjust their genetic programs via alternative ? factors to face new environmental pressures. Here, we analyzed a unique set of paralogous alternative ? factors, termed ?^Is, which fine-tune the regulation of one of the most intricate cellulolytic systems in nature, the bacterial cellulosome, that is involved in degradation of environmental polysaccharides. We combined bioinformatics with experiments to decipher the regulatory networks of five ?^Is in Clostridium thermocellum, the epitome of cellulolytic microorganisms, and one ?^I in Pseudobacteroides cellulosolvens which produces the cellulosomal system with the greatest known complexity. Despite high homology between different ?^Is, our data suggest limited cross-talk among them. Remarkably, the major cross-talk occurs within the main cellulosomal genes which harbor the same ?^I-dependent promoter elements, suggesting a promoter-based mechanism to guarantee the expression of relevant genes. Our findings provide insights into the mechanisms used by ?^Is to differentiate among their corresponding regulons, representing a comprehensive overview of the regulation of the cellulosome to date. Finally, we show the advantage of using a heterologous host system for analysis of multiple ?^Is, since information generated by their analysis in their natural host can be misinterpreted owing to a cascade of interactions among the different ?^Is.

SUBMITTER: Ortiz de Ora L

PROVIDER: S-EPMC6056542 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Regulation of biomass degradation by alternative σ factors in cellulolytic clostridia.

Ortiz de Ora Lizett L Lamed Raphael R Liu Ya-Jun YJ Xu Jian J Cui Qiu Q Feng Yingang Y Shoham Yuval Y Bayer Edward A EA Muñoz-Gutiérrez Iván I

Scientific reports 20180723 1

Bacteria can adjust their genetic programs via alternative σ factors to face new environmental pressures. Here, we analyzed a unique set of paralogous alternative σ factors, termed σ<sup>I</sup>s, which fine-tune the regulation of one of the most intricate cellulolytic systems in nature, the bacterial cellulosome, that is involved in degradation of environmental polysaccharides. We combined bioinformatics with experiments to decipher the regulatory networks of five σ<sup>I</sup>s in Clostridium ...[more]

PMID: 30038431

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Project description: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.

Project description:The extracellular substrate-binding proteins (SBPs) of ATP-binding cassette (ABC) importers tend to be expressed in excess relative to their cognate translocators, but how the stoichiometry of ABC transporters is controlled remains unclear. Here, we elucidated a mechanism contributing to differential gene expression in operons encoding ABC importers by employing cellulolytic Clostridia species, specifically Ruminiclostridium cellulolyticum. We found that there were usually stem-loop structures downstream of SBP genes, which could prematurely terminate the transcription of ABC importers and were putative internal intrinsic terminators, resulting in high transcript levels of upstream SBP genes and low transcript levels of downstream cognate translocator genes. This was determined by their termination efficiencies. Internal terminators had a lower U content in their 3' U-rich tracts and longer GC-rich stems, which distinguishes them from canonical terminators and potentially endows them with special termination efficiencies. The pairing of U-rich tracts and the formation of unpaired regions in these internal terminators contributed to their folding energies, affecting the stability of their upstream SBP transcripts. Our findings revealed a strategy of internal transcriptional terminators controlling in vivo stoichiometry of their flanking transcripts. IMPORTANCE Operons encoding protein complexes or metabolic pathways usually require fine-tuned gene expression ratios to create and maintain the appropriate stoichiometry for biological functions. In this study, a strategy for controlling differential expression of genes in an operon was proposed by utilizing ABC importers from Ruminiclostridium cellulolyticum. We found that a stem-loop structure is introduced into the intergenic regions of operons encoding ABC importers as the putative internal terminator, which results in the premature termination of transcription. Consequently, the stoichiometric ratio of genes flanking terminators is precisely determined by their termination efficiencies and folding energies at the transcriptional level. Thus, it can be utilized as a promising synthetic biology tool to control the differential expression of genes in an operon.

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Dataset Information

Regulation of biomass degradation by alternative ? factors in cellulolytic clostridia.

Publications

Regulation of biomass degradation by alternative σ factors in cellulolytic clostridia.

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