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

PLoS One. 2016 Jan 5;11(1):e0146316. doi: 10.1371/journal.pone.0146316. eCollection 2016.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / genetics
  • Bacillus subtilis / metabolism*
  • Biomass*
  • Cellulosomes / genetics
  • Cellulosomes / metabolism
  • Clostridium thermocellum / genetics
  • Clostridium thermocellum / metabolism*
  • Regulon / physiology*
  • Sigma Factor / metabolism*

Substances

  • Sigma Factor

Grants and funding

IMG was supported with a postdoctoral fellowship by the Consejo Nacional de Ciencia y Tecnologia (CONACyT proposal number 238334) [http://www.conacyt.mx/]. This research was supported by the Israel Science Foundation (grant No. 24/11 to RL and IB; grant Nos. 715/12, 500/10 to YS, and 1349/13 to EAB) [http://www.isf.org.il/] with additional support issued to RL and IB by The Sidney E. Frank Foundation through the ISF. The authors also acknowledge support from the Israeli Center of Research Excellence (I-CORE Center No. 152/11) Program of the Planning and Budgeting Committee, the Ministry of Environmental Protection and the Grand Technion Energy Program, comprising part of The Leona M. and Harry B. Helmsley Charitable Trust reporting on alternative energy series of the Technion – Israel Institute of Technology and the Weizmann Institute of Science. EAB also appreciates the support of the European Union, Area NMP.2013.1.1-2: Self-assembly of naturally occurring nanosystems: CellulosomePlus Project number: 604530 and an ERA-IB Consortium (EIB.12.022), acronym FiberFuel. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.