RRNPP-type quorum-sensing systems regulate solvent formation, sporulation and cell motility in Clostridium saccharoperbutylacetonicum

Jun Feng; Wenming Zong; Pixiang Wang; Zhong-Tian Zhang; Yanyan Gu; Mark Dougherty; Ilya Borovok; Yi Wang

doi:10.1186/s13068-020-01723-x

RRNPP-type quorum-sensing systems regulate solvent formation, sporulation and cell motility in Clostridium saccharoperbutylacetonicum

Biotechnol Biofuels. 2020 May 8:13:84. doi: 10.1186/s13068-020-01723-x. eCollection 2020.

Authors

Jun Feng¹, Wenming Zong^{1

2}, Pixiang Wang¹, Zhong-Tian Zhang¹, Yanyan Gu¹, Mark Dougherty¹, Ilya Borovok³, Yi Wang^{1

4}

Affiliations

¹ 1Department of Biosystems Engineering, Auburn University, 350 Mell Street, Auburn, AL 36849 USA.
² 2School of Engineering, Anhui Agricultural University, Hefei, 230036 China.
³ 3School of Molecular Cell Biology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 6997801 Tel Aviv, Israel.
⁴ 4Center for Bioenergy and Bioproducts, Auburn University, Auburn, AL 36849 USA.

Abstract

Background: Clostridium saccharoperbutylacetonicum N1-4 (HMT) is a strictly anaerobic, spore-forming Gram-positive bacterium capable of hyper-butanol production through the well-known acetone-butanol-ethanol fermentation process. Recently, five putative RRNPP-type QSSs (here designated as QSS1 to QSS5) were predicted in this bacterial strain, each of which comprises a putative RRNPP-type regulator (QssR1 to QssR5) and a cognate signaling peptide precursor (QssP1 to QssP5). In addition, both proteins are encoded by the same operon. The functions of these multiple RRNPP-type QSSs are unknown.

Results: To elucidate the function of multiple RRNPP-type QSSs as related to cell metabolism and solvent production in N1-4 (HMT), we constructed qssR-deficient mutants ΔR1, ΔR2, ΔR3 and ΔR5 through gene deletion using CRISPR-Cas9 and N1-4-dcas9-R4 (with the QssR4 expression suppressed using CRISPR-dCas9). We also constructed complementation strains by overexpressing the corresponding regulator gene. Based on systematic characterization, results indicate that QSS1, QSS2, QSS3, and QSS5 positively regulate the sol operon expression and thus solvent production, but they likely negatively regulate cell motility. Consequently, QSS4 might not directly regulate solvent production, but positively affect cell migration. In addition, QSS3 and QSS5 appear to positively regulate sporulation efficiency.

Conclusions: Our study provides the first insights into the roles of multiple RRNPP-type QSSs of C. saccharoperbutylacetonicum for the regulation of solvent production, cell motility, and sporulation. Results of this study expand our knowledge of how multiple paralogous QSSs are involved in the regulation of essential bacterial metabolism pathways.

Keywords: Acetone–butanol–ethanol (ABE); Butanol; CRISPR–Cas9; Cell motility; Clostridium saccharoperbutylacetonicum; RRNPP-type quorum-sensing systems; Signaling peptide precursor; Sporulation.