A Two-Component System (XydS/R) Controls the Expression of Genes Encoding CBM6-Containing Proteins in Response to Straw in Clostridium cellulolyticum

Hamza Celik,Henri-Pierre Fierobe,Dörte Becher,Birgit Voigt,Valentine Trotter,Jean-Charles Blouzard,Pascale De Philip,Chantal Tardif,Sandrine Pagès,Vladimir N Uversky

doi:10.1371/journal.pone.0056063

Abstract

The composition of the cellulosomes (multi enzymatic complexes involved in the degradation of plant cell wall polysaccharides) produced by Clostridium cellulolyticum differs according to the growth substrate. In particular, the expression of a cluster of 14 hemicellulase-encoding genes (called xyl-doc) seems to be induced by the presence of straw and not of cellulose. Genes encoding a putative two-component regulation system (XydS/R) were found upstream of xyl-doc. First evidence for the involvement of the response regulator, XydR, part of this two-component system, in the expression of xyl-doc genes was given by the analysis of the cellulosomes produced by a regulator overproducing strain when grown on cellulose. Nano-LC MS/MS analysis allowed the detection of the products of all xyl-doc genes and of the product of the gene at locus Ccel_1656 predicted to bear a carbohydrate binding domain targeting hemicellulose. RT-PCR experiments further demonstrated that the regulation occurs at the transcriptional level and that all xyl-doc genes are transcriptionally linked. mRNA quantification in a regulator knock-out strain and in its complemented derivative confirmed the involvement of the regulator in the expression of xyl-doc genes and of the gene at locus Ccel_1656 in response to straw. Electrophoretic mobility shift assays using the purified regulator further demonstrated that the regulator binds to DNA regions located upstream of the first gene of the xyl-doc gene cluster and upstream of the gene at locus Ccel_1656.

Highlights

Degradation of plant cell wall polysaccharides has remarkable practical applications, especially in the renewable bioenergy sector [1,2]
We show that XydR regulates the transcription of another gene encoding the only other CBM6containing protein produced by C. cellulolyticum
Taken together with the fact that many of the products of these genes were detected in the same conditions [11], we studied the transcription of these genes by RTPCR in C. cellulolyticum grown in straw-based medium

Summary

Introduction

Degradation of plant cell wall polysaccharides has remarkable practical applications, especially in the renewable bioenergy sector [1,2]. Clostridium cellulolyticum, an anaerobic, mesophilic and cellulolytic bacterium, isolated from decayed grass [3], is a model organism for such applications [4,5] and for the study of plant cell wall polysaccharide degradation. This bacterium produces extracellular enzymes assembled in high-molecular-mass complexes named cellulosomes [6] that hydrolyze plant cell wall polysaccharides into simple sugars. The assembly of cellulosomes is mediated via the interaction between eight cohesin modules of a non-catalytic scaffolding protein (CipC) and the dockerin modules of eight catalytic enzymes [9,10]. It has been demonstrated that the composition of cellulosomes depends on the growth substrate to be degraded [11] and on the amounts of dockerinbearing proteins present [13]

Methods

Results

Conclusion