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. 2013;8(2):e56063.
doi: 10.1371/journal.pone.0056063. Epub 2013 Feb 13.

A two-component system (XydS/R) controls the expression of genes encoding CBM6-containing proteins in response to straw in Clostridium cellulolyticum

Hamza Celik  1 , Jean-Charles Blouzard, Birgit Voigt, Dörte Becher, Valentine Trotter, Henri-Pierre Fierobe, Chantal Tardif, Sandrine Pagès, Pascale de Philip
Affiliations

A two-component system (XydS/R) controls the expression of genes encoding CBM6-containing proteins in response to straw in Clostridium cellulolyticum

Hamza Celik et al. PLoS One. 2013.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Loci Ccel_1227 to Ccel_1242 of C. cellulolyticum genome.
A) Modular organization of xyl-doc genes products (loci Ccel_1229 to Ccel_1242). Signal sequence are given as black- and dockerin domains as dark grey- boxes. The catalytic domains are the white boxes in which the predicted glycoside-hydrolase (GHx) or carbohydrate esterase (CEx) family when known or UNK for a domain of unknown function are given. A bold unique number under a light grey box indicates the family of the carbohydrate binding domains found in the proteins. The family numbers are given according to CAZy database (http://www.cazy.org). B) Genetic organization of xydS/R (loci Ccel_1227 and Ccel_1228 respectively, shown in light grey) and xyl-doc genes (from loci Ccel_1229 to Ccel_1242, shown in black). The genes are named by their locus tag. Sizes (in bp) of intergenic sequences are indicated on the black triangles. C) RT-PCR analysis of xydS/R and xyl-doc genes. PCR amplification was performed on cDNA after RT with pairs of primers and PCR products were analyzed by electrophoresis in agarose gel (lanes RT). Primers used were localized at the end of the first gene for the direct primer and at the beginning of the second gene for the reverse primers to reveal transcriptional links between two successive genes. The pairs are as follow : 1229rtD/1230rtR (lanes 1), 1230rtD/1231rtR (lanes 2), 1231rtD/1232rtR (lanes 3), 1232rtD/1233rtR (lanes 4), 1233rtD/1234rtR (lanes 5), 1234rtD/1235rtR (lanes 6), 1235rtD/1236rtR (lanes 7), 1236rtD/1237rtR (lanes 8), 1237rtD/1238rtR (lanes 9), 1238rtD/1239rtR (lanes 10), 1239rtD/1240rtR (lanes 11), 1240rtD/1241rtR (lanes 12), 1241rtD/1242rtR (lanes 13), 1242rtD/1243rtR (lanes 14), 1227rtD/1228rtR (lanes 15), 1228rtD/1229rtR (lanes 16). −, PCRs performed on RNAs in the absence of the RT step; +, PCRs performed on genomic DNA templates. D) Genetic organization of xydS/R and xyl-doc genes as in (A) with schematic localizations of promoters (thin arrow) and terminators (stem-loop) predicted by BPROM and FindTerm programs (http://linux1.softberry.com).
Figure 2
Figure 2. Detection of CBM6-containing proteins by western blot in cellulosomes.
25 µg of cellulosomes produced by the control strain H10(pSOSzero) grown in cellulose-based medium (lanes 1) or straw-based medium (lanes 2) and the overproducing truncated regulator strain H10(pSOSΔ116) grown on cellulose-based medium (lanes 3) were subjected to SDS-PAGE and transferred to nylon membranes. Western blots were probed with antibodies directed against (A) GH10 domain of the product of the gene at locus Ccel_1230, (B) product of gene at locus Ccel_1234, (C) Gal27A (product of gene gal27A, locus Ccel_1237), and (D) unknown domain of the product of the gene at locus Ccel_1656. Arrows indicate the expected molecular mass of the corresponding mature proteins.
Figure 3
Figure 3. RT-PCR amplification of mRNA produced by C. cellulolyticum wild type or overproducing constitutive regulator.
mRNA were prepared from cultures of H10, H10(pSOSzero), and H10(pSOS954Δ116) grown on cellulose or straw as indicated and reverse transcribed. PCR was performed on the cDNA with the following pairs of primers (Table S1): RPO-F/RPO-R (Table S1) targeting a housekeeping gene used for standardization and quantification of the induction (RPO), 1229rtD/1229rtR (Table S1) targeting end of the first gene and beginning of the second gene of xyl-doc cluster (29–30), 1230rtD/1231rtR (Table S1) targeting end of the second gene and beginning of the third gene of xyl-doc cluster (30–31), and 1656rtD/1656rtR (Table S1) targeting upstream sequence and beginning of the gene at locus Ccel_1656 (1656).
Figure 4
Figure 4. qRT-PCR analysis of mRNA produced by xydR knock-out strain.
mRNA were prepared from cultures in straw-based medium of wild-type (H10), MTL1228(pSOSzeroTc) and MTL1228(pSOS955Δ116) strains and reverse transcribed. qPCR was performed on the cDNA with the following pairs of primers :1229qRT-F/1229qRT-R (Table S1) targeting the first gene of xyl-doc cluster at locus Ccel_1229, 1656qRT-F/1656qRT-R (Table S1) targeting the gene at locus Ccel_1656. Levels of expression of genes at loci Ccel_1229 and Ccel_1656 are given after standardization with the level of expression of rpoD (using the primers RPO-F and RPO-R, Table S1). Error bars indicate the standard deviation of three independent qPCR reactions. Dark grey represents the results obtained with mRNA from H10 strain, light grey are the ones with mRNA from MTL1228(pSOSzeroTc) strain and grey ones with mRNA from MTL1228(pSOSΔ116) strain.
Figure 5
Figure 5. Binding of a truncated XydR regulator to target regions.
A) Schematic representation of targeted regions [R1 (309 bp), R2 (347 bp), R3 (272 bp), R4 (429 bp), R5 (345 bp), R6 (246 bp), and R7 (240 bp)] upstream genes at loci Ccel_1227, CCel_1228, Ccel_1229, and Ccel_1656. The loci of the different genes are represented as black arrows. B, C and D) Electromobility shift assays were carried out with 20 fmol of 3′ OH biotin-labeled regions (R1, R2, R3, R4, R5, R6, and R7) without any protein or with various amounts of purified MBP-Δ116 which are indicated in nM on top of the lanes. Competitive inhibition of the bindings were performed with 5 nM of MBP-Δ116, 20 fmol of 3′ OH biotin-labeled of regions R3 and R6 and 2 pmol of unlabeled R3 and R6 regions respectively (lanes with+at the bottom).
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This research was supported by funding from the European Community’s Seventh Framework Program FP7/2007–2013 under grant agreement no. 237942 (CLOSTNET), the Centre National de la Recherche Scientifique and Aix-Marseille Université. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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