Causes of Transition from Democracy to Totalitarism

Additional file 6: Table S2. Summary of proteins annotated as substrate-binding proteins (SBP) from ATP-binding cassette (ABC) transporters detected in the proteomes of Paenibacillus O199. Annotation was performed with RAST

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

<div><p>The composition of the cellulosomes (multi enzymatic complexes involved in the degradation of plant cell wall polysaccharides) produced by <em>Clostridium cellulolyticum</em> differs according to the growth substrate. In particular, the expression of a cluster of 14 hemicellulase-encoding genes (called <em>xyl-doc</em>) 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 <em>xyl-doc</em>. First evidence for the involvement of the response regulator, XydR, part of this two-component system, in the expression of <em>xyl-doc</em> 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 <em>xyl-doc</em> 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 <em>xyl-doc</em> 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 <em>xyl-doc</em> 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 <em>xyl-doc</em> gene cluster and upstream of the gene at locus Ccel_1656.</p> </div

Bacterial strains and plasmids used in this study.

<p>Ap^r, ampicilline resistance; Tc^r, tetracycline resistance, Cm/Tm^r, chloramphenicol/thiamphenicol resistance; Em^r, erythromycine resistance.</p

Binding of a truncated XydR regulator to target regions.

<p>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).</p

Common DNA sequences upstream of loci Ccel_1229 and Ccel_1656.

a<p>given from 5′ extremity to 3′extremity.</p>b<p>distance in base pair from predicted −10 consensus sequence (BPROM;<a href="http://linux1.softberry.com" target="_blank">http://linux1.softberry.com</a>).</p

qRT-PCR analysis of mRNA produced by <i>xyd</i>R knock-out strain.

<p>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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056063#pone.0056063.s001" target="_blank">Table S1</a>) targeting the first gene of <i>xyl-doc</i> cluster at locus Ccel_1229, 1656qRT-F/1656qRT-R (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056063#pone.0056063.s001" target="_blank">Table S1</a>) 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 <i>rpo</i>D (using the primers RPO-F and RPO-R, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056063#pone.0056063.s001" target="_blank">Table S1</a>). 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.</p

RT-PCR amplification of mRNA produced by <i>C. cellulolyticum</i> wild type or overproducing constitutive regulator.

<p>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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056063#pone.0056063.s001" target="_blank">Table S1</a>): RPO-F/RPO-R (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056063#pone.0056063.s001" target="_blank">Table S1</a>) targeting a housekeeping gene used for standardization and quantification of the induction (RPO), 1229rtD/1229rtR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056063#pone.0056063.s001" target="_blank">Table S1</a>) targeting end of the first gene and beginning of the second gene of <i>xyl-doc</i> cluster (29–30), 1230rtD/1231rtR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056063#pone.0056063.s001" target="_blank">Table S1</a>) targeting end of the second gene and beginning of the third gene of <i>xyl-doc</i> cluster (30–31), and 1656rtD/1656rtR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056063#pone.0056063.s001" target="_blank">Table S1</a>) targeting upstream sequence and beginning of the gene at locus Ccel_1656 (1656).</p

Detection of the products of <i>xyl-doc</i> and CBM6-containing proteins by LC-MS/MS in cellulosomes^a.

a<p>Cellulosomes were produced by H10, H10(pSOSzero), H10(pSOS954Δ116) grown in cellulose- or straw-based media.</p>b<p>As given in Blouzard et al. 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056063#pone.0056063-Blouzard1" target="_blank">[11]</a>.</p>c<p>Theoretical masses.</p>d<p>Quantitative values given as the spectral counts found by LC MS/MS analysis for identified proteins. ni, not identified.</p

Loci Ccel_1227 to Ccel_1242 of <i>C. cellulolyticum</i> genome.

<p>A) Modular organization of <i>xyl-doc</i> 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 (<a href="http://www.cazy.org" target="_blank">http://www.cazy.org</a>). B) Genetic organization of <i>xyd</i>S/R (loci Ccel_1227 and Ccel_1228 respectively, shown in light grey) and <i>xyl-doc</i> 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 <i>xyd</i>S/R and <i>xyl-doc</i> 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 <i>xyd</i>S/R and <i>xyl-doc</i> genes as in (A) with schematic localizations of promoters (thin arrow) and terminators (stem-loop) predicted by BPROM and FindTerm programs (<a href="http://linux1.softberry.com" target="_blank">http://linux1.softberry.com</a>).</p

Detection of CBM6-containing proteins by western blot in cellulosomes.

<p>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 <i>gal27A</i>, 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.</p