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

<div><p>The Gram-positive, anaerobic, cellulolytic, thermophile <i>Clostridium</i> (<i>Ruminiclostridium</i>) <i>thermocellum</i> 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. <i>C</i>. <i>thermocellum</i> 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 <i>Bacillus subtilis</i> σ^I factor. The aim of the present work was to demonstrate whether individual <i>C</i>. <i>thermocellum</i> σ^I-like factors regulate specific cellulosomal genes, focusing on <i>C</i>. <i>thermocellum</i> σ^I6 and σ^I3 factors. To search for putative σ^I6- and σ^I3-dependent promoters, bioinformatic analysis of the upstream regions of the cellulosomal genes was performed. Because of the limited genetic tools available for <i>C</i>. <i>thermocellum</i>, the functionality of the predicted σ^I6- and σ^I3-dependent promoters was studied in <i>B</i>. <i>subtilis</i> as a heterologous host. This system enabled observation of the activation of 10 predicted σ^I6-dependent promoters associated with the <i>C</i>. <i>thermocellum</i> genes: <i>sigI6</i> (itself, Clo1313_2778), <i>xyn11B</i> (Clo1313_0522), <i>xyn10D</i> (Clo1313_0177), <i>xyn10Z</i> (Clo1313_2635), <i>xyn10Y</i> (Clo1313_1305), <i>cel9V</i> (Clo1313_0349), <i>cseP</i> (Clo1313_2188), <i>sigI1</i> (Clo1313_2174), <i>cipA</i> (Clo1313_0627), and <i>rsgI5</i> (Clo1313_0985). Additionally, we observed the activation of 4 predicted σ^I3-dependent promoters associated with the <i>C</i>. <i>thermocellum</i> genes: <i>sigI3</i> (itself, Clo1313_1911), <i>pl11</i> (Clo1313_1983), <i>ce12</i> (Clo1313_0693) and <i>cipA</i>. Our results suggest possible regulons of σ^I6 and σ^I3 in <i>C</i>. <i>thermocellum</i>, as well as the σ^I6 and σ^I3 promoter consensus sequences. The proposed -35 and -10 promoter consensus elements of σ^I6 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 σ^I3, 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 σ^I factors.</p></div

Identification of conserved elements of σ^I-dependent promoter sequences.

<p>(A) WebLogo generated with the <i>Bacillales sigI</i> promoters shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146316#pone.0146316.s006" target="_blank">S4 Table</a>. (B) WebLogo generated with the <i>C</i>. <i>thermocellum</i> and <i>C</i>. <i>straminisolvens sigI</i> promoters shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146316#pone.0146316.t001" target="_blank">Table 1</a>, and the <i>C</i>. <i>clariflavum</i>, <i>A</i>. <i>cellulolyticus and Pseudobacteroides cellulosolvens sigI</i> promoters shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146316#pone.0146316.s007" target="_blank">S5 Table</a>.</p

Evaluation of σ^I6 promoter sequence validity by mutagenesis.

<p>The activities are shown as relative activities, with the control promoter <i>xyn10Zshort</i> without mutations set to 100%. ND means not detected.</p

Identification of conserved elements of σ^I3-dependent promoter sequences.

<p>WebLogo generated with σ^I3-dependent promoter sequences of <i>C</i>. <i>thermocellum</i> and orthologous promoter sequences of <i>C</i>. <i>straminisolvens</i>.</p

<i>Pseudomonas</i> exotoxin (PE) productions using the S30-T7 CFPS system originated from two different <i>E</i>. <i>coli</i> strains (BL21 and MRE600).

<p>Reactions were performed with and without the presence of DNA template. (A) Western blot analysis of cell-free reactions demonstrated the production of PE ~ 66 kDa. Purified PE served as positive control (described in Appendix F in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165137#pone.0165137.s001" target="_blank">S1 File</a>.). Arrows indicate the position of PE bands. (B) The therapeutic potency of PE was evaluated on 4T1 cell-line. The viability of the cells was determined by MTT assay. Cell viability values obtained without the presence of purified PE or DNA were set as 100%, and the other values were normalized according to them (error bars represent standard deviation from at least three independent samples).</p

<i>In vitro</i> protein synthesis using cell-free system based on S30-T7 lysate.

<p><i>In vitro</i> protein synthesis using cell-free system based on S30-T7 lysate.</p

Identification of conserved elements of σ^I6-dependent promoter sequences.

<p>WebLogo generated with σ^I6-dependent promoter sequences of <i>C</i>. <i>thermocellum</i> and orthologous promoter sequences of <i>C</i>. <i>straminisolvens</i>.</p

Preparation of S30-T7 lysate.

<p>Preparation of S30-T7 lysate.</p

A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

<div><p>Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from <i>E</i>. <i>coli</i>. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 <i>E</i>. <i>coli</i> strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and <i>E</i>. <i>coli</i> strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.</p></div

A historical overview of improvements made to CFPS procedures over time.

<p>A historical overview of improvements made to CFPS procedures over time.</p