Population Level Analysis of Evolved Mutations Underlying Improvements in Plant Hemicellulose and Cellulose Fermentation by <i>Clostridium phytofermentans</i>

<div><p>Background</p><p>The complexity of plant cell walls creates many challenges for microbial decomposition. <i>Clostridium phytofermentans</i>, an anaerobic bacterium isolated from forest soil, directly breaks down and utilizes many plant cell wall carbohydrates. The objective of this research is to understand constraints on rates of plant decomposition by <i>Clostridium phytofermentans</i> and identify molecular mechanisms that may overcome these limitations.</p><p>Results</p><p>Experimental evolution via repeated serial transfers during exponential growth was used to select for <i>C. phytofermentans</i> genotypes that grow more rapidly on cellobiose, cellulose and xylan. To identify the underlying mutations an average of 13,600,000 paired-end reads were generated per population resulting in ∼300 fold coverage of each site in the genome. Mutations with allele frequencies of 5% or greater could be identified with statistical confidence. Many mutations are in carbohydrate-related genes including the promoter regions of glycoside hydrolases and amino acid substitutions in ABC transport proteins involved in carbohydrate uptake, signal transduction sensors that detect specific carbohydrates, proteins that affect the export of extracellular enzymes, and regulators of unknown specificity. Structural modeling of the ABC transporter complex proteins suggests that mutations in these genes may alter the recognition of carbohydrates by substrate-binding proteins and communication between the intercellular face of the transmembrane and the ATPase binding proteins.</p><p>Conclusions</p><p>Experimental evolution was effective in identifying molecular constraints on the rate of hemicellulose and cellulose fermentation and selected for putative gain of function mutations that do not typically appear in traditional molecular genetic screens. The results reveal new strategies for evolving and engineering microorganisms for faster growth on plant carbohydrates.</p></div

Homology models suggest that the selected mutations in an ABC transporter binding protein occur at protein-protein and protein-ligand interfaces.

<p>Three mutations in Cphy 2654 (G457E, Y196N, and Y196S) were found in xylan-adapted populations. A. The structure of the maltose transporter complex with maltose binding protein (3pv0) is shown with the maltose binding protein replaced by a homology model of Cphy 2654 (cyan cartoon, based on 3omb). The model suggests that the Cphy 2654 G457E mutation (red) is near the interface between the binding protein and the transmembrane domains. B. Surface representation of another homology model of Cphy 2654 (based on 2fnc) shows that the Y196N/S mutations (red) is predicted to occur in the ligand binding pocket.</p

Major fermentation product formation by cellulose adapted populations and founder after 10 days of growth.

<p>Major fermentation product formation by cellulose adapted populations and founder after 10 days of growth.</p

Growth, cellobiose utilization and ethanol production of cellobiose adapted populations and the founder.

<p>Growth (A) was measured every four hours as change in optical density in a spectrophotometer. Supernatant was collected every eight hours for measuring cellobiose utilization (B) and ethanol production (C) rates. Cellobiose and ethanol values represent an average of two independent samples.</p

Genes and intergenic regions where multiple mutations were detected.

<p>Mutation hotspots which were identified in multiple evolved lines or in the same population more than once. For example, Cphy 0515 was observed to have a separate SNP (red star), insertion (blue star) and deletion (blue box) in Xyn-B and one insertion in Ceb-B (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086731#pone.0086731.s002" target="_blank">Table S1</a>).</p

Schematic representation of the adaptive evolution process starting from an isogenic founder.

<p>Rep 1, 2 & 3 represent the three replicates in each of the three individual lines.</p

Overview of carbohydrate sensing, saccharification and transport systems with the approximate location of evolved mutations.

<p>The mutations are represented by yellow stars.</p

Homology modeling suggests that a selected mutation in an ABC transporter transmembrane domain (Cphy 2465) in cellulose-adapted populations occurs at a protein-protein interface.

<p>The maltose transporter (3pv0) is shown because its transmembrane domains MalF (green) and MalG (magenta) are the best templates of known structure for Cphy 2465 and Cphy 2464, respectively. A homology model of Cphy 2465 based on MalF places the selected A207V mutation (red) in the coupling helix (arrow and table) that is important in transmitting changes between the transmembrane domains (green and magenta) and the ATPase domains (blue and gold). The mutation occurs in the consensus sequence originally identified in several transporters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086731#pone.0086731-Dassa1" target="_blank">[45]</a>.</p

Localization of SNPs in Cphy 3212 cellulose adapted lines.

<p>T83I in Cel-C and T223I in Cel-B. Predicted transmembrane regions of the protein are highlighted with a grey box, strings of ‘o’ represent the extracytoplasmic regions, while the regions marked ‘i’ are predicted to lie within the cell. Both SNPs are located in the region of the protein predicted to be on the extracellular face.</p

Summary of sequencing results including the average read-depth, number of reads and mutations detected in the adapted populations as well as in the founder line.

<p>Summary of sequencing results including the average read-depth, number of reads and mutations detected in the adapted populations as well as in the founder line.</p