Comparative Analysis of Carbohydrate Active Enzymes in <i>Clostridium termitidis</i> CT1112 Reveals Complex Carbohydrate Degradation Ability

<div><p><i>Clostridium termitidis</i> strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, <i>Nasutitermes lujae</i>. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes), sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in <i>C. termitidis</i>, the recently sequenced strain CT1112 of <i>C. termitidis</i> was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in <i>C. termitidis</i>, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199) and carbohydrate binding modules (95) were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.</p></div

Predicted extracellular glycoside hydrolases of <i>C.termitidis</i> based on PSORTb.3 analysis.

<p>Bold: GH genes with dockerin domain.</p><p>^*: Genes with SLH domain.</p

General features of genomes of select <i>Clostridium</i> species.

<p>*Based on PSORTb 3.0 prediction and includes GHs, PLs, and CEs.</p

Phylogenetic analysis of selected Clostridial species based on <i>cpn60</i> gene sequences.

<p>The phylogenetic tree was obtained using neighbor-joining <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104260#pone.0104260-Saitou1" target="_blank">[29]</a> provided in MEGA version 4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104260#pone.0104260-Tamura1" target="_blank">[30]</a>. Bootstrap tests with 1000 replications were conducted to examine the reliability of the interior branches. Asterisks (*) indicates the other <i>Clostridium</i> species used in CAZy comparison.</p

Cellulosome components of <i>C. thermocellum</i>.

<p>Enzymatic components (colored differently to indicate enzyme variety) produced by anaerobic bacteria contain a dockerin domain. Dockerins bind the cohesins of a non-catalytic scaffoldin, providing a mechanism for cellulosome assembly. Scaffoldins also contain a cellulose-specific family 3 CBM (cellulose binding module) and a C-terminal dockerin domain II that targets the cellulosome to cellulose and the bacterial cell envelope, respectively.</p

Modular structure of putative cohesin I domain containing proteins identified in the <i>C. termitidis</i> CT1112 genome.

<p>(a) Cter_0001; (b) Cter_0520; (c) Cter_0526; (d) Cter_3731, and, (e) Cter_0525. CBM3-carbohydrate binding module. X2- domain of unknown function which may play a role in attachment of the putative cellulosome to the cell wall. Cohesin I proteins have dockerin binding surfaces, which bind cellulosomal enzymes and are considered important in the formation of a cellulosome. Cohesins a, c and d show putative truncated ends. Cohesins b, c and e are components of a putative cellulosome related gene cluster.</p

Comparative analysis of the number of putative CAZy sequences in selected Clostridial species.

<p>Comparative analysis of the number of putative CAZy sequences in selected Clostridial species.</p

Comparison of the number of putative dockerin containing GH sequences in selected Clostridial species.

<p><i>C.phytofermentans</i> ISDg and <i>C.stercorarium</i> DSM 8532 have no detectable dockerin domains.</p

Biomass growth measured by optical density at 600 nm and protein content for <i>C. termitidis</i> cultured on 2 g/L xylan.

<p>Data points represent the means of 3 independent replicates. Bars above and below the means represent the standard deviation.</p