Novel family of carbohydrate-binding modules revealed by the genome sequence of Spirochaeta thermophila DSM 6192

Abstract

Spirochaeta thermophila is a thermophilic, free-living, and cellulolytic anaerobe. The genome sequence data for this organism have revealed a high density of genes encoding enzymes from more than 30 glycoside hydrolase (GH) families and a noncellulosomal enzyme system for (hemi)cellulose degradation. Functional screening of a fosmid library whose inserts were mapped on the S. thermophila genome sequence allowed the functional annotation of numerous GH open reading frames (ORFs). Seven different GH ORFs from the S. thermophila DSM 6192 genome, all putative β-glycanase ORFs according to sequence similarity analysis, contained a highly conserved novel GH-associated module of unknown function at their C terminus. Four of these GH enzymes were experimentally verified as xylanase, β-glucanase, β-glucanase/carboxymethylcellulase (CMCase), and CMCase. Binding experiments performed with the recombinantly expressed and purified GH-associated module showed that it represents a new carbohydrate-binding module (CBM) that binds to microcrystalline cellulose and is highly specific for this substrate. In the course of this work, the new CBM type was only detected in Spirochaeta, but recently we found sequences with detectable similarity to the module in the draft genomes of Cytophaga fermentans and Mahella australiensis, both of which are phylogenetically very distant from S. thermophila and noncellulolytic, yet inhabit similar environments. This suggests a possibly broad distribution of the module in nature.

Fig. 1.

Overview of the central metabolism of S. thermophila deduced from the genome data. Abbreviations: G-1-P, glucose-1-phosphate; F-1-P, fructose-1-phosphate; ADP-G, ADP-glucose; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; 3-PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; KDG, ketodeoxygluconate; KDPG, ketodeoxyphosphogluconate; Fd^red, reduced ferredoxin; MCP, methyl-accepting chemotaxis protein. The numbers indicate the S. thermophila DSM 6192 GenBank protein identification tags (ADNxxxxx).

Fig. 2.

Amino acid sequence alignment (ClustalW2) of the new carbohydrate-binding modules from seven S. thermophila GH ORFs. The numbers on the left are S. thermophila ORF designations (GenBank protein ID tags), and the numbers in parentheses represent the respective amino acid positions used in the alignment. Conservation is measured as a numerical index reflecting the conservation of physicochemical properties for each column in the alignment (19).

Fig. 3.

(A) Double reciprocal plot of the binding of the purified module StX-1787wt to Avicel; (B) binding capacity (bound StX-1787wt protein) plotted against the amount of Avicel used. The binding assay conditions are described in Materials and Methods. P, amount of purified StX-1787wt protein added in the assay; PC, amount of Avicel-bound protein.