An expression cDNA library was produced from the anaerobic ciliate Epidinium ecaudatum living in the rumen of a "monofaunated" sheep. When screened in lambda ZAP Express for the ability to hydrolyse carboxymethyl cellulose (CMC) a clone with sequence similarity to glycosyl hydrolase family 5 cellulases was isolated. After overexpression in E. coli the recombinant protein showed an unusual pH optimum against carboxymethyl cellulose (CMC) at above 8. Since a putative N-terminal signal peptide was identified suggesting that the mature protein was exported into digestive vacuoles in the ciliate's cytoplasm, it had to be concluded this cellulase is active within the alkaline digestive vacuoles of the ciliate. Notwithstanding a distinctive codon usage pattern similar to other rumen ciliate genes, phylogenetic analysis suggested strongly that the isolated sequence was closely related to certain cellulase genes of rumen bacteria. It is concluded that this gene has been acquired by lateral gene transfer from rumen bacteria. It is postulated that this gene underwent ameliorization of its codon usage and an adaptation to an alkaline pH optimum to meet the requirements for a function in the alkaline digestive vacuoles of its host.
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