Molecular cloning and transcriptional and expression analysis of engO, encoding a new noncellulosomal family 9 enzyme, from Clostridium cellulovorans

Clostridium cellulovorans produces a major noncellulosomal family 9 endoglucanase EngO. A genomic DNA fragment (40 kb) containing engO and neighboring genes was cloned. The nucleotide sequence contained reading frames for endoglucanase EngO, a putative response regulator, and a putative sensor histidine kinase protein. The engO gene consists of 2,172 bp and encodes a protein of 724 amino acids with a molecular weight of 79,474. Northern hybridizations revealed that the engO gene is transcribed as a monocistronic 2.6-kb mRNA. 5′ RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) PCR analysis indicated that the single transcriptional start site of engO was located 264 bp upstream from the first nucleotide of the translation initiation codon. Alignment of the engO promoter region provided evidence for highly conserved sequences that exhibited strong similarity to the σ^A consensus promoter sequences of gram-positive bacteria. EngO contains a typical N-terminal signal peptide of 28 amino acid residues, followed by a 149-amino-acid sequence which is homologous to the family 4-9 carbohydrate-binding domain. Downstream of this domain was an immunoglobulin-like domain of 89 amino acids. The C terminus contains a family 9 catalytic domain of glycosyl hydrolase. Mass spectrometry analysis of EngO was in agreement with that deduced from the nucleotide sequence. Expression of engO mRNA increased from early to middle exponential phase and decreased during the early stationary phase. EngO was highly active toward carboxymethyl cellulose but showed no activity towards xylan. It was optimally active at 40 to 50°C and pH 5 to 6. The analysis of the products from the cellulose hydrolysis through thin-layer chromatography indicated its endoglucanase activity.