celS, a gene encoding an extracellular cellulase from Sulfolobus solfataricus

Cellulose is one of the most abundant biopolymer on Earth and represents an important source of renewable energy. It is an unbranched polymer composed of D-glucose units linked by 1,4--D-glucosidic bonds. Enzymatic hydrolysis of cellulose requires a consortium of enzymes, including endo--glucanases, exoglucanases, and -glucosidases. Endoglucanases randomly hydrolyse internal glycosidic linkages and are widely distributed among fungi, Bacteria and Archaea. Cellulase activitiy was detected in the supernatant of S. solfataricus MT4 cultures and specific enzyme staining after SDS-PAGE revealed the presence of two proteins with apparent molecular mass of about 49 kDa and 40 kDa. At the same time we have cloned a DNA fragment from Sulfolobus solfataricus MT4 containing an ORF (CelS) of 322 aminoacids (molecular weight 36703 Da) with significant homology to the Pyrococcus furiosus (1), Thermotoga maritima (2) and T. neapolitana (3) endo-1,4--glucanases. The gene was demonstrated to be transcribed in vivo. In order to optimise the expression of celS we tried to grow MT4 on different -glucans, namely in minimal media containing Avicel, lichenan, carboxylmethylcellulose, cellobiose, but they resulted to be unable to support growth. Therefore enzymatic and transcriptional analysis were performed on cells cultured in more unspecific minimal and rich media. The results obtained suggested a catabolite repression by glucose and in general a down regulation by complex nutrients. The transcription start site was unambiguously identified by primer extension and revealed coincident with translational initiation