THE XYLOSE ISOMERASE-ENCODING GENE (XYLA) OF CLOSTRIDIUM-THERMOSACCHAROLYTICUM - CLONING, SEQUENCING AND PHYLOGENY OF XYLA ENZYMES

The xylose isomerase (XylA)-encoding gene (xylA) of the thermophilic a naerobic bacterium, Clostridium thermosaccharo-lyticum NCIB 9385, was cloned as a 4.0-kb DNA fragment by complementation of the Escherichia coli xylA mutant strain, DS941. The open reading frame of 1317 bp enco ded a protein of 439 amino acids (aa), with a calculated M(r) of 50236 . The gene was preceeded by a typical clostridial Shine-Dalgarno seque nce, and was expressed constitutively in the cloning host. Downstream, the clone appeared to carry a xylB gene (encoding xylulokinase) in th e same orientation as xylA. Comparison of the deduced aa sequence of t he C. thermosaccharolyticum XylA with 18 other XylA showed that this f amily of proteins was separated into two clusters, one comprising prot eins from organisms with G+C-rich DNA, and the other proteins from org anisms with a lower G+C composition. Within the second cluster, the Xy lA of C. thermosaccharolyticum was most closely related to the enzymes from C. thermosulfurogenes (Thermoanaerobacterium thermosulfurigenes) and C. thermohydrosulfuricum (93 and 84% identity, respectively). Ana lysis of the aligned sequences indicated two signatures (VXWGPGREGY STAE and LIVMEPKPXEQP) which may be useful in isolation of novel XylA.