The xylose isomerase-encoding gene ( xylA) of Clostridium thermosaccharolyticum: cloning, sequencing and phylogeny of Xy1A enzymes

Abstract

The xylose isomerase ( xylA)-encoding gene ( xylA) of the thermophilic anaerobic 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 encoded a protein of 439 amino acids (aa), with a calculated M r of 50236. The gene was preceeded by a typical clostridial Shine-Dalgarno sequence, and was expressed constitutively in the cloning host. Downstream, the clone appeared to carry a xylB gene (encoding xylulokinase) in the same orientation as xylA. Comparison of the deduced aa sequence of the C. thermosaccharolyticum XylA with 18 other XylA showed that this family of proteins was separated into two clusters, one comprising proteins from organisms with G + C-rich DNA, and the other proteins from organisms with a lower G + C composition. Within the second cluster, the Xy1A of C. thermosaccharolyticum was most closely related to the enzymes from C. thermosulfurogenes ( Thermoanaerobacterium thermosulfurigenes) and C. thermohydrosulfuricum (93 and 84% identity, respectively). Analysis of the aligned sequences indicated two signatures (VXW[GP]GREG[YSTA]E and [LIVM]EPKPX[EQ]P) which may be useful in isolation of novel XylA.