Cloning and sequencing the Lactobacillus brevis gene encoding xylose isomerase

The gene (xylA) coding for the Lactobacillus brevis xylose isomerase (Xi) has been isolated and its complete nucleotide sequence determined. L. brevis Xi was purified and the N-terminal sequence determined. All attempts to directly clone the intact xylA using a degenerative primer deduced from amino acids (aa) 10-14 were not successful. A fragment coding for the first 462 bp from the 5' end of xylA was isolated by PCR with two primers, one coding for aa M36 to W43 and the second coding for an aa sequence (WGGREG) conserved in a number of Xi's isolated from other bacteria. From the sequence of this fragment, two additional PCR primers were synthesized, which were used in an 'outward' reaction to clone a 546-bp fragment including a region upstream from the N terminus. Finally, the complete xylA gene was cloned in a 0.43-kb NlaIII-SalI fragment and a 1.9-kb SalI-EcoRI fragment. The 449-aa sequence for the L. brevis Xi shows homology with Xis isolated from other bacteria, especially within the primary catalytic domains of the enzyme.