The D-xylose binding protein, XylF, from Thermoanaerobacter ethanolicus 39E:: Cloning, molecular analysis, and expression of the structural gene

Immediately downstream from the Thermoanaerobacter ethanolicus xyL4B operon, comprising genes that encode D-xylose isomerase and D-xylulose kinase, lies a 1,101-bp open reading frame that exhibits 61% amino acid sequence identity to the Escherichia coli D-xylose binding periplasmic receptor, XylF, a component of the high-affinity binding-protein-dependent D-xylose transport, The 25-residue N-terminal fragment of the deduced T. ethanolicus XylF has typical features of bacterial leader peptides, The C-terminal portion of this lender sequence matches the cleavage consensus for lipoproteins and is followed by a 22-residue putative linker sequence rich in serine, threonine, and asparagine. The putative mature 341-amino-acid-residue XylF (calculated molecular mass of 37,069 Da) appears to be a lipoprotein attached to the cell membrane via a lipid anchor covalently linked to the N-terminal cysteine, as demonstrated by metabolic labelling of the recombinant XylF with [C-14]palmitate. The induced E. coli avidly bound D-[C-14]xylose, yielding additional evidence that T. ethanolicus XylF is the D-xylose binding protein. On the basis of sequence comparison of XylFs to other monosaccharide-binding proteins,,ve propose that the sequence signature of binding proteins specific for hexoses and pentoses be refined as (KDQ) (LIVFAG)(3)IX3(DN) (SGP)X-3(GS)X(LIVA)(2)X(2)A. Transcription of the monocistronic 1,3-kb xylF mRNA is inducible by xylose and unaffected by glucose. Primer extension analysis indicated that xq IF transcription initiates from two +1 sites, both situated within the xyL4B operon, Unlike in similar transport systems in other bacteria, the genes specifying the membrane components (e.g., ATP-binding protein and permease) of the high-affinity D-xylose uptake system are not located in the vicinity of xylF in T. ethanolicus, This is the first report of a gene encoding a xylose-binding protein in a gram-positive or thermophilic bacterium.