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 xylAB 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 leader 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 [(14)C]palmitate. The induced E. coli avidly bound d-[(14)C]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, we propose that the sequence signature of binding proteins specific for hexoses and pentoses be refined as (KDQ)(LIVFAG)(3)IX(3)(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 xylF transcription initiates from two +1 sites, both situated within the xylAB 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.