MOLECULAR-GENETICS OF A RECEPTOR PROTEIN FOR D-XYLOSE, ENCODED BY THEGENE XYLF IN ESCHERICHIA-COLI

Mutants of Escherichia coli K12 have been isolated containing either t he 'XylF' or the 'XylE' transport systems for D-xylose. This enabled t he XylF system containing a D-xylose receptor-binding protein to be as sociated with high-affinity transport, apparent K-m 0.2-4 mu M, while the XylE sugar-H+ symporter system exhibited relatively low affinity, apparent K-m 63-169 mu M. Their apparent V-max values were similar. Is olation of a xylose transport-negative mutation in a downstream gene, xylG, and a series of transduction experiments enabled the gene order xylB xylA p/o xyl(FG) mtlA to be proposed near 80 min on the E. coli c hromosome, The xylose receptor protein (XylF) was highly purified from the periplasm and the sequence of its N-terminal 44 amino acids deter mined, From this a mixed oligonucleotide (14 mer) was synthesised of c orresponding DNA sequence, hybridisation of which to restriction fragm ents from the ColE I hybrid plasmid, pLC32-9, which conferred xylose t ransport activity on a xylE(-)xylF(-) double mutant, enabled precise l ocation of the xylF gene next to the xylose isomerase gene, xylA (3984 kbp on the Kohara physical map). The DNA sequence of the 2.5 kb Bgl I I fragment containing the xylF gene was then determined, which confirm ed the above gene order and revealed two divergent operons, probably r egulated in a co-ordinate manner, one containing enzymes for catabolis m of D-xylose, and the other proteins comprising the XylFG receptor-tr ansport system. Analysis of the 330 amino acid sequence of the XylF re ceptor protein located the scission point of its leader peptide betwee n Ala(23) and Lys(24), and revealed significant homologies with the ot her sugar receptor proteins for ribose (RbsB), galactose (MglB) and ar abinose (AraF).