| Popis: |
Mutants of Escherichia coli K12 have been isolated containing either the 'XylF' or the 'XylE' transport systems for D-xylose. This enabled the XylF system containing a D-xylose receptor-binding protein to be associated with high-affinity transport, apparent Km 0.2-4 microM, while the XylE sugar-H+ symporter system exhibited relatively low affinity, apparent Km 63-169 microM. Their apparent Vmax values were similar. Isolation 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 chromosome. The xylose receptor protein (XylF) was highly purified from the periplasm and the sequence of its N-terminal 44 amino acids determined. From this a mixed oligonucleotide (14 mer) was synthesised of corresponding DNA sequence, hybridisation of which to restriction fragments from the ColE I hybrid plasmid, pLC32-9, which conferred xylose transport activity on a xylE- xylF- double mutant, enabled precise location 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 II fragment containing the xylF gene was then determined, which confirmed the above gene order and revealed two divergent operons, probably regulated in a co-ordinate manner, one containing enzymes for catabolism of D-xylose, and the other proteins comprising the XylFG receptor-transport system. Analysis of the 330 amino acid sequence of the XylF receptor protein located the scission point of its leader peptide between Ala23 and Lys24, and revealed significant homologies with the other sugar receptor proteins for ribose (RbsB), galactose (MglB) and arabinose (AraF). |
