Identification of the DNA-binding site for the phosphorylated VanR protein required for vancomycin resistance in Enterococcus faecium

Abstract

The vancomycin resistance operon of Enterococcus faecium encodes a two-component regulatory system comprising VanS and VanR. In vitro experiments showed that about 5% of a labile phosphorylated VanR (P-VanR) was accumulated from ATP and a maltose-binding protein-VanS fusion protein (MBP-VanS). Alternatively, about an 8% abundance of P-VanR was produced with acetyl phosphate. In such incubations, gel shift experiments revealed that P-VanR selectively bound to a 254-bp DNA fragment that contains the vanH promoter for the vanH, vanA, and vanX structural genes. When VanS was added with a mole ratio for VanS:VanR of higher than 1:1, VanS competed with DNA for P-VanR and abolished the gel shift. P-VanR bound 500-fold more tightly to the vanH promoter region, with an estimated EC50 of 40 nM, than the unphosphorylated VanR. A second DNA fragment of 197 bp containing the proposed vanR promoter for the vanR and vanS regulatory genes also exhibited gel shift, but with much lower affinities. A mutant VanR(D53A) was shown to be incompetent for phosphorylation by phosphorylated MBP-VanS or by acetyl phosphate; however, it still bound DNA specifically, albeit with low affinity. DNase I footprinting by P-VanR revealed that a ca. 80-bp region was protected on the vanH promoter and a ca. 40-bp region was protected on the vanR promoter. The unphosphorylated VanR footprinted the same 80 bp on the vanH promoter, but only 20 bp on the vanR promoter.(ABSTRACT TRUNCATED AT 250 WORDS)