IDENTIFICATION OF THE DNA-BINDING SITE FOR THE PHOSPHORYLATED VANR PROTEIN REQUIRED FOR VANCOMYCIN RESISTANCE IN ENTEROCOCCUS-FAECIUM

The vancomycin resistance operon of Enterococcus faecium encodes a two -component regulatory system comprising VanS and VanR. In vitro experi ments showed that about 5% of a labile phosphorylated VanR (P-VanR) wa s accumulated from ATP and a maltose-binding protein-VanS fusion prote in (MBP-VanS). Alternatively, about an 8% abundance of P-VanR was prod uced with acetyl phosphate. In such incubations gel shift experiments revealed that P-VanR selectively bound to a 254-bp DNA fragment that c ontains the vanH promoter for the vanH, vanA; and vanX structural gene s. 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 EC(50) of 40 nM, than the unphosphorylated VanR. A second DN A fragment of 197 bp containing the proposed vanR promoter for the van R and vanS regulatory genes also exhibited gel shift, but with much lo wer affinities. A mutant VanR(D53A) was shown to be incompetent for ph osphorylation by phosphorylated MBP-VanS or by acetyl phosphate; howev er, 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 van R promoter. The unphosphorylated VanR footprinted the same 80 bp on th e vanH promoter, but only 20 bp on the vanR promoter. The markedly inc reased affinity of P-VanR for the vanH promoter over the vanR promoter and the doubling in size of the protected footprint (80-bp vs 40-bp) are consistent with the oligomerization of P-VanR on the vanH promoter . Analysis of the footprinted regions revealed a 12-bp sequence presen t in one copy in the vanR promoter and in two copies in the vanH promo ter; which may serve as the consensus recognition site for P-VanR bind ing, transcriptional activation, and expression of vancomycin resistan ce.