ZntR is an autoregulatory protein and negatively regulates the chromosomalzinc resistance operon znt of Staphylococcus aureus

A chromosomally encoded znt operon of Staphylococcus aureus consists of two consecutive putative genes designated zntR and zntA. The zntA gene encodes a transmembrane protein that facilitates extrusion of Zn2+ and Co2+, where as the zntR gene encodes a putative regulatory protein that controls the ex pression of the znt operon. The zntR gene was amplified using the polymeras e chain reaction, cloned into Escherichia coil for overexpression as His-ta gged ZntR and purified by Ni2+-affinity column. His-tag-free ZntR was purif ied to near homogeneity after digestion with enterokinase. Electrophoretic mobility shift assays (EMSAs) indicated that the ZntR bound to a fragment o f DNA corresponding to the chromosomal znt promoter region with an affinity of about 8.0 x 10(-12) M. Th, addition of 25 mu M Zn2+ or Co2+ in, th, bin ding reaction completely or significantly inhibited association of ZntR wit h the znt promoter. DNase I footprinting assays identified a ZntR binding s ite encompassing 49 nucleotides in the znt promoter region that contained r epeated TGAA sequences. These sequences have been proposed to be the bindin g sites for SmtB, a metallorepressor protein from the cyanobacterium Synech ococcus, to its corresponding operator/promoter. In vitro transcription ass ays, using S, aureus RNA polymerase, revealed that ZntR represses transcrip tion from the znt promoter in a concentration-dependent fashion. The EMSAs, DNase I footprinting and in vitro transcription assays indicate that ZntR is a trans-acting repressor protein that binds to the mt promoter region an d regulates its own transcription together with that of zntA.