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ZntR is an autoregulatory protein and negatively regulates the chromosomal zinc resistance operon znt of Staphylococcus aureus
Author(s) -
Singh Vineet K.,
Xiong Anming,
Usgaard Thomas R.,
Chakrabarti Swarup,
Deora Rajendar,
Misra Tapan K.,
Jayaswal Radheshyam K.
Publication year - 1999
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1999.01466.x
Subject(s) - operon , biology , dna footprinting , transcription (linguistics) , microbiology and biotechnology , rna polymerase , repressor , promoter , footprinting , gene , lac operon , escherichia coli , transcription factor , genetics , gene expression , linguistics , philosophy
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 Zn 2+ and Co 2+ , whereas the zntR gene encodes a putative regulatory protein that controls the expression of the znt operon. The zntR gene was amplified using the polymerase chain reaction, cloned into Escherichia coli for overexpression as His‐tagged ZntR and purified by Ni 2+ ‐affinity column. His‐tag‐free ZntR was purified to near homogeneity after digestion with enterokinase. Electrophoretic mobility shift assays (EMSAs) indicated that the ZntR bound to a fragment of DNA corresponding to the chromosomal znt promoter region with an affinity of about 8.0 × 10 −12 M. The addition of 25 μM Zn 2+ or Co 2+ in the binding reaction completely or significantly inhibited association of ZntR with the znt promoter. DNase I footprinting assays identified a ZntR binding site encompassing 49 nucleotides in the znt promoter region that contained repeated TGAA sequences. These sequences have been proposed to be the binding sites for SmtB, a metallorepressor protein from the cyanobacterium Synechococcus , to its corresponding operator/promoter. In vitro transcription assays, using S. aureus RNA polymerase, revealed that ZntR represses transcription 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 znt promoter region and regulates its own transcription together with that of zntA .