Characterization of MarR superrepressor mutants

Abstract

MarR negatively regulates expression of the multiple antibiotic resistance (mar) locus in Escherichia coli. Superrepressor mutants, generated in order to study regions of MarR required for function, exhibited altered inducer recognition properties in whole cells and increased DNA binding to marO in vitro. Mutations occurred in three areas of the relatively small MarR protein (144 amino acids). It is surmised that superrepression results from increased DNA binding activities of these mutant proteins.

FIG. 1

(A) Sequence of Pmar_II-marO. The locations of the −35 and −10 hexamer sequences and MarR ribosome binding site are indicated, and the SspI restriction enzyme recognition sequence in site I is in boldface. (B) Map of plasmid pSup-Test. Expression of ccdB is positively controlled by the marRAB promoter (Pmar_II) and negatively regulated by the lac (lacO) and marRAB (marO) operators in the presence of their cognate proteins, LacI and MarR. The positions of the SspI sites within the plasmid are indicated.

FIG. 2

Locations of the MarR superrepressor mutations identified in this study. The designations in parentheses consist of the single-letter code for the wild-type residue followed by the location of that amino acid in the full-length MarR and the single-letter code for the mutation isolated at this point. The numbers in parentheses represent the number of independent isolates at that site. The cross-hatched box indicates the region of amino acid homology among the MarR family members.

FIG. 3

MarR repressor activity assayed in the Pmar_II::lacZ reporter strain E. coli SPC105 (marR⁺). Cells were grown at 37°C to mid-logarithmic phase in LB broth, without glucose, containing the appropriate antibiotics and IPTG (50 μM) and sodium salicylate (5 mM) where appropriate. β-Galactosidase assays were performed with cells permeabilized with chloroform-SDS as previously described (17, 20). The relative β-galactosidase activities (±standard deviations) in the absence (open bars) or presence (hatched bars) of 5 mM sodium salicylate are presented. Activities were determined for the host strain alone (None), the wild-type MarR (WT), and other MarR proteins that show a superrepressor phenotype (mutations are indicated below each bar).