A C-terminal 18 amino acid deletion in MarR in a clinical isolate of Escherichia coli reduces MarR binding properties and increases the MIC of ciprofloxacin.
As described recently, the different degree of fluoroquinolone resistance in a pair of sequential clinical isolates of Escherichia coli was due to the increased expression of the regulatory gene marA as a consequence of an 18 amino acid C-terminal deletion in the repressor MarR (MarR Delta). To further investigate the molecular mechanism of the loss of repressor function, we purified recombinant wild-type and mutated MarR, and tested their respective ability to form dimers and their specific DNA binding properties to the operator region marO. The dimerization capacity was analysed by non-reducing SDS-PAGE and by disuccinimidyl suberate-mediated cross-linking of the recombinant proteins. The binding of MarR was studied using the recombinant proteins and DNA probes containing the two identified binding sites in marO in the presence or absence of specific and non-specific DNA fragments. Dimerization of MarR Delta was reduced compared with MarR: the dimer portion was 33.8% (MarR) and 12.4% (MarR Delta) at a protein concentration of 10 IM. In mobility-shift assays MarR Delta showed a highly reduced complex formation. Footprinting analysis confirmed reduced binding of MarR Delta to its target sites, compared with MarR. The biochemical data are in full agreement with the crystal structure of MarR, which shows that the N- and C-terminal regions of MarR contribute to dimer formation. The data also indicate a major role of the MarR dimer as opposed to the monomer in DNA binding.
Notka
Frank
F
Institute of Medical Microbiology, University of Regensburg, Franz-Josef-Strauss Allee 11, D-93053 Regensburg, Germany.
Linde
Hans-Jörg
HJ
Dankesreiter
Arnd
A
Niller
Hans-Helmut
HH
Lehn
Norbert
N
D000595:Amino Acid Sequence / Q000235:genetics*; D000890:Anti-Infective Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D002939:Ciprofloxacin / Q000494:pharmacology*; D019281:Dimerization; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification*; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D006801:Humans; D008826:Microbial Sensitivity Tests; D010446:Peptide Fragments / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*
D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C080004:MarR protein, E coli; D010446:Peptide Fragments; D012097:Repressor Proteins; D002939:Ciprofloxacin