ACTIVATION OF MULTIPLE ANTIBIOTIC-RESISTANCE AND BINDING OF STRESS-INDUCIBLE PROMOTERS BY ESCHERICHIA-COLI ROB PROTEIN

Multiple antibiotic resistance in Escherichia coli can be mediated by induction of the SoxS or MarA protein, triggered by oxygen radicals (i n the soxRS regulon) or certain antibiotics (in the marRAB regulon), r espectively. These small proteins (SoxS; 107 residues; MarA, 127 resid ues) are homologous to the C terminus of the XylS-AraC family of prote ins and are more closely related to a similar to 100-residue segment i n the N terminus of Rob protein, which binds the right arm of the repl ication origin, oriC. We investigated whether the SoxS-MarA homology i n Rob might extend to the regulation of some of the same inducible gen es. Overexpression of Rob indeed conferred multiple antibiotic resista nce similar to that known for SoxS and MarA (against chloramphenicol, tetracycline, nalidixic acid, and puromycin), as welt as resistance to the superoxide-generating compound phenazine methosulfate. The Rob in duced antibiotic resistance depended only partially on the micF antise nse RNA that down-regulates the OmpF outer membrane porin to limit ant ibiotic uptake. Similar antibiotic resistance was conferred by express ion of a Rob fragment containing only the N-terminal 123 residues that constitute the SoxS-MarA homology. Both intact Rob and the N-terminal fragment activated expression of stress genes (inaA, fumC, sodA) but with a pattern distinct from that found for SoxS and MarA. Purified Ro b protein bound a DNA fragment containing the micF promoter (50% bound at similar to 10(-9) M Rob) as strongly as it did oriC, and it bound more weakly to DNA containing the sodA, nfo, or zwf promoter (50% boun d at 10(-8) to 10(-7) M). Rob formed multiple DNA-protein complexes wi th these fragments, as seen previously for SoxS. These data point to a DNA-binding gene activator module used in different protein contexts.