Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein

Abstract

Multiple antibiotic resistance in Escherichia coli can be mediated by induction of the SoxS or MarA protein, triggered by oxygen radicals (in the soxRS regulon) or certain antibiotics (in the marRAB regulon), respectively. These small proteins (SoxS, 107 residues; MarA, 127 residues) are homologous to the C terminus of the XylS-AraC family of proteins and are more closely related to a ϳ100-residue segment in the N terminus of Rob protein, which binds the right arm of the replication origin, oriC. We investigated whether the SoxS-MarA homology in Rob might extend to the regulation of some of the same inducible genes. Overexpression of Rob indeed conferred multiple antibiotic resistance similar to that known for SoxS and MarA (against chloramphenicol, tetracycline, nalidixic acid, and puromycin), as well as resistance to the superoxide-generating compound phenazine methosulfate. The Rob-induced antibiotic resistance depended only partially on the micF antisense RNA that down-regulates the OmpF outer membrane porin to limit antibiotic uptake. Similar antibiotic resistance was conferred by expression 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 Rob protein bound a DNA fragment containing the micF promoter (50% bound at ϳ10 ؊9 M Rob) as strongly as it did oriC, and it bound more weakly to DNA containing the sodA, nfo, or zwf promoter (50% bound at 10 ؊8 to 10 ؊7 M). Rob formed multiple DNA-protein complexes with these fragments, as seen previously for SoxS. These data point to a DNA-binding gene activator module used in different protein contexts.

Characterization of a cationic surfactant-resistant mutant isolated spontaneously from Escherichia coli

“…It is known that permeability to antimicrobial agents, especially to small hydrophilic agents, through the outer membrane in E. coli greatly depends on the contents of OmpF and OmpC porin proteins, and that OmpF deficiency makes cells resistant to some antibiotics (Harder et al 1981;Cohen et al 1989;Ariza et al 1995;Thanassi et al 1995). The finding that the content of OmpF in strain OW66 was lower than that of strain OW6, even in the absence of CTAB, strongly suggests dependency of the resistance of the mutant on the OmpF level in the outer membrane.…”

MarA, SoxS and Rob function as virulence factors in an Escherichia coli murine model of ascending pyelonephritis

“…Experiments with Rob, a MarA and SoxS paralogue, showed that it could function in a similar manner (Ariza et al, 1995). Subsequent data have documented multidrug-resistant clinical strains of E. coli (Linde et al, 2000;Maneewannakul & Levy, 1996) and Salmonella enterica serovar Typhimurium (S. typhimurium) (Koutsolioutsou et al, 2001) that constitutively express AraC family members.…”

A comprehensive library of DNA-binding site matrices for 55 proteins applied to the complete Escherichia coli K-12 genome 1 1Edited by R. Ebright

“…For example, the E. coli proteins MarA, Rob and SoxS all target genes involved in the oxidative stress response and bind to closely related DNA sequences. However, some targets respond differently from these proteins (Ariza et al, 1995;Jair et al, 1995;Li & Demple, 1996).…”