Overexpression of the MarA positive regulator is sufficient to confer multiple antibiotic resistance in Escherichia coli

Abstract

A genetic approach was undertaken to identify normal bacterial genes whose products function to limit the effective concentration of antibiotics. In this approach, a multicopy plasmid library containing cloned Escherichia coli chromosomal sequences was screened for transformants that showed increased resistance to a number of unrelated antibiotics. Three such plasmids were identified, and all contained sequences originating from the mar locus. DNA sequence analysis of the minimal complementation unit revealed that the resistance phenotype was associated with the presence of the marA gene on the plasmids. The putative marA gene product is predicted to contain a helix-turn-helix DNA binding domain that is very similar to analogous domains found in three other E. coli proteins. One such similarity was to the SoxS gene product, the elevated expression of which has previously been associated with the multiple antibiotic resistance (Mar) phenotype. Constitutive expression of marA conferred antibiotic resistance even in cells carrying a deletion of the chromosomal mar locus. We have also found that transformants bearing marA plasmids show a significant reduction in ompF translation but not transcription, similar to previously described mar mutants. However, this reduction in ompF expression plays only a minor role in the resistance mechanism, suggesting that functions encoded by genes unlinked to mar must be affected by marA. These results suggest that activation of marA is the ultimate event that occurs at the mar locus during the process that results in multiple antibiotic resistance.

Identification and analysis of aarP, a transcriptional activator of the 2'-N-acetyltransferase in Providencia stuartii

“…MarA (8,16), and SoxS (1,38). Like the MarA and SoxS proteins, AarP is small in size, 15.9 kDa, relative to other members of the AraC/XyIS family and is missing the domain proposed to bind small molecules.…”

“…2) abolished the ability of this plasmid to activate aac(2Ј)-Ia expression (data not shown). Protein comparisons of the deduced product of this open reading frame, designated AarP, indicated extensive homology to members of the AraC/ XyIS family of transcriptional activators and exhibited 52% amino acid identity to TetD (32), 48% identity to Rob (36), 44% identity to MarA (8,16) (Fig. 3), and 42% identity to SoxS (1,38) (Fig.…”

“…The chromosomal AmpC ␤-lactamase is under the control of the activator AmpR (23a), and genes involved in multiple antibiotic resistance in Escherichia coli are subject to activation by the MarA (8,16) and SoxS (7,26) proteins. To determine if aac(2Ј)-Ia is also subject to regulation by a positive activator(s), we have initiated a search for plasmids containing random fragments of the P. stuartii chromosome which confer increased aac(2Ј)-Ia expression.…”

Genetic definition of the Escherichia coli zwf "soxbox," the DNA binding site for SoxS-mediated induction of glucose 6-phosphate dehydrogenase in response to superoxide

“…Thus, it is highly likely that the putative DNA binding sites recognized by the respective proteins will be found to have nearly identical sequences. Moreover, if all four proteins prove to be transcriptional activators (the function of TetD is unknown [35], Rob was isolated on the basis of its binding to the right border of the E. coli replication origin [37], and MarA is the positive regulator of multiple antibiotic resistance [14]), there should be a considerable amount of cross talk among them. Indeed, 2,4-dinitrophenol, which is an effector of the mar operon (7), has been known for many years to induce glucose 6-phosphate dehydrogenase expression (32), and the soxQ1 mutation, which was initially isolated on the basis of conferring resistance to menadione (16), a redox-cycling compound, is a regulatory mutation of the marAB operon (2) that leads to transcriptional activation of some (e.g., zwf) but not all (e.g., nfo) member genes of the SoxRS regulon (16).…”

“…Indeed, E. coli strains deleted for zwf are hypersensitive to oxidative stress, thus making zwf a pivotal member of the soxRS regulon (18, 21). In addition, treatment of cells with several antibiotics and aromatic weak acids appears to activate zwf transcription through the action of marA, thus making zwf a member of the multiple antibiotic resistance (mar) regulon (2,6,7,14,16).…”