Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon.

Multidrug resistance in bacteria is generally attributed to the acquisition of multiple transposons and plasmids bearing genetic determinants for different mechanisms of resistance (48, 62). However, descriptions of intrinsic mechanisms that confer multidrug resistance have begun to emerge. The first of these was a chromosomally encoded multiple antibiotic resistance (mar) locus (Fig. 1) in Escherichia coli (45, 46). Mar mutants of E. coli arose at a frequency of 10 to 10 and were selected by growth on subinhibitory levels of tetracycline or chloramphenicol (45, 46). These mutants exhibited resistance to tetracyclines, chloramphenicol, penicillins, cephalosporins, puromycin, nalidixic acid, and rifampin (45). Later, the resistance phenotype was extended to include fluoroquinolones (25, 105), oxidative stress agents (7, 51), and, more recently, organic solvents (8, 49, 144). The expression of the Mar phenotype is greater at 30°C than 37°C (45, 127). Continued growth in the same or higher antibiotic concentrations led to increased levels of resistance, thus demonstrating an amplifiable multiple antibiotic resistance phenotype (45). Both highand low-level resistances were decreased or completely reversed by a Tn5 insertion into a single locus at 34 min (1,636.7 kb) on the E. coli chromosome, called the mar locus (46). The genetic basis for high-level resistance is only partially attributed to the mar locus, since transduction of the locus from highor low-level mar mutants produced only a low level of multidrug resistance (94). The mar locus consists of two divergently positioned transcriptional units that flank the operator marO (Fig. 1) in E. coli (22, 24, 112) and Salmonella typhimurium (133). One operon encodes MarC, a putative integral inner membrane protein (Fig. 1) without any yet apparent function, but which appears to contribute to the Mar phenotype in some strains (see below) (49, 143, 144). The other operon comprises marRAB, encoding the Mar repressor (MarR), which binds marO and negatively regulates expression of marRAB (22, 90, 127), an activator (MarA), which controls expression of other genes on the chromosome, e.g., the mar regulon (22, 41, 126), and a putative small protein (MarB) of unknown function (Fig. 1). The marRAB operon responds to a variety of compounds (7, 24, 52, 54, 99, 119, 127) including tetracycline and chloramphenicol (54). Deletion or inactivation of the marRAB operon results in increased susceptibility to multiple antibiotics, a variety of oxidative stress agents, and organic solvents (22, 24, 46, 51, 54, 144). Since salicylate and acetylsalicylate induced a reversible “phenotypic antibiotic resistance” to multiple antibiotics (118), a connection was made between phenotypic antibiotic resistance and mar (24); salicylate induced expression of marRAB (24). These studies also extended the spectrum of inducers to include acetaminophen, sodium benzoate, 2,4-dinitrophenol (an uncoupling agent), and cinnamate (a salicylate precursor in plants) (24). The uncoupling agent carbonyl cyanide m-chlorophenylhydrazone and redox-cycling compounds, menadione and plumbagin, are also inducers of marRAB transcription (127). Initially it was thought that 7.8 kb of chromosomal DNA was needed to generate constitutive mar mutants (mar) in a strain bearing a large (39 kb) chromosomal deletion (54). However, this finding was shown to be strain specific, and ;1.1 kb of mar sequence, containing marO, marR, and marA sequences, was sufficient to select a mar mutant (91, 134). mar mediates tetracycline resistance through an energy-dependent efflux system (45). Mar mutants are resistant to fluoroquinolones through a combined decrease in cell influx, e.g., a decrease in the porin OmpF, and an intrinsic efflux system (25). Chloramphenicol resistance in Mar mutants is also attributed to active efflux, which is enhanced over an intrinsic efflux system (96). The Mar phenotype is linked to overexpression of the acrAB locus; deletion of acrAB confers increased susceptibility to multiple drugs (85, 106) and organic solvents (144) in wild-type or Mar strains (85, 106, 144). These findings suggest that the acrAB efflux system is a major mechanism of Mar-mediated resistance (106). However, since the tetracycline (45) and fluoroquinolone (26) efflux systems were only saturated by the respective drug and not by others, it is probable that other drug-specific efflux systems are involved, particularly in high-level multidrug-resistant mutants.