marA locus causes decreased expression of OmpF porin in multiple-antibiotic-resistant (Mar) mutants of Escherichia coli

Abstract

Mar (multiple antibiotic resistant) mutants of Escherichia coli express chromosomally mediated resistance to a variety of structurally unrelated hydrophilic and hydrophobic antibiotics. Insertion of transposon Tn5 into the marA locus at min 34.05 on the chromosome completely reverses the Mar phenotype (A. M. George and S. B. Levy, J. Bacteriol. 155:531-540, 1983). We found that among changes in the outer membrane of Mar mutants, porin OmpF was greatly reduced, although Mar mutants were more resistant than cells lacking only OmpF. Transduction of the marA region from a Mar strain, but not a wild-type strain, led to loss of OmpF. P1 transduction of marA::Tn5 into a Mar mutant partially restored OmpF levels. Therefore, OmpF reduction required a mutation in the marA region. Mar mutants of an ompF-lacZ operon fusion strain expressed 50 to 75% of the beta-galactosidase activity of the isogenic non-Mar parental strain, while Mar mutants of a protein fusion strain expressed less than 10% of the enzyme activity in the non-Mar strain. These changes were completely reversed by insertion of marA::Tn5. The responsiveness of OmpF-LacZ to osmolarity and temperature changes was similar in Mar and wild-type strains. Although some transcriptional control may have been present, OmpF reduction appeared to occur primarily by a posttranscriptional mechanism. The steady-state levels of ompF mRNA were twofold lower and the mRNA was five times less stable in the Mar mutant than in the wild-type strain. Expression of micF, which lowers ompF mRNA levels, was elevated in Mar strains, as revealed by a micF-lacZ fusion. Studies with strains deleted for the micF locus showed that the marA-dependent reduction of OmpF required an intact micF locus. Our findings suggest that the marA locus directly or indirectly increases micF expression, causing a posttranscriptional decrease in ompF mRNA and reduced amounts of OmpF.

Genotype-by-Environment Interactions Influencing the Emergence of rpoS Mutations in Escherichia coli Populations

“…Resistance to antibiotics is not a known role for RpoS so the argument above-that mutations are less selected when RpoS is needed in stress resistance-does not hold readily. However, previous studies have shown that the major outer membrane porin through which chloramphenicol transverses in E. coli is OmpF (Harder et al 1981;Cohen et al 1988Cohen et al , 1989. RpoS negatively influences ompF transcription (Liu and Ferenci 2001); therefore loss of RpoS would be a disadvantage as high outer membrane permeability to antibiotics would be a liability and hence provide an indirect F sp contribution.…”

Repressor mutations in the marRAB operon that activate oxidative stress genes and multiple antibiotic resistance in Escherichia coli

“…Antibiotic resistance in these cases depends in part on the elevated synthesis of micF RNA (5,8), an antisense transcript that destabilizes the ompF mRNA (2) encoding a major porin of the outer membrane (25). Several oxidative stress genes are also transcriptionally activated by both the mar and the soxRS systems: sodA and zwf, as noted above, as well as the soil71l9 locus (21) and the genes encoding two other, unidentified oxidative stress proteins (14,15).…”

Biological characterization of an Enterobacter cloacae outer membrane protein (OmpX)

“…Exponentially growing cultures in Luria broth (22) without NaCl were harvested at an A565 of 0.8 to 1.0. Total RNA was isolated by the method of Cohen et al (9). A 20-,ug sample of total RNA was denatured with formaldehyde and formamide and subjected to electrophoresis in 1.2% agarose gels containing 2.2 M formaldehyde (22).…”