The periplasmic protein MppA is not involved in regulation of marA in Escherichia coli.

The marRAB operon of Escherichia coli is self-repressed by MarR, while the MarA protein activates a number of promoters, including those for the AcrAB/TolC multidrug efflux pump (1, 4, 5). Inactivation of marR leads to a “Mar” multipleantibiotic resistance phenotype. In 1999, the mppA gene was reported to be a novel factor involved in the regulation of the marRAB operon (3). Strain TP985 had been created by introducing mppA::Tn10Cm into E. coli strain AT980 by P1 transduction (6). AT980 was multidrug susceptible, but TP985 was multidrug resistant in a marAdependent manner. It was initially proposed that mppA, the gene for a periplasmic murein tripeptide binding protein involved in transport, helped repress marRAB in AT980 (3). However, the multidrug resistance of TP985 was later explained by the inadvertent acquisition of an inactivating marR c18 mutation by that strain (2). Complementation with plasmid pTrc99A bearing the mppA gene (pMLD1285) was reported to cause loss of the resistance (3). This effect of pMLD1285 did not occur in a strain reconstructed to be like TP985, namely, strain AT980 marR c18 mppA::Tn10Cm (2). Therefore, a second (unidentified) locus, “X,” altered only in TP985, appeared to help mppA repress marRAB (2). In the present work, all strains were cultured at 37°C in LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter) supplemented with 50 g diaminopimelic acid per ml (for dapD2). The genotypes and phenotypes of the strains and the plasmid used in this study are shown in Table 1. We found that the multidrug resistance of TP985 freshly transformed with pMLD1285 (carrying the mppA gene) was not suppressed, even when mppA transcription was induced with 300 M isopropyl-D-thiogalactopyranoside (IPTG). This was contrary to the previous work, where pMLD1285 was reported to cause a decrease in resistance in TP985 (3). Moreover, clones of mppA in pJP105 (7) or in pACYC177 also failed to suppress resistance (Xiaowen Bina, unpublished data). Therefore, we sequenced the marR locus of strain TP985 and the original TP985/pMLD1285 transformant construct obtained from our laboratory collection, as well as from the original stocks of J. T. Park. All of the strains had been stored at 70°C. We found that TP985 indeed had the expected inactivating marR c18 mutation. However, surprisingly, the original TP985/pMLD1285 strain no longer had the marR c18 mutation but rather was wild type for marR (and marA) (Table 1). That the chromosomal marR gene of the original TP985/ pMLD1285 strain had reverted to the wild type explains completely the loss of multidrug resistance in that strain. No role for the plasmid-borne mppA gene need be presumed. We do not know how this restoration of marR to the wild type occurred. Strain TP985/pMLD1285 was indeed derived from TP985 since, like TP985, it required diaminopimelic acid, had mppA::Tn10Cm, and was lysogenic for bacteriophage phi80 (as was TP985 but not AT980; unpublished data). In conclusion, the proposal that mppA contributes to repression of the marRAB operon (3) is no longer plausible.