We found that cells of Vibrio parahaemolyticus possess an energy-dependent efflux system for norfloxacin. We cloned a gene for a putative norfloxacin efflux protein from the chromosomal DNA ofV. parahaemolyticus by using an Escherichia coli mutant lacking the major multidrug efflux system AcrAB as the host and sequenced the gene (norM). Cells of E. coli transformed with a plasmid carrying the norMgene showed elevated energy-dependent efflux of norfloxacin. The transformants showed elevated resistance not only to norfloxacin and ciprofloxacin but also to the structurally unrelated compounds ethidium, kanamycin, and streptomycin. These results suggest that this is a multidrug efflux system. The hydropathy pattern of the deduced amino acid sequence of NorM suggested the presence of 12 transmembrane domains. The deduced primary structure of NorM showed 57% identity and 88% similarity with that of a hypothetical E. coli membrane protein, YdhE. No reported drug efflux protein in the sequence databases showed significant sequence similarity with NorM. Thus, NorM seems to be a novel type of multidrug efflux protein. We cloned the ydhE gene from E. coli. Cells ofE. coli transformed with the cloned ydhE gene showed elevated resistance to norfloxacin, ciprofloxacin, acriflavine, and tetraphenylphosphonium ion, but not to ethidium, when MICs were measured. Thus, it seems that NorM and YdhE differ somehow in substrate specificity.

Two new genes (mexXY) similar to mexAB,mexCD, and mexEF and mediating multidrug resistance were cloned from the chromosome of Pseudomonas aeruginosa. Elevated ethidium extrusion was observed withEscherichia coli cells harboring the plasmid carryingmexXY. This MexXY system confers higher resistance to fluoroquinolones than the MexAB and MexCD systems, and E. coli TolC or P. aeruginosa OprM is necessary for the function of the MexXY system.

We detected chloramphenicol/H+ antiport activity in membrane vesicles of Escherichia coli and cloned a gene for the antiporter from chromosomal DNA of E. coli. Introduction of the gene into E. coli cells conferred resistance to chloramphenicol and ethidium. A slight increase in resistance to acridine orange was also observed. Elevated chloramphenicol efflux and ethidium efflux were observed in cells harboring a plasmid carrying the gene. Addition of chloramphenicol to the assay mixture reduced the efflux of ethidium. Elevated chloramphenicol/H+ antiport activity was observed in membrane vesicles prepared from cells harboring the plasmid. The pH optimum for the activity was 6.5. We sequenced the gene and deduced the amino acid sequence of its product. A sequence homology search revealed that it was same as that of Cmr (or MdfA). Thus, it became clear that Cmr (MdfA) is the chloramphenicol(and ethidium)/H+ antiporter.