USE OF FLUORESCENCE PROBES TO MONITOR FUNCTION OF THE SUBUNIT PROTEINS OF THE MEXA-MEXB-OPRM DRUG EXTRUSION MACHINERY IN PSEUDOMONAS-AERUGINOSA

The MexA-MexB-OprM efflux pump of Pseudomonas aeruginosa consists of t wo inner membrane proteins, MexA and MexB, and one outer membrane prot ein, OprM, We investigated the role of the components of this drug ext rusion system by evaluating the repercussions of deleting these subuni t components on the accumulation of several fluorescent probes, Fluore scence intensities of positively charged 2-(4-dimethylaminostyryl)-1-e thylpyridinium and uncharged N-phenyl-1-naphtylamine were 7 and 4 time s higher, respectively, in the mutant lacking OprM and 4 and 1.7 times higher, respectively, in the mutants lacking MexA or MexB than in the wild type strain, This order of fluorescence intensity was fully cons istent with a previously reported minimum inhibitory concentration of antibiotics such as tetracycline, chloramphenicol, and fluoroquinolone s, Ethidium bromide accumulation in all the Mex mutants proceeded at a bout 5 times faster than the rate in the wild type cells, This result is in accord with the minimum inhibitory concentration of beta-lactam antibiotics. These results suggest that the fluorescence probes could be successfully used in real time monitoring of the function of the dr ug extrusion machinery in Gram-negative bacteria, The downhill extrusi on kinetics of rimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, whic h orients perpendicular to the inner leaflet of the cytoplasmic membra ne, from preloaded cells lacking the extrusion pump was preceded by a slow increase in fluorescence intensity, whereas the wild type cell im mediately released the dye, This observation was explained by a slow t rans-cytoplasmic membrane crossing of intracellular dye in the mutants , These results reflected higher accumulation of the probe in the cyto plasmic membrane in the mutants and strengthened the hypothesis that e xtrusion of hydrophobic substrate mediated by MexA-MexB-OprM mainly ta kes place from the interior of the cytoplasmic membrane.