Chromosomal multidrug resistance (MDR) in bacteria is a serious clinical problem. Our studies have shown that Escherichia coli becomes resistant to a variety of antibiotics, organic solvents and superoxides when the activities of any of three paralogous, but differently regulated, transcriptional activators, MarA, SoxS and Rob, are increased. These activators bind a sequence called the marbox which lies upstream of the promoters of a set of at least 40 chromosomal genes called the marA/soxS/rob regulon. The expression of three proteins upregulated by MarA, SoxS and Rob is particularly important for MDR in E. coli. 1) TolC, encodes an outer membrane protein that interacts with 2) AcrB, one of nine different inner membrane efflux pumps and 3) AcrA, a cognate periplasmic protein. This AcrAB-TolC tripartite complex forms a trans-periplasmic channel for extruding antibiotics directly out of the cell into the environment. We have recently shown that TolC and several TolC-dependent pumps reduce cellular stress in E. coli, presumably by expelling toxic cellular metabolites. We would like to identify these natural substrates so that we can design chemical analogs that would behave as Efflux Pump Inhibitors that render the pumps ineffective for antibiotic resistance: either by overwhelming their capacity for efflux or by inhibiting the pumps. In an effort to identify these metabolites, we have initiated metabolomic studies (with K. van de Wetering, Amsterdam). Our strategy is to look for compounds that accumulate inside the cells of the mutants but are deficient in the growth medium of the mutant because they cannot be exported from the cell. Thus, wild-type and 3 different, but related, efflux mutants were grown under standardized conditions on 5 successive days and their pellets were separated from the medium by centrifugation. These 40 samples (4 strains, pellets and supernatants, 5 preparations of each) were analyzed by liquid chromatography/mass spectroscopy (LC/MS) in Amsterdam. Analysis of the data shows that 26 different molecular weight species were present in greater amounts (>3-fold) in the pellets of all three mutants compared to the wild-type (p<0.05). In addition, 35 species were present in greater amounts (>3-fold) in the supernatants of the wild-type. These data suggest that the LC/MS method is potentially very useful for finding natural substrates of the pumps. The on-going effort is to identify these species by tandem mass spectrometry (MS/MS) and other techniques.