Our previous results have demonstrated that when added exogenously, DHB and ANT activate the marRAB promoter in vivo. To better correlate the metabolite effector hypothesis of Rosner and Martin to these observations, we disrupted the enzymatic steps in enterobactin and tryptophan metabolism that block either the synthesis or utilization of DHB or ANT. Specifically, mutants lacking the enterobactin synthesis pathway (ΔentCEBAH) or deficient in 2,3-dihydro-2,3-dihydroxybenzoate (DDHB) dehydrogenase (entA), enterobactin synthase (entF), anthranilate synthase (trpE), and phoshoribosyl transferase (the C-terminal region of trpD) activities were constructed in otherwise wild-type and tolC mutant strains and tested for alterations in marRAB promoter activity.
In TolC+ backgrounds, we observed no significant changes in marRAB promoter activity in any of the mutants, presumably due to the ability of these cells to readily excrete accumulated intermediates (data not shown). However, when we repeated these experiments in the absence of TolC, we found that the marRAB promoter was less active in mutants unable to synthesize enterobactin from chorismate (1,559 ± 170 relative fluorescence units per optical density unit [RFU/OD] [ΔtolC mutant] versus 1,387 ± 195 RFU/OD [ΔtolC ΔentCEBAH mutant], P = 0.001). However, when we attempted to accumulate the DHB in the cell by blocking conversion of DHB to enterobactin, we observed a decrease in marRAB promoter activity as opposed to an expected increase (1,559 ± 170 RFU/OD [ΔtolC mutant] versus 1,491 ± 78 RFU/OD [ΔtolC ΔentF mutant], P = 0.04). While statistically significant, the effect is minor and likely not physiologically significant. We suspect that this mutant (ΔtolC ΔentF) does not accumulate significant amounts of DHB. Interestingly, we observed a significant increase in marRAB promoter activity when we blocked the conversion of DDHB to DHB (1,559 ± 170 RFU/OD [ΔtolC mutant] versus 1,737 ± 133 RFU/OD [ΔtolC ΔentA mutant], P = 0.0001). Given the similar chemical structures of DDHB and DHB, differing only by a hydrated 2,3 carbon-carbon bond on the benzyl ring, we suspect that DDHB may accumulate in this mutant and serve as an alternate activator of MarR. Collectively, these results suggest that enterobactin intermediates are physiological activators of the marRAB operon.
We also investigated the effects of tryptophan biosynthesis on marRAB expression in the absence of TolC. We found that mutants unable to convert ANT to tryptophan did not exhibit any significant changes in marRAB promoter activity (data not shown). Interestingly, in the absence of anthranilate synthase, and therefore the ability to synthesize ANT, we observed a statistically significant increase in the marRAB promoter activity (1,516 ± 68 RFU/OD [ΔtolC mutant] versus 1,702 ± 173 RFU/OD [ΔtolC ΔtrpE mutant], P < 0.00002). We suspect that blocking the initial step of tryptophan biosynthesis may redirect metabolic fluxes to other pathways where the metabolite intermediates induce marRAB expression. Taken together, these results indicate that disruption of tryptophan biosynthesis affects marRAB expression, although they suggest that tryptophan intermediates do not contribute directly to the tolC phenotype. Moreover, the effect is not direct nor is the mechanism clear. We were not entirely surprised by this result, as we found that ANT does not directly bind MarR and affect its activity. Rather, the effect appears to be indirect.