The naturally occurring 2-hydroxy-4,7-dimethoxybenzoxazin-3-one (HDMBOA), essentially the sole component of maize seedling organic exudate, was shown to be a potent inhibitor of the VirA-VirG two-component system which mediates host recognition and activates virulence gene transcription in the soil pathogen Agrobacterium tumefaciens. The hydrolytic lability of HDMBOA creates a steady-state zone of inhibition circumscribing the young maize seedling. We now show that rather than the HDMBOA natural product, an o-imidoquinone decomposition intermediate, (3Z)-2,2-dihydroxy-N-(4-methoxy-6-oxocyclohexa-2,4-dienylidene)acetamide, can function as an inhibitor of virulence gene expression in A. tumefaciens. Structural characterization of this o-imidoquinone intermediate clarifies several issues related to the decomposition pathways available to this class of antibiotics. Of direct ecological importance, this species is produced rapidly and quantitatively within the more neutral pH ranges of the A. tumefaciens cytoplasm, while HDMBOA is more persistent at the slightly acidic pH common to many soils. These results suggest the rather intriguing possibility that the physical instability of the benzoxazinone antibiotics may not only create a steady-state local defense, but also enable a "pro-drug" strategy directed against bacterial environmental sensing.
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