Dissecting the VanRS signal transduction pathway with specific inhibitors

The VanRS two-component signal transduction pathway from Enterococcus faeci um was reconstituted in vitro from partially purified components and shown to be inhibited by the halophenyl isothiazolone LY-266,400, inhibitor A, a compound shown previously to reduce expression of the AlgR1-AlgR2 two-compo nent signal transduction pathway in Pseudomonas aeruginosa (S. Roychoudhury , N. A. Zielinski, A. J. Ninfa, N. E. Alien, L. N. Jungheim, T. I. Nicas, a nd A. M. Chakrabarty, Proc. Natl. Acad. Sci. USA 90:965-969, 1993). Inhibit or A attenuates phosphoryl transfer from VanS similar to P to VanR by its a ction on the ability of VanR to accept, We observed an apparent stimulatory effect of inhibitor A on VanS autophosphorylation which is attributable to the accumulation of VanS similar to P as an intermediate unable to transfe r P-i to the inhibited VanR, Thus, inhibitor A acts on the second of two se quential steps which lead to transcriptional activation of the VanHAXYZ gen e cluster and the resultant expression of vancomycin resistance.