THE ACCUMULATION OF GLUTAMATE IS NECESSARY FOR OPTIMAL-GROWTH OF SALMONELLA-TYPHIMURIUM IN MEDIA OF HIGH-OSMOLALITY BUT NOT INDUCTION OF THE PROU OPERON

Synthesis of glutamate can be limited in bacterial strains carrying mu tations to loss of function of glutamate synthase (2-oxoglutarate:glut amine aminotransferase) by using low concentrations of NH4+ in the gro wth medium. By using such gltB/D mutant strains of Salmonella typhimur ium, we demonstrated that: (i) a large glutamate pool, previously obse rved to correlate with growth at high external osmolality, is actually required for optimal growth under these conditions; (ii) the osmoprot ectant glycine betaine (N,N,N-trimethylglycine) apparently cannot subs titute for glutamate; and (iii) accumulation of glutamate is not neces sary for high levels of induction of the proU operon in vivo. Expressi on of the proU operon, which encodes a transport system for the osmopr otectants proline and glycine betaine, is induced >100-fold in the wil d-type strain under conditions of high external osmolality. Ramirez et al. (R. M. Ramirez, W. S. Prince, E. Bremer, and M. Villarejo, Proc. Natl. Acad. Sci. USA 86:1153-1157, 1989) observed and we confirmed tha t in vitro expression of the lacZ gene from the wild-type proU promote r is stimulated by 0.2 to 0.3 M K glutamate. However, we observed a ve ry similar stimulation for lacZ expressed from the lacUV5 promoter and from the proU promoter when an important negative regulatory element downstream of this promoter (the silencer) was deleted. Since the lacU V5 promoter is not osmotically regulated in vivo and osmotic regulatio n of the proU promoter is largely lost as a result of deletion of the silencer, we conclude that stimulation of proU expression by K glutama te in vitro is not a specific osmoregulatory response but probably a m anifestation of the optimization of in vitro transcription-translation at high concentrations of this solute. Our in vitro and in vivo resul ts demonstrate that glutamate is not an obligatory component of the tr anscriptional regulation of the proU operon.