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 mutations to loss of function of glutamate synthase (2-oxoglutarate:glutamine aminotransferase) by using low concentrations of NH4+ in the growth medium. By using such gltB/D mutant strains of Salmonella typhimurium, we demonstrated that: (i) a large glutamate pool, previously observed to correlate with growth at high external osmolality, is actually required for optimal growth under these conditions; (ii) the osmoprotectant glycine betaine (N,N,N-trimethylglycine) apparently cannot substitute for glutamate; and (iii) accumulation of glutamate is not necessary for high levels of induction of the proU operon in vivo. Expression of the proU operon, which encodes a transport system for the osmoprotectants proline and glycine betaine, is induced >100-fold in the wild-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 that in vitro expression of the lacZ gene from the wild-type proU promoter is stimulated by 0.2 to 0.3 M K glutamate. However, we observed a very 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 lacUV5 promoter is not osmotically regulated in vivo and osmotic regulation of the proU promoter is largely lost as a result of deletion of the silencer, we conclude that stimulation of proU expression by K glutamate in vitro is not a specific osmoregulatory response but probably a manifestation of the optimization of in vitro transcription-translation at high concentrations of this solute. Our in vitro and in vivo results demonstrate that glutamate is not an obligatory component of the transcriptional regulation of the proU operon.