Mutations that render the promoter of the histidine operon of Salmonella typhimurium insensitive to nutrient-rich medium repression and amino acid downshift.
The effects of mutations in the promoter of the histidine operon of Salmonella typhimurium were examined in vivo. The wild-type chromosomal copy of the his promoter was replaced with mutations in the -10 hexamer sequence and in the region between the -10 hexamer and the transcriptional start point-termed the discriminator sequence. The substitutions were performed with a phage M13 allele replacement system. Expression of the his operon is known to correlate with levels of guanosine 5',3'-bispyrophosphate (ppGpp) in vivo. Strains containing either the wild-type his promoter or his promoter mutations were grown in both nutrient-rich and minimal media under steady-state conditions known to alter intracellular levels of ppGpp in a predictable way. The effect of the presence or absence of the his attenuator was assessed under these conditions as well. Expression of the his operon was studied by measuring the differential rate of beta-galactosidase synthesis with a his-lac transcriptional fusion. Regulation of the his operon in the promoter mutants was also studied under conditions of a transient amino acid downshift induced by the addition of serine hydroxamate to cultures growing in nutrient-rich medium. These growth conditions cause elevated levels of ppGpp. The results provide physiological confirmation of previous evidence obtained with a coupled transcription-translation system in vitro which indicated that ppGpp regulates interaction of RNA polymerase at the his promoter. More specifically, the in vivo evidence shows that the region of the his promoter that includes the -10 hexamer and discriminator sequences is the target at which ppGpp stimulates transcription.
Da Costa
X J
XJ
Section of Microbiology, University of California, Davis 95616, USA. xdacosta@warren.med.harvard.edu
Artz
S W
SW
D000596:Amino Acids / Q000378:metabolism*; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D006639:Histidine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009876:Operon; D011401:Promoter Regions, Genetic; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism; D012694:Serine / Q000031:analogs & derivatives / Q000494:pharmacology
D000596:Amino Acids; D003470:Culture Media; C028002:serine hydroxamate; D006159:Guanosine Tetraphosphate; D012694:Serine; D006639:Histidine