Regulation of heme biosynthesis in Salmonella typhimurium: activity of glutamyl-tRNA reductase (HemA) is greatly elevated during heme limitation by a mechanism which increases abundance of the protein.
In Salmonella typhimurium and Escherichia coli, the hemA gene encodes the enzyme glutamyl-tRNA reductase, which catalyzes the first committed step in heme biosynthesis. We report that when heme limitation is imposed on cultures of S. typhimurium, glutamyl-tRNA reductase (HemA) enzyme activity is increased 10- to 25-fold. Heme limitation was achieved by a complete starvation for heme in hemB, hemE, and hemH mutants or during exponential growth of a hemL mutant in the absence of heme supplementation. Equivalent results were obtained by both methods. To determine the basis for this induction, we developed a panel of monoclonal antibodies reactive with HemA, which can detect the small amount of protein present in a wild-type strain. Western blot (immunoblot) analysis with these antibodies reveals that the increase in HemA enzyme activity during heme limitation is mediated by an increase in the abundance of the HemA protein. Increased HemA protein levels were also observed in heme-limited cells of a hemL mutant in two different E. coli backgrounds, suggesting that the observed regulation is conserved between E. coli and S. typhimurium. In S. typhimurium, the increase in HemA enzyme and protein levels was accompanied by a minimal (less than twofold) increase in the expression of hemA-lac operon fusions; thus HemA regulation is mediated either at a posttranscriptional step or through modulation of protein stability.
Wang
L Y
LY
Department of Microbiology and Immunology, West Virginia University Health Sciences Center, Morgantown 26506, USA.
Brown
L
L
Elliott
M
M
Elliott
T
T
D000064:Acclimatization; D000445:Aldehyde Oxidoreductases / Q000096:biosynthesis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005838:Genotype; D006418:Heme / Q000096:biosynthesis* / Q000494:pharmacology; D006706:Homeostasis; D019751:Intramolecular Transferases; D011166:Porphyrins / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012486:Salmonella typhimurium / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
D001426:Bacterial Proteins; D011166:Porphyrins; D011993:Recombinant Fusion Proteins; D006418:Heme; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D001616:beta-Galactosidase; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase