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

Abstract

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.

ASalmonella entericaSerovar TyphimuriumhemAMutant Is Highly Susceptible to Oxidative DNA Damage

“…Null mutations in hemA cause an auxotrophy for ALA, and supplementing the medium with ALA can rescue the growth defects of these mutants (5,40). Addition of ALA had no effect on the growth rate of wild-type cells; however, it diminished the generation time of hemA26 cells substantially ( Table 2).…”

“…In this mutant, Tn10 was inserted within the promoter region of hemA, leaving the gene intact. In contrast to hemA-null mutants that can hardly grow (40), the low expression of hemA26 by readthrough from the tetR gene made it possible to examine the phenotypes of this mutant. Here we show that exposure of hemA-deficient cells to hydrogen peroxide results in iron-dependent DNA damage and cell death.…”

Differential Regulation of the Multiple Flagellins in Spirochetes

“…The monoclonal antibody against the B. hyodysenteriae 35-kDa protein was prepared using standard methodology by injecting purified PFs derived from the mutant A204 (53) lacking the FlaA protein into mice. Lymphocyte fusions were carried out with myeloma cell line P3.X63.Ag8.653 (62). Immunoblots were developed by using horseradish peroxidase secondary antibody with an ECL luminol assay (GE Healthcare).…”

Glutamyl-tRNA in Bacteria. Multiple Identities for Multiple Functions

“…The cellular concentration of GluTR can be controlled at the transcriptional level 75−77 and also by the modulation of the enzyme stability in γ-proteobacteria. 78,79 GluTR from E. coli and Salmonella typhimurium are degraded by Lon and ClpAP proteases when there is an excess of heme. 80 The proposed mechanism is that binding of heme to GluTR exposes a hidden proteolysis signal located at the amino terminus of the enzyme enhancing its degradation.…”