Transcription of the glutamyl-tRNA reductase (hemA) gene in Salmonella typhimurium and Escherichia coli: role of the hemA P1 promoter and the arcA gene product

Abstract

In Salmonella typhimurium and Escherichia coli, the hemA gene encodes the enzyme glutamyl-tRNA reductase, which catalyzes the first committed step in the heme biosynthetic pathway. It has recently been reported that a lac operon fusion to the hemA promoter of E. coli is induced 20-fold after starvation for heme. Induction was dependent on the transcriptional regulator ArcA, with a second transcriptional regulator, FNR, playing a negative role specifically under anaerobic conditions (S. Darie and R. P. Gunsalus, J. Bacteriol. 176:5270-5276, 1994). We have investigated the generality of this effect by examining the response to heme starvation of a number of lac operon fusions to the hemA promoters of both E. coli and S. typhimurium. We confirmed that such fusions are induced during starvation of a hemA auxotroph, but the level of induction observed was maximally sixfold and for S. typhimurium fusions it was only two-to fourfold. Sequences required for high-level expression of hemA lie within 129 bp upstream of the major (P1) promoter transcriptional start site. Mutants defective in the P1 promoter had greatly reduced hemA-lac expression both in the presence and in the absence of ALA. Mutations in arcA had no effect on hemA-lac expression in E. coli during normal growth, although the increase in expression during starvation for ALA was half that seen in an arcA ؉ strain. Overexpression of the arcA gene had no effect on hemA-lac expression. Primer extension analysis showed that RNA 5 ends mapping to the hemA P1 and P2 promoters were not expressed at significantly higher levels in induced cultures. These results differ from those previously reported.

Role of the hemA gene product and delta-aminolevulinic acid in regulation of Escherichia coli heme synthesis

“…A control plasmid containing the 244 nucleotides of hemA coding sequence fused to lacZ produced approximately 10% of the ␤-galactosidase activity produced by a similar plasmid containing the weaker of the two hemA promoters (hemA 2 ) fused to lacZ (data not shown). Our results are similar to Choi and coworkers' findings about the importance of the hemA P1 promoter in high level expression of the Salmonella typhimurium hemA (4).…”

“…ALA (Sigma Chemical Co., St. Louis, Mo.) was solubilized in water, filter sterilized, and added to cultures to yield solutions with final concentrations of 0.02, 0.10, 0.39, 1.56, 6.25, 4 . Glucose was added to a final concentration of 1% (wt/vol); thiamine was added to a final concentration of 0.32 mg/ml.…”

ASalmonella entericaSerovar TyphimuriumhemAMutant Is Highly Susceptible to Oxidative DNA Damage

“…The location of this start site corresponds to a site previously denoted P1 (4), as well as to a site that was identified by S1 analysis for the E. coli hemA gene (37). RNA analysis of the plasmid-encoded promoter region revealed two additional bands, of which the minor one corresponds to a site previously denoted P2 (4,37). The third RNA product is presumably a degradation product originating from the P2 transcript.…”

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 our earlier study, a small effect of starvation for heme on the expression of a hemA-lac operon fusion, in both E. coli and S. typhimurium, was noted (15,19). Consistent with these and other unpublished experiments, we find only a 1.5-to 2-fold increase in hemA-lac expression after starvation for heme in a hemB mutant, or during heme limitation in adapted hemL mutants, when the standard method for these experiments is used (46a).…”

“…ALA production in organisms other than the enteric bacteria is known to be regulated at the levels of both gene expression and enzyme activity (see, e.g., references 31a and 51). In previous work, we examined the expression of hemA-lac operon fusions during heme limitation and found only modest effects on expression (15). Furthermore, this effect was very small unless pyruvate was present in the Luria-Bertani (LB) medium.…”