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Journal of Bacteriology, February 1999, p. 1211-1219, Vol. 181, No. 4
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Conditional Stability of the HemA Protein (Glutamyl-tRNA Reductase) Regulates Heme Biosynthesis in Salmonella typhimurium

Liying Wang, Meenal Elliott, and Thomas Elliott*

West Virginia University Health Sciences Center, Morgantown, West Virginia 26506

Received 13 July 1998/Accepted 2 December 1998

In many bacteria, including the enteric species Salmonella typhimurium and Escherichia coli, heme is synthesized starting from glutamate by a pathway in which the first committed step is catalyzed by the hemA gene product, glutamyl-tRNA reductase (HemA). We have demonstrated previously that when heme limitation is imposed on cultures of S. typhimurium, HemA enzyme activity is increased 10- to 25-fold. Western (immunoblot) analysis with monoclonal antibodies reactive with HemA revealed that heme limitation results in a corresponding increase in the abundance of the enzyme. Similar regulation was also observed for E. coli. The near absence of regulation of hemA-lac operon fusions suggested a posttranscriptional control. We report here the results of pulse-labeling and immunoprecipitation studies of this regulation. The principal mechanism that contributes to elevated HemA abundance is protein stabilization. The half-life of HemA protein is sime 20 min in unrestricted cells but increases to >300 min in heme-limited cells. Similar regulation was observed for a HemA-LacZ hybrid protein containing almost all of the HemA protein (416 residues). Sodium azide prevents HemA turnover in vivo, suggesting a role for energy-dependent proteolysis. This was confirmed by the finding that HemA turnover is completely blocked in a lon clpP double mutant of E. coli. Each single mutant shows only a small effect. The ClpA chaperone, but not ClpX, is required for ClpP-dependent HemA turnover. A hybrid HemA-LacZ protein containing just 18 amino acids from HemA is also stabilized in the lon clpP double mutant, but this shorter fusion protein is not correctly regulated by heme limitation. We suggest that the 18 N-terminal amino acids of HemA may constitute a degradation tag, whose function is conditional and modified by the remainder of the protein in a heme-dependent way. Several models are discussed to explain why the turnover of HemA is promoted by Lon-ClpAP proteolysis only when sufficient heme is available.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, P.O. Box 9177, WVU Health Sciences Center, Morgantown, WV 26506-9177. Phone: (304) 293-2676. Fax: (304) 293-7823. E-mail: telliott{at}wvu.edu.

Journal of Bacteriology, February 1999, p. 1211-1219, Vol. 181, No. 4
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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