Joseph M. Calvo

Studies by R. Lin et al. (J. Bacteriol. 174:1948–1955, 1992) suggested that the Escherichia coli leu operon might be a member of the Lrp regulon. Their results were obtained with a leucine auxotroph; in leucine prototrophs grown in a medium lacking leucine, there was little difference in leu operon expression between lrp ⁺ and lrp strains. Furthermore, when leuP-lacZ transcriptional fusions that lacked the leu attenuator were used, expression from the leu promoter varied less than twofold between lrp ⁺ and lrp strains, irrespective of whether or not excess leucine was added to the medium. The simplest explanation of the observations of Lin et al. is that the known elevated leucine transport capacity of lrp strains (S. A. Haney et al., J. Bacteriol. 174:108–115, 1992) leads to very high intracellular levels of leucine for strains grown with leucine, resulting in the superattenuation of leu operon expression.

Lrp (leucine-responsive regulatory protein) activates some Escherichia coli operons that function in anabolism and represses others involved in catabolism (for a review, see J. M. Calvo and R. G. Matthews, Microbiol. Rev. 58:466-490, 1994). This overall pattern suggests that Lrp may help cells adapt to changes in the nutritional environment. Here, we tested the idea that the nutritional richness of the medium determines the amount of Lrp in cells. Lrp was measured directly by Western blotting (immunoblotting) in cells grown in a chemically defined rich medium or in a minimal medium. In addition, transcription from the lrp promoter was assessed with a lacZ reporter gene. The results with these two different measurements were nearly the same, indicating that under the conditions employed, beta-galactosidase measurements can accurately reflect Lrp levels. For cells in a minimal medium, Lrp levels were consistently lowest during the logarithmic phase of growth, but overall, there was not much variation in levels as a function of growth phase (1.3-fold difference between highest and lowest values). However, for cells in a rich medium, Lrp levels dropped 3- to 4-fold during the lag phase, remained constant during the log phase, and then rose to starting levels upon entry into the stationary phase. When cells in the log phase were compared, Lrp levels were 3- to 4-fold higher in cells growing in a minimal medium than those in a rich medium. The levels of lrp expression were the same or slightly higher in strains containing mutations in rpoS, cya, or crp compared with wild-type strains, suggesting that neither RpoS nor the cyclic AMP (cAMP) receptor protein-cAMP complex is required for expression. On the other hand, lrp expression was severely restricted in cells that could not make ppGpp because of mutations in relA and spoT. The reduced expression of lrp during logarithmic growth in a rich medium may be due to low ppGpp levels under these conditions. The repressive effects of rich medium and the stimulatory effects of ppGpp were also observed with a construct having only a minimal lrp promoter (-57 to +21). The results of other experiments suggest that Lrp levels vary inversely with the growth rate of cells instead of being determined by some component of the medium.

Lrp (leucine-responsive regulatory protein) is a global regulator of metabolism in Escherichia coli (J. M. Calvo and R. G. Matthews, Microbiol. Rev. 58:466-490, 1994). The lrp genes from three other enteric microorganisms, Enterobacter aerogenes, Klebsiella aerogenes, and Salmonella typhimurium, were cloned and sequenced. An analysis of these sequences and of the previously determined sequence from E. coli indicated that the vast majority of changes were synonymous rather than nonsynonymous changes. Nucleotide changes occurred at 89 of 492 positions but resulted in amino acid changes at only 2 of 164 positions. This analysis suggests that the Lrp amino acid sequence is highly adapted for function and that almost all amino acid changes lead to a protein that functions less well than the wild-type protein.