Use of an in vivo titration method to study a global regulator: effect of varying Lrp levels on expression of gltBDF in Escherichia coli.
Most studies of global regulatory proteins are performed in vitro or involve phenotypic comparisons between wild-type and mutant strains. We report the use of strains in which the gene for the leucine-responsive regulatory protein (lrp) is transcribed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters for the purpose of continuously varying the in vivo concentration of Lrp. To obtain a broad range of Lrp concentrations, strains were employed that contained the lrp fusion either in the chromosome (I. C. Blomfield, P. J. Calie, K. J. Eberhardt, M. S. McClain, and B. I. Eisenstein, J. Bacteriol. 175:27-36, 1993) or on a multicopy plasmid. Western blot (immunoblot) analysis with polyclonal antiserum to Lrp confirmed that Lrp levels could be varied more than 70-fold by growing the strains in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium containing different amounts of IPTG. Expression of an Lrp-regulated gltB::lacZ operon fusion was measured over this range of Lrp concentrations. beta-Galactosidase activity rose with increasing Lrp levels up to the level of Lrp found in wild-type strains, at which point expression is maximal. The presence of leucine in the medium increased the level of Lrp necessary to achieve half-maximal expression of the gltB::lacZ fusion, as predicted by earlier in vitro studies (B. R. Ernsting, J. W. Denninger, R. M. Blumenthal, and R. G. Matthews, J. Bacteriol. 175:7160-7169, 1993). Interestingly, levels of Lrp greater than those in wild-type cells interfered with activation of gltB::lacZ expression. The growth rate of cultures correlated with the intracellular Lrp concentration: levels of Lrp either lower or higher than wild-type levels resulted in significantly slower growth rates. Thus, the level of Lrp in the cell appears to be optimal for rapid growth in minimal medium, and the gltBDF control region is designed to give maximal expression at this Lrp level.
Borst
D W
DW
Department of Biological Chemistry, The University of Michigan, Ann Arbor 48109-1055, USA.
Blumenthal
R M
RM
Matthews
R G
RG
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003470:Culture Media; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005947:Glucose / Q000378:metabolism; D005970:Glutamate Synthase / Q000096:biosynthesis / Q000235:genetics*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007930:Leucine / Q000494:pharmacology; D051756:Leucine-Responsive Regulatory Protein; D009876:Operon; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D003470:Culture Media; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C497278:Lrp protein, E coli; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051756:Leucine-Responsive Regulatory Protein; D007544:Isopropyl Thiogalactoside; D005970:Glutamate Synthase; D007930:Leucine; D005947:Glucose