PMID: 8955313Dec 1, 1996Paper

Use of an in vivo titration method to study a global regulator: effect of varying Lrp levels on expression of gltBDF in Escherichia coli

Journal of Bacteriology
D W BorstRowena G Matthews

Abstract

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 ne...Continue Reading

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Citations

Dec 21, 2010·Journal of Bacteriology·Benjamin R Hart, Robert M Blumenthal
May 22, 2016·BMC Evolutionary Biology·Yvette UnoarumhiJyl S Matson
Dec 4, 2013·Microbiology and Molecular Biology Reviews : MMBR·Wally C van HeeswijkFred C Boogerd
Feb 10, 2007·Proceedings of the National Academy of Sciences of the United States of America·Chang-Ro LeeYeong-Jae Seok
Jul 15, 2015·Journal of Bacteriology·Elizabeth A HussaHeidi Goodrich-Blair
Mar 8, 2002·Omics : a Journal of Integrative Biology·Denise M Wolf, Adam P Arkin
Jun 7, 2011·Journal of Bacteriology·Benjamin R HartRobert M Blumenthal
May 7, 2003·Current Opinion in Microbiology·Denise M Wolf, Adam P Arkin
Sep 1, 1997·Journal of Bacteriology·B R BelitskyC Von Wachenfeldt
Apr 1, 2007·EcoSal Plus·Ian Blomfield, Marjan van der Woude

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