Home LiteratureArticle Details
PMID: 9335270 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S.

Use of an inducible regulatory protein to identify members of a regulon: application to the regulon controlled by the leucine-responsive regulatory protein (Lrp) in Escherichia coli.

Journal of bacteriology ·Vol. 179 ·No. 20 ·1997-10-00 ·Pages 6254-63

Bhagwat SP, Rice MR, Matthews RG, Blumenthal RM

Abstract

Procedures were developed to facilitate the identification of genes that belong to a given regulon and characterization of their responses to the regulator. The regulon controlled by the Escherichia coli leucine-responsive regulatory protein (Lrp) was studied by isolating random transcriptional fusions to lacZ, using lambda placMu53 and a strain in which lrp is under isopropylthio-beta-D-galactopyranoside (IPTG)-inducible control. Fusions exhibiting IPTG-responsive beta-galactosidase activity were cloned by integrating the suicide vector pIVET1 via homologous recombination at lacZ, followed by self-ligating digested chromosomal DNA. We verified the patterns of lacZ expression after using the plasmid clones to generate merodiploid strains with interrupted and uninterrupted copies of the same sequence. If the merodiploid expression pattern was unchanged from that shown by the original fusion strain, then the cloned fusion was responsible for the regulatory pattern of interest; a difference in the expression pattern could indicate that the original strain carried multiple fusions or that there were autogenous effects of having interrupted the fused gene. Using these procedures, we generated a fusion library of approximately 5 x 10(6) strains; approximately 3,000 of these strains were screened, yielding 84 Lrp-responsive fusions, and 10 of the 84 were phenotypically stable and were characterized. The responses of different fusions in a given operon to in vivo Lrp titrations revealed variations in expression with the position of insertion. Among the newly identified members of the regulon is an open reading frame (orf3) between rpiA and serA. Also, expression of a fusion just downstream of dinF was found to be Lrp dependent only in stationary phase.

MeSH Terms
Bacterial Proteins/genetics,metabolism Bacteriophage mu/genetics Cloning, Molecular DNA-Binding Proteins/genetics,metabolism Escherichia coli/genetics,growth & development,metabolism Escherichia coli Proteins Gene Expression Regulation, Bacterial Gene Library Leucine/pharmacology Leucine-Responsive Regulatory Protein Membrane Transport Proteins Open Reading Frames Operon Regulon Sequence Analysis, DNA Transcription Factors
Chemicals
Bacterial Proteins DNA-Binding Proteins Escherichia coli Proteins Lrp protein, E coli Membrane Transport Proteins Transcription Factors livG protein, E coli Leucine-Responsive Regulatory Protein Leucine
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Bhagwat S P
Department of Microbiology and Immunology, Medical College of Ohio, Toledo 43614-5806, USA.
Rice M R
Matthews R G
Blumenthal R M
References (56)
56 references, click to expand
  1. The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli.
    Microbiol Rev. 1994 Sep;58(3):466-90 PMID: 7968922
  2. The leucine-responsive regulatory protein binds to the fim switch to control phase variation of type 1 fimbrial expression in Escherichia coli K-12.
    J Bacteriol. 1994 Sep;176(18):5665-72 PMID: 7916011
  3. Identification of Lrp-regulated genes by inverse PCR and sequencing: regulation of two mal operons of Escherichia coli by leucine-responsive regulatory protein.
    J Bacteriol. 1995 May;177(10):2679-83 PMID: 7751276
  4. On microbial states of growth.
    Mol Microbiol. 1995 Mar;15(5):789-94 PMID: 7596281
  5. Isolation and sequencing of gene fusions carried by lambda placMu specialized transducing phage.
    Nucleic Acids Res. 1995 Aug 11;23(15):3076-8 PMID: 7659535
  6. A consensus sequence for binding of Lrp to DNA.
    J Bacteriol. 1995 Sep;177(17):4872-80 PMID: 7665463
  7. Analysis of cell size and DNA content in exponentially growing and stationary-phase batch cultures of Escherichia coli.
    J Bacteriol. 1995 Dec;177(23):6791-7 PMID: 7592469
  8. D-histidine utilization in Salmonella typhimurium is controlled by the leucine-responsive regulatory protein (Lrp).
    J Bacteriol. 1996 Jan;178(2):327-31 PMID: 8550449
  9. The leucine-responsive regulatory protein (Lrp) from Escherichia coli. Stoichiometry and minimal requirements for binding to DNA.
    J Biol Chem. 1996 Mar 22;271(12):6611-7 PMID: 8636076
  10. Experimental analysis of global gene regulation in Escherichia coli.
    Prog Nucleic Acid Res Mol Biol. 1996;55:1-86 PMID: 8787606
  11. The leucine-responsive regulatory protein (Lrp) acts as a specific repressor for sigma s-dependent transcription of the Escherichia coli aidB gene.
    Mol Microbiol. 1996 Jun;20(5):947-55 PMID: 8809748
  12. Leucine-responsive regulatory protein-DNA interactions in the leader region of the ilvGMEDA operon of Escherichia coli.
    J Biol Chem. 1996 Oct 25;271(43):26499-507 PMID: 8900118
  13. Use of an in vivo titration method to study a global regulator: effect of varying Lrp levels on expression of gltBDF in Escherichia coli.
    J Bacteriol. 1996 Dec;178(23):6904-12 PMID: 8955313
  14. Effects of nutrition and growth rate on Lrp levels in Escherichia coli.
    J Bacteriol. 1996 Dec;178(23):6930-6 PMID: 8955316
  15. A nucleoprotein activation complex between the leucine-responsive regulatory protein and DNA upstream of the gltBDF operon in Escherichia coli.
    J Mol Biol. 1997 Jul 11;270(2):152-68 PMID: 9236118
  16. From carpet bombing to cruise missiles: the 'second-order' mechanisms used by transcription factors to ensure specific DNA binding in vivo.
    Chem Biol. 1995 May;2(5):267-79 PMID: 9383429
  17. Culture medium for enterobacteria.
    J Bacteriol. 1974 Sep;119(3):736-47 PMID: 4604283
  18. Derivation of glycine from threonine in Escherichia coli K-12 mutants.
    J Bacteriol. 1975 Jun;122(3):810-7 PMID: 1097400
  19. Role of transport systems in amino acid metabolism: leucine toxicity and the branched-chain amino acid transport systems.
    J Bacteriol. 1977 Mar;129(3):1257-65 PMID: 321421
  20. Escherichia coli transport mutants lacking binding protein and other components of the branched-chain amino acid transport systems.
    J Bacteriol. 1977 Apr;130(1):384-92 PMID: 323236
  21. The inhibition of beta-galactosidase (Escherichia coli) by amino sugars and amino alcohols.
    Can J Biochem. 1982 Jun;60(6):608-12 PMID: 6811114
  22. L-Serine deaminase activity is induced by exposure of Escherichia coli K-12 to DNA-damaging agents.
    J Bacteriol. 1982 Nov;152(2):702-5 PMID: 6813312
  23. Transposable lambda placMu bacteriophages for creating lacZ operon fusions and kanamycin resistance insertions in Escherichia coli.
    J Bacteriol. 1985 Jun;162(3):1092-9 PMID: 2987183
  24. The complete nucleotide sequences of the Escherichia coli LIV-BP and LS-BP genes. Implications for the mechanism of high-affinity branched-chain amino acid transport.
    J Biol Chem. 1985 Jul 15;260(14):8257-61 PMID: 3891753
  25. Regulation of peptide transport in Escherichia coli: induction of the trp-linked operon encoding the oligopeptide permease.
    J Bacteriol. 1986 Feb;165(2):428-33 PMID: 3511033
  26. opp-lac Operon fusions and transcriptional regulation of the Escherichia coli trp-linked oligopeptide permease.
    J Bacteriol. 1986 Feb;165(2):434-42 PMID: 3080404
  27. Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K.
    J Mol Biol. 1986 Jan 20;187(2):225-39 PMID: 3009827
  28. Dual level control of the Escherichia coli pheST-himA operon expression. tRNA(Phe)-dependent attenuation and transcriptional operator-repressor control by himA and the SOS network.
    J Mol Biol. 1987 Oct 5;197(3):453-70 PMID: 3326947
  29. A protein that binds to the regulatory region of the Escherichia coli ilvIH operon.
    J Bacteriol. 1989 Mar;171(3):1658-64 PMID: 2646291
  30. Identification of phosphate starvation-inducible genes in Escherichia coli K-12 by DNA sequence analysis of psi::lacZ(Mu d1) transcriptional fusions.
    J Bacteriol. 1990 Jun;172(6):3191-200 PMID: 2160940
  31. The leucine regulon of Escherichia coli K-12: a mutation in rblA alters expression of L-leucine-dependent metabolic operons.
    J Bacteriol. 1990 Aug;172(8):4529-35 PMID: 2165479
  32. The ilvIH operon of Escherichia coli is positively regulated.
    J Bacteriol. 1990 Aug;172(8):4563-70 PMID: 2115869
  33. Chromosome rearrangements induced by recombinant coliphage lambda placMu.
    Gene. 1990 Sep 28;94(1):15-22 PMID: 2146189
  34. Structure-function relationship of the Lrp-binding region upstream of lysU in Escherichia coli.
    J Mol Biol. 1994 Aug 19;241(3):378-89 PMID: 8064854
  35. Lrp stimulates phase variation of type 1 fimbriation in Escherichia coli K-12.
    J Bacteriol. 1993 Jan;175(1):27-36 PMID: 8093239
  36. Lrp, a global regulatory protein of Escherichia coli, binds co-operatively to multiple sites and activates transcription of ilvIH.
    J Mol Biol. 1993 Jan 20;229(2):306-18 PMID: 8429549
  37. Selection of bacterial virulence genes that are specifically induced in host tissues.
    Science. 1993 Jan 29;259(5095):686-8 PMID: 8430319
  38. Mutations affecting the ability of Escherichia coli Lrp to bind DNA, activate transcription, or respond to leucine.
    J Bacteriol. 1993 Feb;175(4):1110-7 PMID: 8432705
  39. Lrp, a major regulatory protein in Escherichia coli, bends DNA and can organize the assembly of a higher-order nucleoprotein structure.
    EMBO J. 1993 Jun;12(6):2495-501 PMID: 8508774
  40. Synthesis of the Escherichia coli K-12 nucleoid-associated DNA-binding protein H-NS is subjected to growth-phase control and autoregulation.
    Mol Microbiol. 1993 May;8(5):875-89 PMID: 8355613
  41. Escherichia coli rpiA gene encoding ribose phosphate isomerase A.
    J Bacteriol. 1993 Sep;175(17):5628-35 PMID: 8366047
  42. Regulation of the gltBDF operon of Escherichia coli: how is a leucine-insensitive operon regulated by the leucine-responsive regulatory protein?
    J Bacteriol. 1993 Nov;175(22):7160-9 PMID: 7901196
  43. Complex transcriptional control of the sigma s-dependent stationary-phase-induced and osmotically regulated osmY (csi-5) gene suggests novel roles for Lrp, cyclic AMP (cAMP) receptor protein-cAMP complex, and integration host factor in the stationary-phase response of Escherichia coli.
    J Bacteriol. 1993 Dec;175(24):7910-7 PMID: 8253679
  44. Mechanism of the down-regulation of cAMP receptor protein by glucose in Escherichia coli: role of autoregulation of the crp gene.
    EMBO J. 1994 Jul 1;13(13):3077-82 PMID: 7518773
  45. Basic local alignment search tool.
    J Mol Biol. 1990 Oct 5;215(3):403-10 PMID: 2231712
  46. micF RNA binds to the 5' end of ompF mRNA and to a protein from Escherichia coli.
    Biochemistry. 1990 Oct 2;29(39):9249-56 PMID: 1702997
  47. Characterization of Lrp, and Escherichia coli regulatory protein that mediates a global response to leucine.
    J Biol Chem. 1991 Jun 15;266(17):10768-74 PMID: 2040596
  48. The tdh and serA operons of Escherichia coli: mutational analysis of the regulatory elements of leucine-responsive genes.
    J Bacteriol. 1991 Oct;173(19):5944-53 PMID: 1917830
  49. Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli.
    J Bacteriol. 1992 Jan;174(1):108-15 PMID: 1729203
  50. Characterization of the regulon controlled by the leucine-responsive regulatory protein in Escherichia coli.
    J Bacteriol. 1992 Feb;174(4):1109-18 PMID: 1346534
  51. The leucine-Lrp regulon in E. coli: a global response in search of a raison d'être.
    Cell. 1992 Feb 21;68(4):617-9 PMID: 1739970
  52. Lambda placMu insertions in genes of the leucine regulon: extension of the regulon to genes not regulated by leucine.
    J Bacteriol. 1992 Mar;174(6):1948-55 PMID: 1532173
  53. The lrp gene product regulates expression of lysU in Escherichia coli K-12.
    J Bacteriol. 1992 May;174(9):2779-84 PMID: 1569010
  54. Leucine-responsive regulatory protein controls the expression of both the pap and fan pili operons in Escherichia coli.
    Proc Natl Acad Sci U S A. 1992 May 15;89(10):4250-4 PMID: 1350087
  55. Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli.
    J Bacteriol. 1992 Dec;174(24):8043-56 PMID: 1459953
  56. The leucine-responsive regulatory protein of Escherichia coli negatively regulates transcription of ompC and micF and positively regulates translation of ompF.
    J Bacteriol. 1995 Jan;177(1):103-13 PMID: 8002608
Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.179.20.6254-6263.1997
Published
1997-10-00
Pages
6254-63
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC179537
Subset
IM
External Links
PubMed source DOI: 10.1128/jb.179.20.6254-6263.1997 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2