Integration host factor is required for anaerobic pyruvate induction of pfl operon expression in Escherichia coli

The expression of the pyruvate formate-lyase gene (pfl) is induced by anaerobic growth, and this is increased further by growth in pyruvate. Previous work has shown that anaerobic induction is strongly dependent on the activator FNR and partially dependent on a second transcription factor, ArcA, while pyruvate induction only required FNR. Anaerobic and pyruvate regulation both require the presence of a 5' nontranslated regulatory sequence which spans approximately 500 bp of DNA. A mobility shift assay was developed to identify proteins that bind to this regulatory region. Several binding activities were separated by heparin agarose chromatography, and one of these activities was characterized and shown to be integration host factor (IHF). Mobility shift and DNase I footprinting experiments defined a single IHF binding site in the pfl promoter-regulatory region. With pfl-lacZ fusions, it could be shown that introduction of a himD mutation abolished pyruvate-dependent induction of anaerobic expression in vivo. The same result was observed when the pfl IHF binding site was mutated. In addition, the partial anaerobic induction of expression found in an fnr strain was completely blocked in an fnr himD double mutant and in an fnr IHF binding site double mutant. Taken together, these data suggest that IHF is necessary for both pyruvate induction and the anaerobic induction mediated by ArcA.

[1]  F. A. Neugebauer,et al.  Post-translational activation introduces a free radical into pyruvate formate-lyase. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[2]  D. Crothers,et al.  Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. , 1981, Nucleic acids research.

[3]  K. Appelt,et al.  The integration host factor of Escherichia coli binds to multiple sites at plasmid R6K gamma origin and is essential for replication. , 1988, Nucleic acids research.

[4]  A. Sirko,et al.  The role of integration host factor In gene expression in Escherichia coli , 1992, Molecular microbiology.

[5]  P. Youderian,et al.  The phi 80 and P22 attachment sites. Primary structure and interaction with Escherichia coli integration host factor. , 1985, The Journal of biological chemistry.

[6]  S. Cohen,et al.  Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[7]  D. Hartl,et al.  Genetic applications of an inverse polymerase chain reaction. , 1988, Genetics.

[8]  F. A. Neugebauer,et al.  The free radical in pyruvate formate-lyase is located on glycine-734. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[9]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[10]  W. Gilbert,et al.  Sequencing end-labeled DNA with base-specific chemical cleavages. , 1980, Methods in enzymology.

[11]  W. McClure,et al.  Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF). , 1990, Nucleic acids research.

[12]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[13]  J. Guest,et al.  FNR and its role in oxygen-regulated gene expression in Escherichia coli. , 1990, FEMS microbiology reviews.

[14]  A. Böck,et al.  Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[15]  G. Sawers,et al.  A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli. , 1990, FEMS microbiology reviews.

[16]  U. Henning,et al.  Regulation of pyruvate dehydrogenase complex synthesis in Escherichia coli K 12. Identification of the inducing metabolite. , 1970, European journal of biochemistry.

[17]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[18]  H. Nash,et al.  Purification and properties of the Escherichia coli protein factor required for lambda integrative recombination. , 1981, The Journal of biological chemistry.

[19]  D. Friedman,et al.  Integration host factor: A protein for all reasons , 1988, Cell.

[20]  P. Datta,et al.  Catabolite gene activator protein and integration host factor act in concert to regulate tdc operon expression in Escherichia coli , 1992, Journal of bacteriology.

[21]  R. Gunsalus,et al.  Activation of the Escherichia coli nitrate reductase (narGHJI) operon by NarL and Fnr requires integration host factor. , 1993, The Journal of biological chemistry.

[22]  H I Miller,et al.  Primary structure of the himA gene of Escherichia coli: homology with DNA-binding protein HU and association with the phenylalanyl-tRNA synthetase operon. , 1984, Cold Spring Harbor symposia on quantitative biology.

[23]  T. Kunkel.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[24]  D. Galas,et al.  DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. , 1978, Nucleic acids research.

[25]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[26]  R. Weisberg,et al.  Overproduction of Escherichia coli integration host factor, a protein with nonidentical subunits , 1987, Journal of bacteriology.

[27]  J. Demoss,et al.  Upstream sequence elements required for NarL-mediated activation of transcription from the narGHJI promoter of Escherichia coli. , 1992, The Journal of biological chemistry.

[28]  B. Wanner.  Novel regulatory mutants of the phosphate regulon in Escherichia coli K-12. , 1986, Journal of molecular biology.

[29]  B. Haddock,et al.  The identification of mutants of Escherichia coli deficient in formate dehydrogenase and nitrate reductase activities using dye indicator plates , 1977 .

[30]  B. Magasanik,et al.  Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[31]  V. Stewart,et al.  In vivo requirement of integration host factor for nar (nitrate reductase) operon expression in Escherichia coli K-12. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[32]  E. Lin,et al.  arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[33]  J. Guest,et al.  Biochemical genetics of the alpha-keto acid dehydrogenase complexes of Escherichia coli K12: isolation and biochemical properties of deletion mutants. , 1977, Journal of general microbiology.

[34]  U. Henning,et al.  Mutants of Escherichia coli K-12 which synthesize the pyruvate dehydrogenase complex constitutively. , 1971, Archives of biochemistry and biophysics.

[35]  S. Kustu,et al.  The integration host factor stimulates interaction of RNA polymerase with NIFA, the transcriptional activator for nitrogen fixation operons , 1990, Cell.

[36]  A. Böck,et al.  Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression , 1989, Journal of bacteriology.

[37]  G. Sawers,et al.  Anaerobic induction of pyruvate formate-lyase gene expression is mediated by the ArcA and FNR proteins , 1992, Journal of bacteriology.

[38]  R. Weisberg,et al.  Primary structure of the hip gene of Escherichia coli and of its product, the beta subunit of integration host factor. , 1985, Journal of molecular biology.