CadC-Mediated Activation of the cadBA Promoter in Escherichia coli

The transcriptional activator CadC in Escherichia coli, a member of the ToxR-like proteins, activates transcription of the cadBA operon encoding the lysine decarboxylase CadA and the lysine-cadaverine antiporter CadB. cadBA is induced under conditions of acidic external pH and exogenous lysine; anoxic conditions raise the expression level up to 10 times. To characterize the binding mechanism of CadC, procedures for the purification of this membrane-integrated protein and its reconstitution into proteoliposomes were established. The binding sites of CadC upstream of the cadBA promoter region were determined by in vitro DNaseI protection analysis. Two regions were protected during DNaseI digestion, one from –144 to –112 bp, designated Cad1, and another one from –89 to –59 bp, designated Cad2. Binding of purified CadC to Cad1 and Cad2 was further characterized by DNA-binding assays, indicating that CadC was able to bind to both DNA fragments. Genetic analysis with promoter-lacZ fusions confirmed that both sites, Cad1 and Cad2, are essential for activation of cadBA transcription. Moreover, these experiments revealed that binding of H-NS upstream of the CadC-binding sites is necessary for repression of cadBA expression at neutral pH and under aerobic conditions. Based on these results, a model for transcriptional regulation of the cadBA operon is proposed, according to which H-NS is involved in the formation of a repression complex under non-inducing conditions. This complex is dissolved by binding of CadC to Cad1 under inducing conditions. Upon binding of CadC to Cad2 cadBA expression is activated.

[1]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[2]  C. Sasakawa,et al.  Transcriptional control of the invasion regulatory gene virB of Shigella flexneri: activation by virF and repression by H-NS , 1993, Journal of bacteriology.

[3]  H. Ingmer,et al.  H‐NS: a modulator of environmentally regulated gene expression , 1997, Molecular microbiology.

[4]  M. Neely,et al.  Kinetics of expression of the Escherichia coli cad operon as a function of pH and lysine , 1996, Journal of bacteriology.

[5]  P. W. Holloway,et al.  A simple procedure for removal of Triton X-100 from protein samples. , 1973, Analytical biochemistry.

[6]  W. Ansorge,et al.  Nonradioactive, solid-phase DNase I footprints analyzed on an A.L.F. DNA Sequencer. , 1994, BioTechniques.

[7]  K. Altendorf,et al.  The K+-translocating Kdp-ATPase from Escherichia coli. Purification, enzymatic properties and production of complex- and subunit-specific antisera. , 1988, European journal of biochemistry.

[8]  E. Fischer,et al.  Purification and physical properties of inducible Escherichia coli lysine decarboxylase. , 1974, Biochemistry.

[9]  D. Goodsell,et al.  Bending and curvature calculations in B-DNA. , 1994, Nucleic acids research.

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

[11]  G. Bennett,et al.  Regulation of the Escherichia coli cad operon: location of a site required for acid induction , 1992, Journal of bacteriology.

[12]  F. Studier,et al.  Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. , 1986, Journal of molecular biology.

[13]  E. Bremer,et al.  Synthesis of the Escherichia coli K‐12 nucleoid‐associated DNA‐binding protein H‐NS is subjected to growth‐phase control and autoregulation , 1993, Molecular microbiology.

[14]  A. Kuraishi,et al.  Excretion and uptake of cadaverine by CadB and its physiological functions in Escherichia coli , 2004, Molecular microbiology.

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

[16]  K. Jung,et al.  Purification, Reconstitution, and Characterization of KdpD, the Turgor Sensor of Escherichia coli * , 1997, The Journal of Biological Chemistry.

[17]  J. Mekalanos,et al.  Cholera toxin transcriptional activator ToxR is a transmembrane DNA binding protein , 1987, Cell.

[18]  G. Bennett,et al.  Modulation of acid-induced amino acid decarboxylase gene expression by hns in Escherichia coli , 1993, Journal of bacteriology.

[19]  M. Uhlén,et al.  Immobilization and affinity purification of recombinant proteins using histidine peptide fusions. , 1989, European journal of biochemistry.

[20]  C. Higgins,et al.  The chromatin-associated protein H-NS interacts with curved DNA to influence DNA topology and gene expression , 1992, Cell.

[21]  R. Gunsalus,et al.  Regulation of Escherichia coli fumarate reductase (frdABCD) operon expression by respiratory electron acceptors and the fnr gene product , 1987, Journal of bacteriology.

[22]  R. Wagner,et al.  Evidence for a regulatory function of the histone‐like Escherichia coli protein H‐NS in ribosomal RNA synthesis , 1994, Molecular microbiology.

[23]  Miguel Antonio Aon,et al.  Altered topoisomerase activities may be involved in the regulation of DNA supercoiling in aerobic-anaerobic transitions inEscherichia coli , 1993, Molecular and Cellular Biochemistry.

[24]  Ronald K. Taylor,et al.  Vibrio cholerae H-NS Silences Virulence Gene Expression at Multiple Steps in the ToxR Regulatory Cascade , 2000, Journal of bacteriology.

[25]  M. Neely,et al.  Roles of LysP and CadC in mediating the lysine requirement for acid induction of the Escherichia coli cad operon , 1994, Journal of bacteriology.

[26]  G. Bennett,et al.  Nucleotide sequence of the Escherichia coli cad operon: a system for neutralization of low extracellular pH , 1992, Journal of bacteriology.

[27]  B. Pitard,et al.  Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins. , 1995, Biochimica et biophysica acta.

[28]  G. Peterson,et al.  A simplification of the protein assay method of Lowry et al. which is more generally applicable. , 1977, Analytical biochemistry.

[29]  H. Gross,et al.  Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels , 1987 .

[30]  B. Colonna,et al.  Involvement of FIS in the H‐NS‐mediated regulation of virF gene of Shigella and enteroinvasive Escherichia coli  ‡ , 2001, Molecular microbiology.

[31]  J. Slonczewski,et al.  Identification of elements involved in transcriptional regulation of the Escherichia coli cad operon by external pH , 1992, Journal of bacteriology.

[32]  Hiroshi Kobayashi,et al.  Escherichia coli cad operon functions as a supplier of carbon dioxide , 1994, Molecular microbiology.

[33]  R. Simons,et al.  Improved single and multicopy lac-based cloning vectors for protein and operon fusions. , 1987, Gene.

[34]  N. W. Davis,et al.  The complete genome sequence of Escherichia coli K-12. , 1997, Science.

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

[36]  C. Richardson,et al.  A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[37]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. 1977. , 1992, Biotechnology.

[38]  D. Clark,et al.  Effect of chelating agents and respiratory inhibitors on regulation of the cadA gene in Escherichia coli , 1997, Archives of Microbiology.

[39]  M. Neely,et al.  Altered pH lysine signalling mutants of cadC, a gene encoding a membrane‐bound transcriptional activator of the Escherichia coli cadBA operon , 1994, Molecular microbiology.

[40]  F. Neidhardt,et al.  Culture Medium for Enterobacteria , 1974, Journal of bacteriology.

[41]  D. Lane,et al.  Expression of the second lysine decarboxylase gene of Escherichia coli. , 1998, Microbiology.

[42]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[43]  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.

[44]  G. Bennett,et al.  Construction of lac fusions to the inducible arginine‐and lysine decarboxylase genes of Escherichia coli K12 , 1989, Molecular microbiology.