title: Purification of Vibrio cholerae fur and estimation of its intracellular abundance by antibody sandwich enzyme-linked immunosorbent assay.
abstract: The Vibrio cholerae fur gene was previously cloned and sequenced. A putative Fur box was identified in the divergent promoters of irgA, a virulence factor of V. cholerae, and irgB, a transcriptional activator of irgA. In this work, V. cholerae Fur was overexpressed in Escherichia coli and purified to approximately 95% homogeneity. The purified protein bound a DNA fragment containing the irgA-irgB promoter in a gel shift assay. The purified protein was used to raise monoclonal and polyclonal antibodies to V. cholerae Fur, and a Fur sandwich enzyme-linked immunosorbent assay was developed to estimate the intracellular abundance of Fur under a variety of growth conditions. The number of Fur molecules per cell during exponential growth was approximately 2,500, which is higher than most measurements for other bacterial repressors but comparable to the intracellular concentration of the leucine-responsive regulatory protein. The number of Fur molecules per cell increased in the late logarithmic and stationary phases. Growth of V. cholerae in low-iron medium did not alter the intracellular abundance of Fur significantly. Growth under microaerophilic conditions resulted in a significant, approximately twofold decrease in the intracellular levels of Fur. The measurements of intracellular Fur abundance indicate that a large amount of this repressor is produced constitutively and that the concentration of Fur in the cell varies by less than a factor of 2 under the conditions studied. We hypothesize that the high constitutive expression of Fur is necessary for its role as an iron-responsive regulator.
authors.affiliation: Infectious Disease Unit, Massachusetts General Hospital, Boston 02114, USA.
authors.forename: P I
authors.initials: PI
authors.lastname: Watnick
authors.forename: T
authors.initials: T
authors.lastname: Eto
authors.forename: H
authors.initials: H
authors.lastname: Takahashi
authors.forename: S B
authors.initials: SB
authors.lastname: Calderwood
mesh_terms: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D008970:Molecular Weight; D011401:Promoter Regions, Genetic / Q000235:genetics; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D014734:Vibrio cholerae / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C068477:ferric uptake regulating proteins, bacterial; C074789:irgA protein, Vibrio cholerae; C074788:irgB protein, Vibrio cholerae