PURIFICATION OF VIBRIO-CHOLERAE FUR AND ESTIMATION OF ITS INTRACELLULAR ABUNDANCE BY ANTIBODY SANDWICH ENZYME-LINKED-IMMUNOSORBENT-ASSAY

The Vibrio cholerae fur gene was previously cloned and sequenced. A pu tative Fur box was identified in the divergent promoters of irgA, a vi rulence factor of V. cholerae, and irgB, a transcriptional activator o f irgA. In this work, V. cholerae Fur was overexpressed in Escherichia coli and purified to approximately 95% homogeneity. The purified prot ein bound a DNA fragment containing the irgA-irgB promoter in a gel sh ift assay. The purified protein was used to raise monoclonal and polyc lonal antibodies to V. cholerae Fur, and a Fur sandwich enzyme-linked immunosorbent assay was developed to estimate the intracellular abunda nce of Fur under a variety of grow-th conditions. The number of Fur mo lecules per cell during exponential growth was approximately 2,500, wh ich is higher than most measurements for other bacterial repressors bu t comparable to the intracellular concentration of the leucine respons ive 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 si gnificantly. Growth under microaerophilic conditions resulted in a sig nificant, approximately twofold decrease in the intracellular levels o f Fur, The measurements of intracellular Fur abundance indicate that a large amount of this repressor is produced constitutively and that th e concentration of Fur in the cell varies by less than a factor of 2 u nder the conditions studied. We hypothesize that the high constitutive expression of Fur is necessary for its role as an iron-responsive reg ulator.