J. Bacteriol., Jan 1997, 243-247, Vol 179, No. 1
Copyright © 1997, American Society for Microbiology

Purification of Vibrio cholerae fur and estimation of its intracellular abundance by antibody sandwich enzyme-linked immunosorbent assay

PI Watnick, T Eto, H Takahashi and SB Calderwood
Infectious Disease Unit, Massachusetts General Hospital, Boston 02114, USA.

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.

This article has been cited by other articles:

• Coutard, F., Lozach, S., Pommepuy, M., Hervio-Heath, D. (2007). Real-Time Reverse Transcription-PCR for Transcriptional Expression Analysis of Virulence and Housekeeping Genes in Viable but Nonculturable Vibrio parahaemolyticus after Recovery of Culturability. Appl. Environ. Microbiol. 73: 5183-5189 [Abstract] [Full Text]  
• Lee, H.-J., Bang, S. H., Lee, K.-H., Park, S.-J. (2007). Positive Regulation of fur Gene Expression via Direct Interaction of Fur in a Pathogenic Bacterium, Vibrio vulnificus. J. Bacteriol. 189: 2629-2636 [Abstract] [Full Text]  
• Quatrini, R., Lefimil, C., Holmes, D. S., Jedlicki, E. (2005). The ferric iron uptake regulator (Fur) from the extreme acidophile Acidithiobacillus ferrooxidans. Microbiology 151: 2005-2015 [Abstract] [Full Text]  
• Oram, D. M., Avdalovic, A., Holmes, R. K. (2004). Analysis of Genes That Encode DtxR-Like Transcriptional Regulators in Pathogenic and Saprophytic Corynebacterial Species. Infect. Immun. 72: 1885-1895 [Abstract] [Full Text]  
• Delany, I., Ieva, R., Alaimo, C., Rappuoli, R., Scarlato, V. (2003). The Iron-Responsive Regulator Fur Is Transcriptionally Autoregulated and Not Essential in Neisseria meningitidis. J. Bacteriol. 185: 6032-6041 [Abstract] [Full Text]  
• Sha, J., Lu, M., Chopra, A. K. (2001). Regulation of the Cytotoxic Enterotoxin Gene in Aeromonas hydrophila: Characterization of an Iron Uptake Regulator. Infect. Immun. 69: 6370-6381 [Abstract] [Full Text]  
• Park, S. Y., Kelminson, K. L., Lee, A. K., Zhang, P., Warner, R. E., Rehkopf, D. H., Calderwood, S. B., Koehler, J. E. (2001). Identification, Characterization, and Functional Analysis of a Gene Encoding the Ferric Uptake Regulation Protein in Bartonella Species. J. Bacteriol. 183: 5751-5755 [Abstract] [Full Text]  
• Butterton, J. R., Choi, M. H., Watnick, P. I., Carroll, P. A., Calderwood, S. B. (2000). Vibrio cholerae VibF Is Required for Vibriobactin Synthesis and Is a Member of the Family of Nonribosomal Peptide Synthetases. J. Bacteriol. 182: 1731-1738 [Abstract] [Full Text]  
• Escolar, L., Pérez-Martín, J., de Lorenzo, V. (1999). Opening the Iron Box: Transcriptional Metalloregulation by the Fur Protein. J. Bacteriol. 181: 6223-6229 [Full Text]  
• Zheng, M., Doan, B., Schneider, T. D., Storz, G. (1999). OxyR and SoxRS Regulation of fur. J. Bacteriol. 181: 4639-4643 [Abstract] [Full Text]  
• Escolar, L., Perez-Martin, J., de Lorenzo, V. (2000). Evidence of an Unusually Long Operator for the Fur Repressor in the Aerobactin Promoter of Escherichia coli. J. Biol. Chem. 275: 24709-24714 [Abstract] [Full Text]