ANALYSIS OF THE COMPLEXITY OF GENE-REGULATION BY FUR IN VIBRIO-CHOLERAE

Iron concentration influences the expression of a number of genes invo lved in iron uptake and virulence in bacteria. In Escherichia coli, co ordinate regulation of these genes by iron depends on the product of t he fur gene, which acts as an iron-responsive, DNA-binding repressor p rotein. Several genes in Vibrio cholerae are also repressed by iron; a nd a fur gene, homologous to E. coli fur, has been previously cloned f rom this organism. The present study was undertaken to define the role s of Fur and iron in regulating gene expression in V. cholerae. V. cho lerae strains with a mutation in fur by virtue of suicide plasmid inte gration into this gene showed derepressed expression of two previously characterized, iron-regulated genes, irgA and viuA, in high concentra tions of iron; even in the fur mutants, however, residual two- to thre efold regulation by iron persisted. The fur mutant strains constructed by suicide plasmid integration required antibiotic selection to maint ain the mutation. To analyze further the effect of Fur and iron on gen e regulation in V. cholerae without the need for antibiotic selection, we used in vivo marker exchange to construct a nonrevertible V. chole rae fur mutant. This V. cholerae fur mutant grew significantly less we ll in Luria-Bertani medium than the wild-type parent but grew slightly better than the wild type under iron-restricted conditions. The V. ch olerae fur mutant was unable to utilize a number of carbon sources inc luding glycerol, acetate, succinate, lactate, and fumarate, that suppo rted growth of the wild-type strain on minimal media. We utilized two- dimensional gel electrophoresis of whole-cell protein extracts from th e fur mutant and wild-type strains following growth in conditions of e ither low or high concentrations of iron to identify proteins regulate d by iron and/or Fur. Twenty-two proteins were negatively regulated by iron in the wild-type strain but constitutively expressed in the fur mutant, consistent with the model of Fur as an iron-dependent represso r. However, many other proteins were regulated in a different manner b y iron and/or Fur. Seventeen proteins were negatively regulated by iro n but independent of Fur, suggesting the presence of an additional iro n-dependent repressor(s). Six proteins were strongly iron regulated in the fur mutant but hardly expressed at all in the wild-type strain re gardless of the iron concentration, suggesting an interaction between Fur and another iron regulatory mechanism. There were 11 proteins that were induced rather than repressed by iron, in four different regulat ory classes. Gene regulation in V. cholerae by Fur and iron is much mo re complex than previously thought and is reminiscent of the Lrp regul on in E. coli.