Vibrio cholerae, a natural inhabitant of aquatic environments and the causative agent of the diarrheal disease cholerae, requires iron for survival. Since one of the key factors in the survival of V. cholerae in the environment is the formation of biofilms, we determined the effect of iron on this aspect of the pathogens lifestyle. Since wild type V. cholerae forms a much more robust biofilm in the presence of exogenous iron we tested mutants in iron transport and regulation and found that a mutation in the gene encoding an iron-regulated small RNA, RyhB, was clearly attenuated in the biofilm assay. We determined through microarray analysis that the ryhB mutant has altered regulation of genes involved in many systems that may be involved in biofilm formation including amino acid biosynthesis, the TCA cycle, motility and chemotaxis, and the expression of outer membrane proteins. Due to the pleiotropic regulatory effects of RyhB, it is unlikely that any one individual gene or system regulated by RyhB is the cause of the biofilm defect, but rather the sum effect of the regulatory changes is decreased biofilm formation. Additionally, we discovered that the outer membrane protein, OmpT, is positively regulated by iron and Fur. Generally, when Fur has acted as a positive regulator in previous studies, it has been ultimately shown to do so by negatively regulating the negative regulator, RyhB. However, the positive regulation of ompT by Fur is independent of RyhB. While CRP, a positive regulator of ompT expression, did not affect iron-dependent regulation of ompT, over-expression of the negative regulator ToxR abolishes the iron and Fur dependent regulation. Sequence analysis has revealed a possible Fur box approximately 70 base pairs upstream of the transcriptional start site in a region that overlaps both a ToxR binding site and a CRP binding site in the ompT promoter. We propose the model that in iron-replete environments under ToxR repressing conditions, such as when amino acids are limiting, Fur can further increase the expression of ompT. / text