Growth phenotype of mutant PBR840 (Δ pvdL ) in iron-limited media. A: Growth of SBW25 and PBR840 on LB agar for two days, after which the mutant produced no visible PVD and colonies were smaller than wild-type. B: Growth in CAA broth (SBW25, clear circle; PBR840, filled circle) and in CAA broth supplemented with 450 μM FeCl3 (SBW25, clear square; PBR840, filled square). Results are means and standard errors from at least five independent cultures (error bars are contained within the symbols). Data were collected at 5 minute intervals, but 2 hourly time points are shown for clarity. Data from each medium were analyzed separately using a multivariate model (MANOVA) with repeated measures (2 h intervals): no significant difference was detected between genotypes grown in CAA supplemented with FeCl3 [F1,8 = 0.03, P = 0.862], however, a significant genotype effect was evident in iron deficient medium [F1,8 = 37.22, P < 0.001]. An analysis at each time point revealed a highly significant difference between genotypes from (and including) the 14 h time point [F1,8 = 28.02, P < 0.0001]. C: PBR840 cells were plated on CAA media with EDDHA and overlaid with filter paper disks impregnated with sterile water (left) and 50 nmol purified SBW25 PVD (right). PBR840 was not able to grow on the strongly iron-chelated media, growth was restored when supplemented with PVD.