In contrast the pvd- strain was sensitive to almost 3 orders of magnitude less EDDHA, with an IC50 of only 0.57 ± 0.02 μg/ml, demonstrating that achromobactin cannot completely compensate for the absence of pyoverdine. However, the IC50 for the pvd-/acr- double mutant strain (0.31 ± 0.01 μg/ml) was reproducibly lower yet, verifying that in the absence of pyoverdine achromobactin still makes a small contribution to fitness during

iron starvation. At 28°C the selleck chemicals llc IC50 for WT and acr- strains were essentially unchanged, but the difference between the pvd- mutant (0.38 ± 0.01) and pvd-/acr- double mutant (0.26 ± 0.01) was less marked. Assessment of pathogenicity in Phaseolus vulgaris In order to assess the pathogenicity in the natural host of P. syringae 1448a each of the mutant strains (including the pvd-/acr-/ybt- triple mutant) was subjected to the standard ‘bean prick’ pathogenicity test using bean pods [44]. All mutant strains were still able to cause characteristic water soaked lesions after inoculation and incubation in bean pods (Figure 6), irrespective of temperature and whether

or not the beans were picked or still attached to the parental plant. This indicates that neither pyoverdine nor achromobactin is essential in enabling P. syringae 1448a to cause halo blight in the bean plant Phaseolus vulgaris. Figure 6 Assessment of pathogenicity of mutant strains in Phaseolus vulgaris. Three replicates are indicated each Sirolimus mouse containing, in order from left to right, WT, pvd-, acr/pvd- https://www.selleckchem.com/products/fosbretabulin-disodium-combretastatin-a-4-phosphate-disodium-ca4p-disodium.html and acr-/pvd-/ybt- strains. Each strain was inoculated from a single colony, using a hypodermic needle. The pod was then incubated in a humid chamber at room temperature for 48 hours. All strains display characteristic water-soaked lesions indicating successful establishment of pathogenicity in Phaseolus vulgaris. Discussion Unlike P. aeruginosa, P. syringae does not appear to exhibit a high degree of variability in pyoverdine structure from strain to strain, with all fluorescent P. syringae pathovars tested thus far having been found to produce an identical pyoverdine molecule

[35, 36]. Our bioinformatic studies suggested that P. syringae 1448a would not be any different in this regard; and MALDI-TOF and MS/MS analyses demonstrated that the same pyoverdine is indeed made by this strain. However, these analyses also indicated that P. syringae 1448a is able to make an additional pyoverdine variant that was fundamentally similar in most aspects, but with an overall mass 71 Da greater. The most plausible interpretation of the fragmentation pattern in Figure 2C is that an extra monomer is incorporated into the pyoverdine side chain. If so, the B-ion pattern suggests that this monomer appears as the first residue of the side chain, falling between the chromophore and L-lysine, and increasing the mass by 71 Da.