Relationship of siderophore-mediated iron assimilation to virulence in crown gall disease

Three classes of mutants defective in the biosynthesis of the siderophore agrobactin were isolated from Agrobacterium tumefaciens A217 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Class I mutants produced uniquely the catechol 2,3-dihydroxybenzoic acid, whereas classes II and III produced no detectable catechol. Class II differed from class III mutants in that exogenous 2,3-dihydroxybenzoic acid was utilized only by the former to synthesize agrobactin. Growth of strains B6 and A217, under iron starvation, led to enhanced production of several envelope proteins migrating in the 80,000-dalton range upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One mutant, defective in agrobactin iron utilization, lacked one of these proteins. This protein may represent a siderophore receptor or fragment or subunit thereof. With a single exception, all of the mutants obtained in this work were capable of initiating tumorous growth in sunflower plants and on carrot root disks, provided pTiB6806 was present. Comparison of the catechols produced by strain B6806 and its nononcogenic, Ti-plasmid-deficient derivative A217, indicated that the genes encoding agrobactin synthesis are not associated with the virulence plasmid of A. tumefaciens B6806. Analysis of gall tissue for agrobactin did not reveal the presence of this siderophore. Finally, citrate, an iron-carrier in plants, enhanced significantly the growth of the agrobactin-deficient mutants in a low-iron medium. These results suggest that the production of agrobactin in planta is not requisite to infection and that citrate may serve as an alternative carrier of iron for A. tumefaciens within the host.