Iron-binding compounds from Agrobacterium spp.: Biological control strain Agrobacterium rhizogenes K84 produces a hydroxamate siderophore

Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the speci es A. tumefaciens, A. rhizogenes, and A. vitis. The crown gall biocontrol a gent A. rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described a ntibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was exp ressed only when susceptible cells were tested in low-iron media. Five inde pendent Tn5-induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84. One of these mutants, M8-10, was completely deficient in the product ion of both agents and grew poorly compared to the wild type under iron-lim iting conditions. Thus, the hydroxamate compound has siderophore activity. A 9.1-kb fragment of chromosomal DNA containing the Tn5 insertion from this mutant was cloned and marker exchanged into wild-type strain K84. The homo genote lost the ability to produce the hydroxamate siderophore and also ALS 84. A cosmid clone was isolated from a genomic library of strain K84 that r estored to strain M8-10 the ability to produce of the siderophore and ALS84 , as well as growth in iron-deficient medium. This cosmid clone contained t he region in which Tn5 was located in the mutant. Sequence analysis showed that the Tn5 insert in this mutant was located in an open reading frame cod ing for a protein that has similarity to those of the gramicidin S syntheta se repeat superfamily. Some such proteins are required for synthesis of hyd roxamate siderophores by other bacteria. Southern analysis revealed that th e biosynthetic gene from strain K84 is present only in isolates of A. rhizo genes that produce hydroxamate-type compounds under low-iron conditions. Ba sed on physiological and genetic analyses showing a correlation between pro duction of a hydroxamate siderophore and ALS84 by strain K84, we conclude t hat the two activities share a biosynthetic route and may be the same compo und.