Iron-Binding Compounds from Agrobacterium spp.: Biological Control Strain Agrobacterium rhizogenes K84 Produces a Hydroxamate Siderophore

Abstract

Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the species A. tumefaciens, A. rhizogenes, and A. vitis. The crown gall biocontrol agent A. rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media. Five independent 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 production of both agents and grew poorly compared to the wild type under iron-limiting 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 homogenote lost the ability to produce the hydroxamate siderophore and also ALS84. A cosmid clone was isolated from a genomic library of strain K84 that restored 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 the 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 coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily. Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria. Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of A. rhizogenes that produce hydroxamate-type compounds under low-iron conditions. Based on physiological and genetic analyses showing a correlation between production of a hydroxamate siderophore and ALS84 by strain K84, we conclude that the two activities share a biosynthetic route and may be the same compound.Siderophores are relatively low-molecular-weight, iron-chelating agents produced by bacteria and fungi growing under low-iron stress conditions (42). These compounds bind ferric iron (Fe 3ϩ ) with high affinity and transport the metal into the cell via dedicated uptake systems (41). Microbial siderophores vary widely in their overall structure, but most of them contain hydroxamate or catechol groups, which are involved in chelating the ferric ion (42). Apart from their role in the active transport of iron, siderophores may act as growth factors, and some show potent antimicrobial activity (41).The capacity to utilize siderophores is important to the growth of bacteria in the rhizosphere (27) and on plant surfaces (34). Specific siderophore-producing Pseudomonas strains rapidly colonize plant roots of several crops, and this colonization can result in significant yield increas...

Gene cluster for ferric iron uptake in Agrobacterium tumefaciens MAFF301001.

“…were no positive signals in the macro-fragments from strain C58 (lanes 3 and 4). This is consistent with the previous report that strain C58 does not produce detectable siderophore activity even in iron-limiting condition (Penyalver et al, 2001). …”

“…A. tumefaciens strain B6 produces agrobactin which is a typical catechol-type siderophore (Ong et al, 1979). Some strains of A. tumefaciens produce either catechol-type or hydroxamate-type siderophores or both (Penyalver et al, 2001). In E. coli, V. cholerae and P. fluorescens, the genes involved in production of siderophores have also been identified.…”

“…The genome sequence was recently determined completely in A. tumefaciens strain C58. Though six putative different iron uptake systems were found in C58 genome (Goodner et al, 2001;Wood et al, 2001), both function and expression of the systems remain unknown because strain C58 does not produce either catechol or hydroxamate siderophores (Penyalver et al, 2001).…”

Antimicrobial Properties of Pyridine-2,6-Dithiocarboxylic Acid, a Metal Chelator Produced by Pseudomonas spp

“…There are a variety of biochelators produced by microorganisms which do not function as siderophores (23), and some rather well-known siderophores appear to have additional activities apart from transport of metals, such as antioxidant and antibiotic action (10,25,28,30). The antimicrobial activity of siderophores can have significant ecological effects.…”

Exploiting inter-organismal chemical communication for improved inoculants

“…trifolii CB782 and Rhizobium tropici WSM1385, among others, produce siderophores capable of binding available Fe 3+ , then transporting it to citrate (Carson et al 2000). One of the agents responsible for controlling crown gall disease, Agrobacterium rhizogenes K84 produces an iron chelator, responsible for indirect antibiotic activity, when cultured in a low-Fe medium (Penyalver et al 2001). Plants are able to withstand lower levels of Fe than prokaryotes (Johnson et al 2002).…”