Agrobacterium tumefaciens fur Has Important Physiological Roles in Iron and Manganese Homeostasis, the Oxidative Stress Response, and Full Virulence

Abstract

In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the fur mutant was more sensitive than the wild type to an iron chelator, 2,2-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations. A. tumefaciens sitA, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (sitABCD), was strongly repressed by Mn 2؉ and, to a lesser extent, by Fe 2؉ , and this regulation was Fur dependent. Moreover, the fur mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the fur mutant showed constitutive up-expression of the manganese uptake sit operon. Fur At showed a regulatory role under ironlimiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The fur mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the fur mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4.

Antiparallel and Interlinked Control of Cellular Iron Levels by the Irr and RirA Regulators of Agrobacterium tumefaciens

“…In particular, increased sensitivity to H 2 O 2 correlates with increased available cellular iron (66). The iron-activated antimicrobial streptonigrin has also been used to approximate the iron state of several bacterial species, with an increased SNG sensitivity implying an increased available cellular iron (33,43,70). In A. tumefaciens, it has previously been shown that a disruption of rirA results in an increased sensitivity to both H 2 O 2 and SNG, and it has been suggested that increased sensitivity is due to increased iron concentrations in the cell (43).…”

“…tumefaciens encodes homologues of fur, rirA, and irr, as well as a large cluster of genes that directs the synthesis of a siderophore (20,54,72). The A. tumefaciens fur homologue is reported to primarily regulate manganese uptake and plays only a minor role in response to iron levels (33,53). The RirA and Irr proteins are in fact the probable iron-responsive regulatory candidates.…”

Roles of Agrobacterium tumefaciens RirA in Iron Regulation, Oxidative Stress Response, and Virulence

“…The disruption of the rirA gene (Atu0201) (34) was performed by the method described previously (14). The internal DNA fragment of the rirA coding region was amplified by PCR with primers BT1201 and BT1202, using Pfu DNA polymerase and genomic DNA isolated from wild-type NTL4 as the template.…”

“…In some experiments, cells were grown in media with 50 M AS in the presence of either 50 M FeCl 3 or an iron chelator, 50 M Dy. Cells were further incubated at 28°C with shaking for 24 h. Cells were harvested, and ␤-galactosidase activity was measured as previously described (14). ␤-Galactosidase activity is presented in units per milligram of protein.…”

“…A. tumefaciens strains containing pCMA1 plasmid were used to infect tobacco (Nicotiana tabacum) leaves according to the method described previously (14,17).…”