The genes involved in organic hydroperoxide protection in Agrobacterium tumefaciens were functionally evaluated. Gene inactivation studies and functional analyses have identified ohr, encoding a thiol peroxidase, as the gene primarily responsible for organic hydroperoxide protection in A. tumefaciens. An ohr mutant was sensitive to organic hydroperoxide killing and had a reduced capacity to metabolize organic hydroperoxides. ohr is located next to, and is divergently transcribed from, ohrR, encoding a sensor and transcription regulator of organic hydroperoxide stress. Transcription of both ohr and ohrR was induced by exposure to organic hydroperoxides but not by exposure to other oxidants. This induction required functional ohrR. The results of gel mobility shift and DNase I footprinting assays with purified OhrR, combined with in vivo promoter deletion analyses, confirmed that OhrR regulated both ohrR and ohr by binding to a single OhrR binding box that overlapped the ohrR and ohr promoters. ohrR and ohr are both required for the establishment of a novel cumene hydroperoxide-induced adaptive response. Inactivation or overexpression of other Prx family genes (prx1, prx2, prx3, bcp1, and bcp2) did not affect either the resistance to, or the ability to degrade, organic hydroperoxide. Taken together, the results of biochemical, gene regulation and physiological studies support the role of ohrR and ohr as the primary system in sensing and protecting A. tumefaciens from organic hydroperoxide stress.

Thioredoxin-like protein (TlpA) is a membrane-anchored periplasmic protein that contains a thioredoxin domain at its C-terminus. An Agrobacterium tumefaciens mutant lacking functional tlpA shows increased sensitivity to a superoxide generator, increased resistance to H2O2, and reduced cytochrome c oxidase activity. Whereas the levels of antioxidant enzymes, including total superoxide dismutase (SOD) and catalases, are unaltered, high expression of periplasmic SOD partially restores the superoxide hypersensitivity of the mutant, suggesting an accumulation of superoxide anions in the periplasm. The change in the ability of the mutant to cope with H2O2 stress is independent of OxyR, an H2O2 sensor and transcription regulator. The presented data indicate that TlpA not only is involved in the proper assembly of cytochrome c but is also implicated in the bacterial oxidative stress response.

Exposure of Agrobacterium tumefaciens to menadione, cumene hydroperoxide, and diamide strongly induced trxA expression. The trxA mutant showed a reduction in the aerobic growth rate and plating efficiency and was cytochrome c oxidase negative. Atypically, the mutant has decreased resistance to menadione but an increased H 2 O 2 resistance phenotype.

In the legend to Fig. 5A, the third sentence should be replaced with the following: "The C, T, A, and G lanes of a dideoxy sequencing ladder using the pUC/M13 forward 24-mer primer and pGEM-3Zf(ϩ) as the template are shown. The primers 5ЈCGGTGGCGGATGCTTTGG3Ј and 5ЈCTGCGCCGTAAAGGGCAA3Ј were used in ohr and ohrR primer extension experiments, respectively."