Analysis of the sequence immediate upstream of ohr revealed an open reading frame, designated ohrR, with the potential to encode a 17-kDa peptide with moderate amino acid sequence homology to the MarR family of negative regulators of gene expression. ohrR was transcribed as bicistronic mRNA with ohr, while ohr mRNA was found to be 95% monocistronic and 5% bicistronic with ohrR. Expression of both genes was induced by tert-butyl hydroperoxide (tBOOH) treatment. High-level expression of ohrR negatively regulated ohr expression. This repression could be overcome by tBOOH treatment. In vivo promoter analysis showed that the ohrR promoter (P1) has organic peroxide-inducible, strong activity, while the ohr promoter (P2) has constitutive, weak activity. Only P1 is autoregulated by OhrR. ohr primer extension results revealed three major primer extension products corresponding to the 5 ends of ohr mRNA, and their levels were strongly induced by tBOOH treatment. Sequence analysis of regions upstream of these sites showed no typical Xanthomonas promoter. Instead, the regions can form a stem-loop secondary structure with the 5 ends of ohr mRNA located in the loop section. The secondary structure resembles the structure recognized and processed by RNase III enzyme. These findings suggest that the P1 promoter is responsible for tBOOH-induced expression of the ohrR-ohr operon. The bicistronic mRNA is then processed by RNase III-like enzymes to give high levels of ohr mRNA, while ohrR mRNA is rapidly degraded.

The analysis of genetics and physiological functions of Agrobacterium tumefaciens RirA (rhizobial iron regulator) has shown that it is a transcription regulator and a repressor of iron uptake systems. The rirA mutant strain (NTLrirA) overproduced siderophores and exhibited a highly constitutive expression of genes involved in iron uptake (fhuA, irp6A, and fbpA) compared to that of the wild-type strain (NTL4). The deregulation in the iron control of iron uptake in NTLrirA led to iron overload in the cell, which was supported by the observation that the NTLrirA mutant was more sensitive than wild-type NTL4 to an iron-activated antibiotic, streptonigrin. The NTLrirA mutant was more sensitive than the parental strain to oxidants, including hydrogen peroxide, organic hydroperoxide, and a superoxide generator, menadione. However, the addition of an iron chelator, 2,2-dipyridyl, reversed the mutant hypersensitivity to H 2 O 2 and organic hydroperoxide, indicating the role of iron in peroxide toxicity. Meanwhile, the reduced level of superoxide dismutase (SodBIII) was partly responsible for the menadione-sensitive phenotype of the NTLrirA mutant. The NTLrirA mutant showed a defect in tumorigenesis on tobacco leaves, which likely resulted from the increased sensitivity of NTLrirA to oxidants and the decreased ability of NTLrirA to induce virulence genes (virB and virE). These data demonstrated that RirA is important for A. tumefaciens during plant-pathogen interactions.

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.

We constructed and characterized a Xanthomonas campestris pv. phaseoli oxyR mutant. The mutant was hypersensitive to H2O2 and menadione killing and had reduced aerobic plating efficiency. The oxidants’ induction of the catalase and ahpC genes was also abolished in the mutant. Analysis of the adaptive responses showed that hydrogen peroxide-induced protection against hydrogen peroxide was lost, while menadione-induced protection against hydrogen peroxide was retained in the oxyR mutant. These results show that X. campestris pv. phaseoli oxyR is essential to peroxide adaptation and revealed the existence of a novel superoxide-inducible peroxide protection system that is independent of OxyR.

Agrobacterium tumefaciens membrane-bound ferritin (MbfA) is a member of the erythrin (Er)–vacuolar iron transport family. The MbfA protein has an Er or ferritin-like domain at its N terminus and has been predicted to have five transmembrane segments in its C-terminal region. Analysis of protein localization using PhoA and LacZ reporter proteins supported the view that the N-terminal di-iron site is located in the cytoplasm whilst the C-terminal end faces the periplasm. An A. tumefaciens mbfA mutant strain had 1.5-fold higher total iron content than the WT strain. Furthermore, multi-copy expression of mbfA reduced total iron content two- and threefold in WT and mbfA mutant backgrounds, respectively. These results suggest that MbfA may function as an iron exporter rather than an iron storage protein. The mbfA mutant showed 10-fold increased sensitivity to the iron-activated antibiotic streptonigrin, implying that the mutant had increased accumulation of intracellular free iron. Growth of the mbfA mutant was reduced in the presence of high iron under acidic conditions. The expression of mbfA was induced highly in cells grown in iron-replete medium at pH 5.5, further supporting the view that mbfA is involved in the response to iron under acidic conditions. A. tumefaciens MbfA may play a protective role against increased free iron in the cytoplasm through iron binding and export, thus preventing iron-induced toxicity via the Fenton reaction.

The repressor for an organic peroxide-inducible operon is uniquely regulated at multiple levels duced cells, the concentration of OhrR is maintained at low levels by the autoregulation mechanism at the transcriptional levels and by the ohrR mRNA instability coupled with inefficient translation at the posttranscriptional level. Upon exposure to an organic peroxide, the compound probably interacts with OhrR and prevents it from repressing the P1 promoter, thus allowing high-level expression of the ohrR-ohr operon. The rapid processing of bicistronic mRNA gives highly stable ohr mRNA and corresponding high levels of Ohr, which remove an organic peroxide. Once the peroxide has been removed, the autoregulation mechanism feeds back to inhibit the expression of the operon. IntroductionXanthomonas is a soil bacterium that causes diseases in plants. In the environment, Xanthomonas is exposed to reactive oxygen species (ROS) from a variety of sources (Baker and Orlandi, 1995;Gonzalez-Flecha and Demple, 1997). These ROS (e.g. superoxide, H 2 O 2 and organic peroxide) are highly toxic to biological systems (Halliwell and Gutteridge, 1984). Bacteria have evolved multiple protective pathways to ensure their detoxification. Bacterial defence against organic peroxide is a complex process involving several structural and regulatory genes (Storz and Imlay, 1999).Alkyl hydroperoxide reductase is the best characterized organic peroxide detoxification enzyme in bacteria (Poole, 1996;Poole and Ellis, 1996). ahpC encodes the catalytic subunit of the enzyme and is regulated by OxyR, the global regulator of peroxide stress response (Storz and Imlay, 1999). ahpC expression is induced by exposure of bacteria to various oxidants (Bsat et al., 1997;Loprasert et al., 1997;Rocha and Smith, 1999;Storz and Imlay, 1999). Inactivation of ahpC results in pleiotropic changes in oxidative stress response, suggesting that it involves processes other than organic peroxide detoxification (Bsat et al., 1996;Rocha and Smith, 1999;Mongkolsuk et al., 2000;Seaver and Imlay, 2001).We discovered a novel family of genes in Xanthomonas designated ohr that are involved in organic peroxide resistance (Mongkolsuk et al., 1998). ohr homologues have subsequently been found in both Gram-positive and Gram-negative bacteria Fuangthong et al., 2001;Ochsner et al., 2001; Rince Molecular Microbiology (2002) SummaryohrR encodes a novel organic peroxide-inducible transcription repressor, and we have demonstrated that ohrR is regulated at the transcriptional and the post-transcriptional levels. Primer extension results show that ohrR transcription initiates at the A residue of the ATG translation initiation codon for the ohrR coding sequence. Thus, the gene has a leaderless mRNA. The ohrR promoter (P1) has high homology to the consensus sequence for Xanthomonas promoters, which is reflected in the high in vivo promoter activity of P1. Deletion of a 139 bp fragment containing the P1 promoter showed that the sequences upstream of -35 regions were required for neither the ...

Control of zinc homeostasis in Agrobacterium tumefaciens via zur and the zinc uptake genes znuABC and zinT The Agrobacterium tumefaciens zinc uptake regulator (Zur) was shown to negatively regulate the zinc uptake genes znuABC, encoding a zinc transport system belonging to the ATP-binding cassette (ABC) transporter family, and zinT, which encodes a periplasmic zinc-binding protein.The expression of znuABC and zinT was inducible when cells were grown in medium containing a metal chelator (EDTA), and this induction was shown to be specific for zinc depletion. The expression of znuABC was reduced in response to increased zinc in a dose-dependent manner, and zinT had a less pronounced but similar pattern of zinc-regulated expression. The inactivation of zur led to constitutively high expression of znuABC and zinT. In addition, a zur mutant had an increased total zinc content compared to the WT NTL4 strain, whereas the inactivation of zinT caused a reduction in the total zinc content. The zinT gene is shown to play a dominant role and to be more important than znuA and znuB for A. tumefaciens survival under zinc deprivation. ZinT can function even when ZnuABC is inactivated. However, mutations in zur, znuA, znuB or zinT did not affect the virulence of A. tumefaciens.

An Agrobacterium tumefaciens membrane-bound ferritin (mbfA) mutant was generated to assess the physiological functions of mbfA in response to iron and hydrogen peroxide (H(2) O(2) ) stresses. Wild-type and the mbfA mutant strains showed similar growth under high- and low-iron conditions. The mbfA mutant was more sensitive to H(2) O(2) than wild-type strain. Expression of a functional mbfA gene could complement the H(2) O(2) -hypersensitive phenotype of the mbfA mutant and a rhizobial iron regulator (rirA) mutant, suggesting that MbfA protects cells from H(2) O(2) toxicity by sequestering intracellular free iron, thus preventing the Fenton reaction. The expression of mbfA could be induced in response to iron and to H(2) O(2) treatment. The iron response regulator (irr) also acted as a repressor of mbfA expression. An irr mutant had high constitutive expression of mbfA, which partly contributed to the H(2) O(2) -hyperresistant phenotype of the irr mutant. The data reported here demonstrate an important role of A. tumefaciens MbfA in the cellular defence against iron and H(2) O(2) stresses.