Agrobacterium tumefaciens iron superoxide dismutases have protective roles against singlet oxygen toxicity generated from illuminated Rose Bengal.

Singlet oxygen is a highly reactive form of molecular oxygen that is harmful to biological systems. Here, the role of three iron-containing superoxide dismutase (sodB) genes is clearly shown in protecting Agrobacterium tumefaciens against singlet oxygen toxicity. A sodBI mutant was more sensitive to singlet oxygen than both wild-type bacteria and a double sodBII-sodBIII mutant strain. Moreover, a sodBI-sodBII double mutant had higher sensitivity to singlet oxygen than a single sodBI mutant, although the double mutant was comparable to a sodB null mutant. High-level expression of sodBI and sodBII fully complemented the singlet oxygen hypersensitivity phenotype of the sodB null mutant, while high-level expression of sodBIII encoding a periplasmic SOD only partially restored the phenotype. Taken together, our data suggest that SodBI and SodBII have novel protective roles against singlet oxygen toxicity through unknown mechanisms.

Important role for methionine sulfoxide reductase in the oxidative stress response of Xanthomonas campestris pv. phaseoli.

A methionine sulfoxide reductase gene (msrA) from Xanthomonas campestris pv. phaseoli has unique expression patterns and physiological function. msrA expression is growth dependent and is highly induced by exposure to oxidants and N-ethylmaleimide in an OxyR- and OhrR-independent manner. An msrA mutant showed increased sensitivity to oxidants but only during stationary phase.

OxyR mediated compensatory expression between ahpC and katA and the significance of ahpC in protection from hydrogen peroxide in Xanthomonas campestris.

katA and ahpC, encoding monofunctional catalase and alkyl hydroperoxide reductase, respectively, play important protective roles against peroxide toxicity in Xanthomonas campestris pv. phaseoli (Xp). The expression of both katA and ahpC is controlled by the global peroxide sensor and transcriptional activator, OxyR. In Xp, these two genes have compensatory expression patterns. Inactivation of katA leads to an increase in the level of AhpC and a concomitant increase in resistance to tert-butyl hydroperoxide (tBOOH). High-level expression of katA from an expression vector in Xp also lowered the level of ahpC expression. The compensatory regulation of katA and ahpC was mediated by OxyR, since the compensatory response was not observed in an oxyR mutant background. ahpC and katA play important but unequal roles in protecting Xp from H(2)O(2) toxicity. These observations, taken together with a previous observation that an ahpC mutant expresses high levels of KatA and is hyper-resistant to H(2)O(2), suggest the possibility that inactivation of either gene leads to accumulation of intracellular H(2)O(2). This in turn oxidizes reduced OxyR and converts the regulator to the oxidized form that then activates expression of genes in the OxyR regulon.

Genetic and physiological analysis of the major OxyR-regulated katA from Xanthomonas campestris pv. phaseoli.

katA encodes the major catalase that accounts for 90 % of the total catalase activity present in Xanthomonas campestris pv. phaseoli. katA is located upstream of an ORF designated ankA encoding a cytoplasmic membrane protein homologous to eukaryotic ankyrin. Transcriptional analysis of katA and ankA identified two katA transcripts: a major monocistronic katA transcript and a minor bicistronic katA-ankA transcript. KatA expression was induced in the presence of various oxidants including H2O2, organic hydroperoxides and the superoxide-generating agent menadione, in an OxyR-dependent manner. Analysis of the katA promoter region showed a putative OxyR binding site located upstream of an Escherichia coli-like sigma70 -35 region that is likely to be responsible for transcription activation in response to oxidant treatment. Gel mobility shift experiments confirmed that purified OxyR specifically binds to the katA promoter. A katA mutant was highly sensitive to H2O2 during both the exponential and stationary phases of growth. This phenotype could be complemented by functional katA, confirming the essential role of the gene in protecting X. campestris from H2O2 toxicity. Unexpectedly, inactivation of ankA also significantly reduced resistance to H2O2 and the phenotype could be complemented by plasmid-borne expression of ankA. Physiological analyses showed that katA plays an important role in, but is not solely responsible for, both the adaptive and menadione-induced cross-protective responses to H2O2 killing in X. campestris.

The repressor for an organic peroxide-inducible operon is uniquely regulated at multiple levels.

ohrR 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 promoter activity nor OhrR autoregulation. In vitro, purified OhrR specifically binds to the P1 promoter. DNase I footprinting of OhrR binding to the P1 revealed a 44 bp region of protection on both DNA strands. The protected regions include the -35 and -10 regions of P1. We suggest that OhrR represses gene expression by blocking RNA polymerase binding to the promoter. There are two steps in the post-transcriptional regulation of ohrR, namely differential stability and inefficient translation of the mRNA. The bicistronic ohrR-ohr mRNA was highly labile and underwent rapid processing in vivo to give only stable monocistronic ohr mRNA and undetectable ohrR mRNA. Furthermore, the ohrR mRNA was inefficiently translated. We propose that, in uninduced 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 post-transcriptional 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 per-oxide. Once the peroxide has been removed, the autoregulation mechanism feeds back to inhibit the expression of the operon.