Danièle Touati

Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator

The Sm‐like protein Hfq is involved in post‐transcriptional regulation by small, noncoding RNAs in Escherichia coli that act by base pairing. Hfq stabilises the small RNAs and mediates their interaction with the target mRNA by an as yet unknown mechanism. We show here a novel chaperoning use of Hfq in the regulation by small RNAs. We analysed in vitro and in vivo the role of Hfq in the interaction between the small RNA RyhB and its sodB (iron superoxide dismutase) mRNA target. Hfq bound strongly to sodB mRNA and altered the structure of the mRNA, partially opening a loop. This gives access to a sequence complementary to RyhB and encompassing the translation initiation codon. RyhB binding blocked the translation initiation codon of sodB and triggered the degradation of both RyhB and sodB mRNA. Thus, Hfq is a critical chaperone in vivo and in vitro, changing the folding of the target mRNA to make it subject to the small RNA regulator.

Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction.

Reactive oxygen species are produced as an early event in plant defense response against avirulent pathogens. We show here that alfalfa responds to infection with Sinorhizobium meliloti by production of superoxide and hydrogen peroxide. This similarity in the early response to infection by pathogenic and symbiotic bacteria addresses the question of which mechanism rhizobia use to counteract the plant defense response.

Critical protective role of bacterial superoxide dismutase in Rhizobium–legume symbiosis

In nitrogen‐poor soils, rhizobia elicit nodule formation on legume roots, within which they differentiate into bacteroids that fix atmospheric nitrogen. Protection against reactive oxygen species (ROS) was anticipated to play an important role in Rhizobium–legume symbiosis because nitrogenase is extremely oxygen sensitive. We deleted the sodA gene encoding the sole cytoplasmic superoxide dismutase (SOD) of Sinorhizobium meliloti. The resulting mutant, deficient in superoxide dismutase, grew almost normally and was only moderately sensitive to oxidative stress when free living. In contrast, its symbiotic properties in alfalfa were drastically affected. Nitrogen‐fixing ability was severely impaired. More strikingly, most SOD‐deficient bacteria did not reach the differentiation stage of nitrogen‐fixing bacteroids. The SOD‐deficient mutant nodulated poorly and displayed abnormal infection. After release into plant cells, a large number of bacteria failed to differentiate into bacteroids and rapidly underwent senescence. Thus, bacterial SOD plays a key protective role in the symbiotic process.

Reactive oxygen species, nitric oxide and glutathione: a key role in the establishment of the legume-Rhizobium symbiosis?

Reactive oxygen species are generated in the first steps of the Rhizobium–legume symbiosis. Superoxide radicals and hydrogen peroxide have been detected in infection threads and there is also evidence of the presence of nitric oxide in young alfalfa nodules. Moreover, rhizobial mutants, with a reduced antioxidant defense, exhibit an impaired capacity to nodulate. The oxidative burst generated in response to symbiotic infection can be consistent with rhizobia being initially perceived as invaders by the plant; in this framework, it may be correlated with the existence of abortive infections. However, the burst appears to be also involved in the expression of early nodulins associated with successful infections. Thus, in parallel to its involvement in defense processes, a positive role for the oxidative burst (including nitric oxide) in the establishment of the symbiotic interaction can also be proposed. The burst could trigger the expression of plant and/or bacterial genes which are essential for the nodulation process. In this framework, glutathione and homoglutathione could be key intermediates for gene expression, via the modification of the redox balance. Thus, the oxidative burst may have a dual role in the establishment of the symbiosis.

Involvement of Superoxide Dismutases in the Response of Escherichia coli to Selenium Oxides

Selenium can provoke contrasting effects on living organisms. It is an essential trace element, and low concentrations have beneficial effects, such as the reduction of the incidence of cancer. However, higher concentrations of selenium salts can be toxic and mutagenic. The bases for both toxicity and protection are not clearly understood. To provide insights into these mechanisms, we analyzed the proteomic response of Escherichia coli cells to selenate and selenite treatment under aerobic conditions. We identified 23 proteins induced by both oxides and ca. 20 proteins specifically induced by each oxide. A striking result was the selenite induction of 8 enzymes with antioxidant properties, particularly the manganese and iron superoxide dismutases (SodA and SodB). The selenium inductions of sodA and sodB were controlled by the transcriptional regulators SoxRS and Fur, respectively. Strains with decreased superoxide dismutase activities were severely impaired in selenium oxide tolerance. Pretreatment with a sublethal selenite concentration triggered an adaptive response dependent upon SoxRS, conferring increased selenite tolerance. Altogether, our data indicate that superoxide dismutase activity is essential for the cellular defense against selenium salts, suggesting that superoxide production is a major mechanism of selenium toxicity under aerobic conditions.

Essential Role of Superoxide Dismutase on the Pathogenicity of Erwinia chrysanthemi Strain 3937

The sodA gene from Erwinia chrysanthemi strain 3937 was cloned by functional complementation of an Escherichia coli sodA sodB mutant and sequenced. We identified a 639-bp open reading frame, which encodes a protein that is 85% identical to the E. coli manganese-containing superoxide dismutase MnSOD. Promoter elements of this gene were identified by transcriptional mapping experiments. We constructed an E. chrysanthemi deltasodA mutant by reverse genetics. The deltasodA mutation resulted in the absence of a cytoplasmic SOD, which displays the same characteristics as those of MnSOD. The deltasodA mutant was more sensitive to paraquat than the wild-type strain. This mutant could macerate potato tubers, similar to the wild-type strain. In contrast, when inoculated on African violets, the mutant produced, at most, only small necrotic lesions. If the inoculum was supplemented with the superoxide anion-scavenging metalloporphyrin MnTMPyP or purified SOD and catalase, the deltasodA mutant was able to macerate the inoculated zone. Generation of superoxide anion by African violet leaves inoculated with E. chrysanthemi was demonstrated with nitroblue tetrazolium as an indicator. Therefore, at the onset of infection, E. chrysanthemi cells encounter an oxidative environment and require active protective systems against oxidative damages such as MnSOD to overcome these types of conditions.

Activation of SoxR by Overproduction of Desulfoferrodoxin: Multiple Ways To Induce the soxRS Regulon

The soxRS response, which protects cells against superoxide toxicity, is triggered by the oxidation of SoxR, a transcription factor. Superoxide excess and NADPH depletion induce the regulon. Unexpectedly, we found that the overproduction of desulfoferrodoxin, a superoxide reductase from sulfate-reducing bacteria, also induced this response. We suggest that desulfoferrodoxin interferes with the reducing pathway that keeps SoxR in its inactive form.