Proteomic and Mutant Analysis of Hydrogenase Maturation Protein Gene hypE in Symbiotic Nitrogen Fixation of Mesorhizobium huakuii.

Hydrogenases catalyze the simple yet important redox reaction between protons and electrons and H2, thus mediating symbiotic interactions. The contribution of hydrogenase to this symbiosis and anti-oxidative damage was investigated using the M. huakuii hypE (encoding hydrogenase maturation protein) mutant. The hypE mutant grew a little faster than its parental 7653R and displayed decreased antioxidative capacity under H2O2-induced oxidative damage. Real-time quantitative PCR showed that hypE gene expression is significantly up-regulated in all the detected stages of nodule development. Although the hypE mutant can form nodules, the symbiotic ability was severely impaired, which led to an abnormal nodulation phenotype coupled to a 47% reduction in nitrogen fixation capacity. This phenotype was linked to the formation of smaller abnormal nodules containing disintegrating and prematurely senescent bacteroids. Proteomics analysis allowed a total of ninety differentially expressed proteins (fold change > 1.5 or <0.67, p < 0.05) to be identified. Of these proteins, 21 are related to stress response and virulence, 21 are involved in transporter activity, and 18 are involved in energy and nitrogen metabolism. Overall, the HypE protein is essential for symbiotic nitrogen fixation, playing independent roles in supplying energy and electrons, in bacterial detoxification, and in the control of bacteroid differentiation and senescence.

The Mesorhizobium huakuii transcriptional regulator AbiEi plays a critical role in nodulation and is important for bacterial stress response.

BACKGROUND: Bacterial abortive infection (Abi) systems are type IV toxin-antitoxin (TA) system, which could elicit programmed cell death and constitute a native survival strategy of pathogenic bacteria under various stress conditions. However, no rhizobial AbiE family TA system has been reported so far. Here, a M. huakuii AbiE TA system was identified and characterized. RESULTS: A mutation in M. huakuii abiEi gene, encoding an adjacent GntR-type transcriptional regulator, was generated by homologous recombination. The abiEi mutant strain grew less well in rich TY medium, and displayed increased antioxidative capacity and enhanced gentamicin resistance, indicating the abiEi operon was negatively regulated by the antitoxin AbiEi in response to the oxidative stress and a particular antibiotic. The mRNA expression of abiEi gene was significantly up-regulated during Astragalus sinicus nodule development. The abiEi mutant was severely impaired in its competitive ability in rhizosphere colonization, and was defective in nodulation with 97% reduction in nitrogen-fixing capacity. The mutant infected nodule cells contained vacuolation and a small number of abnormal bacteroids with senescence character. RNA-seq experiment revealed it had 5 up-regulated and 111 down-regulated genes relative to wild type. Of these down-regulated genes, 21 are related to symbiosis nitrogen fixation and nitrogen mechanism, 16 are involved in the electron transport chain and antioxidant responses, and 12 belong to type VI secretion system (T6SS). CONCLUSIONS: M. huakuii AbiEi behaves as a key transcriptional regulator mediating root nodule symbiosis.

Corrigendum: Rhizobium leguminosarum Glutathione Peroxidase Is Essential for Oxidative Stress Resistance and Efficient Nodulation.

[This corrects the article DOI: 10.3389/fmicb.2021.627562.].

Rhizobium leguminosarum Glutathione Peroxidase Is Essential for Oxidative Stress Resistance and Efficient Nodulation.

Glutathione (GSH) plays a key role in regulating the cellular Redox Homeostasis, and appears to be essential for initiation and development of root nodules. Glutathione peroxidase (Gpx) catalyzes the reduction of H2O2 and organic hydroperoxides by oxidation of GSH to oxidized GSH (GSSG), which in turn is reduced by glutathione reductase (GR). However, it has not been determined whether the Rhizobium leguminosarum Gpx or GR is required during symbiotic interactions with pea. To characterize the role of glutathione-dependent enzymes in the symbiotic process, single and double mutants were made in gpxA (encoding glutathione peroxidase) and gshR (encoding glutathione reductase) genes. All the mutations did not affect the rhizobial growth, but they increased the sensitivity of R. leguminosarum strains to H2O2. Mutant in GpxA had no effect on intracellular GSH levels, but can increase the expression of the catalase genes. The gshR mutant can induce the formation of normal nodules, while the gpxA single and double mutants exhibited a nodulation phenotype coupled to more than 50% reduction in the nitrogen fixation capacity, these defects in nodulation were characterized by the formation of ineffective nodules. In addition, the gpxA and gshR double mutant was severely impaired in rhizosphere colonization and competition. Quantitative proteomics using the TMT labeling method was applied to study the differential expression of proteins in bacteroids isolated from pea root nodules. A total of 27 differentially expressed proteins were identified in these root bacteroids including twenty down-regulated and seven up-regulated proteins. By sorting the down-regulated proteins, eight are transporter proteins, seven are dehydrogenase, deoxygenase, oxidase, and hydrolase. Moreover, three down-regulating proteins are directly involved in nodule process.