Antioxidation and symbiotic nitrogen fixation function of prxA gene in Mesorhizobium huakuii

Abstract Peroxiredoxins (Prxs) play an essential role in the antioxidant activity and symbiotic capacity of Mesorhizobium huakuii. A mutation in the M. huakuii prxA gene (encoding a Prx5‐like peroxiredoxin) was generated by homologous recombination. The mutation of prxA did not affect M. huakuii growth, but the strain displayed decreased antioxidative capacity under organic cumene hydroperoxide (CUOOH) conditions. The higher resistance of the prxA mutant strain compared with the wild‐type strain to more than 1 mmol/L H2O2 was associated with a significantly higher level of glutathione reductase activity and a significantly lower level of intracellular hydrogen peroxide content. Real‐time quantitative PCR showed that under 1 mmol/L H2O2 conditions, expression of the stress‐responsive genes katG and katE was significantly upregulated in the prxA mutant. Although the prxA mutant can form nodules, the symbiotic ability was severely impaired, which led to an abnormal nodulation phenotype coupled to a 53.25% reduction in nitrogen fixation capacity. This phenotype was linked to an absence of bacteroid differentiation and deregulation of the transcription of the symbiotic genes nifH, nifD, and fdxN. Expression of the prxA gene was induced during symbiosis. Thus, the PrxA protein is essential for antioxidant capacity and symbiotic nitrogen fixation, playing independent roles in bacterial differentiation and cellular antioxidative systems.

molecule (flavonoid) that is perceived by the bacterium Rhizobium. In response, the Rhizobium activates transcription of nod genes and produces lipo-chitooligosaccharides (Nod factors), which provoke tight root hair curls (Gressent et al., 2002). Subsequently, rhizobia enter their host cells via endocytosis and develop infection threads that are filled with proliferating bacteria. The infection threads elongate toward root hairs and penetrate the root layers. Bacteria differentiate into large pleiomorphic, nitrogen-fixing bacteroids, leading to cell division and the consequent formation of nodules in which bacteroids reduce atmospheric nitrogen to ammonia, which is used by leguminous plants as a nitrogen source (Brewin, 2004).
During the infection process and nitrogen-fixing symbiosis, rhizobia are greatly dependent on antioxidant systems to protect themselves against oxidant damage (Dombrecht et al., 2010).
Numerous antioxidative systems can contribute to maintaining certain levels of reactive oxygen species (ROS), which include antioxidant enzymes such as catalases, glutathione (GSH), glutathione peroxidases (Gpxs), superoxide dismutases and a recently identified rapidly growing family of peroxiredoxins (Prxs) (Duan et al., 2013). Prxs are cysteine-dependent peroxidases of the thioredoxin family that react with hydrogen peroxide, larger hydroperoxide substrates, and peroxynitrite through the formation of a cysteine sulfenic acid (R-SOH) at the active site (Parsonage et al., 2015). An intriguing biophysical property of Prxs is the redox-dependent modulation of their oligomeric state between decamers and dimers within the physiological concentration range (Parsonage et al., 2005). Prxs are ubiquitously distributed in all organisms, including plants and bacteria as well as animals, to protect cells against oxidative stress-induced damage (Umate, 2010). Thus far, six isoforms of Prxs have been characterized in mammals, and all of the Prxs are implicated, via their antioxidant activity, in intracellular signaling and in the regulation of processes such as cell proliferation and differentiation and protection of other proteins from oxidative damage (Lim et al., 1998).
Moreover, Prxs have also been shown to be induced by oxidative stress and to be associated with the development, progression, and drug resistance of cancer (Duan et al., 2013). In Rhizobium etli, the peroxiredoxin gene prxS is strongly expressed under microaerobic conditions and plays an important role against oxidative stress during symbiotic interactions with the host Phaseolus vulgaris (Dombrecht et al., 2010). In rhizobia-Pisum sativum symbiosis, a reduction in the nodule cytosolic peroxiredoxin contributes to nodule senescence (Groten et al., 2006).
Astragalus sinicus (Chinese milk vetch) is an important wintergrowing green manure that has also been used to fertilize rice fields in China and Japan for 2,000 years. Mesorhizobium huakuii nodulated only on its host plant, A. sinicus, forming indeterminate nodules (Cheng, Li, & Zhou, 2006). Mesorhizobium huakuii 7653R prxA encodes a protein with peroxiredoxin activity. In this study, mutations in the prxA gene were constructed by homologous recombination, and the role of M. huakuii prxA in oxidative stress and during nitrogen-fixing symbiosis was investigated by analyzing the phenotypes of the mutant strain.
huakuii strains were grown at 28°C with shaking at 200 rpm, and the optical density at 600 nm (OD 600 ) was measured during the culture period.

| Construction and complementation of the prxA gene mutant of M. huakuii 7653R
Primers MKprxUP and MKprxLW were used to PCR amplify the prxA region from M. huakuii 7653R genomic DNA. The 640-bp prxA PCR product was cloned into the BamHI and XbaI sites of pK-19mob-producing plasmid pKprxA. The plasmid pKprxA was conjugated with M. huakuii strain 7653R using pRK2013 as a helper plasmid as previously described (Poole et al., 1994). Insertions into the prxA gene of strain 7653R were confirmed by PCR using prxAMP and a pK19mob-specific primer (either pK19A or pK19B) (Karunakaran et al., 2010).
To complement the prxA mutant, primers prxAhbUP and prxAh-bLW were used to amplify the complete prxA gene from 7653R. The PCR product was digested with BamHI and XbaI and cloned into pBBR1MCS-5, resulting in plasmid pBBRprxA. Plasmid pBBRprxA was conjugated into the mutant strain HKprxA using pRK2013 as a helper plasmid to provide the transfer genes as previously described (Karunakaran et al., 2010).

| Antioxidation experiments
The logarithmic phase (OD 600 : 0.3-0.6) mutant strain HKprxA and wild-type 7653R were collected and washed twice in sterile phosphate-buffered saline (PBS) (1X; 136 mM NaCl, 2.6 mM KCl, 8.0 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 ). Cells were treated with H 2 O 2 and CUOOH at different concentrations (0, 0.5 mmol/L, mmol/L, 5 mmol/L) for 1 hr. Strains were thoroughly washed with distilled water to remove any remaining oxidants, and the diluted TY plate method was used to evaluate the bacterial survival rate. The experiment consisted of three repetitions for each treatment.

| Enzyme activity experiments
For analysis of enzymatic and nonenzymatic antioxidant activities, PBS cells were collected by centrifugation at 5,000 rpm for 5 min at 4°C. The cells were held in an ice-water bath and sonicated for 15 min. The sonicate was centrifuged at 12,000 rpm for 10 min at 4°C. Glutathione reductase activity was determined according to the method of Di Ilio, Polidoro, Arduini, Muccini, & Federici, 1983. GSH content was determined according to the method of Irfan, Aruna, and Saibal (2006). Hydrogen peroxide content was measured as previously described (Maisonneuve, Fraysse, Lignon, Capron, & Dukan, 2008), and peroxidase activity was determined using a peroxidase assay kit (Beyotime, China). rinsed 10 times with sterile water. Inoculation with M. huakuii was performed on 7-day-old seedlings. Plants were incubated in a controlled-environment chamber with an 18-hr photoperiod (day/night temperature, 22°C and 20°C) and were harvested at 4 weeks postinoculation (Poole et al., 1994). The acetylene reduction in the plants was detected by gas chromatographic measurement as previously described (Allaway et al., 2010).

| Construction and antioxidation analysis of the M. huakuii prxA mutant
To confirm the function of the prxA gene in antioxidation and symbiotic ability, a single-crossover integration mutation in prxA was constructed by the homologous recombination method. In liquid TY or AMS minimal medium with succinate or glucose as a carbon source, there is no significant difference in growth between the prxA mutant and wild-type 7653R (data not shown).  (1.0 ± 0.6) × 10 8 (7.9 ± 1.2) × 10 7 (9.2 ± 3.3) × 10 6 (6.8 ± 1.4) × 10 3
conditions. The results showed that the peroxidase activity and GSH content of mutant HKprxA were not different from that of wild-type strain 7653R, but its hydrogen peroxide content was significantly lower (Table 3). The glutathione reductase activity of mutant HKprxA was higher than that of wild-type strain 7653R and displayed a significant difference between mutant HKprxA and wild-type 7653R (Table 3). These results indicated that the prxA gene played an important role in the glutathione redox balance of rhizobia.

| Plant properties of the prxA mutant and wild-type strain
To assess the nodulation and nitrogen-fixing capacity of the prxA mutant strain, A. sinicus seedlings were inoculated with the prxA mutant HKprxA or wild-type 7653R, and 28 days later, nodule numbers per plant and acetylene reduction activity were measured. No significant difference in the number of nodules was observed between plants inoculated with the prxA mutant strain and plants inoculated with the wild-type 7653R strain (Table 4). A highly significant feature of our study was that the prxA mutant induced partially effective nodules on A. sinicus. The prxA mutant elicited more spherical, rather than elongated, nodules compared to the wild-type and showed a 53.25% decrease in acetylene reduction activity compared to the wild-type (Table 4). When prxA on an environmentally stable plasmid (pBBR1MCS-5) was introduced into mutant HKprxA, plants inoculated with the resulting strain HKprxA(pBBRprxA) formed normal nodules and showed nitrogen-fixing ability at the same rate as did 7653R-inoculated plants (Table 4).  Values in each column followed by the same letter are not significantly different (p ≤ 0.05).

| RNA isolation and quantitative RT-PCR analysis
PrxA-deficient mutant. The expression of nifH, nifD, and fdxN, three genes involved in nitrogen fixation metabolism and induced in mature bacteroids (Capela, Filipe, Bobik, Batut, & Bruand, 2006), was analyzed in 4-week-old nodules by qRT-PCR (Figure 1b). A significant overexpression of the katG gene was also detected in prxA mutant bacteroids, suggesting that the nitrogen fixation process induces the transcription of the other antioxidant genes against strong oxidative stress (Figure 1b). In contrast, nifH, nifD, and fdxN were found to be significantly downregulated in HKprxA compared with control nodules. These results confirm the impairment of bacteroid differentiation and N 2 -fixation function observed in the HKprxA strain.

| Quantification of prxA gene expression in nodules induced by 7653R
The Although a single prxS mutant is not affected in its symbiotic abilities or defence response against oxidative stress, a prxS-katG double mutant displays only half of the normal nitrogen fixation capacity (Dombrecht et al., 2010). Here, we focus on a Prx5-like prxA single-mutant strain of M. huakuii that is affected with regard to its nitrogen fixation capacity and oxidative stress response.
Phylogenetic analysis and secondary and tertiary structure predictions indicate that M. huakuii PrxA belongs to the peroxiredoxin-5-like subfamily, which includes a novel thioredoxin peroxidase mainly identified in mammals (Yuan et al., 2004).
A study of human peroxiredoxin 5 family members suggested that they are involved not only in oxidative stress protection F I G U R E 1 Relative expression of genes involved in prxA mutant in hydrogen peroxide stresses (a) and 4-week-nodule bacteroids (b) in prxA mutant compared with wild-type 7653R measured by qRT-PCR. Data are the average from three independent biological samples (each with three technical replicates). Statistical analysis of data sets was performed using REST (Pfaffl, Horgan, & Dempfle, 2002). Superscript asterisk indicates significant difference in relative expression (>2-fold, p ≤ 0.05) F I G U R E 2 Expression patterns of prxA gene in symbiotic nodules. Gene expression levels were examined by real-time RT-PCR. Nodules were collected on different days after inoculation with Mesorhizobium huakuii 7653R. Relative expression of genes involved in nodule bacteroids compared with 7653R cells growth in AMS Glc. Data are the average from three independent biological samples (each with three technical replicates). Asterisk (*) indicates a significant difference (>2-fold, p ≤ 0.05). AMS, acid minimal salt medium mechanisms by reducing apoptosis but also in cell differentiation and signal induction (Yuan et al., 2004). Mutation of M. huakuii prxA did not affect the growth of free-living bacteria but displayed decreased antioxidative capacity under the conditions of the organic oxide CUOOH. In eukaryotes, peroxiredoxins participate in the antioxidant response by catalyzing the reduction of organic hydroperoxides (Abbas, Riquier, & Drapier, 2013 (Table 3).
The nitrogen fixation phenotype of PrxA-deficient mutants was assessed, and the prxA mutant showed a large reduction in the nitrogen-fixing activity of root nodules (reduced to approx. 50%), although the total number of nodules/plant (p ≤ 0.05) was not affected. In contrast, the symbiotic phenotype of nodules on plants inoculated with R. etli prxS mutants was reported to be similar to those inoculated with wild-type strains, but a prxS and katG double mutant has significantly reduced (>40%) nitrogen fixation capacity (Yuan et al., 2004). Moreover, a notable feature of our study was the concomitant existence of partially effective nod- Thus, considering the lack of bacterial differentiation in the prxA mutant, it is possible that PrxA reacts directly with these peptides to regulate their activity via thioredoxin reductase activity (Benyamina et al., 2012).

ACK N OWLED G M ENTS
This study was supported by the National Natural Science Foundation of China (grant no. 31772399) and the Fundamental Research Funds for the Central Universities, South-Central University for Nationalities (grant no. CZY18022).

CO N FLI C T O F I NTE R E S T S
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
G.C conceived and designed experiments and S.W., T. L., Q. X., and G. C. contributed to the writing of the manuscript. S.W., T. L., Q. X., and K. X. conducted experiments.

E TH I C S S TATEM ENT
None required.

DATA ACCE SS I B I LIT Y
All data are provided in full in the results section of this paper apart from Mesorhizobium huakuii 7653R genome sequence which is available at https ://www.ncbi.nlm.nih.gov/nucco re/NZ_CP006581.