Levels of Periplasmic Nitrate Reductase during Denitrification are Lower in Bradyrhizobium japonicum than in Bradyrhizobium diazoefficiens

Soybean plants host endosymbiotic dinitrogen (N2)-fixing bacteria from the genus Bradyrhizobium. Under oxygen-limiting conditions, Bradyrhizobium diazoefficiens and Bradyrhizobium japonicum perform denitrification by sequentially reducing nitrate (NO3–) to nitrous oxide (N2O) or N2. The anaerobic reduction of NO3– to N2O was previously shown to be lower in B. japonicum than in B. diazoefficiens due to impaired periplasmic nitrate reductase (Nap) activity in B. japonicum. We herein demonstrated that impaired Nap activity in B. japonicum was due to low Nap protein levels, which may be related to a decline in the production of FixP and FixO proteins by the cbb3-type oxidase.

Bradyrhizobium diazoefficiens (reclassified from Bradyrhizobium japonicum [Delamuta et al., 2013]; type strain USDA 110) may completely reduce NO 3 to N 2 . These reactions depend on napEDABC, which encodes periplasmic NO 3 -reductase (Nap); nirK, which encodes copper--containing NO 2 reductase (NirK); norCBQD, which encodes c-type NO reductase (Nor); and nosRZDYFLX, which encodes N 2 O reductase (Nos) (Sánchez et al., 2011). While B. diazoefficiens is a complete denitrifier, B. japonicum (type strain USDA 6) lacks the nos gene cluster and is, thus, unable to reduce N 2 O to N 2 (Sameshima-Saito et al., 2006b;Itakura et al., 2009;Kaneko et al., 2011;Siqueira et al., 2014). We previously screened the growth of 11 strains of B. japonicum and 15 strains of B. diazoefficiens and found that anaerobic (≈0% O 2 ) growth by B. japonicum with NO 3 -as the electron acceptor was significantly lower than that by B. diazoefficiens; however, no significant differences were observed between the growth rates of B. japonicum and B. diazoefficiens strains under microaerobic (≈2% O 2 ) and aerobic (≈12% O 2 ) growth conditions in the presence of -NO 3 (Siqueira et al., 2017). The lower growth rate of B. japonicum in anaerobiosis was not explained by the absence of nos, but by markedly impaired Nap activity that had a negative impact on the reduction of NO 3 -to N 2 O (Siqueira et al., 2017). B. japonicum conserved the FixLJ-FixK 2 regulatory cascade, which mediates the response of the nap operon to low oxygen and NO 3 -; the binding sites for FixK/FNR (fumarate and nitrate reductase) regulators upstream of napE were also conserved (Robles et al., 2006;Bueno et al., 2017;Siqueira et al., 2017). Additionally, napA transcript levels were similar in B. japonicum and B. diazoefficiens (Siqueira et al., 2017). Thus, we proposed that impaired Nap activity in B. japonicum may be due to posttranscriptional events (Siqueira et al., 2017).
In growth experiments, precultured cells were inoculated into 34-mL test tubes containing 5 mL of HMMN medium. The initial optical density at 660 nm (OD 660 ) was adjusted to 0.01. In the competition experiment, initial cell numbers Article ME19129 were adjusted to 3×10 7 cells mL -1 and cells were used at a 1:1 ratio. Aerobic (≈12% O 2 ), microaerobic (≈2% O 2 ), and anaerobic (≈0% O 2 ) treatments were prepared and monitored as previously described (Siqueira et al., 2017). Cells were grown at 30°C with reciprocal shaking at 300 rpm. Growth was monitored daily by measuring the OD 660 of the cultures, and the number of cells was directly counted using a 20-μm-deep hemocytometer (Sunlead Glass) and BX51 Fluorescence Microscope (Olympus).
To construct Bd-USDA 110 tagged with GFP and Bj-USDA 6 tagged with DsRed, pRJPaph-gfp (Ledermann et al., 2015) and pBjGroEL4::DsRed2 (Hayashi et al., 2014) plasmids were transferred by triparental mating using pRK2013 as a helper plasmid (Figurski and Helinski, 1979). Single recombination events, indicating the integration of pRJPaph-gfp into the Bd-USDA 110 chromosome or pBjGroEL4::DsRed2 into the Bj-USDA 6 chromosome, were selected based on their acquisition of resistance to Tc or Sp/Sm, respectively. Single recombinant strains were further confirmed using the BX51 Fluorescence Microscope (Olympus).
In immunoblotting and heme-staining analyses, precultured Bradyrhizobium cells were inoculated into 500-mL flasks containing 100 mL of HMNN (OD 660 ≈0.03) and incubated under microaerobic or anaerobic conditions at 30°C, with reciprocal shaking at 100 rpm. Soluble and membrane fractions were prepared as described previously (Fischer et al., 2001;Torres et al., 2014a). Soluble fractions were concentrated using Amicon Ultra-15 centrifugal filter units (Merck). In immunoblotting experiments, rabbit polyclonal antibodies were raised against the synthetic NapA peptide, CPLQKKATGAAKAND (GenScript Japan), which is common between the Nap of Bd-USDA 110 and Bj-USDA 6. Soluble fractions (10 μg) were separated by SDS-PAGE (10% polyacrylamide) and transferred to P plus PVDF membranes (ATTO) using a HorizeBLOT 4M-R semi-dry transfer system (ATTO). Membranes were incubated with an anti-NapA antibody (1:10,000) at room temperature for 30 min and then with HRP-conjugated anti-rabbit IgG (1:2,500; Promega) for 30 min. Immunoreactive bands were detected using the chemiluminescent substrate, Promega TM ECL Western Blotting Substrate. Regarding heme staining, membrane fractions (10 μg) were separated by SDS-PAGE at 4°C (stacking phase at 5% acrylamide and resolving phase at 12% acrylamide), transferred to a 0.45 μm Hybond-P membrane (GE Healthcare), and stained with the Promega TM ECL Western Blotting Substrate to detect hemedependent activity by chemiluminescence (Vargas et al., 1993). Blots were visualized using a Molecular Imager Gel Doc XR+ System with Image Lab software (Bio-Rad). Relative band intensities were calculated using ImageJ software (Schneider et al., 2012). Experiments were performed with at least two independent biological replicates.
The impaired activity of Nap in B. japonicum during anaerobic growth in the presence of NO 3 -may rely on posttranscriptional effects that affect the structure, function, or level of Nap (Siqueira et al., 2017). The napEDABC gene cluster of B. diazoefficiens encodes NapE, a membrane protein of 6.6 kDa with unknown function; NapD (11.8 kDa), a chaperone involved in the maturation of NapA; NapA (94.5 kDa), the catalytic subunit that contains a Mobis-MGD active site and [4Fe4S] cluster; and NapB (16.9 kDa), the electron transfer subunit, which is a diheme cytochrome c 552 . NapA and NapB form the heterodimeric subunit NapAB, which is present in the periplasm. NapC (25 kDa) is a membrane-anchored, tetraheme, c-type cytochrome that is responsible for the transfer of electrons from the quinol pool to the NapAB complex (Zumft, 1997;Delgado et al., 2003). A sequence comparison of the napEDABC gene cluster of Bd-USDA 110 and Bj-USDA 6 showed high identity (>85%) at the nucleotide level (Fig. S1). The amino acid sequences of Bj-USDA 6 NapEDABC were conserved, exhibiting 89-97% identity with Bd-USDA 110 (Fig. S1). In NapA of Bj-USDA 6, the twin arginine motif, the residues involved in the binding of the Mobis-MGD cofactor, and the [4Fe4S] cluster were conserved, except in some cases in which there was a substitution with a similar residue. In Bj-USDA 6 NapB, both the cytochrome c-binding sites and motif for translocation via the general secretory pathway were conserved. The structural similarity of the Bj-USDA 6 and Bd-USDA 110 Nap proteins was high, with a TM-score of 0.81, which indicated the same type of folding (Fig. S1). These results suggested that the structure and function of Nap were conserved in Bj-USDA 6.
We compared the amount of Nap in Bj-USDA 6 and Bd-USDA 110 cells incubated under microaerobic and anaero--bic conditions in the presence of NO 3 . An immunoblotting analysis of soluble fractions revealed a bradyrhizobial NapA protein band at ≈100 kDa (Fig. 1A), which was consistent with the expected molecular weight of 94.5 kDa (Delgado et al., 2003). This was further confirmed by the absence of this band in soluble fractions of the B. diazoefficiens ΔnapA mutant (Fig. 1A). The intensity of the NapA band was weaker in Bj-USDA 6 than in Bd-USDA 110, in cells incubated microaerobically (≈0.57 times; Fig. 1A and Table S1), and particularly in cells incubated anaerobically (≈0.33 times; Fig. 1A and Table S1). This result suggested that Bj-USDA 6 produced less NapA than Bd-USDA 110 under low-oxygen conditions (i.e. ≤2% O 2 ) in the presence of -NO 3 . Additionally, we detected NapC in the membrane fractions of bradyrhizobial cells by heme staining; the staining of heme covalently bound to c-type cytochromes (Vargas et al., 1993). Bj-USDA 6 and Bd-USDA 110 showed the typical profile of five stained bands previously identified in membrane fractions of Bd-USDA 110 (Fig. 1B) (Preisig et al., 1993;Mesa et al., 2002;Delgado et al., 2003;Bueno et al., 2008;Torres et al., 2014a). The 32-kDa and 28-kDa bands, almost co-migrating, corresponded to the FixP and FixO proteins, respectively, of cbb 3 -type high affinity cytochrome oxidase, encoded by the fixNOQP operon (Preisig et al., 1993(Preisig et al., , 1996; the 25-kDa band corre- sponded to NapC (Delgado et al., 2003); the 20-kDa band corresponded to cytochrome CycM (Bott et al., 1991); and the 16-kDa band corresponded to the NorC subunit of Nor (Mesa et al., 2002). We confirmed the identities of bands by analyzing soluble fractions of the B. diazoefficiens ΔnapA mutant under microaerobic conditions; bands correspondig to NapC and NorC were absent (Fig. 1B), as described previously (Delgado et al., 2003). Under anaerobic conditions, the intensity of the NapC band was ≈0.3-fold weaker in Bj-USDA 6 than in Bd-USDA 110 ( Fig. 1B and Table S1). Thus, immunoblotting and heme-staining results indicated that Bj-USDA 6 produced lower levels of Nap under low--oxygen conditions in the presence of NO 3 . Therefore, impaired Nap activity in B. japonicum during NO 3 -dependent anaerobic growth, which was reported previously (Siqueira et al., 2017), may be caused by a low level of Nap.
Based on the results of heme staining, the amount of NorC was higher in Bj-USDA 6 than in Bd-USDA 110 under microaerobic and anaerobic conditions (≈1.3-and 1.6--fold, respectively) in the presence of NO 3 (Fig. 1B and Table S1). This result was supported by a previous finding showing that norB transcript levels were higher in Bj-USDA 6 than in Bd-USDA 110 under anaerobic NO 3 --dependent growth (Siqueira et al., 2017). Heme staining also revealed that besides NapC, FixP and FixO were the only other cytochromes affected in Bj-USDA 6 under low-oxygen conditions; the amounts of FixP and FixO were lower in Bj-USDA 6 than in Bd-USDA 110 by ≈0.45-fold under microaerobic conditions and ≈0.8-fold under anaerobic conditions ( Fig. 1B and Table S1). This result suggested that cbb 3 oxidase is a critical enzyme for the adaptation process to NO 3 respiration under low-oxygen conditions in Bradyrhizobium. Accordingly, the maximal expression of Nap does not appear to occur until the oxygen concentration becomes very low, and this is only observed after cbb 3 oxidase has consumed the oxygen present in the growth medium (Bueno et al., 2008). Since the transcription of nap genes appeared to be unaffected in B. japonicum cells -grown anaerobically in the presence of NO 3 (Siqueira et al., 2017), the remaining oxygen in the growth medium may affect the amount of the Nap protein at the posttranscriptional level by inhibiting the translation of nap messenger RNA or inducing the degradation of Nap proteins.
The mechanisms responsible for low levels of cbb 3 oxidase in B. japonicum under low-oxygen conditions in the presence of NO 3 may be related to B. japonicum being unable to make an effective switch to denitrification in the absence of oxygen, similar to other bacteria (Aida et al., 1986;Bergaust et al., 2011;Torres et al., 2014b;Siqueira et al., 2017). We tested the NO 3 --dependent anaerobic growth of bradyrhizobial cells preincubated microaerobically, instead of aerobically as previously reported (Siqueira et al., 2017). The microaerobic preincubation resulted in a reduced growth rate of Bj-USDA 6, to a similar extent as the aerobic preincubation (Fig. S2), indicating the absence of a significant difference between a rapid (aerobic preincubation) and gradual (microaerobic preincubation) transition from aerobiosis to anaerobiosis. However, further studies are needed to elucidate the mechanisms responsible for the changes observed in cbb 3 oxidase levels in B. japonicum under denitrifying conditions.
Our previous findings prompted the hypothesis that B. japonicum may be less competitive than B. diazoefficiens due to energy depletion under anaerobic denitrifying growth (Siqueira et al., 2017). We examined the competitive growth of Bd-USDA 110 and Bj-USDA 6 tagged with GFP and DsRed proteins, respectively. The tagged strains were tested to exclude the possible effects of tagging on growth rates (Fig. S3). Under microaerobic and anaerobic conditions, in the presence of NO 3 , the growth of DsRed-tagged Bj-USDA 6 was lower than that of GFP-tagged Bd-USDA 110, whereas the aerobic growth rates of these two species were similar (Fig. 2). This result indicated that Bd-USDA 110 is more competitive than Bj-USDA 6 at low-oxygen levels in the presence of NO 3 . In Bradyrhizobium, growth at lowoxygen levels in the presence of NO 3 -is supported by both cbb 3 oxidase, which is active during free-living microaerobic growth and in N 2 -fixing bacteroids, and the denitrification pathway (Preisig et al., 1993(Preisig et al., , 1996Bueno et al., 2008). The weaker competitiveness of B. japonicum USDA 6 under low-oxygen conditions in the presence of NO 3 -may correlate with low levels of Nap ( Fig. 1 and Table S1).
In conclusion, the present study demonstrated that the level of Nap under low-oxygen conditions in the presence of -NO 3 was lower in B. japonicum USDA 6 than in B. diazoefficiens USDA 110 and suggests that the capacity to maintain a sufficient quantity of Nap is an advantage under denitrifying conditions. The lower levels of Nap in B. japonicum USDA 6 presumably resulted in a competitive disadvantage against B. diazoefficiens USDA 110 due to energy depletion during denitrifying growth. Ecologically, this advantage of B. diazoefficiens may be an important factor that influences the predominance of B. diazoefficiens over B. japonicum in soils exposed to low-oxygen conditions (Shiina et al., 2014).