Abstract Reduction of nitrite to nitric oxide gas by respiratory nitrite reductases (NiRs) is the key step of denitrification. Denitrifiers are strictly divided into two functional groups based on whether they possess the copper-containing nitrite reductase (CuNiR) encoded by nirK or the cytochrome cd1 nitrite reductase (cdNiR) encoded by nirS. Recently, some organisms carrying both nirK and nirS genes have been found. Bradyrhizobium oligotrophicum S58 is a nitrogen-fixing oligotrophic bacterium that carries a set of genes for complete denitrification of nitrate to dinitrogen, including nirK and nirS genes. We show that denitrification in S58 is functional under low-oxygen conditions (anaerobiosis and microaerobiosis), but not under aerobiosis. Under denitrifying conditions, the ΔnirK and ΔnirS single S58 mutants grew normally and their NiR activity was not affected. However, the ΔnirKS double mutant grew more slowly, presumably because the impaired NiR activity resulted in nitrite accumulation in the medium. These results suggest a redundant role for nirK and nirS genes in B. oligotrophicum S58 denitrification. In addition, we found that the nirS gene product, but not that of nirK, maintains swimming motility of S58 under aerobic and low-oxygen conditions in the presence of nitrate. Bradyrhizobium, denitrification, copper-containing nitrite reductase, cytochrome cd1 nitrite reductase, swimming motility INTRODUCTION Denitrification is commonly defined as the respiratory reduction of the nitrogen oxyanions nitrate (NO3−) or nitrite (NO2−) to the gaseous nitrogen oxides nitric oxide (NO) and nitrous oxide (N2O), which may be further reduced to dinitrogen (N2). The committed step of this process, the reduction of NO2− to NO, is catalyzed by two non-homologous nitrite reductases (NiRs) that are located in the periplasm. CuNiR is a homotrimeric enzyme that contains type I and type II copper centers, whereas cdNiR is a cytochrome cd1-type nitrite reductase, which is a homodimer with a c-heme- and d1-heme-binding domain in each monomer (Zumft 1997; van Spanning 2011). The synthesis of CuNiR requires a single gene, nirK, sometimes accompanied by nirV, which may be required for the proper insertion of the copper catalytic center (van Spanning 2011). In contrast, a complex gene cluster is responsible for the synthesis of cdNiR, which requires at least ten genes (nirSECFDLGHJN); nirS gene encodes the functional subunits of cdNiR. All other genes are required for proper synthesis, maturation and assemblage of the unique d1-heme cofactor in the reaction center (reviewed by van Spanning 2011 and Rinaldo, Giardina and Cutruzzolà 2016): nirC encodes a specific cytochrome c; NirE and NirJ are involved in the maturation of the d1-heme in the cytoplasm; the NirN and NirF proteins are periplasmic and act prior to insertion of the d1-heme into NirS. NirD, NirL, NirG and NirH compose a siroheme decarboxylase involved in d1-heme biosynthesis. The lack of ancillary genes results in non-functional cdNiR when nirS is heterologously expressed (Zumft 1997; Rinaldo, Giardina and Cutruzzolà 2016). In some bacteria, nirS is found in a more complex locus, which also includes nirXI genes encoding factors that regulate the expression and activity of cdNiR (van Spanning 2011). The presence of nirK and nirS in an organism was thought to be mutually exclusive, and communities of denitrifiers with nirS respond differently to environmental gradients than those with nirK, which suggests that these communities occupy different ecological niches (Jones and Hallin 2010). The recent identification of organisms harboring both nirK and nirS genes (Campbell et al.2011; Graf, Jones and Hallin 2014) may provide new insights into the relative importance of the two enzymes in adaptation to different niches. Denitrification is a common trait in the Bradyrhizobiaceae and it has been studied in depth in the soybean symbiont Bradyrhizobium diazoefficiens USDA 110 (Bedmar, Robles and Delgado 2005; Sameshima-Saito, Chiba and Minamisawa 2006; Siqueira, Minamisawa and Sánchez 2017). The respiratory reduction of NO2− to NO in bradyrhizobia typically depends on nirK (Table S1, Supporting Information), but recently, some strains harboring nirS gene have been identified (Ishii et al.2011). Inspection of the annotated genome of Bradyrhizobium oligotrophicum S58 (Okubo et al.2013) showed that it carries gene clusters for the synthesis of all denitrification reductases: periplasmic NO3− reductase (napEDABC), CuNiR (nirKV), c-type NO reductase (norECBQD), N2O reductase (nosRZDFYLX) and a complete gene cluster for the synthesis of cdNiR (nirSECFDGHJN; Fig. 1). Bradyrhizobium oligotrophicum S58 (reclassified from Agromonas oligotrophica S58 by Ramírez-Bahena et al.2013) is an oligotrophic bacterium that was isolated from a paddy field soil in Japan (Ohta and Hattori 1983). It is classified into the photosynthetic Bradyrhizobium clade, which includes, among others, Bradyrhizobium sp. ORS 278 and Bradyrhizobium sp. BTAi1 (Okubo et al.2013). Members of the photosynthetic Bradyrhizobium clade form N2-fixing nodules on stems and roots of aquatic legumes of the genus Aeschynomene (Okubo, Fukushima and Minamisawa 2012, 2013). Some members of this clade, including B. oligotrophicum S58, also form an endophytic association with rice roots, where they may provide fixed nitrogen to the host plant, as suggested for Bradyrhizobium sp. ORS 278 (Ohta and Hattori 1983; Chaintreuil et al.2000; Okubo et al.2013). Figure 1. View largeDownload slide Organization of denitrification genes in B. oligotrophicum S58. (A) Organization of nap, nirK, nor and nos gene clusters in B. oligotrophicum S58, Bradyrhizobium diazoefficiens USDA 110 and Bradyrhizobium sp. BTAi1. (B) Organization of the nirS gene cluster in B. oligotrophicum S58 and P. denitrificans PD1222. Percentages indicate identity at the protein level. Figure 1. View largeDownload slide Organization of denitrification genes in B. oligotrophicum S58. (A) Organization of nap, nirK, nor and nos gene clusters in B. oligotrophicum S58, Bradyrhizobium diazoefficiens USDA 110 and Bradyrhizobium sp. BTAi1. (B) Organization of the nirS gene cluster in B. oligotrophicum S58 and P. denitrificans PD1222. Percentages indicate identity at the protein level. It has not yet been demonstrated whether the two types of NiR are functional when present in the same microorganism. Thus, the main aim of this work was to assess whether B. oligotrophicum S58 is able to denitrify and, if so, to characterize the function of the nirK and nirS genes in denitrification in free-living S58 cells. In addition, we examined the role of the nirS gene in the swimming motility of S58. RESULTS AND DISCUSSION Denitrification genes in B. oligotrophicum S58 We found that the organization of napEDABC, nirKV, norECBQD and nosRZDFYLX gene clusters in Bradyrhizobium oligotrophicum S58 was similar to those of Bradyrhizobium diazoefficiens USDA 110 (Kaneko et al.2002) and Bradyrhizobium sp. BTAi1 (Giraud et al.2007; Fig. 1A). The catalytic subunits NapA, NirK, NorB and NosZ of S58 had higher sequence identity to those of Bradyrhizobium sp. BTAi1 than to those of B. diazoefficiens USDA 110 (Fig. 1A, Table S2, Supporting Information), which agrees with the phylogenetic proximity between S58 and BTAi1 (Okubo et al.2013). S58 NirK contained the TAT (twin arginine translocation system) motif, residues that bind copper in the type 1 and type 2 centers and residues involved in catalysis (Velasco et al.2001; Fig. S1, Supporting Information), suggesting functional conservation of S58 CuNiR. The nirS cluster in S58 includes the nirSECFDGHJN genes and the regulators nirIX, which are transcribed divergently (Fig. 1B). This organization was similar to that of the soil bacterium Paracoccus denitrificans PD1222, a well-known model for denitrification studies (Zumft 1997; van Spanning 2011; Fig. 1B). S58 NirS shared 66% identity with P. denitrificans NirS, although the highest identity was observed with the NirS proteins of the heterotrophic bacterium Prosthecomicrobium hirschii (73%), which was isolated from a muskrat pond (Daniel et al.2015), and the moderately thermophilic bacterium Albidovulum xiamenense (71%), which was isolated from a terrestrial hot spring (Yin et al.2012; Table S3, Supporting Information). The presence of genes required for the synthesis of the specialized d1-heme cofactor of cdNir, along with the conservation in the NirS sequence of c-heme ligands, d1-heme ligands and His residues involved in catalysis (Fig. S2, Supporting Information; Rinaldo, Giardina and Cutruzzolà 2016), suggests functional conservation of this enzyme in B. oligotrophicum S58. Bradyrhizobium oligotrophicum S58 growth in the presence of nitrate The presence of nap, nir, nor and nos gene clusters suggests that S58 is able to denitrify NO3− to N2. In bradyrhizobia, denitrification is induced under low oxygen in the presence of NO3− (Bedmar, Robles and Delgado 2005; Siqueira, Minamisawa and Sánchez 2017). Thus, we assessed the growth of S58 in the absence and in the presence of NO3− under different oxygen regimes. Under aerobic and microaerobic conditions, NO3− did not affect bacterial growth; optical density reached approximately 0.9 (Fig. 2A). Under anaerobic conditions, bacteria grew only upon addition of NO3− (final optical density, ∼0.19; Fig. 2A). In aerobic cultures, NO3− was not consumed, suggesting the absence of denitrification, whereas 60% of NO3− in microaerobic cultures and all NO3− in anaerobic cultures was consumed by the end of the growth period (Fig. 2B), likely because of its reduction through denitrification. It seems that anaerobic growth was supported by NO3− respiration, whereas microaerobic growth was mainly supported by oxygen respiration (Fig. 2). The presence of N2O reductase activity in S58 free-living cells under anaerobic conditions (T. Kumei and K. Minamisawa, unpublished results) suggests that this bacterium completely denitrifies NO3− to N2. Taken together, these results indicate that B. oligotrophicum S58 is able to denitrify under microaerobic and anaerobic conditions. Figure 2. View largeDownload slide Growth of B. oligotrophicum S58. (A) Growth of B. oligotrophicum S58 in the absence and in the presence of nitrate. Growth was measured by recording optical density at 660 nm on a daily basis. (B) Extracellular nitrate (NO3−) concentrations. Cells were grown as in (A) in the presence of nitrate. The results presented are the mean of three biological replicates ± standard deviation. A, aerobic; A + N, aerobic + NO3−; M, microaerobic; M + N, microaerobic + NO3−; An, anaerobic; An + N, anaerobic + NO3−. Figure 2. View largeDownload slide Growth of B. oligotrophicum S58. (A) Growth of B. oligotrophicum S58 in the absence and in the presence of nitrate. Growth was measured by recording optical density at 660 nm on a daily basis. (B) Extracellular nitrate (NO3−) concentrations. Cells were grown as in (A) in the presence of nitrate. The results presented are the mean of three biological replicates ± standard deviation. A, aerobic; A + N, aerobic + NO3−; M, microaerobic; M + N, microaerobic + NO3−; An, anaerobic; An + N, anaerobic + NO3−. We analyzed the methyl viologen (MV)-dependent nitrite reductase (MV-NiR) activity in S58. By measuring β-galactosidase activity in cells carrying PnirK-lacZ and PnirS-lacZ transcriptional fusions, we also assessed the activity of nirK and nirS promoters. Values of MV-NiR activity and β-galactosidase gene expression followed a similar pattern after incubation for 48 h (data not shown) and 96 h (Table 1), but were higher at 96 h. Both MV-NiR activity and nirK and nirS promoter activity were induced in response to low oxygen and NO3−, and were highest under anaerobic conditions in the presence of NO3− (Table 1). Similarly, in B. diazoefficiens, maximal induction of the nirK gene requires simultaneously low-oxygen conditions and the presence of NO3− (Velasco et al.2001). Expression of nir genes in different bacteria depends on FNR (fumarate and nitrate reductase)-like regulators (Zumft 1997; van Spanning 2011). In B. diazoefficiens, the FNR-like protein FixK2 induces the expression of nirK in response to a low-oxygen signal that is perceived and transduced by the FixLJ two-component regulatory system (Bedmar, Robles and Delgado 2005). In S58, we found that the region upstream of nirK and the intergenic region between nirI and nirS contain sites resembling the consensus FNR-binding site TTGAT-N4-ATCAA; these sites are located 67 bp upstream of nirK (TTGTT-N4-CACAA) and 89 bp upstream of nirS (TTAAC-N4-GTCAA). This suggests that both genes respond to low-oxygen conditions via FNR-like regulators (Zumft 1997; van Spanning 2011). Under all the conditions tested, the expression of nirS was markedly higher than that of nirK, and nirK expression was not detected under aerobic conditions (Table 1). The expression of nirS in aerobiosis has been reported previously (Zumft 1997; Rinaldo, Giardina and Cutruzzolà 2016). Table 1. MV-dependent nitrite reductase (NiR) activities and transcriptional activities of nirK and nirS promoter regions in B. oligotrophicum S58. β-galactosidase activityb Condition NiR activitya PnirK PnirS Aerobic 89 ± 27 ND 34 ± 10 –KNO3 Microaerobic 130 ± 16 1.3 ± 0.7 50 ± 11 Anaerobic NM 0.6 ± 0.2 8.5 ± 3.7 Aerobic 89 ± 38 ND 30 ± 10 +KNO3 Microaerobic 171 ± 74 12 ± 1 86 ± 31 Anaerobic 275 ± 51 16 ± 6 470 ± 118 β-galactosidase activityb Condition NiR activitya PnirK PnirS Aerobic 89 ± 27 ND 34 ± 10 –KNO3 Microaerobic 130 ± 16 1.3 ± 0.7 50 ± 11 Anaerobic NM 0.6 ± 0.2 8.5 ± 3.7 Aerobic 89 ± 38 ND 30 ± 10 +KNO3 Microaerobic 171 ± 74 12 ± 1 86 ± 31 Anaerobic 275 ± 51 16 ± 6 470 ± 118 a Values (nmol NO2− consumed mg protein−1 h−1) are means ± standard deviation (n = 3). b Values (Miller units) are means ± standard deviation (n = 3). ND, not detected; NM, not measured. View Large Effect of nirK and nirS mutations on growth under denitrifying conditions We constructed ΔnirK and ΔnirS single mutants and a ΔnirKS double mutant of B. oligotrophicum S58. Under aerobic conditions, the wild-type S58 and the ΔnirKS mutant grew similarly (Fig. 3A) and exhibited similar values of MV-NiR activity after incubation for 96 h (Table 2), which indicates that nirK and nirS genes are not involved in aerobic growth in the presence of NO3−. Under microaerobic and anaerobic conditions, wild-type S58 and both single mutants grew similarly (Fig. 3A) and consumed similar amounts of NO3− (Fig. 3B). Nitrite was not accumulated in the medium, except transient accumulation in ΔnirS mutant anaerobic cultures, where it disappeared by the end of the growth period (Fig. 3C). MV-NiR activity was similar in the single mutants and wild type (Table 2). Because MV does not easily traverse the cytoplasmic membrane (Jones, Gray and Garland 1976), NiR activity likely resulted exclusively from the periplasmic enzymes: CuNiR, cdNiR or both. The growth of the ΔnirKS double mutant was reduced slighty under microaerobiosis and strongly under anaerobiosis (Fig. 3A). MV-NiR activity was low in microaerobic cultures and undetectable in anaerobic cultures of the ΔnirKS mutant (Table 2). NO2− accumulated in the medium by the end of the growth period (∼3 mM in microaerobic cultures and ∼10 mM in anaerobic cultures; Fig. 3C). Thus, while CuNiR and cdNiR may totally account for periplasmic NiR activity under anaerobiosis, an additional periplasmic NiR enzyme may be active under microaerobiosis (Table 2). The growth defect in the ΔnirKS mutant was probably due to the toxicity of NO2− that accumulated in the medium (Fig. 3C); indeed, NO2− (0.25 mM or higher) reduced the growth of wild-type S58 (Fig. S3, Supporting Information). Together, these results suggest that CuNiR and cdNiR fulfill the same role under denitrifying conditions and the lack of one NiR seems to be compensated by the other. Likewise, Zumft, Braun and Cuypers (1994) demonstrated that the lack of cdNiR on Pseudomonas stutzeri can be complemented by CuNiR from Pseudomonas aureofaciens. In the ΔnirS and ΔnirK cells carrying PnirK-lacZ and PnirS-lacZ transcriptional fusions, respectively, β-galactosidase activity after incubation for 96 h was similar to that in the wild type carrying the respective constructs (Table S4, Supporting Information). This suggests that compensation does not involve the induction of nirS or nirK gene expression. Figure 3. View largeDownload slide Growth of nir mutants. (A) Growth of B. oligotrophicum S58 and ΔnirK, ΔnirS and ΔnirKS mutant strains under aerobic, microaerobic and anaerobic conditions in the presence of nitrate. Growth was measured by recording optical density at 660 nm on a daily basis. Extracellular concentrations of (B) nitrate (NO3−) and (C) nitrite (NO2−) are shown for the cultures shown in (A). The results presented are the mean of three biological replicates ± standard deviation. Values significantly different from those of B. oligotrophicum S58 are indicated with * (t-test, P < 0.01; n = 3). Figure 3. View largeDownload slide Growth of nir mutants. (A) Growth of B. oligotrophicum S58 and ΔnirK, ΔnirS and ΔnirKS mutant strains under aerobic, microaerobic and anaerobic conditions in the presence of nitrate. Growth was measured by recording optical density at 660 nm on a daily basis. Extracellular concentrations of (B) nitrate (NO3−) and (C) nitrite (NO2−) are shown for the cultures shown in (A). The results presented are the mean of three biological replicates ± standard deviation. Values significantly different from those of B. oligotrophicum S58 are indicated with * (t-test, P < 0.01; n = 3). Table 2. MV-dependent NiR activity in B. oligotrophicum S58 and ΔnirK, ΔnirS and ΔnirKS mutants in HMMN medium. NiR activitya Strain Aerobic Microaerobic Anaerobic S58 71 ± 17 186 ± 83 254 ± 52 ΔnirK 71 ± 17 124 ± 62 201 ± 70 ΔnirS 77 ± 28 124 ± 13 221 ± 52 ΔnirKS 87 ± 36 54 ± 25 ND NiR activitya Strain Aerobic Microaerobic Anaerobic S58 71 ± 17 186 ± 83 254 ± 52 ΔnirK 71 ± 17 124 ± 62 201 ± 70 ΔnirS 77 ± 28 124 ± 13 221 ± 52 ΔnirKS 87 ± 36 54 ± 25 ND a Values (nmol NO2− consumed mg protein−1 h−1) are means ± standard deviation (n = 3). ND, not detected. View Large Although our results show a redundant function of CuNiR and cdNiR in B. oligrotrophicum S58 denitrification, we would like to emphasize that our experiments were performed in the presence of sufficient copper and iron (see Materials and Methods). As CuNiR and cdNiR depend on copper and iron, respectively, it will be interesting to explore the exchangeability of these enzymes under copper or iron deficit. Effect of the nirS mutation on swimming motility Similar to other members of the photosynthetic Bradyrhizobium clade, S58 has a complete set of genes for a polar flagellum (Okubo et al.2013; Ramírez-Bahena et al.2013; Quelas et al.2016). A structural role of NirS in the control of flagella production under anaerobic conditions has been reported; NirS forms a periplasmic complex with the chaperone DnaK and the flagellar protein FliC (Borrero-de Acuña et al.2015). In addition, several studies have shown a coupling between denitrification and motility through NO produced by cdNiR (Cutruzzolà and Frankenberg-Dinkel 2016). To examine the role of the B. oligotrophicum S58 nirS gene in swimming motility, we tested the ΔnirK, ΔnirS and ΔnirKS mutants in motility assays under aerobic and low-oxygen conditions. Under both conditions, swimming ability of wild-type S58 on agar plates decreased in the presence of NO3− (Figs S4 and S5, Supporting Information). At a concentration of 10 mM or higher, the inhibition was stronger in the ΔnirS and ΔnirKS mutants than in the wild-type S58 and ΔnirK mutant (Fig. 4), which indicates specific involvement of nirS. Importantly, planktonic growth of the ΔnirS mutant in the presence of the same concentration of NO3− was indistinguishable from that of the wild type (Fig. 3A), suggesting that the failure in swimming is independent of a failure of the ΔnirS mutant to grow. Thus, the phenotype of the ΔnirS mutant on plates may be due to a specific effect on swimming. Such involvement of nirS under both aerobic and low-oxygen conditions seems to be reasonable as we detected the expression of nirS under both conditions (Table 1). Further research is needed to clarify whether the role of nirS in S58 swimming motility in the presence of NO3− is enzymatic, structural or both (Borrero-de Acuña et al.2015; Cutruzzolà and Frankenberg-Dinkel 2016). Figure 4. View largeDownload slide Swimming motility of B. oligotrophicum S58 and ΔnirK, ΔnirS and ΔnirKS mutant strains. (A) Soft agar plates with HMM containing the indicated concentrations of KNO3 were inoculated in the middle and incubated aerobically or under low oxygen at 30°C for 13 days. (B) The diameters of the halos of bacterial growth, representing the distance migrated by the strain via swimming. Bars indicate mean halo diameters ± standard deviation. Values significantly different from those of B. oligotrophicum S58 are indicated with * (t-test, P < 0.01; n = 5) or ** (t-test, P < 0.05; n = 5). Figure 4. View largeDownload slide Swimming motility of B. oligotrophicum S58 and ΔnirK, ΔnirS and ΔnirKS mutant strains. (A) Soft agar plates with HMM containing the indicated concentrations of KNO3 were inoculated in the middle and incubated aerobically or under low oxygen at 30°C for 13 days. (B) The diameters of the halos of bacterial growth, representing the distance migrated by the strain via swimming. Bars indicate mean halo diameters ± standard deviation. Values significantly different from those of B. oligotrophicum S58 are indicated with * (t-test, P < 0.01; n = 5) or ** (t-test, P < 0.05; n = 5). The presence of nirS gene in bradyrhizobia has been reported only in oligotrophic strains isolated from rice paddy soils, and among them, only B. oligrotrophicum S58 also carries nirK (Ishii et al.2011; Okubo et al.2013). A better understanding of the ecology of S58 could explain why it is advantageous for these bradyrhizobia to have acquired and kept the nirS gene cluster. The specific involvement of nirS in swimming motility points to additional functions of cdNiR in S58 beyond denitrification. Maintenance of swimming ability in the presence of NO3− may be essential for survival in paddy fields under fluctuating oxygen conditions as a result of alternate waterlogged and drained conditions. By supporting S58 motility, nirS may also play a role in the colonization of rice and Aeschynomene roots (Capdevila et al.2004; Sessitsch et al.2012). Evolutionary aspects of nir genes in B. oligotrophicum S58 The common NiR in the Bradyrhizobiaceae seems to be CuNiR (nirK, Table S1, Supporting Information). This suggests that this group have inherited nirKV genes from a common ancestor, and later B. oligrotrophicum S58 acquired the nirS gene cluster. Bradyrhizobial genomes have many trn elements, which are genomic islands that were inserted into tRNA genes with target duplication (Kaneko et al.2002, 2011). Interestingly, an Arg-tRNA gene (S58_68290) is located close to the coding region of the nirS gene cluster (S58_68110–S58_68230), suggesting that this cluster is part of a genomic island that has been horizontally transferred into the S58 genome, although the Arg-tRNA gene contains no obvious duplicated fragment. The observation of nearly identical nirS sequences in phylogenetically distantly related Bradyrhizobium strains supports the acquisition of nirS sequences by horizontal transfer (Ishii et al.2011). MATERIALS AND METHODS Bacterial strains and growth conditions Bacterial strains and plasmids are listed in Table S5, Supporting Information. Cells of Bradyrhizobium oligotrophicum were cultured at 30°C in HM salt medium (Cole and Elkan 1973) supplemented with 0.1% (w/v) arabinose and 0.025% (w/v) yeast extract. For denitrification assays, HM medium was also supplemented with trace metals (HMM medium; Sameshima-Saito, Chiba and Minamisawa 2006) and 10 mM KNO3 (HMMN medium). Escherichia coli cells were grown at 37°C in Luria–Bertani medium (Miller 1972). The following antibiotics were added: for B. oligotrophicum, kanamycin (Km; 100 μg mL−1), tetracyclin (Tc; 100 μg mL−1) and polymyxin B (100 μg mL−1); for E. coli, Km (50 μg mL−1) and Tc (10 μg mL−1). For growth experiments, cells were inoculated into 5 mL of HMM medium (optical density at 660 nm ∼0.01) in 35-mL tubes and were reciprocally shaken (300 rpm, 30°C) aerobically, microaerobically or anaerobically. For assays under microaerobic conditions, the tubes were sealed and the gas phase was replaced daily with a gas mixture containing 98% N2 and 2% O2 (Siqueira, Minamisawa and Sánchez 2017). For assays under anaerobic conditions, the tubes were sealed and the gas phase was replaced once with 100% N2 (Siqueira, Minamisawa and Sánchez 2017). Growth was measured daily by recording optical density at 660 nm. Construction of ΔnirK and ΔnirS mutants Bradyrhizobium oligotrophicum ΔnirK and ΔnirS mutants were constructed by overlap extension. This approach involves PCR to independently generate DNA fragments that contain incorporated complementary oligonucleotide primers. The fragments can then be effectively ‘fused’ anywhere along the gene sequence by combining them in a second primer extension reaction (Ho et al.1989). First, in separate PCRs, two fragments (700–800 nucleotides each) of the target sequence were amplified by using PrimeSTAR Max DNA Polymerase (TaKaRa Bio Inc., Shiga, Japan) and the primer sets nirK_01/nirK_02 (to generate nirK-A), nirK_03/nirK_04 (nirK-B), nirS_01/nirS_02 (nirS-A) and nirS_03/nirS_04 (nirS-B); the primer sequences are listed in Table S6, Supporting Information. Then, nirK-A and nirK-B, and nirS-A and nirS-B fragments were fused in a second PCR with the same polymerase and the primer sets nirK_01/nirK_04 and nirS_01/nirS_04 (Table S6, Supporting Information), respectively. The PCR products were cloned as ∼1.6-kb EcoRI–BamHI fragments into the pK18mobsacB vector (Schäfer et al.1994), and the resulting plasmids (pΔnirK and pΔnirS; Table S5, Supporting Information) were transferred by conjugation from E. coli DH5α to B. oligotrophicum to generate markerless deletions as described previously (Schäfer et al.1994). Triparental matings were conducted using pRK2013 as a helper plasmid (Figurski and Helinski 1979). Kanamycin-resistant transconjugants were selected and grown in the presence of 10% sucrose to force the loss of the vector-encoded sacB gene. The resulting colonies were checked for Km sensitivity. The desired deletions were confirmed by PCR. To obtain the ΔnirKS double mutant, the plasmid pΔnirS was transferred by conjugation from E. coli DH5α to B. oligotrophicum ΔnirK. The desired deletion in the nirS region was confirmed by PCR. Analysis of gene expression using lacZ-reporter fusions Chromosomally integrated transcriptional lacZ fusions with the nirK and nirS promoters were used. To construct the plasmids pBo-PnirKlz and pBo-PnirSlz, DNA fragments corresponding to the promoter regions of nirK and nirS were cloned into the pSUP3535 vector (Mesa et al.2003) as 0.74-kb EcoRI–PstI and 0.8-kb EcoRI–PstI fragments, respectively. pBo-PnirKlz was transferred into B. oligotrophicum S58 and the ΔnirS mutant strain, and pBo-PnirSlz was transferred into B. oligotrophicum S58 and the ΔnirK mutant strain. Triparental matings were conducted using pRK2013 as a helper plasmid (Figurski and Helinski 1979). Acquisition of Tc resistance indicated that the suicide plasmid containing the fusion had been integrated into the chromosome after a single recombination event, which generated a tandem duplication. Integration of the fusion plasmid was confirmed by PCR. For the β-galactosidase assay, cells were inoculated into 5 mL of HMM or HMMN medium (optical density at 660 nm ∼0.02) in 35-mL tubes and were reciprocally shaken (300 rpm, 30°C, 96 h) aerobically, microaerobically or anaerobically. β-galactosidase activity was determined in a microplate assay as described previously (Griffith and Wolf 2002). Analytical methods Cells were inoculated into 15 mL of HMMN medium (optical density at 660 nm, ∼0.02) in 80-mL tubes and were reciprocally shaken (300 rpm, 30°C, 96 h) aerobically, microaerobically or anaerobically. MV-dependent NiR activity was measured as described previously (Sánchez et al.2010). Protein concentrations were estimated by using the BioRad Protein Assay (Bio-Rad, CA, USA) with a standard curve obtained with varying bovine serum albumin concentrations. Extracellular nitrate and nitrite concentrations were determined using a Dionex ICS-1100 Basic Integrated Ion Chromatography System (Thermo Scientific, MA, USA). The Anion Mixed Standard Solution IV (Kanto Chemical Co., Inc., Tokyo, Japan) was used as a standard. Samples were diluted with Milli-Q water and passed through a 0.2 μm syringe filter before injection. Swimming assay Swimming motility was tested on soft agar (0.3%) plates with HMM medium supplemented with 0.5, 2, 10 or 20 mM KNO3. The plates were inoculated in the middle with 2 μL of cell suspensions at an optical density at 660 nm of ∼0.4. Plates were incubated aerobically or under low oxygen in anaerobic jars in which the gas phase was replaced once with 100% N2. The plates were incubated bottom up at 30°C for 13 days. Swimming motility was determined by measuring the colony halo diameter. Bioinformatics Basic local alignment search tool (BLASTP) searches were performed at the GenomeNet site (http://www.genome.jp/en/). Amino acid sequences were obtained from the UniProt database (http://www.uniprot.org/) by using the following accession numbers: B. oligotrophicum S58 NirK, M4Z2Q6; Bradyrhizobium diazoefficiens USDA 110 NirK, Q89EJ6; Rhodopseudomonas palustris CGA009 NirK, Q6N2A5; B. oligotrophicum S58 NirS, M4ZG55; Magnetospirillum magneticum NirS, Q2VZK6; Paracoccus denitrificans PD1222 NirS, Q51700. DNA sequences were aligned with the ClustalΩ algorithm (Sievers et al.2011). SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online. Acknowledgements We thank S. Mesa and M.J. Delgado (EEZ-CSIC, Granada, Spain) for kindly providing pSUP3535. We thank H. Mitsui and S. Hara (Graduate School of Life Sciences, Tohoku University, Sendai, Japan) for helpful technical discussions. 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Published: Mar 1, 2018
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