Spotlight | Plant Microbiology

Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants

Yue Zhen Li, Dan Wang, Xue Ying Feng, Jian Jiao, Wen Xin Chen, Chang Fu Tian
H. Goodrich-Blair, Editor
DOI: 10.1128/AEM.03454-15

ABSTRACT

The nitrogen phosphotransferase system (PTSNtr) consists of EINtr, NPr, and EIIANtr. The active phosphate moiety derived from phosphoenolpyruvate is transferred through EINtr and NPr to EIIANtr. Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EINtr and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIANtr) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-β-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIANtr in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EINtr with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTSNtr in symbiosis was also observed when related PTSNtr mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTSNtr in addition to the negative role of unphosphorylated EIIANtr.

INTRODUCTION

In bacteria, the phosphotransferase system (PTS) is important for transport and signal transduction in cellular metabolism (1). Two general types of PTSs have been identified (Fig. 1): the sugar PTS, dedicated to carbohydrate transport, and the nitrogen PTS (PTSNtr), which exerts regulatory functions (2). In the canonical model of both PTSs, described in Gram-negative bacteria, the active phosphate moiety derived from phosphoenolpyruvate (PEP) is transferred through two general phosphotransferase proteins: enzyme I (EI, or EINtr) and histidine protein (HPr or NPr) (2, 3). In the sugar PTS, HPr can then phosphorylate sugar-specific EIIA, which will pass the phosphate to the corresponding EIICB transport protein, allowing uptake of the sugar (3). With PTSNtr, NPr can phosphorylate EIIANtr, which is a homolog of EIIA but is not active in sugar transport, as the required EIIB- and EIIC-like domains are lacking (2). Accumulating evidence has suggested that EIIANtr seems to exclusively serve regulatory functions. In Escherichia coli, for example, unphosphorylated EIIANtr directly binds the TrkA subunit of the low-affinity potassium transporter, thereby inhibiting uptake of potassium at high concentrations, while it stimulates autophosphorylation of KdpD through protein-protein interactions when the potassium level is low, enhancing the transcription of kdpFABC, which encode the high-affinity potassium transporter (4, 5). In addition to potassium homeostasis, many other processes are either directly or indirectly regulated by EIIANtr, such as the phosphate starvation response in E. coli (6), expression of nitrogen fixation genes (nif) in Klebsiella pneumoniae (7), polyhydroxyalkanoate (PHA) accumulation in Azotobacter vinelandii (8), and virulence of Legionella pneumophila (9). Moreover, glutamine and α-ketoglutarate can regulate the phosphorylation state of PTSNtr by binding to the GAF domain of EINtr in E. coli, suggesting that the PTSNtr senses nitrogen availability (10).

FIG 1
FIG 1

Model of parallel phosphotransferase systems in bacteria. Genes encoding key components of a sugar PTS (left) and nitrogen PTS (PTSNtr; right) are shown in brackets. Various EII complexes, each specific for a given substrate, in the sugar PTS have been found, and the corresponding genes are not shown here.

Rhizobia induce the formation, on legume roots, of new organs called nodules, where rhizobia colonize intracellularly and atmospheric nitrogen is reduced to ammonium in specialized rhizobial cells (bacteroids). Accumulating information on genome sequences suggests that PTS transport components (i.e., EIIB or EIIC) are absent in many rhizobial strains: Sinorhizobium meliloti 1021, Mesorhizobium loti MAFF303099, Bradyrhizobium diazoefficiens USDA110, Rhizobium leguminosarum bv. viciae 3841, and Sinorhizobium fredii strains (11–17). However, proteins homologous to the core PTS components (EINtr, HPr, NPr, EIIANtr, and a mannose-type EIIAMan), as well as an HPr kinase (HPrK), can be identified in all of these rhizobia (18–20). In R. leguminosarum bv. viciae 3841 and S. meliloti 1021, phosphorylation of NPr (named HPr in S. meliloti 1021) by EINtr has been demonstrated (18, 20). The GAF domain of EINtr is not required for PEP-dependent autophosphorylation (20), but it can sense the nitrogen signal glutamine, inhibiting EINtr phosphorylation of NPr (18). EINtr, NPr, and EIIANtr mutants of R. leguminosarum bv. viciae 3841 show a rough colony morphology and are impaired in activities of a wide range of ABC transporters (19, 20). In B. diazoefficiens I110 (a derivative of strain USDA110), EINtr physically interacts with the aspartokinase AspK and regulates oligopeptide transport (21), and this AspK is encoded by the conserved lysC adjacent to ptsP in rhizobial genomes (18–20). In S. meliloti 1021, an NPr homolog can be also phosphorylated by HPrK and has been linked to succinate-mediated catabolite repression (22, 23). In R. etli, a mutant strain lacking EIIANtr exhibited reduced nifH expression (free-living cells under 0.5% oxygen), melanin synthesis, and growth on dicarboxylates (24). Notably, none of the tested strains with mutations in core PTS components (EINtr, HPr/NPr, EIIANtr, or EIIAMan) was impaired in symbiotic performance, though the hprK mutants of S. meliloti 1021 form nodules that do not fix nitrogen on alfalfa (19, 20, 22, 23).

In an exploratory project screening S. fredii mutants with symbiotic defects on the legume crop Glycine max, we found that a mutant strain of S. fredii carring ptsP::Tn5 induced ineffective nodules. As ptsP, ptsO, and ptsN encode EINtr, NPr, and EIIANtr, respectively, this suggests a potential relevance of core PTSNtr components in rhizobium-legume symbiosis (18, 19, 23). Further reverse genetics analyses were carried out to investigate the role of these PTSNtr components in establishing effective symbiosis with G. max (determinate nodules) and Cajanus cajan (indeterminate nodules).

MATERIALS AND METHODS

Bacterial strains and growth conditions.Bacterial strains and plasmids used in this study are listed in Table 1. S. fredii CCBAU 45436 is resistant to nalidixic acid (NA) and trimethoprim (TMP). The concentrations of antibiotics used for S. fredii cultures were 20 μg/ml for NA, 10 μg/ml for TMP, 50 μg/ml for kanamycin (Km), 30 μg/ml for gentamicin (Gm), and 10 μg/ml for tetracycline (Tc). S. fredii strains were grown at 28°C in TY medium (tryptone at 5 g/liter, yeast extract at 3 g/liter, and CaCl2 at 0.6 g/liter) (25).

View this table:
TABLE 1

Strains and plasmids

Construction of genetically modified strains.The plasmid pRL1063a containing Tn5 was previously introduced into S. fredii CCBAU 45436 by conjugation to generate a random mutant library containing 25,500 mutants (26, 27). A mutant forming rough colonies was found to be defective in symbiosis with G. max. To identify the Tn5 insertion site of this mutant, its DNA was digested with EcoRI, then self-ligated, and transferred into E. coli DH5α by electroporation. The resultant positive clones were used for sequencing the transposon-genome junction region, using the primer PM (5′-TCATCTAATGCTAAGGCTGC-3′) (27). For deletion of ptsP, ptsO, ptsN, ptsI (encoding an EI homolog), and ptsH (encoding an HPr homolog) (Fig. 1), the cre-lox system was used (28). DNA fragments containing the upstream/amino-terminal coding region and the downstream/carboxyl-terminal coding region of ptsP were obtained by PCR using primers PupL-PupR and PdownL-PdownR, respectively. These two fragments were digested by EcoRI/KpnI and ApaI/AgeI and cloned into EcoRI/KpnI and ApaI/AgeI restriction sites of pCM351, respectively. The resulting plasmid with correct fragment sequences was introduced into the CCBAU 45436 wild-type background by conjugation, and transconjugants sensitive to tetracycline and resistant to gentamicin were screened. The plasmid pCM157 expressing Cre recombinase, which catalyzes in vivo excision of the DNA region (containing a gentamicin resistance gene) flanked by codirectional loxP recognition sites provided by pCM351 (28), was introduced into this ptsP mutant by conjugation, and transconjugants sensitive to gentamicin were screened. Then, pCM157 was cured by selecting tetracycline-sensitive clones, resulting in the ΔptsP mutant lacking the resistance marker gene. Deletion mutant strains, including ptsO (Gmr), ptsN (Gmr), ptsH (Gmr), ptsI (Gmr), ΔptsO, and ΔptsN, were constructed with the same method, using the corresponding primers and restriction endonucleases (listed in Table 2). Moreover, the ptsPN (Gmr) and ptsON (Gmr) double mutants were obtained by deleting ptsN in the backgrounds of ΔptsP and ΔptsO, respectively. The ΔptsPN mutant strain was also constructed by removing the gentamicin resistance cassette by using pCM157, which was then cured as described above. Genetic modifications in these constructed mutants were verified by using PCR examination and Sanger sequencing.

View this table:
TABLE 2

Primers used in this study

The ΔptsP/ptsP strain was generated as follows. Primers lysCL and ptsPR were used to amplify a PCR fragment encompassing the carboxyl-terminal coding region of lysC (659 bp), the intergenic region between lysC and ptsP (92 bp), the ptsP coding region (2,268 bp), and the downstream fragment of ptsP (76 bp). This fragment was digested with HindIII/XbaI and cloned into pVO155, which does not replicate in Sinorhizobium (29). The resulting pVO155::ptsP construct with correct sequences was conjugated into the ΔptsP mutant, and the integration of this plasmid at the homologous site in the S. fredii genome was verified by PCR and Sanger sequencing. The ΔptsPN/ptsN strain was generated by using the same procedure with the corresponding cloning primers FptsN and RptsN (Table 2) and verified by PCR and Sanger sequencing.

The ΔptsP/ptsPΔGAF mutant strain was constructed as follows. The plasmid pVO155::ptsP mentioned above was used as the template, and a fragment corresponding to pVO155::ptsPΔGAF was amplified by using gafL and gafR as primers in the reverse PCR. The resulting PCR product was treated with T4 polynucleotide kinase (NEB) at 37°C for 30 min and then subjected to overnight ligation using T4 ligase (NEB) at 16°C. The resulting plasmid, pVO155::ptsPΔGAF with correct sequences, was introduced into the ΔptsP mutant by conjugation. The integration of this plasmid at the homologous site in the S. fredii genome and the absence of the region coding for the GAF domain were verified by PCR and Sanger sequencing.

Plant assays, cytological observation, and nodule occupancy.Seeds of soybean and pigeonpea were surface sterilized in 3% (vol/vol) NaClO solution, allowed to germinate, and inoculated with 1 ml (optical density at 600 nm [OD600] of 0.2) of the appropriate bacterial culture. At 30 days postinoculation (dpi), leaf chlorophyll concentrations were determined by using a SPAD-502 meter (Konica Minolta) (30). To minimize the sampling error, three leaflets of the third leaf were tested for each plant. Plant shoots were dried at 65°C for 5 days and then used for determining shoot dry weight per plant. For electron microscopy, ultrathin sections of 1-month nodules from soybeans were prepared and observed in a JEM-1230 transmission electron microscope as described earlier (31). To check nodule occupation, S. fredii mutants were individually mixed with the wild-type strain (or the mutant indicated) in an equal quantity (OD600, 0.2; 1-ml volume) and inoculated on soybeans. At 30 dpi, nodules were sequentially rinsed in 95% ethanol for 30 s, then 3% NaClO for 5 min, and then washed with sterilized water eight times. Subsequently, crude extract of each nodule (5 μl) was inoculated on TY plates with or without the corresponding antibiotics.

Carbon source utilization.In the exploratory test, CCBAU 45436 and the strain with the ptsP::Tn5 insertion were compared for their carbon source utilization abilities by using the Biolog GN2 MicroPlate. Then, growth of all PTSNtr mutants was tested in White's medium (32, 33) containing 0.1% (wt/vol) of the sole carbon sources that the strain with ptsP::Tn5 could not use as efficiently as CCBAU 45436 (i.e., d-glucosaminic acid, γ-aminobutyric acid, N-acetyl-d-galactosamine, l-carnitine, and l-alanine). Three independent experiments were carried out. Briefly, all strains were grown on TY plates with the corresponding antibiotics at 28°C for 4 days. Then, colonies were resuspended in 0.8% NaCl solution, and the centrifuged pellets were washed by using 0.8% NaCl and centrifuged again. Three washing cycles were done. The final suspension was further diluted, resulting in inoculant suspensions with calculated OD600s of 0.1, 0.01, 0.001, 0.0001, 0.0001, and 0.000001. Then, 5 μl of each inoculant suspension was cultured on a White medium plate without antibiotics at 28°C for 6 days. Finally, the plate with all test strains was photographed.

Nucleotide sequences.The GenBank accession number for the S. fredii CCBAU 45436 draft genome assembly is GCA_000261885.1 (17). Sequences of related genes described in this study are available in the supplemental material.

RESULTS AND DISCUSSION

S. fredii mutants of ptsP or ptsO induce ineffective nodules on soybean.S. fredii strains can establish symbiosis with diverse legumes, including the important crops soybean (G. max, with determinate nodules) and pigeonpea (C. cajan, with indeterminate nodules) (34–37). CCBAU 45436 represents a dominant sublineage of S. fredii that nodulates soybeans in alkaline-saline soils (17, 38, 39). A mutant forming rough colonies was found in a Tn5 mutant library of CCBAU 45436 constructed previously in our lab (27). Soybean plants inoculated with this mutant showed a typical nitrogen starvation phenotype (data not shown). Sequencing the DNA flanking the ends of the Tn5 from this mutant, as described in Materials and Methods, revealed that the transposon had inserted 2,211 bp downstream of the start of ptsP (total length, 2,268 bp). The link between this Tn5 insertion and the symbiosis phenotype was demonstrated by using ptsP deletion mutants with or without the replacing fragment, which provides gentamicin resistance (Fig. 2A and data not shown). To minimize potential contamination during the study, the ptsP mutant resistant to gentamicin was used in all experiments unless indicated. Nodule number per plant induced by this ptsP mutant on soybeans was around 170% of that formed by CCBAU 45436 wild type (Fig. 2B). However, the relative chlorophyll concentration and shoot dry weight of soybean inoculated with the ptsP mutant was 69% and 77%, respectively, of that with CCBAU 45436 and slightly higher than the uninoculated control (116% and 104%, respectively) (Fig. 2C and D). As shown in Fig. S1A in the supplemental material, this PtsP showed high similarity to the published EINtr proteins of Sinorhizobium meliloti (96.1%) and Rhizobium leguminosarum (82.6%) (18, 20), though no symbiotic defects have been found for ptsP mutants of S. meliloti and R. leguminosarum. Further analysis of the CCBAU 45436 genome identified ptsO, which encodes a protein with high similarity to the NPr homologs in S. meliloti (90.4%) and R. leguminosarum (80.7%) (18, 20) (see Fig. S1B). The ptsO mutant generally showed symbiotic defects on soybeans similar to the ptsP mutant (Fig. 2A to D). Although the shoot dry weight of soybean inoculated with the ptsO mutant was not significantly different from uninoculated control plants, its difference with shoot dry weight of plants inoculated with CCBAU 45436 was also not statistically significant (Duncan's test, α = 0.05; t test, P = 0.32).

FIG 2
FIG 2

Symbiotic performance of PTSNtr mutant strains on soybeans. (A) Soybean shoot and nodule morphology. Bar, 1 mm. (B) Nodule number per plant. (C) Relative concentration of chlorophyll, determined using a SPAD-502 chlorophyll meter. (D) Shoot dry weight per plant. Means ± standard errors of the means of results for each treatment are based on 13 to 33 scored plants from multiple independent experiments. Different letters above the error bar indicate a significant difference between means based on Duncan's test (α = 0.05).

Results of the acetylene reduction assay (ARA) (see Fig. S2 in the supplemental material) suggested that the nitrogenase activity was reduced by the ptsP mutation to 44% (t test, P < 0.001) of CCBAU 45436, while ARA was impaired by the mutation of ptsO to a lesser extent (62% of CCBAU 45436; t test, P = 0.071). Impaired but detectable nitrogenase activities in the ptsP or ptsO mutants were consistent with a higher leaf chlorophyll content of soybean plants inoculated with these two mutants than the uninoculated control plants (Fig. 2C).

In S. meliloti, HPrK is essential for effective symbiosis with alfalfa and can phosphorylate the NPr homolog at the serine-53 residue (18, 22); however, the deletion mutant missing the gene encoding NPr exhibited wild-type nodulation and nitrogen fixation on alfalfa (23). In addition to the conserved EINtr and NPr, a gene cluster coding for the putative EI (ptsI), HPr (ptsH), and EIIAMan is also present in the genome of S. fredii strains (CCBAU 45436/HH103) (17, 40). However, the ptsI and ptsH mutants were not impaired in symbiotic performance in terms of the relative chlorophyll concentration and shoot dry weight of soybean (Fig. 2A to D).

Negative and positive roles of ptsN in symbiosis of S. fredii and soybean.Although multiple homologs of PtsN can be found in the genomes of S. fredii (CCBAU 45436 and HH103) and also R. leguminosarum and R. etli (11, 17, 40, 41), phylogenetic analysis suggested that only one of them is the putative ortholog of EIIANtr in proteobacteria (see Fig. S1C in the supplemental material). This view is also supported by its conserved genomic location, i.e., ptsN encoding this EIIANtr ortholog is located downstream of rpoN and yhbH in CCBAU 45436, as in other proteobacteria (2). However, the ptsN mutant formed effective nodules, as indicated by the chlorophyll concentration and shoot dry weight, both of which were slightly higher than those of plants inoculated with CCBAU 45436 (Fig. 2C and D) (t test, P < 0.05). Since EINtr and NPr catalyze the PEP-dependent phosphorylation of EIIANtr, these results imply that EIIANtr might not be essential for symbiosis, but the nonphosphorylated EIIANtr in the ptsP or ptsO mutants could play unidentified negative regulation roles, as proposed in earlier studies of PTSNtr in other bacteria (2). To test this hypothesis, ptsN was further deleted in the ptsP or ptsO mutant backgrounds, resulting in ptsPN and ptsON double mutants, respectively. As shown in Fig. 2, the nodule numbers per plant formed by ptsPN or ptsON were significantly reduced compared to the ptsP or ptsO mutants, while chlorophyll concentration was increased (Duncan's test, P < 0.05), indicating that the nodules formed by the ptsPN or ptsON mutant strains are more effective than those induced by the ptsP or ptsO mutants. Therefore, nonphosphorylated EIIANtr in the ptsP or ptsO mutants may negatively regulate symbiotic processes in nodules. Moreover, introducing the wild-type ptsN back into the chromosome of ΔptsPN generates ΔptsPN/ptsN, which performs similarly to the ptsP mutant (Fig. 2A to D). These results suggest that ptsN within the conserved rpoN loci of bacteria could be the major EIIANtr coding gene, though additional ptsN copies are present in CCBAU 45436 (see Fig. S1C).

As the efficiency of nodules is closely related to the development and persistence of bacteroids (42), 30-dpi nodules induced by PTSNtr mutants were collected and observed by using TEM. In contrast to CCBAU 45436, the number of bacteroids within each symbiosome and the production of the carbon storage polymer poly-β-hydroxybutyrate (PHB, a representative PHA), were decreased in nodules induced by the ptsP or ptsO mutants but not in the ptsN, ptsPN, or ptsON mutants (Fig. 3). Consistent with these results, it has been reported that loss of ptsP or ptsO significantly diminishes PHA accumulation in A. vinelandii and Pseudomonas putida, while loss of ptsN can increase PHA accumulation (8, 43, 44). S. meliloti strains with mutations in phbC and phaP1-phaP2, genes involved in PHB synthesis and granule formation, respectively, exhibited reduced symbiotic performance on Medicago truncatula (45). The phbC gene of Azorhizobium caulinodans is required for nitrogen fixation (46). However, the phbC mutant of Rhizobium etli showed increased symbiotic performance (47). Although PHB has been proposed as a store of both carbon and reductant, the role of PHB during symbiosis remains elusive, particularly when comparing different rhizobium-legume systems (48). Moreover, it has been demonstrated that nonphosphorylated EIIANtr can directly interact with KdpD (from E. coli and S. meliloti) and PhoR (from E. coli) in vitro; these two proteins are involved in regulating K+ homeostasis and the phosphate starvation response, respectively (4, 6, 19). Involvement of K+ homeostasis and phosphate transporters in rhizobium-legume symbiosis has been described elsewhere (49–51).

FIG 3
FIG 3

Ultrathin sections of soybean nodules formed by PTSNtr mutant strains. Pictures of ultrathin sections of 30-dpi nodules induced by CCBAU 45436 and the ptsP, ptsO, ptsN, ptsPN, and ptsON mutants were obtained by using transmission electron microscopy. Bar, 1 μm.

It should be noted that the symbiotic abilities of ptsPN or ptsON were still not comparable to that of CCBAU 45436 in terms of either chlorophyll concentration or shoot dry weight (Fig. 2C and D). This might be explained by the functions of PtsP and PtsO that involve more than transferring a phosphoryl group to PtsN. The PtsP homolog in B. diazoefficiens I110 physically interacts with LysC, which encodes an aspartokinase that catalyzes the phosphorylation of aspartate, and this interaction could regulate oligopeptide transport (21). In E. coli, the dephosphorylated PtsO has been demonstrated to interact with and inhibit LpxD, which is involved in biosynthesis of lipid A of the lipopolysaccharide layer (52). Homologs of LysC and LpxD are also present in the CCBAU 45436 genome.

Alternatively, a potential unknown positive regulation role of EIIANtr might also be involved in optimizing symbiosis. To test these possibilities, nodulation occupancy experiments were carried out by equally mixing representative strains in inoculants. As expected, the ptsP and ptsO mutants occupied only 3% and 7% of nodules, respectively, when inoculated equally with CCBAU 45436 (P < 0.05, t test). The ptsN mutant showed a smaller but also significant reduction in nodule occupancy compared to CCBAU 45436 (33% versus 67%; P < 0.05, t test), indicating a positive role of EIIANtr in symbiosis. Moreover, ptsPN occupied 5% and 8% of nodules when equally inoculated with CCBAU 45436 and the ΔptsN mutant, respectively (P < 0.05, t test), implying an essential role of cellular processes other than the transfer of the phosphoryl group to EIIANtr.

The GAF domain of EINtr is dispensable for S. fredii-soybean symbiosis.As described above, we demonstrated that the PTSNtr core components EIIANtr, EINtr, and NPr are essential for modulation of symbiosis between S. fredii and soybean plants. Although this phosphotransferase system has been historically named the nitrogen-related PTS due to the conserved localization of its genes in the rpoN gene cluster, signals modulating the phosphorylation state of the PTSNtr were not known for a long time (2). Recently, glutamine and α-ketoglutarate, the canonical signals of nitrogen availability, were shown to regulate the phosphorylation state of EINtr in E. coli, and the GAF signal transduction domain of EINtr is necessary for this process (10). In S. meliloti, in vitro experiments suggest that glutamine rather than α-ketoglutarate can bind the GAF domain and inhibit EINtr activity (18). Therefore, it is very likely that the GAF domain of EINtr is involved in regulating PTSNtr phosphorylation by sensing nitrogen availability. To study the potential role of the GAF domain of EINtr in symbiosis, a ptsP fragment with or without the GAF domain was introduced into the genome of the ptsP deletion mutant (ΔptsP) in such a way that the transcription of the gene fragment was under the control of the wild-type ptsP promoter, resulting in ΔptsP/ptsP and ΔptsP/ptsPΔGAF, respectively (Table 1 and Fig. 4A). The nodule number induced by either ΔptsP/ptsP or ΔptsP/ptsPΔGAF was significantly less than that formed by the ptsP mutant but similar to CCBAU 45436 (Fig. 4B) (Duncan's test, α = 0.05). On the other hand, ΔptsP/ptsP and ΔptsP/ptsPΔGAF showed similar symbiotic capacities as CCBAU 45436 as measured by soybean chlorophyll concentration (Fig. 4C) (Duncan's test, α = 0.05) and shoot dry weight (Fig. 4D) (Duncan's test, α = 0.05), which were significantly higher than in plants inoculated with the ptsP mutant (Duncan's test, P < 0.05). Moreover, their bacteroids were also indistinguishable from those of wild-type strain CCBAU 45436 (Fig. 4E and 3). In short, symbiotic defects of the ptsP mutant can be complemented by the wild-type ptsP or the one lacking GAF domain sequences, in contrast to the partial recovery in ptsPN and ptsON double mutants (Fig. 2). Thus, EINtr lacking the GAF domain is still functional in bacteroids. This is supported by the in vitro evidence that the N-terminal GAF domain of EINtr from R. leguminosarum is not required for PEP-dependent autophosphorylation (20). These results also indicate that the concentration of glutamine in bacteroids might be below the level that could inhibit the activity of EINtr through binding the GAF domain (18). This view is consistent with the downregulation of NH4+ uptake (AmtB) and assimilation system (glutamine synthetase) in bacteroids and the export of fixed nitrogen by rhizobia to support plant growth (53).

FIG 4
FIG 4

The GAF domain of EINtr is dispensable for symbiosis. (A) Soybean shoot and nodule morphology. Bar, 1 mm. (B) Nodule number per plant. (C) Relative concentration of chlorophyll, determined by using a SPAD-502 chlorophyll meter. (D) Shoot dry weight per plant. Means ± standard errors of the means for results of each treatment are based on 16 scored plants from two independent experiments. Different letters above the error bars indicate significant differences between means, based on Duncan's test (α = 0.05). (E) Ultrathin sections of soybean nodules formed by ΔptsP/ptsPΔGAF (ΔptsP complemented with a ptsP gene lacking the fragment coding for the GAF domain) and ΔptsP/ptsP (ΔptsP complemented with a ptsP gene). Pictures of 30-dpi nodules were obtained using transmission electron microscopy. Bar, 1 μm.

Role of PTSNtr in symbiotic interactions between S. fredii and pigeonpea.Despite the high similarity of core PTSNtr components among rhizobia (see Fig. S1 in the supplemental material), no symbiotic defects have been found for related PTSNtr mutants in tested species, such as R. leguminosarum and S. meliloti, when they were inoculated on corresponding legume hosts pea and alfalfa (19, 23). This further strengthens the view that diverse rhizobium recipes are used in establishing symbiosis with legumes (54). Given the broad host range of S. fredii (34), representative mutants of PTSNtr were inoculated on pigeonpea, which forms indeterminate nodules. As shown in Fig. 5, compared to CCBAU 45436 and the ptsN mutant, the ptsP or ptsO mutants formed a greater number of ineffective nodules on pigeonpea (Fig. 5A and B), and both the chlorophyll concentration and shoot dry weight of host plants were significantly reduced when the ptsP or ptsO mutants were inoculated (Fig. 5C and D). In contrast to the ptsP mutant, ptsPN formed fewer but effective nodules and performed better in improving the chlorophyll concentration and shoot dry weight of pigeonpea (Fig. 5A to D). Moreover, the ptsN mutant was slightly better than CCBAU 4536 regarding shoot dry weight, though this was not statistically significant (Fig. 5D) (Duncan's test α = 0.05; t test, P = 0.68). These results suggest that nonphosphorylated EIIANtr may play a negative regulation role in symbiosis between S. fredii and pigeonpea. Therefore, PTSNtr is required by S. fredii for establishing effective symbiosis with both soybean and pigeonpea. Notably, bacteroids in soybean and pigeonpea are of the nonswollen type, whereas those in alfalfa and pea belong to the swollen type characterized by limited PHB accumulation, enlarged cell size, genome amplification, and terminal differentiation (55). It remains elusive whether these differences could explain the inconsistences regarding the symbiotic phenotype of PTSNtr mutants between this study and those focused on R. leguminosarum/pea and S. meliloti/alfalfa.

FIG 5
FIG 5

Symbiotic performance of PTSNtr mutant strains on pigeonpea. (A) Pigeonpea shoot and nodule morphology. Bar, 1 mm. (B) Nodule number per plant. (C) Relative concentration of chlorophyll, determined by using a SPAD-502 chlorophyll meter. (D) Shoot dry weight per plant. Means ± standard errors of the means of results with each treatment are based on 10 to 29 scored plants from multiple independent experiments. Different letters above the error bars indicate a significant difference between means, based on Duncan's test (α = 0.05).

Relevance of PTSNtr components to carbon metabolism and exopolysaccharide production.The reduced PHB accumulation in bacteroids of the ptsP and ptsO mutants in soybean nodules (Fig. 3) implies potential defects in carbon metabolism compared to CCBAU 45436, ptsN, ptsPN, and ptsON mutants. In the exploratory test of carbon source utilization using the Biolog GN2 MicroPlate, which provides simultaneous tests of 95 carbon sources, the ptsP::Tn5 mutant showed impaired growth on d-glucosaminic acid, γ-aminobutyric acid, N-acetyl-d-galactosamine, l-carnitine, and l-/d-alanine. Therefore, utilization of these carbon sources was further tested for other PTSNtr mutants (Fig. 6). The ptsP, ptsO, ptsN, and ptsPN mutants showed notable defects in utilizing l-alanine compared to the ptsON mutant or CCBAU 45436, implying either a positive (in the background of the wild type or the ptsP mutant) or a negative (in the ptsO mutant background) regulatory role of EIIANtr. It was initially reported that l-alanine, not ammonia, is excreted from bacteroids inside soybean nodules as the principle export product of nitrogen fixation (56), but this view was later disputed based on a reassessment of products secreted by soybean bacteroids (57). De novo l-alanine synthesis by alanine dehydrogenase is not essential for nitrogen fixation by R. leguminosarum or M. loti in nodules of pea or Lotus corniculatus, while secretion of l-alanine, generated by bacterial transamination, from bacteroids was detected (58, 59). Although the role of l-alanine in symbiosis remains elusive, apparently differences in l-alanine utilization by mutants of PTSNtr components (Fig. 6) do not correlate well with their symbiotic capacity described above (Fig. 2 to 4).

FIG 6
FIG 6

Carbon source utilization by PTSNtr mutants. Dashed lines separate White's medium plates containing different carbon sources. Colonies formed by strains at the same dilution level are shown.

In contrast to l-alanine, the other four carbon sources can be utilized by all strains, though ptsP, ptsO, and ptsPN formed relatively rugous colonies, indicating reduced exopolysaccharide (EPS) production (Fig. 6) (35, 60). The mutation of ptsP or ptsO in R. leguminosarum also resulted in rough colonies (19, 20). It has been demonstrated that the production of EPS could vary upon different carbon sources available to rhizobia (61). This view is also supported by the fact that CCBAU 45436 colonies utilizing d-glucosaminic acid, l-carnitine, or N-acetyl-d-galactosamine as sole carbon source are smoother than those utilizing l-alanine or γ-aminobutyric acid. Moreover, the ptsN mutant generally shows normal colonies compared with CCBAU 45436, except when utilizing l-carnitine. It is noteworthy that the ptsON mutant seems to produce more EPS than the ptsO mutant, while colonies of the ptsPN mutant are indistinguishable from those of the ptsP mutant. However, when cultured on medium containing yeast extract and mannitol, the ptsPN mutant formed mucoid colonies similar to CCBAU 45436, in contrast to the rugous colonies of the ptsP mutant (see Fig. S3 in the supplemental material). It has been reported that the symbiotic capacity of an S. fredii exoA mutant unable to produce EPS was not significantly altered on soybean and pigeonpea (62, 63). Therefore, the alteration of EPS production might not be the main cause of the contrasting symbiotic performance exhibited by mutants of PTSNtr components.

Although molecular determinants underlying these growth patterns remain elusive, as described above, apparently the linear transfer of a phosphoryl group to EIIANtr through EINtr and NPr should not be the only cue. Indeed, an interaction of PtsP with LysC in B. diazoefficiens I110 and regulation of the PtsO homolog by HPrK in S. meliloti have been demonstrated (21, 22), indicating multiple input signals into the PTSNtr system. Consequently, the effect of ptsN deletion might be also condition dependent, as demonstrated in the utilization of l-alanine by the ptsN, ptsPN, and ptsON mutant strains. Moreover, ΔptsP/ptsPΔGAF and ΔptsP/ptsP are similar to CCBAU 45436 regarding carbon source utilization ability and colony morphology, suggesting that the GAF domain is not essential for PEP-dependent PTSNtr phosphorylation under the tested conditions, as demonstrated with R. leguminosarum bv. viciae 3841 (20). Taken together with the evidence from earlier studies on the GAF domain of EINtr (10, 18, 20), it is likely that the GAF domain is mainly involved in the inhibition of PTSNtr when a signal such as a high level of glutamine is present, i.e., when integrating nitrogen and carbon metabolism (18).

Conclusions.The nitrogen PTSNtr (containing three core components: EINtr, NPr, and EIIANtr) is conserved in many proteobacteria, including rhizobia. Although the GAF domain of EINtr is dispensable for PEP-dependent autophosphorylation of EINtr, as shown in E. coli and R. leguminosarum (10, 20), the inhibition of EINtr by the binding of glutamine (a canonical signal of nitrogen availability) to its GAF domain has been demonstrated in both E. coli and S. meliloti (10, 18). In this study, ΔptsP/ptsPΔGAF was indistinguishable from CCBAU 45436 and ΔptsP/ptsP with regard to symbiotic performance, possibly due to an unchanged phosphate transfer process in PTSNtr. This implies that the signal (putatively glutamine) inhibiting EINtr might be limited in bacteroids. However, mutants with defects in phosphate transfer of PTSNtr (ptsP or ptsO) showed reduced carbon storage (PHB) in nodules that are ineffective in providing nitrogen to plants. These defects were significantly recovered in the ptsPN and ptsON mutants, indicating a negative role of unphosphorylated EIIANtr in PHB synthesis and symbiosis. On the other hand, reduced nodule occupancy by the ptsN mutant implied an unidentified essential role of phosphorylated EIIANtr. Mutants of core PTSNtr components in CCBAU 45436 showed a consistent symbiotic phenotype on soybean (determinate nodules) and pigeonpea (indeterminate nodules), whereas no symbiotic defects have been found for mutants of their homologs in rhizobia nodulating pea and alfalfa (indeterminate nodules). It would be interesting to study whether PTSNtr is differentially integrated into the regulation network in contrasting types of bacteroids: nonswollen bacteroids in soybean and pigeonpea and swollen bacteroids in pea and alfalfa.

ACKNOWLEDGMENTS

We thank J. Peter W. Young for language revision.

This work was supported by the National Basic Research Program of China (973 Program 2015CB158300) and the Innovative Project of State Key Laboratory of Agrobiotechnology (2014SKLAB4-1).

FOOTNOTES

    • Received 21 October 2015.
    • Accepted 10 December 2015.
    • Accepted manuscript posted online 18 December 2015.

  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03454-15.

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