Alfalfa Motility, and Symbiotic Nodulation on Involved ...

The Sinorhizobium meliloti ntrX Gene Is Involved in Succinoglycan Production, Motility, and Symbiotic Nodulation on Alfalfa Dong Wang, Haiying Xue, Yiwen Wang, Ruochun Yin, Fang Xie and Li Luo Appl. Environ. Microbiol. 2013, 79(23):7150. DOI: 10.1128/AEM.02225-13. Published Ahead of Print 13 September 2013.

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The Sinorhizobium meliloti ntrX Gene Is Involved in Succinoglycan Production, Motility, and Symbiotic Nodulation on Alfalfa Dong Wang,a,b Haiying Xue,a Yiwen Wang,c Ruochun Yin,a Fang Xie,b Li Luob School of Life Science, Anhui University, Heifei, Anhui, Chinaa; State Key Lab of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, Chinab; School of Life Science, East China Normal University, Shanghai, Chinac

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nder combined nitrogen starvation conditions, Sinorhizobium meliloti infects alfalfa plants to induce the formation of indeterminate root nodules where atmospheric dinitrogen is fixed as ammonia in exchange for photosynthesis products. This very complex process is regulated by many factors. At the early stage, S. meliloti NodD regulators sense specific secondary metabolic signals, such as luteolin, and activate the expression of nodulation (nod) genes to synthesize nodulation factors (NFs), a group of lipo-chitin-oligosaccharides (1–3). NFs initiate rhizobial infection and the development of nodule primodia on host plants (4, 5). S. meliloti cells colonize the tips of alfalfa root hairs and induce the formation of infection threads, through which they enter the polyploidy cells of the nodule primodia (6). Sinorhizobium polysaccharides, including exopolysaccharides (EPS) and cyclic-␤-glucan, are required for the formation of infection threads, for which biosynthesis is strictly regulated by some two-component regulatory systems (TCSs) (7). Succinoglycan, one S. meliloti EPS, is composed of variable units of an octosaccharide repeat (8, 9). More than 22 (exo) genes are involved in S. meliloti succinoglycan biosynthesis, including those of glycosyltransferases (exoY, exoU, exoW, exoL, and exoA), modification enzymes (exoZ, exoH, and exoV), and a transporter (exoT) (10, 11). Succinoglycan biosynthesis is modulated by several regulators, including SyrA, MucR, ExoX, NtrB/NtrC, PhoB, ExoR, ExoS/ChvI, CbrA, ExpR, and EmmABC (12–22). Among them, NtrB/NtrC, PhoB, ExoS/ChvI, CbrA, and EmmB/EmmC belong to two-component systems. It is well known that the ExoS/ChvI system directly regulates exo gene expression together with the periplasmic repressor ExoR (23, 24). S. meliloti cyclic-␤-glucan is synthesized by NdvB, and its export requires the permease NdvA. The expression of ndvA is regulated by the TCS FeuP/FeuQ (25). Rhizobial cells differentiate into bacteroids in which the expression of nitrogenase genes (such as nifHDK) is activated by the positive regulator NifA. Transcription of nifA is upregulated under microaerobic conditions by FixL/FixJ, an oxygen-sensing TCS

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(26). Moreover, the transport of dicarboxylic acids (carbon sources of bacteroids) from host cells to bacteroids is regulated by the TCS DctB/DctD (27). Therefore, TCSs play key roles in regulating symbiotic interactions between rhizobia and legumes. A TCS is usually composed of one histidine kinase (HK) and one response regulator (RR) (28). Their encoding genes are present in almost all bacteria and are involved in all kinds of physiological processes, including nitrogen metabolism, polysaccharide production, and flagellum formation (29). A total of 102 TCS genes (44 HK and 58 RR) have been reported in S. meliloti 1021 (30). Among these, seven HK genes and 12 RR genes have been studied using genetic mutations, including the TCS NtrB/NtrC, which is required for nitrogen fixation and nif gene expression in Klebsiella pneumoniae and the free-living Azorhizobium caulinodans (31). However, NtrB/NtrC is not essential for symbiotic nitrogen fixation in S. meliloti or Bradyrhizobium japonicum, suggesting that unknown regulators participating in symbiotic nitrogen metabolism exist in these rhizobia (31). In A. caulinodans, the TCS NtrY/NtrX, identified by Tn5 transposon insertion, regulates free-living nitrogen metabolism and symbiotic nodulation on Sesbania rostrata (32). A. caulinodans is a special Rhizobium species because it fixes dinitrogen under both free-living and symbiotic conditions. The homolog of NtrY/NtrX also regulates nif gene expression in Azospirillum brasilense (33). However, the function of NtrY/NtrX homologs in typical rhizobia (with nitrogen fixation only under symbiotic conditions) such as S. meliloti is unclear. In this study, we constructed ntrX/ntrY mu-

Received 5 July 2013 Accepted 5 September 2013 Published ahead of print 13 September 2013 Address correspondence to Li Luo, [email protected]. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.02225-13

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Rhizobia establish a symbiotic relationship with their host legumes to induce the formation of nitrogen-fixing nodules. This process is regulated by many rhizobium regulators, including some two-component regulatory systems (TCSs). NtrY/NtrX, a TCS that was first identified in Azorhizobium caulinodans, is required for free-living nitrogen metabolism and symbiotic nodulation on Sesbania rostrata. However, its functions in a typical rhizobium such as Sinorhizobium meliloti remain unclear. Here we found that the S. meliloti response regulator NtrX but not the histidine kinase NtrY is involved in the regulation of exopolysaccharide production, motility, and symbiosis with alfalfa. A plasmid insertion mutant of ntrX formed mucous colonies, which overproduced succinoglycan, an exopolysaccharide, by upregulating its biosynthesis genes. This mutant also exhibited motility defects due to reduced flagella and decreased expression of flagellins and regulatory genes. The regulation is independent of the known regulatory systems of ExoR/ExoS/ChvI, EmmABC, and ExpR. Alfalfa plants inoculated with the ntrX mutant were small and displayed symptoms of nitrogen starvation. Interestingly, the deletion mutant of ntrY showed a phenotype similar to that of the parent strain. These findings demonstrate that the S. meliloti NtrX is a new regulator of succinoglycan production and motility that is not genetically coupled with NtrY.

New Functions of the Sinorhizobium ntrX Gene

TABLE 1 Strains and plasmids Relevant properties

Reference/source

E. coli strains DH5␣ MT616

F⫺ ␾80lacZ⌬M15 ⌬(lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (rK⫺ mK⫹) phoA supE44 ␭⫺ thi-1 gyrA96 relA1 MM294 pRK600; Cmr

Lab stock 34

S. meliloti strains 1021 (Rm1021) Rm8530 EC69 Rm7095 SmHC12 SmLL1 SmLL2 SmLL3

SU47 but also str-2; Smr Rm1021 expR⫹ (formerly expR101) Rm1021 chvIK124T Rm1021 exoR95::Tn5; Nmr Rm1021 exoR108::pK19mob2⍀HMB chvI109 Rm1021 ntrX::pK18mobsacB; Nmr Rm1021 ⌬ntrY Rm8530 ntrX::pK18mobsacB; Nmr

19 44 24 18 19 This study This study This study

Plasmids pK18mobsacB pLL1 pLL2 pSRK-Tc pLL3 pLL4 pLL5 pLL6 pLL7 pLL8 pLL9 pLL10 pHC510

Suicide vector; Kanr pK18mobsacB carrying a 1,600-bp joint DNA fragment from S. meliloti ntrY and trkA; Kanr pK18mobsacB carrying a 1.600-bp joint DNA fragment from S. meliloti ntrC and ntrX; Kanr Expressing vector under the control of lac promoter; Tetr pSRK-Tc carrying S. meliloti ntrX gene; Tetr pSRK-Tc carrying S. meliloti ntrY gene; Tetr pSRK-Tc carrying S. meliloti ntrXY genes; Tetr pSRK-Tc carrying S. meliloti trkA gene; Tetr pSRK-Tc carrying S. meliloti chvI gene; Tetr pSRK-Tc carrying S. meliloti phoB gene; Tetr pSRK-Tc carrying S. meliloti cbrA gene; Tetr pSRK-Tc carrying S. meliloti emmC gene; Tetr pMB393 carrying S. meliloti exoR gene; Sper

36 This study This study 37 This study This study This study This study This study This study This study This study 19

tants in the background of S. meliloti 1021. We analyzed the freeliving and symbiotic phenotypes of the mutants and characterized succinoglycan production and motility under free-living conditions as well as symbiotic nodulation on alfalfa. Our new findings reveal that the S. meliloti ntrX (not ntrY) gene is pleiotropic for succinoglycan biosynthesis, motility, and symbiosis. MATERIALS AND METHODS Bacterial strains and growth conditions. Escherichia coli DH5␣ and MT616 were grown in Luria-Bertani (LB) medium at 37°C. Sinorhizobium meliloti strains (Table 1) were grown in LB medium supplemented with 2.5 mM MgSO4 and 2.5 mM CaCl2 (LB/MC) at 28°C (34). M9 medium was prepared as described by Charusanti et al. (35). The following antibiotics were used at the indicated concentrations: chloramphenicol, 10 ␮g ml⫺1; neomycin, 200 ␮g ml⫺1; kanamycin, 25 ␮g ml⫺1; tetracycline, 10 ␮g ml⫺1; and streptomycin, 500 ␮g ml⫺1. The S. meliloti phage ␾M12 was used for the general transduction (34). Construction of plasmids and the ntrX/Y mutants. Two 800-bp DNA fragments flanking the S. meliloti ntrX or ntrY open reading frame (ORF) were amplified with KOD plus DNA polymerase (Toyobo, Osaka, Japan), and both PCR products were purified using a Gel Extraction System B kit (Biodev-Tech, Beijing, China). The purified PCR products (molar ratio ⫽ 1:1) were used to amplify a 1.6-kb joining fragment with TransStart Taq DNA polymerase (Transgen, Beijing, China). All four primers (P1 to P4 or P1-1 to P4-1) are listed in Table 2. The resulting PCR product was digested with HindIII and XbaI (TaKaRa, Dalian, China) at 37°C overnight. The digested DNA was purified using an EasyPure PCR purification kit (Transgen) and then ligated to pK18mobsacB by incubation with T4 DNA ligase (TaKaRa) overnight at 16°C. The ligation product was used to transform E. coli DH5␣ competent cells (36). The recombinant plasmid pLL1 or pLL2 was extracted and purified using an EasyPure Plasmid MiniPrep kit (Transgen). Positive clones were identified by EcoRI and


XbaI digestion. The resulting plasmid was then transferred into S. meliloti 1021 by triparental mating with the helper strain E. coli MT616 (34). The plasmid insertion mutants were named SmLL1 and SmLL2-1 (Table 1). The streptomycin- and neomycin-resistant colonies were spread onto LB/MC agar plates containing 5% sucrose and streptomycin. The resulting colonies were screened for sensitivity to neomycin and identified by PCR with the primers P1/P4 or P1-1/P1-4 (Table 2). The specific PCR products were sequenced to confirm deletion of the ntrX or ntrY ORF (Invitrogen, Shanghai, China). The S. meliloti strain without the ntrY ORF was named SmLL2 (Table 1). The ORFs of ntrX, ntrY, ntrYX, trkA, chvI, phoB, cbrA, and emmC were also amplified using the primers P4/P5, P6/ P7, P5/P7, P8/P9, P53/P54, P55/P56, P57/P58, and P59/P60, respectively (Table 2), digested by NdeI and XbaI, and cloned into pSRK-Tc under the control of the E. coli lac promoter and produced the recombinant plasmids pLL3 to pLL10 (Table 1), respectively. These plasmids were transferred into the ntrX mutant by triparental mating. A total of 0.25 mM isopropyl ␤-D-1-thiogalactopyranoside (IPTG) (Sigma, St. Louis, MO, USA) was used to induce expression of the cloned gene (37). Nodulation test and nitrogenase activity assay. Assays of alfalfa nodulation on plates were conducted as described previously (38). Assays of alfalfa nodulation in a vermiculite-perlite (3:1) mixture were performed as described by Wang et al. (39). Alfalfa nodule nitrogenase activity was assayed using an acetylene (C2H2) reduction method (38). Assay for succinoglycan or EPS levels. Fresh S. meliloti cultures on LB/MC plates were restreaked onto LB/MC solid medium containing 0.02% calcofluor white M2R (Sigma) and 0.01 M HEPES (Sigma), pH 7.5. After 3 days of incubation at 28°C, succinoglycan production was visualized under visible and UV light (18). Photographs were taken with a Coolpix 4500 digital camera (Nikon, Tokyo, Japan). The fluorescence intensity was analyzed by the free BandScan program. Next, 0.25 ml of the supernatant was collected from the overnight subculture of Sinorhizobium strains in LB/MC broth (optical density at 600 nm [OD600] ⬇ 0.8), and

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Strain or plasmid

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TABLE 2 Primers Sequence

Purpose

P1 P2 P3 P4 P1-1 P2-1 P3-1 P4-1 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 P25 P26 P27 P28 P29 P30 P31 P32 P33 P34 P35 P36 P37 P38 P39 P40 P41 P42 P43 P44 P45 P46 P47 P48 P49 P50 P51 P52 P53 P54 P55 P56 P57 P58 P59 P60

5=-AAGCTTGACGAATCGCATGGCTCCTAT-3= 5=-CATTGGTATTACCCTTATCCTTCAAATT-3= 5=-AATTTGAAGGATAAGGGTAATACCAATGTGATCAGAAACCCATAACGAGCC-3= 5=-GCTCTAGACGTTATAGCCGATAAAGCAGC-3= 5=-CCCAAGCTTTGACGGCACTTTATCCACAGGATG⫺3= 5=-AGTTGATGTGGCTGTTGCCCATCCATCCACCATGAAAAGTGCTACC-3= 5=-TGGGCAACAGCCACATCAACT-3= 5=-GCTCTAGACCGAAAGGATTCCCGAGACG-3= 5=-ggaattccatatgGCGGCTGATATTCTGGTTGTGGATGACGA-3= 5=-ggaattccatatgGCCTTGCCATTGGGCACGGAGGACG-3= 5=-GCTCTAGATCACACGCCCAACGACTTCAGCTTGCGA-3= 5=-GCTCTAGAGAAGTCGTTGGGCGTGTGATGA-3= 5=-CCGCTCGAGTCAGAAATATTGAATGCTGACGC-3= 5=-GAAAACTCAGAGCTGAGACGGAAGT-3= 5=-CATGATACGGCTGTTGGTGGG-3= 5=-CATGGCTCCTATGTCATCACCG-3= 5=-AAGACCGCACGGTCCTCCTG-3= 5=-GAAAGGCACCGTCCAACTACTATCT-3= 5=-ACTGGGATTGCGAAAGCGTC-3= 5=-GCTTTGCCCGTCTCGCTTTC-3= 5=-CCGCCATTGTTCCATCCGTC-3= 5=-AAGATCATCCACAGTTGCGGCTAT-3= 5=-GTCGCTGCTCAGTCGGCTCC-3= 5=-AACCCCGACGCCTATGAAGAAT-3= 5=-GTTGAGGAGCTGCGGCAACTC-3= 5=-AGGTGGGCGAGTCGGTCAA-3= 5=-TGTCGGGCAGTCCGTAGGTC-3= 5=-GTCGCAGGCCAGGCAGTTGT-3= 5=-CGCCCGGTTCAGCCATTTC-3= 5=-TTCCCGGAACTGGTTGAGATACATG-3= 5=-CTGTTCGGCAATGCGGTCTTCT-3= 5=-TGGACGGCATGGAGCTGTTG-3= 5=-CGCTGTGAGAAGGGCTTGGT-3= 5=-GCAGGTCGGTTACGGCATCG-3= 5=-GCGTCTCGGTTATGTAGGCAATCA-3= 5=-CCGCTGCTTCGCTTGGTTC-3= 5=-GCGGGTCGATTCTTCGTTCATAT-3= 5=-ACCACGCCTGTGACGAAGAGC-3= 5=-CGTATCGAAGAGGACGGAGTTGC-3= 5=-CGGTTCTCCTTGATGCTGCTCTT-3= 5=-TCGGTCAGTTCGCATTCCCTATC-3= 5=-GCTCACCTGCGTCCTCCACAA-3= 5=-CGGTCTGATGCTCGCCCTGA-3= 5=-GATCGTCATGTAGCGCAGGA-3= 5=-CCTCGCTCGGCAGGACAT-3= 5=-CGTACATGACGATGCGGGATA⫺3= 5=-GTGCGTCGTGCAGTTCCAGTT-3= 5=-GGCTGATATTCTGGTTGTGGATGA-3= 5=-CAGGCCGTCGAGCTTGCTT-3= 5=-CCGAACGAGGTTGGGGACA-3= 5=-TGCGATGAAGGGCGGAGC-3= 5=-GCAGGTCGGTTACGGCATCG-3= 5=-CAGCGTCTCGGTTATGTAGGCAAT-3= 5=-CCGCATCACCATCTTCAATC-3= 5=-CGATGTTGCCGTGACCTG-3= 5=-GAGCGAGGAAGCGGAAGA-3= 5=-CGGAATTCCATATG CAGACCATCGCGCTTGTC-3= 5=-CGCTCTAGA CGCATTCGCCCGTTCCC-3= 5=-CGGAATTCCATATGTTGCCGAAGATTGCCGTAGTC-3= 5=-CGCTCTAGATGCACGACGAAAAATGGGGC-3= 5=-GCTCTAGAGCCCGAAAGACAGTACCCCTTCAT-3= 5=-GGGGTACCCCCTCAAGAACCGACAAACTGG-3= 5=-CGCCATATGACGGGAATTCGAGTAGCC-3= 5=-GGGGTACCCCGGAGACGGCTTCTGTCCTT-3=

Construction of ntrX deletion mutant Construction of ntrX deletion mutant Construction of ntrX deletion mutant Construction of ntrX deletion mutant Construction of ntrY deletion mutant Construction of ntrY deletion mutant Construction of ntrY deletion mutant Construction of ntrY deletion mutant Construction of pLL3 Construction of pLL4 Construction of pLL4 Construction of pLL6 Construction of pLL6 qRT-PCR of ntrX qRT-PCR of ntrX qRT-PCR of ntrY qRT-PCR of ntrY qRT-PCR of exoN qRT-PCR of exoN qRT-PCR of exoK qRT-PCR of exoK qRT-PCR of exoU qRT-PCR of exoU qRT-PCR of exoY qRT-PCR of exoY qRT-PCR of exoB qRT-PCR of exoB qRT-PCR of exoR qRT-PCR of exoR qRT-PCR of exoS qRT-PCR of exoS qRT-PCR of chvI qRT-PCR of chvI qRT-PCR of trkA qRT-PCR of trkA qRT-PCR of flaA qRT-PCR of flaA qRT-PCR of flaD qRT-PCR of flaD qRT-PCR of visN qRT-PCR of visN qRT-PCR of visR qRT-PCR of visR qRT-PCR of rpsF qRT-PCR of rpsF RT-PCR of ntrY RT-PCR of ntrY RT-PCR of ntrX RT-PCR of ntrX RT-PCR of ntrX RT-PCR of ntrX RT-PCR of trkA RT-PCR of trkA Identification of SmLL1 Identification of SmLL1 Identification of SmLL1 Construction of pLL7 Construction of pLL7 Construction of ppLL8 Construction of ppLL8 Construction of pLL9 Construction of pLL9 Construction of pLL10 Construction of pLL10

0.75 ml of isopropanol was used to precipitate the EPS. The pellet was washed once with 70% ethanol, air dried, redissolved in 0.25 ml of distilled water, and then used for total EPS measurement using the sulfateanthrone method (39).

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Motility test on soft agar plates and microscopy. Cell motility was examined as described by Yao et al. (18). Briefly, each Sinorhizobium inoculation from fresh LB/MC plates with appropriate antibiotics was dipped into LB/MC soft agar (0.25%) medium with a toothpick and in-



Primer


RESULTS

Construction of S. meliloti ntrX/Y mutants. On the S. meliloti genome, SMc01044 and SMc01045 (annotated as ntrY and ntrX) are homologous to A. caulinodans ntrY/ntrX (32). Analyzing the homology of NtrY and NtrX between S. meliloti and A. caulinodans using the BLAST protein program revealed that NtrX proteins share higher identity (67%) than NtrY proteins (48%), suggesting that the RR gene ntrX is more conserved than the HK gene ntrY in Rhizobium species. To determine the function of ntrX/ntrY genes in S. meliloti, we tried to construct the in-frame deletion mutants of both genes. However, the ntrX-deleted colonies could not be screened without the plasmid expressing the ntrX gene, while the ntrY deletion mutant (SmLL2) was obtained. These results suggest that deletion of the ntrX gene is lethal under the tested conditions. Therefore, single insertion mutants of ntrX (SmLL1) were collected instead for phenotype analysis. PCR and DNA sequencing indicated that the suicide plasmid was inserted into the fifth codon of ntrX in these mutants (Fig. 1A). To analyze the polar effect of the plasmid insertion in the ntrX mutant, transcription of the ntrY/ntrX genes together with trkA in S. meliloti 1021 was first assayed. RT-PCR showed that the transcripts containing both ntrY and ntrX, as well as ntrX and trkA, were detectable (Fig. 1B), suggesting that ntrY, ntrX, or trkA could be cotranscribed and share a single promoter. The transcript levels of ntrY, ntrX, and trkA were also assayed in the ntrX mutant by


FIG 1 Verification of the Sinorhizobium meliloti ntrX mutant. (A) Scheme of the ntrYX genomic organization from the wild-type and ntrX mutant (SmLL1) strains. The mutant was identified by PCR using one primer (k) specific to the suicide plasmid pK18mobsacB and another (i) identical to the ntrY gene; the specific PCR product was sequenced. (B) Transcripts of ntrX, ntrY, and trkA were detected by reverse transcription-PCR (RT-PCR). a to h, PCR primers P42 to P49; RNA and cDNA, templates for PCR. (C) Expression of ntrY, ntrX, and trkA in the ntrX mutant detected using qualitative RT-PCR. S. meliloti strains were grown to an OD600 of ⬇0.8. The experiment was repeated three times. SmLL1⫹ntrX, SmLL1 expressing the ntrX gene from the pSRK-Tc plasmid. Error bars stand indicate standard deviations (SD). P values were calculated using one-tailed t tests.*, P ⬍ 0.05; **, P ⬍ 0.001.

qRT-PCR, showing that ntrX and trkA transcription was differentially decreased (only 14% and 44% of the wild-type level, respectively), but there was a 4.4-fold increase for ntrY compared to that in Rm1021 (Fig. 1C). The expression level of ntrY, but not trkA, was partially restored after introduction of the plasmid carrying the expressible ntrX gene into the ntrX mutant (Fig. 1C). These data indicate that the ntrX gene is expressed at a low level in the plasmid insertion mutant. Moreover, the plasmid insertion possibly has a polar effect on trkA expression. S. meliloti NtrX negatively regulates succinoglycan production. The ntrX mutant formed mucous colonies on LB/MC or M9 agar plates, which prompted us to evaluate EPS production. The fluorescent stain calcofluor white M2R can specifically bind to the acidic EPS succinoglycan, meaning that it can be used for analysis of succinoglycan produced by S. meliloti (8). Calcofluor staining showed that the ntrX mutant colony produced stronger fluorescence than the wild type and the ntrY deletion mutant (Fig. 2A and 3B). Quantitative analysis of the fluorescence intensity revealed that 4-fold more succinoglycans were produced by the ntrX mu-

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cubated at 28°C for 3 days. Photographs were taken with the Coolpix 4500 digital camera (Nikon). Transmission electron microscopy (TEM) was used to observe S. meliloti flagella (40). In brief, Sinorhizobium strains were grown in soft LB/MC agar (0.25%) at 28°C for 3 days. The bacterial cells were adsorbed to freshly glow-discharged, carbon-coated grids (no. 300) and incubated in fresh 2% (wt/vol) uranyl acetate for 10 s. The excess solution was absorbed by a piece of filter paper, and the dried grids were then examined under TEM (JEM-2100; Jeol, Tokyo, Japan). Measurement of S. meliloti gene expression levels. Sinorhizobium cells (OD600 ⬇ 0.8) were collected from LB/MC subcultures. Total RNA was extracted using a TransZol kit (Transgen), treated with genomic DNA Eraser (TaKaRa) to remove any remaining genomic DNA, and then transcribed into cDNA using a PrimeScript RT reagent kit (Transgen). Quantitative real-time reverse transcription-PCR (qRT-PCR) was performed using a SYBR Premix Ex Taq II (Perfect Real Time) kit (TaKaRa) and the Bio-Rad CFX manager. The qPCR system (20 ␮l) was composed of the following components: SYBR green real-time PCR master mix, 10 ␮l; each primer, 0.5 ␮l; diluted cDNA sample, 1 ␮l (50 ng); and sterile water, 9 ␮l. All readings were performed in triplicate using a real-time PCR system (Bio-Rad, Hercules, CA, USA). The program consisted of a denaturing cycle at 95°C for 3 min, 40 cycles comprising 95°C for 10 s, 62°C for 30 s, and 72°C for 30 s, and a final step in which the temperature was elevated on a gradient from 65°C to 95°C to dissociate double-stranded DNA products. Primers used for the amplification of ntrX, ntrY, trkA, exoN, exoK, exoY, exoB, exoU, exoR, exoS, chvI, flaA, flaD, visN, visR, and rpsF fragments are listed in Table 2. Sequence analysis. The deduced protein sequences of NtrY/NtrX were downloaded from the Sinorhizobium meliloti genome website (http: //iant.toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi). Homologs of NtrY/NtrX were aligned and downloaded from the NCBI homepage using the BLAST with microbial genomes program (http://www.ncbi.nlm.nih .gov/sutils/genom_table.cgi). The putative promoter DNA sequences were downloaded from the S. meliloti genome server, and possible promoters were predicted by the Berkeley Drosophila Genome Project program (http://www.fruitfly.org/seq_tools/promoter.html).

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FIG 2 Succinoglycan is overproduced by the ntrX mutant. (A) Succinoglycan produced by Sinorhizobium meliloti strains. Sinorhizobium strains were streaked onto LB/MC agar plates containing 0.02% calcofluor white M2R; after 2 to 3 days of incubation at 28°C, they were observed under UV light. Fluorescence intensity was analyzed with the BandScan program. (B) Quantification of the total exopolysaccharide produced by S. meliloti strains in LB/MC broth. S. meliloti strains were grown to an OD600 of ⬇0.8. The sulfateanthrone method was used in the test. Each experiment was repeated three times. Error bars indicate SD. P values were calculated using one-tailed t tests. *, P ⬍ 0.05.

tant than by the wild-type strain (Fig. 2A). The plasmids carrying ntrX and ntrYX genes under the control of the lac promoter suppressed succinoglycan overproduction of the ntrX mutant, whereas the plasmid-borne ntrY or trkA did not (Fig. 2A). The empty vector did not affect succinoglycan production in either the ntrX mutant or the wild-type strain (Fig. 3). Total EPS quantification from the supernatant of LB/MC liquid cultures using the sulfate-anthrone method showed that the ntrX mutant produced 2-fold more EPS than the parent and complementation strains (Fig. 2B). These results suggest that S. meliloti NtrX represses succinoglycan production. To exclude the possibility that high-level ntrY expression leads to succinoglycan overproduction in the ntrX mutant (Fig. 1C), ntrY and ntrX were overexpressed individually in Rm1021. Both strains produced as much succinoglycan as the parent strain, Rm1021 (Fig. 3), suggesting that succinoglycan overproduction by the ntrX mutant results from low-level expression of ntrX. Therefore, the S. meliloti response regulator NtrX negatively regulates succinoglycan production, whereas the HK NtrY does not. NtrX is required for S. meliloti motility and flagellum production. S. meliloti ntrX mutant cells precipitated at the bottom of the tubes after the LB/MC cultures were allowed to settle for 30

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FIG 3 Phenotypes of Sinorhizobium meliloti strains with ntrY gene deletion or overexpression. (A) S. meliloti strains used in the test; (B) succinoglycan production on LB/MC calcofluor agar plates; (C) motility of S. meliloti on LB/MC soft agar plates. Vector, pSRK-Tc; Rm1021⫹ntrY, Rm1021 overexpressing the ntrY gene from the plasmid. The experiment was repeated three times.

min at room temperature, suggesting that the mutant was defective for flagellar motility. To test this theory, the motility of S. meliloti ntrX/ntrY mutants was analyzed on soft LB/MC agar plates. The ntrX mutant is less motile than the parent, complementation, and ntrY deletion strains (Fig. 3C and 4A). The motile phenotype of Rm1021 overexpressing ntrY or ntrX was also checked but was not found to differ from that of the parent strain (Fig. 3C). These results indicate that NtrX (not NtrY) is required for S. meliloti motility.



FIG 4 Defective motility and flagellum formation of the ntrX mutant. (A) Swimming of Sinorhizobium meliloti strains on LB/MC soft agar (0.25%) plates. (B to D) Flagella of the wild type (B) and the ntrX mutant (C and D) under transmission electron microscopy. Arrows, flagella; bars, 1 ␮m.

dance of exoB, exoY, exoK, exoN, and exoU was increased to various degrees in the ntrX mutant compared with the parent and complementation strains (Fig. 5A). Therefore, S. meliloti NtrX represses succinoglycan biosynthesis by negatively regulating exo gene expression. NtrX positively regulates the expression of flagellin and regulatory genes. To analyze why the ntrX mutant assembles fewer flagella, the expression of the flagellum biosynthetic genes was assayed using qRT-PCR. Our findings indicated that the expression of flagellin genes flaA and flaD and the regulatory genes visN and visR were remarkably decreased (Fig. 5B), suggesting that S.

FIG 5 Expression of succinoglycan and flagellum biosynthetic genes in the ntrX mutant. (A) The expression of succinoglycan biosynthetic genes was upregulated in the ntrX mutant. (B) Downregulation of the flagella biosynthetic genes was found in the ntrX mutant. S. meliloti strains were grown to an OD600 of ⬇0.8. Total RNA was extracted, transcribed, and subjected to qualitative reverse transcription-PCR with the internal standard of rpsF. All data were from one representative experiment that was repeated three times. Error bars indicate SD. P values were calculated using one-tailed t tests. *, P ⬍ 0.05; **, P ⬍ 0.001.


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The motility defect of the ntrX mutant prompted us to examine flagella of S. meliloti cells using TEM. Fewer or no flagella were visible on the ntrX mutant cell (Fig. 4C and D), whereas the wild-type cell carried several flagella (Fig. 4B). These data reveal that S. meliloti NtrX is required for efficient flagellum formation. NtrX negatively regulates succinoglycan biosynthetic gene transcription. To determine whether excess succinoglycan produced by the ntrX mutant results from the elevated exo gene expression, the transcript levels of succinoglycan biosynthetic genes were examined. The qRT-PCR data showed that the mRNA abun-

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meliloti NtrX positively regulates the expression of flagellin and regulatory genes to control flagellum formation. S. meliloti NtrX is not associated with the ExoR/ExoS/ChvI regulatory system. It is well known that the ExoR/ExoS/ChvI system regulates succinoglycan biosynthesis and flagellum formation (18, 23, 24). To analyze whether succinoglycan overproduction and the motility defect of the ntrX mutant is associated with this regulatory system, we tried to construct the chvIK214T and ntrX:: pK18mobsacB double mutant. However, no resulting colonies were isolated without the plasmid expressing an ntrX gene, suggesting that the double mutation of ntrX and chvI was lethal under the tested conditions. Subsequently, the transcription of exoR, exoS, and chvI was analyzed by qRT-PCR, which showed that the transcript level of exoR did not differ between the ntrX mutant and the wild-type or complementation strain (Fig. 5A). We noticed that transcription of exoS was increased and that of chvI was decreased in the ntrX mutant compared with the wild-type strain (Fig. 5A). To exclude that NtrY, the level of which was high in the ntrX mutant, interacts with ChvI and thereby deregulates the synthesis of succinoglycan and flagella, the plasmid (pLL7) expressing chvI under the control of the lac promoter was introduced into the mutant. Phenotype analysis showed that chvI overexpression did not rescue the defects of the ntrX mutant (Fig. 6), suggesting that ChvI is not acting on succinoglycan production and motility instead of NtrX. To determine whether ExoR, the repressor of ExoS/ChvI, interacts with the TCS of NtrY/NtrX, the plasmid (pHC510) expressing the exoR gene was introduced into the ntrX mutant and also into the two exoR null mutants (Rm7095 and RmHC12) that were also deregulated for succinoglycan production and flagellum formation. Although pHC510 suppressed succinoglycan overproduction and the motility defects of exoR null mutants, it did not rescue the defects of the ntrX mutant (Fig. 3), implying that ExoR could not be coupled with the TCS of NtrY/NtrX. Taken together, these data reveal that S. meliloti NtrX is independent of the ExoR/ExoS/ChvI regulatory system. S. meliloti NtrX is linked with neither the EmmABC nor the ExpR regulatory system. S. meliloti emmABC genes are associated with EPS and motility, and emmBC encode an HK and a RR of a putative TCS (22). To evaluate the association between NtrX and EmmB/EmmC, the emmC gene was overexpressed in the ntrX mutant. The resulting strain showed succinoglycan production and motility similar to those of the ntrX mutant (Fig. 6), suggesting that NtrX is not associated with EmmABC. The ExpR/Sin system is also involved in low-molecular-weight succinoglycan production and motility (41–44). The expR plus strain (Rm8530) produces more EPSII and is less motile than Rm1021 (expR minus). The ntrX mutant in an expR plus background produced more succinoglycan (stronger florescence) and was less motile than Rm8530, which was similar to the ntrX mutant (SmLL1) (Fig. 6). These data reveal that NtrX is not dependent on the ExpR/Sin system. The ntrX mutant establishes defective symbiosis on host alfalfa. Since A. caulinodans ntrX mutants display symbiotic defects (32), we performed nodulation tests of the S. meliloti ntrX mutant on alfalfa. Four weeks after inoculation, the alfalfa plants inoculated with the ntrX mutant were smaller and had more yellow leaves than those plants inoculated with Rm1021. Moreover, the biomass of the alfalfa shoots and the nitrogenase activity of the

FIG 6 Phenotypes of the ntrX mutant overexpressing regulator genes or in the expR plus background. (A) Sinorhizobium meliloti strains used in the test; (B) succinoglycan production on LB/MC calcofluor agar plates; (C) motility of S. meliloti strains on LB/MC soft agar plates. SmLL3, the ntrX mutant in the Rm8530 (expR plus) background. The experiment was repeated three times.

root nodules induced by the mutant were significantly decreased, whereas the number of nodules per plant was apparently elevated compared with that in plants inoculated with the wild-type strain (Fig. 7A to C). Introduction of the plasmid-borne ntrX rescued the symbiotic defects of the ntrX mutant (Fig. 7). These data prove that NtrX is required for different rhizobia to symbiotically interact with their host plants.



alfalfa, as measured by biomass (fresh weight) of the alfalfa shoots (A), the average number of alfalfa nodules after 3 weeks inoculation (B), and the nitrogenase activity of alfalfa nodules per plant as detected by C2H2 reduction (C). The figure shows results from one representative experiment. A total of 21 to 25 alfalfa seedlings were inoculated with each Sinorhizobium strain. Three independent experiments were performed, and P values were calculated using one-tailed t tests. *, P ⬍ 0.05.

DISCUSSION

The TCS of NtrY/NtrX in A. caulinodans, A. brasilense, and Brucella species has been reported to be involved in nitrogen metabolism, symbiotic nodulation, low-oxygen response, and persistent survival in host cells (32, 33, 45). In the present work, we found that the S. meliloti response regulator NtrX regulates succinoglycan production and flagellum formation in a manner independent of the HK NtrY. Our findings not only demonstrate that NtrX is a new player in the regulation of EPS production and motility but also prove that the ntrX gene is pleiotropic in rhizobia. The organization of ntrY, ntrX, and trkA is conserved on the


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FIG 7 The Sinorhizobium meliloti ntrX mutant is defective for symbiosis with

chromosomes of alphaproteobacteria as shown by genomic DNA sequence analyses. Based on the data from Brucella species (45) and the analysis of their transcripts in S. meliloti (Fig. 1B), these genes could be located within a single operon. According to the BDPG program, the transcriptional start site of ntrY could be located at nucleotide 1570912 on the S. meliloti chromosome, similar to that in Brucella species (45). NtrX may be required for the duplication or proliferation of alphaproteobacteria. First, the S. meliloti ntrX deletion mutant as well as mutants with point mutations on the phosphorylation or dimerization sites of NtrX (D. Wang, D. Lu, and L. Luo, unpublished data) could not be obtained under our test conditions. Second, the NtrY/NtrX homolog has been suggested to be linked with the cell cycle in Caulobacter crescentus (46). Third, no ntrX null mutants have been reported from Brucella species until now. It is possible that the Tn5 insertion mutants from A. caulinodans could be mutants with either partial loss of function of the ntrX gene or low-level expression of the genes, like our S. meliloti ntrX mutant (SmLL1) (32). The free-living and symbiotic defects of the S. meliloti ntrX mutant (SmLL1) result mainly from low-level expression of ntrX. First, the expression of ntrX from a plasmid was able to rescue almost all of the defects of the mutant (Fig. 2, 4A, 6, and 7). Second, the low-level expression of trkA is not responsible for the defects of the ntrX mutant (Fig. 2A). The trkA gene encodes a putative low-affinity potassium ion channel. We have not obtained its null mutant using the suicide plasmid insertion, implying that it is required for survival of S. meliloti. Expression of trkA could be controlled by two different promoters (PntrY and PtrkA) in the wild-type strain, whereas it could be affected by the plasmid insertion and its own promoter, PtrkA, in the ntrX mutant (Fig. 1B and C). Third, elevated ntrY expression is probably not responsible for the defects of the ntrX mutant, as overexpression of the ntrY gene in the wild-type strain (Rm1021) did not affect succinoglycan production or motility (Fig. 3). In contrast to the case for A. caulinodans and Brucella species, S. meliloti ntrY could not be genetically coupled with ntrX. The null mutants of the A. caulinodans ntrY homolog induced only ineffective nodules on S. rostrata and were not able to efficiently utilize nitrate as a nitrogen source (32). Brucella NtrY is a sensor of cellular redox status and is associated with persistent survival in host cells (45). It is surprising that the deletion mutant (including other two plasmid insertion mutants [H. Xue, Y. Wang, and L. Luo, unpublished data]) of the S. meliloti ntrY gene showed the same phenotypes as the parent strain, Rm1021 (Fig. 3) (Wang et al., unpublished data on symbiotic nodulation). Therefore, we propose that an unknown HK could interact with NtrX in S. meliloti. It is well known that succinoglycan production and flagellum formation are jointly regulated by the regulatory systems of ExoR/ ExoS/ChvI, EmmA/EmmB/EmmC, ExpR/Sin, or CbrA (18, 19, 22, 24, 40, 41, 42, 46, 47, 48). A question was raised regarding whether NtrX is dependent on these systems. First, we found that NtrX is not associated with the ExoR/ExoS/ChvI pathway, because overexpression of exoR or chvI did not affect succinoglycan production and motility of the ntrX mutant (Fig. 6). Similarly, NtrX could not be linked with the S. meliloti quorum-sensing system, for the ntrX mutant overproduced succinoglycan and was less motile in both the plus and minus expR backgrounds (Fig. 6). Third, the RR EmmC from the EmmABC regulatory pathway was not regulating succinoglycan production and flagellum formation

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ACKNOWLEDGMENTS We are grateful to Esther J Chen for the gift of CE69. This work was supported by grants from the National Key Program for Basic Research (2010CB126501 and 2011CB100702) and the Natural Science Foundation of China (31070218 and 31370277) to L.L.

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instead of NtrX (Fig. 6). Fourth, S. meliloti cbrA encodes a hybrid HK and has pleiotropic effects on motility, succinoglycan production, stress adaptation, and symbiosis with alfalfa (47). The cbrA Tn5 insertion mutant produced more succinoglycan and exhibited less motility, and the exo and fla genes were differentially expressed (48). Therefore, S. meliloti CbrA could be genetically coupled with NtrX. We tried to construct the cbrA and ntrX double mutant, but no mutants were obtained under our tested conditions. We overexpressed the cbrA gene in the ntrX mutant; however, it did not suppress the defects of the mutant (Fig. 6). Thus, whether CbrA directly interacts with NtrX will be further studied in future experiments. S. meliloti NtrX could indirectly regulate flagellin gene expression. The expression of flagellin genes in S. meliloti is regulated by the VisN/VisR pathway (41). From our data, NtrX positively regulated transcription of visN/visR together with flaA and flaD (Fig. 5B). Therefore, NtrX could positively regulate the expression of flagellin genes through the VisN/VisR pathway. We noticed that the complementation strain did not completely restore the transcript levels of the vis and fla genes (Fig. 5B). A possible explanation for this is that the expression of these genes could be NtrX dosage dependent and the amount of IPTG used for inducing expression of the ntrX gene from the plasmid may not be optimal in this study. NtrX could directly regulate the transcription of the exo genes. The expression of genes encoding known regulators (including some transcriptional factors) of EPS biosynthesis was not correspondingly changed in the ntrX mutant (Fig. 5) (Wang et al., unpublished). Moreover, some key regulator genes (such as chvI and emmC) were not acting on succinoglycan production and flagellum formation instead of ntrX (Fig. 6). These inferences will be further verified using chromatin immunoprecipitation and DNA sequencing. NtrX homologs regulate the expression of nifA and nifH, which are required for nitrogenase biosynthesis in free-living A. caulinodans and A. brasilense (32, 33). The ntrX mutants from A. caulinodans induced ineffective nodules with low nitrogenase activity, similarly to the S. meliloti ntrX mutant (Fig. 7). However, whether this resulted from the downregulation of nif genes is not clear. In this work, we introduced nifA and nifH promoter-GUS fusions into S. meliloti strains. Surprisingly, the activity of each promoter in alfalfa nodules did not differ between the ntrX mutant and the wild-type strain (Xue et al., unpublished data). These data suggest that the expression of nif genes was not regulated by NtrX in S. meliloti bacteroids. Therefore, the ineffective alfalfa nodules induced by the S. meliloti ntrX mutant could have resulted from other factors.


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