Transcriptome Response to Heavy Metals in Sinorhizobium meliloti CCNWSX0020 Reveals New Metal Resistance Determinants That Also Promote Bioremediation by Medicago lupulina in Metal-Contaminated Soil

ABSTRACT

The symbiosis of the highly metal-resistant Sinorhizobium meliloti CCNWSX0020 and Medicago lupulina has been considered an efficient tool for bioremediation of heavy metal-polluted soils. However, the metal resistance mechanisms of S. meliloti CCNWSX00200 have not been elucidated in detail. Here we employed a comparative transcriptome approach to analyze the defense mechanisms of S. meliloti CCNWSX00200 against Cu or Zn exposure. Six highly upregulated transcripts involved in Cu and Zn resistance were identified through deletion mutagenesis, including genes encoding a multicopper oxidase (CueO), an outer membrane protein (Omp), sulfite oxidoreductases (YedYZ), and three hypothetical proteins (a CusA-like protein, a FixH-like protein, and an unknown protein), and the corresponding mutant strains showed various degrees of sensitivity to multiple metals. The Cu-sensitive mutant (ΔcueO) and three mutants that were both Cu and Zn sensitive (ΔyedYZ, ΔcusA-like, and ΔfixH-like) were selected for further study of the effects of these metal resistance determinants on bioremediation. The results showed that inoculation with the ΔcueO mutant severely inhibited infection establishment and nodulation of M. lupulina under Cu stress, while inoculation with the ΔyedYZ and ΔfixH-like mutants decreased just the early infection frequency and nodulation under Cu and Zn stresses. In contrast, inoculation with the ΔcusA-like mutant almost led to loss of the symbiotic capacity of M. lupulina to even grow in uncontaminated soil. Moreover, the antioxidant enzyme activity and metal accumulation in roots of M. lupulina inoculated with all mutants were lower than those with the wild-type strain. These results suggest that heavy metal resistance determinants may promote bioremediation by directly or indirectly influencing formation of the rhizobium-legume symbiosis.

IMPORTANCE Rhizobium-legume symbiosis has been promoted as an appropriate tool for bioremediation of heavy metal-contaminated soils. Considering the plant-growth-promoting traits and survival advantage of metal-resistant rhizobia in contaminated environments, more heavy metal-resistant rhizobia and genetically manipulated strains were investigated. In view of the genetic diversity of metal resistance determinants in rhizobia, their effects on phytoremediation by the rhizobium-legume symbiosis must be different and depend on their specific assigned functions. Our work provides a better understanding of the mechanism of heavy metal resistance determinants involved in the rhizobium-legume symbiosis, and in further studies, genetically modified rhizobia harboring effective heavy metal resistance determinants may be engineered for the practical application of rhizobium-legume symbiosis for bioremediation in metal-contaminated soils.

INTRODUCTION

Copper (Cu) and zinc (Zn) are transition metals and essential micronutrients required as enzymatic cofactors for many biological processes in most living organisms (1). However, Cu and Zn in excess are usually highly cytotoxic to cells (2). Accordingly, even relatively simple bacteria have evolved different strategies for Cu and Zn homeostasis in terms of uptake, intracellular distribution, and efflux (3, 4). For instance, in the model organism Escherichia coli, the Cu(I) sensor CueR is required for Cu(I) detection in the cytoplasm and induces the transcription of genes encoding Cu resistance proteins (5), including the Cu(I)-exporting P_1B-type ATPase CopA, which exports excessive Cu(I) from the cytoplasm to the periplasm (6, 7), and the multicopper oxidase CueO, which oxidizes Cu(I) to less toxic Cu(II) in the periplasm (8). Likewise, the two-component regulatory system CusRS, located in the cytoplasmic membrane but with a sensory domain extending into the periplasm, regulates the multicomponent Cu(I) efflux system CusCFBA, which exports excessive Cu(I) from the periplasm to the extracellular space (9). Zn homeostasis differs markedly from Cu resistance, so E. coli has evolved both import and export systems to strictly maintain intracellular Zn(II) levels. Zn import is mediated by the low-affinity ZupT transporter, a member of the ZIP (ZRT/IRT-like protein) transporter family (10), and the high-affinity ZnuABC transport system, an ABC transporter that is regulated by the zinc-responsive regulator Zur (11). Zn is taken up by ZupT under conditions of moderate Zn availability, whereas it is taken up by ZnuACB in zinc-deficient environments (12). Zn efflux is catalyzed mainly by the cation diffusion facilitator (CDF) ZitB, which is constitutively expressed to act as a first line of defense against Zn influx (13), and the P_1B-type ATPase ZntA, which is upregulated by the Zn(II) sensor ZntR to combat high extracellular Zn concentrations (14, 15). In addition, the RND-type efflux pump MdtABC and the major facilitator superfamily (MFS) transporter MdtD, as well as the periplasmic protein Spy, all controlled by the BaeSR two-component system, are also involved in Zn detoxification (16). So far, varied mechanisms of Cu and Zn resistance have been found in the microbial world and vary significantly between species (3, 17).

Soil contamination by heavy metals has become a serious worldwide environmental problem. As a cost-effective and environmentally friendly alternative to physical and chemical methods, the use of phytoremediation has been shown to be a promising approach to clean up contaminated soils (18). Furthermore, nitrogen-fixing rhizobia with high intrinsic metal resistance have been investigated widely for their potential to improve plant growth, reduce metal toxicity, and change metal availability in soil, which may help in the development of microbe-assisted phytoremediation (19–22). In recent years, people have paid more attention to the potential use of legume-rhizobium symbiosis for bioremediation of contaminated soil and to the responsible biochemical and molecular mechanisms, thereby promoting further development of this powerful bioremediation approach into a widely accepted technique (23–25).

Sinorhizobium meliloti CCNWSX0020 was isolated from root nodules of Medicago lupulina plants growing on mine tailings in northwestern China (26). It displayed tolerance to high levels of multiple metals, such as Cu, Zn, Cd, and Pb. Moreover, it could promote the growth, metal uptake, and antioxidant responses of M. lupulina in copper-contaminated soil (21, 22, 26). In our previous work, a number of Cu and Zn tolerance mechanisms of S. meliloti CCNWSX0020 were investigated. (i) Extracellular polymeric substances were found to immobilize Cu²⁺ and were predicted to play a role as a first protective barrier to prevent Cu²⁺ from reaching the cytoplasm (27). (ii) Several genes conferring copper resistance were identified, including merR, encoding a MerR family transcriptional regulator resembling CueR; lpxXL, encoding an LpxXL C₂₈ acyltransferase; and omp, encoding an outer membrane protein (21). (iii) A putative Cu⁺-transporting P_1B-type ATPase (CopA1b) and a Zn²⁺-transporting P_1B-type ATPase (ZntA) were identified and were shown to be involved in Zn, Cd, and Pb resistance (28). Additionally, the genome sequence of S. meliloti CCNWSX0020 revealed several putative molecular chaperones, metal binding proteins, and unspecific divalent cation transporters predicted to have a role in Cu and Zn resistance (29). However, the molecular mechanisms and regulatory networks responsible for Cu and Zn resistance in S. meliloti CCNWSX0020 have not been elucidated completely and experimentally verified so far. In this study, the transcriptome profiles of the S. meliloti CCNWSX0020 responses to Cu and Zn stresses were analyzed using transcriptome sequencing (RNA-Seq) to investigate S. meliloti CCNWSX0020 Cu and Zn resistance mechanisms. Moreover, Cu- and Zn-sensitive mutants were used to further investigate the positive effects of S. meliloti CCNWSX0020 Cu and Zn resistance determinants on growth and thereby the bioremediation potential of legume-rhizobium symbiosis in metal-contaminated soils.

RESULTS

Experimental design and overview of the RNA-Seq data.

To study the Cu and Zn resistance mechanisms of S. meliloti CCNWSX0020, we carried out a preliminary experiment to find a sublethal dose that did not cause mortality due to metal exposure by measuring the growth curves of S. meliloti CCNWSX0020 in the presence of different CuSO₄ and ZnSO₄ concentrations (for CuSO₄, 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM; and for ZnSO₄, 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mM) (see Fig. S1 in the supplemental material). Thereafter, S. meliloti CCNWSX0020 strains were exposed to 0.6 mM CuSO₄ or 0.2 mM ZnSO₄ and then analyzed in comparison with untreated (control) strains by next-generation sequencing using an Illumina HiSeq 4000 system. Moreover, to investigate the different responses of S. meliloti CCNWSX0020 to short-term and long-term metal stresses, we set two metal treatment time points. One condition of interest was long-term exposure, where the cells were incubated for 24 h, to mid-exponential phase, in the presence of 0.6 mmol/liter CuSO₄ or 0.2 mmol/liter ZnSO₄. In contrast, the other condition of interest was short-term exposure, where preincubated cells from mid-exponential phase were exposed to 0.6 mmol/liter CuSO₄ or 0.2 mmol/liter ZnSO₄ for 30 min. Total RNAs were extracted from three independent biological replicates and then prepared for RNA sequencing. On average, 10 million clean reads were generated for each sample, and in each sample, >98% of all reads were mapped to the reference genome. The genes whose expression levels were significantly (adjusted P < 0.05) changed were identified as differentially expressed genes (DEGs) responsive to heavy metal stress. DEGs with log₂(fold change) values of >1 were considered upregulated genes in response to Cu or Zn, while genes with log₂(fold change) values of <1 were considered downregulated genes in response to Cu or Zn, and we further analyzed these differentially expressed genes.

To validate the results obtained by RNA-Seq, the expression levels of 10 selected genes with different expression patterns were measured using quantitative real-time PCR (qPCR). The log₂-transformed mean value for each gene for three biological replicates was in good agreement with the log₂-transformed fold change from RNA-Seq (Fig. 1), suggesting that reliable expression results were generated by RNA sequencing.

FIG 1.

Validation of RNA-Seq data using qPCR. Ten genes commonly upregulated under Cu and Zn exposures for 30 min were chosen to validate the RNA-Seq data by qPCR. The light gray and medium gray bars represent mean log₂-transformed fold changes obtained for three biological qPCR replicates, with error bars showing standard deviations. Dark gray and black bars represent RNA-Seq data.

Gene expression patterns differ for short-term and long-term metal exposures.

The gene expression patterns of S. meliloti CCNWSX0020 under four conditions (0.6 mM CuSO₄ exposure for 30 min, 0.6 mM CuSO₄ exposure for 24 h, 0.2 mM ZnSO₄ exposure for 30 min, and 0.2 mM ZnSO₄ exposure for 24 h) are illustrated in Fig. S2. The complete list of DEGs and their expression patterns under these four conditions are shown in Table S3. Irrespective of the differences in transcript expression between Cu-exposed and Zn-exposed samples, there were more transcripts induced by short-term metal exposure than by long-term metal exposure, suggesting that immediate early response genes play a role in the first line of protection against heavy metal stress, while continuous expression of genes gives the ability to cope with continuous exposure to excessive amounts of heavy metals. Significantly upregulated genes induced by Cu for 24 h included two operons. One we labeled the multicopper oxidase operon (MCO), and it encodes the outer membrane protein Omp (SM0020_RS14550), the multicopper oxidase CueO (SM0020_RS14545), a blue copper azurin-like protein (SM0020_RS14540), and a copper chaperone (SM0020_RS14535) that was previously reported to be involved in Cu homeostasis (21, 30). The other operon was labeled the CopG operon, encoding a CopG family protein and three hypothetical proteins (HPs) that will be discussed in more detail later. Moreover, one gene (SM0020_RS0132600), encoding an adenylate cyclase (AC) that can synthesize cyclic AMP (cAMP) as a central second messenger, was also significantly upregulated by 24 h of Cu exposure. Previous studies reported that AC plays a regulatory role in multiple stress responses of fungi (31), and the YHS domain of AC from Mycobacterium phlei appears to be a copper sensor module (32). In contrast, the significantly upregulated gene induced by 24 h of Zn exposure was a gene (SM0020_RS10845) encoding the P_1B-type ATPase ZntA, previously shown to be required for Zn(II), Cd(II), and Pb(II) efflux (28). These genes showed long-term elevated expression under Cu/Zn stress and might play vital roles in Cu/Zn resistance and adaptation mechanisms in S. meliloti CCNWSX0020.

Consortium of genes responsible for global and specific responses to short-term Cu and Zn exposures.

More genes were differentially expressed in response to short-term metal treatment, indicating that most short-term Cu- and Zn-responsive genes were transiently expressed for coping with stressful environmental conditions. We next determined whether these genes were induced specifically by Cu or Zn or by a more global, unspecific response. Venn diagrams showed that 54.8% of the upregulated genes of the 30-min Zn-treated strain were also upregulated in the 30 min Cu-treated strain, while there were no downregulated genes for both the 30 min Cu-treated strain and the 30 min Zn-treated strain (Fig. 2). Although 34 genes were upregulated by both short-term Cu and Zn exposures, their expression levels were a little different (Table 1). For instance, the MCO operon and the gene encoding AC showed higher expression levels under Cu-exposed conditions than under Zn-exposed conditions, while zntA was significantly more highly expressed in response to Zn exposure than in response to Cu exposure. These common DEGs upregulated by Cu and Zn could be grouped by functional category. The first group included gene products related to ion transport, including ZntA, the outer membrane protein, and a nickel/cobalt transporter regulator (SM0020_RS01870), revealing that S. meliloti CCNWSX00200 may employ diverse transport systems to avoid excessive metal ion toxicity. The second group encompassed genes encoding gene products involved in the oxidative stress response, including the sulfite oxidoreductase YedYZ (SM0020_RS02490/SM0020_RS02495), which is known to reduce free methionine sulfoxide in vitro (33), and methionine sulfoxide reductase B (SM0020_RS12930), which has been reported to protect cells against oxidative damage (34). The last group contained genes encoding proteins involved in carbohydrate and amino acid metabolism, including serine peptidase (SM0020_RS24280), polyketide synthase (SM0020_RS28565), gluconate 2-dehydrogenase (SM0020_RS01225), diacylglycerol kinase (SM0020_RS00560), carboxyl-terminal processing protease (SM0020_RS20795), and others. The products of these commonly upregulated genes were probably required for the defense against nonspecific metal injury or might play a central role in the global stress response.

FIG 2.

Venn diagrams displaying differentially expressed genes up- or downregulated after exposure of S. meliloti CCNWSX0020 to CuSO₄ or ZnSO₄ for 30 min and 24 h.

TABLE 1.

Commonly upregulated genes in both Cu- and Zn-treated S. meliloti strains

Gene ID	Gene product	Log2(fold change)
		Cu	Zn
SM0020_RS14550	Outer membrane protein	9.048	4.3547
SM0020_RS14545	Multicopper oxidase	9.045	4.707
SM0020_RS14540	Blue copper azurin-like protein	8.9623	4.7258
SM0020_RS14535	Copper chaperone	8.2981	4.077
SM0020_RS0132600	Adenylate cyclase	7.0539	3.6661
SM0020_RS24280	Serine peptidase	4.7365	2.7584
SM0020_RS03630	Hypothetical protein	4.6972	2.5258
SM0020_RS02495	Sulfite oxidase YedY	4.5899	4.3153
SM0020_RS02490	Sulfite reductase subunit YedZ	4.5086	3.9248
SM0020_RS28565	Polyketide synthase	4.2965	4.2513
SM0020_RS09985	Glucose sorbosone dehydrogenase	4.1793	4.0022
SM0020_RS27630	Transpeptidase	4.0795	3.9581
SM0020_RS01870	Periplasmic regulator RcnB of Ni and Co efflux	3.8281	2.6931
SM0020_RS01220	Cytochrome C556	3.7605	4.4727
SM0020_RS17320	Hypothetical protein	3.7505	3.2336
SM0020_RS12930	Methionine sulfoxide reductase B	3.6927	3.5204
SM0020_RS19575	AraC family transcriptional regulator	3.5494	3.1952
SM0020_RS01225	Gluconate 2-dehydrogenase	3.4479	3.8922
SM0020_RS17820	Hypothetical protein	3.4355	3.3273
SM0020_RS00560	Diacylglycerol kinase	3.11	3.5504
SM0020_RS10845	P1B-type ATPase ZntA	2.9793	7.7
SM0020_RS21600	Carboxyl-terminal processing protease	2.96	2.7324
SM0020_RS09340	Cytochrome c biogenesis protein	2.7267	2.5949
SM0020_RS26615	Hypothetical protein	2.4944	3.5307
SM0020_RS06440	Hypothetical protein	2.2822	2.198
SM0020_RS06445	RNA-binding protein	2.2498	2.2537
SM0020_RS30430	ABC transporter permease	2.235	2.4258
SM0020_RS30435	GCN5 family acetyltransferase	2.2026	2.3376
SM0020_RS11190	Hypothetical protein	2.1998	2.1715
SM0020_RS20795	Carbon monoxide dehydrogenase	2.1546	2.2666
SM0020_RS06450	Membrane protein	2.1114	2.1734
SM0020_RS12060	ABC transporter substrate-binding protein	1.9899	1.9832
SM0020_RS30425	ABC transporter permease	1.9798	1.9649
SM0020_RS17855	Glycosyltransferase family 1 protein	1.6898	2.0224

Nevertheless, except for the common DEGs, metal-specific stress responses were also induced. For example, several genes, encoding the CopG metal binding protein (SM0020_RS03645), a CsoR-like transcriptional regulator (SM0020_RS15490) known to respond to Cu(I) (35), and the multidrug efflux RND transporter AcrB (SM0020_RS05505), were specifically upregulated, while genes encoding cytochrome O ubiquinol oxidase (SM0020_RS27740) and cytochrome D ubiquinol oxidase (SM0020_RS14675) were specifically downregulated, following Cu exposure. In contrast, the gene encoding the transcriptional regulator RcnB (SM0020_RS01870), which might control downstream genes encoding a zinc ABC transporter, a putative MFS-type efflux transporter (SM0020_RS18595), the divalent ion tolerance protein CutA (SM0020_RS07340) and four GroEL (SM0020_RS00235 and SM0020_RS21385) and GroES (SM0020_RS00240 and SM0020_RS21390) molecular chaperones, was specifically upregulated, while genes encoding iron transporters (SM0020_RS02755 and SM0020_RS02760) were specifically downregulated, following Zn exposure (Table S3). The Cu and Zn network conferring resistance to S. meliloti CCNWSX0020 did show that the Cu and Zn homeostatic systems are multilayered and intricate as analyzed by transcriptome profiling, and their specific resistance mechanisms were also distinguishable from each other.

Phenotypic characterization of heavy metal resistance gene deletion mutants.

Eight genes (SM0020_RS14545, SM0020_RS14550, SM0020_RS03630, SM0020_RS03635, SM0020_RS03640, SM0020_RS03645, SM0020_RS02490, and SM0020_RS02495) from three operons highly induced by Cu or Zn stress were deleted for functional identification. Figure 3 shows a physical map of these genes and their expression levels under Cu and Zn exposures as determined by qPCR. SM0020_RS14545, encoding a multicopper oxidase (CueO), and SM0020_RS14550, encoding an outer membrane protein (Omp), are part of the MCO operon, and their deletion mutants were designated the ΔcueO and Δomp mutants, respectively. The other four, contiguous genes (SM0020_RS03630, SM0020_RS03635, SM0020_RS03640, and SM0020_RS03645), encoding four hypothetical proteins, were induced as one transcript by Cu or Zn, since a cotranscription test showed that these four genes formed a transcriptional unit and were therefore presumed to form an operon (Fig. S3). Based on sequence homology, the SM0020_RS03640-encoded protein was 97% identical to the Cu(I)/Ag(I) efflux system membrane protein CusA from S. meliloti RU11/001, while the SM0020_RS03635-encoded hypothetical protein showed 60% similarity to the FixH family protein in Rhizobium etli bv. mimosae strain Mim1, SM0020_RS03630 encoded an unknown protein, and SM0020_RS03645 encoded a CopG metal binding protein. These four deletion mutants were designated the ΔcusA-like, ΔfixH-like, ΔHP, and ΔcopG mutants, respectively. SM0020_RS02490 encoded a sulfite reductase heme-binding subunit (YedZ) and SM0020_RS02495 a sulfite oxidase (YedY) that together formed a sulfite oxidoreductase, and therefore they were deleted together and the corresponding mutant designated the ΔyedYZ mutant.

FIG 3.

Three operons were all involved in Cu and Zn resistance in S. meliloti CCNWSX0020. (a) Physical map of the YedYZ operon, the CopG operon, and the MCO operon. (b) Transcriptional responses of the YedYZ operon (left), the CopG operon (middle), and the MCO operon (right) after CuSO₄ or ZnSO₄ exposure for 30 min or 24 h. qPCR was used to determine the transcription levels of the three operons. Error bars represent standard deviations for three repeats.

To further explore the roles of the mutated genes, the maximum tolerable concentrations (MTCs) of various metal ions [AgNO₃, CuSO₄, ZnSO₄, CdSO₄, Pb(NO₃)₂, NiSO₄, and CoCl₂] in the S. meliloti CCNWSX0020 wild-type strain and seven mutants (Δomp, ΔcueO, ΔcopG, ΔcusA-like, ΔfixH-like, ΔHP, and ΔyedYZ) were determined on solid tryptone-yeast extract (TY) medium. For the S. meliloti CCNWSX0020 wild-type strain, the MTCs of the various tested metals were 1.6, 1.2, 0.26, and 3.4 mM for CuSO₄, ZnSO₄, CdSO₄, and Pb(NO₃)₂, respectively (Fig. 4). The various mutant strains displayed intriguing responses. The ΔcueO mutant showed sensitivity only to Cu, with an MTC much lower (1.2 mM) than that for the wild-type strain (1.6 mM). In contrast, the Δomp, ΔcusA-like, ΔfixH-like, ΔHP, and ΔyedYZ mutants exhibited various degrees of decreased tolerance toward Cu, Zn, Cd, and Pb in comparison to that of the wild-type strain (Fig. 4). In addition, no difference in tolerance to Cu, Zn, Cd, and Pb was observed in comparing the ΔcopG mutant and the wild-type strain, and the AgNO₃, CoCl₂, and NiSO₄ tolerances of the seven mutants did not show any remarkable difference compared to those of the wild-type strain (data not shown). These results suggest that CueO plays a specific role in Cu detoxification, while the Omp, YedYZ, CusA-like, FixH-like, and hypothetical proteins are all important for tolerance to Zn, Pb, Cd, and Cu, in S. meliloti CCNWSX0020.

FIG 4.

Influence of deletions in selected genes on metal tolerance of S. meliloti CCNWSX0020. The wild-type and mutant strains of S. meliloti CCNWSX0020 were grown on TY solid medium in the presence of the indicated concentrations of CuSO₄, ZnSO₄, Pb(NO₃)₂, and CdSO₄. The MTC values are depicted on the right. Five 10-fold dilutions were spotted from left to right, and the OD₆₀₀ of the first 10-fold dilution was 0.1. The experiment was repeated three times.

Deletion of genes involved in heavy metal resistance affects root nodule symbiosis.

In our previous greenhouse studies, the metal resistance determinants in rhizobia were shown to affect the functional symbiosis formation under heavy metal stress (20, 21, 23). Therefore, the Cu-sensitive mutant (ΔcueO) and three mutants that were both Cu and Zn sensitive (ΔyedYZ, ΔcusA-like, and ΔfixH-like) were selected for further study of the effects of the metal resistance determinants on root nodule symbiosis. The root lengths and lateral root numbers of the plants inoculated with these four mutant strains were not significantly changed compared to those of control plants inoculated with the wild-type strain at 7 days postinoculation (dpi) and 14 dpi (data not shown). However, nodule and infection thread numbers of plants inoculated with the ΔcusA-like mutant were decreased significantly (P < 0.05) in both uncontaminated and contaminated soil. Meanwhile, nodule and infection thread numbers of plants inoculated with the ΔcueO mutant decreased dramatically (P < 0.05) only under Cu stress compared to those of control plants (Fig. 5a to d). Significant decreases could also be observed in the much lower shoot and root dry weights and N contents of plants inoculated with the ΔcusA-like mutant in both uncontaminated and contaminated soil and those of plants inoculated with the ΔcueO mutant in Cu-contaminated soil (Table 2). In addition, we found that most infection events elicited by the ΔcueO mutant were inhibited in the presence of Cu. The infection threads of M. lupulina inoculated with the S. meliloti CCNWSX0020 wild-type strain proceeded in the following stages: the infection threads initiated from curled root hairs, extended through the epidermal cells, and finally ramified into the cortex (Fig. 5e). However, during the infection process generated by the ΔcueO mutant, the progression of infection threads was blocked, or rhizobia were observed within root hairs but outside infection threads (Fig. 5f). We also observed the ultrastructure of the nodules formed on plant roots inoculated with the wild-type and mutant strains. We determined that the nodules from plant roots inoculated with the ΔcueO mutant had fewer infected cells in Cu-contaminated soil than the nodules from plant roots inoculated with the wild-type strain (Fig. 5g). These results suggest that the Cu resistance conferred by CueO is important for successful infection and functional symbiosis formation by S. meliloti CCNWSX0020 under Cu stress. No obvious differences in shoot and root dry weights and N contents of plants inoculated with the ΔyedYZ and ΔfixH-like mutants were found with or without Cu or Zn stress in comparison with those of plants inoculated with the S. meliloti CCNWSX0020 wild-type strain (Table 2). However, the numbers of infection threads and nodules produced with the ΔyedYZ and ΔfixH-like mutants were significantly (P < 0.05) decreased at 7 dpi under Cu and Zn stress compared to those for plants inoculated with the S. meliloti CCNWSX0020 wild-type strain (Fig. 5a and c), while no big differences were found at 14 dpi (Fig. 5b and d). The infection frequency and nodulation were decreased with inoculation by the S. meliloti CCNWSX0020 ΔyedYZ and ΔfixH-like mutants at an earlier stage, suggesting that deletion of the yedYZ and fixH-like genes did not impair symbiosis development but affected the infection efficiency under metal stress.

FIG 5.

Deletions of heavy metal resistance genes affected root rhizobial infection and nodulation development in M. lupulina. Plants were grown in uncontaminated or metal-contaminated soil, inoculated with the S. meliloti CCNWSX0020 wild-type strain or one of four metal-sensitive mutant strains (ΔyedYZ, ΔfixH-like, ΔcusA-like, and ΔcueO) harboring an EGFP reporter, and assayed for nodulation at 7 dpi (a) and 14 dpi (b) and for rhizobial infection events at 7 dpi (c) and 14 dpi (d). (e) Normal formation of infection threads as visualized by fluorescence microscopy of the S. meliloti CCNWSX0020 wild-type strain expressing EGFP under Cu stress. (f) Representative images of infection events that were inhibited in M. lupulina inoculated with the ΔcueO mutant under Cu stress. (g) Light microscopy images of nodule sections produced by the S. meliloti CCNWSX0020 wild-type strain (left) and the S. meliloti CCNWSX0020 ΔcueO mutant strain (right) under Cu stress. The experiments were repeated three times, and error bars represent standard errors. Asterisks indicate significant decreases by Duncan's test for plants inoculated with mutants compared to those inoculated with the wild-type strain (*, P < 0.05; ***, P < 0.005).

TABLE 2.

Effects of mutants on dry weight and N content of Medicago lupulina grown in uncontaminated or contaminated soils^a

Strain	Dry wt (mg)						N content (g/kg dry wt)
	Roots			Shoots			Roots			Shoots
	No treatment	200 mg/kg Cu	200 mg/kg Zn	No treatment	200 mg/kg Cu	200 mg/kg Zn	No treatment	200 mg/kg Cu	200 mg/kg Zn	No treatment	200 mg/kg Cu	200 mg/kg Zn
Noneb	1.1 ± 0.2b	0.8 ± 0.1c	0.8 ± 0.2c	3.9 ± 0.1d	3.0 ± 0.6c	3.1 ± 0.3c	25.8 ± 0.1c	22.6 ± 0.7b	23.1 ± 0.4b	30.3 ± 0.2c	26.1 ± 0.8b	28.1 ± 0.4b
Wild type	1.8 ± 0.1a	1.3 ± 0.1a	1.3 ± 0.1a	7.3 ± 0.9a	5.8 ± 0.5a	5.6 ± 0.6a	30.2 ± 0.7a	28.7 ± 0.2a	28.9 ± 1.0a	38.2 ± 0.8a	35.8 ± 0.7a	37.0 ± 0.5a
ΔyedYZ mutant	1.7 ± 0.3a	1.2 ± 0.2a	1.2 ± 0.1a	6.7 ± 1.8ab	5.7 ± 0.3a	5.2 ± 0.1a	29.5 ± 0.7a	27.1 ± 0.1a	27.1 ± 0.6a	37.6 ± 0.5a	34.0 ± 1.0a	36.3 ± 0.3a
ΔfixH-like mutant	1.8 ± 0.3a	1.2 ± 0.1a	1.2 ± 0.1a	7.1 ± 0.1a	5.6 ± 0.3a	5.2 ± 0.6a	29.8 ± 0.4a	27.4 ± 0.1a	27.8 ± 0.9a	37.8 ± 0.7a	35.1 ± 0.8a	36.1 ± 0.9a
ΔcusA-like mutant	1.1 ± 0.2b	0.8 ± 0.1bc	0.9 ± 0.1b	4.8 ± 0.3c	3.6 ± 1.3bc	3.5 ± 0.2b	27.4 ± 0.1b	23.0 ± 0.3b	23.1 ± 1.2b	32.7 ± 0.3b	26.8 ± 0.7b	28.8 ± 0.8b
ΔcueO mutant	1.6 ± 0.2a	0.9 ± 0.1b	1.2 ± 0.1a	6.7 ± 1.5ab	4.7 ± 0.3b	5.4 ± 1.4a	30.0 ± 0.2a	23.1 ± 0.2b	27.6 ± 0.9a	38.1 ± 0.7a	26.5 ± 0.5b	36.5 ± 0.6a

^aValues are means ± SD for three replicates. Different letters (a to d) represent significant differences between values in the same column (P < 0.05 by Duncan's test).

^bPlants without inoculation.

Deletion of heavy metal resistance genes affects heavy metal content and antioxidant enzyme activity of the Sinorhizobium-Medicago symbiosis.

Rhizobial symbioses with the S. meliloti CCNWSX0020 wild-type strain or any of the four metal-sensitive mutant strains (ΔcueO, ΔyedYZ, ΔcusA-like, and ΔfixH-like) displayed various effects on Cu or Zn content in the roots and shoots of M. lupulina (Fig. 6a). The Cu and Zn contents were considerably greater in roots than in shoots for both inoculated and noninoculated M. lupulina plants exposed to excess Cu and Zn. Moreover, Cu and Zn accumulation was higher in the plants inoculated with the wild-type strain and the four mutants than in noninoculated plants. The Cu and Zn accumulation in roots of M. lupulina plants inoculated with the mutants showed different degrees of reduction in comparison to the accumulation in control plants inoculated with the wild-type strain. These results indicated that the heavy metal resistance determinants of S. meliloti CCNWSX0020 could aid in phytoremediation by M. lupulina plants grown in metal-contaminated soil and that the efficiency of phytoremediation was dependent to various degrees on the genes that were deleted.

FIG 6.

Effects of four heavy metal resistance gene deletions on the heavy metal content and antioxidant enzyme activity of Medicago lupulina. M. lupulina plants inoculated with the S. meliloti CNWSX0020 wild-type, ΔyedYZ, ΔfixH-like, ΔcusA-like, and ΔcueO strains were grown in uncontaminated or 200-mg/kg Cu- or Zn-contaminated soils and harvested at 21 dpi for Cu or Zn content determination (a) and at 7 dpi and 14 dpi for CAT and SOD activity assays (b). Error bars represent standard deviations for three biological repeats. Asterisks indicate significant decreases by Duncan's test for plants inoculated with mutants compared to those inoculated with the wild-type strain (*, P < 0.05). Fw, fresh weight of sample.

Excess metal ions can generate an increase in reactive oxygen species (ROS) and oxidative stress in plants, such that plants have to use their own antioxidant defense systems to scavenge ROS and alleviate oxidative damage (36). In a previous study conducted by our group, inoculation with the S. meliloti CCNWSX0020 wild-type strain not only increased the expression levels of M. lupulina antioxidant genes (i.e., CuZnSODc, CuZnSODp, CAT, APXc, and GRc) but also elevated the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) of M. lupulina in the presence of excess Cu (22). To investigate the influence of the four metal-sensitive mutants on the antioxidant defense response to excess Cu and Zn in M. lupulina, the activities of SOD, CAT, and peroxidase (POD) of M. lupulina inoculated with the wild-type and mutant strains were determined. The results showed that the activity of CAT at 7 dpi and the activity of SOD at 14 dpi for plants inoculated with the four metal-sensitive mutant strains exhibited various degrees of decreasing antioxidant activity compared to that of control plants inoculated with the wild-type strain (Fig. 6b). In terms of POD activity, no significant changes were observed for plants inoculated with the wild-type or mutant strains (data not shown).

DISCUSSION

RNA-Seq is a convenient and efficient high-throughput sequencing method that can provide valuable information at the transcriptome level for differentially expressed genes induced by various environmental stresses, as evidenced from the various processes and pathways that are missed by microarray analysis (37). The transcriptome of S. meliloti under Cu and Zn exposure is presented here and is intended to provide the molecular basis for a better understanding of the toxicity of Cu and Zn and the response to this toxicity in S. meliloti CCNWSX0020. Furthermore, a major advantage of RNA-Seq is the direct estimation of gene expression levels. Several studies on the expression levels of genes in different rhizobia under heavy metal stress were conducted previously (38, 39). However, there is still limited information on transcriptome profiling of gene networks and identifying regulatory mechanisms. The present study, using the RNA-Seq method on S. meliloti CCNWSX0020, yielded a dynamic range of genes with both predicted functions and hypothetical functions (uncharacterized functions) that were differentially expressed in S. meliloti CCNWSX0020 under Cu or Zn exposure, which may provide a broad basis for the further elucidation of Cu and Zn resistance mechanisms.

Based on our RNA-Seq analysis, the gene expression patterns in S. meliloti CCNWSX0020 were very different between short-term and long-term exposures to Cu and Zn. Many more DEGs were identified under short-term metal stress than under long-term metal stress. This result is reasonable, since most immediate response genes are rapidly induced by a variety of stimuli, and their elevated expression may not last long. The long-term expression of genes induced by heavy metals may be involved in restructuring the central metabolism and other nontransient changes which bacterial organisms have evolved for defense strategies. Genes encoding a P_1B-type ATPase, a multicopper oxidase, an outer membrane protein, and a metal binding protein were significantly upregulated by Cu or Zn exposure for 24 h, indicating that S. meliloti CCNWSX0020 relies on efflux, intracellular sequestration, and Cu transformation for adaptation and long-term detoxification. Commonly upregulated genes under short-term exposure to Cu and Zn are predicted to encode proteins involved in ion transport, the oxidative stress response, and carbohydrate and amino acid metabolism, suggesting that S. meliloti CCNWSX0020 responds to nonspecific metal injury through multiple avenues and adjusts its metabolism by the global stress response, as it needs to produce more energy and synthesize functional proteins to cope with these stressful environmental conditions. In addition, some transcriptional regulators, transporters, or molecular chaperones were induced specifically by Cu or Zn. The results presented in this report reveal that the regulation of intracellular Cu and Zn homeostasis is both complicated and multilayered. In addition, the specific Cu and Zn detoxification or resistance mechanisms in S. meliloti CCNWSX0020 are quite different.

Analysis of deletion mutants revealed that three operons were all involved in further alleviating Cu and Zn toxicity in S. meliloti CCNWSX0020 (Fig. 5). The ΔcueO mutant, with a deletion of a gene located within the MCO operon, displayed a specific sensitivity to Cu, showing a critical role of this multicopper oxidase in Cu resistance. It is well known that in E. coli CueO usually oxidizes Cu(I) to Cu(II), which is extruded by CopA from the cytoplasm into the periplasm, thereby reducing copper toxicity (8). CueO of S. meliloti CCNWSX0020 showed 40% similarity to CueO of E. coli. It is thus proposed that cueO may play a similar function in Cu detoxification, e.g., CueO may oxidize Cu(I) to Cu(II) in the periplasm and Omp might transport excessive Cu(II) into the extracellular medium. The Δomp mutant not only was sensitive to Cu but also showed differing sensitivities to Zn, Cd, and Pb, suggesting that the outer membrane protein encoded by omp might act as a transmembrane channel that is vital for efflux of multiple divalent metal ions from S. meliloti CCNWSX0020.

In contrast, the ΔcusA-like, ΔfixH-like, and ΔHP mutants, with deletions from the CopG operon of S. meliloti CCNWSX0020, displayed differing degrees of sensitivity to multiple metal ions. The ΔcusA-like mutant was more sensitive to Zn, Pb, and Cd than to Cu. The CusA-like protein from S. meliloti CCNWSX0020 contains only 146 amino acids, while the entire CusA transporter from other bacteria is usually more than 1,000 amino acids, and the SM0020_RS03640-encoded CusA-like protein is only 5.8% similar to CusA from E. coli. It is well known that CusA works in conjunction with the membrane fusion protein CusB and the outer membrane channel CusC and that these three components form the CusCBA efflux complex, which spans the entire cell envelope of E. coli to export Cu(I) and Ag(I) (9). CusA from E. coli has 12 transmembrane helices (TM1 to TM12). E. coli CusA is predicted to form a Cu(I) and Ag(I) ion channel spanning the entire transmembrane region up to the periplasmic space, with the channel including four methionine (Met) pairs as well as the three-methionine specific binding site, and this channel works with three charged residues (Asp, Glu, and Lys) essential for transport functions (40). The CusA-like protein from S. meliloti CCNWSX0020 is 43.5% similar to the terminal membrane component (TM10 to TM12) of E. coli CusA, and it contains one conserved set of Met, Glu, and Lys in the transmembrane helices (Fig. S4). Therefore, we predict that the CusA-like protein might function as a metal binding protein, possibly a chaperone, in the periplasm of S. meliloti CCNWSX0020. The ΔfixH-like mutant was also sensitive to Cu, Zn, Pb, and Cd. It has been suggested that FixH associated with FixG, FixI, and FixS may participate in a membrane-bound complex coupling the FixI cation pump with a redox process catalyzed by FixG (41). In contrast to the FixH family proteins, the sensitivity to multiple metal ions of the ΔfixH-like mutant suggests that the FixH-like protein in S. meliloti CCNWSX0020 is involved in metal resistance, but the resistance mechanism remains unknown. In addition, whether the CusA-like, FixH-like, and hypothetical proteins in S. meliloti CCNWSX0020 function separately or conjointly for metal detoxification still needs to be explored.

The third operon shown to confer various degrees of metal resistance to S. meliloti CCNWSX0020 encodes YedYZ. YedYZ from E. coli was the first molybdenum-containing oxidoreductase identified in bacteria. Although YedY was characterized as a sulfite oxidase, biochemical assays showed that it was a reductase, not an oxidase, and was able to reduce free methionine sulfoxide in vitro (33, 42). However, the optimal substrate or physiological role in vivo for YedY in E. coli remains unknown. Previous transcriptomic studies showed upregulation of yedYZ or a yedYZ homolog in response to chlorite stress in E. coli O157:H7 and Shewanella algae ACDC (43, 44), indicating a possibly physiological role of YedYZ in perchlorate reduction. It has never been reported that YedYZ may be involved in heavy metal resistance, except for one report showing upregulation of yedYZ in Salmonella enterica serovar Typhimurium in response to Cu excess (45). In the present study, the S. meliloti CCNWSX0020 ΔyedYZ mutant showed sensitivities to Cu, Zn, Pb, and Cd (Fig. 5). Therefore, we have shown resistance to multiple heavy metals conferred by YedYZ. In S. meliloti 1021, the expression of the YedY-related gene SMc01281 was highly induced during growth on taurine and thiosulfate, indicating a possible role for this enzyme in sulfur metabolism (46). It has been reported that sulfite may cause cytotoxicity by increasing free radical formation and lipid peroxidation and disabling the cellular antioxidant defense system by depleting reduced glutathione (GSH) and lowering the activities of catalase and glutathione peroxidase (47–49). Therefore, we speculated that a yedYZ deletion may influence S. meliloti CCNWSX0020 sulfite metabolism and potentially further impair the antioxidant defense which copes with reactive oxygen species (ROS) generated by excess heavy metals. Future biochemical studies should bear out the precise function of YedYZ from S. meliloti CCNWSX0020 during sulfur metabolism and the correlation between YedYZ and cellular oxidative stress defense mechanisms.

Our previous studies showed that several metal resistance determinants in rhizobia were required for effective symbiosis formation or could enhance phytohormone indole-3-acetic acid (IAA) production (20, 21, 23). However, the exact molecular mechanisms explaining how the rhizobial metal resistance determinants interact with the synthesis of growth-promoting substances, nodulation and nitrogen fixation, and, ultimately, phytoremediation processes still remain largely elusive. Interestingly, four heavy metal resistance determinants identified in S. meliloti CCNWSX0020 showed different influences on the symbiotic process. The cusA-like deletion in S. meliloti CCNWSX0020 severely inhibited infection and nodulation with M. lupulina grown in uncontaminated or Cu- and Zn-contaminated soils (Fig. 5a to d), indicating that the cusA-like gene is vital during initial infection. The CusA efflux pump had never been reported to be related to the rhizobium-legume symbiosis due to its specific role in Cu(I)/Ag(I) transport. The NolGHI proteins of S. meliloti, belonging to the RND-type efflux family, have been reported to be involved in the export of nodulation signals (50). The CusA-like protein from S. meliloti CCNWSX0020 appears to be a metal binding protein and is not the entire CusA transport protein as in E. coli but rather the C terminus of CusA. The C terminus contains conserved metal binding residues, suggesting that the CusA-like protein might function in metal binding or sequestration or act as a chaperone. Whether the CusA-like protein can play a role in an unknown and earlier symbiotic signaling pathway crucial for the occurrence of infection and nodulation with M. lupulina needs further investigation. In contrast, the cueO deletion in S. meliloti CCNWSX0020 severely inhibited infection and nodulation with M. lupulina only under Cu stress (Fig. 5a to d, f, and g), suggesting the importance of this heavy metal resistance determinant for rhizobial survival and symbiosis formation in metal-contaminated soil. In addition, the ΔyedYZ and ΔfixH-like mutants did not affect the symbiotic nodulation capacity of S. meliloti with M. lupulina (Fig. 5b and d) but decreased the infection efficiency under Cu and Zn stress at an earlier stage of infection (Fig. 5a and c). These results indicate that heavy metal resistance determinants can protect rhizobia and assist them to establish infection under metal-stressed conditions.

Furthermore, inoculation with four metal-sensitive mutants led to an obvious decrease in Cu or Zn content in roots accompanied by lower activities of plant CAT and SOD (Fig. 6). ROS are continuously produced in response to biotic and abiotic stresses. Referring to the rhizobium-legume symbiosis, large amounts of ROS are generated in root nodules during the lifetime of symbiosis development, so both symbiotic partners use a set of antioxidant enzymes to cope with these stresses (51). A previous study showed significantly increased activities of antioxidant enzymes in plants inoculated with S. meliloti CCNWSX0020 compared to those in noninoculated plants in the presence of excess Cu, suggesting that promotion of plant antioxidant defenses by rhizobia may improve symbiotic performance in alleviating heavy metal toxicity (22). The results of the present study showed that inoculation with four metal-sensitive mutants led to significant decreases of CAT and SOD activities in the M. lupulina plants compared to those in control plants inoculated with the S. meliloti CCNWSX0020 wild-type strain in the presence of Cu and Zn stress. These results suggest that the heavy metal resistances of S. meliloti CCNWSX0020 conferred by heavy metal resistance determinants are important for increasing plant health by promoting the antioxidant defenses in metal-contaminated soil and subsequently improve bioremediation by the rhizobium-legume symbiosis.

In conclusion, through RNA-Seq technology and gene deletion mutagenesis, the MCO operon, the YedYZ operon, and a putative CopG operon have been proved to be involved in Cu, Zn, Pb, and Cd tolerance of S. meliloti CCNWSX00200. Moreover, the cueO, cusA-like, fixH-like, and yedYZ genes from these three operons are vital for promoting bioremediation by directly or indirectly influencing the rhizobium-legume symbiosis in metal-contaminated soils.

MATERIALS AND METHODS

Bacterial strain culture and metal treatments.

Sinorhizobium meliloti CCNWSX0020 was used in all experiments. The draft genome sequence of S. meliloti CCNWSX0020 is available under GenBank accession number AGVV00000000.1 (52). S. meliloti CCNWSX0020 was grown in TY liquid medium (28) at 28°C with shaking at 150 rpm, and cell suspensions were prepared to an optical density at 600 nm (OD₆₀₀) of 1.0. One-percent aliquots of the cell suspensions were added to fresh TY medium and divided into two groups for Cu and Zn treatments. The metal concentrations used were sublethal doses that did not cause mortality for at least 24 h of metal exposure. In the first group, the cells were incubated to mid-exponential phase for 24 h in the presence of 0.6 mmol/liter CuSO₄ or 0.2 mmol/liter ZnSO₄, and in the second group, the cells were preincubated for 24 h without initially adding any metal, followed by addition of 0.6 mmol/liter CuSO₄ or 0.2 mmol/liter ZnSO₄ and incubation for another 30 min. Strains incubated to mid-exponential phase without metal exposure served as controls. The cells were harvested by centrifugation at 8,000 × g for 5 min for RNA isolation. Three biological replicates were prepared under each condition.

RNA extraction and cDNA library preparation.

Total RNA was extracted according to the protocol of Rivas et al. (53) and was treated with RNase-free DNase I following the manufacturer's instructions (Ambion, USA). The quality of purified RNAs was assessed using an Agilent Bioanalyzer 2100 system, and those with RNA integrity numbers of 8.0 or above were used for rRNA depletion. The rRNA was removed by use of a Ribo-Zero rRNA removal kit for Gram-negative bacteria according to the manufacturer's instructions (Epicentre, USA). The cDNA library was generated using an NEBNext Ultra Directional RNA library prep kit for Illumina (NEB, USA) following the manufacturer's instructions, as previously described (54), and library quality was assessed on an Agilent Bioanalyzer 2100 system.

Illumina sequencing and data analysis.

The library preparations were sequenced on an Illumina HiSeq 4000 platform at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China), and 150-bp paired-end reads were generated.

Clean data (clean reads) were obtained by removing reads containing adapters, reads containing poly-N results, and low-quality reads from the raw data, and the reads were then mapped to the reference genome by use of Bowtie2-2.2.3. The expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) was used to estimate gene expression levels (55). Differential gene expression analysis between two assigned groups was performed using the DESeq R package (1.18.0) (56). The resulting P values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate. The differentially expressed genes (DEGs) were considered to be induced or repressed if the adjusted P value was <0.05 (57).

Unique transcripts and transcripts common to each of the heavy metals (P < 0.05) were analyzed by Venn diagrams, using the VennDiagram package (58) in the R environment (http://www.r-project.org). Heat maps were used to visualize transcript fold changes on a log₂ scale for each metal versus the control and were generated using the Lattice package (59).

Validation of RNA-Seq data by qPCR.

To validate the RNA-Seq data, the expression levels of some representative genes were examined by quantitative real-time PCR (qPCR). The primers used for qPCR are listed in Table S1 in the supplemental material. All qPCRs were performed with SYBR Premix Ex Taq (TaKaRa Japan) in a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA). The qPCR conditions were as follows: the thermal profile was 95°C for 30 s, 40 amplification cycles of 95°C for 5 s and 60°C for 30 s, a dissociation cycle of 95°C for 10 s and 60°C for 5 s, and then a return to 95°C. All reactions were performed in triplicate, and mean values were calculated. To standardize results, the 16S rRNA gene was used as an internal standard, and the relative levels of transcription were calculated using the 2^−ΔΔCT method (60).

Construction of genetic deletion mutants in S. meliloti CCNWSX0020.

For gene deletion mutant construction, the bacterial strains and plasmids used are listed in Table S2, and all primers used are listed in Table S1. An in-frame deletion strain of S. meliloti CCNWSX0020 was constructed by a method using the suicide vector pK18mobsacB (61) and double homologous recombination as described in our previous study (28). First, the upstream and downstream fragments of the target gene were amplified and then ligated by crossover PCR to generate an in-frame deletion (62). Second, the resulting fragment was digested with appropriate restriction enzymes and inserted into the same site of the pK18mobsacB plasmid. Third, the resulting plasmid was transformed into Escherichia coli DH5α competent cells. Finally, a triparental mating procedure was conducted to transform the plasmid from E. coli into S. meliloti CCNWSX0020, helped by E. coli DH5α cells containing pRK2013, and the deletion was obtained through double homologous recombination. Double-crossover recombinants were confirmed by PCR, and the correct PCR product was sequenced.

Metal sensitivity assays of seven deletion mutants.

Metal sensitivity assays of the S. meliloti CCNWSX0020 wild-type strain and the deletion mutants were performed on TY solid medium with different concentrations of heavy metals. Cells were grown to exponential phase in TY liquid medium at 28°C with shaking at 150 rpm, and cell suspensions were prepared to an OD₆₀₀ of 1.0. Series of 10-fold dilutions were carried out and spotted from left to right onto TY agar plates supplemented with seven different metals, at the following concentrations: AgNO₃, 0.05, 0.1, 0.15, and 0.17 mM; CuSO₄, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.5, and 1.6 mM; ZnSO₄, 0.2, 0.4, 0.6, 0.8, 1.0, 1.1, and 1.2 mM; CdSO₄, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, and 0.26 mM; Pb(NO₃)₂, 2.4, 2.6, 2.8, 3.0, 3.2, 3.3, and 3.4 mM; CoCl₂, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mM; and NiSO₄, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mM. Each experiment was repeated three times.

Plant growth, nodulation conditions, and N content and Cu/Zn content determinations.

Medicago lupulina seeds were surface sterilized and germinated using previously described procedures (21). Germinated seedlings were sown in plastic pots filled with 100 g of a sterilized perlite-vermiculite (3:2 [vol/vol]) mixture supplied with CuSO₄ or ZnSO₄ to produce a concentration of Cu²⁺ or Zn²⁺ (respectively) of 200 mg kg⁻¹ and were incubated in the greenhouse at 25°C. Six seedlings were planted in each pot, and at least three replicates were conducted for each treatment. When the first main leaf grew out, seedlings were inoculated with cell suspensions of S. meliloti CCNWSX0020 or the different mutants (approximately 10⁸ CFU/ml) that had been transformed with plasmid pMP2444, expressing enhanced green fluorescent protein (EGFP), corresponding to zero days postinoculation (0 dpi). Seedlings without inoculation were included as blank controls. The numbers of infection threads and nodules were scored at 7 dpi and 14 dpi. The plant tissues were also collected and weighed at 7 dpi and 14 dpi, and samples were then quickly stored in liquid nitrogen for antioxidant enzyme activity assays. The catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities were assayed as previously described (22, 28). Finally, plants were harvested at 21 dpi, and the lengths and dry weights of shoots and roots were recorded. The total numbers of nodules were counted, and nodules were collected for microscopic observation. For final observation with a BX53 biological microscope (Olympus, Japan), paraffin-embedded nodule sections (5 to 10 μm) were stained with 0.05% toluidine blue for 5 min. Dried shoots and roots were ground, homogenized, and then prepared for determination of total N, Cu, and Zn content. Total N content was measured by the Kjeldahl method on a Kjeltec 8400 analyzer unit (Foss-Tecator AB, Hoganas, Sweden). The Cu or Zn content was analyzed by atomic absorption spectrophotometry (AAS) (Z-5000; Hitachi, Tokyo, Japan).

Statistical analyses.

All statistical analyses were performed with SPSS 18.0. Data were subjected to statistical evaluation using analysis of variance (ANOVA) followed by Duncan's test (P < 0.05). All data were means ± standard deviations (SD) for three independent replicates and were analyzed using Origin Pro v8.0 (Origin Lab, USA) to create the figures.

Accession number(s).

The clean sequencing data were deposited in the NCBI Sequence Read Archive (SRA) under accession number SRS1751249 (BioSample accession number SAMN05921096; BioProject accession number PRJNA344820).