Article Figures & Data
Figures
- FIG 1
Expression of Zur under zinc excess and depleted conditions. (A) Cotranscription of zur with znuC and znuB. Transcription of four intergenic regions was tested by RT-PCR on total RNA isolated from wild-type CCBAU45436 in the M9 medium using the indicated primer pairs (see Table S2 in the supplemental material). Reverse transcriptase was added to the reaction in lanes 1, 4, 7, and 10 (RT+) but omitted from reactions in lanes 2, 5, 8, and 11 (RT−) to rule out the possibility of contamination by genomic DNA. Genomic DNA was amplified as a positive control in lanes 3, 6, 9, and 12 (DNA). M, 100 bp marker. (B) Western blot analysis of Zur. Exponentially grown CCBAU45436_3×Myc cells were either untreated or treated with various concentrations of chelator TPEN (4, 2, and 1 μM) or ZnSO4 (200, 100, and 50 μM) for 12 h before cell harvest. (C) qRT-PCR analysis of zur gene of wild-type CCBAU45436 in M9 medium supplemented with 200 μM ZnSO4 or 4 μM TPEN. ***, P < 0.001 by t test. Values are given as means ± standard deviations (SDs) from biological triplicates in three independent experiments.
- FIG 2
RNA-seq and qRT-PCR analyses of the Zur regulon of S. fredii. (A) RNA-seq analysis of transcriptomes of the wild-type CCBAU45436 (WT) and Mzur under low-zinc (4 μM TPEN) and high-zinc (200 μM ZnSO4) conditions (see Table S1A). The numbers of down- and upregulated genes are shown for WT under low-zinc conditions compared with those under high-zinc conditions (left), and for Mzur compared with WT under either high-zinc or low-zinc conditions (right). RNA-seq (B) and qRT-PCR (C) revealed transcriptional profiles of putative zinc transporter genes in WT and Mzur under low- and high-zinc conditions. Results from three biological replicates are shown. Subcellular locations of these putative zinc transporter proteins are shown on the right of panel B. (D) qRT-PCR analysis of znuA transcription in wild-type CCBAU45436 and Mzur grown under conditions of low or high zinc. qRT-PCR analysis of transcription of zinc transporter genes under high levels of zinc (E) and cobalt (200 μM CoCl2) (H). qRT-PCR analysis of c06450 (F) and znuA (G) genes in CCBAU45436 in the presence of 200 μM metals (FeCl3, CoCl2, NiSO4, or ZnSO4) or 25 μM EDTA. Mean transcription values were compared with that in the medium with EDTA. (C to H) Values are given as means ± SDs from biological triplicates in three independent experiments. *, P < 0.05; ***, P < 0.001 by t test.
- FIG 3
Dose-dependent regulation of znuA by zinc and Zur, and EMSA validation of Zur-binding sites. (A) β-galactosidase (β-Gal) assays showing expression of znuA promoter-lacZ translational fusion in wild-type CCBAU45436 and Mzur grown in different concentrations of ZnSO4. (B) Zur binds the znuA promoter in a zinc-dependent manner. Biotin-labeled znuA promoter fragment was incubated with increasing concentrations (0.33, 0.66, 1.32, and 2.64 μM) of purified Zur of CCBAU45436. (C) DNA sequence logos derived from 17 predicted Zur-binding sites with a score of >5 (Table S1B). The consensus sequence contains a 15-bp palindromic motif (7-1-7 inverted repeat). (D) Biotin-labeled promoter fragment containing predicted Zur-binding sites was incubated with purified Zur (2.64 μM). For panels B and D, 200 μM ZnSO4 was present in the reaction buffer of all samples. + and − indicate the presence and absence, respectively, of Zur protein, a 100-fold excess of unlabeled znuA promoter fragment (specific competitor), unlabeled mutated znuA promoter DNA fragment (nonspecific competitor), or 50 μM TPEN (zinc chelator).
- FIG 4
Impaired infection ability of mutants lacking znu. Number of infected nodules per plant of G. max (A), G. soja (B) and C. cajan (C). Means ± SDs are based on more than 20 to 40 scored plants from multiple independent experiments. Different lowercase letters indicate significant difference (Duncan test, alpha = 0.05). Representative photos showing infected nodules (red arrows) and bumps (blue arrows) observed on roots of G. max (D), G. soja (E), and C. cajan (F) inoculated with corresponding strains. Light microscopy pictures (F) and transmission election microscopy pictures (G) of sections for infected nodules (red border) and bumps (blue border) on C. cajan inoculated with MznuA. Bumps were rarely infected by rhizobia.
- FIG 5
Replete zinc recovers nodulation defects of mutants lacking znu. (A) Representative photos showing infected nodules (red arrows) and bumps (blue arrows) observed on roots of G. max inoculated with corresponding strains with or without supplement of 700 μM ZnSO4 in vermiculite. pB-znuA and pB-zip1 carrying functional znuA and zip1, respectively, were used in complementary experiments. Numbers of infected nodules per plant of G. max (B), G. soja (C), and C. cajan (D) inoculated with corresponding strains under conditions with or without 700 μM ZnSO4 in vermiculite moistened with low-N nutrient solution. (E) Number of infected nodules per plant of G. max inoculated with MznuAzip1 and its derivatives carrying pB-znuA or pB-zip1 on G. max. (F) Root surface colonization by CCBAU45436, MznuAzip1zip2, and MznuAc06450 on G. max. CFU were counted 5 days after plating the suspension of cells collected by using ultrasound. (G) Maximum likelihood phylogenic tree based on 2,166 core genes of representative Sinorhizobium strains. The filled circles on the branches indicate >95% bootstrap support. The scale bar represents 0.02 nucleotide substitution per site. The presence of conserved zinc transporter genes in the corresponding genome is indicated. (H) Numbers of infected nodules per plant of G. soja inoculated with CCBAU25509, CCBAU05684, CCBAU05631, and their znuA-insertion mutants under conditions supplemented with or without 700 μM ZnSO4 in vermiculite moistened with low-N nutrient solution. (B to F and H) Means ± SDs are based on more than 20 to 40 scored plants from multiple independent experiments. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by t test.
- FIG 6
Transcription of rhizobial nod and T3SS genes. (A) qRT-PCR analysis of nod and T3SS gene transcription in wild-type CCBAU45436 and MznuAzip1 or MznuAzip1zip2 mutants grown in M9 medium with 12-h induction by 1 μM genistein. (B) qRT-PCR analysis of nod and T3SS genes in wild-type CCBAU45436 and MznuAc06450zip1zip2 grown in M9 medium (with or without 200 μM ZnSO4) with 12-h induction by 1 μM genistein. Mean transcription values were compared with the wild-type CCBAU45436. ns, P > 0.05; *, P < 0.05; **, P < 0.01 by t test. Values are means ± SDs from biological triplicates in three independent experiments.
- FIG 7
Working model for the role of zinc starvation machinery in modulating symbiotic compatibility. Under zinc replete conditions (−, left), znu and c06450 were repressed by Zur. Zip1, Zip2, and an unknown zinc transporter(s) import zinc. Under zinc-deplete conditions during symbiosis establishment (+, right), transcriptional repression of znu and c06450 by Zur was released due to decreased intracellular zinc level. Znu together with accessory c06450/Zip1/Zip2 and an unknown zinc transporter(s) in the Sinorhizobium pangenome were essential for proper transcription of T3SS genes and nodD2, which are involved in regulating symbiotic compatibility during both infection and nodule organogenesis. IM, inner membrane; OM, outer membrane; ?m and ?n indicate that pools of zinc-associated proteins under zinc-replete and -deplete conditions, respectively, may differ as implied by the transcriptomes.
Supplemental Material
FIG S1
Sequence alignment of c18210 with other bacterial Zur proteins. The protein sequence of SF45436_c18210 was aligned with Zur proteins from the following bacteria: Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, Yersinia pestis, Vibrio cholerae, Pseudomonas aeruginosa, Xanthomonas campestris, Bacillus subtilis, ans Staphylococcus aureus using ClustalW. Five conserved residues (H77, C87, C90, C128, and C130) that are critical for the specific binding to zinc ions are indicated with triangles. Download FIG S1, PDF file, 0.1 MB.
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TABLE S1
RNA-seq analysis of Zur regulon and predicted Zur-box. (A) RNA-seq analysis of Sinorhizobium fredii CCBAU45436 and its Mzur mutant under zinc-replete (high_zinc) and zinc-deplete (TPEN) conditions. (B) Zur-box identified in the genome of Sinorhizobium fredii CCBAU45436 and transcription profiles of associated genes in RNA-seq analysis. Download Table S1, XLSX file, 2.9 MB.
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FIG S2
Putative zinc uptake transporters encodedby the multipartite genome of S. fredii CCBAU45436. (A) Genes encoding putative transporters ZnuABC, Zip1, and Zip2 are shown in black. These genes are located on three of the five replicons of CCBAU45436: cSF45436 (chromosome), pSF45436b (chromid), and pSF45436d (an accessory plasmid). (B) Zip1 and Zip2 of CCBAU45436 harbor two characterized conserved regions (shaded) in two transmembrane domains of ZIP family proteins. The partial protein sequences of Zip1 (SF45436_b54490) and Zip2 (SF45436_d66670) were aligned with ZIP protein homologs from Arabidopsis thaliana, Saccharomyces cerevisiae, Glycine max, and Escherichia coli. Download FIG S2, PDF file, 0.2 MB.
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FIG S3
Znu as the major zinc uptake system under zinc-deplete conditions. (A) Intracellular cobalt content of strains grown in M9 medium supplied with 10 μM CoCl2. (B andC) Intracellular zinc content of strains grown in M9 medium supplied with 10 μM ZnSO4. (D to G) Growth curves determined in M9 medium without (C and F) or with (D and G) 100 μM ZnSO4. Values are mean ± SDs from biological triplicates. Different lowerletters indicate significant difference (Duncan test, alpha = 0.05). Download FIG S3, PDF file, 0.5 MB.
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FIG S4
Znu is essential for low zinc adaptation. (A) Cell suspension at an OD600 of 1.0 was serially diluted and spotted onto plates with the M9 minimal medium with or without 50 μM ZnSO4 or onto plates with the YMA medium supplemented with 50 μM EDTA or 50 μM EDTA and 50 μM ZnSO4. Plasmids pB-znuA, pB-zip1, and pB-c06450 were used in complementation experiments. (B) Growth defects of znu mutants on M9 plates was partially restored by adding CoCl2. Cell suspension at an OD600 of 1.0 was serially diluted and spotted onto plates with the M9 medium supplemented with 20 μM or 2 μM CoCl2. Download FIG S4, PDF file, 1.1 MB.
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FIG S5
Comparative analysis of nodulation phenotypes of mutants lacking different znu genes. (A) Representative photos showing infected nodules (red arrows) and bumps (blue arrows) observed on roots of G. max and G. soja inoculated with corresponding strains. Numbers of infected nodules per plant of G. max (B), G. soja (C), and C. cajan (D). Means ± SDs are based on 20 to 30 scored plants from three independent experiments. Different lowercase letters indicate significant difference (Duncan test, alpha = 0.05). Numbers of infected nodules per plant of G. max (E), G. soja (F), and C. cajan (G) inoculated with corresponding strains under conditions with or without 700 μM ZnSO4 in vermiculite moistened with low-N nutrient solution. Means ± SDs are based on 20 to 30 scored plants from three independent experiments. **, P < 0.01; ***, P < 0.001 by t test. Download FIG S5, PDF file, 2.2 MB.
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FIG S6
Ultrathin sections of G. max (A), G. soja (B), and C. cajan (C) nodules. Pictures of ultrathin sections of 40-day postinoculation (dpi) nodules infected by CCBAU45436 and representative mutants were obtained by transmission electron microscopy. Download FIG S6, PDF file, 1.1 MB.
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FIG S7
Symbiotic performance of mutants lacking individual or multiple zinc transporter genes. Shoot dry weight of G. max (A), G. soja (B), and C. cajan (C) plants inoculated with S. fredii CCBAU45436 and its derivatives lacking individual or multiple zinc transporter genes. (D) Shoot dry weight of G. soja plants inoculated with CCBAU25509/CCBAU05684/CCBAU05631 and their znuA mutants. Means ± SDs are based on 20 to 30 scored plants from three independent experiments. Different lowercase letters indicate significant difference (Duncan test, alpha = 0.05). ***, P < 0.001 by t test of the mean comparison with that of wild-type S. fredii CCBAU25509, S. sojae CCBAU05684, or Sinorhizobium sp. CCBAU05631; ns, nonsignificant. Download FIG S7, PDF file, 0.3 MB.
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FIG S8
Nodulation defects of mutants lacking znu cannot be recovered by replete cobalt. Nodule number per plant of Glycine max and Glycine soja were recorded under conditions with different concentrations of cobalt being supplied. Means ± SDs are based on more than 20 to 30 scored plants. ns, P > 0.05; ***, P < 0.001 by t test. There were no nodules formed on Cajanus cajan plants inoculated with either WT or the mutant when cobalt was added. Download FIG S8, PDF file, 0.1 MB.
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TABLE S2
Strains, plasmids, and primers used in this study. Download Table S2, XLSX file, 0.02 MB.
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