Breaking the Salinity-Nitrogen Fixation Trade-Off: Engineering A Synthetic Nitrogen-Fixing Vibrio natriegens Strain

20 Vibrio natriegens, nitrogen fixation, nitrogen metabolism, saline stress, soybean 21 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint INTRDUCTION 22 Soil salinization is a major environmental stress that impairs plant growth and reduces crop 23 yield, affecting approximately 400 million hectares worldwide, including 40% of irrigated land.1 24 In legumes, saline stress suppresses rhizosphere diazotroph activity,2 impairs rhizobia infection, 25 inhibits nodule formation,3 and reduces nitrogenase activity,4 collectively diminishing biological 26 nitrogen fixation. Enhancing nitrogen fixation under saline stress is crucial for improving 27 agricultural productivity and addressing food security, yet excessive chemical fertilizer use 28 exacerbates soil salinization and environmental degradation.5 29 In addition to accessing soil nitrogen for growth, legumes can acquire fixed nitrogen through 30 symbiosis with bacteria hosted in root organs known as nodules.6 However, conventional 31 symbiotic nitrogen fixation is limited by host specificity and saline stress inhibition. Synthetic 32 biology offers a powerful solution to improve nitrogen fixation under saline stress. Since Dixon's 33 groundbreaking transfer of the K. oxytoca nitrogen fixation (nif) gene cluster to E. coli in 1972,7 34 precise cross-species transplantation of nif gene clusters has been achieved.8,9,10 Gene-editing 35 tools now allow the integration of nif gene clusters into halotolerant bacterial chassis, creating 36 saline-tolerant nitrogen-fixing engineered bacteria as a sustainable alternative to chemical 37 fertilizers. 38 As a synthetic biology platform, Vibrio natriegens exhibits rapid growth, high halotolerance, and 39 a broad substrate spectrum, making it an ideal chassis for nitrogen fixation.11,12 With ribosomal 40 densities reaching 115,000 per cell in the logarithmic phase, V. natriegens has significant 41 potential for heterologous protein expression.13 Recent advances in genetic tools (e.g., 42 SWAPnDROP,14 INTIMATE15 and NT-CRISPR,16,17 have expanded their engineering 43 capabilities, yet their role in plant growth promotion and nodulation under saline stress remains 44 unexplored. 45 Here, we engineered V. natriegens into a multifunctional plant growth-promoting bacteria 46 (PGPB), which can enhance plant growth and nitrogen assimilation by reprogramming metabolic 47 signaling and activating host-microbiome interactions. Concomitantly, engineered nitrogen-48 fixing V. natriegens (ENF V. natriegens) upregulated flavonoids biosynthesis in soybean roots, 49 which modulated the expression of nitrogen metabolism genes and symbiotic nitrogen fixation 50 genes, ultimately boosting soybean nitrogen-fixing capacity under saline stress. 51 52

53 Engineering and Functional Characterization of ENF V. natriegens 54 Native nif gene clusters exhibit complex phylogenetic distributions, necessitating the 55 identification of core functional cluster elements. Our engineering strategy prioritized 56 evolutionarily stable nif clusters from Enterobacterales, Bacillales, Pseudomonadales, and 57 Rhizobiales. Redundant genomic regions (e.g., Pst1307-Pst1312 in Pseudomonas stutzeri 58 A1501) were excised,18 while essential electron transport components (rnf/fix from Azotobacter 59 vinelandii DJ) and molybdate transporter genes (mod) were incorporated (Fig. 1A).19 The nif 60 clusters (13-65 kb) from Paenibacillus polymyxa WLY78, Klebsiella oxytoca M5al, Azotobacter 61 vinelandii DJ, and Pseudomonas stutzeri A1501 were constructed via de novo DNA synthesis or 62 PCR amplification, assembled in yeast, and cloned into host-compatible plasmids. Each gene 63 cluster (13-65 kb) was separately integrated into the chr2_297 site of V. natriegens (Vmax) using 64 multiplex genome editing by optimized INTIMATE method, generating ENF V. natriegens 65 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint 3 strains W1, W2, W34, and W78 (Fig. S1). Additionally, a nif gene cluster knockout mutant 66 (Δnif) was generated in the wild type V. natriegens (VCOD-2) as a control (Fig. S2).15 67 68 To assess whether these heterologous nif clusters were functional in V. natriegens, we evaluated 69 growth dynamics and nitrogenase activity across ENF V. natriegens. Under aerobic conditions, 70 no discernible differences in growth rates were observed, although the Δnif mutant displayed 71 slightly improved growth, suggesting a metabolic burden associated with nif cluster 72 maintenance. However, under anaerobic conditions, ENF V. natriegens strains exhibited 73 improved growth relative to wild type V. natriegens (Fig. 1B). Nitrogenase activity 74 measurements followed a similar trend, confirming that heterologous nif clusters enhanced 75 functional nitrogen fixation capability (Fig. 1C). 76 77 To investigate the nitrogen metabolic reprogramming induced by nif cluster integration, we 78 performed transcriptome analyses under three growth conditions: aerobic with nitrogen 79 supplementation (YYYN), anaerobic without nitrogen (WYWN), and anaerobic with nitrogen 80 supplementation (WYYN). Gene expression profiles displayed marked divergence across these 81 conditions (Fig. S3A). Principal component analysis (PCA) revealed high intra-group 82 consistency and clear inter-group separation (Fig. S3B) (ADONIS: YYYN R² = 0.91, p = 0.001; 83 WYWN R² = 0.84, p = 0.001; WYYN R² = 0.87, p = 0.001), identifying nitrogen availability as a 84 key regulator of native nif cluster expression (Fig. S4). Subsequent qPCR analysis confirmed that 85 heterologous nif clusters were actively expressed in all ENF V. natriegens strains (Fig. S5). 86 87 Interestingly, we observed that the integration of K. oxytoca M5al nif clusters upregulated 88 endogenous nif gene expression in V. natriegens, while A. vinelandii DJ and P. stutzeri A1501 89 nif cluster insertions downregulated native nif expression (Fig. 1D). This regulatory divergence 90 likely reflects the differing aerobic and anaerobic adaptations of donor organisms, which 91 differentially modulate nitrogen metabolic pathways in V. natriegens. To further elucidate how 92 heterologous nif clusters influence nitrogen metabolism, we conducted a comparative 93 transcriptomic profiling focusing on nitrogen-associated pathways. Notably, in W2, genes 94 involved in dissimilatory and assimilatory nitrate reduction were downregulated relative to other 95 variants. In contrast, W34 exhibited marked upregulation of glutamate metabolism regulators 96 (gltBD, gdh, glnA), carbamate kinase (arcC) and carbonic anhydrase (cah) gene (Fig. 1E). 97 Meanwhile, expression of amino acid biosynthesis genes was increased (Fig. S6). These findings 98 demonstrate that native nif integration enhances both nitrogen fixation capacity and nitrogen 99 metabolic flux in ENF V. natriegens. 100 101 Growth promotion of soybean by ENF V. natriegens under saline stress 102 We observed that the integration of the nif gene cluster in ENF V. natriegens modulated the 103 expression of genes associated with chemotaxis and cell motility (Fig. S7), which are critical for 104 root colonization capacity in bacteria.20 Specifically, nif gene cluster integration in W34 105 upregulated plant growth-promoting genes (Fig. S8) and markedly improved the secretion of 106 phytohormones, including indole-3-acetic acid (IAA) and siderophores (Fig. S9). These findings 107 aligned with soybean seed germination results under saline stress (Fig. S10). Pot experiments 108 further confirmed that inoculation with W34 and W78 significantly promoted soybean seedling 109 growth under saline stress (Fig. S11). 110 111 Given that V. natriegens lacks nodulation genes, we hypothesized that co-inoculation with 112 Bradyrhizobium diazoefficiens USDA110 could enhance root development in legumes.21 ENF V. 113 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint natriegens W34/W78 conducted pot experiments were selected (Fig. 2A). Under 500 mM NaCl 114 condition, negative control plants (non-inoculated) displayed severe saline stress phenotypes, 115 including leaf curling and wilting. Soybeans inoculated with USDA110 alone exhibited 116 substantial growth inhibition, with similar suppression observed in the WT+USDA110 group. In 117 contrast, experimental groups co-inoculated with ENF V. natriegens (W34/W78) exhibited 118 robust growth (Fig. 2B), with shoot fresh weight increasing by 102.7-284.6%, dry weight 119 elevating by 146-275.7%, and plant height improving by 87-180% relative to the WT+USDA110 120 group (Fig. 2C-E). 121 122 Root system development was also significantly enhanced under saline stress in plants inoculated 123 with ENF V. natriegens (Fig. 2F). The W34+USDA110 group exhibited a 90.5-160.9% increase 124 in root fresh weight, a 145.1-229.9 % elevation in dry weight, and a 61.9-126.9% extension in 125 root length compared to WT+USDA110 group (Fig. 2G-I). Additionally, chlorophyll content 126 (SPAD) and photosynthetic rates were significantly enhanced in the ENF V. natriegens co-127 inoculation group (Fig. 2J, K). These results demonstrate that ENF V. natriegens mitigates saline 128 stress in soybeans, enhances photosynthetic efficiency and improves soybean productivity. 129 130 ENF V. natriegens enhances soybean nodulation and nitrogenous compound accumulation 131 Nodule nitrogenase activity, nodule number, and nodule fresh weight are key metrics for 132 assessing symbiotic nitrogen fixation in legumes. Under saline stress, only a very limited number 133 of root nodules were observed in negative controls or soybean inoculated with USDA110 alone. 134 However, soybean inoculated with ENF V. natriegens exhibited significantly enhanced nodule 135 formation and nitrogenase activity (Fig. 3A). The W34-inoculated group developed an average 136 of 45 nodules with a total biomass of 1.05 g, representing 10.3-fold increases in nodule number 137 and 8.6-fold enhancements in nodule biomass compared to the WT+USDA110 group. These 138 increases were accompanied by a 5.25-fold elevation in nitrogenase activity (Fig. 3B-D). 139 Paraffin sectioning further confirmed the beneficial effects of ENF V. natriegens on nodule 140 growth and biological nitrogen fixation. Nodules from the experimental groups inoculated with 141 ENF V. natriegens were deep blue, with round and fully developed infected cells and a uniform 142 distribution of rhizobia, indicative of high metabolic activity. These characteristics were 143 comparable to those observed in the positive control group (+ Control). In contrast, nodules from 144 the WT+USDA110 group exhibited smaller infected cell areas and an uneven distribution of 145 rhizobia (Fig. 3A). These findings suggest that ENF V. natriegens enhances soybean growth by 146 promoting nodulation and nitrogenase activity under saline stress. 147 148 Nitrogenous compounds, including nitrate nitrogen, ammonium nitrogen, and ureides, play 149 essential roles in soybean nitrogen metabolism by enhancing carbon utilization efficiency and 150 facilitating amino acid assimilation.22 As such, their accumulation levels serve as direct 151 indicators of nitrogen fixation capability. Under saline stress, negative control plants exhibited 152 significantly lower nitrogen compound concentrations than those inoculated with ENF V. 153 natriegens. The W34+USDA110 group showed a 1.16- to 1.27-fold increase in nitrate nitrogen, 154 a 1.5- to 2.2-fold elevation in ammonium nitrogen, and a 1.96- to 2.38-fold enhancement in 155 ureide content compared to the WT+USDA110 group (Fig. 3E), suggesting that ENF V. 156 natriegens contribute to enhanced nitrogen compound accumulation under saline stress. 157 158 Soil inorganic nitrogen serves as a key plant-available nutrient, with nitrite reductase activity 159 directly reflecting soil nitrogen transformation efficiency. The highest nitrite reductase activity 160 was observed in the W34+USDA110 group, showing a 1.3-fold increase relative to the 161 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint 5 USDA110 mono-inoculated group and a 1.08-fold increase compared to the WT+USDA110 162 group (Fig. 3F), suggesting that W34 exhibits higher nitrogenase activity, which, in turn, 163 enhances soil nitrite reductase activity. Furthermore, soybean plants inoculated with ENF V. 164 natriegens exhibited reduced saline-induced cellular damage, as evidenced by increased 165 flavonoid exudation (Fig. 3G) and diminished malondialdehyde (MDA) accumulation (Fig. 3H). 166 Mantel test revealed a statistically significant correlation between V. natriegens abundance in 167 soil and increased ammonium/nitrate nitrogen levels and flavonoid (p < 0.05) (Fig. 3I). These 168

confirm that ENF V. natriegens promotes soybean nitrogen fixation through enhanced 169 nodulation and increased nitrogenous compound accumulation under saline stress. 170 171 ENF V. natriegens activated nitrogen fixation capability of rhizosphere bacteria 172 To investigate how ENF V. natriegens enhances nitrogen fixation in soybeans, we investigated 173 its interaction with soybean roots. GFP-tagged V. natriegens maintained a stable rhizosphere 174 population density of 3× 10³ CFU/g for 10 days post-inoculation (dpi) (Fig. S12). The 175 stereomicroscopic analysis confirmed surface colonization of soybean roots by GFP-tagged V. 176 natriegens (Fig. S13). However, confocal laser scanning microscopy detected root 177 autofluorescence in the epidermal layers and vascular bundles (Fig. S14), indicating limited 178 endophytic colonization capacity of V. natriegens despite stable rhizosphere persistence under 179 saline stress. 180 181 Given its colonization on root surfaces and in saline soils, we hypothesized that V. natriegens 182 might influence soybean nitrogen fixation by regulating the rhizosphere bacteria. To test this, we 183 analyzed bacterial community composition at three time points (0 dpi, 14 dpi, and 28 dpi) using 184 Illumina MiSeq sequencing of the V5-V7 region of the 16S rRNA gene. Alpha diversity analysis 185 showed the Chao1 index significantly increased (p < 0.01) in the ENF V. natriegens groups (Fig. 186 S15A). Principal Coordinate Analysis (PCoA) based on Bray-Curtis dissimilarity matrices 187 further revealed significant shifts in microbial community composition at both 14 dpi (ADONIS: 188 R2 = 0.9795, p = 0.001) and 28 dpi (ADONIS: R2 = 0.9727, p = 0.001) (Fig. S15B). These 189 findings suggest that ENF V. natriegens play a role in shaping the rhizosphere bacteria. To 190 further explore the effect of V. natriegens on the rhizosphere bacteria, we analyzed the genus-191 level compositional profiles. Inoculation with ENF V. natriegens led to a significant increase in 192 the relative abundance of nitrogen-fixing bacteria, including Rhizobium, Bradyrhizobium, and 193 Paraburkholderia (Fig. S15C). 194 195 Functional annotation using the Ncyc database revealed a progressive increase in nitrogen cycle 196 gene abundance. Rhizosphere soils inoculated with W34 exhibited elevated nifHDK gene copy 197 numbers and upregulated expression of genes associated with nitrification, nitrogen fixation, and 198 nitrate reduction pathway (Fig. S15D). Taxonomic annotation of nitrogen-fixation-associated 199 genes in W34-treated rhizosphere soil, based on metagenomic assembled ORFs, showed distinct 200 diversity and distribution patterns. The nifHDK genes were predominantly expressed in 201 Burkholderiales, Hyphomicrobiales, Rhodospirillales and Sphingomonadales, with high 202 annotation proportions at the phylum (96%), class (94%), and order (78%) levels (Fig. S15E). 203 These results demonstrate that ENF V. natriegens increases rhizosphere biodiversity and 204 enhances nitrogen fixation efficiency of rhizosphere bacteria. 205 206 Next, we hypothesize that ENF V. natriegens affects the rhizosphere species composition by 207 stimulating the secretion of metabolites from soybean roots. To identify key metabolite factors in 208 soybeans influenced by ENF V. natriegens W34. Weighted Gene Co-expression Network 209 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint Analysis (WGCNA) was used to integrate differentially accumulated metabolites (DAMs) from 210 soybean roots with rhizosphere bacteria abundance profiles. The relative abundance of V. 211 natriegens displayed the highest connectivity with the M-antiquewhite4, which was selected for 212 further analysis (Fig. S16A, B). Using filtering criteria of R² > 0.6 and p 0.8) and microbial 214 significance (GS > 0.5) (Fig. S16C). KEGG (Kyoto Encyclopedia of Genes and Genomes) 215 pathway analysis revealed that metabolites associated with V. natriegens were enriched in 216 isoflavonoid biosynthesis, phenylpropanoid biosynthesis, flavone and flavonol biosynthesis, and 217 flavonoid biosynthesis pathways (Fig. S16D). Paraburholderia, a nitrogen-fixing bacteria with 218 increased relative abundance in ENF V. natriegens-treated rhizosphere soil, exhibited significant 219 positive correlations (p < 0.05) with coniferyl aldehyde (MN9636), trans-cinnamate (MP2946), 220 and apigenin (MN882) (Fig. S17). These finding suggest that ENF V. natriegens enhances the 221 abundance of rhizosphere nitrogen-fixing bacteria by stimulating flavonoid metabolites secretion 222 from soybean roots. 223 224

225 Nitrogen is a key element for microbial and plant growth, thereby mediating plant resistance to 226 abiotic stress.28 In this study, we demonstrated that ENF V. natriegens carrying the nif gene 227 cluster from A. vinelandii DJ enhanced rhizosphere nitrogen fixation by stimulating flavonoid 228 secretion. Concomitantly, ENF V. natriegens upregulated neohesperidin biosynthesis in soybean 229 roots, which modulated the expression of nitrogen metabolism genes and symbiotic nitrogen 230 fixation genes, ultimately boosting soybean nitrogen-fixing capacity under saline stress. 231 232 Naturally occurring nitrogen-fixing genes are typically clustered, with sizes ranging from 13 kb 233 in P. polymyxa WLY78 to 65 kb in P. stutzeri A1501. Larger gene clusters tend to encode 234 bioactive nitrogenase, electron transport chains, and oxygen protection mechanisms,29 allowing 235 for more effective nitrogen fixation under diverse conditions. However, transferring nif gene 236 clusters across species using inducible regulatory systems presents challenges in real-world 237 applications due to safety concerns, unpredictable induction timing, and unstable nitrogenase 238 expression.30,31 To overcome these limitations, four nif gene clusters with high heterologous 239 nitrogenase activity in E. coli were selected: those from P. polymyxa WLY78, K. oxytoca M5al, 240 A. vinelandii DJ, and P. stutzeri A1501.32 Compared to P. polymyxa WLY78 and K. oxytoca 241 M5al, A. vinelandii DJ contain longer nif clusters (65 kb), which may contribute to their 242 enhanced functionality (Fig. 1A). Although all four clusters conferred nitrogen fixation 243 capability, the W34 carrying A. vinelandii DJ gene clusters, demonstrated superior growth rates 244 and nitrogenase activity (Fig. 1B, C), which was consistent with previous research.32 As obligate 245 aerobes that fix nitrogen under ambient conditions, A. vinelandii DJ demonstrate improved 246 oxygen resilience, facilitating nitrogen fixation in soil environments.33,34 247 248 Notably, the A. vinelandii DJ gene cluster enhances the expression of upstream glutamate 249 metabolism genes (gltBD, gdh, glnA) in ENF V. natriegens W34(Fig. 1E). Glutamine synthetase 250 (GS) and glutamate synthase (GOGAT) constitute the core pathway of microbial nitrogen 251 assimilation. Improving the efficiency of the GS/GOGAT cycle is considered an effective 252 approach to enhance nitrogen utilization efficiency,35,36 thereby supplying carbon skeletons for 253 the TCA cycle and maintaining the carbon-nitrogen balance in the bacteria.37 Since GS is a key 254 enzyme for ammonium assimilation in soybean, its overexpression is known to enhance plant 255 nitrogen use efficiency.38,39 Inoculation with ENF V. natriegens W34 significantly upregulated 256 glutamine synthetase (GS) genes (SoyZH13_14G150200, SoyZH13_19G060300) in soybean, 257 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint 7 with expression levels exceeding those in other treatment groups at both 14 and 28 dpi (Fig. 258 S18). Consistent with these findings, the relative abundance of ENF V. natriegens showed a 259 positive correlation with SoyZH13_06G10900 (K10534: nitrate reductase) and 260 SoyZH13_08G267800 (K02575: nitrate transporter) (Fig. S19), suggesting that ENF V. 261 natriegens plays a critical role in enhancing soybean nitrogen metabolism efficiency. 262 263 Synthetic biology presents novel solutions for sustainable agriculture. In this study, ENF V. 264 natriegens maintained its growth-promoting effects under 500 mM NaCl stress (Fig. S20), 265 whereas naturally saline-tolerant PGPBs and synthetic microbial communities (SynComs) 266 exhibited significant growth-promoting effects only at NaCl concentrations ≤ 200 mM (table. 267 S4). Consistent results were obtained from peanut pot trials under 500 mM NaCl stress (Fig. 268 S21), demonstrating the broad applicability of this approach in sustainable agriculture. LefSe 269 analysis identified microbial species differences across treatments.40 At 14 dpi, major species 270 shifts were observed between the positive and negative control groups, likely due to saline-271 induced changes in the rhizosphere community. However, species differences expanded across 272 all treatment groups, with Vibrio, Soliminas, and Verrucomicrobiaceae dominating in the 273 W34+USDA110 group at 28 dpi (Fig. S22). This suggests that W34 is a broad-host-range 274 nitrogen-fixing bacteria and establishes a stable presence in the rhizosphere. 275 276 This study establishes a synthetic biology framework for engineering next-generation 277 multifunctional plant growth-promoting bacteria (PGPB). It also provides insights into how ENF 278 V. natriegens enhances soybean growth and nitrogen fixation under saline stress. These 279 discoveries serve as a foundation for advancing precision agriculture through synthetic biology-280 driven microbial engineering, offering a sustainable and transformative approach to improving 281 crop stress resilience in saline farmland ecosystems. 282 283 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint

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Plant, Cell & Environment 24, 141 –153. https://doi.org/10.1111/j.1365 -458 3040.2001.00669.x. 459 460 Acknowledgments: 461 Funding: 462 National Key Research and Development Program (China) 2021YFA0909500 463 National Natural Science Foundation (China) 32030004 464 National Natural Science Foundation (China) 32370106 465 National Natural Science Foundation (China) U22A20444 466 Shanghai Municipal Science and Technology Major Project 467 Author contributions: 468 Conceptualization: Hongzhi Tang, Wanjing Wu 469 Methodology: Wanjing Wu 470 Investigation: Wanjing Wu 471 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint 13 Visualization: Wanjing Wu 472 Funding acquisition: Hongzhi Tang, 473 Project administration: Hongzhi Tang 474 Supervision: Hongzhi Tang 475 Writing – original draft: Wanjing Wu 476 Writing – review & editing: Wanjing Wu 477 Declaration of interests: 478 The authors declare that they have no competing interests. 479 Data and materials availability: 480 The clean reads of RNA-seq in this paper have been deposited in the SRA database (SRA 481 Accession No.: PRJNA1255886, PRJNA1256108, PRJNA1256128). The raw metabolomics 482 data reported in this paper have been deposited in the MTBLS, MetaboLights 483 (https://www.ebi.ac.uk/metabolights/; Accession No.: REQ20250427210173). All other data 484 are included in the manuscript and/or supporting information. 485 Supplementary Materials 486

487 Figures. S1 to S28 488 Tables. S1 to S7 489 Table S2. Excel file containing additional data too large to fit in a PDF 490 Table S3. Excel file containing additional data too large to fit in a PDF 491

(52-80) 492 493 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint FIGURE LEGENDS 494 Fig. 1 Engineering and functional characterization of ENF V. natriegens. 495 (A) Alignment of four nitrogen fixation (nif) gene clusters from free-living nitrogen-fixing 496 bacteria based on 16S rRNA phylogenetic relationships. Genes are color-coded by function and 497 operon type: red, structural components; purple, cofactor biosynthesis; blue, electron transport; 498 green, regulatory genes; orange, rnf and fix operons; and grey, unknown nitrogen fixation-related 499 genes. Dots on the DNA line indicate where multiple genomic regions were cloned and 500 combined to form a single plasmid-borne nif cluster. The boundaries of all of the nif clusters are 501 provided in table. S1, and a complete list of strain genotypes is in table. S2 502 (B) Growth curves of different ENF V. natriegens strains under aerobic and anaerobic 503 conditions. Data are presented as the mean ± SD from three biological replicates (*p < 0.05, **p 504 < 0.01, Student’s t-test). 505 (C) Nitrogenase activity of V. natriegens strains carrying native nif clusters. Data are presented 506 as mean ± SD from three biological replicates, with error bars indicating standard deviations (*p 507 < 0.05, **p < 0.01, ***p < 0.001). 508 (D) Effect of different exogenous nif gene clusters on endogenous nif gene expression in V. 509 natriegens under different culture conditions. Bubble size represents gene expression levels; 510 bubble color indicates different strain types; rectangle color denotes culture conditions. 511 (E) Expression of genes associated with nitrogen metabolism pathways in different ENF V. 512 natriegens. Compared to wild type V. natriegens, ENF V. natriegens W34 shows upregulated 513 expression of genes associated with the upstream glutamate metabolic pathway. Green highlights 514 indicate relevant pathways and red boxes denote associated genes. 515 516 Fig. 2 Growth promotion of soybean by ENF V. natriegens under saline stress. 517 (A) Schematic representation of the experimental design. Soybeans were divided into six 518 treatment groups: (1) positive control group (+Control), normal culture conditions without saline 519 or bacterial inoculation; (2) negative control (-Control), saline-stressed (500 mM NaCl) without 520 bacterial inoculation; (3) Bradyrhizobium diazoefficiens USDA110 only; (4) wild type V. 521 natriegens WT + USDA110; (5) ENF V. natriegens W34 + USDA110; (6) ENF V. natriegens 522 W78 + USDA110. Root tissues and rhizosphere soil were collected at 0, 14, and 28 dpi for 523 untargeted metabolomics, transcriptomics, and metagenomic analyses (n = 10 biologically 524 independent samples). Created with BioRender.com. 525 (B) Soybean shoot phenotype under 500 mM saline stress. Scale bars = 12 cm. 526 (C, D, E) Plant height (C), plant fresh weight (D), and dry weight (E) under 500 mM saline 527 stress. 528 (F) Soybean root phenotype under 500 mM saline stress. Scale bars = 4 cm. 529 (G, H, I) Root length (G), root fresh weight (H), and root dry weight (I) under 500 mM saline 530 stress. 531 (J) Plant photosynthetic rate. (K) Plant chlorophyll (SPAD). 532 For boxplot: the center line represents the media, the box bounds represent the lower (Q1) and 533 upper (Q3) quartiles, and the whiskers indicate the minimum and maximum values. Data are 534 presented as mean ± SD (n = 10). Error bars represent standard deviations of ten replicates (*p < 535 0.05, **p < 0.01, ***p < 0.001, Student’s t-test. 536 537 Fig. 3 ENF V. natriegens enhances soybean nodulation and nitrogenous compound 538 accumulation. 539 (A) Effect of different ENF V. natriegens inoculations on soybean nodulation phenotypes under 540 500 mM saline stress. Scale bars = 1 cm. The color of root nodule paraffin sections indicates the 541 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint 15 presence of living cells within the root nodules. Dark blue staining represents high metabolic 542 activity of rhizobia and well-developed nodules. Scale bars = 500 μm. 543 (B, C, D) Nodule number (B), nodule weight (C), and nitrogenase activity (D) of soybean under 544 500 mM saline stress across treatments. Data are presented as mean ± SD (n = 10). Statistical 545 significance was determined using Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). 546 (E) Accumulation of nitrogen-containing compounds in the roots and leaves of soybeans under 547 500 mM saline stress across treatments. 548 (F) Nitrate reductase activity in the rhizosphere soil of soybeans under 500 mM saline stress 549 across treatments. Data are presented as mean ± SD (n = 3). Statistical significance was 550 determined using Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). 551 (G, H) Malondialdehyde (MDA) concentration (G) and flavonoid content (H) of soybeans under 552 500 mM saline stress across treatments. Data are presented as mean ± SD (n = 3). Statistical 553 significance was determined using Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). The 554 boxplot displays the median at the center, with the box bounds representing the lower (Q1) and 555 upper (Q3) quartiles. The whiskers indicate the minimum and maximum values. 556 (I) Relationship between ENF V. natriegens abundance and nitrogen-containing compounds in 557 soybean, analyzed using Mantel tests. Pearson’s correlations were used to assess the 558 relationships between ENF V. natriegens abundance and nitrogenous compounds (nitrate 559 nitrogen, ammonium nitrogen, ureide, flavonoids, MDA, and soil nitrate reductase activity). 560 Asterisks indicate the significance levels of correlations. The width of the lines represents the 561 magnitude of the absolute value of Mantel's r, while the color of the lines corresponds to the p-562 value significance range (*p < 0.05, **p < 0.01, ***p < 0.001). 563 564 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint 565 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint 17 566 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint 567 568 569 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2025. ; https://doi.org/10.1101/2025.07.16.665069doi: bioRxiv preprint