Engineering a Root-Associated Bacterium Pseudomonas stutzeri  A1501 with Nitrogen Fixation and Phosphate Solubilization Activities for Enhanced Growth of Host Rice

Engineering a Root-Associated Bacterium Pseudomonas stutzeri A1501 with Nitrogen Fixation and Phosphate Solubilization Activities for Enhanced Growth of Host Rice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Engineering a Root-Associated Bacterium Pseudomonas stutzeri A1501 with Nitrogen Fixation and Phosphate Solubilization Activities for Enhanced Growth of Host Rice Changyan Yin, Dongqi Wang, Yaoyao Liu, Yuhua Zhan, Wei Lu, Haichao Feng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9387281/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Aims Both nitrogen (N) and phosphorus (P) are essential nutrients for plant growth but is often limiting in high yielding agricultural production systems. This study investigated the synergistic effect of nitrogen fixation and organic phosphate mineralization by rhizospheric microorganisms, thereby promoting the growth of host plants. Methods After assessing phosphate-solubilizing activities of five Pseudomonas strains and identifying putative phosphatase genes in silico , selected genes were expressed in nitrogen-fixing P. stutzeri A1501, and the resulting recombinants were evaluated for growth, nitrogenase activity, organic P solubilization (pure and rice co-culture), root colonization, and rice growth-promotion under different N regimes. Results Four phosphatase genes were introduced into A1501 and the resulted recombinant strains displayed significantly elevated extracellular phosphatase activity. Three recombinant strains, i.e. A15PAALP1, A15PAALP2, and A15NapA, exhibited enhanced nitrogenase activity, while six recombinant strains displayed reduced nitrogenase activity. Notably, nitrogenase activity of A15NapA was approximately 7500 nmol ethylene mg − 1 protein h − 1 , representing an obvious increase of 17%. Lecithin solubilization assay indicated that A1510ACP increased the concentration of available phosphorus by 10.98% relative to A1501. In a rice co-culture system, A1510ACP displayed superior root colonization ability, while enhanced hydrolysis of organic phosphorus increased available phosphorus levels were observed compared to A1501. Pot experiments demonstrated that inoculation with A1510ACP significantly promoted rice growth, particularly under nitrogen-supplemented conditions. Conclusion This study presents the first successful example of engineering a phosphate-solubilizing and nitrogen-fixing strain for enhanced growth of host rice, highlighting the potential of multifunctional engineered strains as the new generation of biological fertilizers. Pseudomonas stutzeri A1501 Genetic engineering Nitrogen fixation Phosphate-solubilization Phosphatase Host rice Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Global food demand is rising rapidly, with particularly pronounced needs in developing countries (Ashfaq et al. 2023 ; Elhaissoufi et al. 2022 ). Rice, wheat, and maize represent the world's most crucial food crops, whose production has played a pivotal role in sustaining global population growth and mitigating food crises (Seck et al. 2012 ; Shiferaw et al. 2011 ). In China, rice is the most extensively cultivated cereal crop, and enhancing its productivity is critical to meeting the rising domestic demand (Fan et al. 2022 ; Peng et al. 2009 ). Nitrogen (N) and phosphorus (P) are the two most limiting nutrients for crop productivity, and their availability directly influences rice yield. Fertilizer applications have been estimated to increase rice production by approximately 30–50% and 10–15% due to N and P supplementation, respectively (Igiehon and Babalola 2018 ). Phosphorus is essential for crop growth, accounting for approximately 0.2% of plant dry weight and participating in key physiological processes including biosynthesis, photosynthesis, and symbiotic nitrogen fixation in legumes (Elhaissoufi et al. 2022 ). Despite its abundance in many soils, phosphorus is often present in forms unavailable to plants. Only inorganic orthophosphate (Pi) can be directly assimilated, whereas organophosphorus compounds accounting for 30–65% of total soil phosphorus, remain largely inaccessible (George et al. 2018 ). These organic pools primarily consist of monophosphate esters, di-phosphoesters (up to 90%), and phytates (up to 50%) (George et al. 2018 ; Lidbury et al. 2017 ). Consequently, improving the mobilization and uptake of organic phosphorus represents a critical strategy for alleviating phosphorus deficiency in crops (Alori et al. 2017 ). The coupled cycling of N and P is fundamental to ecological processes (Jiao et al. 2026 ). Previous research has demonstrated that long-term nitrogen addition changes phosphorus availability and reshapes phosphate-solubilizing bacterial community, suggesting that key N and P cycling processes could be mediated by microorganisms (Zhou et al. 2025 ). Metagenomic approaches were used to investigate microbially-driven phosphorus cycling and its coupling mechanisms with nitrogen cycling, indicating the effect of N availability on P availability via changing microbial structure and stimulating microbial growth and activity (Liu et al. 2025 ). While it is well established that soil microbes significantly mediate both N and P cycling processes, the specific microbial interactions underlying these two nutrient cycles, as well as the synergistic effect of nitrogen fixation and phosphate solubilization on the growth of host plants remain yet poorly understood. The heavy reliance on chemical N and P fertilizers has raised environmental concerns, including greenhouse gas emissions and water eutrophication, prompting interest in sustainable agricultural alternatives. Plant growth-promoting rhizobacteria (PGPR) have emerged as promising candidates due to their capacity to enhance nutrient availability and support plant health through mechanisms such as biological nitrogen fixation, phosphate solubilization, phytohormone production, induced systemic resistance, and bioremediation (Ahmad et al. 2022 ; Igiehon and Babalola 2018 ; Rahman and Singh 2020 ; Vejan et al. 2016 ). Among PGPR, phosphate-solubilizing bacteria (PSB) improve phosphorus availability by solubilizing inorganic phosphates and mineralizing organic phosphorus compounds via the production of organic acids (which lower soil pH) and the secretion of phosphatases. Prominent PSB includes genera Pseudomonas (Peng et al. 2025 ), Bacillus (de la Paz-Osorio et al. 2025 ), Rhizobia (Nascimento et al. 2024 ), Burkholderia (Guo et al. 2025 ), and Acinetobacter , with Pseudomonas and Bacillus being particularly effective. The mineralization of organic phosphorus into plant-available Pi is primarily catalyzed by enzymes such as acid phosphatases (ACP), alkaline phosphatases (ALP), and phytases, whose genetic determinants have been widely characterized in diverse soil microorganisms (Alori et al. 2017 ; Bargaz et al. 2021 ; Rezakhani et al. 2019 ; Richardson and Simpson 2011 ). The potential of bacterial secretory phosphatases for enhancing phosphorus solubilization has been explored in several studies. Previous studies identified and cloned several key acid phosphatases, including the non-specific acid phosphatase NapD and NapE from Sinorhizobium meliloti 104A14 (Mihara et al. 2000 ), as well as the phosphate-repressible acid phosphatase PhoC and NapA from Morganella morganii (Thaller et al. 1994 ). Subsequent research demonstrated that heterologous expression of these enzymes in PGPR strains could enhance their ability to mineralize organic phosphorus. For instance, the introduction of napA or phoC gene into Burkholderia cepacia or Azospirillum spp., respectively, resulted in elevated phosphatase activity and improved phosphate solubilization (Rodríguez et al. 2006 ). These findings supported the feasibility of engineering PGPR with enhanced phosphorus-mobilizing traits for agricultural applications (Hakim et al. 2021 ; Haskett et al. 2021 ). Pseudomonas stutzeri A1501 has emerged as a promising chassis strain for such engineering efforts (Yan et al. 2008 ; Zhan et al. 2016 ). This bacterium efficiently colonizes the rice rhizosphere, exhibits nitrogen-fixing activity, and promotes plant growth (Jiang et al. 2022 ; Zhang et al. 2019 ). Its ecological adaptability and plant-beneficial properties make it an ideal candidate for further genetic improvement. The present study therefore aimed to engineer P. stutzeri A1501 for synergistic nitrogen fixation and phosphate solubilization. We successfully constructed a set of recombinant strains that simultaneously exhibited nitrogen-fixing activity and enhanced organic phosphorus mineralization. Our results demonstrated that the engineered strain displayed superior root colonization and increased available phosphorus levels in the rhizosphere, thereby significantly promoting rice growth. These results strongly suggested that engineered bacterial strains with enhanced phosphorus solubilization and nitrogen fixation capabilities would have a synergistic effect when applied to crops in the field. Materials and Methods Strains culture and determination of phosphate solubilization ability The bacterial strains used in this study included five wild-type strains of Pseudomonas genus: P. stutzeri A1501, P. stutzeri ATCC 17588, P. syringae pv. tomato DC3000, P. aeruginosa PAO1 and P. fluorescens ACCC 10190. P. stutzeri A1501, P. stutzeri ATCC 17588 and P. syringae pv. tomato DC3000 strains were preserved in this laboratory. P. aeruginosa PAO1 (accession No. P. aeruginosa ATCC 15692) and P. fluorescens ACCC 10190 (accession No. P. fluorescens ATCC 13525) strains were obtained from China Agricultural Microbial Culture Preservation Center and China General Microbial Culture Preservation Center, respectively. All strains were routinely cultured in Luria-Bertani (LB) broth. The organophosphorus-solubilizing capacity of the five Pseudomonas strains was preliminarily evaluated using a modified plate assay on PVK agar (Sanchez-Gonzalez et al. 2023 ) containing tricalcium phosphate, lecithin or phytic lecithin as the sole phosphorus source (Kalayu 2019 ). Briefly, single colonies were inoculated into LB medium and incubated at 30 ℃ for 12 h. Cells were harvested by centrifugation, washed with 0.85% (w/v) NaCl, and resuspended to an density at 600 nm (OD 600 ) of 1.0. A 10 µL aliquot of each bacterial suspension was spotted onto the center of the PVK agar plates. After 3 days incubation, the colony diameter (d) and the solubilization halo diameter (D) were measured. The phosphate solubilization index (PSI) was calculated for each replicate (n = 3) according to the following formula: PSI = (Colony diameter + Halo zone diameter)/Colony diameter Bioinformatic identification of putative secretory phosphatases To identify potential secretory phosphatases in Pseudomonas species, a bioinformatic screening pipeline was established. Putative phosphatase sequences were retrieved from the Pseudomonas Genome Database ( https://www.pseudomonas.com/ ) and functionally annotated using the National Center for Biotechnology Information (NCBI) non-redundant protein database (Winsor et al. 2016 ). The secretion potential of each candidate was evaluated by predicting the presence of signal peptides and cleavage sites using SignalP 6.0 (Teufel et al. 2022 ). Transmembrane helices were subsequently analyzed with TMHMM-2.0 to infer the likelihood of extracellular localization (Krogh et al. 2001 ). Construction of recombinant strain The target genes 10ACP , 10ALP , DCACP , PAALP1 , and PAALP2 were amplified using genomic DNA from P. fluorescens ACCC 10190, P. syringae pv. tomato DC3000, and P. aeruginosa PAO1 as templates, respectively. The amplified fragments were cloned into linearized plasmids (Vazyme Biotech Co., Ltd., China) to generate recombinant constructs. These constructs were first introduced into Escherichia coli DH5α via heat shock and subsequently mobilized into P stutzeri A1501 by tri-parental conjugation using the helper plasmid pRK2013 (Shang et al. 2021 ). All primers, strains and plasmids used in this study were listed in Table S1 and S2, respectively, along with the corresponding recombinant strain names. Antibiotics were applied at the following concentrations: 50 µg mL − 1 kanamycin and 50 µg mL − 1 tetracycline for both recombinant P. stutzeri and E. coli . Detection of phosphatase gene expression in recombinant strains To assess the expression of heterologous phosphatase genes in the recombinant strains, overnight cultures of single colonies in LB broth at 30°C were diluted with sterile 0.85% NaCl to an optical density (OD 600 =0.1) and incubated for additional 8 h under the same conditions. Total RNA was extracted from 1 mL of the bacterial culture using the RNA Easy Fast Kit (TIANGEN, China). Residual genomic DNA was removed, and cDNA was synthesized using the PrimeScript RT reagent Kit with gDNA Eraser (TAKARA, Japan). Reverse transcription qPCR (RT-qPCR) was performed using cDNA templates. Gene-specific primers (Table S3) were designed based on the corresponding genome sequences. The 16S rRNA gene was used as an internal reference to normalize the expression levels of the target genes. Growth kinetics assay A single colony of each strain was inoculated into LB broth or K medium (containing 0.4 g L − 1 KH 2 PO 4 , 0.1 g L − 1 K 2 HPO 4 , 0.1 g L − 1 NaCl, 0.2 g L − 1 MgSO 4 ·7H 2 O, 0.01 g L − 1 MnSO 4 ·H 2 O, 0.01 g L − 1 Fe 2 (SO 4 ) 3 ·H 2 O, and 0.01 g L − 1 Na 2 MoO 4 ·H 2 O, pH 6.8) supplemented with sodium lactate (50 mM) as the sole carbon source and no nitrogen source and cultured at 30°C for 12 h. The cells were harvested, washed with 0.85% NaCl, and resuspended to an OD 600 of 0.1. Aliquots of the bacterial suspension were dispensed into a 96-well microplate (Corning, USA), and growth was monitored using an automated Bioscreen Plate Reader (Labsystems, Finland). The plate was incubated at 30°C with continuous shaking, and OD 600 measurements were recorded at 2 h intervals over a 48 h period to construct growth curves. Quantitative analysis of organic phosphorus (lecithin) solubilization The organic phosphorus-solubilizing capacity of the recombinant strains was quantitatively evaluated using a modified NBRIP liquid medium containing lecithin as the sole phosphorus source. The medium composition per liter was as follows: 10 g glucose, 0.5 g (NH₄)₂SO₄, 0.3 g KCl, 0.3 g NaCl, 0.03 g MnSO₄·4H₂O, 0.03 g FeSO₄·7H₂O, 0.4 g yeast extract, and 0.4 g lecithin (pH 6.75). Bacterial strains were pre-cultured in LB medium at 30°C for 12 h, harvested by centrifugation, washed with sterile 0.85% NaCl, and adjusted to an OD 600 of 1.0. A 10 mL aliquot of bacterial suspension was inoculated into 90 mL of the NBRIP medium in a 250 mL flask, yielding an initial OD 600 of approximately 0.1. Cultures were incubated at 30°C with shaking at 220 rpm. for 5 days. Uninoculated medium served as the negative control. Samples were collected every 24 h from each biological replicate (n = 3). The culture supernatant was obtained by centrifugation at 12,000 × g for 10 min, and the soluble phosphate concentration was determined using the molybdenum blue method (King 1932 ). Determination of phosphorus-solubilizing capacity in a rice co-culture system To evaluate the ability of recombinant strains to solubilize organic phosphorus in the presence of rice plants, a hydroponic co-culture system was established. Bacterial strains were grown overnight in LB broth, harvested by centrifugation (6000 rpm., 4°C, 8 min), washed twice with 0.85% NaCl, and resuspended in sterile distilled water to an OD 600 of 1.0. Rice seedlings were pre-grown hydroponically in a nutrient solution containing lecithin (320 mg L − 1 ) as the sole phosphorus source, without nitrogen supplementation. Each culture vessel received 8 mL of the bacterial suspension, while control seedlings were treated with an equal volume of sterile nutrient solution. At 2 and 4 days post-inoculation, aliquots of the culture solution were sampled, and the soluble phosphorus content was determined using the molybdenum blue method. Rice root colonization efficiency of inoculants To evaluate the root colonization ability of the phosphate-solubilizing recombinant strains, rice seedlings were inoculated with either the recombinant strains or the wild-type P. stutzeri A1501. Surface-sterilized rice seeds were germinated on 1/2 Murashige and Skoog (MS) semi-solid agar medium in sterile flasks and cultivated in a growth chamber for one week (Yimam et al. 2025 ). The seedlings were then transferred to sterile tubes containing 72 mL of Hoagland nutrient solution supplemented with lecithin (320 mg L − 1 ) as the sole phosphorus source, without nitrogen. Each seedling was inoculated with 8 mL of bacterial suspension (OD 600 =1.0), resulting in a final OD 600 of approximately 0.1 in the culture medium. To monitor root colonization dynamics, root samples were collected weekly. Roots were placed in 10 mL of sterile 0.85% NaCl, and the attached bacteria were dislodged by vortexing. The bacterial suspensions were serially diluted and plated onto selective medium for colony enumeration. Greenhouse experiment setup A pot experiment was conducted under the greenhouse conditions to evaluate the effects of bacterial inoculation on rice plant growth under nitrogen-deficient and nitrogen-sufficient regimes, respectively. Bacterial inoculants were cultured in LB medium at 30°C for 12 h, harvested by centrifugation, and resuspended in sterile deionized water to a final concentration of 10⁸ cells mL − 1 . The growth substrate consisted of a 1:1 (w/w) mixture of diatomite and vermiculite. To eliminate residual soluble phosphorus, the substrate was thoroughly washed with deionized water and air-dried prior to use. Each pot was filled with 250 g of the prepared substrate. Rice seedlings were inoculated by applying 10 mL of bacterial suspension via soil drenching. Two fertilization treatments were established: (1) N + P treatment, in which NH 4 NO 3 (100 mg kg − 1 ) and lecithin (320 mg kg − 1 ) were supplied as nitrogen and phosphorus source, respectively); and (2) P-only treatment, in which lecithin (320 mg kg − 1 ) was provided sole phosphorus source without nitrogen supplementation. Nutrient solutions were prepared accordingly and applied as follows: an initial 100 mL of the respective nutrient solution was administered at the time of inoculation, followed by supplemental applications at weekly intervals. After 30 days of cultivation, rice seedlings were sampled. Roots were carefully cleaned, and shoot and root lengths were recorded. The dry weights of shoots and roots were determined after oven-drying to constant weight. Statistical analysis Spearman's correlation analysis was performed to evaluate pairwise relationships among the measured parameters. Differences between treatment groups were assessed using one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) post-hoc test for multiple comparisons. All analyses were conducted using SPSS software (version 31.0, IBM Corp., USA). Statistical significance was set at p < 0.05. Data were presented as means ± standard deviation (SD) from three independent biological replicates. Results Phosphate solubilization ability of selected Pseudomonas strains under different culture conditions In this study, five representative strains including P. stutzeri A1501, P. stutzeri ATCC 17588, P. syringae pv. tomato DC3000, P. aeruginosa PAO1 and P. fluorescens ACCC 10190, were evaluated for their phosphate-solubilizing capacities using plate assays on modified Pikovskaya (PVK) medium (Fig. 1 a). All five strains exhibited organophosphorus-solubilizing activity. On lecithin-supplemented medium, the phosphate solubilization indices (PSI) of the five strains were 1.26, 1.58, 2.48, 2.53, and 2.64, respectively (Fig. 1 b). The PSI values of four strains were significantly higher than that of P. stutzeri A1501, with P. syringae pv. tomato DC3000, P. aeruginosa PAO1, and P. fluorescens ACCC 10190 exhibiting 1.97-, 2.00-, and 2.09-fold increases, respectively. On phytate-containing medium, the PSI values of P. aeruginosa PAO1 and P. fluorescens ACCC 10190 were 2.00-and 1.36-fold higher than that of A1501. In contrast, only P. aeruginosa PAO1 and P. fluorescens ACCC 10190 demonstrated the ability to solubilize inorganic phosphorus in the form of tricalcium phosphate. Construction of recombinant strains expressing a heterologous phosphatase In K medium supplemented with various concentrations of phosphorus, A1501 exhibited optimal growth at a phosphorus concentration of 0.2 g/L, while its growth capacity was significantly reduced at concentrations either above or below this level (Fig. 2 a). Comparative genomic analysis revealed distinct profiles of phosphatase-encoding genes among the evaluated Pseudomonas strains. P. fluorescens ACCC 10190 carried genes encoding an acid phosphatase (ACP, B0A76_RS14940 ) and an alkaline phosphatase (ALP, B0A76_RS21295 ). P. syringae pv. tomato DC3000 contained an ACP-encoding gene ( PSPTO_3648 ), while P. aeruginosa PAO1 harbored two ALP genes ( PA0689 , PA3296 ). Notably, no homologous ACP or ALP genes were identified in the genome of P. stutzeri A1501. However, a unique phytase gene ( PST_1945 ) was present in both P. stutzeri A1501 and P. stutzeri ATCC 17588. Interestingly, the expression of PST_1945 gene was significantly upregulated in A1501 under phosphorus-limited conditions (Fig. 2 b). The predicted structural features and homology of PST_1945 were shown in Fig. 2 c and 2 d. A summary of the key phosphatases involved in organophosphorus hydrolysis across the four Pseudomonas strains was presented in Table 1 . Table 1 Distribution of the key enzymes involved in organophosphorus hydrolysis among the five Pseudomonas strains. P. stutzeri A1501 Alkaline phosphatase (ALP) Acid phosphatase (AP) Phytase - - + P. stutzeri ATCC 17588 - - + P. syringae pv. Tomato DC3000 + + + P. aeruginosa PAO1 + - - P. fluorescens ACCC 10190 + + - +: Phosphatase genes identified in the genome; -: None of phosphatase genes identified in the genome. Based on the screening described above, a total of nine phosphatases-coding genes were selected for heterologous expression in P. stutzeri A1501. These included four genes from previously characterized sources ( napD and napE from S. meliloti ; phoC and napA from M. morganii ) and five genes identified in the Pseudomonas strains analyzed in this study (Table 2 ). Signal peptide prediction confirmed that all selected phosphatases contain putative secretion signal, supporting their potential for extracellular localization and function. Nine recombinant strains were successfully constructed via tri-parental mating and designated as A1510ACP, A1510ALP, A15DCACP, A15PAALP1, A15PAALP2, A15NapD, A15NapE, A15PhoC, and A15NapA (Fig. 3 a). Transcriptional expression of the integrated phosphatase genes was confirmed by reverse transcription quantitative PCR (RT-qPCR). As shown in Fig. 3 b, all nine heterologous phosphatases genes were successfully expressed at the mRNA level in their respective recombinant strains. Table 2 Genes selected for construction of recombinant strains in this study and the predicted signal peptides of the encoded target proteins. Strain Gene origin Gene(homologous gene) Gene ID Signal peptide sequence A1510ACP P. fluorescens ACCC 10190 10ACP (phoC) B0A76_RS14940 MTEETEDPKATQNAADTLPDSSRRRFLGGVAVLGAGATLSAC A1510ALP 10ALP (phoD) B0A76_RS21295 MSHFDLGRRRVMQAVGAGLLLPGLAPAVIA A15DCACP P. syringae pv. tomato DC3000 DCACP(phoC) PSPTO_3648 MSDKPEDENTNENPGRRRFLGGMAALGAGVTLSGYVSA A15PAALP1 P. aeruginosa PAO1 PAALP1 (phoA) PA0689 MTPGYPLALSLAVSMAVLGSALPAQA A15PAALP2 PAALP2 (pstS) PA3296 MLKPMFGVVALTLLTSHTTLG A15NapD Sinorhizobium meliloti napD U74652 MFQVTRRSAMKIAALAAAASSMGQAAFA A15NapE napE AF354653 MFAIRNSLKLVRDAALAVAAGLIFAVSALA A15PhoC Morganella morganii phoC AB035805 MKKNIIAGCLFSLFSLSALA A15NapA napA X78328 MRKLTLTLSALALALSLNSVADA Organic phosphate solubilization ability of recombinant strains The capacity of P. stutzeri A1501 and its recombinant derivatives to solubilize organic phosphorus was quantitatively evaluated by measuring soluble phosphate levels in liquid medium containing lecithin as the sole phosphorus source over a 5-day incubation period. As shown in Fig. 4 , strain A15PLA (A1501 carrying the empty vector pLAFR3) showed no significant difference in lecithin solubilization compared to the wild-type strain. In contrast, heterologous expression of acid phosphatase gene 10ACP (derived from P. fluorescens ACCC 10190) and alkaline phosphatase gene PAALP1 (from P. aeruginosa PAO1) significantly enhanced soluble phosphate release, whereas their respective paralogs ( 10ALP and PAALP2 ) conferred no such improvement. Phosphatases genes originating from S. meliloti ( napD , napE ) failed to enhance solubilization activity, while those from M. morganii ( napA , phoC ) led to a significant increase. Overall, four recombinant strains, i.e. A1510ACP, A15PAALP1, A15NapA and A15PhoC, exhibited substantially enhanced organic phosphorus solubilization capacity. Root colonization and nitrogenase activities of recombinant strains To assess potential physiological impacts of heterologous gene expression, the growth performances of the engineered P. stutzeri strains were evaluated. As shown in Fig. 5 a, heterologous expression of phosphatases did not impair bacterial growth in LB medium over a 48h period. nor significantly altered nitrogenase activity compared to the wild-type control (Fig. 5 b). Besides, heterologous expression of various phosphatases positively or negatively affected nitrogenase activity of corresponding recombinant strains. As shown in Fig. 5 b, three recombinant strains, i.e. A15PAALP1, A15PAALP2, and A15NapA, exhibited enhanced nitrogenase activity, while six recombinant strains displayed significantly reduced nitrogenase activity. Markedly, nitrogenase activity of A15NapA was approximately 7500 nmol ethylene mg − 1 protein h − 1 , representing an obvious increase in nitrogenase activity of 17% compared to A1501. The root colonization dynamics of A1501 and its recombinant derivatives were monitored over a two-week period. Except for A1510ACP, all recombinant strains exhibited colonization rates comparable to the wild-type strain, with population densities generally declining over time (Fig. 5 c and d). Notably, A1510ACP maintained a stable population throughout the experimental period. At 7 and 14 days post-inoculation, the population of A1510ACP on rice roots reached 5.7 × 10 7 CFU g − 1 and 5.9 × 10 7 CFU g − 1 root fresh weight, respectively, indicating superior root colonization. These results strongly suggested that engineered bacterial strains with enhanced phosphorus solubilization and nitrogen fixation capabilities would have a synergistic effect when applied to crops. Phosphorus solubilization by recombinant strains co-cultured with rice The organic phosphorus-mineralizing capacity of the wildtype strain A1501 and recombinant strains was assessed in a hydroponic rice co-culture system with. Across all sampling time points, inoculation with bacterial strains significantly increased soluble phosphorus concentrations in the culture medium (Fig. 6 ). Among the tested strains, A1510ACP, A15PAALP1, and A15NapA exhibited the highest phosphorus-solubilizing activities compared to the wild-type strain and non-inoculated rice. At two days post-inoculation, the available phosphorus concentrations in cultures inoculated with three strains reached 2.05, 2.11 and 1.96 mg L − 1 , respectively. After four days of co-cultivation, the corresponding phosphorus levels increased further to 2.13, 2.12 and 2.37 mg L − 1 , respectively, which was 2.0-fold higher than wild-type strain A1501. These results demonstrated that the enhanced organic phosphorus solubilization capacity of the engineered strains was maintained in the presence of rice plants, highlighting their potential for improving phosphorus availability in the rhizosphere (Fig. 6 ). Promoting effects of recombinant strain inoculation on rice growth The interactive effects of nitrogen and phosphorus availability on rice development were evident, as varying levels of N and P supply significantly influenced plant growth parameters (Fig. 7 ). To evaluate the plant-growth-promoting effects of engineered strains, shoot and root lengths as well as dry weights were measured and compared between plants inoculated with recombinant strains and those inoculated with the wild-type A1501 or uninoculated. After 30 days of growth, inoculation with recombinant strains generally resulted in improved agronomic traits compared to the non-inoculated or wild-type-treated controls under both fertilization regimes (Table 3 ). Notably, under nitrogen-supplemented conditions (100 mg kg − 1 NH 4 NO 3 ) with lecithin as the sole phosphorus source (320 mg kg − 1 ), the maximum values for the shoot and root dry weight of the rice seedling inoculated with strain A1510ACP were 0.29 and 0.19 g plant − 1 , which exhibited significant increases in shoot and root dry weight by 16% and 26.6%, respectively, relative to those inoculated with the wild-type strain. These results indicated a synergistic effect of nitrogen fixation and phosphorus solubilization on plant growth. Table 3 Growth promotion of rice by recombinant strains under soil culture conditions. Treatments Lecithin (320 mg kg − 1 ) and ammonium nitrate (100 mg kg − 1 ) as P and N source, respectively Lecithin (320mg kg − 1 ) as P source, no nitrogen input Shoot length (cm) Root length (cm) Shoot Dry weight (g/plant) Root Dry weight (g/plant) Shoot length (cm) Root length (cm) Shoot Dry weight (g/plant) Root Dry weight (g/plant) non-inoculation 41.17 ± 2.12 13.00 ± 0.50 0.22 ± 0.06 0.13 ± 0.05 28.00 ± 2.30 11.50 ± 1.41 0.071 ± 0.013 0.025 ± 0.006 A1501 40.83 ± 1.70 14.33 ± 0.58 0.25 ± 0.06 0.15 ± 0.02 28.67 ± 1.44 11.25 ± 0.99 0.085 ± 0.009 0.035 ± 0.006 A1510ACP 42.08 ± 3.08 15.33 ± 0.58 0.29 ± 0.06* 0.19 ± 0.04* 29.25 ± 1.86 12.71 ± 1.60 0.087 ± 0.011 0.039 ± 0.005 A15PAALP1 42.83 ± 2.29 15.67 ± 0.76 0.25 ± 0.04 0.16 ± 0.04 29.33 ± 2.02 11.42 ± 0.99 0.087 ± 0.014 0.039 ± 0.008 A15NapA 41.83 ± 2.25 14.17 ± 0.29 0.25 ± 0.05 0.19 ± 0.04* 29.08 ± 1.51 11.83 ± 0.58 0.084 ± 0.008 0.043 ± 0.006 A15PhoC 43.58 ± 4.06 13.50 ± 0.87 0.23 ± 0.06 0.16 ± 0.05 27.00 ± 1.20 10.94 ± 0.56 0.083 ± 0.011 0.040 ± 0.005 Data are presented as mean ± standard deviation from three biological replicates. Asterisks indicate significant differences compared to the wild-type A1501 strain as determined by one-way ANOVA (*p < 0.05). Discussion Phosphorus (P) and nitrogen (N) are the most vital macronutrients for plant growth and development. Despite being extremely abundant, N 2 in air and most of total P in soils are inaccessible to plants. Soil microorganisms play a vital role in transforming soil N and P into bioavailable forms by various mechanisms, including nitrogen fixation by the enzyme nitrogenase capable of reducing atmospheric nitrogen into ammonia, and organic P mineralization by phosphatase capable of hydrolysing simple phosphate monoesters to acquire orthophosphate. Thus, it is promising to develop environmental-friendly methods to increase both N and P bioavailability by N 2 -fixing or P-solubilizing bacteria. However, the use of genetic engineering techniques to enhance the synergistic effect of nitrogen fixation and organic phosphate mineralization by microorganisms in rhizosphere environments, thereby promoting the growth of host plants, has not yet been reported. On the other hand, most naturally occurring phosphate-solubilizing strains typically exhibit only a single P-solubilization function. To overcome this limitation, approaches such as constructing synthetic microbial consortia have been explored to leverage the metabolic diversity of beneficial microorganisms. Nevertheless, these strategies remain limited in their scalability and practical applicability for field practice. Engineering nitrogen-fixing bacteria with enhanced phosphorus-solubilizing capacity offers an innovative alternative. Organic phosphorus constitutes 30% to 65% of total phosphorus in most agricultural soils and remains largely inaccessible to plants (Rodríguez et al. 2006 ). Therefore, endowing nitrogen-fixing bacteria with the ability to mineralize organic phosphates could simultaneously improve nitrogen and phosphorus availability, thereby reducing reliance on chemical fertilizers while maintaining stable crop yields. The design of our experiment was to enhance the organic phosphate-solubilizing and plant-growth-promoting capabilities of a nitrogen-fixing bacterium through genetic engineering. We successfully constructed engineered derivatives with dual functions of nitrogen fixation and organic phosphate solubilization. This modification significantly improved the phosphate-solubilizing activity of A1501 and conferred enhanced growth-promoting effects on rice. Our findings demonstrate the feasibility of engineering multifunctional biofertilizers by integrating complementary metabolic traits into a single plant-beneficial bacterial chassis (Fig. 8 ). Phosphatase gene function and construction of dual-function engineered bacteria for phosphate solubilization and nitrogen fixation Unavailable organic phosphorus in soil primarily comprises phospholipids and phytic acid. Phosphatases, including acid phosphatases (AP) and alkaline phosphatases (ALP), are key enzymes secreted by microorganisms to hydrolyze phospholipids and are predominantly distributed among γ- and α-proteobacteria. Our results demonstrated that P. stutzeri A1501 possessed a moderate ability to hydrolyze phytate but exhibits weak hydrolytic activity toward inorganic phosphorus and lecithin. Bioinformatic analysis revealed that the genome of A1501 lacked genes encoding AP or ALP, although a phytase-encoding gene ( PST_1945 ) was identified. In liquid culture, the optimal phosphorus concentration for A1501 growth was 0.2 g L − 1 (Fig. 2 a), with lower concentrations inhibiting growth. Under phosphorus-limited conditions, the expression of PST_1945 was significantly upregulated by 1.73-fold compared to normal conditions (Fig. 2 b). Further bioinformatic analysis indicated that the phytase encoded by PST_1945 shared 55.73% amino acid identity with the P. ogarae phytase WP014338283.1. Based on these findings, we hypothesize that the absence of endogenous phosphatase is the primary reason for the weak lecithin-hydrolyzing activity of A1501. Consequently, heterologous expression of phosphatases represents a key strategy for enhancing its phosphate-solubilizing capacity. Pseudomonas species are widely recognized as effective solubilizers of soil organophosphorus (Peng et al. 2025 ; Rasul et al. 2024 ), with strains such as Pseudomonas sp. WS32 and P. asiatica JP233 (Ou et al. 2022 ; Wang et al. 2022 ), albeit their hydrolytic capacities vary considerably among strains. In this study, three Pseudomonas strains (i.e. P. aeruginosa PAO1, P. fluorescens ACCC 10190 and P. syringae pv. tomato DC3000) displaying strong lecithin-hydrolyzing activity were identified. Genomic analysis revealed that P. fluorescens ACCC 10190 harbored one acid phosphatase and one alkaline phosphatase, P. syringae pv. tomato DC3000 contained one acid phosphatase, and P. aeruginosa PAO1 carried two alkaline phosphatases. Among these, PAALP1 was known as alkaline phosphatase secreted via the type II secretion system of P. aeruginosa (Ball et al. 2002 ). All five identified phosphatases contained signal peptides and were subsequently selected for heterologous expression in P. stutzeri A1501. In addition to these Pseudomonas -derived genes, we also included four phosphatases previously characterized in other genera: NapD and NapE (periplasmic acid phosphatases from Sinorhizobium meliloti ), as well as PhoC and NapA (acid phosphatases from Morganella morganii ). Introduction of napA into Burkholderia spp. has previously been shown to confer organic phosphorus-degrading capacity (Fraga et al. 2001 ). All four enzymes contain signal peptides and were therefore selected for expression in A1501. Among the nine recombinant strains constructed, four strains i.e. A1510ACP, A15PAALP1, A15NapA, and A15PhoC exhibited substantially enhanced organic phosphorus solubilization. Specifically, A1510ACP (expressing 10ACP from P. fluorescens ), A15PAALP1 (expressing PAALP1 from P. aeruginosa ), and the two M. morganii -derived phosphatase-expressing strains (A15NapA and A15PhoC) significantly increased soluble phosphorus release, with available phosphorus concentrations in the culture medium elevated by 10.9% to 43.3% compared to the wild-type strain. These increases were accompanied by detectable extracellular phosphatase activity (Fig. 4 ), confirming successful secretion of the heterologous enzymes. This finding is consistent with prior reports demonstrating enhanced extracellular phosphatase activity in E. coli and Burkholderia cepacia harboring plasmids encoding the M. morganii NapA phosphatase (Thaller et al. 1995 ). Similarly, the recombinant strain Phosphobacterium SDLiuTP02 released over 50 µmol mL − 1 soluble phosphorus after 72 h when cultured with insoluble calcium phytate as the sole phosphate source (Liu et al. 2015 ), and three phytase-expressing strains ( P. simiae , Ralstonia sp., and P. putida ) released up to 500 µM orthophosphate after 10 days (Shulse et al. 2019 ). The varying levels of phosphatase activity observed among the transformants may be attributed to several factors. First, the intrinsic catalytic efficiencies of the source phosphatases likely differ. Second, heterologous expression levels may vary within the A1501 host strain due to differences in transcriptional regulation, mRNA stability, or protein folding. Although expression of all introduced phosphatases in A1501 was confirmed at the mRNA level (Fig. 3 b), the resulting functional output is influenced by the combination of these factors. Importantly, the introduction of heterologous phosphatases did not impose a detectable metabolic burden on the host strain. Growth kinetics and root colonization capacity of the engineered strains remained comparable to those of the wild-type A1501 (Fig. 5 ). This observation aligns with previous research; for instance, introduction of a pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase gene into the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae Z67 did not adversely affect growth, biofilm formation, or exopolysaccharide secretion (Wagh et al. 2014 ). To our knowledge, this study represents the first report of an engineered bacterial strain combining both nitrogen-fixing and organic phosphate-solubilizing capabilities. Synergistic effects of nitrogen and phosphorus on plant growth promotion Nitrogen and phosphorus deficiencies severely impair crop growth and development. These two nutrients interact synergistically: nitrogen availability influences phosphorus uptake, while phosphorus status modulates nitrogen assimilation in plants (Jiang et al. 2019 ). Preliminary experimental in this study confirmed this synergistic relationship, demonstrating that combined application of nitrogen and phosphorus significantly enhanced rice plant height, shoot fresh weight, and root fresh weight compared to either nutrient applied alone (Fig. 7 ). These observations underscore the potential value of engineering nitrogen-fixing bacteria with additional phosphate-solubilizing capacity to simultaneously address both nutrient limitations. Previous studies have identified naturally occurring microorganisms possessing dual plant-growth-promoting functions. For instance, the Ensifer sp. EN1, isolated from leguminous plants, exhibited both nitrogen-fixing and phosphate-solubilizing activities (mineralizing calcium phytate and tricalcium phosphate), and inoculation experiments confirmed its ability to enhance plant phosphorus acquisition (Xu et al. 2025 ). Although other nitrogen-fixing genera including Brevundimonas , Sphingomonas , Microbacterium , and Arthrobacter , have also been reported to solubilize phytin and tricalcium phosphate, the phosphate-solubilizing capacities of these natural isolates are generally low. Current research has partially addressed this limitation by constructing synthetic microbial consortia that combine nitrogen-fixing and phosphate-solubilizing functions (Faller et al. 2024 ). In the present study, under co-cultivation with rice, the engineered phosphate-solubilizing strains A1510ACP, A15PAALP1, and A15NapA increased available phosphorus concentrations in the culture medium by 9.7%, 9.4%, and 22.1%, respectively, compared to the wild-type A1501 treatment, while maintaining nitrogenase activity comparable to the parental strain. These results demonstrate that the engineered strains successfully integrate both functions without compromising their native nitrogen-fixing capacity. Consistent with their enhanced phosphorus-solubilizing activity, the engineered strains conferred superior plant growth-promoting effects. Under both nitrogen-free and nitrogen-sufficient conditions, inoculation with either the wild-type or engineered strains increased rice shoot and root dry weights compared to non-inoculated controls, confirming that the engineered strains retain their ability to support plant growth under nitrogen-limited conditions. Notably, under nitrogen-sufficient conditions, rice seedlings inoculated with the engineered strain A1510ACP exhibited significantly greater biomass accumulation compared to those inoculated with the wild-type A1501. This suggests that under adequate nitrogen supply, the enhanced phosphate-solubilizing function of the engineered strains becomes operational, providing additional phosphorus to support plant growth. This synergistic effect of nitrogen and phosphorus is consistent with observations in forest ecosystems, where nutrient co-limitation and synergistic interactions have been widely documented (Liu et al. 2013 ). Importantly, the engineered strains display superior root colonization capacities comparable to the wild-type strain (Fig. 5 c and d). Previous studies have demonstrated that P. stutzeri A1501 effectively colonizes the rhizosphere of various crops, including maize and Pakchoi (Ke et al. 2019 ; Li et al. 2025 ). Taken together, these findings indicate that the engineered dual-function strains developed in this study possess enhanced plant growth-promoting potential and represent promising candidates for biofertilizer development to improve agricultural productivity. Conclusion In this study, we successfully constructed a series of engineered strains with significantly enhanced ability to mineralize organic phosphorus. These strains exhibited exhibit enhanced nitrogen fixation, phosphate solubilization activities, and superior root colonization, thereby significantly increasing available phosphorus levels in the rhizosphere and consequently rice growth. Furthermore, pot experiments demonstrated that inoculation with these engineered strains significantly promoted rice growth, particularly under nitrogen-sufficient conditions where the enhanced phosphorus supply contributed to increased plant biomass. This work provides insights and preliminary attempts for engineering strains capable of both nitrogen fixation and organophosphorus mineralization, offering a promising strategy for sustainable agricultural productivity. Declarations The authors have no competing interests to declare. Funding We thank the financial support for the research provided by Hainan Seed Industry Laboratory and China National Seed Group (project of ZZGS-ZNBM-2025-230), and by National Natural Science Foundation of China (32270067 and 32370091), Agricultural Science and Technology Innovation Program and the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA28030201). Author Contributions All authors contributed to the study conception and design. Xiubin Ke and Min Lin conceived the study, planned and designed the research. Changyan Yin, Dongqi Wang, Yaoyao Liu, and Haicao Feng performed the experiments. Changyan Yin analyzed the data, prepared the figures, and wrote the original manuscript with assistance from Yongliang Yan and Wei Lu. Yuhua Zhan, Wei Lu, Yongliang Yan, and Xiubin Ke reviewed and edited the manuscript. Min Lin supervised the research. All authors contributed critically to the drafts and gave final approval for publication. Data availability The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. References Ahmad A, Zafar U, Khan A et al (2022) Effectiveness of compost inoculated with phosphate solubilizing bacteria. J Appl Microbiol 133:1115–1129. https://doi.org/10.1111/jam.15633 Alori ET, Glick BR, Babalola OO (2017) Microbial phosphorus solubilization and its potential for use in sustainable agriculture. 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Environ Technol Innov 40:104353. https://doi.org/10.1016/j.eti.2025.104353 Supplementary Files SupportingInformation20260405.doc Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 15 Apr, 2026 Reviewers invited by journal 15 Apr, 2026 Editor invited by journal 14 Apr, 2026 Editor assigned by journal 13 Apr, 2026 First submitted to journal 13 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9387281","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":623693330,"identity":"5a23a327-aa3e-4ca0-b01b-e0ac76811395","order_by":0,"name":"Changyan Yin","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Changyan","middleName":"","lastName":"Yin","suffix":""},{"id":623693331,"identity":"a3682d13-7e3c-4311-9de1-5354a44eb148","order_by":1,"name":"Dongqi Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dongqi","middleName":"","lastName":"Wang","suffix":""},{"id":623693332,"identity":"910da0b9-49c0-4c8c-b6ae-39c7f89bcefc","order_by":2,"name":"Yaoyao Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yaoyao","middleName":"","lastName":"Liu","suffix":""},{"id":623693333,"identity":"db3d1f14-3b21-4fe5-857a-9d242f850c8f","order_by":3,"name":"Yuhua Zhan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yuhua","middleName":"","lastName":"Zhan","suffix":""},{"id":623693334,"identity":"097a1518-255e-4c68-987d-574a5a27aa5a","order_by":4,"name":"Wei Lu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Lu","suffix":""},{"id":623693336,"identity":"c9d239fd-ef62-44c7-9b8d-4527ac4d6d47","order_by":5,"name":"Haichao Feng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Haichao","middleName":"","lastName":"Feng","suffix":""},{"id":623693337,"identity":"50a70022-2b9e-4f43-9e0f-63228e4a2caf","order_by":6,"name":"Yongliang Yan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yongliang","middleName":"","lastName":"Yan","suffix":""},{"id":623693338,"identity":"f94b3b33-d076-4fa8-aa33-576d380f62f3","order_by":7,"name":"Xiubin Ke","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiubin","middleName":"","lastName":"Ke","suffix":""},{"id":623693339,"identity":"17ddc048-ef01-492c-92d5-022ab613668e","order_by":8,"name":"Min Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYBACPmYgkcBgkwDhshGhhQ2iJY0ULRDqMCla2HmPbni443wev3SPAcOHssMM/LMbCDmML+1G4pnbxZJzzhgwzjh3mEHizgFCWnjMbiS23U7ccCPHgJm37TCDgUQCUVrOJe4HaflLgpYDiRskgFoYSdCSnDjjRlrBwZ5z6TwSNwho4ec/Y3bzZ5tdYv+M5I0PfpRZy/HPIKAFBRwAYh4S1I+CUTAKRsEowAUAhkY/fynS8pIAAAAASUVORK5CYII=","orcid":"","institution":"Henan University","correspondingAuthor":true,"prefix":"","firstName":"Min","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2026-04-11 11:08:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9387281/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9387281/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107706401,"identity":"bc927798-c6bd-4472-8f53-cef12ca637af","added_by":"auto","created_at":"2026-04-24 09:18:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19939960,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Phosphate solubilization activity of five selected \u003cem\u003ePseudomonas \u003c/em\u003estrains on PVK plates containing tricalcium phosphate, lecithin or phytic acid as the sole phosphorus source. (b) Quantitative assessment of phosphate solubilization activity. The phosphate solubilization index (PSI) was calculated as (colony diameter + halo zone diameter) / colony diameter. Data are presented as mean ± standard deviation (n=3). Asterisks indicate significant differences compared to the wild-type strain A1501 (one-way ANOVA; *p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9387281/v1/632cd28d25a248519919fb77.png"},{"id":107631298,"identity":"a76196dd-688b-4657-9a28-085c65fbd4ca","added_by":"auto","created_at":"2026-04-23 11:43:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3515700,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Growth of \u003cem\u003eP. stutzeri\u003c/em\u003e A1501 at varying phosphorus concentrations. (b) Expression analysis of \u003cem\u003ePST_1945\u003c/em\u003e in A1501. P\u003csup\u003e+\u003c/sup\u003e (supplemented with 320 mg kg\u003csup\u003e-1\u003c/sup\u003e lecithin) and P\u003csup\u003e- \u003c/sup\u003e(no lecithin) conditions. \u0026nbsp;(c) Predicted structure of PST_1945. (d) Homologous analysis of PST_1945.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9387281/v1/8601e69932492f4bd7b00b70.png"},{"id":107707346,"identity":"7348ef68-bd80-466e-b630-3d30d39554e6","added_by":"auto","created_at":"2026-04-24 09:20:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7008522,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Construction of the recombinant plasmid pLA10ACP. (b)Transcriptional expression analysis of nine selected phosphatase genes detected by RT-PCR. M, Trans2K Plus II DNA Marker. Lanes 1, 3, 5, 7, 9, 11, 13, 15, 17: negative controls (cDNA from wild-type A1501). Lanes 2, 4, 6, 8, 10, 12, 14, 16, 18: cDNA from the corresponding recombinant strains. All amplifications were performed using gene-specific quantitative PCR primers.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9387281/v1/aca32e547dd0f48e4ff1a14e.png"},{"id":107705965,"identity":"57c46975-5edd-41bc-accf-e4a8a5e0d6ad","added_by":"auto","created_at":"2026-04-24 09:16:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1244100,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of lecithin solubilization by recombinant strains. (a-f) Soluble phosphate concentration in culture supernatants of the wild-type A1501 and recombinant strains: (a) A15PLA (A1501 carrying the empty vector pLAFR3); (b) A15DCACP; (c) A1510ACP and A1510ALP; (d) A15PAALP1 and A15PAALP2; (e) A15NapD and A15NapE; (f) A15NapA and A15PhoC. Error bars represent standard deviation (n=3). Asterisks indicate significant differences compared to the wild-type strain A1501 by one-way ANOVA (*p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9387281/v1/c9f7f03227750a71c8b5397b.png"},{"id":107869181,"identity":"b0faa497-b527-4972-899c-9ea3eb50713a","added_by":"auto","created_at":"2026-04-27 07:36:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":651036,"visible":true,"origin":"","legend":"\u003cp\u003eRoot colonization and nitrogenase activities of recombinant strains. (a) Growth kinetics of \u003cem\u003eP. stutzeri\u003c/em\u003e A1501 and recombinant strains. (b) Nitrogenase activities of strains following heterologous phosphatase expression. (c) Root colonization of rice by \u003cem\u003eP. stutzeri\u003c/em\u003e A1501 and recombinant strains at one-week post-inoculation. (d) Root colonization of rice by \u003cem\u003eP. stutzeri\u003c/em\u003e A1501 and recombinant strains at two-weeks post-inoculation. Values are presented as mean ± standard deviation (n=3).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9387281/v1/d29fced04a5cd06c900dec3b.png"},{"id":107707639,"identity":"a36bfaa4-b308-4f57-a8eb-e9d92f146c28","added_by":"auto","created_at":"2026-04-24 09:20:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":785299,"visible":true,"origin":"","legend":"\u003cp\u003eTime-course analysis of phosphate solubilization in a rice co-culture system. Soluble phosphate concentrations in the culture supernatant were measured at (a) day 2 and (b) day 4 post-inoculation. Error bars represent the standard deviation (n=3). At each time point, bars denoted with different lowercase letters indicate significant differences by one-way ANOVA (p ≤ 0.05).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9387281/v1/514f6722fad4a0e0a95f10db.png"},{"id":107706969,"identity":"48b3d0aa-9268-459b-b151-45535f13a586","added_by":"auto","created_at":"2026-04-24 09:19:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":983411,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of nitrogen and phosphorus on the growth of rice seedling (Zhonghua 11) in pot experiments. (a) Growth morphology of rice seedlings after 30 days of cultivation. (b) Plant height. (c) Plant fresh weight. (d) Root length. (e) Root fresh weight. ns, not significant. *p ≤ 0.05, **p ≤ 0.01.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9387281/v1/5848227eee950093bb117d99.png"},{"id":107707638,"identity":"deb8b2d0-9324-41f7-a2de-d02bb97dd224","added_by":"auto","created_at":"2026-04-24 09:20:48","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":356529,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of plant-microbe interactions in the rhizosphere, tentatively illustrating the synergistic effects of nitrogen and phosphorus on plant growth promotion by dual-function engineered bacteria. In the natural environments, plants interact with diverse rhizosphere microorganisms, some of which exhibit nitrogen fixation via nitrogenase or organic P mineralization via phosphatase. The red arrow and red circle highlight the engineered strains, which exhibit enhanced nitrogen fixation, phosphate solubilization activities, and superior root colonization, thereby significantly increasing available phosphorus levels in the rhizosphere and consequently rice growth.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9387281/v1/1e7fd15c021705104e621190.jpeg"},{"id":108491225,"identity":"12ad7812-d3e3-45f4-9305-7024d0afb852","added_by":"auto","created_at":"2026-05-05 09:52:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28829024,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9387281/v1/8d597eeb-2581-436a-be96-a947a30b10fc.pdf"},{"id":107706159,"identity":"4e620630-d010-4718-86bc-fefc43427af1","added_by":"auto","created_at":"2026-04-24 09:17:32","extension":"doc","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":442334,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation20260405.doc","url":"https://assets-eu.researchsquare.com/files/rs-9387281/v1/8ab8bb656f5455076a91e915.doc"}],"financialInterests":"","formattedTitle":"Engineering a Root-Associated Bacterium Pseudomonas stutzeri A1501 with Nitrogen Fixation and Phosphate Solubilization Activities for Enhanced Growth of Host Rice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlobal food demand is rising rapidly, with particularly pronounced needs in developing countries (Ashfaq et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Elhaissoufi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Rice, wheat, and maize represent the world's most crucial food crops, whose production has played a pivotal role in sustaining global population growth and mitigating food crises (Seck et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Shiferaw et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In China, rice is the most extensively cultivated cereal crop, and enhancing its productivity is critical to meeting the rising domestic demand (Fan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Peng et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Nitrogen (N) and phosphorus (P) are the two most limiting nutrients for crop productivity, and their availability directly influences rice yield. Fertilizer applications have been estimated to increase rice production by approximately 30\u0026ndash;50% and 10\u0026ndash;15% due to N and P supplementation, respectively (Igiehon and Babalola \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhosphorus is essential for crop growth, accounting for approximately 0.2% of plant dry weight and participating in key physiological processes including biosynthesis, photosynthesis, and symbiotic nitrogen fixation in legumes (Elhaissoufi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Despite its abundance in many soils, phosphorus is often present in forms unavailable to plants. Only inorganic orthophosphate (Pi) can be directly assimilated, whereas organophosphorus compounds accounting for 30\u0026ndash;65% of total soil phosphorus, remain largely inaccessible (George et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These organic pools primarily consist of monophosphate esters, di-phosphoesters (up to 90%), and phytates (up to 50%) (George et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lidbury et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Consequently, improving the mobilization and uptake of organic phosphorus represents a critical strategy for alleviating phosphorus deficiency in crops (Alori et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe coupled cycling of N and P is fundamental to ecological processes (Jiao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). Previous research has demonstrated that long-term nitrogen addition changes phosphorus availability and reshapes phosphate-solubilizing bacterial community, suggesting that key N and P cycling processes could be mediated by microorganisms (Zhou et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Metagenomic approaches were used to investigate microbially-driven phosphorus cycling and its coupling mechanisms with nitrogen cycling, indicating the effect of N availability on P availability via changing microbial structure and stimulating microbial growth and activity (Liu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). While it is well established that soil microbes significantly mediate both N and P cycling processes, the specific microbial interactions underlying these two nutrient cycles, as well as the synergistic effect of nitrogen fixation and phosphate solubilization on the growth of host plants remain yet poorly understood.\u003c/p\u003e \u003cp\u003eThe heavy reliance on chemical N and P fertilizers has raised environmental concerns, including greenhouse gas emissions and water eutrophication, prompting interest in sustainable agricultural alternatives. Plant growth-promoting rhizobacteria (PGPR) have emerged as promising candidates due to their capacity to enhance nutrient availability and support plant health through mechanisms such as biological nitrogen fixation, phosphate solubilization, phytohormone production, induced systemic resistance, and bioremediation (Ahmad et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Igiehon and Babalola \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rahman and Singh \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Vejan et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Among PGPR, phosphate-solubilizing bacteria (PSB) improve phosphorus availability by solubilizing inorganic phosphates and mineralizing organic phosphorus compounds via the production of organic acids (which lower soil pH) and the secretion of phosphatases. Prominent PSB includes genera \u003cem\u003ePseudomonas\u003c/em\u003e (Peng et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), \u003cem\u003eBacillus\u003c/em\u003e (de la Paz-Osorio et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), \u003cem\u003eRhizobia\u003c/em\u003e (Nascimento et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), \u003cem\u003eBurkholderia\u003c/em\u003e (Guo et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and \u003cem\u003eAcinetobacter\u003c/em\u003e, with \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e being particularly effective. The mineralization of organic phosphorus into plant-available Pi is primarily catalyzed by enzymes such as acid phosphatases (ACP), alkaline phosphatases (ALP), and phytases, whose genetic determinants have been widely characterized in diverse soil microorganisms (Alori et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bargaz et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rezakhani et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Richardson and Simpson \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe potential of bacterial secretory phosphatases for enhancing phosphorus solubilization has been explored in several studies. Previous studies identified and cloned several key acid phosphatases, including the non-specific acid phosphatase NapD and NapE from \u003cem\u003eSinorhizobium meliloti\u003c/em\u003e 104A14 (Mihara et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), as well as the phosphate-repressible acid phosphatase PhoC and NapA from \u003cem\u003eMorganella morganii\u003c/em\u003e (Thaller et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Subsequent research demonstrated that heterologous expression of these enzymes in PGPR strains could enhance their ability to mineralize organic phosphorus. For instance, the introduction of \u003cem\u003enapA\u003c/em\u003e or \u003cem\u003ephoC\u003c/em\u003e gene into \u003cem\u003eBurkholderia cepacia\u003c/em\u003e or \u003cem\u003eAzospirillum\u003c/em\u003e spp., respectively, resulted in elevated phosphatase activity and improved phosphate solubilization (Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). These findings supported the feasibility of engineering PGPR with enhanced phosphorus-mobilizing traits for agricultural applications (Hakim et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Haskett et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e A1501 has emerged as a promising chassis strain for such engineering efforts (Yan et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhan et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This bacterium efficiently colonizes the rice rhizosphere, exhibits nitrogen-fixing activity, and promotes plant growth (Jiang et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Its ecological adaptability and plant-beneficial properties make it an ideal candidate for further genetic improvement. The present study therefore aimed to engineer \u003cem\u003eP. stutzeri\u003c/em\u003e A1501 for synergistic nitrogen fixation and phosphate solubilization. We successfully constructed a set of recombinant strains that simultaneously exhibited nitrogen-fixing activity and enhanced organic phosphorus mineralization. Our results demonstrated that the engineered strain displayed superior root colonization and increased available phosphorus levels in the rhizosphere, thereby significantly promoting rice growth. These results strongly suggested that engineered bacterial strains with enhanced phosphorus solubilization and nitrogen fixation capabilities would have a synergistic effect when applied to crops in the field.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains culture and determination of phosphate solubilization ability\u003c/h2\u003e \u003cp\u003eThe bacterial strains used in this study included five wild-type strains of \u003cem\u003ePseudomonas\u003c/em\u003e genus: \u003cem\u003eP. stutzeri\u003c/em\u003e A1501, \u003cem\u003eP. stutzeri\u003c/em\u003e ATCC 17588, \u003cem\u003eP. syringae pv. tomato\u003c/em\u003e DC3000, \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 and \u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190. \u003cem\u003eP. stutzeri\u003c/em\u003e A1501, \u003cem\u003eP. stutzeri\u003c/em\u003e ATCC 17588 and \u003cem\u003eP. syringae pv. tomato\u003c/em\u003e DC3000 strains were preserved in this laboratory. \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 (accession No. \u003cem\u003eP. aeruginosa\u003c/em\u003e ATCC 15692) and \u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190 (accession No. \u003cem\u003eP. fluorescens\u003c/em\u003e ATCC 13525) strains were obtained from China Agricultural Microbial Culture Preservation Center and China General Microbial Culture Preservation Center, respectively. All strains were routinely cultured in Luria-Bertani (LB) broth.\u003c/p\u003e \u003cp\u003eThe organophosphorus-solubilizing capacity of the five \u003cem\u003ePseudomonas\u003c/em\u003e strains was preliminarily evaluated using a modified plate assay on PVK agar (Sanchez-Gonzalez et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) containing tricalcium phosphate, lecithin or phytic lecithin as the sole phosphorus source (Kalayu \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Briefly, single colonies were inoculated into LB medium and incubated at 30 ℃ for 12 h. Cells were harvested by centrifugation, washed with 0.85% (w/v) NaCl, and resuspended to an density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of 1.0. A 10 \u0026micro;L aliquot of each bacterial suspension was spotted onto the center of the PVK agar plates. After 3 days incubation, the colony diameter (d) and the solubilization halo diameter (D) were measured. The phosphate solubilization index (PSI) was calculated for each replicate (n\u0026thinsp;=\u0026thinsp;3) according to the following formula: PSI = (Colony diameter\u0026thinsp;+\u0026thinsp;Halo zone diameter)/Colony diameter\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBioinformatic identification of putative secretory phosphatases\u003c/h3\u003e\n\u003cp\u003eTo identify potential secretory phosphatases in \u003cem\u003ePseudomonas\u003c/em\u003e species, a bioinformatic screening pipeline was established. Putative phosphatase sequences were retrieved from the \u003cem\u003ePseudomonas\u003c/em\u003e Genome Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pseudomonas.com/\u003c/span\u003e\u003cspan address=\"https://www.pseudomonas.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and functionally annotated using the National Center for Biotechnology Information (NCBI) non-redundant protein database (Winsor et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The secretion potential of each candidate was evaluated by predicting the presence of signal peptides and cleavage sites using SignalP 6.0 (Teufel et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Transmembrane helices were subsequently analyzed with TMHMM-2.0 to infer the likelihood of extracellular localization (Krogh et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eConstruction of recombinant strain\u003c/h3\u003e\n\u003cp\u003eThe target genes \u003cem\u003e10ACP\u003c/em\u003e, \u003cem\u003e10ALP\u003c/em\u003e, \u003cem\u003eDCACP\u003c/em\u003e, \u003cem\u003ePAALP1\u003c/em\u003e, and \u003cem\u003ePAALP2\u003c/em\u003e were amplified using genomic DNA from \u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190, \u003cem\u003eP. syringae\u003c/em\u003e pv. \u003cem\u003etomato\u003c/em\u003e DC3000, and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 as templates, respectively. The amplified fragments were cloned into linearized plasmids (Vazyme Biotech Co., Ltd., China) to generate recombinant constructs. These constructs were first introduced into \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α via heat shock and subsequently mobilized into \u003cem\u003eP stutzeri\u003c/em\u003e A1501 by tri-parental conjugation using the helper plasmid pRK2013 (Shang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). All primers, strains and plasmids used in this study were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2, respectively, along with the corresponding recombinant strain names. Antibiotics were applied at the following concentrations: 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e kanamycin and 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e tetracycline for both recombinant \u003cem\u003eP. stutzeri\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003eDetection of phosphatase gene expression in recombinant strains\u003c/h3\u003e\n\u003cp\u003eTo assess the expression of heterologous phosphatase genes in the recombinant strains, overnight cultures of single colonies in LB broth at 30\u0026deg;C were diluted with sterile 0.85% NaCl to an optical density (OD\u003csub\u003e600\u003c/sub\u003e=0.1) and incubated for additional 8 h under the same conditions. Total RNA was extracted from 1 mL of the bacterial culture using the RNA Easy Fast Kit (TIANGEN, China). Residual genomic DNA was removed, and cDNA was synthesized using the PrimeScript RT reagent Kit with gDNA Eraser (TAKARA, Japan). Reverse transcription qPCR (RT-qPCR) was performed using cDNA templates. Gene-specific primers (Table S3) were designed based on the corresponding genome sequences. The 16S rRNA gene was used as an internal reference to normalize the expression levels of the target genes.\u003c/p\u003e\n\u003ch3\u003eGrowth kinetics assay\u003c/h3\u003e\n\u003cp\u003eA single colony of each strain was inoculated into LB broth or K medium (containing 0.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.1 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.1 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl, 0.2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 0.01 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO, 0.01 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Fe\u003csub\u003e2\u003c/sub\u003e(SO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO, and 0.01 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO, pH 6.8) supplemented with sodium lactate (50 mM) as the sole carbon source and no nitrogen source and cultured at 30\u0026deg;C for 12 h. The cells were harvested, washed with 0.85% NaCl, and resuspended to an OD\u003csub\u003e600\u003c/sub\u003e of 0.1. Aliquots of the bacterial suspension were dispensed into a 96-well microplate (Corning, USA), and growth was monitored using an automated Bioscreen Plate Reader (Labsystems, Finland). The plate was incubated at 30\u0026deg;C with continuous shaking, and OD\u003csub\u003e600\u003c/sub\u003e measurements were recorded at 2 h intervals over a 48 h period to construct growth curves.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative analysis of organic phosphorus (lecithin) solubilization\u003c/h2\u003e \u003cp\u003eThe organic phosphorus-solubilizing capacity of the recombinant strains was quantitatively evaluated using a modified NBRIP liquid medium containing lecithin as the sole phosphorus source. The medium composition per liter was as follows: 10 g glucose, 0.5 g (NH₄)₂SO₄, 0.3 g KCl, 0.3 g NaCl, 0.03 g MnSO₄\u0026middot;4H₂O, 0.03 g FeSO₄\u0026middot;7H₂O, 0.4 g yeast extract, and 0.4 g lecithin (pH 6.75). Bacterial strains were pre-cultured in LB medium at 30\u0026deg;C for 12 h, harvested by centrifugation, washed with sterile 0.85% NaCl, and adjusted to an OD\u003csub\u003e600\u003c/sub\u003e of 1.0. A 10 mL aliquot of bacterial suspension was inoculated into 90 mL of the NBRIP medium in a 250 mL flask, yielding an initial OD\u003csub\u003e600\u003c/sub\u003e of approximately 0.1. Cultures were incubated at 30\u0026deg;C with shaking at 220 rpm. for 5 days. Uninoculated medium served as the negative control. Samples were collected every 24 h from each biological replicate (n\u0026thinsp;=\u0026thinsp;3). The culture supernatant was obtained by centrifugation at 12,000 \u0026times; g for 10 min, and the soluble phosphate concentration was determined using the molybdenum blue method (King \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1932\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of phosphorus-solubilizing capacity in a rice co-culture system\u003c/h3\u003e\n\u003cp\u003eTo evaluate the ability of recombinant strains to solubilize organic phosphorus in the presence of rice plants, a hydroponic co-culture system was established. Bacterial strains were grown overnight in LB broth, harvested by centrifugation (6000 rpm., 4\u0026deg;C, 8 min), washed twice with 0.85% NaCl, and resuspended in sterile distilled water to an OD\u003csub\u003e600\u003c/sub\u003e of 1.0. Rice seedlings were pre-grown hydroponically in a nutrient solution containing lecithin (320 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) as the sole phosphorus source, without nitrogen supplementation. Each culture vessel received 8 mL of the bacterial suspension, while control seedlings were treated with an equal volume of sterile nutrient solution. At 2 and 4 days post-inoculation, aliquots of the culture solution were sampled, and the soluble phosphorus content was determined using the molybdenum blue method.\u003c/p\u003e\n\u003ch3\u003eRice root colonization efficiency of inoculants\u003c/h3\u003e\n\u003cp\u003eTo evaluate the root colonization ability of the phosphate-solubilizing recombinant strains, rice seedlings were inoculated with either the recombinant strains or the wild-type \u003cem\u003eP. stutzeri\u003c/em\u003e A1501. Surface-sterilized rice seeds were germinated on 1/2 Murashige and Skoog (MS) semi-solid agar medium in sterile flasks and cultivated in a growth chamber for one week (Yimam et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The seedlings were then transferred to sterile tubes containing 72 mL of Hoagland nutrient solution supplemented with lecithin (320 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) as the sole phosphorus source, without nitrogen. Each seedling was inoculated with 8 mL of bacterial suspension (OD\u003csub\u003e600\u003c/sub\u003e=1.0), resulting in a final OD\u003csub\u003e600\u003c/sub\u003e of approximately 0.1 in the culture medium. To monitor root colonization dynamics, root samples were collected weekly. Roots were placed in 10 mL of sterile 0.85% NaCl, and the attached bacteria were dislodged by vortexing. The bacterial suspensions were serially diluted and plated onto selective medium for colony enumeration.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGreenhouse experiment setup\u003c/h2\u003e \u003cp\u003eA pot experiment was conducted under the greenhouse conditions to evaluate the effects of bacterial inoculation on rice plant growth under nitrogen-deficient and nitrogen-sufficient regimes, respectively. Bacterial inoculants were cultured in LB medium at 30\u0026deg;C for 12 h, harvested by centrifugation, and resuspended in sterile deionized water to a final concentration of 10⁸ cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The growth substrate consisted of a 1:1 (w/w) mixture of diatomite and vermiculite. To eliminate residual soluble phosphorus, the substrate was thoroughly washed with deionized water and air-dried prior to use. Each pot was filled with 250 g of the prepared substrate. Rice seedlings were inoculated by applying 10 mL of bacterial suspension via soil drenching. Two fertilization treatments were established: (1) N\u0026thinsp;+\u0026thinsp;P treatment, in which NH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e (100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and lecithin (320 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were supplied as nitrogen and phosphorus source, respectively); and (2) P-only treatment, in which lecithin (320 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was provided sole phosphorus source without nitrogen supplementation. Nutrient solutions were prepared accordingly and applied as follows: an initial 100 mL of the respective nutrient solution was administered at the time of inoculation, followed by supplemental applications at weekly intervals. After 30 days of cultivation, rice seedlings were sampled. Roots were carefully cleaned, and shoot and root lengths were recorded. The dry weights of shoots and roots were determined after oven-drying to constant weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSpearman's correlation analysis was performed to evaluate pairwise relationships among the measured parameters. Differences between treatment groups were assessed using one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) post-hoc test for multiple comparisons. All analyses were conducted using SPSS software (version 31.0, IBM Corp., USA). Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from three independent biological replicates.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePhosphate solubilization ability of selected\u003c/b\u003e \u003cb\u003ePseudomonas\u003c/b\u003e \u003cb\u003estrains under different culture conditions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, five representative strains including \u003cem\u003eP. stutzeri\u003c/em\u003e A1501, \u003cem\u003eP. stutzeri\u003c/em\u003e ATCC 17588, \u003cem\u003eP. syringae\u003c/em\u003e pv. tomato DC3000, \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 and \u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190, were evaluated for their phosphate-solubilizing capacities using plate assays on modified Pikovskaya (PVK) medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). All five strains exhibited organophosphorus-solubilizing activity. On lecithin-supplemented medium, the phosphate solubilization indices (PSI) of the five strains were 1.26, 1.58, 2.48, 2.53, and 2.64, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The PSI values of four strains were significantly higher than that of \u003cem\u003eP. stutzeri\u003c/em\u003e A1501, with \u003cem\u003eP. syringae\u003c/em\u003e pv. \u003cem\u003etomato\u003c/em\u003e DC3000, \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, and \u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190 exhibiting 1.97-, 2.00-, and 2.09-fold increases, respectively. On phytate-containing medium, the PSI values of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 and \u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190 were 2.00-and 1.36-fold higher than that of A1501. In contrast, only \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 and \u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190 demonstrated the ability to solubilize inorganic phosphorus in the form of tricalcium phosphate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of recombinant strains expressing a heterologous phosphatase\u003c/h2\u003e \u003cp\u003eIn K medium supplemented with various concentrations of phosphorus, A1501 exhibited optimal growth at a phosphorus concentration of 0.2 g/L, while its growth capacity was significantly reduced at concentrations either above or below this level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Comparative genomic analysis revealed distinct profiles of phosphatase-encoding genes among the evaluated \u003cem\u003ePseudomonas\u003c/em\u003e strains. \u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190 carried genes encoding an acid phosphatase (ACP, \u003cem\u003eB0A76_RS14940\u003c/em\u003e) and an alkaline phosphatase (ALP, \u003cem\u003eB0A76_RS21295\u003c/em\u003e). \u003cem\u003eP. syringae\u003c/em\u003e pv. \u003cem\u003etomato\u003c/em\u003e DC3000 contained an ACP-encoding gene (\u003cem\u003ePSPTO_3648\u003c/em\u003e), while \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 harbored two ALP genes (\u003cem\u003ePA0689\u003c/em\u003e, \u003cem\u003ePA3296\u003c/em\u003e). Notably, no homologous \u003cem\u003eACP\u003c/em\u003e or \u003cem\u003eALP\u003c/em\u003e genes were identified in the genome of \u003cem\u003eP. stutzeri\u003c/em\u003e A1501. However, a unique phytase gene (\u003cem\u003ePST_1945\u003c/em\u003e) was present in both \u003cem\u003eP. stutzeri\u003c/em\u003e A1501 and \u003cem\u003eP. stutzeri\u003c/em\u003e ATCC 17588. Interestingly, the expression of \u003cem\u003ePST_1945\u003c/em\u003e gene was significantly upregulated in A1501 under phosphorus-limited conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The predicted structural features and homology of PST_1945 were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed. A summary of the key phosphatases involved in organophosphorus hydrolysis across the four \u003cem\u003ePseudomonas\u003c/em\u003e strains was presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of the key enzymes involved in organophosphorus hydrolysis among the five \u003cem\u003ePseudomonas\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP. stutzeri\u003c/em\u003e A1501\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlkaline phosphatase (ALP)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcid phosphatase (AP)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePhytase\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. stutzeri\u003c/em\u003e ATCC 17588\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. syringae pv. Tomato\u003c/em\u003e DC3000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e+: Phosphatase genes identified in the genome; -: None of phosphatase genes identified in the genome.\u003c/p\u003e \u003cp\u003eBased on the screening described above, a total of nine phosphatases-coding genes were selected for heterologous expression in \u003cem\u003eP. stutzeri\u003c/em\u003e A1501. These included four genes from previously characterized sources (\u003cem\u003enapD\u003c/em\u003e and \u003cem\u003enapE\u003c/em\u003e from \u003cem\u003eS. meliloti\u003c/em\u003e; \u003cem\u003ephoC\u003c/em\u003e and \u003cem\u003enapA\u003c/em\u003e from \u003cem\u003eM. morganii\u003c/em\u003e) and five genes identified in the \u003cem\u003ePseudomonas\u003c/em\u003e strains analyzed in this study (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Signal peptide prediction confirmed that all selected phosphatases contain putative secretion signal, supporting their potential for extracellular localization and function.\u003c/p\u003e \u003cp\u003eNine recombinant strains were successfully constructed via tri-parental mating and designated as A1510ACP, A1510ALP, A15DCACP, A15PAALP1, A15PAALP2, A15NapD, A15NapE, A15PhoC, and A15NapA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Transcriptional expression of the integrated phosphatase genes was confirmed by reverse transcription quantitative PCR (RT-qPCR). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, all nine heterologous phosphatases genes were successfully expressed at the mRNA level in their respective recombinant strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenes selected for construction of recombinant strains in this study and the predicted signal peptides of the encoded target proteins.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene origin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGene(homologous gene)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGene ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSignal peptide sequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA1510ACP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e10ACP (phoC)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eB0A76_RS14940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMTEETEDPKATQNAADTLPDSSRRRFLGGVAVLGAGATLSAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA1510ALP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e10ALP (phoD)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eB0A76_RS21295\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMSHFDLGRRRVMQAVGAGLLLPGLAPAVIA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA15DCACP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP. syringae\u003c/em\u003e pv. tomato DC3000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDCACP(phoC)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePSPTO_3648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMSDKPEDENTNENPGRRRFLGGMAALGAGVTLSGYVSA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA15PAALP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePAALP1 (phoA)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePA0689\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMTPGYPLALSLAVSMAVLGSALPAQA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA15PAALP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePAALP2 (pstS)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePA3296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMLKPMFGVVALTLLTSHTTLG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA15NapD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eSinorhizobium meliloti\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003enapD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eU74652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMFQVTRRSAMKIAALAAAASSMGQAAFA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA15NapE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003enapE\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAF354653\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMFAIRNSLKLVRDAALAVAAGLIFAVSALA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA15PhoC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eMorganella morganii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ephoC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAB035805\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMKKNIIAGCLFSLFSLSALA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA15NapA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003enapA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX78328\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMRKLTLTLSALALALSLNSVADA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOrganic phosphate solubilization ability of recombinant strains\u003c/h2\u003e \u003cp\u003eThe capacity of \u003cem\u003eP. stutzeri\u003c/em\u003e A1501 and its recombinant derivatives to solubilize organic phosphorus was quantitatively evaluated by measuring soluble phosphate levels in liquid medium containing lecithin as the sole phosphorus source over a 5-day incubation period. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, strain A15PLA (A1501 carrying the empty vector pLAFR3) showed no significant difference in lecithin solubilization compared to the wild-type strain. In contrast, heterologous expression of acid phosphatase gene \u003cem\u003e10ACP\u003c/em\u003e (derived from \u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190) and alkaline phosphatase gene \u003cem\u003ePAALP1\u003c/em\u003e (from \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1) significantly enhanced soluble phosphate release, whereas their respective paralogs (\u003cem\u003e10ALP\u003c/em\u003e and \u003cem\u003ePAALP2\u003c/em\u003e) conferred no such improvement. Phosphatases genes originating from \u003cem\u003eS. meliloti\u003c/em\u003e (\u003cem\u003enapD\u003c/em\u003e, \u003cem\u003enapE\u003c/em\u003e) failed to enhance solubilization activity, while those from \u003cem\u003eM. morganii\u003c/em\u003e (\u003cem\u003enapA\u003c/em\u003e, \u003cem\u003ephoC\u003c/em\u003e) led to a significant increase. Overall, four recombinant strains, i.e. A1510ACP, A15PAALP1, A15NapA and A15PhoC, exhibited substantially enhanced organic phosphorus solubilization capacity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRoot colonization and nitrogenase activities of recombinant strains\u003c/h2\u003e \u003cp\u003eTo assess potential physiological impacts of heterologous gene expression, the growth performances of the engineered \u003cem\u003eP. stutzeri\u003c/em\u003e strains were evaluated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, heterologous expression of phosphatases did not impair bacterial growth in LB medium over a 48h period. nor significantly altered nitrogenase activity compared to the wild-type control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Besides, heterologous expression of various phosphatases positively or negatively affected nitrogenase activity of corresponding recombinant strains. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, three recombinant strains, i.e. A15PAALP1, A15PAALP2, and A15NapA, exhibited enhanced nitrogenase activity, while six recombinant strains displayed significantly reduced nitrogenase activity. Markedly, nitrogenase activity of A15NapA was approximately 7500 nmol ethylene mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, representing an obvious increase in nitrogenase activity of 17% compared to A1501.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe root colonization dynamics of A1501 and its recombinant derivatives were monitored over a two-week period. Except for A1510ACP, all recombinant strains exhibited colonization rates comparable to the wild-type strain, with population densities generally declining over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and d). Notably, A1510ACP maintained a stable population throughout the experimental period. At 7 and 14 days post-inoculation, the population of A1510ACP on rice roots reached 5.7 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 5.9 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e root fresh weight, respectively, indicating superior root colonization. These results strongly suggested that engineered bacterial strains with enhanced phosphorus solubilization and nitrogen fixation capabilities would have a synergistic effect when applied to crops.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePhosphorus solubilization by recombinant strains co-cultured with rice\u003c/h2\u003e \u003cp\u003eThe organic phosphorus-mineralizing capacity of the wildtype strain A1501 and recombinant strains was assessed in a hydroponic rice co-culture system with. Across all sampling time points, inoculation with bacterial strains significantly increased soluble phosphorus concentrations in the culture medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Among the tested strains, A1510ACP, A15PAALP1, and A15NapA exhibited the highest phosphorus-solubilizing activities compared to the wild-type strain and non-inoculated rice. At two days post-inoculation, the available phosphorus concentrations in cultures inoculated with three strains reached 2.05, 2.11 and 1.96 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. After four days of co-cultivation, the corresponding phosphorus levels increased further to 2.13, 2.12 and 2.37 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, which was 2.0-fold higher than wild-type strain A1501. These results demonstrated that the enhanced organic phosphorus solubilization capacity of the engineered strains was maintained in the presence of rice plants, highlighting their potential for improving phosphorus availability in the rhizosphere (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePromoting effects of recombinant strain inoculation on rice growth\u003c/h2\u003e \u003cp\u003eThe interactive effects of nitrogen and phosphorus availability on rice development were evident, as varying levels of N and P supply significantly influenced plant growth parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). To evaluate the plant-growth-promoting effects of engineered strains, shoot and root lengths as well as dry weights were measured and compared between plants inoculated with recombinant strains and those inoculated with the wild-type A1501 or uninoculated. After 30 days of growth, inoculation with recombinant strains generally resulted in improved agronomic traits compared to the non-inoculated or wild-type-treated controls under both fertilization regimes (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, under nitrogen-supplemented conditions (100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e) with lecithin as the sole phosphorus source (320 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the maximum values for the shoot and root dry weight of the rice seedling inoculated with strain A1510ACP were 0.29 and 0.19 g plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which exhibited significant increases in shoot and root dry weight by 16% and 26.6%, respectively, relative to those inoculated with the wild-type strain. These results indicated a synergistic effect of nitrogen fixation and phosphorus solubilization on plant growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrowth promotion of rice by recombinant strains under soil culture conditions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eLecithin (320 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and ammonium nitrate (100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) as P and N source, respectively\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eLecithin (320mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) as P source, no nitrogen input\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShoot length (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoot length (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShoot Dry weight (g/plant)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRoot Dry weight (g/plant)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eShoot length (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRoot length (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eShoot Dry weight (g/plant)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eRoot Dry weight (g/plant)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003enon-inoculation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e41.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e28.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e11.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.071\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e0.025\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA1501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e40.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e14.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e28.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e11.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.085\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e0.035\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA1510ACP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e42.08\u0026thinsp;\u0026plusmn;\u0026thinsp;3.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e15.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e29.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e12.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.087\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e0.039\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA15PAALP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e42.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e15.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e29.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e11.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.087\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e0.039\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA15NapA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e41.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e14.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e29.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e11.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.084\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e0.043\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA15PhoC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e43.58\u0026thinsp;\u0026plusmn;\u0026thinsp;4.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e27.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e10.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.083\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e0.040\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation from three biological replicates. Asterisks indicate significant differences compared to the wild-type A1501 strain as determined by one-way ANOVA (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePhosphorus (P) and nitrogen (N) are the most vital macronutrients for plant growth and development. Despite being extremely abundant, N\u003csub\u003e2\u003c/sub\u003e in air and most of total P in soils are inaccessible to plants. Soil microorganisms play a vital role in transforming soil N and P into bioavailable forms by various mechanisms, including nitrogen fixation by the enzyme nitrogenase capable of reducing atmospheric nitrogen into ammonia, and organic P mineralization by phosphatase capable of hydrolysing simple phosphate monoesters to acquire orthophosphate. Thus, it is promising to develop environmental-friendly methods to increase both N and P bioavailability by N\u003csub\u003e2\u003c/sub\u003e-fixing or P-solubilizing bacteria. However, the use of genetic engineering techniques to enhance the synergistic effect of nitrogen fixation and organic phosphate mineralization by microorganisms in rhizosphere environments, thereby promoting the growth of host plants, has not yet been reported. On the other hand, most naturally occurring phosphate-solubilizing strains typically exhibit only a single P-solubilization function.\u003c/p\u003e \u003cp\u003eTo overcome this limitation, approaches such as constructing synthetic microbial consortia have been explored to leverage the metabolic diversity of beneficial microorganisms. Nevertheless, these strategies remain limited in their scalability and practical applicability for field practice. Engineering nitrogen-fixing bacteria with enhanced phosphorus-solubilizing capacity offers an innovative alternative. Organic phosphorus constitutes 30% to 65% of total phosphorus in most agricultural soils and remains largely inaccessible to plants (Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Therefore, endowing nitrogen-fixing bacteria with the ability to mineralize organic phosphates could simultaneously improve nitrogen and phosphorus availability, thereby reducing reliance on chemical fertilizers while maintaining stable crop yields. The design of our experiment was to enhance the organic phosphate-solubilizing and plant-growth-promoting capabilities of a nitrogen-fixing bacterium through genetic engineering. We successfully constructed engineered derivatives with dual functions of nitrogen fixation and organic phosphate solubilization. This modification significantly improved the phosphate-solubilizing activity of A1501 and conferred enhanced growth-promoting effects on rice. Our findings demonstrate the feasibility of engineering multifunctional biofertilizers by integrating complementary metabolic traits into a single plant-beneficial bacterial chassis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePhosphatase gene function and construction of dual-function engineered bacteria for phosphate solubilization and nitrogen fixation\u003c/h2\u003e \u003cp\u003eUnavailable organic phosphorus in soil primarily comprises phospholipids and phytic acid. Phosphatases, including acid phosphatases (AP) and alkaline phosphatases (ALP), are key enzymes secreted by microorganisms to hydrolyze phospholipids and are predominantly distributed among γ- and α-proteobacteria. Our results demonstrated that \u003cem\u003eP. stutzeri\u003c/em\u003e A1501 possessed a moderate ability to hydrolyze phytate but exhibits weak hydrolytic activity toward inorganic phosphorus and lecithin. Bioinformatic analysis revealed that the genome of A1501 lacked genes encoding AP or ALP, although a phytase-encoding gene (\u003cem\u003ePST_1945\u003c/em\u003e) was identified. In liquid culture, the optimal phosphorus concentration for A1501 growth was 0.2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), with lower concentrations inhibiting growth. Under phosphorus-limited conditions, the expression of \u003cem\u003ePST_1945\u003c/em\u003e was significantly upregulated by 1.73-fold compared to normal conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Further bioinformatic analysis indicated that the phytase encoded by \u003cem\u003ePST_1945\u003c/em\u003e shared 55.73% amino acid identity with the \u003cem\u003eP. ogarae\u003c/em\u003e phytase WP014338283.1. Based on these findings, we hypothesize that the absence of endogenous phosphatase is the primary reason for the weak lecithin-hydrolyzing activity of A1501. Consequently, heterologous expression of phosphatases represents a key strategy for enhancing its phosphate-solubilizing capacity.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePseudomonas\u003c/em\u003e species are widely recognized as effective solubilizers of soil organophosphorus (Peng et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Rasul et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), with strains such as \u003cem\u003ePseudomonas\u003c/em\u003e sp. WS32 and \u003cem\u003eP.\u003c/em\u003e asiatica JP233 (Ou et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), albeit their hydrolytic capacities vary considerably among strains. In this study, three \u003cem\u003ePseudomonas\u003c/em\u003e strains (i.e. \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, \u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190 and \u003cem\u003eP. syringae pv. tomato\u003c/em\u003e DC3000) displaying strong lecithin-hydrolyzing activity were identified. Genomic analysis revealed that \u003cem\u003eP. fluorescens\u003c/em\u003e ACCC 10190 harbored one acid phosphatase and one alkaline phosphatase, \u003cem\u003eP. syringae pv. tomato\u003c/em\u003e DC3000 contained one acid phosphatase, and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 carried two alkaline phosphatases. Among these, PAALP1 was known as alkaline phosphatase secreted via the type II secretion system of \u003cem\u003eP. aeruginosa\u003c/em\u003e (Ball et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). All five identified phosphatases contained signal peptides and were subsequently selected for heterologous expression in \u003cem\u003eP. stutzeri\u003c/em\u003e A1501. In addition to these \u003cem\u003ePseudomonas\u003c/em\u003e-derived genes, we also included four phosphatases previously characterized in other genera: NapD and NapE (periplasmic acid phosphatases from \u003cem\u003eSinorhizobium meliloti\u003c/em\u003e), as well as PhoC and NapA (acid phosphatases from \u003cem\u003eMorganella morganii\u003c/em\u003e). Introduction of \u003cem\u003enapA\u003c/em\u003e into \u003cem\u003eBurkholderia\u003c/em\u003e spp. has previously been shown to confer organic phosphorus-degrading capacity (Fraga et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). All four enzymes contain signal peptides and were therefore selected for expression in A1501. Among the nine recombinant strains constructed, four strains i.e. A1510ACP, A15PAALP1, A15NapA, and A15PhoC exhibited substantially enhanced organic phosphorus solubilization. Specifically, A1510ACP (expressing 10ACP from \u003cem\u003eP. fluorescens\u003c/em\u003e), A15PAALP1 (expressing PAALP1 from \u003cem\u003eP. aeruginosa\u003c/em\u003e), and the two \u003cem\u003eM. morganii\u003c/em\u003e-derived phosphatase-expressing strains (A15NapA and A15PhoC) significantly increased soluble phosphorus release, with available phosphorus concentrations in the culture medium elevated by 10.9% to 43.3% compared to the wild-type strain. These increases were accompanied by detectable extracellular phosphatase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), confirming successful secretion of the heterologous enzymes. This finding is consistent with prior reports demonstrating enhanced extracellular phosphatase activity in \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eBurkholderia cepacia\u003c/em\u003e harboring plasmids encoding the \u003cem\u003eM. morganii\u003c/em\u003e NapA phosphatase (Thaller et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Similarly, the recombinant strain \u003cem\u003ePhosphobacterium\u003c/em\u003e SDLiuTP02 released over 50 \u0026micro;mol mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e soluble phosphorus after 72 h when cultured with insoluble calcium phytate as the sole phosphate source (Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and three phytase-expressing strains (\u003cem\u003eP. simiae\u003c/em\u003e, \u003cem\u003eRalstonia\u003c/em\u003e sp., and \u003cem\u003eP. putida\u003c/em\u003e) released up to 500 \u0026micro;M orthophosphate after 10 days (Shulse et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The varying levels of phosphatase activity observed among the transformants may be attributed to several factors. First, the intrinsic catalytic efficiencies of the source phosphatases likely differ. Second, heterologous expression levels may vary within the A1501 host strain due to differences in transcriptional regulation, mRNA stability, or protein folding. Although expression of all introduced phosphatases in A1501 was confirmed at the mRNA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), the resulting functional output is influenced by the combination of these factors.\u003c/p\u003e \u003cp\u003eImportantly, the introduction of heterologous phosphatases did not impose a detectable metabolic burden on the host strain. Growth kinetics and root colonization capacity of the engineered strains remained comparable to those of the wild-type A1501 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This observation aligns with previous research; for instance, introduction of a pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase gene into the endophytic nitrogen-fixing bacterium \u003cem\u003eHerbaspirillum seropedicae\u003c/em\u003e Z67 did not adversely affect growth, biofilm formation, or exopolysaccharide secretion (Wagh et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). To our knowledge, this study represents the first report of an engineered bacterial strain combining both nitrogen-fixing and organic phosphate-solubilizing capabilities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSynergistic effects of nitrogen and phosphorus on plant growth promotion\u003c/h2\u003e \u003cp\u003eNitrogen and phosphorus deficiencies severely impair crop growth and development. These two nutrients interact synergistically: nitrogen availability influences phosphorus uptake, while phosphorus status modulates nitrogen assimilation in plants (Jiang et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Preliminary experimental in this study confirmed this synergistic relationship, demonstrating that combined application of nitrogen and phosphorus significantly enhanced rice plant height, shoot fresh weight, and root fresh weight compared to either nutrient applied alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These observations underscore the potential value of engineering nitrogen-fixing bacteria with additional phosphate-solubilizing capacity to simultaneously address both nutrient limitations. Previous studies have identified naturally occurring microorganisms possessing dual plant-growth-promoting functions. For instance, the \u003cem\u003eEnsifer sp.\u003c/em\u003e EN1, isolated from leguminous plants, exhibited both nitrogen-fixing and phosphate-solubilizing activities (mineralizing calcium phytate and tricalcium phosphate), and inoculation experiments confirmed its ability to enhance plant phosphorus acquisition (Xu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Although other nitrogen-fixing genera including \u003cem\u003eBrevundimonas\u003c/em\u003e, \u003cem\u003eSphingomonas\u003c/em\u003e, \u003cem\u003eMicrobacterium\u003c/em\u003e, and \u003cem\u003eArthrobacter\u003c/em\u003e, have also been reported to solubilize phytin and tricalcium phosphate, the phosphate-solubilizing capacities of these natural isolates are generally low. Current research has partially addressed this limitation by constructing synthetic microbial consortia that combine nitrogen-fixing and phosphate-solubilizing functions (Faller et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In the present study, under co-cultivation with rice, the engineered phosphate-solubilizing strains A1510ACP, A15PAALP1, and A15NapA increased available phosphorus concentrations in the culture medium by 9.7%, 9.4%, and 22.1%, respectively, compared to the wild-type A1501 treatment, while maintaining nitrogenase activity comparable to the parental strain. These results demonstrate that the engineered strains successfully integrate both functions without compromising their native nitrogen-fixing capacity.\u003c/p\u003e \u003cp\u003eConsistent with their enhanced phosphorus-solubilizing activity, the engineered strains conferred superior plant growth-promoting effects. Under both nitrogen-free and nitrogen-sufficient conditions, inoculation with either the wild-type or engineered strains increased rice shoot and root dry weights compared to non-inoculated controls, confirming that the engineered strains retain their ability to support plant growth under nitrogen-limited conditions. Notably, under nitrogen-sufficient conditions, rice seedlings inoculated with the engineered strain A1510ACP exhibited significantly greater biomass accumulation compared to those inoculated with the wild-type A1501. This suggests that under adequate nitrogen supply, the enhanced phosphate-solubilizing function of the engineered strains becomes operational, providing additional phosphorus to support plant growth. This synergistic effect of nitrogen and phosphorus is consistent with observations in forest ecosystems, where nutrient co-limitation and synergistic interactions have been widely documented (Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Importantly, the engineered strains display superior root colonization capacities comparable to the wild-type strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and d). Previous studies have demonstrated that \u003cem\u003eP. stutzeri\u003c/em\u003e A1501 effectively colonizes the rhizosphere of various crops, including maize and \u003cem\u003ePakchoi\u003c/em\u003e (Ke et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Taken together, these findings indicate that the engineered dual-function strains developed in this study possess enhanced plant growth-promoting potential and represent promising candidates for biofertilizer development to improve agricultural productivity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we successfully constructed a series of engineered strains with significantly enhanced ability to mineralize organic phosphorus. These strains exhibited exhibit enhanced nitrogen fixation, phosphate solubilization activities, and superior root colonization, thereby significantly increasing available phosphorus levels in the rhizosphere and consequently rice growth. Furthermore, pot experiments demonstrated that inoculation with these engineered strains significantly promoted rice growth, particularly under nitrogen-sufficient conditions where the enhanced phosphorus supply contributed to increased plant biomass. This work provides insights and preliminary attempts for engineering strains capable of both nitrogen fixation and organophosphorus mineralization, offering a promising strategy for sustainable agricultural productivity.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003cb\u003eThe authors have no competing interests to declare.\u003c/b\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eWe thank the financial support for the research provided by Hainan Seed Industry Laboratory and China National Seed Group (project of ZZGS-ZNBM-2025-230), and by National Natural Science Foundation of China (32270067 and 32370091), Agricultural Science and Technology Innovation Program and the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA28030201).\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eAll authors contributed to the study conception and design. Xiubin Ke and Min Lin conceived the study, planned and designed the research. Changyan Yin, Dongqi Wang, Yaoyao Liu, and Haicao Feng performed the experiments. Changyan Yin analyzed the data, prepared the figures, and wrote the original manuscript with assistance from Yongliang Yan and Wei Lu. Yuhua Zhan, Wei Lu, Yongliang Yan, and Xiubin Ke reviewed and edited the manuscript. Min Lin supervised the research. All authors contributed critically to the drafts and gave final approval for publication.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmad A, Zafar U, Khan A et al (2022) Effectiveness of compost inoculated with phosphate solubilizing bacteria. 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Environ Technol Innov 40:104353. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.eti.2025.104353\u003c/span\u003e\u003cspan address=\"10.1016/j.eti.2025.104353\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pseudomonas stutzeri A1501, Genetic engineering, Nitrogen fixation, Phosphate-solubilization, Phosphatase, Host rice","lastPublishedDoi":"10.21203/rs.3.rs-9387281/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9387281/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eAims\u003c/em\u003e Both nitrogen (N) and phosphorus (P) are essential nutrients for plant growth but is often limiting in high yielding agricultural production systems. This study investigated the synergistic effect of nitrogen fixation and organic phosphate mineralization by rhizospheric microorganisms, thereby promoting the growth of host plants.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMethods\u003c/em\u003e After assessing phosphate-solubilizing activities of five \u003cem\u003ePseudomonas\u003c/em\u003e strains and identifying putative phosphatase genes \u003cem\u003ein silico\u003c/em\u003e, selected genes were expressed in nitrogen-fixing \u003cem\u003eP. stutzeri\u003c/em\u003e A1501, and the resulting recombinants were evaluated for growth, nitrogenase activity, organic P solubilization (pure and rice co-culture), root colonization, and rice growth-promotion under different N regimes.\u003c/p\u003e \u003cp\u003e \u003cem\u003eResults\u003c/em\u003e Four phosphatase genes were introduced into A1501 and the resulted recombinant strains displayed significantly elevated extracellular phosphatase activity. Three recombinant strains, i.e. A15PAALP1, A15PAALP2, and A15NapA, exhibited enhanced nitrogenase activity, while six recombinant strains displayed reduced nitrogenase activity. Notably, nitrogenase activity of A15NapA was approximately 7500 nmol ethylene mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, representing an obvious increase of 17%. Lecithin solubilization assay indicated that A1510ACP increased the concentration of available phosphorus by 10.98% relative to A1501. In a rice co-culture system, A1510ACP displayed superior root colonization ability, while enhanced hydrolysis of organic phosphorus increased available phosphorus levels were observed compared to A1501. Pot experiments demonstrated that inoculation with A1510ACP significantly promoted rice growth, particularly under nitrogen-supplemented conditions.\u003c/p\u003e \u003cp\u003eConclusion\u003c/p\u003e \u003cp\u003eThis study presents the first successful example of engineering a phosphate-solubilizing and nitrogen-fixing strain for enhanced growth of host rice, highlighting the potential of multifunctional engineered strains as the new generation of biological fertilizers.\u003c/p\u003e","manuscriptTitle":"Engineering a Root-Associated Bacterium Pseudomonas stutzeri A1501 with Nitrogen Fixation and Phosphate Solubilization Activities for Enhanced Growth of Host Rice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-23 11:43:22","doi":"10.21203/rs.3.rs-9387281/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-04-15T13:59:26+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-15T13:51:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2026-04-14T22:12:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-14T02:24:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2026-04-13T10:13:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"75d1d859-d7bf-4840-b371-92a0ddf9db9b","owner":[],"postedDate":"April 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T11:43:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-23 11:43:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9387281","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9387281","identity":"rs-9387281","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}