An engineered Pseudomonas stutzeri strain overexpressing the sigma factor AlgU promotes maize growth under salt stress

Abstract

Abstract

Soil salinization poses a significant threat to global agriculture, necessitating the development of sustainable strategies to increase crop resilience. Plant growth-promoting rhizobacteria (PGPR) offer a promising solution; however, their effectiveness in saline soils is often limited by their own salt sensitivity. The role of the extracytoplasmic function of the sigma factor AlgU in the salt stress adaptation of the nitrogen-fixing PGPR Pseudomonas stutzeri A1501 was investigated in this study. Through the construction of isogenic algU knockout (ΔalgU) and overexpression (OE-algU) strains, combined with phenotypic assays, transcriptomic profiling, and plant experiments, we demonstrate that AlgU acts as a master regulator of salinity tolerance. AlgU increased bacterial survival under acute salt shock, promoted biofilm formation, and, crucially, protected nitrogenase activity from salt inhibition. RNA-seq analysis revealed that AlgU orchestrates a comprehensive transcriptional reprogramming, upregulating the expression of genes involved in exopolysaccharide synthesis, osmoprotection (otsA), and central carbon metabolism (zwf, fumC). This coordinated response ensures an adequate supply of energy and reducing equivalents while maintaining cellular homeostasis. Consequently, inoculation with the OE-algU strain significantly alleviated salt stress in maize, improving seedling growth in pot experiments and outperforming the wild-type strain by increasing grain yield in saline–alkali field trials. Our findings establish AlgU as a key genetic determinant for engineering salt-tolerant PGPR, providing a mechanistic framework for the development of effective microbial inoculants to improve crop productivity in saline soils.

Key points

• AlgU orchestrates biofilm formation, osmoprotection, and energy metabolism to confer salt tolerance in P. stutzeri.

• Engineering an AlgU overexpression strain increases maize yield under saline–alkali field conditions.

• AlgU safeguards nitrogenase activity under salt stress by reprogramming energy and osmoprotective pathways, thus linking bacterial resilience to improved crop yield.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00253-026-13813-1.

Introduction

Soil salinization is among the most severe abiotic stresses that threaten global agriculture, seriously endangering food security and sustainable agricultural development (Sultan et al. 2023). Statistics show that more than 20% of irrigated farmlands and nearly 10% of terrestrial surfaces worldwide are affected by salt accumulation, a problem exacerbated by climate change and irrational irrigation practices (Devkota et al. 2022; Hassani et al. 2021). High salt concentrations directly inhibit plant water and nutrient uptake through osmotic stress and ionic toxicity, disrupt cellular structure and metabolic balance, and ultimately lead to retarded growth, sharply reduced yields, or even plant death (Atta et al. 2023; Liu et al. 2022; Zia-ur-Rehman et al. 2023). Traditional saline‒alkali soil improvement methods, such as physical leaching and chemical amendment application, can alleviate salt damage to a certain extent but are associated with high costs, excessive water consumption, and potential secondary pollution and fail to meet the demands of modern green agriculture (Gao et al. 2024).

In this context, the use of plant growth-promoting rhizobacteria (PGPR) to enhance crop salt tolerance has shown great application potential (Jiang et al. 2025). PGPR promote plant growth through multiple core mechanisms, including biological nitrogen fixation (Zhou et al. 2025), the production of plant hormones, the secretion of exopolysaccharides (EPS) for biofilm formation (Sharma et al. 2021), and the synthesis of 1-aminocyclopropane-1-carboxylate (ACC) deaminase to alleviate stress (Bal and Adhya 2021). However, high-salt environments impose stress on PGPR themselves, disrupting osmotic balance, inhibiting metabolic activity, and impairing key functions such as nitrogenase (which is highly sensitive to salt) (Rasheed et al. 2025). Thus, deciphering the mechanisms underlying the regulation of salt stress by PGPR and enhancing their performance via genetic engineering are crucial for overcoming application bottlenecks.

Among these PGPR, Pseudomonas stutzeri A1501 is an associative nitrogen-fixing bacterium isolated from the rice rhizosphere and serves as a model strain for plant‒microbe interaction studies due to its efficient nitrogen fixation and diverse metabolism (Desnoues et al. 2003; Vermeiren et al. 1999; Zhan et al. 2016). It harbours an approximately 49-kb nitrogen-fixation gene island with complete genes for nitrogenase synthesis and function, and field inoculation significantly promotes maize growth and nitrogen accumulation (Yan et al. 2008; Yang et al. 2021). Additionally, A1501 forms complex biofilms that facilitate rhizosphere colonization and protect nitrogenase from oxygen inhibition (Wang et al. 2017), making it an ideal candidate for saline‒alkali soil inoculation. However, like those of other PGPR, the functions of A1501 are severely inhibited under salt stress—environmental stresses reduce the expression of nitrogenase genes (nifA and nifH) and impair root colonization and biofilm formation (Lu et al. 2023). Therefore, elucidating its salt stress regulatory network and identifying key regulators are essential to exploit its agricultural potential.

Bacteria adapt to changing environments via sophisticated transcriptional regulatory networks, with sigma (σ) factors playing a central role in initiating stress-responsive gene transcription (Anjou et al. 2024; Ponath et al. 2022). Extracellular function (ECF) σ factors are the most diverse alternative σ factors and sense periplasmic/membrane stress signals to maintain cellular homeostasis (Lonetto et al., 1994). AlgU (AlgT/σ^E/σ22) is a highly conserved ECF σ factor in gram-negative bacteria that acts as a master regulator of cell envelope stress responses. Extensive studies in Pseudomonas aeruginosa have shown that AlgU regulates responses to osmotic, oxidative, and desiccation stress, as well as biofilm formation (Sivakumar et al. 2022; Wang et al. 2024). However, its functions in nonpathogenic, agriculturally important PGPR such as P. stutzeri A1501—especially in the coordinated regulation of salt tolerance, nitrogen fixation, and plant growth promotion—remain largely unknown. A recent study revealed that AlgU regulates A1501 EPS production and biofilm formation via pslA activation (Shao et al. 2022); however, its global role in the salt stress response and effects on nitrogenase activity are unknown. The potential of engineering AlgU to enhance the saline‒alkali adaptation of A1501 also needs verification.

This study aims to systematically elucidate the fundamental role of AlgU in the salt stress response, maintenance of physiological functions, and promotion of plant growth in A1501. By developing algU knockout (ΔalgU) and overexpression (OE-algU) strains and performing phenotyping, RNA sequencing, and pot/field experiments, our objectives are to (1) clarify the role of AlgU in A1501 salt tolerance, biofilm formation, and nitrogenase protection; (2) elucidate the global gene network regulated by AlgU under salt stress and elucidate its mechanism for protecting against nitrogen fixation through the remodelling of energy metabolism, osmotic protection, and biofilm synthesis; and (3) assess the ability of the engineered OE-algU strain to promote maize growth and yield under salt stress conditions. This research enhances the understanding of the mechanisms underlying the salt tolerance of PGPR, expands the functional knowledge of AlgU, and identifies key genetic targets and engineered strains for the development of novel microbial fertilizers, thereby offering innovative solutions for sustainable agriculture on saline‒alkali soils.

Materials and methods

Bacterial strains and genetic manipulation

The algU gene (locus tag PST1223, encoding the 193-aa σ factor AlgU) of P stutzeri A1501 was selected for genetic manipulation. To construct the deletion strain (ΔalgU), approximately 500-bp DNA fragments flanking the algU gene were amplified, fused with a tetracycline resistance (Tcʳ) gene, and cloned and inserted into the suicide vector pK18mobsacB (Windgassen et al. 2000). The recombinant plasmid was introduced into wild-type A1501 via triparental mating. Double-crossover mutants were selected on LB plates supplemented with tetracycline and 10% sucrose, which is lethal to strains retaining the sacB-containing vector. The overexpression strain (OE-algU) was generated by integrating a DNA fragment containing the algU gene along with its native upstream 500-bp regulatory region (including the promoter) into the neutral chromosomal amtB2 locus of A1501, a site previously shown to be dispensable for growth and nitrogen fixation (Zhang et al. 2012), thereby creating a dual-copy algU strain. All the mutant strains were verified by PCR and confirmed by DNA sequencing.

Phenotypic characterization of mutant strains

Freshly activated bacterial cultures were inoculated into LB medium and shaken to mid-exponential phase (OD₆₀₀ = 0.6–0.8). Bacterial cells were harvested by centrifugation at 1500 × g for 10 min, washed twice with phosphate-buffered saline (PBS), and processed for subsequent experiments. For salt stress tolerance analysis, the resuspended cells were incubated in 2.0 M NaCl at 30 °C with shaking (220 rpm) for 1 h, then serially diluted (10⁻1–10⁻5) and spotted on LB agar plates, after which their growth was determined after they were incubated at 30 °C for 1–3 days. For growth curve determination, the cells were adjusted to an OD₆₀₀ = 0.1 with K medium (composed of 0.4 g/L KH₂PO₄, 0.1 g/L K₂HPO₄, 0.1 g/L Na₂MoO₄·H₂O, 0.1 g/L NaCl, 0.2 g/L MgSO₄·7H₂O, 0.01 g/L MnSO₄·H₂O, and 0.01 g/L Fe₂(SO₄)₃·H₂O) and cultured at 30 °C with shaking for OD₆₀₀ monitoring. For biofilm determination, the bacterial cells were adjusted to an OD₆₀₀ = 0.5 with K medium supplemented with 50 mM sodium lactate, 150 μL of the cell suspension was added to 96-well plates and incubated at 30 °C for 48 h, after which the bacterial suspension was removed, the unadhered cells were washed with 160 μL of distilled water, 160 μL of 1% crystal violet was added to each well for 10 min, the crystal violet solution was discarded, the plates were thoroughly rinsed with distilled water, and lateral photography was performed; finally, the bound crystal violet was solubilized with 30% acetic acid for absorbance measurement at 560 nm. Nitrogenase activity was determined using the acetylene reduction assay (Zhang et al. 2019). Briefly, cells were adjusted to an OD₆₀₀ = 0.1 with K medium (10 mL) and then transferred to 100-mL sterile nitrogen-fixation vials, which were sealed, purged with argon for 5 min to remove air, and injected with 1 mL of O₂ and 10 mL of acetylene. After incubation at 30 °C with shaking (220 rpm), 0.25 mL samples were collected 4 h after incubation and every 2 h thereafter for ethylene detection by gas chromatography; nitrogenase activity was calculated based on changes in the area of the ethylene peak.

Transcriptomic analysis

For transcriptomic profiling, mid-exponential phase cells (OD₆₀₀ = 0.6–0.8) were harvested by centrifugation at 1500 × g for 10 min, washed twice with PBS, and subjected to 2.0 M NaCl stress for 1 h, followed by snap-freezing in liquid nitrogen. Total RNA was extracted using TRIzol Reagent, and ribosomal RNA was depleted with an Illumina Ribo-Zero™ Magnetic Kit (Bacteria, Epicentre Technologies, Madison, Wisconsin, USA). Sequencing libraries were constructed with the Illumina TruSeq Stranded mRNA LT Sample Prep Kit, and paired-end sequencing was performed on an Illumina NextSeq™500 platform using the High Output Kit v2. Raw reads were filtered to remove adapters and low-quality sequences, with clean reads mapped to the P. stutzeri A1501 reference genome via HISAT2. Gene expression levels were quantified as FPKMs, and differentially expressed genes (DEGs) were identified using edgeR with |log₂(fold change)|≥ 1 and adjusted padj < 0.01. GO and KEGG enrichment analyses of DEGs were conducted with KOBAS, and data visualization was performed using R software.

Plant growth promotion assays

Pot experiment

Maize seeds were surface-sterilized with 75% ethanol (5 min) and 5% NaClO (10 min), rinsed 8 times with sterile water, and germinated on water agar. Uniform seedlings were transplanted to plastic pots (10 cm × 10 cm × 9 cm) containing approximately 60 g of sterilized vermiculite, with five seedlings per pot. Seven days after transplantation, the seedlings were inoculated with 1.0 mL bacterial suspensions of either the wild-type (WT) A1501 or the OE-algU strain, prepared in Hoagland nutrient solution at an OD₆₀₀ of 0.2 (corresponding to approximately 2.1 × 10⁸ CFU/mL), which were inoculated directly to the base of the plant stems. Non-inoculated plants receiving sterile Hoagland solution served as controls. Salt stress was imposed with 100 mM NaCl, and the seedlings were grown under controlled conditions (16 h light/8 h dark, 30 °C). After 20–25 days of cultivation, key growth parameters, including plant height, fresh weight, and dry weight (85 °C oven-dried for 24 h), were measured, with three biological replicates per treatment, and 30 seedlings were included in each treatment group (n = 30).

Field experiment

A field experiment was conducted at the experimental base of the Biotechnology Research Institute, Chinese Academy of Agricultural Sciences (Dongying City, Shandong Province). The maize cultivar “Zhengdan 958” was used as the test crop. Sowing was performed from June 16–18, 2025, and yield determination was carried out on November 4, 2025. A randomized complete block design was employed with three treatments: a noninoculated control, root inoculation with wild-type A1501, and root inoculation with the OE-algU strain. For inoculation, bacterial cultures were grown in LB medium for 24 h and adjusted to an OD₆₀₀ of approximately 1.0. Maize seeds were immersed in the respective bacterial suspensions for 2 h before sowing; control seeds were treated with sterile LB medium. The seeds were sown at a spacing of 60 cm between rows and 25 cm within rows, with approximately 250 plants per plot. At maturity, three 2 m² sampling points were randomly selected per plot to determine the planting density. Representative plants were randomly chosen from each plot to measure agronomic traits, including ear length, ear diameter, single-ear weight, total number of kernels per ear, and 100-kernel weight. The grain yield per hectare was calculated based on plant density, kernel number per ear, and kernel weight.

Statistical analysis

All experiments were performed with at least three biological replicates. The data are presented as the mean ± standard error. Statistical significance was determined using Student’s t test or one-/two-way analysis of variance (ANOVA) followed by Tukey’s test in GraphPad Prism (P < 0.05).

Results

AlgU enhances salt stress tolerance and maintains key physiological functions

Prior to the phenotypic analysis, the successful construction of the ΔalgU and OE-algU strains was confirmed by PCR and sequencing (Supplementary Fig. S1). Phenotypic characterization under salt stress revealed that AlgU is a central regulator of both basal growth and acute tolerance in P. stutzeri. Growth analysis revealed that although all the strains grew similarly at NaCl concentrations ≤ 0.6 M, compared with the WT and ΔalgU strains, the OE-algU strain exhibited accelerated entry into the logarithmic phase at 0.8 M NaCl and was uniquely capable of substantial growth at the severely inhibitory concentration of 1.0 M NaCl (Fig. 1A). This requirement for the sustained growth of AlgU under high salinity was mirrored in acute survival assays. Following exposure to a gradient of high NaCl concentrations (2.0–5.0 M), the OE-algU strain consistently demonstrated the highest survival rate, whereas the ΔalgU mutant was the most sensitive, establishing a clear tolerance hierarchy: OE-algU > WT > ΔalgU (Fig. 1B).

Fig. 1.

Growth curves and acute salt shock tolerance of P. stutzeri wild-type, ΔalgU, and OE-algU strains under salt stress. A Growth curves of the wild-type (WT), ΔalgU, and OE-algU strains in K medium with increasing NaCl concentrations. Data points represent the mean OD₆₀₀ from three biological replicates. B Acute salt shock tolerance assay. Cells were exposed to the indicated NaCl concentrations for 1 h. Representative images of serial dilutions spotting on LB agar plates are shown

In addition to conferring cellular resilience, AlgU was essential for maintaining key plant-beneficial functions under saline conditions. Nitrogenase activity, which was unaffected by genotype in the absence of salt, strongly increased in an AlgU-dependent manner under stress. At 0.2 M NaCl, the OE-algU strain maintained activity comparable to those of the nonstressed strains, which was significantly greater than the diminished activity of the WT and ΔalgU strains; activity was nearly abolished in all strains at 0.4 M NaCl (Fig. 2A). Similarly, biofilm formation was fundamentally reliant on AlgU. The ΔalgU mutant was severely deficient in biofilm production even in the absence of stress. Although increasing salt concentrations universally inhibited biofilm formation, the OE-algU strain consistently outperformed the WT strain, resulting in greater biomass at each concentration, indicating that AlgU overexpression partially mitigated the salt-induced suppression of this critical colonization trait (Fig. 2B).

Fig. 2.

Nitrogenase activity and biofilm formation of P. stutzeri wild-type, ΔalgU, and OE-algU strains under salt stress. A Nitrogenase activity of the WT, ΔalgU, and OE-algU strains under the indicated NaCl stress conditions. Activity was measured in an acetylene reduction assay and is presented as the mean ± SE (n = 3). B Biofilm biomass quantified by crystal violet staining under increasing NaCl stress. Data are presented as the mean ± SE (n = 3). Asterisks indicate statistical significance: * denotes a significant difference (P < 0.05), and ** denotes an extremely significant difference (P < 0.01), as determined by one-way ANOVA followed by Tukey’s post hoc test

Transcriptome analysis reveals AlgU-mediated molecular mechanisms underlying salt stress adaptation

To elucidate the molecular mechanism by which AlgU regulates key phenotypes (biofilm formation, salt tolerance, and biological nitrogen fixation) of P. stutzeri under salt stress, RNA-seq analysis was performed on four experimental groups: WT (unstressed control), WT + NaCl, OE-algU + NaCl, and ΔalgU + NaCl. Transcriptome quality was validated by strict metrics (Supplementary Table S1), with all samples showing Q30 values ≥ 90%, ensuring reliability for downstream analyses. Principal component analysis revealed that PC1 (88.22% variance) and PC2 (11.78%) effectively captured the transcriptional differences (Fig. 3A). Although the biological replicates clustered tightly, the samples separated primarily along PC1 according to algU genotype, with OE-algU and ΔalgU forming discrete clusters distinct from the overlapping WT groups. To identify core AlgU-regulated genes, we focused on genes whose expression differed between OE-algU and the WT under NaCl stress (|log₂ FoldChange|≥ 1, padj < 0.01; Fig. 3B). A total of 987 differentially expressed genes (DEGs) were identified, including 742 upregulated and 245 downregulated genes. These DEGs were prominently enriched in four functional modules. Key genes involved in biofilm synthesis and regulation (wzc, pslA, algA, and dgc) and osmoprotection (otsA and osmC) were among the most significantly upregulated genes. Furthermore, a suite of genes central to energy metabolism, including those encoding rate-limiting enzymes in the TCA cycle (fumC) and the pentose phosphate pathway (zwf), were strongly upregulated.

Fig. 3.

AlgU orchestrates global transcriptional reprogramming under salt stress. A Principal component analysis (PCA) of RNA-seq data from the wild-type (WT), WT with NaCl stress, ΔalgU mutant with NaCl stress, and OE-algU strain with NaCl stress. B Volcano plot depicting genes whose expression differed between the OE-algU and WT strains under NaCl stress (|log₂FoldChange|≥ 1, padj < 0.01). Significantly upregulated and downregulated genes are coloured red and blue, respectively. Labelled genes with different filled colours correspond to four functional modules: energy metabolism (green), osmotic protection (orange), and biofilm synthesis and regulation (purple)

Building upon the genotype-driven transcriptional separation revealed by PCA, we further examined the expression patterns of key DEGs across the ΔalgU, WT, and OE-algU strains under NaCl stress. A Z score-normalized heatmap of 18 core DEGs clearly revealed AlgU-dependent expression gradients (Fig. 4A). Genes involved in critical functional modules—including energy metabolism (acnA, fumC, zwf), biofilm synthesis (rmlA, xcpR, xcpS), and osmotic protection (otsA, amiC)—displayed a consistent trend of progressive upregulation in the order ΔalgU < WT < OE-algU. In contrast, putP (encoding a sodium/proline symporter) and purK (involved in purine biosynthesis) were distinctly downregulated in the OE-algU strain.

Fig. 4.

Core genes and pathways regulated by algU. A Z score-normalized expression heatmap of core genes from key functional modules in ΔalgU with NaCl, WT with NaCl, and OE-algU with NaCl. Rows represent DEGs, and columns represent samples. The colour gradient reflects relative expression levels. Pathways and genes are grouped by functional modules (marked with different colours on the left: energy metabolism, biofilm synthesis, osmotic protection, and metabolic fine-tuning). B KEGG enrichment bubble plot of all the DEGs (both up- and down-regulated) between WT with NaCl and OE-algU with NaCl. The x-axis represents the Rich factor (the ratio of DEGs to total genes in the pathway), and the y-axis represents specific KEGG pathways (annotated with level 1 and level 2 functional classifications). Bubble size corresponds to the number of DEGs in the pathway, and bubble colour indicates enrichment significance (− log10(adjusted P value))

To define the functional pathways coordinated by AlgU, KEGG enrichment analysis was performed on the DEG sets identified between the WT and OE-algU strains under salt stress. The results, presented as a bubble plot (Fig. 4B), highlighted significant enrichment across several key categories. Central carbon metabolism pathways were strongly induced, with “starch and sucrose metabolism” (23 DEGs), “pentose phosphate pathway” (19 DEGs), and “glycolysis/gluconeogenesis” (14 DEGs) being prominently represented. Concurrently, pathways underlying biofilm matrix production and export were enriched, including “amino sugar and nucleotide sugar metabolism” (9 DEGs) and “bacterial secretion system” (12 DEGs). Furthermore, modules essential for osmotic homeostasis, such as the “two-component system” (53 DEGs) and “ABC transporters” (22 DEGs), were significantly activated.

Taken together, these transcriptional profiles demonstrate that AlgU orchestrates a coherent reprogramming of gene expression under salt stress, simultaneously upregulating the expression of genes responsible for energy generation, biofilm formation, and osmoprotection while selectively fine-tuning specific metabolic and transport functions.

Beyond the core stress-adaptation pathways, algU overexpression co-ordinately upregulated a broad repertoire of genes associated with plant growth-promoting (PGP) functions (Supplementary Table S2). These included genes encoding antioxidant enzymes (osmC, sodC, katE) and osmoprotectant synthesis/uptake systems (otsAB, betS), which enhance bacterial fitness and metabolic activity under salt stress, thereby enabling sustained rhizosphere colonization and continuous delivery of plant-beneficial functions (Liang et al. 2024; Mitra et al. 2025). Genes involved in nutrient mobilization were also upregulated, including the pyoverdine exporter opmQ for iron acquisition and the glucose dehydrogenase gene gcd for phosphate solubilization. Furthermore, AlgU activated numerous genes implicated in rhizosphere colonization, such as those involved in exopolysaccharide biosynthesis (rmlA-D, algA, wzc) and chemotaxis (mcpU). In total, we identified 60 AlgU-upregulated genes with potential roles in plant–microbe interactions, spanning diverse functional categories including antioxidant defence, osmoprotection, nutrient acquisition, cell envelope biogenesis, and signalling. This comprehensive transcriptional reprogramming positions AlgU as a master switch that converts P. stutzeri into a multifunctional biofertilizer optimized for salt-stressed environments.

Interestingly, within the 59-gene nitrogen fixation island, only three core structural genes—nifH gene coding for the nitrogenase iron protein and the nifDK genes coding for the molybdenum-iron protein—were significantly upregulated in the OE-algU strain under salt stress (Supplementary Table S2). In contrast, genes involved in nitrogenase maturation (nifM, nifU, nifS), FeMo-cofactor biosynthesis (nifB, nifE, nifN), and electron transport (nifF, nifJ) showed no significant changes. Whether the upregulation of these three nif genes is directly mediated by AlgU or via crosstalk with other regulators requires further investigation.

AlgU-overexpressing P. stutzeri increases maize salt stress tolerance in pot experiments

The effects of the WT and OE-algU strains on maize seedling growth under non-salt and 100 mM NaCl stress conditions are shown in Fig. 5. In the absence of salt stress, compared with no inoculation treatment, the inoculation with P. stutzeri (WT or OE-algU) promoted maize seedling growth, but no significant differences were detected between the WT and OE-algU groups (Fig. 5A, B). NaCl stress (100 mM) significantly inhibited maize seedling development, as evidenced by reduced plant size in noninoculated plants (Fig. 5A) and decreased growth parameter values (Fig. 5B). Inoculation with WT or OE-algU alleviated salt stress inhibition, particularly in terms of plant height, root length, leaf fresh weight, and leaf dry weight. Notably, under 100 mM NaCl stress, plants inoculated with the OE-algU strain exhibited significantly greater plant height compared to those inoculated with the WT strain (Fig. 5B), which indicated that OE-algU conferred an additional growth benefit beyond that provided by the WT under saline conditions.

Fig. 5.

Effects of Pseudomonas stutzeri strains on maize seedling growth under salt stress in pot experiments. A Representative photographs of maize seedlings under non-salt stress and 100 mM NaCl stress. B Box plots showing the results of the quantitative analysis of growth parameters, including plant height, root length, leaf fresh weight, leaf dry weight, root fresh weight, and root dry weight. Statistical significance is indicated by * (P < 0.05) and ** (P < 0.01) determined by one-way ANOVA with Tukey’s post hoc test

AlgU-overexpressing P. stutzeri improves maize yield in saline–alkali fields

Field experiments were conducted in saline‒alkali soil to verify the plant growth-promoting potential of AlgU-overexpressing P. stutzeri under natural stress conditions (Supplementary Table S3). Yield-associated trait profiling revealed distinct phenotypic differences in maize ear performance between the two inoculation groups, with OE-algU-inoculated maize exhibiting more robust ear phenotypes than the WT-inoculated group did. Quantitative analysis of yield-related parameters further confirmed these phenotypic differences (Fig. 6): among the six measured traits, ear weight, total grains per ear, and relative grain yield were significantly greater in the OE-algU group than in the WT group, whereas no significant differences were detected in ear length, ear diameter, or 100-grain weight between the two strains. Notably, compared with the WT strain (483.8 ± 18.4), the OE-algU strain presented significantly more total grains per ear (513.5 ± 15.7), which directly contributed to its significantly increased single-ear weight. With respect to relative grain yield (%), the OE-algU strain exhibited an 8.84% increase. These results demonstrate that AlgU increases the yield-promoting capacity of P. stutzeri in saline‒alkali fields, with the OE-algU strain exhibiting superior performance to that of the WT strain.

Fig. 6.

Statistical analysis of maize yield traits in response to P. stutzeri inoculation under saline‒alkali field conditions. Statistical comparison of yield-associated traits in maize inoculated with P. stutzeri wild-type (WT) and algU-overexpressing (OE-algU) strains under saline‒alkali field conditions. The evaluated traits included ear length, ear diameter, single-ear weight, total grain number per ear, 100-grain weight, and grain yield. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, where * denotes P < 0.05 and ** denotes P < 0.01

Discussion

This study reveals a comprehensive regulatory role for the extracytoplasmic function of the sigma factor AlgU in enabling the diazotrophic rhizobacterium P. stutzeri A1501 to adapt to salt stress. Through the generation and phenotypic analysis of isogenic algU knockout and overexpression strains, we demonstrated that AlgU functions as a master regulator, coordinating a multifaceted physiological response that encompasses enhanced biofilm formation, osmoprotection, and energy metabolism. Crucially, this AlgU-directed reprogramming directly safeguards the activity of nitrogenase—a core plant-beneficial function highly sensitive to salt. This molecular and cellular adaptation translated into superior plant growth promotion under saline conditions, as evidenced by the results of pot experiments and, pivotally, by increased grain yield in field trials with the engineered OE-algU strain.

Our findings significantly expand the functional understanding of AlgU beyond its well-characterized roles in virulence and stress responses in pathogenic pseudomonads, establishing its importance for beneficial traits in a plant growth-promoting rhizobacterium (PGPR) (Li et al. 2021; Pezzoni et al. 2022). Under salt stress, AlgU activates a synergistic defence network by upregulating exopolysaccharide biosynthesis (pslA) and c-di-GMP signalling genes to increase biofilm formation (a physical barrier against ionic intrusion) (Shao et al. 2022), inducing osmoprotective pathways (otsA) and antioxidant systems to maintain cellular homeostasis, and reinforcing central carbon metabolism (zwf, fumC) to supply ATP and NADPH, which are essential for nitrogenase activity under stress (Chevalier et al. 2022). Concomitant downregulation of putP and purK in the OE-algU strain also reflects a refined metabolic strategy prioritizing stress adaptation over nonessential anabolism. Moreover, AlgU-mediated upregulation of energy metabolism and osmoprotection genes complements the function of NtrC—a global nitrogen regulator that controls nitrogen metabolism and oxidative stress responses—with both regulators likely cooperating to safeguard nitrogenase under adverse conditions (Wang et al. 2023). AlgU also engages in hierarchical crosstalk with other global regulators: RpoN directly activates algU transcription (Shao et al. 2022), whereas RsmA may posttranscriptionally modulate algU mRNA stability (Lv et al. 2024), resulting in the formation of a regulatory network that dynamically balances nitrogen fixation, biofilm formation, and stress adaptation in the rhizosphere. Notably, increasing salt tolerance and promoting plant growth via overexpression of AlgU is a complementary strategy to metabolic engineering approaches such as the construction of ammonium-excreting strains (Jiang et al. 2022). Combining transcriptional regulation engineering (AlgU overexpression) with metabolic pathway engineering could generate next-generation PGPR inoculants with stronger competitiveness and functional resilience, synergistically improving crop yield and nutrient use efficiency in saline‒alkali soils.

A key element of the AlgU-mediated strategy is the direct mitigation of the primary components of salinity stress: osmotic imbalance and oxidative damage (Hasanuzzaman et al. 2024). The strong induction of the otsAB operon, responsible for trehalose biosynthesis, is particularly compelling. Trehalose is a potent osmoprotectant that enhances bacterial survival in hyperosmotic environments (Wang et al. 2024). More importantly, when exuded into the rhizosphere, it can directly stabilize plant cell membranes and proteins, thereby alleviating osmotic stress in the host plant (He et al. 2018). Concurrently, the upregulation of genes encoding antioxidant enzymes such as catalase (katE) and superoxide dismutase (sodC), along with the osmotically inducible peroxidase (osmC), suggests the creation of a protective microbial shield in the rhizosphere. This shield can detoxify reactive oxygen species (ROS) generated by salt stress, which are a primary cause of root cell damage and growth inhibition (Kumar et al. 2024). This dual approach of managing both osmotic and oxidative stress at the root-soil interface is a hallmark of effective PGPR and is consistent with the multifaceted mechanisms employed by diverse PGPR to confer induced systemic tolerance in plants (Etesami and Maheshwari 2018; Luo et al. 2024).

Beyond direct stress mitigation, the AlgU regulon encompasses traits that actively promote plant growth by enhancing nutrient availability, a critical function as salinity often impairs nutrient uptake (Lin et al. 2025). The upregulation of opmQ, a component of the pyoverdine secretion system, indicates that the OE-algU strain maintains or enhances siderophore production. Siderophores are crucial for chelating ferric iron (Fe3⁺) in the soil, making it available for uptake by both the bacterium and the plant. This phenomenon is particularly important in saline soils, which are often alkaline and have low iron bioavailability. The ability to provide iron is a well-documented PGP trait (Gupta and Pandey 2019; Rokhbakhsh-Zamin et al. 2011). Furthermore, the modest but significant upregulation of gcd, which encodes glucose dehydrogenase, points to an enhanced capacity for mineral phosphate solubilization via the production of gluconic acid. This is another cornerstone PGP mechanism that converts insoluble soil phosphates into forms that are accessible to plants (Alotaibi et al. 2024; El-Sayed et al. 2014). The co-regulation of nitrogen fixation, iron acquisition, and phosphate solubilization under a single transcriptional regulator highlights an elegant and efficient system for comprehensive nutritional support, likely underpinning the superior growth observed in inoculated plants under challenging field conditions.

A key novelty of our work lies in directly linking AlgU to the preservation of biological nitrogen fixation—a core plant-beneficial function—under salt stress. AlgU is traditionally associated with pathogenesis (Shahrokhi et al. 2022; Shang et al. 2021; Zhang et al. 2024); however, here, it appears “recruited” to safeguard against agriculturally vital processes in a PGPR (Wang et al. 2023). These findings indicate that AlgU-mediated adaptation creates a protected and energetically favourable environment for nitrogen fixation, while simultaneously activating a suite of complementary PGP traits that address osmotic stress, oxidative damage, and nutrient limitation. The practical relevance of this mechanism was confirmed in field trials, where the OE-algU strain significantly outperformed the wild type, demonstrating its superior yield-promoting capacity under field conditions. This successful translation from molecular mechanisms to field efficacy underscores the potential of engineering master regulators such as AlgU for developing next-generation, stress-resilient microbial inoculants, offering a sustainable strategy for improving crop productivity in saline‒alkali soils (Heng et al. 2022).

Despite these advances, the limitations warrant future investigation. Although the survival dynamics and colonization activity of the parental strain P. stutzeri A1501 in the rice rhizosphere under saline stress have been well characterized (Wu et al. 2000), the survival rate and metabolic activity of the OE-algU strain in the maize rhizosphere of saline–alkali soils remain to be elucidated and will be a key focus of our subsequent work. Our transcriptomic analysis, conducted at a single time point (1 h after salt stress), provides a critical snapshot but not the dynamic progression of the AlgU regulon; time series omics studies will elucidate the temporal hierarchy of this stress response. Furthermore, distinguishing direct from indirect AlgU targets via ChIP-seq will precisely map its sigmulon—an approach successfully applied in other pseudomonads (Huang et al. 2019). The potential cross-talk between AlgU and other global regulators (RpoN and Hfq) involved in the integration of environmental signals also remains unexplored, as recent work suggests that Hfq interacts with algU mRNA to regulate nitrogen fixation in A1501 (Lv et al. 2024). Addressing these fundamental questions will refine our understanding of the AlgU regulatory network and enable more rational engineering of the stress-resilient PGPR.

The agricultural application of engineered microbial strains has long been a topic of considerable public concern, and different countries and organizations have established corresponding regulatory frameworks and guidelines for genetically modified microorganisms (Chemla et al. 2025; Shams et al. 2024). In China, a comprehensive regulatory system governs agricultural genetically modified microorganisms, with strict approval and supervision implemented by the competent departments of the Ministry of Agriculture and Rural Affairs. Genetically modified organisms that pass safety assessments are granted safety certificates and permitted for environmental release. Notably, several agricultural microbial strains have already been approved as successful examples. For instance, Bacillus thuringiensis G033A obtained a transgenic safety certificate in 2024 (Agricultural GMO Safety Certificate No. 2024–299). As early as 2000, the ammonium-tolerant engineered strain AC1541, derived from the parental strain A1501 used in this study, received a commercial production certificate in Liaoning Province, China (Agricultural GMO Safety Approval No. 2000A-02–09). The field trials of the OE-algU strain developed in this study were conducted under official approval and filed with the Ministry of Agriculture and Rural Affairs (Agricultural GMO Office Report No. 2025–1154). The present study demonstrates that the OE-algU strain exhibits significant growth-promoting effects on maize in saline-alkali soils. Accordingly, we will proceed with the application for a transgenic safety certificate in accordance with the regulatory requirements of China’s Ministry of Agriculture and Rural Affairs, with commercialization to be implemented following official approval. For other countries and regions, including the United States, European nations, and African countries, the strain will be applied in agriculture only after completing standardized approval procedures in full compliance with local regulatory requirements.

Conclusion

In conclusion, this study establishes the ECF-σ factor AlgU as a master regulator that orchestrates a multi-layered adaptive strategy in P. stutzeri A1501, enabling it to withstand salt stress while preserving and even enhancing its plant-beneficial functions. By integrating biofilm formation, osmoprotection, energy metabolism, and—crucially—the coordinated activation of multiple plant growth-promoting traits (including antioxidant defencse, iron acquisition, and phosphate solubilization), AlgU transforms a naturally competent PGPR into a robust biofertilizer optimized for saline environments. The OE-algU strain significantly increased maize grain yield in coastal saline–-alkali soil, demonstrating that targeted modulation of a single master regulator can yield agriculturally significant outcomes. Beyond its practical implications, this work expands the functional paradigm of AlgU from its well-known roles in pathogenesis to beneficial plant–microbe interactions, highlighting the evolutionary plasticity of stress-responsive regulators. Collectively, our findings provide a mechanistic framework and a proof-of-concept for engineering next-generation, stress-resilient PGPR inoculants, offering a sustainable strategy to enhance crop productivity in salt-affected agricultural systems.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

X.L. performed the investigation, data analysis, and contributed to manuscript preparation. C.T. conducted the experiments, formal analysis, and drafted the original manuscript. J.Z. and X.K. participated in the investigation and visualization. Y.Z. conceived and supervised the study, secured funding, and critically revised the manuscript. W.L. designed and conducted the field experiments, performed data analysis, and contributed to manuscript review and editing. M.L. provided essential research resources. L.C. conceived the research framework. H.W. constructed the mutant strains, performed phenotypic characterization, and engaged in manuscript review and editing. Y.Y. was involved in conceptualization, research supervision, and funding acquisition. All authors reviewed and approved the final manuscript.

Funding

This work was supported by grants from the National Key R&D Program of China (2024YFA0918200), National Natural Science Foundation of China (32270067 and 32370091), Agricultural Science and Technology Innovation Program (CAAS-ZDRW202308), Hainan Seed Industry Laboratory and China National Seed Group (project of ZZGS-ZNBM-2025-230) and the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA28030201).

Data availability

The RNA-Seq data were deposited into the NCBI database under accession number PRJNA1405500.

Declarations

Ethical approval

Not applicable, as this article does not contain any studies with human participants or animals performed by any of the authors.

Competing interests

The authors declare no competing interests.