Members of the Microvirga genus are metabolically versatile and widely distributed in Nature. However, knowledge of the bacteria that belong to this genus is currently limited to biochemical characteristics. Herein, a novel thermo-tolerant bacterium named Microvirga thermotolerans HR1 was isolated and identified. Based on the 16S rRNA gene sequence analysis, the strain HR1 belonged to the genus Microvirga and was highly similar to Microvirga sp. 17 mud 1-3. The strain could grow at temperatures ranging from 15 to 50 °C with a growth optimum at 40 °C. It exhibited tolerance to pH range of 6.0–8.0 and salt concentrations up to 0.5% (w/v). It contained ubiquinone 10 as the predominant quinone and added group 8 as the main fatty acids. Analysis of 11 whole genomes of Microvirga species revealed that Microvirga segregated into two main distinct clades (soil and root nodule) as affected by the isolation source. Members of the soil clade had a high ratio of heat- or radiation-resistant genes, whereas members of the root nodule clade were characterized by a significantly higher abundance of genes involved in symbiotic nitrogen fixation or nodule formation. The taxonomic clustering of Microvirga strains indicated strong functional differentiation and niche-specific adaption.

Diazotroph mutants designed using metabolic engineering to excrete surplus ammonium were used to enhance nitrogen fixation and plant growth, as the levels of nitrogen fixation attained with diazotrophs are insufficient for the plant’s needs. In this study, wild-type (A1501) and engineered ammonium-excreting (1568/pVA3) strains of nitrogen-fixing Pseudomonas stutzeri strains were tested in vitro based on plant growth-promoting traits, such as phosphate solubilization ability, indole acetic acid (IAA) production and nitrogenase activities, as well as ammonium excretion as affected by mannitol-mediated osmotic stress. The maize plant growth-promoting effect of the A1501 and 1568/pVA3 strains was evaluated in pots and in the field, and the ¹⁵N-dilution technique was employed to assess the proportion of plant nitrogen derived from nitrogen fixation. The results demonstrate that the 1568/pVA3 strain displayed higher IAA production and nitrogenase activity than A1501 and released significant quantities of ammonium. After 50 days, in all of the conditions assayed, maize inoculated with 1568/pVA3 accumulated more plant biomass (3.3% on average) and fixed N (39.4% on average) than plants inoculated with A1501. In the field experiment, the grain yield of maize was enhanced by 5.6% or 5.9% due to the inoculation of seeds with 1568/pVA3 in the absence or presence of exogenous N fertilizer, respectively. Therefore, the engineered P. stutzeri strain tested in the greenhouse and field was shown to perform better than the wild-type strain with respect to maize growth parameters and biologically fixed nitrogen.

Pseudomonas stutzeri A1501, a plant-associated diazotrophic bacterium, prefers to conform to a nitrogen-fixing biofilm state under nitrogen-deficient conditions. The extracytoplasmic function (ECF) sigma factor AlgU is reported to play key roles in exopolysaccharide (EPS) production and biofilm formation in the Pseudomonas genus; however, the function of AlgU in P. stutzeri A1501 is still unclear. In this work, we mainly investigated the role of algU in EPS production, biofilm formation and nitrogenase activity in A1501. The algU mutant ΔalgU showed a dramatic decrease both in the EPS production and the biofilm formation capabilities. In addition, the biofilm-based nitrogenase activity was reduced by 81.4% in the ΔalgU mutant. The transcriptional level of pslA, a key Psl-like (a major EPS in A1501) synthesis-related gene, was almost completely inhibited in the algU mutant and was upregulated by 2.8-fold in the algU-overexpressing strain. A predicted AlgU-binding site was identified in the promoter region of pslA. The DNase I footprinting assays indicated that AlgU could directly bind to the pslA promoter, and β-galactosidase activity analysis further revealed mutations of the AlgU-binding boxes drastically reduced the transcriptional activity of the pslA promoter; moreover, we also demonstrated that AlgU was positively regulated by RpoN at the transcriptional level and negatively regulated by the RNA-binding protein RsmA at the posttranscriptional level. Taken together, these data suggest that AlgU promotes EPS production and nitrogen-fixing biofilm formation by directly activating the transcription of pslA, and the expression of AlgU is controlled by RpoN and RsmA at different regulatory levels.

EPSP synthase is the target enzyme of glyphosate herbicides. Due to the extensive use of glyphosate, it is very important to obtain EPSPS genes with high glyphosate resistance for the development of transgenic crops. GR79-EPSPS is a class I EPSP synthase with certain glyphosate resistance isolated from glyphosate-contaminated soil. After more than 1000 generations, a Y40I substitution was identified, and the enzyme had a nearly 1.8-fold decrease in Km [PEP] and a 1.7-fold increase in Ki[glyphosate] compared to the wild-type enzyme. Enzyme dynamics and molecular dynamics analysis showed that the substitution was near the hinge region of EPSPS, and the affinity of glyphosate binding to amino acid residues of the active site decreased due to Y40I substitution, resulting in an increase in glyphosate resistance. These results provide more evidence for the combination of directed evolution and rational design of protein engineering.

Nitrogen acquisition is pivotal for plant growth. In soil ecosystems, bacterial interactions promote nitrogen assimilation and rhizosphere colonization. However, the mechanisms underlying the interactions between nitrogen-fixing microorganisms and their neighboring organisms in the environment remain unclear. Here, we demonstrate that Bacillus velezensis BRI3, a rhizosphere-derived strain, forms microbial synergy with Stutzerimonas stutzeri A1501, functioning as a facilitator. This microbial synergy greatly increases the nitrogen-fixation by 3.2-fold and rhizosphere colonization capabilities by 2.3-fold of A1501, collectively promoting plant growth in the rhizosphere. In this study, for the first time, we propose that surfactin produced by BRI3 regulates interactions among this bacterial consortium by stimulating A1501 biofilm formation. This discovery enhances the understanding of metabolic interactions between nitrogen-fixing bacteria and their neighboring organisms. Overall, we propose a strain interaction paradigm that offers a novel framework for improving nitrogen utilization and crop yield. IMPORTANCE: Nitrogen is essential for crop productivity because it directly participates in the construction of proteins and nucleic acids. Associative diazotrophs convert N₂ into NH₄ ⁺ , yet require root biofilms and stable colonization. Owing to the complexity of the rhizospheric microbiota, a systematic understanding of microbe–microbe interactions and their impact on nitrogen-fixation capacity is still lacking. This study uncovers a novel intergeneric synergism in which B. velezensis BRI3 secretes surfactin that triggers S. stutzeri A1501 biofilm formation and concurrently elevates nif gene expression, thereby facilitating the integration of microbe–microbe interaction, biofilm development, and nitrogen-fixation efficiency into a single linear pathway. This phenomenon also provides a portable molecular-to-phenotypic blueprint for designing composite inoculants. Second, field trials revealed that coinoculation of these strains boosted maize growth, allowing partial synthetic-N replacement without transgenes or high costs, merely via rational strain formulation. This study highlights a transition from focusing on the ecological features of associative bacteria toward the development of deployable technology, offering a theory and prototype for sustainable agriculture.

A Gram-stain-negative, aerobic, rod-shaped, oxidase- and catalase-positive and non-motile bacterium, named MINF-07-Sa-05^T, was isolated from sediment samples collected from Niya Fish Salt Lake in Xinjiang, northwest China. Phylogenetic tree based on 16S rRNA gene sequences showed that strain MINF-07-Sa-05^T consistently fell within the genus Nitrincola and formed a clade together with Nitrincola alkalilacustris ZV-19^T (98.97%) and Nitrincola lacisaponensis 4CA^T (96.16%). Genomic average nucleotide identity values for strain MINF-07-Sa-05^T with the type strains within the genus Nitrincola ranged from 69.8 to 87.8%, while the in silico DNA–DNA hybridization values for strain MINF-07-Sa-05^T with these strains ranged from 20.4 to 33.7%. The genome sequence size of the strain was 4.81 Mb, with a DNA G+C content of 52.6 mol%. Its growth occurred at 4–40 °C, at pH 6.0–10.0, and tolerated up to 9% (w/v) NaCl. The major fatty acids contained summed feature 8 (C_18:1  ω7c/C_18:1  ω6c; 59.8%), summed feature 3 (C_16:1  ω7c/C_16:1  ω6c; 20.4%) and C_16:0 (13.8%). The primary respiratory quinone was ubiquinone-8. The main polar lipids were phosphatidylglycerol and phosphatidylethanolamine. Genomic analyses showed a high abundance of genes involved in the nitrogen, sulphur cycle and antibiotic resistance. According to the genotypic, phylogenetic and chemotaxonomic data, strain MINF-07-Sa-05^T represents a novel species within the genus Nitrincola, for which the name Nitrincola salilacus sp. nov. is proposed. The type strain is MINF-07-Sa-05^T (=GDMCC 1.4420^T=JCM 37233^T).