A bacterial ally for nitrogen-fixing biofilm: enhancing the rhizosphere colonization of <i>Stutzerimonas stutzeri</i> A1501 with surfactin-producing <i>Bacillus velezensis</i> BRI3

ABSTRACT 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.