Small non-coding RNA <i>fen36</i> : a novel positive regulator of biofilm formation and swarming motility in <i>Bacillus amyloliquefaciens</i>

ABSTRACT Biofilm formation is a critical developmental process for the survival of Bacillus , its environmental adaptation, and host colonization. This study investigated the regulatory function of the novel small non-coding RNA (sRNA) fen36 in biofilm architecture and swarming motility in Bacillus amyloliquefaciens . A comparative transcriptomic analysis was carried out between the wild-type B. amyloliquefaciens LPB-18 and a fenSr3 deletion mutant (LPB-18N). This analysis, integrated with IntaRNA-based thermodynamic predictions, identified fen36 as a highly upregulated sRNA. Subsequently, functional assays utilizing isogenic fen36 -knockout (LPB-18NΔ fen36 ) and overexpression (LPB-18N:: fen36 ) strains established fen36 as a positive regulator of biofilm formation. Specifically, the overexpression of fen36 yielded a 3.59-fold increase in biofilm biomass, induced a hyper-swarming phenotype, and generated highly wrinkled biofilm topologies, as confirmed by scanning electron microscopy (SEM). Crucially, these phenotypic enhancements occurred without compromising planktonic growth kinetics or the biosynthesis of the antifungal lipopeptide fengycin. In vitro dual-culture assays further demonstrated that the fen36 overexpression strain maintained potent antagonistic efficacy against the phytopathogen Fusarium oxysporum . Mechanistic investigations employing a dual-plasmid reporter system and RT-qPCR revealed that fen36 targets the 5′-untranslated region of tasA , upregulating this core matrix gene. Furthermore, the transcription of fen36 is negatively regulated by the stress-responsive sRNA fenSr3 . Collectively, these findings elucidate a novel fenSr3-fen36-tasA regulatory cascade that governs biofilm architecture and motility independent of secondary metabolism, offering a precise genetic target for optimizing Bacillus biocontrol performance. IMPORTANCE Bacillus amyloliquefaciens is extensively harnessed in agriculture for its robust rhizosphere colonization and antimicrobial lipopeptide synthesis. Understanding the genetic networks uncoupling physical colonization from secondary metabolism is critical for engineering superior biocontrol agents. This study elucidates a novel post-transcriptional regulatory cascade, fenSr3-fen36-tasA, governing multicellular behavior. The newly identified sRNA fen36 significantly enhances biofilm formation and hyper-swarming motility by upregulating the matrix gene tasA. Crucially, this enhancement occurs without disrupting fengycin biosynthesis, maintaining potent antagonism against phytopathogens such as Fusarium oxysporum . By mapping this dual-sRNA hierarchy, our research provides crucial mechanistic insights into bacterial environmental adaptation, offering refined genetic targets to optimize Bacillus strains for sustainable agricultural applications.