Identification of distinct N-acyl homoserine lactone profiles in non-hemolytic plant-associated symbiotic and non-symbiotic rhizobacteria.

Quorum sensing (QS) is a critical bacterial communication system in the plant rhizosphere regulating interactions and biofilm formation, which are essential for survival. While AHL profiles in rhizobacteria are important for sustainable agriculture, a comprehensive understanding of their diversity, particularly in beneficial, non-hemolytic strains, remains limited. This study addresses this gap by providing a comparative analysis of N-acyl homoserine lactone (AHL) profiles and biofilm capabilities in a panel of symbiotic Ensifer meliloti and non-symbiotic Klebsiella pneumoniae and Klebsiella quasi-pneumoniae strains. For definitive taxonomic identification, the two MTCC strains (Ensifer meliloti RM and SINO) were subjected to 16S rRNA gene sequencing and phylogenetic analysis. Initial biosensor screening confirmed AHL production in all tested strains. Four strong AHL producers were selected for in-depth analysis. While Autoinducer Type-II (AI-2) signaling was absent, all strains demonstrated robust biofilm formation. Detailed LC-MS/MS profiling revealed distinct AHL profiles for each strain, highlighting significant intra-species diversity. Notably, this study reports the first identification of N-3-oxo-hexanoyl-HSL (C6-oxo-HSL) in Ensifer meliloti strain RM (10499). Furthermore, the non-hemolytic Klebsiella strains exhibited a profile dominated by 3-O-C8-HSL and 3-O-C12-HSL, which significantly differs from profiles reported in pathogenic Klebsiella strains, suggesting a link between QS systems and bacterial lifestyle. The identification of these specific AHL signals in robust biofilm-forming, non-hemolytic bacteria underscores their potential as safe and effective bioinoculants for enhancing rhizosphere colonization and supporting sustainable agriculture.