RDAR biofilm formation and in silico analysis of putative amyloid proteins in Azospirillum baldaniorum Sp245

Abstract Azospirillum baldaniorum Sp245 is a plant growth-promoting rhizobacterium (PGPR) whose ability to benefit host crops depends on robust root colonization, a process driven by the transition from a planktonic state to a sessile, biofilm-associated lifestyle. This study investigated the capacity of A. baldaniorum Sp245 to form Red, Dry, and Rough (RDAR) morphotype biofilms. Through macroscopic evaluation and confocal laser scanning microscopy (CLSM), we visualized the presence of exopolysaccharides, putative amyloid proteins, and extracellular DNA (eDNA) within the RDAR biofilm matrix using Calcofluor White (CFW), Thioflavin S (TS), and Propidium Iodide (PI) fluorescent probes, respectively. Furthermore, formic acid treatment of extracellular matrix proteins extracts suggests the presence of resistant, putative amyloid proteins. Because genomic screening revealed the absence of homologs of a canonical curli-dependent amyloid system, we performed a genome-wide in silico analysis to identify genes encoding alternative biofilm-associated proteins. Using the Staphylococcus aureus Biofilm-Associated Protein (Bap protein) as a reference, we identified 20 candidate proteins characterized by extensive domain reiteration and diverse architectures, including bacterial immunoglobulin (Ig-like) domains, VCBS repeats, SWM motifs, DUF4347, PKD domains, and RTX β-roll structures. Three-dimensional modeling indicates that these candidates possess elongated, filament-like conformations with repetitive β-strand motifs typical of amyloid-like assembly and calcium-dependent adhesion. These findings suggest that in the absence of traditional curli machinery, A. baldaniorum Sp245 could be using a diverse, domain-rich repertoire of alternative matrix proteins to facilitate cell surface interactions, coordinate structural stability, and establish the RDAR biofilm morphotype. Ultimately, this study establishes a foundation for understanding the role of amyloid proteins in Azospirillum biofilm matrix formation and offers new insights into the potential mechanisms of bacterial persistence and ecological fitness in the rhizosphere.