Study of the genome of Bacillus amyloliquefaciens subsp. plantarum S499 and role of its plasmid (pS499) in rhizosphere interactions

Understanding how soil-borne microorganisms can modulate the plant defence responses and which factors affect rhizosphere multitrophic interactions is crucial to improve the efficacy of biopesticides in agriculture. From this perspective, whole-genome sequencing is a powerful tool to characterize the bacterial strains of agronomic interest. Among these, Bacillus amyloliquefaciens subsp. plantarum strain S499 is a plant-beneficial rhizobacterium that shows direct antagonistic properties against phytopathogenic fungi and, in addition, a remarkable efficacy as elicitor of induced systemic resistance (ISR) in plants. In these activities, the production of bioactive secondary metabolites, such as cyclic lipopeptides belonging to the fengycin, iturin and surfactin families, is involved. By sequencing, assembling and annotating S499 genome, we identified the principal genes involved in root colonization, plant-growth promotion and biocontrol activities. These genes share a high percentage of nucleotide identity with their homologs in the strain FZB42, the type strain of the bacterial subspecies. One of the main genetic elements distinguishing S499 from FZB42 is the presence of extrachromosomal DNA (plasmid pS499). This small rolling circle plasmid was unknown before S499 genome sequencing, which also allowed to identify on pS499 the genes encoding a Rap-Phr regulatory system involved in quorum sensing. Through a plasmid-curing approach, we carried out a functional characterization of pS499. First, we studied the impact of the plasmid loss on the bacterial physiology, by comparing the behaviours of S499, its plasmid-cured derivative, S499 P-, and FZB42 on Luria-Bertani (LB) medium. Growth rate, extracellular proteolytic activity and the regulation of lipopeptide production were significantly affected in S499 P-. In agreement with an increased release of surfactins, swarming motility improved after curing, whereas biofilm production was reduced in vitro. When the evolution of bacterial populations was compared in planta, pS499 seemed not to influence the root colonization ability, although we observed an over-production of surfactins by S499 Palso on tomato roots. The quantification of the relative expression of srfA and rap genes suggested an inhibitory effect of the plasmidencoded Rap-Phr system on surfactin synthesis. Moreover, on LB, the antagonistic effect against phytopathogenic fungi was limited for S499 P-, most probably due to a verified reduction of iturin secretion. Although less clearly, an impact of plasmid curing on the biocontrol ability was observed also on a medium (RE) that reproduced the typical composition of plant root exudates. Globally, our results show that pS499 differently modulates S499 phenotype depending on the nutritional context. More evidences are required to prove that pS499 is relevant for the fitness of the rhizobacterium in its natural environment.