Azospirillum baldaniorum SP245 INDUCE BIOFILM FORMATION AND SWITCH TO A MICROAEROBIC METABOLISM WHEN CO-CULTURED WITH Pseudomonas fluorescens A506

AMENTA MB; MARONICHE GM; BONATO P; DIAZ P; PAGNUSSAT LA; SOUZA EM; CREUS CECILIA

Congreso; SAMIGE 2022; 2022

Azospirillum and Pseudomonas are two genera of rhizobacteria that are often used together todevelop crop inoculants. The success of the interaction between them depends on the straincompatibility and the culture conditions. Previous results from our laboratory indicated that in mixedmacrocolony biofilms P. fluorescens A506 is able to stimulate the growth of A. baldaniorum Sp245,while Sp245 negatively affects the growth of A506. In this work, we investigated the underlyingmechanisms of this interaction through RNA-seq focusing mainly on changes in the transcriptionprofile of Sp245. Total RNA was isolated from single and mixed Sp245 and A506 cultures grown inpolystyrene Petri dishes and NFb-NO3 liquid media for 2 days under static conditions, in triplicates.The mRNA was enriched, fragmented and converted to cDNA, which was then amplified andsequenced on an Ion Proton platform. Output reads were filtered, fractionated by mapping to genomic sequences with Bowtie2, and analyzed for differential expression with feature Counts and DESeq2.The results allowed us to identify 245 genes from Sp245 that were differentially expressed in mixedcultures (FDR±1.5). When co-cultured with A506, Sp245 upregulated several genesrelated to carbon uptake and metabolism, stress response, T6SS secretion system and nitrogenfixation; and downregulated genes associated to cellular cycle, motility, chemotaxis, aerobiccatabolism and denitrification, among others. These results suggest that Sp245 adapts to the presenceof A506 switching to a microaerobic metabolism and inducing a state of resistance/endurance,possibly by increasing biofilm formation. To test this hypothesis, we analyzed the number of total,planktonic and attached cells of Sp245 and A506 in static single and mixed cultures. We observed thattotal growth of Sp245 is not influenced by the presence of A506, while A506 decreases its growth byan order of magnitude when cultured with Sp245. Furthermore, we found that Sp245 tends to developmore biofilm when cultivated with A506 as opposed to when grown alone. When we swapped outSp245 for Faj164, an isogenic mutant of Sp245 deficient in a periplasmic nitrate reductase (NapA-) that struggles to thrive under microerophilic conditions when using nitrate as N source, that tendency was reversed. Taken together, these evidences indicate that A. baldaniorum Sp245 and P. fluorescens A506 compete when co-cultured in static culture biofilms. As a consequence, Azospirillum induce stresscoping mechanisms, such as biofilm formation, and Pseudomonas reduces its growth. In addition, our results suggest that oxygen plays an important role in modulating the interaction between both strains in static culture biofilms.