Journal Pre-proof Priority changes between the synthesis of biofilm exopolysaccharides and rhamnolipids are mediated by a cyclic di-GMP-specific phosphodiesterase NbdA in Pseudomonas aeruginosa

The synthesis of biofilm exopolysaccharides and rhamnolipids (RLs) are two interrelated 18 processes in Pseudomonas aeruginosa , but how bacteria coordinate these two processes 19 remains unclear. We collected a P. aeruginosa KT1115 with rugose small colony variant 20 (RSCV) phenotype from soil, and used it to study the dynamic regulation mechanism of 21 biofilm polysaccharide and RLs synthesis. The results showed that the overproduction of 22 biofilm exopolysaccharides at biofilm stage ultimately contributed the surge of RLs 23 production at RLs stage. This phenomenon was further verified by comparing PAO1 with 24 its engineered RSCV mutant, PAO1Δ wspF . Further genomic, transcriptomic analyses and 25 gene deletion revealed that downregulation of c-di-GMP level was the key to switch 26 biofilm exopolysaccharides accumulation to RLs surge, by transcriptionally upregulating a 27 c-di-GMP phosphodiesterase NbdA. Overall, this study demonstrates the importance of c- 28 di-GMP in coordinating biofilm exopolysaccharides and RLs synthesis, and provides an 29 inspiration for enhancing RLs production through regulating c-di-GMP level. monitored the dynamic changes in exopolysaccharides and RLs production by using the P. aeruginosa RSCV strain KT1115 during a long fermentation process. We found that the formation of RSCV phenotype (overproduction of biofilm exopolysaccharides, Psl and Pel) at the early stage benefits the production of RLs at the late stage. This phenomenon was also verified by comparing PAO1 with its engineered RSCV mutant PAO1Δ wspF . In addition, we systematically analyzed their intracellular c-di- GMP and gene expression patterns using transcriptome data. Our data imply that c-di- GMP is a key player involved in the switch between the biofilm stage and the RLs stage by targeting genes in both exopolysaccharides and RLs synthesis pathways. Finally, our results reveal that the c-di-GMP-related PDE, NbdA, plays a key role in regulating the switch between the biofilm and RLs stages by controlling the c-di-GMP level. Our study sheds new light on the relationship between biofilm exopolysaccharides formation and RLs synthesis and provides strong inspiration for the application of microbial biofilm engineering in fermentation processes as an alternative strategy to improve the yields of RLs and other metabolites. Our previous work showed that P. aeruginosa KT1115 produces a high ratio of di-RLs after fermentation 18,19 . Here, we examined several other phenotypes of KT1115, including colony morphology, biofilm formation, and total RLs production. KT1115 showed rugose colonies with darker colors on Congo red agar and formed robust biofilms with approximately 1.6-fold higher levels of biofilm exopolysaccharides than PAO1, a model laboratory strain 1A and 1D). A high level of biofilm exopolysaccharides usually results in bacterial aggregation in planktonic culture 20,21 . We then used bacterial aggregation to estimate the biofilm formation of KT1115 in liquid culture. The results show that the auto-aggregation of KT1115 cells was a macroscopic level in LB medium, whereas PAO1 did not show any auto-aggregation under same 1C S1C). In rapeseed oil nitrate (RON ) medium, when the aggregation was a coacervate 100 200 S1A). These results also confirm that strain KT1115 exhibits a typical RSCV phenotype along with hyper-biofilm formation ability 22,23 overproduction of exopolysaccharides and decrease RLs production by competing for common sugar precursors. At the RLs stage, upregulation of the PDE gene nbdA caused a 305 significant reduce of the c-di-GMP levels, led to transition from the biofilm stage to the RLs stage along with a rapid accumulation of RLs. These findings provide novel insights regarding how RSCV strains coordinate biofilm formation and RLs synthesis and form a theoretical basis for improving the understanding of RLs fermentation behavior, which can be applied for regulating the production cycle of RLs in natural producers and enhancing RLs accumulation in heterologous hosts.