INVESTIGATING THE ROLE OF 16550 IN BIOFILM FORMATION OF PSEUDOMONAS AERUGINOSA PA14

Abstract

Pseudomonas aeruginosa is a pathogenic bacterium that commonly infects hospitalized patients and individuals with cystic fibrosis. This bacterium is particularly known for its ability to form biofilms, which are communities of bacterial cells enveloped by a self-produced extracellular matrix. This matrix, composed of various extracellular polymers such as proteins, DNA, and exopolysaccharides (EPS), serves as a defense mechanism to protect resident cells. Within P. aeruginosa biofilms, the EPS, notably Pel, acts as a principal component of the matrix. The regulation of Pel biosynthesis involves a combination of transcriptional and post- translational processes, with cyclic-di-GMP playing a central role. Cyclic-di-GMP, a significant secondary messenger, regulates the transition from a planktonic lifestyle to the biofilm state. Its synthesis is facilitated by diguanylate cyclases (DGCs) and its degradation is carried out by phosphodiesterases (PDEs). Elevated levels of intracellular cyclic-di-GMP is usually associated with biofilm formation, while low levels correlate with a planktonic lifestyle. In our previous investigations, we identified that the PA14 16550 protein, a TetR-type DNA repressor, is required for biofilm formation in P. aeruginosa PA14. Deleting 16550 resulted in a significant reduction in EPS production and cyclic di-GMP levels across multiple biofilm-overproducing strain backgrounds, including a moderately hyper-biofilm-forming strain. However, the precise mechanism by which 16550 regulates cyclic di-GMP and exopolysaccharide production within the biofilm matrix remains unknown. Through transcriptomics analysis, we observed significant changes in the expression of only six genes upon deletion of 16550. To assess their specific impact on biofilm formation, we constructed clean deletions of these genes. Our findings revealed that two of these genes exerted a negative impact on biofilm formation, while the remaining genes exhibited a positive impact on swarming. Additionally, we employed transposon mutagenesis to identify genes whose deletion can reverse the reduction of biofilm formation in a Δ16550 strain. Surprisingly, our investigations uncovered the involvement of the recA gene, which encodes a recombination protein. Given that RecA functions in both homologous recombination and in the SOS response, we genetically dissected these functions to delineate their contribution to biofilm formation. Ultimately, our analysis revealed that abrogation of either function leads to increased levels of biofilm formation.