A—Biofilm formation of wild-type (WT) AB5075 compared to three ABUW_2208::T26 transposon mutants labelled with their corresponding number from the Manoil transposon mutant library. B—Proposed cell localisation and function of CavA adenylate cyclase based on domains present in the protein. The transmembrane domains anchor the protein to the inner membrane the cells. The catalytic domain forms a dimer and converts ATP molecules to cyclic AMP (cAMP). Figure was created using PyMOL and BioRender.com. C–Biofilm levels of ΔcavA clean deletion mutant and ΔcavA+cavA complemented strain. D–cAMP concentrations in ΔcavA mutant and complemented ΔcavA+cavA strain, as well as ΔcavB and ΔcavAΔcavB double mutant which was complemented with either cavA (ΔcavAΔcavB+cavA) or cavB (ΔcavAΔcavB+cavB). Cultures grown in LB broth were harvested and lysed via sonication. Bradford assay was used to quantify the total protein concentration and cAMP concentrations in each strain were determined using Cyclic Nucleotide XP Enzymatic Immunoassay kit. Data shown is the mean cAMP concentration per milligram protein from three independent repeats. E—Biofilm formation of ΔcavB deleted mutant compared to WT. Biofilm biomass was assessed after 24 h incubation at 37°C shaking and is presented as the optical density measured at 570 nm (OD570). All data represents the averages of three biological replicates ± standard deviations (SD). Bacterial growth was assessed at OD600 prior to staining the biofilms (S1 Fig). ns p>0.05, *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001—One-Way ANOVA with Dunnett (A) or Tukey (C, D) post-hoc tests and Unpaired t-test (D). Strains with empty miniTn7 were used as controls (S1 Fig).

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