Validation of ABUW_2208 (cavA) transposon mutants as well as schematic representation of CavA and CavB domains, and their mutants effect on growth, biofilm formation and cAMP concentrations in A. baumannii AB5075 [Dataset]

A–PCR validating T26 insertion in the ABUW_2208 gene in five strains annotated as ABUW_2208::T26 transposon mutants, labelled with their corresponding number from the Manoil transposon mutant library. C.ABUW_2208 fw/rv (annealing at the beginning and end of the ABUW_2208 gene) were used. Wild-type ABUW_2208 gene is 1.5-kb in size (C+), while the cavA gene with T26 transposon (1.8-kb) is ~3.2-kb. Genomic DNA (20 ng) from AB5075 was used as positive control (C+) and water was used as a negative one (C-). Only AB05781, AB05784 and AB05783 were confirmed to be ABUW_2208::T26 mutants. The other two strains (AB05780 and AB05782) did not harbour the T26 transposon in the ABUW_2208 gene and thus, were incorrectly assigned as ABUW_2208::T26 mutants. B–Schematic representation of CavA protein (489 aa) showing its predicted transmembrane (TM) regions and the adenylate/guanylate cyclase domain (PF00211). C & D–Growth measured as optical density at 600 nm (OD600) (C) and biofilm formation measured as optical density at 570 nm (OD570) (D) after 24 h at 37°C shaking, demonstrating that both phenotypes remained unchanged in the controls bearing chromosomal insertion of the empty miniTn7 (EV) in the wild-type (WT) and ΔcavA backgrounds. E—Intracellular cAMP concentrations presented as μmol per milligram protein of wild-type (WT), ΔcavA and ΔcavB single and ΔcavAΔcavB double mutants and their derivatives with EV showing that the empty miniTn7 did not alter cAMP production in these strains. ns p>0.05, *p<0.05, **p<0.01, ****p<0.0001 One-Way ANOVA with Tukey post-hoc test. F–Representation of CavB protein (487 aa) with its CYTH (PF01928) and CHAD (PF05235) domains. T26 transposon insertion positions in the transposon mutants from the Manoil mutant library [24] are presented by red triangles. G–Growth (OD600) of ΔcavB mutant and WT after 24 h period incubation at 37°C shaking. ns p>0.05—Unpaired t-test.