FIG 1 .

Complementation of increased biofilm formation in the A. tumefaciens dcpA mutant requires an intact DcpA EAL catalytic motif. (A) Diagram of PruR-DcpA genetic locus. Atu3497 is a conserved hypothetical protein with no annotated domains. (B) Protein topology of DcpA. Protein domains predicted by the BLAST database (NCBI) are shown. Domains are drawn to scale. TM, transmembrane; DGC, diguanylate cyclase; PDE, phosphodiesterase. (C) A. tumefaciens quantitative biofilm formation on PVC coverslips after 48 h of static growth at 28°C. Adherent biomass was quantified by staining with crystal violet (CV). CV absorbance was quantified by absorbance at 600 nm (A₆₀₀). In parallel, the optical density at 600 nm (OD₆₀₀) of planktonic culture was determined. CV absorbance was normalized to culture growth by calculating the A₆₀₀/OD₆₀₀ ratio. IPTG (400 µM) was added to all strains. The wild-type (WT) strain, ΔdcpA mutant with no plasmid inserted (-), and ΔdcpA mutant strain with P_lac-dcpA derivatives are shown. A wild-type copy of dcpA provided on a plasmid expressed from the lacZ promoter (P_lac-dcpA; DGC⁺PDE⁺) was introduced into a ΔdcpA mutant. The P_lac-dcpA derivatives include a DcpA variant containing catalytic site GGDEF→GGDAF mutation (DGC−) and a DcpA variant containing catalytic site EAL→AAL mutation (PDE−). Values are results of three independent biological replicates consisting of three technical replicates each. The error bars show 1 standard deviation (SD).