Fig. 2: PA0744 and DspI are co-expressed in a cell density-dependent manner and indispensable for CDA biosynthesis.

A Relative expression of PA0744 and dspI was measured by RT-qPCR in the PAO1 WT(EV), ΔPA0744(EV), ΔdspI(EV), ΔPA0744(PA0744), and ΔdspI(dspI) strains. N.D., not detected. The 50S ribosomal protein gene rplU was used as internal control. B β-galactosidase activity of the PPA0747-lacZ transcriptional fusions was measured in the PAO1 WT(EV), ΔPA0744(EV), ΔdspI(EV), ΔPA0744(PA0744), and ΔdspI(dspI) strains. C Relative expression of PA0744 and dspI in the PAO1 WT strain was measured by RT-qPCR at different growth stages with different OD₆₀₀ values of the cell culture compared to the samples collected at OD₆₀₀ of 0.3. (D) Biofilm biomass was measured in PAO1 WT, ΔPA0744, and the mixed cultures containing ΔPA0744 and WT in different ratios (1:1, 1:2, 1:3). E CDA production in the PAO1 WT(EV), ΔPA0744(EV), ΔPA0744(PA0744), ΔdspI(EV), and ΔdspI(dspI) strains was measured by LC-MS. N.D., not detected. F Biofilm biomass was measured in the PAO1 WT(EV), ΔPA0744(EV), and ΔPA0744(PA0744) strains when the strains were cultured with exogenous addition of CDA at concentrations of 1 nM, 50 nM, 500 nM, 5 μM, and 50 μM. G LC-MS quantification of CDA in the PAO1 WT, ΔdspII, ΔdspI, ΔdspIIΔdspI strains and strains with complemented expression of dspII or/and dspI. H, I Biofilm biomass (H) and swarming motility (I) were measured in the PAO1 WT, ΔdspII, ΔdspI, ΔdspIIΔdspI strains and the strains with complemented expression of dspII or/and dspI. J Domain structures of DspI and DspII. The conserved enzymatic residues of enoyl-CoA hydratase/isomerases (Glu126 and Glu146 in DspI and Glu143 in DspII) were labeled. K CDA production in ΔdspII and ΔdspI mutants with the complementation of their enzymatically dead variants. L, M Biofilm biomass (L) and swarming motility (M) were measured in ΔdspII and ΔdspI mutants with the complementation of their enzymatically dead variants. For panels A to I and K to M: data are presented as mean values  ±  SEM of n = 3 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test (A, F), one-way ANOVA with Tukey’s multiple comparisons test (B, D), or one-way ANOVA with Dunnett’s multiple comparisons test compared to the WT(EV) group (E, G, H, I) or the indicated groups (L, M). ***, p < 0.0001. Source data are provided as a Source Data file.