Fig. 4: The C-terminal WTF motif of DimA is required for its function.
a Phylogenetic distribution and presence/absence of select genes across Pseudomonas spp. Rings surrounding the tree depict metadata and gene presence/absence across genomes − 4 innermost rings: presence (colored) or absence (gray) of four regulatory genes: dimA (purple), algB (brown), bphP (teal), and algW (orange), as determined by reciprocal BLAST against the P. aeruginosa PA14 genome. Outer ring: classification of P. aeruginosa strains as clinical (red) or environmental (gray). Colored branches in the tree represent P. aeruginosa (black), other Pseudomonas spp. (blue), and outgroup genomes (gray). b AlphaFold2 structure prediction model for DimA (PA14_20480) protein. The N- and C- termini are shown by their respective arrows. The structure is colored according to the per-residue model confidence score (pLDDT). c Protter visualization plot of DimA amino acid sequence. The predicted signal peptide is colored red, and the rest of the sequence is outlined in purple. d Confocal microscopy analysis of DimA and its N-terminal deletion mutant in P. aeruginosa. The cells were grown overnight in LB medium supplemented with 100 μg/ml tetracycline and 1 mM IPTG. Top row: PA14 ΔdimA strain harboring the plasmid pME6032-dimA-mNeonGreen, bottom row: PA14 ΔdimA strain harboring the plasmid pME6032-‘dimA -mNeonGreen fusion under induced conditions. Left to right: Membrane stained with FMTM4-64 (red), DimA-mNeonGreen fusion protein (green), and an overlay of both channels. Scale bar is 3 μm for all images. Representative images shown of three independent experiments. e Measurement of alkaline phosphatase activity. Overnight cultures of P. aeruginosa WT and ΔkinB strains bearing the dimA-phoA translational fusion were normalized for their growth at OD600 = 1. The phosphatase activity was measured at OD405 by the production of a yellow-colored product of the pNPP substrate. f Different truncation mutants of DimA were assayed for pyocyanin production. g Alanine substitutions in the WTF motif of DimA were tested for pyocyanin production. f, g Pyocyanin production (OD695) was measured and normalized to growth (OD600) in WT PA14 and the designated mutants grown overnight under ambient light conditions. Pyocyanin levels in WT PA14 were set to 100%. e–g Error bars represent SEM of three biological replicates (n = 3). Only pairwise comparisons that had P-value < 0.05 are denoted. Statistical significance was determined using unpaired, two-tailed t tests for pairwise comparisons in GraphPad Prism software (version 10.6.0). **** P < 0.0001, *** P < 0.001, ** P < 0.005. Significant P-values were calculated as (left to right): For 4e 0.0066, < 0.0001, for 4 f 0.0007, < 0.0001, < 0.0001, for 4 g 0.0003, 0.0004, 0.0044, 0.0004, 0.0004.
