Fig. 7: A highly conserved ARG box in the rmp promoter is required for ArgR-dependent mucoidy regulation.
From: Arginine regulates the mucoid phenotype of hypervirulent Klebsiella pneumoniae
A K. pneumoniae genomes with intact rmpADC loci (n = 1572) were analyzed for ARG box sequence diversity using Geneious v2023.1.1 and WebLogo v3.7.9. Diversity is shown as percentage and minor allele frequency. Asterisks indicate highly conserved nucleotides. B KPPR1 carrying either a Prmp-GFP reporter plasmid or a mutated ARG box (Prmp∆ARG-GFP) was cultured in M9 + All, M9-Arg, M9 + Glyc, or M9 + Glyc + Arg. GFP expression measured promoter activity. C DNA-protein interaction ELISA assessed ArgR binding to the ARG box in Prmp. Purified His6-tagged ArgR was incubated in wells coated with DNA oligomers containing either the predicted (Prmp) or mutated binding site (Prmp∆ARG). Binding was detected using an HRP-conjugated anti-poly-histidine antibody. OD450 values reflect ArgR-DNA binding. D, E KPPR1S or chromosomal ARG box mutant (∆ARGbox) were cultured in the four media conditions. D Mucoidy was assessed by sedimentation assay. E Capsule abundance was measured by uronic acid quantification. F−H KPPR1S and ∆ARGbox mutant were cultured in M9+All. F Transcript levels of rmpA, rmpD, and rmpC were measured by qRT-PCR and normalized to gap2. G Capsular polysaccharides (CPS) were separated by SDS-PAGE and stained with Alcian blue and silver stain; representative image of three independent gels shown. H Quantification was done with ImageJ with background subtraction. Data presented are the mean, and error bars represent SEM. Statistical tests: unpaired two-tailed t-test (B, F); two-tailed Mann-Whitney test (C); two-way ANOVA with Bonferroni’s post-test (D, E); two-tailed one sample t-test (H). p-values: **p < 0.01; #p < 0.0001. Exact P-values left to right: (B): <0.0001, <0.0001, <0.0001, <0.0001; (C): 0.0087, 0.0022, 0.0022, 0.0022, 0.0022, 0.0022, 0.0022; (D): <0.0001, <0.0001; (E): 0.0025, <0.0001, 0.0085, 0.0061; (F): 0.0098, 0.0007, <0.0001; (H): 0.0188. Experiments were performed > 3 independent times (n = 9), in triplicate, except (G) (n = 3) and (C) (duplicates, n = 6). Source data are provided as a Source Data file.
