Article Figures & Data
Figures
- FIG. 1.
Cyanide production is controlled by AlgR and elevated in laboratory mucoid P. aeruginosa. Quantification of HCN (micromoles per milligram of protein) produced in 4 h from nonmucoid parent strains PAO1 and PAO381 (horizontally lined bars), mucoid derivative strains PAO568, PAO6857, and PDO300 (solid bars), subsequent isogenic algR deletion strains PAR568, PAR6857, and PDR300 (vertically lined bars), and complemented algR deletion strains PDR300+algR, PAR6857+algR, and PAR568+algR (open bars). All of the parent mucoid strains produced elevated levels of HCN compared to their relative algR deletion counterparts. *, P of <0.05 when compared to the parent strain, as determined by unpaired t test.
- FIG. 2.
AlgR controls the expression of hcnA in mucoid P. aeruginosa. S1 nuclease protection assay of the hcnA promoter region. A. Western blot analysis of PAO1, PDO300 (mucA2), PDR300 (mucA2 ΔalgR), PAO6857 (mucB::Tcr), and PAR6857 (mucB::Tcr ΔalgR) using anti-AlgR 3H-9 confirmed the deletion of the 29-kDa transcriptional regulator, AlgR, in strains PAR6857, PDR300, and PAR568. MW, molecular weight. B. Mapped transcriptional start sites T1 and T2 of the mucoid strains PAO6857 and PAO568 and their algR deletion strains PAR6857 and PAR568. C. Schematic of the hcnA promoter sequence. T1 and T2, mapped transcriptional start sites (indicated with arrows); ANR box, ANR binding site; lux α and lux β, LasR and RhlR binding sites.
- FIG. 3.
AlgR binds specifically to the hcnA promoter. Competitive gel mobility shift assay of AlgR on the hcnA promoter region. The probe-alone lane contains only labeled hcnA promoter (99 bp, −495 to −396 in relation to the translational start site) from plasmid pCRhcnA. All other lanes contain the same amount of labeled hcnA promoter plus 100 ng of purified AlgR. For the specific competitor, the given micrograms of undigested pCRhcnA was added to the DNA/AlgR mix. For the nonspecific competitor, the given micrograms of undigested pUC12 was added to the DNA/AlgR mix.
- FIG. 4.
Mucoid clinical CF isolates produce more HCN than nonmucoid isolates. A. Mean quantification of HCN produced in 4 h from 20 mucoid and 21 nonmucoid CF clinical isolates. Mucoid isolates produce approximately twofold more HCN than nonmucoid isolates. *, P < 0.05, as determined by unpaired t test. B. Western blot analysis of clinical isolates FRD1, TUMC92, and TUMC197 and their algR deletion strains FRD1R (algR::Smr), TUMC-92R (ΔalgR), and TUMC-197R (ΔalgR) using anti-AlgR. C. Quantification of HCN produced in 4 h from clinical isolates TUMC197 and TUMC92 and their algR deletion strains, TUMC-197R and TUMC-92R, and from the mucoid CF isolate FRD1 and its nonmucoid algR strain FRD1R. Solid bar, mucoid strain; horizontally lined bars, nonmucoid strains; vertically lined bars, algR deletion strains. *, P < 0.05, as determined by unpaired t test. **, P < 0.01, as determined by unpaired t test.
- FIG. 5.
Schematic representing possible mechanisms for AlgR activation. Transcriptional profiling experiments have shown that AlgR is capable of repressing transcription of hcnA and other genes (30). We have shown that AlgR activates hcnA transcription and HCN production in mucoid P. aeruginosa. Mutations in mucA (4, 34), mucB (33), or mucD (3) result in release of the alternative sigma factor AlgU, resulting in increased transcription of algR (35). AlgZ/FimS has been shown to be a repressor of alginate production (60) and an activator of twitching motility (51, 52). However phosphorylation does not play a role in AlgR activation of the alginate system (31) but is necessary for activation of twitching motility (52). These results imply that there may be more than one way to activate AlgR in P. aeruginosa. The hcnA promoter is the only known example where AlgR acts as a repressor in the nonmucoid state and as an activator in the mucoid phenotype. Rounded star, repressor AlgR; pointed star, activated AlgR.
Tables
- TABLE 1.
Strains used in this study
P. aeruginosa Genotype Phenotypea Reference or source PAO1 Wild type NM 27 PAO381 leu NM 17 PDO300 mucA2 M 32 PDR300 mucA2 ΔalgR NM This study PAO6857 mucB::Tcr M 47 PAR6857 mucB::Tcr ΔalgR NM This study PA0568 mucA2 M 17 PAR568 mucA2 ΔalgR NM This study FRD1 mucA M 21 FRD1R mucA algR::Smr NM 31 TUMC92 Clinical isolate NM This study TUMC-92R ΔalgR NM This study TUMC197 Clinical isolate NM This study TUMC-197R ΔalgR NM This study Escherichia coli DH-5α Invitrogen ↵ a M, mucoid; NM, nonmucoid.
