Article Figures & Data
Figures
- Fig. 1.
Analysis of protein expression patterns by SDS-PAGE. Whole-cell proteins of the PhoP− (lane 1) strain, the PhoP− strain with plasmid pPC3-2 (lane 2), and the PhoQ-constitutive (pho24) (lane 3) strain were separated on a 10% polyacrylamide gel. The expression pattern of lane 2 containing proteins of the PhoP− strain with plasmid pPC3-2 is similar to that of lane 3 containing proteins of the PhoQ-constitutive (pho24) strain and dissimilar from that of lane 1, further demonstrating the constitutive phenotype imparted by the pPC3-2 constitutive phoP allele. Arrows point to the most obvious protein differences between those with the PhoP-constitutive pattern (lanes 2 and 3) and the PhoP− pattern (lane 1). Lane M contains the molecular mass standards, the sizes of which are given to the left in daltons.
- Fig. 2.
Protein alignment of OmpR-type regulators with known constitutive mutations. The protein sequences aligned include those of PhoP (GenBank accession no. M24424), PmrA (accession no. L13395), OmpR (accession no. J01656), and VirG (29). Circled residues with arrows denote those changes that have been shown to result in a constitutive phenotype. Highly conserved consensus regions located near these sites, including the aspartic acid residue (D) predicted to be a site of phosphorylation, are boxed.
- Fig. 3.
Activation of pagD transcription by arabinose-inducible PhoP production. PhoP− strains carrying a pagD::TnphoA reporter and the pPCBAD3-2 plasmid were grown to log phase and then not supplemented or supplemented with arabinose. Alkaline phosphatase activity was monitored over time. Lines with squares are results from cultures with arabinose added at time zero, and those with diamonds are results from cultures without arabinose added.
- Fig. 4.
Activation of 2-hydroxymyristate lipid A (2-OH C14:0) modification by arabinose-inducible PhoP production. A PhoP− strain carrying the pPCBAD1 plasmid was grown to log phase and then not supplemented or supplemented with arabinose (0.2%). The percentage of 2-hydroxymyristate lipid A modification was monitored over time by GC analysis. Solid circles represent a culture with arabinose added at time zero, and solid squares represent a culture without arabinose added. The experiment presented was representative of three separate experiments.
Tables
- Table 1.
Effect of phoP-constitutive alleles onpag transcriptional activity
Background strain Relevant genotype Relevant phenotype Plasmid Reporter activity (U)a JSG208 pho24 pagB::MudJ PhoPQ constitutive,pagB-lacZ NAb 321 ± 16 (β) JSG465 pagB::MudJphoP::Tn10d-Tet PhoP−,pagB-lacZ pWSK200c 6.2 ± 2 (β) JSG465 pagB::MudJphoP::Tn10d-Tet PhoP−,pagB-lacZ pPC3-2d 390 ± 14 (β) JSG225 pagD::TnphoA phoP::Tn10d-Tet phoN2 zxx::6251Tn10d-Cam PhoP−,pagD-phoA pWSK200 8.3 ± 2 (A) JSG225 pagD::TnphoA phoP::Tn10d-Tet phoN2 zxx::6251Tn10d-Cam PhoP−,pagD-phoA pPC3-2 784 ± 22 (A) JSG225 pagD::TnphoA phoP::Tn10d-Tet phoN2 zxx::6251Tn10d-Cam PhoP−,pagD-phoA pPSK200-691e 124 ± 8 (A) JSG225 pagD::TnphoA phoP::Tn10d-Tet phoN2 zxx::6251Tn10d-Cam PhoP−,pagD-phoA pPSK200-7974f 540 ± 10 (A) JSG208 pho24 pagD::TnphoA phoN2 zxx::6251Tn10d-Cam PhoPQ constitutive,pagD-phoA NA 674 ± 19 (A) ↵a Units reported as described by Miller (22) for β-galactosidase (β) or alkaline phosphatase (A).
↵b NA, not applicable.
↵c pWSK29 with the wild-type phoPgene.
↵d pWSK29 with the double-mutant, constitutivephoP gene.
↵e pWSK29 with the single-mutant (784 A→G; Q203R), constitutive phoP gene.
↵f pWSK29 with the single-mutant (418 G→A; S93N), constitutive phoP gene.
