Article Figures & Data
Figures
- FIG. 1.
(A) Alignment of the amino-terminal phosphorylation domains of AlgR, YehT, CheY, and NtrC. X, variable sequences between conserved domains of each protein. Overlined sequences are highly conserved among all response regulators, and conserved aspartates and lysines are depicted in red. In CheY and NtrC, the aspartate which aligns with AlgR D54 is the site of phosphorylation by CheA or NtrB, respectively (33, 34). (B) A ribbon diagram for AlgR model. Homology-based model structure for the AlgR receiver domain was constructed using the structure of the NarL nitrate response regulator protein as a template. The α-helices are blue, β-sheets are red, and loops are gray. Asp54 and Asp85 are depicted in stick form and colored according to standard CPK. Two views are shown: the first is down the axis of a barrel formed by the peripheral helices and the second is at right angles to this and shows the two aspartates lying in the same plane as the mouth of the barrel. (C) Comparison of wild-type AlgR and AlgR D54N receiver domain structures. The panel on the left is an overlay of wild-type AlgR receiver domain (yellow) with AlgR D54N (blue) showing very little structural disturbance of the domain structure. The position of residue 54 in these structures is indicated in red. The right panel is a ribbon diagram of the region surrounding residue 54 (shown in stick form) with the wild-type aspartate depicted in blue and the substituted asparagine overlaid in red.
- FIG. 2.
(A) AlgR phosphorylation studies. Phosphorylation assays were conducted as described in Materials and Methods. Lanes contain 100 ng of CheA and/or 1 μg of AlgR or AlgR derivatives (+, addition of protein). The positions of the phosphorylated forms of CheA and AlgR are indicated on the side. Note that CheA can phosphorylate AlgR and AlgR D85N but not AlgR D54N or AlgR D54N D85N. (B) AlgR D54N competes with wild-type AlgR for access to CheA. A competition experiment was performed using 1 μg of wild-type AlgR and different amounts of AlgR D54N (0.5, 1, and 5 μg). The conditions used in the autophosphorylation of CheA, phosphotransfer from CheA to AlgR, and detection are identical to those described for panel A. Lane 1, 100 ng of CheA alone; lanes 2, 3, and 4, 100 ng of CheA and 0.5, 1, or 5 μg of AlgR D54N, respectively; lane 5, 100 ng of CheA and 1 μg of wild-type AlgR; lanes 6, 7, and 8, 100 ng of CheA, 1 μg of wild-type AlgR, and either 0.5, 1, or 5 μg of AlgR D54N, respectively. (C) EMSA of AlgR or AlgR derivatives binding to algD sequences. The fragment used in this assay contains two AlgR-binding sites and is located from −324 to −424 relative to the algD transcription start site. Duplicate samples of AlgR or the various AlgR derivatives (100 ng; protein source depicted below the gel) were tested for binding to algD sequences. The positions of unbound algD as well as the AlgR-algD complexes are indicated.
- FIG. 3.
Twitching motility phenotypes of PAKalgR mutants. (A) Subsurface twitching motility assay of P. aeruginosa PAK (wild type) and mutants PAKpilA::Tcr, S39 (algR), PAKalgR7, PAKalgR10, PAKalgR11, and PAKalgD::Tcr. Bars, 1 cm. (B) Light microscopy of zones of twitching motility showing colony expansion zones obtained at the interstitial surface between the glass coverslip and Gelgro media for PAK (wild type), PAKpilA::Tcr, and S39. Micrographs were taken after 2 to 4 h of incubation (PAK) or 20 h (PAKpilA::Tcr and S39) at 37°C. Bar, 50 μm. In all images, the colony is situated to the left of the image.
- FIG. 4.
Type IV fimbrial subunit production in algR mutants. (A) ELISA against whole-cell samples of the following P. aeruginosa strains: PAK (▪), PAKpilA::Tcr (▴), S39 (×), PAKalgR7 (|), PAKalgR10 (•), and PAKalgR11 (♦). Type IV fimbriae were detected using antipilin antiserum and are indicative of levels of surface fimbriae in these strains. Shown are immunoblots of the PilA subunit of sheared surface fimbriae (B) and PilA subunit (C) remaining in whole-cell samples after surface fimbriae had been sheared off indicated strains. The pilV mutant (R306), which is defective in assembly of the fimbrial structure, was included in these assays to control for fimbrial subunit contribution to surface samples as a result of cell lysis.
- FIG. 5.
AlgR is required for biofilm formation. (A) Subsurface twitching motility assay of P. aeruginosa PAO1 (wild type) and the indicated algR mutant strains. (B) Biofilm formation was assayed every 2 h during initiation using the microtiter plate assay (29). Surface-attached cells were stained with crystal violet, the stain was solubilized in ethanol, and the absorbance was analyzed at 600 nm (A600). The wild-type (WT) strain and strains expressing the various AlgR mutant proteins are indicated.
- FIG. 6.
Electrostatic potential diagrams for the wild-type and D85N mutant AlgR receiver domains. Homology-based model structure for the AlgR receiver domain was constructed using the structure of the NarL nitrate response regulator protein as a template. Electrostatic potential is represented as a mesh contoured to yield isosurfaces with charges of ±3.0. Negative potential is red and positive potential is blue. There is very little positive potential visible in this image. Electrostatic potentials have also been mapped to a solvent-accessible molecular surface constructed with a 1.4-Å probe to show AlgR surface topology. Accessible surface is colored as for electrostatic potential except that uncharged areas are white. The positions of Asp/Asn85 are indicated. Viewpoint is along the helical barrel.
Tables
- TABLE 1.
Strains, plasmids, and primers used in this study
Strain, plasmid, or oligonucleotide Description or sequencea Source or reference E. coli DH5α 80lacZΔM15 Promega JM109 e14−mcrA recA1 endA1 gyrA96 thi-1 hsdR17(rK− mK+) supE44 relA1 Δ(lac-proAB) [F′ traD36 proAB lacIqZΔM15] Promega MV1184 41 P. aeruginosa PAK Nonmucoid P. aeruginosa strain K D. Bradley PAKpilA::Tcr Previously referred to as AWK 44 R306 PAKpilV::Tn5-B21 2 S39 PAKalgR::Tn5-B21 45 PAKalgD::Tcr Tcr cassette insertion in EcoRI site of algD This study PAKalgR7 PAK encoding AlgR D54N This study PAKalgR10 PAK encoding AlgR D85N This study PAKalgR11 PAK encoding AlgR D54N D85N This study PAO1 Nonmucoid P. aeruginosa strain O1 WFPA1 algD::tet in PAO1 This study WFPA12 algR::aac1 in PAO1 This study WFPA8 algR7 in PAO1 (encoding AlgR D54N) This study WFPA13 algR10 in PAO1 (encoding AlgR D85N) This study WFPA16 algR11 in PAO1 (encoding AlgR D54N D85N) This study WFPA6 algR gene replacement of WFPA12 This study Plasmids pSM-TET Source of Tcr cassette 27 pRIC380 P. aeruginosa suicide vector 1 pUK21 Kmr cloning vector 42 pUCPKS P. aeruginosa-E. coli shuttle vector 43 pGEMT-easy E. coli cloning vector Promega pUCPAlgR algR cloned into pUCPKS 45 pGemAlgRD54N algR7 cloned into pGEMT-easy This study pUCPAlgRD54N algR7 cloned into pUCPKS This study pDJW106 algR cloned in pALTER-1 21 pJK223R1 Source of AlgR 19 pUS152 algR7 in pALTER-1 21 pUS164 algR10 in pALTER-1 21 pUS165 algR11 in pALTER-1 21 pUS162 algR7 in pJK223R1 (source of AlgR D54N) This study pUS170 algR10 in pJK223R1 (source of AlgR D85N) This study pUS172 algR11 in pJK223R1 (source of AlgR D54N D85N) This study pUS150 Wild-type algR in gene replacement vector pEX100T 21 pUS157 algR7 in gene replacement vector pEX100T 21 pUS166 algR10 in gene replacement vector pDJW525 21 pUS168 algR11 in gene replacement vector pDJW525 21 pRS1 Source of His-CheA R. Bourret pDJW220 algD cloned in pALTER-1 5 Primers algR-Forward 5′-TAAAGCGAGTCTCAGCGTCG-3′ This study algR-Reverse 5′-GGGACGACATGGGATATTCC-3′ This study algD-Forward 5′-CGCGGATCCGAGGTGAATGCGATGCG-3′ This study algD-Reverse 5′-TGCTCTAGACTAGGAGCAGATGCCCTC-3′ This study algD33 5′-AGCCCTTGTGGCGAATAGGC-3′ This study algD36 5′-GAATTGGGGAAAAGTCTGTG-3′ This study ↵ a Tcr, tetracycline resistance; aac1, gentamicin resistance; Kmr, kanamycin resistance.
