Article Figures & Data
Figures
- FIG. 1.
Partial alignment of AraC, XylS, and UreR. Leucine residues 150, 151, and 161 in AraC are critical for AraC dimerization (39). Leucines 147, 148, and 157 in UreR are critical for dimerization of UreR (30). Leucines 193 and 194 and isoleucine 205 in XylS align with the leucine residues of AraC and UreR that are critical for dimerization.
- FIG. 2.
Cross-linking of MBP-N-XylS. Top panel: SDS-PAGE (8% [wt/vol]) of MBP. Bottom panel: SDS-PAGE (6% [wt/vol]) of MBP-N-XylS. Lane M, molecular size markers, with sizes shown on the left or the right (in kilodaltons). The concentrations of 3MBz are indicated along the top. The + and − symbols indicate whether the samples were incubated or not with glutaraldehyde, as described in Materials and Methods.
- FIG. 3.
Cross-linking of MBP-N-XylS*. E. coli bearing plasmids that will produce MBP-N-XylSL193A, MBP-N-XylSL194A, and MBP-N-XylSI205A were grown in the absence and in the presence of 3MBz. Proteins were purified as described in Materials and Methods, and samples were incubated in the presence (+) and in the absence (−) of 0.005% (vol/vol) glutaraldehyde.
Tables
- TABLE 1.
Strains and plasmids used in this study
Strains or plasmid Relevant characteristicsa Source or reference E. coli DH5α supE44 ΔlacU169 (φ80 lacZΔM15) hsdR17 recA1 gyrA96 relA1 8 JL1436 deoC1 ptsF25 rbsR PsulA::′lacZ 1 MC4100 F−araD139 Δ(argF-lac)U169 rpsL150 (Strr) relA1 flbB5301 18 Plasmids pCMX2 pSELECT-1, xylS ori F1, ColE1, Tcr 22 pERD100 pMP220, Pm:′lacZ, IncP1, Tcr 33 pGB002 pSE380, LexA DNA binding domain (amino acids 1-87) 1 pLOW2 pACYC177, p15A, Kmr 9 pLRRA1 pLOW2 derivative bearing xylS mutant allele encoding XylSL193A This study pLRRA2 As pLLAR1 but encoding XylSL194A This study pLRRA3 As pLLAR1 but encoding XylSI205A This study pLRRA4 As pLLAR1 but encoding XylSL193A, L194A This study pLRRA5 As pLLAR1 but encoding XylS193A, 1205A This study pLRRA6 As pLLAR1 but encoding XylSL194A, 1205A This study pLRRA7 As pLLAR1 but encoding XylSL193A, L194A, I205A This study pLRRA8 pGB002 derivative encoding chimeric N-XylS-LexA protein This study pLRRA9 As pLRRA8 but encoding N-XylS L193A-LexA protein This study pLRRA10 As pLRRA8 but encoding N-XylS L194A-LexA protein This study pLRRA11 As pLRRA8 but encoding N-XylS I205A-LexA protein This study pLRRA12 As pLRRA8 but encoding N-XylS L193A, L194A-LexA protein This study pLRRA13 As pLRRA8 but encoding N-XylS L193A, I205A-LexA protein This study pLRRA14 As pLRRA8 but encoding N-XylS L194A,I205A-LexA protein This study pLRRA15 As pLRRA8 but encoding N-XylS L193A,L194A,I205A-LexA protein This study pMAL-pV pMAL-C2, PtalmalE lacIq 28, 29 ↵ a Apr, Kmr, Smr, and Tcr stand for resistance to ampicillin, kanamycin, streptomycin, and tetracycline, respectively.
- TABLE 2.
β-Galactosidase activity of E. coli JL1436 expressing the N-terminal domain of XylS and the XylS mutants in the LexA-based two-hybrid systema
XylS protein β-Galactosidase (Miller units) Without 3MBz With 3MBz None 4,280 ± 400 4,380 ± 400 N-XylS (wild type) 4,100 ± 300 1,300 ± 100 N-XylSL193→A 4,100 ± 300 3,000 ± 250 N-XylSL194→A 3,950 ± 200 3,800 ± 30 N-XylSI205→A 3,890 ± 284 1,250 ± 80 N-XylSL193,L194→A,A 4,450 ± 350 4,750 ± 100 N-XylSL193,I205→A,A 4,200 ± 280 4,050 ± 300 N-XylSL194,I205→A,A 4,150 ± 300 4,000 ± 300 N-XylSL193,L194,I205→A,A,A 4,200 ± 400 4,400 ± 350 ↵ a E. coli Jl1436 bearing a plasmid encoding LexA, LexA-N-Xyls, or LexA-N-XylS* protein was grown, and β-galactosidase was assayed as described in Materials and Methods. Data (values are rounded) are the averages and standard deviations of at least four independent assays performed in triplicate.
- TABLE 3.
Induction of Pm by variants of XylS proteina
N-XylS protein β-Galactosidase (Miller units) Induction ratio, +3MBz/−3MBz (fold increase) Without 3MBz With 3MBz Wild type 50 ± 2 1,510 ± 80 30 N-XylSL193A 45 ± 1 450 ± 30 10 N-XylSL194A 50 ± 1 50 ± 5 1 N-XylSI205A 45 ± 3 1,650 ± 60 37 N-XylSL193A,L194A 45 ± 4 50 ± 8 1 N-XylSL193A,I205A 45 ± 2 60 ± 2 1 N-XylSL194A,I205A 45 ± 2 50 ± 1 1 N-XylSL193A,L194A,I205A 45 ± 2 45 ± 2 1 ↵ a E. coli MC4100(pERD100) bearing a derivative of pLOW2 that encodes the indicated XylS protein was cultured as indicated in Materials and Methods. Data (values are rounded) are the averages of at least three independent assays performed in triplicate.
