Fig 1.
Protein sequence comparison of QS LuxR family members and the non-AHL sensors PluR and PauR.
(A) Modular domain structure of LuxR-type regulators, with a N-terminal signal-binding domain (SBD) and a C-terminal DNA-binding domain (DBD) [8], containing the helix-turn-helix "HTH LUXR" motif (SMART00421) [25]. (B) Comparison of the protein sequence identity of PluR from P. luminescens, PauR from P. asymbiotica and TraR from A. tumefaciens. The identity was compared either of the full-length protein sequence, only the signal-binding domain (SBD) and only the DNA-binding domain (DBD). To calculate identity of the protein sequences the LALIGN software from SIB (Swiss Institute of Bioinformatics) was used [26]. (C) Sequence alignment of the protein sequences of PauR from P. asymbiotica (P.a.), PluR from P. luminescens (P.l.), QscR from Pseudomonas aeruginosa (P.a.), SdiA from Escherichia coli (E.c.), TraR from Agrobacterium tumefaciens (A.t.) and LuxR from Vibrio fischeri (V.f.). The SBD is depicted with a blue bar and the DBD with a green bar. Within the SBD the six conserved amino acids, displaying the WYDPWG-motif of AHL-sensors, are marked with red asterisks and the three conserved amino acids in the DBD are marked with blue asterisks. Amino acids with a consensus of 60–100% are shown, positions with a lower coverage are marked with a dot. The RasMol colouring of the amino acids and the alignment was generated with CLC Mainworkbench 7 (CLC Bio Qiagen, Hilden, Germany).
Fig 2.
Specificity of PluR and PauR towards different signaling molecules.
PluR (A) senses its cognate signaling molecule PPYD, whereas unrelated signaling molecules, like C8-HSL or DAR, CHDA, CHDB and IPS, are not sensed. To test the specificity of PluR the reporter system pBAD24-His-pluR and pBBR1-pcfAP.l.-lux was used. Similarly, PauR (B) specifically senses its native signaling molecules with the highest specificity towards DAR compared to the DAR-precursors, CHDA, CHDB and IPS. The PauR-specific reporter plasmid system composed of pBAD24-His-pauR and pBBR1-pcfAP.a.-lux was used. Cells harboring the promoter-less reporter plasmid in combination with each PluR and PauR did not exhibit significant pcfA promoter activity. Furthermore, cells harboring the empty pBAD24 plasmid, and therefore no pluR or pauR, with the respective reporter plasmid as well did not exhibit significant pcfA promoter activity. RLUs are shown for 2 h after addition of the depicted signaling molecule. Reference line was set to 370 RLUs to underline the background of the system. RLU, relative light units. (C) Comparison of the structures of the signaling molecules used in this study.
Fig 3.
Amino acid replacements within the SBD of PluR caused either functionality or impaired PPYD-sensing.
The PluR derivatives D75E and S115G dramatically decreased functionality and hence decreased its ability to bind and activate pcfAP.l. promoter (lower left quadrant). Replacements within the TYDQCS-motif of PluR decreased the ability of PluR to sense PPYD. The most drastic influence on PPYD-sensing is detectable with the replacement of Y66A, D75A, D75N, Q76A, Q76P and S115A in PluR (lower right quadrant). Only the replacement T62W showed no effect and same induction levels as PluR wild type (upper right quadrant). The activity of the pcfAP.l. promoter was measured via luminescence as read-out and the depicted values were taken 2 h after addition of 0.1% (w/v) arabinose (lower axis) or 3.5 nM PPYD (left axis) and compared to PluR wild type, which values were set to 100%. To evaluate the different PluR derivatives, a cut-off of 70% was set for each value. RLU (relative light units) values for all PluR derivatives and PluR wild type are depicted in S3 Table.
Fig 4.
The TYDQYI-motif in the SBD of PauR is essential for the overall functionality of the receptor and DAR-sensing.
The most drastic effects on DAR-sensing were gained with the replacement of S38A, T62A, Y66A, D75A and D75N in the SBD of PauR and a decreased effect on DAR-sensing were gained with the replacement of Y90C and I113S in PauR (lower right quadrant). The PauR derivatives Y40A, Y40F, D75E and Q76A dramatically influenced the structure of PauR and decrease its ability to bind and activate pcfAP.a. promoter (lower left quadrant). The activity of pcfAP.a. promoter was measured via luminescence as read-out and pictured values were taken 2 h after addition of 0.1% arabinose (lower axis) or 3.5 nM DAR (left axis) and compared to PauR wild type, which values were set to 100% (upper right quadrant). To evaluate the different derivatives, a cut-off of 70% was set for each value. RLU (relative light units) values for all PauR derivatives and PauR wild type are depicted in S4 Table.
Fig 5.
The conserved motifs in the SBD of PluR and PauR are essential but not sufficient for ligand-binding specificity.
(A) Stepwise replacement of the non-conserved amino acids in PluR to the conserved WYDPWG-motif of AHL-sensors effects the conformation and decreases its ability to activate pcfAP.l. promoter compared to PluR wild type (wt). The quadruple replacement of PluR-T62W/Q76P/C90W/S115G effects most dramatically the conformation compared to PluR wild type. (B) Stepwise replacement of the non-conserved amino acids of PluR decreases its ability to sense its native signaling molecule PPYD, however C8-HSL-sensing could not be gained. (C) Replacement of Y90C and I113S in PauR decreased the ability of PauR-Y90C/I113S to activate pcfAP.a. promoter activity approximately to 50% compared to PauR wild type. (D) DAR-sensing was decreased approximately about 70% in the PauR-Y90C/I113S derivative compared to PauR wild type (wt), however, PPYD-sensing could not gained. (A) and (C): RLUs are shown 2 h after induction and value of PluR or PauR wild type was set to 100% and compared to the respective derivatives. (B) and (D): The RLU values are depicted 2 h after the addition of the distinct signaling molecules, either 3.5 nM PPYD, 3.5 nM DAR or 100 nM C8-HSL. Reference line was set to 370 RLUs to compare better with Fig 2.