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Specificity of Signal-Binding via Non-AHL LuxR-Type Receptors.

Brameyer S, Heermann R - PLoS ONE (2015)

Bottom Line: However, PluR and PauR both harbor substitutions in the conserved amino acid motif compared to that in AHL sensors, which appeared to be important for binding the corresponding signaling molecules.Here we analyze the role of the conserved amino acids in the signal-binding domain of these two non-AHL LuxR-type receptors for their role in signal perception.Our studies reveal that the conserved amino acid motif alone is essential but not solely responsible for ligand-binding.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, Bereich Mikrobiologie, Ludwig-Maximilians-Universität München, Martinsried/München, Germany.

ABSTRACT
Quorum sensing is a typical communication system among Gram-negative bacteria used to control group-coordinated behavior via small diffusible molecules dependent on cell number. The key components of a quorum sensing system are a LuxI-type synthase, producing acyl-homoserine lactones (AHLs) as signaling molecules, and a LuxR-type receptor that detects AHLs to control expression of specific target genes. Six conserved amino acids are present in the signal-binding domain of AHL-sensing LuxR-type proteins, which are important for ligand-binding and -specificity as well as shaping the ligand-binding pocket. However, many proteobacteria possess LuxR-type regulators without a cognate LuxI synthase, referred to as LuxR solos. The two LuxR solos PluR and PauR from Photorhabdus luminescens and Photorhabdus asymbiotica, respectively, do not sense AHLs. Instead PluR and PauR sense alpha-pyrones and dialkylresorcinols, respectively, and are part of cell-cell communication systems contributing to the overall virulence of these Photorhabdus species. However, PluR and PauR both harbor substitutions in the conserved amino acid motif compared to that in AHL sensors, which appeared to be important for binding the corresponding signaling molecules. Here we analyze the role of the conserved amino acids in the signal-binding domain of these two non-AHL LuxR-type receptors for their role in signal perception. Our studies reveal that the conserved amino acid motif alone is essential but not solely responsible for ligand-binding.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0124093.g003: 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.

Mentions: Amino acid substitutions in PluR or PauR might affect the spatial structure of the proteins and influence their functionality to bind the cognate pcfA promoter. To quantify the structural influence of amino acid replacements in the signal-binding domain (SBD) of PluR or PauR, the ability of PluR wild type or PauR wild type and different derivatives to activate pcfA promoter activity was determined. Therefore, the similar method was used as described above, however 0.1% (w/v) arabinose was added and also derivatives of PluR or PauR were used. For better comparison, the values of PluR wild type or PauR wild type was set as 100% in the Figs 3 and 4.


Specificity of Signal-Binding via Non-AHL LuxR-Type Receptors.

Brameyer S, Heermann R - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414361&req=5

pone.0124093.g003: 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.
Mentions: Amino acid substitutions in PluR or PauR might affect the spatial structure of the proteins and influence their functionality to bind the cognate pcfA promoter. To quantify the structural influence of amino acid replacements in the signal-binding domain (SBD) of PluR or PauR, the ability of PluR wild type or PauR wild type and different derivatives to activate pcfA promoter activity was determined. Therefore, the similar method was used as described above, however 0.1% (w/v) arabinose was added and also derivatives of PluR or PauR were used. For better comparison, the values of PluR wild type or PauR wild type was set as 100% in the Figs 3 and 4.

Bottom Line: However, PluR and PauR both harbor substitutions in the conserved amino acid motif compared to that in AHL sensors, which appeared to be important for binding the corresponding signaling molecules.Here we analyze the role of the conserved amino acids in the signal-binding domain of these two non-AHL LuxR-type receptors for their role in signal perception.Our studies reveal that the conserved amino acid motif alone is essential but not solely responsible for ligand-binding.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, Bereich Mikrobiologie, Ludwig-Maximilians-Universität München, Martinsried/München, Germany.

ABSTRACT
Quorum sensing is a typical communication system among Gram-negative bacteria used to control group-coordinated behavior via small diffusible molecules dependent on cell number. The key components of a quorum sensing system are a LuxI-type synthase, producing acyl-homoserine lactones (AHLs) as signaling molecules, and a LuxR-type receptor that detects AHLs to control expression of specific target genes. Six conserved amino acids are present in the signal-binding domain of AHL-sensing LuxR-type proteins, which are important for ligand-binding and -specificity as well as shaping the ligand-binding pocket. However, many proteobacteria possess LuxR-type regulators without a cognate LuxI synthase, referred to as LuxR solos. The two LuxR solos PluR and PauR from Photorhabdus luminescens and Photorhabdus asymbiotica, respectively, do not sense AHLs. Instead PluR and PauR sense alpha-pyrones and dialkylresorcinols, respectively, and are part of cell-cell communication systems contributing to the overall virulence of these Photorhabdus species. However, PluR and PauR both harbor substitutions in the conserved amino acid motif compared to that in AHL sensors, which appeared to be important for binding the corresponding signaling molecules. Here we analyze the role of the conserved amino acids in the signal-binding domain of these two non-AHL LuxR-type receptors for their role in signal perception. Our studies reveal that the conserved amino acid motif alone is essential but not solely responsible for ligand-binding.

No MeSH data available.


Related in: MedlinePlus