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Characterisation of the putative effector interaction site of the regulatory HbpR protein from Pseudomonas azelaica by site-directed mutagenesis.

Vogne C, Bisht H, Arias S, Fraile S, Lal R, van der Meer JR - PLoS ONE (2011)

Bottom Line: Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown.We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR.This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

View Article: PubMed Central - PubMed

Affiliation: Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ(54)-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate P(C) promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

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Exemplary effects of HbpR A-domain mutations on inducible expression.Measured fluorescence intensities of Escherichia coli cells carrying a plasmid with a promoterless egfp under control of the HbpR-dependent PC-promoter in the presence or absence of 20 µM 2-HBP as inducer. EGFP expression was measured on whole cells at two time points and corrected for culture turbidity. Type I to IV correspond to differently shaded entries in Table 1. Note the delayed response in Type II mutants and the higher background in the absence of inducer in type IV mutants.
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pone-0016539-g003: Exemplary effects of HbpR A-domain mutations on inducible expression.Measured fluorescence intensities of Escherichia coli cells carrying a plasmid with a promoterless egfp under control of the HbpR-dependent PC-promoter in the presence or absence of 20 µM 2-HBP as inducer. EGFP expression was measured on whole cells at two time points and corrected for culture turbidity. Type I to IV correspond to differently shaded entries in Table 1. Note the delayed response in Type II mutants and the higher background in the absence of inducer in type IV mutants.

Mentions: An overview of all mutants constructed in the first and the second group is presented in Table 1. All mutations in the HbpR A-domain were constructed by PCR with mutated oligonucleotides and verified by DNA sequencing. Subsequently, the mutated A-domain sequences were used to replace the gene region for the native A-domain in hbpR on an expression vector in E. coli, with which we could test 2-HBP inducible egfp expression under control of the HbpR-dependent PC promoter. This expression vector results in the addition of a His6-tag to the N-terminal end of the protein. All mutants were tested in E. coli for EGFP expression during exponential growth in the presence or absence of 20 µM 2-HBP, which is the cognate effector for the HbpR-PC system. Table 1 gives representative EGFP induction values after 2 and 4 h induction time compared to those of the strain carrying wild-type HbpR. In general and for all mutants, we observed four types of effects: (i) complete loss of activation with respect to the wild-type (type I), (ii) two-fold loss of induction potential in 2 h but not 4 h incubation periods (type II), (iii) no effect compared to the wild-type (type III), and (iv) considerable increase of background expression (type IV, Table 1, Fig. 3). Protein extracts of the same strains were analyzed by Western blotting using an M13-VHH camel antibody to verify (mutant) HbpR expression (Fig. 4). Surprisingly, all Westerns showed two bands, which likely correspond to His6-tagged HbpR (or mutant, 64.1 kDa) and HbpR (mutant) without His6-tag (62.8 kDa). The reason for the production of two N-terminally different HbpR proteins probably lies in the use of an alternative start codon further downstream. The expression level of most HbpR mutant proteins in E. coli was similar to the wild-type, except for L207F (low outlier) and E203P (high outlier) (Fig. 4; Text S1).


Characterisation of the putative effector interaction site of the regulatory HbpR protein from Pseudomonas azelaica by site-directed mutagenesis.

Vogne C, Bisht H, Arias S, Fraile S, Lal R, van der Meer JR - PLoS ONE (2011)

Exemplary effects of HbpR A-domain mutations on inducible expression.Measured fluorescence intensities of Escherichia coli cells carrying a plasmid with a promoterless egfp under control of the HbpR-dependent PC-promoter in the presence or absence of 20 µM 2-HBP as inducer. EGFP expression was measured on whole cells at two time points and corrected for culture turbidity. Type I to IV correspond to differently shaded entries in Table 1. Note the delayed response in Type II mutants and the higher background in the absence of inducer in type IV mutants.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040749&req=5

pone-0016539-g003: Exemplary effects of HbpR A-domain mutations on inducible expression.Measured fluorescence intensities of Escherichia coli cells carrying a plasmid with a promoterless egfp under control of the HbpR-dependent PC-promoter in the presence or absence of 20 µM 2-HBP as inducer. EGFP expression was measured on whole cells at two time points and corrected for culture turbidity. Type I to IV correspond to differently shaded entries in Table 1. Note the delayed response in Type II mutants and the higher background in the absence of inducer in type IV mutants.
Mentions: An overview of all mutants constructed in the first and the second group is presented in Table 1. All mutations in the HbpR A-domain were constructed by PCR with mutated oligonucleotides and verified by DNA sequencing. Subsequently, the mutated A-domain sequences were used to replace the gene region for the native A-domain in hbpR on an expression vector in E. coli, with which we could test 2-HBP inducible egfp expression under control of the HbpR-dependent PC promoter. This expression vector results in the addition of a His6-tag to the N-terminal end of the protein. All mutants were tested in E. coli for EGFP expression during exponential growth in the presence or absence of 20 µM 2-HBP, which is the cognate effector for the HbpR-PC system. Table 1 gives representative EGFP induction values after 2 and 4 h induction time compared to those of the strain carrying wild-type HbpR. In general and for all mutants, we observed four types of effects: (i) complete loss of activation with respect to the wild-type (type I), (ii) two-fold loss of induction potential in 2 h but not 4 h incubation periods (type II), (iii) no effect compared to the wild-type (type III), and (iv) considerable increase of background expression (type IV, Table 1, Fig. 3). Protein extracts of the same strains were analyzed by Western blotting using an M13-VHH camel antibody to verify (mutant) HbpR expression (Fig. 4). Surprisingly, all Westerns showed two bands, which likely correspond to His6-tagged HbpR (or mutant, 64.1 kDa) and HbpR (mutant) without His6-tag (62.8 kDa). The reason for the production of two N-terminally different HbpR proteins probably lies in the use of an alternative start codon further downstream. The expression level of most HbpR mutant proteins in E. coli was similar to the wild-type, except for L207F (low outlier) and E203P (high outlier) (Fig. 4; Text S1).

Bottom Line: Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown.We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR.This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

View Article: PubMed Central - PubMed

Affiliation: Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ(54)-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate P(C) promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

Show MeSH
Related in: MedlinePlus