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Systematic mutational analysis of the LytTR DNA binding domain of Staphylococcus aureus virulence gene transcription factor AgrA.

Nicod SS, Weinzierl RO, Burchell L, Escalera-Maurer A, James EH, Wigneshweraraj S - Nucleic Acids Res. (2014)

Bottom Line: Most DNA-binding bacterial transcription factors contact DNA through a recognition α-helix in their DNA-binding domains.Here, for the first time, we have systematically investigated the role of amino acid residues in transcription activation in a LytTR domain-containing transcription factor.Our analysis, which involves in vivo and in vitro analyses and molecular dynamics simulations of S. aureus AgrA identifies a highly conserved tyrosine residue, Y229, as a major amino acid determinant for maximal activation of transcription by AgrA and provides novel insights into structure-function relationships in S. aureus AgrA.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Molecular Microbiology and Infection, Imperial College London, London, UK.

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Systematic mutational analysis of Staphylococcus aureus AgrA LytTR domain. (A) Graph showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD600) relative to the SH1000−agr IR P3-GFP + pSN-P2-agrA (WT) for each single alanine mutant after 8 h of growth in TSB. AgrA mutants displaying more than 60% activity compared to the wild-type AgrA are shown in white, the AgrA mutants displaying less than 60% activity compared to the wild-type AgrA are shown in grey and the controls (SH1000−agr IR P3-GFP + pSN-P2-agrAH174L, SH1000−agr IR P3-GFP + pSN-P2-empty, SH1000−agr IR P3-GFP + pSN-P2-agrA and SH1000−agr IR P3-GFP + pSN-P2-agrAR233A) are shown in black. (B) Graphs showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD600) over time for SH1000−agr IR P3-GFP + pSN-itet-agrA (green lines) and SH1000−agr IR P3-GFP + pSN-itet-agrAH174L (red lines) strains grown in TSB with and without anhydrotetracycline. The bar chart in the insert represents the GFP expression of the same samples (color coded accordingly) at the 8 h time point. (C) Graph showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD600) relative to the SH1000−agr IR P3-GFP + pSN-itet-agrA (WT) for each of the 21 single alanine mutant displaying less than 60% wild-type activity after 8 h of growth in TSB. A section of the western blot image indicating AgrA detected in whole cell lysates for each mutant is shown under the graph. For the 13 mutants that are detectably expressed, the quantification of the intensity of the band corresponding to AgrA mutants relative to the intensity of the band corresponding to the wild-type AgrA level is shown on the graph. Data for (A–C) were obtained from at least three biological replicates. (D) As in Figure 1B with the six of the eight aa residues (Y156, F161, F196, N206, I210 and N224), which when changed to alanine appear to impair the gross structural stability of AgrA indicated in red. (E) Multiple sequence alignment of the AgrA LytTR domain of representative staphylococci strains. Conserved residues are represented by a dot. The aa residues displaying similar colors have similar properties. The putative TAD mutants are highlighted in yellow.
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Figure 2: Systematic mutational analysis of Staphylococcus aureus AgrA LytTR domain. (A) Graph showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD600) relative to the SH1000−agr IR P3-GFP + pSN-P2-agrA (WT) for each single alanine mutant after 8 h of growth in TSB. AgrA mutants displaying more than 60% activity compared to the wild-type AgrA are shown in white, the AgrA mutants displaying less than 60% activity compared to the wild-type AgrA are shown in grey and the controls (SH1000−agr IR P3-GFP + pSN-P2-agrAH174L, SH1000−agr IR P3-GFP + pSN-P2-empty, SH1000−agr IR P3-GFP + pSN-P2-agrA and SH1000−agr IR P3-GFP + pSN-P2-agrAR233A) are shown in black. (B) Graphs showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD600) over time for SH1000−agr IR P3-GFP + pSN-itet-agrA (green lines) and SH1000−agr IR P3-GFP + pSN-itet-agrAH174L (red lines) strains grown in TSB with and without anhydrotetracycline. The bar chart in the insert represents the GFP expression of the same samples (color coded accordingly) at the 8 h time point. (C) Graph showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD600) relative to the SH1000−agr IR P3-GFP + pSN-itet-agrA (WT) for each of the 21 single alanine mutant displaying less than 60% wild-type activity after 8 h of growth in TSB. A section of the western blot image indicating AgrA detected in whole cell lysates for each mutant is shown under the graph. For the 13 mutants that are detectably expressed, the quantification of the intensity of the band corresponding to AgrA mutants relative to the intensity of the band corresponding to the wild-type AgrA level is shown on the graph. Data for (A–C) were obtained from at least three biological replicates. (D) As in Figure 1B with the six of the eight aa residues (Y156, F161, F196, N206, I210 and N224), which when changed to alanine appear to impair the gross structural stability of AgrA indicated in red. (E) Multiple sequence alignment of the AgrA LytTR domain of representative staphylococci strains. Conserved residues are represented by a dot. The aa residues displaying similar colors have similar properties. The putative TAD mutants are highlighted in yellow.

Mentions: To indentify aa residues in the LytTR domain involved in transcription activation, we generated a mutant library of AgrA by alanine-scanning mutagenesis of the LytTR domain using pSN-P2-agrA as the template. Based on the crystal structure of the AgrA LytTR domain–DNA complex (4), we only targeted residues for alanine substitution that were neither implicated in DNA binding, nor known to be important for maintaining the structrual fold of the LytTR domain. We also constructed the previously described DNA-binding defective mutant AgrAR233A to use as a negative control. SH1000−agr IR P3-GFP cells were transformed with the library comprising a total of 74 AgrA mutants in pSN-P2-agrA and mutant AgrA activity was determined by measuring GFP fluorescence as a function of cell density (OD600 nm) after 8 h of growth in rich media. As expected, GFP activity was bareley detected in SH1000−agr IR P3-GFP containing pSN-P2-agrAR233A and pSN-P2-agrAH174L in comparison to SH1000−agr IR P3-GFP containing pSN-P2-agrA (Figure 2A). Based on the spread of the activities of the mutant AgrA library in SH1000−agr IR P3-GFP, the AgrA mutants were categorized into two activity groups: <60% (21 mutants) and >60% (51 mutants) activity relative to wild-type AgrA (Figure 2A). Mutants in the first group, which hereafter are referred to as putative transcription-activation-defective (TAD) mutants, were selected for further analysis.


Systematic mutational analysis of the LytTR DNA binding domain of Staphylococcus aureus virulence gene transcription factor AgrA.

Nicod SS, Weinzierl RO, Burchell L, Escalera-Maurer A, James EH, Wigneshweraraj S - Nucleic Acids Res. (2014)

Systematic mutational analysis of Staphylococcus aureus AgrA LytTR domain. (A) Graph showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD600) relative to the SH1000−agr IR P3-GFP + pSN-P2-agrA (WT) for each single alanine mutant after 8 h of growth in TSB. AgrA mutants displaying more than 60% activity compared to the wild-type AgrA are shown in white, the AgrA mutants displaying less than 60% activity compared to the wild-type AgrA are shown in grey and the controls (SH1000−agr IR P3-GFP + pSN-P2-agrAH174L, SH1000−agr IR P3-GFP + pSN-P2-empty, SH1000−agr IR P3-GFP + pSN-P2-agrA and SH1000−agr IR P3-GFP + pSN-P2-agrAR233A) are shown in black. (B) Graphs showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD600) over time for SH1000−agr IR P3-GFP + pSN-itet-agrA (green lines) and SH1000−agr IR P3-GFP + pSN-itet-agrAH174L (red lines) strains grown in TSB with and without anhydrotetracycline. The bar chart in the insert represents the GFP expression of the same samples (color coded accordingly) at the 8 h time point. (C) Graph showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD600) relative to the SH1000−agr IR P3-GFP + pSN-itet-agrA (WT) for each of the 21 single alanine mutant displaying less than 60% wild-type activity after 8 h of growth in TSB. A section of the western blot image indicating AgrA detected in whole cell lysates for each mutant is shown under the graph. For the 13 mutants that are detectably expressed, the quantification of the intensity of the band corresponding to AgrA mutants relative to the intensity of the band corresponding to the wild-type AgrA level is shown on the graph. Data for (A–C) were obtained from at least three biological replicates. (D) As in Figure 1B with the six of the eight aa residues (Y156, F161, F196, N206, I210 and N224), which when changed to alanine appear to impair the gross structural stability of AgrA indicated in red. (E) Multiple sequence alignment of the AgrA LytTR domain of representative staphylococci strains. Conserved residues are represented by a dot. The aa residues displaying similar colors have similar properties. The putative TAD mutants are highlighted in yellow.
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Figure 2: Systematic mutational analysis of Staphylococcus aureus AgrA LytTR domain. (A) Graph showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD600) relative to the SH1000−agr IR P3-GFP + pSN-P2-agrA (WT) for each single alanine mutant after 8 h of growth in TSB. AgrA mutants displaying more than 60% activity compared to the wild-type AgrA are shown in white, the AgrA mutants displaying less than 60% activity compared to the wild-type AgrA are shown in grey and the controls (SH1000−agr IR P3-GFP + pSN-P2-agrAH174L, SH1000−agr IR P3-GFP + pSN-P2-empty, SH1000−agr IR P3-GFP + pSN-P2-agrA and SH1000−agr IR P3-GFP + pSN-P2-agrAR233A) are shown in black. (B) Graphs showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD600) over time for SH1000−agr IR P3-GFP + pSN-itet-agrA (green lines) and SH1000−agr IR P3-GFP + pSN-itet-agrAH174L (red lines) strains grown in TSB with and without anhydrotetracycline. The bar chart in the insert represents the GFP expression of the same samples (color coded accordingly) at the 8 h time point. (C) Graph showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD600) relative to the SH1000−agr IR P3-GFP + pSN-itet-agrA (WT) for each of the 21 single alanine mutant displaying less than 60% wild-type activity after 8 h of growth in TSB. A section of the western blot image indicating AgrA detected in whole cell lysates for each mutant is shown under the graph. For the 13 mutants that are detectably expressed, the quantification of the intensity of the band corresponding to AgrA mutants relative to the intensity of the band corresponding to the wild-type AgrA level is shown on the graph. Data for (A–C) were obtained from at least three biological replicates. (D) As in Figure 1B with the six of the eight aa residues (Y156, F161, F196, N206, I210 and N224), which when changed to alanine appear to impair the gross structural stability of AgrA indicated in red. (E) Multiple sequence alignment of the AgrA LytTR domain of representative staphylococci strains. Conserved residues are represented by a dot. The aa residues displaying similar colors have similar properties. The putative TAD mutants are highlighted in yellow.
Mentions: To indentify aa residues in the LytTR domain involved in transcription activation, we generated a mutant library of AgrA by alanine-scanning mutagenesis of the LytTR domain using pSN-P2-agrA as the template. Based on the crystal structure of the AgrA LytTR domain–DNA complex (4), we only targeted residues for alanine substitution that were neither implicated in DNA binding, nor known to be important for maintaining the structrual fold of the LytTR domain. We also constructed the previously described DNA-binding defective mutant AgrAR233A to use as a negative control. SH1000−agr IR P3-GFP cells were transformed with the library comprising a total of 74 AgrA mutants in pSN-P2-agrA and mutant AgrA activity was determined by measuring GFP fluorescence as a function of cell density (OD600 nm) after 8 h of growth in rich media. As expected, GFP activity was bareley detected in SH1000−agr IR P3-GFP containing pSN-P2-agrAR233A and pSN-P2-agrAH174L in comparison to SH1000−agr IR P3-GFP containing pSN-P2-agrA (Figure 2A). Based on the spread of the activities of the mutant AgrA library in SH1000−agr IR P3-GFP, the AgrA mutants were categorized into two activity groups: <60% (21 mutants) and >60% (51 mutants) activity relative to wild-type AgrA (Figure 2A). Mutants in the first group, which hereafter are referred to as putative transcription-activation-defective (TAD) mutants, were selected for further analysis.

Bottom Line: Most DNA-binding bacterial transcription factors contact DNA through a recognition α-helix in their DNA-binding domains.Here, for the first time, we have systematically investigated the role of amino acid residues in transcription activation in a LytTR domain-containing transcription factor.Our analysis, which involves in vivo and in vitro analyses and molecular dynamics simulations of S. aureus AgrA identifies a highly conserved tyrosine residue, Y229, as a major amino acid determinant for maximal activation of transcription by AgrA and provides novel insights into structure-function relationships in S. aureus AgrA.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Molecular Microbiology and Infection, Imperial College London, London, UK.

Show MeSH
Related in: MedlinePlus