Systematic mutational analysis of the LytTR DNA binding domain of Staphylococcus aureus virulence gene transcription factor AgrA.
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.
Affiliation: MRC Centre for Molecular Microbiology and Infection, Imperial College London, London, UK.Show MeSH
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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.
Affiliation: MRC Centre for Molecular Microbiology and Infection, Imperial College London, London, UK.