Abstract
Most DNA-binding bacterial transcription factors contact DNA through a recognition α-helix in their DNA-binding domains. An emerging class of DNA-binding transcription factors, predominantly found in pathogenic bacteria interact with the DNA via a relatively novel type of DNA-binding domain, called the LytTR domain, which mainly comprises β strands. Even though the crystal structure of the LytTR domain of the virulence gene transcription factor AgrA from Staphylococcus aureus bound to its cognate DNA sequence is available, the contribution of specific amino acid residues in the LytTR domain of AgrA to transcription activation remains elusive. 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.
Highlights
Bacteria predominantly rely on two-component signal transduction systems (TCS) to sense and adapt gene expression patterns to constantly changing environments
Since the nucleotide sequence upstream of agrA including intergenic region and the P2 and P3 promoter seqences were intact in SH1000−, these results are consistent with the view that the H174L mutation destablizes the structural integrity of accessory gene regulator A (AgrA) in SH1000− and thereby makes it unavailable to activate transcription from P2 and P3 promoters of the agr operon
Since agr dysfunction is associated with reduced -hemolytic activity, we used a blood agar plate hemolysis assay to confirm that -hemolytic activity is restored in SH1000−agr IR P3-GFP containing pSNP2-agrA, but not in the SH1000−agr IR P3-GFP containing pSN-P2-agrAH174L or pSN-P2-empty (Figure 1E)
Summary
Bacteria predominantly rely on two-component signal transduction systems (TCS) to sense and adapt gene expression patterns to constantly changing environments. The typical bacterial TCS comprises signal input and response output components, typically represented by a histidine kinase (HK) and a response regulator (RR), respectively. The majority of RRs are transcription factors (hereafter referred to as RR-TF) with their carboxyl-terminal domain containing a DNA-binding motif, which allows recognition of tandem or inverted repeating DNA elements located upstream of promoters of genes and determinants for interaction with the RNA polymerase for modulating the transcriptional response. The carboxyl-terminal domain of the majority of RR-TFs contains DNA-binding motifs that belong to extensively characterized structural families and include the winged-helix motif of the OmpR/PhoB family, helix-turn-helix motif of the NtrC family and four-helix-helix-turn-helix of the NarL/FixJ family [1,2]. LytTR domain containing RR-TFs are disproportionately involved in the regulation of virulence gene expression. The molecular interactions between the LytTR domain and DNA have been recently elucidated from the crystal structure of the DNAbound complex of the LytTR domain of Staphylococcus aureus accessory gene regulator A (AgrA), which is a celldensity (quorum) responsive global virulence-associated RR-TF [4,8]
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