Expanding the Staphylococcus aureus SarA Regulon to Small RNAs

Area of Science:

• Microbiology
• Molecular Biology
• Genomics

Background:

• Staphylococcus aureus is a significant human and animal pathogen responsible for diverse infections.
• Gene expression regulation is crucial for S. aureus adaptation during infection, involving transcriptional factors (TFs) and small regulatory RNAs (sRNAs).
• The SarA transcriptional regulator is a key global regulator influencing virulence, metabolism, biofilm formation, and antibiotic resistance in S. aureus.

Purpose of the Study:

• To comprehensively characterize the SarA regulon in S. aureus, including both messenger RNAs (mRNAs) and sRNAs.
• To identify novel SarA-regulated genes and elucidate the regulatory mechanisms.
• To establish a novel consensus DNA binding sequence for SarA.

Main Methods:

• Transcriptome sequencing (RNA-Seq) to identify differentially expressed mRNAs and sRNAs in a ΔsarA mutant.
• Chromatin immunoprecipitation sequencing (ChIP-Seq) to identify direct SarA binding targets across the genome.
• Northern blotting and electrophoretic mobility shift assays (EMSAs) to validate novel SarA targets.
• Bioinformatic analysis, including genome-wide scanning using a deduced SarA motif, to predict new targets.

Main Results:

• RNA-Seq identified 390 differentially expressed mRNAs and 51 sRNAs in the ΔsarA mutant.
• ChIP-Seq revealed 354 mRNA and 55 sRNA targets directly bound by SarA.
• The study identified 85 unprecedented mRNA and sRNA targets, with at least 14 being primary targets.
• Repression of sprG2, encoding a type I toxin-antitoxin system toxin, was characterized, revealing a multilayered toxin expression control.
• A novel SarA consensus DNA binding sequence was determined, enabling prediction of additional target genes.

Conclusions:

• The combination of RNA-Seq, ChIP-Seq, and bioinformatic analyses provides a robust method for characterizing transcriptional factor regulons.
• This study significantly expands the known SarA regulon, identifying numerous novel mRNA and sRNA targets.
• The findings reveal a complex regulatory network, including a novel multilayered repression of toxin expression by SarA and its antitoxin SprF2.