Abstract
An adequate supply of biotin is vital for the survival and pathogenesis of Staphylococcus aureus. The key protein responsible for maintaining biotin homeostasis in bacteria is the biotin retention protein A (BirA, also known as biotin protein ligase). BirA is a bi-functional protein that serves both as a ligase to catalyse the biotinylation of important metabolic enzymes, as well as a transcriptional repressor that regulates biotin biosynthesis, biotin transport and fatty acid elongation. The mechanism of BirA regulated transcription has been extensively characterized in Escherichia coli, but less so in other bacteria. Biotin-induced homodimerization of E. coli BirA (EcBirA) is a necessary prerequisite for stable DNA binding and transcriptional repression. Here, we employ a combination of native mass spectrometry, in vivo gene expression assays, site-directed mutagenesis and electrophoretic mobility shift assays to elucidate the DNA binding pathway for S. aureus BirA (SaBirA). We identify a mechanism that differs from that of EcBirA, wherein SaBirA is competent to bind DNA as a monomer both in the presence and absence of biotin and/or MgATP, allowing homodimerization on the DNA. Bioinformatic analysis demonstrated the SaBirA sequence used here is highly conserved amongst other S. aureus strains, implying this DNA-binding mechanism is widely employed.
Highlights
IntroductionDespite the intrinsic DNA-binding activity of the apo enzyme, S. aureus BirA (SaBirA) has been shown to function as a biotin-dependent transcriptional repressor[26,27]
E. coli is a part of the intestinal microflora which contributes to the synthesis and release of substantial amounts of biotin
We propose that S. aureus BirA has evolved unique properties that allow the bacteria to adapt to low biotin environments
Summary
Despite the intrinsic DNA-binding activity of the apo enzyme, SaBirA has been shown to function as a biotin-dependent transcriptional repressor[26,27] These observations are significantly different to EcBirA, where apo-protein is devoid of DNA-binding activity. Engineered protein mutants designed to disrupt homodimerization, namely EcBirA-R119W and the S. aureus equivalent SaBirA-F123G (Supporting Fig. S1), were employed to address the requirement for protein dimerization on DNA-binding (in vitro) and transcriptional repression (in vivo). Together these data revealed that monomeric SaBirA is competent to bind DNA and, once bound, can recruit a second protein subunit. We propose that this DNA binding mechanism may be widely used amongst S. aureus
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