In vitro and in vivo characterization of Staphylococcus aureus biotin protein ligase transcriptional repressor function

Abstract

Staphylococcus aureus is a versatile and potentially dangerous human pathogen. One of the traits of S. aureus that is crucial for its survival during pathogenesis is its ability to quickly adapt to changes in the microenvironment, including an ability to adapt to the limited availability of micronutrients such as biotin. Biotin is a co-factor required for important metabolic enzymes such as pyruvate carboxylase (PC) and acetyl CoA carboxylase (ACC). In certain bacteria like S. aureus, the protein that is responsible for managing biotin homeostasis is the biotin retention protein, BirA (also known as biotin protein ligase or BPL). BirA is a bi-functional protein that serves as both the enzyme responsible for protein biotinylation and a transcriptional repressor that regulates biotin biosynthesis and import. Escherichia coli BirA (EcBirA) has been well studied, however, less extensive studies have been performed on S. aureus BirA (SaBirA). Whilst EcBirA regulates transcription of the biotin biosynthesis operon (bioO), SaBirA has multiple targets including bioO, the biotin transporter (SabioY) and genes involved in fatty acid synthesis (SayhfS-SayhfT). For both EcBirA and SaBirA, homodimerization is a pre-requisite for DNA binding and subsequent repressor activity. In the absence of protein requiring biotinylation, and when cellular demand for biotin is low, BirA will dimerize, bind to its target DNA and repress expression of biotin biosynthetic enzymes. Previous studies in our laboratory revealed clear differences between EcBirA and SaBirA. One of these differences is that dimerization and DNA binding of EcBirA only takes place when the protein is in complex with the reaction intermediate biotinyl-5ʹ-AMP (i.e. the holo-enzyme), whereas SaBirA was able to dimerize and bind DNA in both the holo (KD²⁻¹= 29 μM, KD DNA = 108 nM) and non-liganded (i.e. apo) states (KD²⁻¹ = 30 μM, KD DNA = 649 nM). I hypothesized that there are clear distinctions in the DNA binding interaction between SaBirA and the well-studied EcBirA. These differences allow S. aureus to elegantly orchestrate biotin synthesis and transport in response to external biotin availability. This study aims to define SaBirA-regulated gene expression using in vitro and in vivo methods. In addition, the effect of extracellular biotin concentration on biotin uptake and gene expression in both S. aureus and E. coli were also investigated in this study. The result showed that within 30 minutes, biotin starved S. aureus could sense changes in exogenous biotin and responded with increased biotin uptake and down regulation of biotin synthesis (>100-fold). These rapid responses were not observed in E. coli. Furthermore, the DNA-binding activity of SaBirA was also probed in vivo. Since S. aureus is not naturally competent to transformation, it can be technically difficult to genetically manipulate this bacteria. To overcome this problem, reporter strains were constructed in E. coli containing chromosomally integrated SaBirA and EcBirA, as well as their target promoters fused to a lacZ reporter gene. Here I confirmed that birA from both bacteria are biotin-responsive transcription factors. Moreover, based on the dimerization constant of apo- SaBirA (KD²⁻¹ = 30 μM) and apo-EcBirA (KD²⁻¹ = 2 mM), and the predicted intracellular concentration of BirA (2nM – 100nM), it is estimated that these apo proteins are predominantly monomeric in growing cells. Therefore, mutant proteins with abolished in vitro dimerization ability were included as mimics of the monomeric apo-state, namely SaBirA F123G and EcBirA R119W . The results obtained from the in vivo assays showed that SaBirA F123G repressed the target promoters, whereas EcBirA R119W was devoid of repressor activity. These results were confirmed in vitro by gel-shift assays. Cross-linking studies added further evidence that DNA promotes dimerization of SaBirA F123G, but not E. coli R119W. In vitro analysis also revealed the affinity for DNA binding varies between SaBirA-target promoters. This suggested a hierarchy of SaBPL regulated genes, with the biotin biosynthesis operon being the most responsive to exogenous biotin concentration. Taken together, the outcomes from in vivo and in vitro analyses performed in this study have validated the hypothesis that SaBirA uses different DNA binding mechanisms to EcBirA. As a consequence, SaBirA provides S. aureus with one avenue to adapt in response to its environment. Finally, this study also investigated the role of a novel SaBirA inhibitor, BPL199, as a co-repressor in DNA binding and its effect on gene transcription. Quantitative Real-Time PCR experiments revealed that BPL199 was able to act as a co-repressor to down-regulate expression of biotin-regulated genes in vivo, with similar kinetics as biotin. EMSA analysis showed that the affinity of SaBPL:BPL199 for DNA binding was similar to that of the natural substrate, biotinyl-5ʹ-AMP. This supported the proposal that BPL199 successfully mimics the action of biotinyl-5ʹ-AMP in initiating transcriptional repression. In addition, a BPL199-resistant strain of S. aureus generated in our laboratory, was also investigated. DNA sequencing revealed a single point mutation in SaBirA (D200E) that mapped within its dimerization interface. The ability of SaBirA D200E to bind DNA, and down regulate gene expression, was subsequently addressed. The results indicated that SaBirA D200E was compromised in the SaBirA:DNA interaction in vivo. The most susceptible target was the SabioY promoter, suggesting that increased transport of exogenous biotin is one mechanism that can be employed by the bacteria to overcome compounds that target BPL. Thesis layout: The thesis will be presented as a combination one published literature review, one manuscript to be submitted for publication as well as conventional chapters. Each manuscript will be a chapter with its own references. A general introduction and discussion will also be included to link together all the research conducted during this candidature. A publishing agreement with all co-authors involved with the work is also included.