Biotin protein ligase (BPL) is a bi-functional protein that serves both as a ligase to catalyze the biotinylation of important metabolic enzymes, as well as a transcriptional repressor that regulates expression of gene products involved in biotin biosynthesis and transport. In Staphylococcus aureus, an adequate supply of biotin during pathogenesis is vital for the bacteria’s survival. Therefore, BPL plays a critical role in controlling the uptake and synthesis of biotin. The mechanism of BPL binding to DNA has been extensively characterized in Escherichia coli, but not for S. aureus. For both bacteria, BPL dimerization is a prerequisite for DNA binding. For E. coli BPL (EcBPL), the non-liganded enzyme does not form a dimer (K_DIM >1 mM) and is unable to bind DNA. In contrast, analytical ultracentrifugation experiments together with electromobility shift assays and small angle X-ray scattering studies revealed S. aureus BPL (SaBPL) can dimerize in the absence of ligands (K_DIM = 29 μM) and binds DNA. This data suggests that SaBPL exhibits more stringent control of gene expression than EcBPL. However, genetic manipulation of S. aureus is challenging. In order to further characterize the mechanisms of DNA binding and transcription by SaBPL in vivo, an E. coli strain will be engineered to facilitate further work. Two-step recombineering will be used to eliminate the endogenous EcBPL and replace this with an R33G mutant unable to bind DNA. LacZ reporter constructs under the control of promoters containing SaBPL recognition sequences will be chromosomally integrated into one of five phage attachment sites available in E. coli. A second phage attachment site will be employed to provide controlled expression of SaBPL, or variants of the protein. This approach will enable us to better understand BPL-regulated gene expression in vivo and in the context of a bacterial chromosome.