Project Summary/Abstract Despite their small size, bacterial cells precisely synchronize cellular processes in space and time. The cell envelopes of bacteria are complex and dynamic structures that are coordinately assembled during the cell cycle. Cell wall anchored surface proteins of Gram-positive bacteria are major cell envelope components, which are secreted across the cytoplasmic membrane and covalently attached to cell wall peptidoglycan by sortase A (SrtA). Strikingly, many surface proteins contain a specific YSIRK/G-S signal peptide that targets proteins to the cross-wall during cell division. Coordinated with cell division and cell wall synthesis, cross-wall targeting promotes efficient incorporation of surface proteins to the newly synthesized cross-wall peptidoglycan; however, the mechanisms remain unknown. It has been proposed that the YSIRK/G-S signal peptide promotes localized secretion at the division septum. However, by developing a new microscopy method in our model organism of Staphylococcus aureus, we now provide evidence that in contrast to the prevailing model, the targeting does not occur during secretion, but rather is SrtA-dependent. We further discovered that cross-wall targeting is regulated by another important cell envelope component: LtaS-mediated lipoteichoic acid (LTA) synthesis and D- alanylation of teichoic acids. Intriguingly, LTA synthesis and D-alanylation regulate different biogenesis stages: LTA regulates SrtA-mediated septal anchoring whereas D-alanylation modulates cross-wall deposition. Collectively, these recent discoveries from my own lab form the foundation of my independent research program for this MIRA application. We will elucidate the distinct mechanisms by which the YSIRK/G-S signal peptide, LTA synthesis and D-alanylation spatially regulate surface protein biogenesis during the cell cycle. Successful completion of the research projects will not only reveal novel mechanisms underlying surface protein biogenesis in Gram-positive bacteria, but also uncover novel functions of LTA and D-alanylation in cell envelope assembly, providing fundamental insight into how bacterial cells precisely coordinate cell envelope assembly during growth and cell division.