Serves as the substrate for the sorting reaction, which is the tethering of the C-terminal threonine of the surface protein to lipid II by an amide bond. Lipid II tethered with the surface proteins is finally incorporated into mature peptidoglycan. Previously, we have described that the N-terminal signal peptides of staphylococcal lipases harbor a conserved motif – Ser, Ile, Arg and Lys. This motif is later found conserved in many, but not all surface proteins. SP with the YSIRK/GS motif promotes the secretion of surface proteins. In Streptococcus pyogenes and in S. aureus, the SP has a function in directing surface proteins to different surface localizations. In S. aureus, SP directs the secretion and anchoring of surface proteins at septum, while the SP leads the secretion and anchoring of surface proteins more to the cell pole. It has also been shown that three transmembrane proteins, namely Spd proteins, are involved in the surface display of protein A, one of the predominant surface proteins carrying SP. The expression level and surface display of protein A are largely reduced in each spd mutant. Moreover, spd mutants affect the expression of surface proteins with SP. Interestingly, the spd mutants exhibit an increased abundance of visible cross walls and thickened cross walls. Yet, how cross wall formation affects the surface display of surface proteins remains unclear. Conventionally, immunofluorescence microscopy has been applied to surface proteins localization studies. However, immunofluorescence microscopy has a certain intrinsic limitation that especially impedes the subcellular and high throughput studies. For example, antibodies cannot penetrate into the septum without cell wall permeabilization; yet cell wall permeabilization using cell wall hydrolase or detergents often leads to the release of surface proteins with the risk of artifacts. Further, a large numbers of specific antibodies are needed in order to study various surface proteins’ localization, which is laborious and time consuming. Particularly in S. aureus immunofluorescence is extremely hindered by protein A, the IgG binding protein. In this study, we developed a direct visualization method for monitoring the surface proteins anchoring process. The red fluorescent protein mCherry was fused with different signal sequences and targeted as cytoplasmic, secreted, and cell wall anchored. Cell wall anchored mCherry enabled us to visualize the cross and peripheral wall localization pattern rather than using immunofluorescence microscopy. Intriguingly, independent of different signal peptides, treatment with sub-lethal concentrations of cell wall biosynthesis antibiotics led to strong accumulation of mCh-cw at the cross wall which MDV3100 customer reviews correlated with the increased Van-FL binding at the cross wall. Our results show that mCherry is a useful tool to localize and follow the anchoring or secretion processes in staphylococci. So far, immunofluorescence microscopy and immunoelectron microscopy have been used for surface proteins localization studies in the last decades. To our knowledge, there is no direct visualization method to be applied in this field yet. In this study, we aimed to develop a direct method for monitoring surface proteins’ subcellular distribution.