We also addressed the question of which role SrtA might play in targeting. In DsrtA, proteins remain at least transiently in the membrane via their C-terminal CWS domain. In the absence of antibiotics a similar distribution of mCh-cw was observed in DsrtA, as in WT. In DsrtA-mCh-cw1, mCh was more accumulated in the cross wall and in DsrtA-mCh-cw2, mCh was more abundant in the side wall. The effect of penicillin and moenomycin in the DsrtA mutant was, however, not as pronounced as in the WT. In the presence of penicillin or moenomycin, not only mCh-cw but also Van-FL was concentrated in the cross wall, indicating that there is an increased content of free D-Ala-D-Ala residues, which represent the substrates for the SrtA transpeptidation reaction. Such an accumulation of uncross-linked peptidoglycan precursors can be postulated since penicillin and moenomycin are known to bind to the active site of PBPs, thus blocking the transpeptidation and transglycosylation, respectively. It was surprising that vancomycin had little effect on mCh-cw distribution, as theoretically vancomycin inhibits both transpeptidation and transglycosylation. The previously described inhibiting effect of vancomycin is most likely due to the 10-times higher concentration used in their studies R428 causing a complete inhibition of transpeptidation or transglycosylation. This paper is more than the introduction of a new experimental approach. We used this new tool to directly follow the targeting and anchoring of various mCh-hybrid constructs. We found that the SPs with or without YSIRK motif targeted proteins to different subcellular localizations. However, in the presence of sub-lethal concentrations of penicillin and moenomycin the influence of SP in targeting was abrogated as all anchored mCh-cw was concentrated at the cross wall. We assume that the antibiotics cause accumulation of SrtA substrates at the cross wall, which attract SrtA to incorporate the mCh-cw almost exclusively at the cross wall, irrespective of SP type. With this study we contribute to better understanding the influence of different signal peptide types in targeting anchored and secreted proteins and the role of cell wall antibiotics. Bladder cancers develop along two “tracks” producing tumors with very different clinical characteristics. One leads to the formation of papillary tumors with high recurrence rates that rarely metastasize or cause death, whereas the other leads to the development of non-papillary, muscle-invasive tumors, of which a subset progresses rapidly and is fatal. At present it is impossible to prospectively identify the lethal muscle-invasive tumors; however, accumulating evidence suggests that molecular reprogramming characteristic of a developmental process known as epithelial-tomesenchymal transition is involved. Muscle-invasive cancers are characterized by downregulation of E-cadherin and p63, two “epithelial” markers uniformly expressed in normal urothelium and in non muscle-invasive cancers. These changes are accompanied by upregulation of mesenchymal markers Zeb-1, Zeb-2, vimentin, and MMP9, leading to increased invasion and migration. A role for EMT in driving bladder cancer progression and metastases is consistent with a large body of evidence emerging in other solid tumors, particularly breast cancer.