The peptidoglycan (PG) layers surrounding bacterial cells play an important role 
in determining cell shape. The machinery controlling when and where new PG is 
made is not understood, but is proposed to involve interactions between 
bacterial actin homologs such as Mbl, which forms helical cables within cells, 
and extracellular multiprotein complexes that include penicillin-binding 
proteins. It has been suggested that labeled antibiotics that bind to PG 
precursors may be useful for imaging PG to help determine the genes that control 
the biosynthesis of this polymer. Here, we compare the staining patterns 
observed in Bacillus subtilis using fluorescent derivatives of two PG-binding 
antibiotics, vancomycin and ramoplanin. The staining patterns for both probes 
exhibit a strong dependence on probe concentration, suggesting 
antibiotic-induced perturbations in PG synthesis. Ramoplanin probes may be 
better imaging agents than vancomycin probes because they yield clear staining 
patterns at concentrations well below their minimum inhibitory concentrations. 
Under some conditions, both ramoplanin and vancomycin probes produce helicoid 
staining patterns along the cylindrical walls of B. subtilis cells. This 
sidewall staining is observed in the absence of the cytoskeletal protein Mbl. 
Although Mbl plays an important role in cell shape determination, our data 
indicate that other proteins control the spatial localization of the 
biosynthetic complexes responsible for new PG synthesis along the walls of B. 
subtilis cells.