Protein secretion zones during overexpression of amylase within the Gram-positive cell wall.

BACKGROUND: Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE within two different Bacillus strains, B. subtilis and B. licheniformis. RESULTS: We show that a C-terminal fusion of AmyE with the fluorescent reporter mCherry is secreted via discrete patches showing very low dynamics. These are visible at many places within the cell wall for many minutes. Expression from a high copy number plasmid was required to be able to see these structures we term "secretion zones". Zones corresponded to visualized AmyE activity on the surface of cells, showing that they release active enzymes. They overlapped with SecA signals but did not frequently co-localize with the secretion ATPase. Single particle tracking showed higher dynamics of SecA and of SecDF, involved in AmyE secretion, at the cell membrane than AmyE. These experiments suggest that SecA initially translocates AmyE molecules through the cell membrane, and then diffuses to a different translocon. Single molecule tracking of SecA suggests the existence of three distinct diffusive states of SecA, which change during AmyE overexpression, but increased AmyE secretion does not appear to overwhelm the system. CONCLUSIONS: Because secretion zones were only found during the transition to and within the stationary phase, diffusion rather than passive transport based on cell wall growth from inside to outside may release AmyE and, thus, probably secreted proteins in general. Our findings suggest active transport through the cell membrane and slow, passive transition through the cell wall, at least for overexpressed proteins, in bacteria of the genus Bacillus.

Ca2+-Daptomycin targets cell wall biosynthesis by forming a tripartite complex with undecaprenyl-coupled intermediates and membrane lipids.

The lipopeptide daptomycin is used as an antibiotic to treat severe infections with gram-positive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA) and drug-resistant enterococci. Its precise mechanism of action is incompletely understood, and a specific molecular target has not been identified. Here we show that Ca2+-daptomycin specifically interacts with undecaprenyl-coupled cell envelope precursors in the presence of the anionic phospholipid phosphatidylglycerol, forming a tripartite complex. We use microbiological and biochemical assays, in combination with fluorescence and optical sectioning microscopy of intact staphylococcal cells and model membrane systems. Binding primarily occurs at the staphylococcal septum and interrupts cell wall biosynthesis. This is followed by delocalisation of components of the peptidoglycan biosynthesis machinery and massive membrane rearrangements, which may account for the pleiotropic cellular events previously reported. The identification of carrier-bound cell wall precursors as specific targets explains the specificity of daptomycin for bacterial cells. Our work reconciles apparently inconsistent previous results, and supports a concise model for the mode of action of daptomycin.