D-Alanine substitution of teichoic acids as a modulator of protein foldingand stability at the cytoplasmic membrane/cell wall interface of Bacillus subtilis

The extracytoplasmic folding of secreted proteins in Gram-positive bacteria is influenced by the microenvironment of the compartment into which they a re translocated, namely the negatively charged matrix of the cell wall poly mers. In this compartment, the PrsA lipoprotein facilitates correct post-tr anslocational folding or prevents misfolding of secreted proteins. In this study, a secretion mutant of B, subtilis (prsA3) encoding a defective PrsA protein was mutagenized and screened for restored secretion of the AmyQ alp ha-amylase, One mini-Tn10 insertion, which partially suppressed the secreti on deficiency, was found to interrupt dht, the operon involved in the D-ala nylation of teichoic acids. The inactivation of dlt rescued the mutant PrsA 3 protein from degradation, and the increased amount of PrsA3 was shown to enhance the secretion of PrsA-dependent proteins. Heterologous or abnormal secreted proteins, which are prone to degradation after translocation, were also stabilized and secreted in increased quantities from a dlt prsA(+) st rain, Furthermore, the dlt mutation partially suppressed the lethal effect of PrsA depletion, suggesting that the dlt deficiency also leads to stabili zation of an essential cell wall protein(s), Our results suggest that main influence of the increased net negative charge of the wall caused by the ab sence of D-alanine is to increase the rate of post-translocational folding of exported proteins.