The influence of secretory-protein charge on late stages of secretion fromthe Gram-positive bacterium Bacillus subtilis

Following their secretion across the cytoplasmic membrane, processed secret ory proteins of Bacillus subtilis must fold into their native conformation prior to translocation through the cell wall and release into the culture m edium. The rate and efficiency of folding are critical in determining the y ields of intact secretory proteins. The B. subtilis membrane is surrounded by a thick cell wall comprising a heteropolymeric matrix of peptidoglycan a nd anionic polymers. The latter confer a high density of negative charge on the wall, endowing it with ion-exchange properties, and secretory proteins destined for the culture medium must traverse the wall as the last stage i n the export process. To determine the influence of charge on late stages i n the secretion of proteins from this bacterium, we have used sequence data from two related alpha-amylases, to engineer the net charge of AmyL, an al pha-amylase from Bacillus licheniformis that is normally secreted efficient ly from B. subtilis. While AmyL has a pI of 7.0, chimaeric enzymes with pi values of 5.0 and 10.0 were produced and characterized. Despite the enginee red changes to their physico-chemical properties, the chimaeric enzymes ret ained many of the enzymic characteristics of AmyL, We show that the positiv ely charged protein interacts with the cell wall in a manner that influence s its secretion.