Characterization of Enterococcus faecalis alkaline phosphatase and use in identifying Streptococcus agalactiae secreted proteins

We have identified and characterized an Enteracoccus faecalis alkaline phosphatase (AP, encoded by phoZ). The predicted gene product shows homology with alkaline phosphatases from a variety of species; it has especially high similarity with two alkaline phosphatases from Bacillus subtilis, Expression of phoZ in Escherichia coli, E.faecalis, Streptacoccus agalactine (group B streptococcus [GBS]), or Streptococcus pyogenes (group A streptococcus [GAS]) produces a blue-colony phenotype on-plates containing a chromogenic substrate, 5-bromo-4-chloro-3-indolylphosphate (XP or BCIP), Two tests were made to determine if the activity of the enzyme is dependent upon the enzyme's subcellular location. First, elimination of the signal sequence reduced AP activity to 3% of the wild-type activity (or less) in three species of gram-positive bacteria. Restoration of export, using the signal sequence from C5a peptidase, restored AP activity to at least 50% of that of the wild type. Second, rye engineered two chimeric proteins in which AP was fused to either a periplasmic domain or a cytoplasmic domain of lactose permease (a membrane protein). In E coli, the periplasmic fusion had 17-fold-higher AP activity than the cytoplasmic fusion, We concluded that AI, activity is export dependent. The signal sequence deletion mutant, phoZ Delta ss, was used to identify random genomic fragments from GBS that encode exported proteins or integral membrane proteins. Included in this set of fragments were genes that exhibited homology with the Rib protein (a cell wall protein from GBS) or with DppB (an integral membrane protein from GAS). AP acts as a reporter enzyme in GBS, GAS, and E.faecalis and is expected to he useful in a variety of gram-positive bacteria.