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

We have identified and characterized an Enteracoccus faecalis alkaline phos phatase (AP, encoded by phoZ). The predicted gene product shows homology wi th alkaline phosphatases from a variety of species; it has especially high similarity with two alkaline phosphatases from Bacillus subtilis, Expressio n of phoZ in Escherichia coli, E.faecalis, Streptacoccus agalactine (group B streptococcus [GBS]), or Streptococcus pyogenes (group A streptococcus [G AS]) produces a blue-colony phenotype on-plates containing a chromogenic su bstrate, 5-bromo-4-chloro-3-indolylphosphate (XP or BCIP), Two tests were m ade 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 g ram-positive bacteria. Restoration of export, using the signal sequence fro m C5a peptidase, restored AP activity to at least 50% of that of the wild t ype. 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 coil, the periplasmic fusion had 17-fold-higher AP activity than the cytoplasmic fusion, We concluded that AI, activity is exp ort dependent. The signal sequence deletion mutant, phoZ Delta ss, was used to identify random genomic fragments from GBS that encode exported protein s or integral membrane proteins. Included in this set of fragments were gen es that exhibited homology with the Rib protein (a cell wall protein from G BS) or with DppB (an integral membrane protein from GAS). AP acts as a repo rter enzyme in GBS, GAS, and E.faecalis and is expected to he useful in a v ariety of gram-positive bacteria.