Characterization of Enterococcus faecalis alkaline phosphatase and use in identifying Streptococcus agalactiae secreted proteins.
We have identified and characterized an Enterococcus 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, Streptococcus agalactiae (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, we 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 AP activity is export dependent. The signal sequence deletion mutant, phoZDeltass, 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 be useful in a variety of gram-positive bacteria.
Lee
M H
MH
Department of Pediatrics, Children's Hospital Regional Medical Center CH-31, University of Washington, Seattle, Washington 98105, USA.
Nittayajarn
A
A
Ross
R P
RP
Rothschild
C B
CB
Parsonage
D
D
Claiborne
A
A
Rubens
C E
CE
D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D013293:Enterococcus faecalis / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008561:Membrane Fusion; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010641:Phenotype; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013292:Streptococcus agalactiae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D027981:Symporters; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D021382:Protein Sorting Signals; D027981:Symporters; C021499:lactose permease; D000469:Alkaline Phosphatase