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Characterization of Enterococcus faecalis alkaline phosphatase and use in identifying Streptococcus agalactiae secreted proteins.
- Source :
-
Journal of bacteriology [J Bacteriol] 1999 Sep; Vol. 181 (18), pp. 5790-9. - Publication Year :
- 1999
-
Abstract
- 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.
- Subjects :
- Amino Acid Sequence
Bacterial Proteins chemistry
Cloning, Molecular
Enterococcus faecalis genetics
Escherichia coli genetics
Membrane Fusion
Membrane Transport Proteins chemistry
Molecular Sequence Data
Phenotype
Protein Sorting Signals chemistry
Protein Sorting Signals genetics
Protein Structure, Secondary
Sequence Deletion
Streptococcus agalactiae classification
Streptococcus agalactiae genetics
Transformation, Bacterial
Alkaline Phosphatase genetics
Alkaline Phosphatase metabolism
Bacterial Proteins metabolism
Enterococcus faecalis enzymology
Escherichia coli Proteins
Genes, Bacterial
Membrane Transport Proteins metabolism
Monosaccharide Transport Proteins
Streptococcus agalactiae isolation & purification
Symporters
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9193
- Volume :
- 181
- Issue :
- 18
- Database :
- MEDLINE
- Journal :
- Journal of bacteriology
- Publication Type :
- Academic Journal
- Accession number :
- 10482522
- Full Text :
- https://doi.org/10.1128/JB.181.18.5790-5799.1999