This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, M. H.
Right arrow Articles by Rubens, C. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, M. H.
Right arrow Articles by Rubens, C. E.

 Previous Article  |  Next Article 

Journal of Bacteriology, September 1999, p. 5790-5799, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Characterization of Enterococcus faecalis Alkaline Phosphatase and Use in Identifying Streptococcus agalactiae Secreted Proteins

Martin H. Lee,1 Aphakorn Nittayajarn,1 R. Paul Ross,2,dagger Cynthia B. Rothschild,2 Derek Parsonage,2 Al Claiborne,2 and Craig E. Rubens1,*

Department of Pediatrics, Children's Hospital Regional Medical Center CH-31, University of Washington, Seattle, Washington 98105,1 and Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157-10162

Received 30 March 1999/Accepted 7 June 1999

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, phoZDelta 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 be useful in a variety of gram-positive bacteria.

* Corresponding author. Mailing address: Department of Pediatrics, Children's Hospital Regional Medical Center CH-31, University of Washington, 4800 Sand Point Way NE, Seattle, WA 98105. Phone: (206) 528-2767. Fax: (206) 527-3890. E-mail: cruben{at}chmc.org.

dagger Present address: National Dairy Products Research Center, Moorepark, Fermoy, County Cork, Ireland.

Journal of Bacteriology, September 1999, p. 5790-5799, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Shelburne, S. A. III, Sumby, P., Sitkiewicz, I., Okorafor, N., Granville, C., Patel, P., Voyich, J., Hull, R., DeLeo, F. R., Musser, J. M. (2006). Maltodextrin utilization plays a key role in the ability of group a streptococcus to colonize the oropharynx.. Infect. Immun. 74: 4605-4614 [Abstract] [Full Text]  
  • Rajagopal, L., Clancy, A., Rubens, C. E. (2003). A Eukaryotic Type Serine/Threonine Kinase and Phosphatase in Streptococcus agalactiae Reversibly Phosphorylate an Inorganic Pyrophosphatase and Affect Growth, Cell Segregation, and Virulence. J. Biol. Chem. 278: 14429-14441 [Abstract] [Full Text]  
  • Wojciechowski, C. L., Kantrowitz, E. R. (2002). Altering of the Metal Specificity of Escherichia coli Alkaline Phosphatase. J. Biol. Chem. 277: 50476-50481 [Abstract] [Full Text]  
  • Gibson, C. M., Caparon, M. G. (2002). Alkaline Phosphatase Reporter Transposon for Identification of Genes Encoding Secreted Proteins in Gram-Positive Microorganisms. Appl. Environ. Microbiol. 68: 928-932 [Abstract] [Full Text]  
  • Berlutti, F., Passariello, C., Selan, L., Thaller, M. C., Rossolini, G. M. (2001). The Chryseobacterium meningosepticum PafA enzyme: prototype of a new enzyme family of prokaryotic phosphate-irrepressible alkaline phosphatases?. Microbiology 147: 2831-2839 [Abstract] [Full Text]  
  • Lukomski, S., Nakashima, K., Abdi, I., Cipriano, V. J., Shelvin, B. J., Graviss, E. A., Musser, J. M. (2001). Identification and Characterization of a Second Extracellular Collagen-Like Protein Made by Group A Streptococcus: Control of Production at the Level of Translation. Infect. Immun. 69: 1729-1738 [Abstract] [Full Text]  
  • Granok, A. B., Parsonage, D., Ross, R. P., Caparon, M. G. (2000). The RofA Binding Site in Streptococcus pyogenes Is Utilized in Multiple Transcriptional Pathways. J. Bacteriol. 182: 1529-1540 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»