This Article
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 Framson, P. E.
Right arrow Articles by Rubens, C. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Framson, P. E.
Right arrow Articles by Rubens, C. E.
Agricola
Right arrow Articles by Framson, P. E.
Right arrow Articles by Rubens, C. E.

 Previous Article  |  Next Article 

Appl. Environ. Microbiol., Sep 1997, 3539-3547, Vol 63, No. 9
Copyright © 1997, American Society for Microbiology

New genetic techniques for group B streptococci: high-efficiency transformation, maintenance of temperature-sensitive pWV01 plasmids, and mutagenesis with Tn917

PE Framson, A Nittayajarn, J Merry, P Youngman and CE Rubens
Virginia Mason Research Center, University of Washington, Seattle, USA.

Three techniques were developed to improve the genetic manipulation of group B streptococci (GBS). We first optimized a protocol for transformation of GBS by electroporation, which provided transformation efficiencies of 10(5) CFU/microgram. Variables that influenced the transformation efficiency were the glycine content of the competent cell growth media, the electric field strength during electroporation, the electroporation buffer composition, the host origin of the transforming plasmid, and the concentration of selective antibiotic at the final plating. Our transformation protocol provides an efficiency sufficient for cloning from ligation reactions directly into GBS, obviating an intermediate host such as Escherichia coli. Second, temperature-sensitive plasmids of the pWV01 lineage were shown to transform GBS, and their temperature-sensitive replication was confirmed. Lastly, the temperature-sensitive pWV01 plasmid pTV1OK, which contains Tn917, was used as a transposon delivery vector for the construction of genomic Tn917 mutant libraries. We have shown, for the first time, that Tn917 transposes to the GBS chromosome and at a frequency of 10(-3)/CFU. Furthermore, representative clones from a Tn917 library contained single transposon insertions that were randomly located throughout the chromosome. These techniques should provide useful methods for cloning, mutagenesis, and characterization of genes from GBS.


This article has been cited by other articles:

  • Bryan, J. D., Shelver, D. W. (2009). Streptococcus agalactiae CspA Is a Serine Protease That Inactivates Chemokines. J. Bacteriol. 191: 1847-1854 [Abstract] [Full Text]  
  • Kimura, K., Suzuki, S., Wachino, J.-i., Kurokawa, H., Yamane, K., Shibata, N., Nagano, N., Kato, H., Shibayama, K., Arakawa, Y. (2008). First Molecular Characterization of Group B Streptococci with Reduced Penicillin Susceptibility. Antimicrob. Agents Chemother. 52: 2890-2897 [Abstract] [Full Text]  
  • Dahesh, S., Hensler, M. E., Van Sorge, N. M., Gertz, R. E. Jr., Schrag, S., Nizet, V., Beall, B. W. (2008). Point Mutation in the Group B Streptococcal pbp2x Gene Conferring Decreased Susceptibility to {beta}-Lactam Antibiotics. Antimicrob. Agents Chemother. 52: 2915-2918 [Abstract] [Full Text]  
  • Maisey, H. C., Quach, D., Hensler, M. E., Liu, G. Y., Gallo, R. L., Nizet, V., Doran, K. S. (2008). A group B streptococcal pilus protein promotes phagocyte resistance and systemic virulence. FASEB J. 22: 1715-1724 [Abstract] [Full Text]  
  • Puopolo, K. M., Klinzing, D. C., Lin, M. P., Yesucevitz, D. L., Cieslewicz, M. J. (2007). A composite transposon associated with erythromycin and clindamycin resistance in group B Streptococcus. J Med Microbiol 56: 947-955 [Abstract] [Full Text]  
  • Locke, J. B., Colvin, K. M., Datta, A. K., Patel, S. K., Naidu, N. N., Neely, M. N., Nizet, V., Buchanan, J. T. (2007). Streptococcus iniae Capsule Impairs Phagocytic Clearance and Contributes to Virulence in Fish. J. Bacteriol. 189: 1279-1287 [Abstract] [Full Text]  
  • Seepersaud, R., Needham, R. H. V., Kim, C. S., Jones, A. L. (2006). Abundance of the {delta} Subunit of RNA Polymerase Is Linked to the Virulence of Streptococcus agalactiae. J. Bacteriol. 188: 2096-2105 [Abstract] [Full Text]  
  • Nallapareddy, S. R., Singh, K. V., Murray, B. E. (2006). Construction of Improved Temperature-Sensitive and Mobilizable Vectors and Their Use for Constructing Mutations in the Adhesin-Encoding acm Gene of Poorly Transformable Clinical Enterococcus faecium Strains. Appl. Environ. Microbiol. 72: 334-345 [Abstract] [Full Text]  
  • Mora, M., Bensi, G., Capo, S., Falugi, F., Zingaretti, C., Manetti, A. G. O., Maggi, T., Taddei, A. R., Grandi, G., Telford, J. L. (2005). Group A Streptococcus produce pilus-like structures containing protective antigens and Lancefield T antigens. Proc. Natl. Acad. Sci. USA 102: 15641-15646 [Abstract] [Full Text]  
  • Buchanan, J. T., Stannard, J. A., Lauth, X., Ostland, V. E., Powell, H. C., Westerman, M. E., Nizet, V. (2005). Streptococcus iniae Phosphoglucomutase Is a Virulence Factor and a Target for Vaccine Development. Infect. Immun. 73: 6935-6944 [Abstract] [Full Text]  
  • Chaffin, D. O., Mentele, L. M., Rubens, C. E. (2005). Sialylation of Group B Streptococcal Capsular Polysaccharide Is Mediated by cpsK and Is Required for Optimal Capsule Polymerization and Expression. J. Bacteriol. 187: 4615-4626 [Abstract] [Full Text]  
  • Fontaine, M. C., Perez-Casal, J., Willson, P. J. (2004). Investigation of a Novel DNase of Streptococcus suis Serotype 2. Infect. Immun. 72: 774-781 [Abstract] [Full Text]  
  • Adderson, E. E., Takahashi, S., Wang, Y., Armstrong, J., Miller, D. V., Bohnsack, J. F. (2003). Subtractive Hybridization Identifies a Novel Predicted Protein Mediating Epithelial Cell Invasion by Virulent Serotype III Group B Streptococcus agalactiae. Infect. Immun. 71: 6857-6863 [Abstract] [Full Text]  
  • Shelver, D., Rajagopal, L., Harris, T. O., Rubens, C. E. (2003). MtaR, a Regulator of Methionine Transport, Is Critical for Survival of Group B Streptococcus In Vivo. J. Bacteriol. 185: 6592-6599 [Abstract] [Full Text]  
  • Jones, A. L., Needham, R. H. V., Rubens, C. E. (2003). The Delta Subunit of RNA Polymerase Is Required for Virulence of Streptococcus agalactiae. Infect. Immun. 71: 4011-4017 [Abstract] [Full Text]  
  • Jeng, A., Sakota, V., Li, Z., Datta, V., Beall, B., Nizet, V. (2003). Molecular Genetic Analysis of a Group A Streptococcus Operon Encoding Serum Opacity Factor and a Novel Fibronectin-Binding Protein, SfbX. J. Bacteriol. 185: 1208-1217 [Abstract] [Full Text]  
  • Chen, Q., Wu, H., Fives-Taylor, P. M. (2002). Construction of a Novel Transposon Mutagenesis System Useful in the Isolation of Streptococcus parasanguis Mutants Defective in Fap1 Glycosylation. Infect. Immun. 70: 6534-6540 [Abstract] [Full Text]  
  • Areschoug, T., Linse, S., Stalhammar-Carlemalm, M., Heden, L.-O., Lindahl, G. (2002). A Proline-Rich Region with a Highly Periodic Sequence in Streptococcal {beta} Protein Adopts the Polyproline II Structure and Is Exposed on the Bacterial Surface. J. Bacteriol. 184: 6376-6383 [Abstract] [Full Text]  
  • Fuller, J. D., Camus, A. C., Duncan, C. L., Nizet, V., Bast, D. J., Thune, R. L., Low, D. E., de Azavedo, J. C. S. (2002). Identification of a Streptolysin S-Associated Gene Cluster and Its Role in the Pathogenesis of Streptococcus iniae Disease. Infect. Immun. 70: 5730-5739 [Abstract] [Full Text]  
  • Beckmann, C., Waggoner, J. D., Harris, T. O., Tamura, G. S., Rubens, C. E. (2002). Identification of Novel Adhesins from Group B Streptococci by Use of Phage Display Reveals that C5a Peptidase Mediates Fibronectin Binding. Infect. Immun. 70: 2869-2876 [Abstract] [Full Text]  
  • Tamura, G. S., Nittayajarn, A., Schoentag, D. L. (2002). A Glutamine Transport Gene, glnQ, Is Required for Fibronectin Adherence and Virulence of Group B Streptococci. Infect. Immun. 70: 2877-2885 [Abstract] [Full Text]  
  • Cheng, Q., Stafslien, D., Purushothaman, S. S., Cleary, P. (2002). The Group B Streptococcal C5a Peptidase Is Both a Specific Protease and an Invasin. Infect. Immun. 70: 2408-2413 [Abstract] [Full Text]  
  • Areschoug, T., Stalhammar-Carlemalm, M., Karlsson, I., Lindahl, G. (2002). Streptococcal beta Protein Has Separate Binding Sites for Human Factor H and IgA-Fc. J. Biol. Chem. 277: 12642-12648 [Abstract] [Full Text]  
  • Cvitkovitch, D.G. (2001). Genetic Competence and Transformation in Oral Streptococci. CROBM 12: 217-243 [Abstract] [Full Text]  
  • Chaffin, D. O., Beres, S. B., Yim, H. H., Rubens, C. E. (2000). The Serotype of Type Ia and III Group B Streptococci Is Determined by the Polymerase Gene within the Polycistronic Capsule Operon. J. Bacteriol. 182: 4466-4477 [Abstract] [Full Text]  
  • Nizet, V., Beall, B., Bast, D. J., Datta, V., Kilburn, L., Low, D. E., De Azavedo, J. C. S. (2000). Genetic Locus for Streptolysin S Production by Group A Streptococcus. Infect. Immun. 68: 4245-4254 [Abstract] [Full Text]  
  • Lee, M. H., Nittayajarn, A., Ross, R. P., Rothschild, C. B., Parsonage, D., Claiborne, A., Rubens, C. E. (1999). Characterization of Enterococcus faecalis Alkaline Phosphatase and Use in Identifying Streptococcus agalactiae Secreted Proteins. J. Bacteriol. 181: 5790-5799 [Abstract] [Full Text]  
  • Spellerberg, B., Pohl, B., Haase, G., Martin, S., Weber-Heynemann, J., Lütticken, R. (1999). Identification of Genetic Determinants for the Hemolytic Activity of Streptococcus agalactiae by ISS1 Transposition. J. Bacteriol. 181: 3212-3219 [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»