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

research-article

Use of the "blue halo" assay in the identification of genes encoding exported proteins with cleavable signal peptides: cloning of a Borrelia burgdorferi plasmid gene with a signal peptide.

Department of Medicine, UCLA School of Medicine 90024.

ABSTRACT

We have recently reported a phoA expression vector, termed pMG, which, like TnphoA, is useful in identifying genes encoding membrane-spanning sequences or signal peptides. This cloning system has been modified to facilitate the distinction of outer membrane and periplasmic alkaline phosphatase (AP) fusion proteins from inner membrane AP fusion proteins by transforming pMG recombinants into Escherichia coli KS330, the strain utilized in the "blue halo" assay first described by Strauch and Beckwith (Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988). The lipoprotein mutation lpp-5508 of KS330 results in an outer membrane that is leaky to macromolecules, and its degP4 mutation greatly reduces periplasmic proteolytic degradation of AP fusion proteins. pMG AP fusions containing cleavable signal peptides, including the E. coli periplasmic protein beta-lactamase, the E. coli and Chlamydia trachomatis outer membrane proteins OmpA and MOMP, respectively, and Tp 9, a Treponema pallidum AP recombinant, diffused through the leaky outer membrane of KS330 and resulted in blue colonies with blue halos. In contrast, inner membrane AP fusions derived from E. coli proteins, including leader peptidase, SecY, and the tetracycline resistance gene product, as well as Tp 70, a T. pallidum AP recombinant which does not contain a signal peptide, resulted in blue colonies without blue halos. Lipoprotein-AP fusions, including the Borrelia burgdorferi OspA and T. pallidum Tp 75 and TmpA showed halo formation, although there was significantly less halo formation than that produced by either periplasmic or outer membrane AP fusions. In addition, we applied this approach to screen recombinants constructed from a 9.0-kb plasmid isolated from the B31 virulent strain of B. burgdorferi. One of the blue halo colonies identified produced an AP fusion protein which contained a signal peptide with a leader peptidase I cleavage recognition site. The pMG/KS330r- cloning and screening approach can identify genes encoding proteins with cleavable signal peptides and therefore can serve as a first step in the identification of genes encoding potential virulence factors.

This article has been cited by other articles:

• Haake, D. A., Matsunaga, J. (2002). Characterization of the Leptospiral Outer Membrane and Description of Three Novel Leptospiral Membrane Proteins. Infect. Immun. 70: 4936-4945 [Abstract] [Full Text]  
• Skare, J. T., Foley, D. M., Hernandez, S. R., Moore, D. C., Blanco, D. R., Miller, J. N., Lovett, M. A. (1999). Cloning and Molecular Characterization of Plasmid-Encoded Antigens of Borrelia burgdorferi. Infect. Immun. 67: 4407-4417 [Abstract] [Full Text]  
• Kornacki, J. A., Oliver, D. B. (1998). Lyme Disease-Causing Borrelia Species Encode Multiple Lipoproteins Homologous to Peptide-Binding Proteins of ABC-Type Transporters. Infect. Immun. 66: 4115-4122 [Abstract] [Full Text]