New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis.
We have created new complementation constructs for use in Neisseria gonorrhoeae and Neisseria meningitidis. The constructs contain regions of homology with the chromosome and direct the insertion of a gene of interest into the intergenic region between the genes iga and trpB. In order to increase the available options for gene expression in Neisseria, we designed the constructs to contain one of three different promoters. One of the constructs contains the isopropyl-β-d-thiogalactopyranoside-inducible lac promoter, which has been widely used in Neisseria. We also designed a construct that contains the strong, constitutive promoter from the gonococcal opaB gene. The third construct contains a tetracycline-inducible promoter, a novel use of this promoter in Neisseria. We demonstrate that anhydrotetracycline can be used to induce gene expression in the pathogenic Neisseria at very low concentrations and without negatively affecting the growth of the organisms. We use these constructs to complement an arginine auxotrophy in N. gonorrhoeae as well as to express a translational fusion of alkaline phosphatase with TraW. TraW is a component of the gonococcal type IV secretion system, and we demonstrate that TraW localizes to the periplasm.
Department of Medical Microbiology and Immunology, University of Wisconsin—Madison School of Medicine and Public Health, Madison, Wisconsin, USA.
Meghan E
ME
Ramsey
Kathleen T
KT
Hackett
Chaitra
C
Kotha
Joseph P
JP
Dillard
D015870:Gene Expression; D005816:Genetic Complementation Test; D005818:Genetic Engineering / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D016254:Mutagenesis, Insertional; D009344:Neisseria gonorrhoeae / Q000235:genetics*; D009345:Neisseria meningitidis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D015533:Transcriptional Activation