New Complementation Constructs for Inducible and Constitutive Gene Expression in Neisseria gonorrhoeae and Neisseria meningitidis

Abstract

ABSTRACTWe have created new complementation constructs for use inNeisseria gonorrhoeaeandNeisseria 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 genesigaandtrpB. In order to increase the available options for gene expression inNeisseria, we designed the constructs to contain one of three different promoters. One of the constructs contains the isopropyl-β-d-thiogalactopyranoside-induciblelacpromoter, which has been widely used inNeisseria. We also designed a construct that contains the strong, constitutive promoter from the gonococcalopaBgene. The third construct contains a tetracycline-inducible promoter, a novel use of this promoter inNeisseria. We demonstrate that anhydrotetracycline can be used to induce gene expression in the pathogenicNeisseriaat very low concentrations and without negatively affecting the growth of the organisms. We use these constructs to complement an arginine auxotrophy inN. gonorrhoeaeas 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.