Localization, expression, and regulation of the type IV secretion system in Neisseria gonorrhoeae

The Neisseria gonorrhoeae type IV secretion system (T4SS) secretes chromosomal DNA into the environment where it can be used for natural transformation. I developed new tools to study the T4SS including plasmids for gene complementation and monoclonal antibodies against T4SS proteins. I used these tools to show that TraK and TraB, predicted structural components of the secretion apparatus, localize to the outer membrane, and that TraK interacts with the lipoprotein TraV. These results suggest that TraK, TraB, and TraV contribute to the structure of the outer membrane pore, making the gonococcal T4SS structurally similar to other characterized T4SSs despite its distinct contact-independent mechanism of DNA secretion. TraK and TraB were not detectable by Western blot in protein extracts of laboratory-grown wild-type cells, although traK is transcribed, suggesting that very low levels of these structural proteins are sufficient for type IV secretion. An epitope-tagged variant of TraK (TraK-FLAG3) was detectable when expressed from the native locus, perhaps due to signal amplification from the tandem epitope. In immunogold electron microscopy studies, TraK-FLAG3 was detected as a single gold particle in the envelope, and flow cytometry experiments showed that there is not detectable cell-to-cell variation in TraK-FLAG3 levels.;To better understand the expression and regulation of the T4SS genes, which are contained in the gonococcal genetic island (GGI), we generated mutants that overexpress T4SS genes and investigated mechanisms that govern gene expression in wild-type cells. Targeted mutagenesis of two predicted promoter elements resulted in increased expression of downstream genes, but did not result in increased levels of DNA secretion, perhaps because T4SS proteins are degraded by a tail-specific protease homolog in this strain. Regulation of another transcript in the GGI depended on two stem-loops in the 5' UTR. One of the stem-loops occludes the ribosome binding site, while the other is involved in the formation of an alternative permissive structure. Finally, I showed that the transcriptional regulator FarR binds to the yaf-ltgX intergenic region. FarR represses the farAB-encoded antimicrobial efflux pump in N. gonorrhoeae, and it is possible that expression of the T4SS may be coordinated with expression of antimicrobial efflux pumps.