B-291. TraG Is a Variable Inner Membrane Protein Essential for Function of the Neisseria gonorrhoeae Type IV Secretion System

P. L. Kohler, H. L. Hamilton, J. P. Dillard;
Univ. of Wisconsin, Madison, WI.

The Neisseria gonorrhoeae type IV secretion system (T4SS) secretes chromosomal DNA into the culture supernatant and is encoded on a genetic island called the gonococcal genetic island (GGI). The GGI is found in 80% of gonococcal strains and in some strains of N. meningitidis. We have previously shown that activity of a GGI-encoded peptidoglycanase, AtlA, is required for DNA secretion. The gene directly upstream of atlA encodes a protein called TraG that is similar to TraG encoded on the Escherichia coli F-plasmid. Gonococcal TraG is a putative inner membrane protein that is necessary for DNA secretion. Using Southern blot and sequence analysis, we found that the traG-atlA region of the GGI is variable among gonococcal strains. Some gonococcal and meningococcal strains lack atlA and encode a different allele of traG where TraG is fused to a C-terminal, putative peptidoglycan endopeptidase (TraG-EP). We found that this putative peptidoglycanase region of the protein can efficiently bind peptidoglycan but that traG-EP cannot functionally replace the traG-atlA region for DNA secretion. In addition, a gonococcal strain that lacks atlA and carries traG-EP does not secrete DNA. A third allele of traG is found in some strains that also contain atlA. This third allele of traG can functionally replace the originally characterized allele of traG for DNA secretion. Together, these results suggest that atlA is essential for DNA secretion and that traG-EP may function in a process other than DNA secretion. traG genes encoded on conjugative plasmids including the F-plasmid have been shown to be inner membrane proteins with several transmembrane domains and a large C-terminal domain found in the cytoplasm or periplasm. Using cellular fractionation and Western blot analysis we found that gonococcal TraG is a membrane protein. Translational fusions revealed that TraG is an inner membrane protein with several transmembrane domains and a soluble C-terminal region with no transmembrane domains. We hypothesize that this C-terminal domain might be important for interactions with other T4SS components.