The Wzx translocase is an essential protein in the biosynthetic pathway of O antigens, which is the immune-dominant antigen in most Gram-negative bacteria. It is responsible for the transport of short oligosaccharide repeat units (O units) across the inner membrane, these O units are assembled onto the Lipid-A core and exported the cell surface as Lipopolysaccharide. The O antigen structure varies between strains based on the composition of the O unit and has evolved this way to avoid host immune recognition. Like the O units, there is also diversity among the translocases, most likely to accommodate the various structures it is required to flip. Previous studies on the translocase have found that they have specificity for particular O unit structures, with preference for their cognate O unit. Over expression of the translocase however has been shown to improve the efficiency of translocation making it a challenge to compare specificity between different O unit structures.
I aim to address this issue of specificity by measuring translocation rate on a quantitative basis and at low levels of expression to mirror chromosomal expression. I therefore have to develop a sensitive assay to detect and quantify very low levels of Wzx to compare rates between the different structures. I designed a system to express Wzx with a cleavable GFP tag to quantify the translocase, however at low expression it was difficult to detect by either fluorescence microscopy or flow cytometry. The biggest issue was the background auto-fluorescence and to work around this I cleaved off the GFP tag and purified the tag to measure fluorescence without any background. By quantifying the Wzx, I can then directly compare the translocation rates of different O units by compensating for the different expression levels. This work will further expand on the specificity and mechanisms of Wzx.