Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis

The major pseudopilin XpsG is an essential component of type II secretion apparatus of Xanthomonas campestris pv. campestris. Along with other ancillary pseudopilins, it forms a pilus-like structure spanning between cytoplasmic and outer membranes. Associations of pseudopilins with non-pseudopilin members of type II secretion apparatus were not well documented, probably due to their dynamic or unstable nature. In this study, by treating intact cells with a cleavable crosslinker dithiobis(succinimidylpropionate) (DSP), followed by metal chelating chromatography and immuno-blotting on secretion-positive strains of X. campestris pv. campestris, we discovered associations of XpsGh with XpsN (GspC), as well as XpsD. These associations were detectable in a strain missing all components, but XpsO, of the type II secretion apparatus. However, chromosomal non-polar mutation in each gene exerted different effects upon the association between the other two. The XpsGh/XpsD association is undetectable in xpsN mutant; however, it was restored to a limited extent by overproducing XpsD protein. The XpsGb/XpsN association is unaltered by a lack of XpsD protein or an elevation of its abundance. Co-immune precipitation between XpsN and XpsD, while being independent of XpsG, was nonetheless enhanced by raising XpsG protein level. These observations agree with the proposition that the type II secretion apparatus in a cell may exist as an integrated multiprotein complex with all components working in concert. Moreover, in functional machinery, the association of the major pseudopilin XpsG with secretin XpsD appears strongly dependent on the existence of XpsN, the GspC protein.