Modularization of the type II secretion gene cluster from Xanthomonas euvesicatoria facilitates the identification of a structurally conserved XpsCLM assembly platform complex

Abstract

Many bacterial pathogens depend on a type II secretion (T2S) system to secrete virulence factors from the periplasm into the extracellular milieu. T2S systems consist of an outer membrane secretin channel, a periplasmic pseudopilus and an inner membrane-associated assembly platform including a cytoplasmic ATPase. The components of T2S systems are often conserved in different bacterial species, however, the architecture of the assembly platform is largely unknown. Here, we analysed predicted assembly platform components of the Xps-T2S system from the plant-pathogenic bacterium Xanthomonas euvesicatoria. To facilitate these studies, we generated a modular xps-T2S gene cluster by Golden Gate assembly of single promoter and gene fragments. The modular design allowed the efficient deletion and replacement of T2S genes and the insertion of reporter fusions. Mutant approaches as well as interaction and crosslinking studies showed that the predicted assembly platform components XpsC, XpsL and XpsM form a trimeric complex which is essential for T2S and associates with the cytoplasmic ATPase XpsE and the secretin XpsD. Structural modeling revealed a similar trimeric architecture of XpsCLM homologs from Pseudomonas, Vibrio and Klebsiella species, despite overall low amino acid sequence similarities. In X. euvesicatoria, crosslinking and fluorescence microscopy studies showed that the formation of the XpsCLM complex is independent of the secretin and vice versa, suggesting that the assembly of the T2S system is a dynamic process which involves the association of preformed subcomplexes.