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

## TL;DR

This study reveals how a key complex in a bacterial secretion system is structured and assembled, offering insights into how pathogens spread virulence factors.

## Contribution

The modular gene cluster approach enabled the identification of a conserved trimeric XpsCLM complex in T2S systems.

## Key findings

- XpsC, XpsL, and XpsM form a trimeric complex essential for T2S function.
- The XpsCLM complex associates with the ATPase XpsE and secretin XpsD.
- Structural modeling shows similar XpsCLM architecture across diverse bacterial species.

## 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.

The plant-pathogenic bacterium Xanthomonas euvesicatoria secretes degradative enzymes via a type II secretion (T2S) system into the extracellular milieu to facilitate infection of its host plants, pepper and tomato. T2S systems are composed of an outer membrane secretin channel, a periplasmic pseudopilus and an inner membrane-associated assembly platform with a cytoplasmic ATPase. The architecture of the assembly platform, which is required for substrate recognition in the periplasm, is still largely uncharacterized. In the present study, we used a modular gene cluster approach to study the assembly platform components XpsC, XpsL and XpsM from X. euvesicatoria. We show that they form a trimeric complex, which associates with the cytoplasmic ATPase XpsE and the secretin XpsD. Structural modeling suggests the presence of similar trimeric complexes in related bacteria, despite low amino acid sequence similarity of the individual proteins. Additionally, our studies indicate that the formation of the XpsCLM complex is independent of the secretin, highlighting the dynamic nature of T2S assembly, which likely involves the association of preformed subcomplexes. This research enhances our understanding of bacterial T2S systems and their contribution to the secretion of virulence factors.