Function-related positioning of the type II secretion ATPase of Xanthomonas campestris pv. campestris.

Gram-negative bacteria use the type II secretion (T2S) system to secrete exoproteins for attacking animal or plant cells or to obtain nutrients from the environment. The system is unique in helping folded proteins traverse the outer membrane. The secretion machine comprises multiple proteins spanning the cell envelope and a cytoplasmic ATPase.

Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation.

Membrane-associated ATPase constitutes an essential element common to all secretion machineries in Gram-negative bacteria. How ATP hydrolysis by these ATPases is coupled to secretion process remains unclear. Here we identified R286 as a key residue in the type II secretion system (T2SS) ATPase XpsE of Xanthomonas campestris that plays a pivotal role in coupling ATP hydrolysis to protein translocation.

XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL.

GspE belongs to a secretion NTPase superfamily, members of which are involved in type II/IV secretion, type IV pilus biogenesis and DNA transport in conjugation or natural transformation. Predicted to be a cytoplasmic protein, GspE has nonetheless been shown to be membrane-associated by interacting with the N-terminal cytoplasmic domain of GspL. By taking biochemical and genetic approaches, we observed that ATP binding triggers oligomerization of Xanthomonas campestris XpsE (a GspE homolog) as well as its association with the N-terminal domain of XpsL (a GspL homolog).

Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system.

Secretion of fully folded extracellular proteins across the outer membrane of Gram-negative bacteria is mainly assisted by the ATP-dependent type II secretion system (T2SS). Depending on species, 12-15 proteins are usually required for the function of T2SS by forming a trans-envelope multiprotein secretion complex. Here we report crystal structures of an essential component of the Xanthomonas campestris T2SS, the 21-kDa N-terminal domain of cytosolic secretion ATPase XpsE (XpsEN), in two conformational states.

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.

Functional dissection of the XpsN (GspC) protein of the Xanthomonas campestris pv. campestris type II secretion machinery.

Type II secretion machinery is composed of 12 to 15 proteins for translocating extracellular proteins across the outer membrane. XpsL, XpsM, and XpsN are components of such machinery in the plant pathogen Xanthomonas campestris pv. campestris.

Crystallization and preliminary X-ray crystallographic analysis of the N-terminal domain of XpsE protein from Xanthomonas campestris, an essential component of the type II protein-secretion machinery.

Secretion of pre-folded extracellular proteins across the outer membrane of Gram-negative bacteria is mainly assisted by the type II secretion machinery composed of 12-15 proteins. Here, the crystallization and preliminary analysis of one of the essential components of Xanthomonas campestris secretion machinery, the 21 kDa N-terminal domain of XpsE protein (XpsE(N)), are reported. XpsE(N) has been crystallized at 277 K using PEG 400 as precipitant.

A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus.

The cytoplasmic membrane proteins XpsL, XpsM and XpsN are components required for type II secretion in Xanthomonas campestris pv. campestris. We performed metal-chelating chromatography to partially purify the His(6)-tagged XpsM (XpsMh)-containing complex.

XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes.

GspG, -H, -I, -J and -K proteins are members of the pseudopilin family. They are the components required for the type II secretion pathway, which translocates proteins across the outer membrane of Gram-negative bacteria to the extracellular milieu. They were predicted to form a pilus-like structure, and this has been shown for PulG of Klebsiella oxytoca by using electron microscopy.

Insertion mutagenesis of XpsD, an outer-membrane protein involved in extracellular protein secretion in Xanthomonas campestris pv. campestris.

XpsD is an outer-membrane protein required for extracellular protein secretion in Xanthomonas campestris pv. campestris. Cross-linking and gelfiltration chromatography analyses have suggested that it forms a multimer.

Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris.

The last ORF of an xps gene cluster, designated xpsD, is required for the secretion of extracellular enzymes across the outer membrane in Xanthomonas campestris pv. campestris. It could encode a protein of 759 amino acid residues.