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. In the present study, we performed biochemical analyses of the XpsG protein of Xanthomonas campestris pv. campestris and observed that it is a pillar-like structure spanning the cytoplasmic and outer membranes. Subcellular fractionation revealed a soluble form (SF) of XpsG, in addition to the membrane form. Chromatographic analysis of SF XpsG in the absence of a detergent indicated that it is part of a large complex (>440 kDa). In vitro studies indicated that XpsG is prone to aggregate in the absence of a detergent. We isolated and characterized a non-functional mutant defective in forming the large complex. It did not interfere with the function of wild-type XpsG and was not detectable in the SF. Moreover, unlike wild-type XpsG, which was distributed in both the cytoplasmic and outer membranes, it appeared only in the cytoplasmic membrane. When wild-type XpsG was co-expressed with His6-tagged XpsH but not with untagged XpsH, SF XpsG bound to nickel and co-eluted with XpsH. This result suggests the presence of other pseudopilin components in the XpsG-containing large-sized molecules.
Hu
Nien-Tai
NT
Institute of Biochemistry, National Chung Hsing University, Taichung 402, Taiwan.
Leu
Wei-Ming
WM
Lee
Meng-Shiunn
MS
Chen
Avon
A
Chen
Shu-Chung
SC
Song
Yu-Ling
YL
Chen
Ling-Yun
LY
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D010861:Fimbriae, Bacterial / Q000737:chemistry / Q000235:genetics; D026901:Membrane Transport Proteins; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D016959:Xanthomonas campestris / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D026901:Membrane Transport Proteins; C071567:general secretion pathway protein, Xanthomonas campestris