Polar secretion of proteins via the Xcp type II secretion system in Pseudomonas aeruginosa

Abstract

The subcellular localization of the major type II secretion system of Pseudomonas aeruginosa, the Xcp system, was studied microscopically using a biarsenical ligand that becomes fluorescent upon binding to a tetracysteine motif (Lumio tag), which was fused to several Xcp components. Fusion of the Lumio tag to the C termini of the XcpR and XcpS proteins did not affect the functionality of these proteins. Fluorescence microscopy showed that they were predominantly localized to the poles of P. aeruginosa cells, when produced at levels comparable to chromosomally encoded XcpR and XcpS. In most labelled cells, the proteins were found at one of the poles, although bipolar localization was also observed. When produced in the absence of other Xcp components, labelled XcpS was still found to locate at the poles, whereas XcpR was evenly distributed in the cell. These data suggest that XcpS, but not XcpR, contains information required for polar localization. The polar location of the Xcp machinery was further confirmed by the visualization of protease secretion with an intramolecularly quenched casein conjugate.

Protein Secretion Systems in Pseudomonas aeruginosa: An Essay on Diversity, Evolution, and Function

“…The visualization of these impressive macromolecular complexes always raises the question on how the cell envelope, and particularly the peptidoglycan, are reorganized to make them fit. One obvious answer is the use of specific transglycosylases, or the assembly of the nanomachine at the pole (Senf et al, 2008), region where the peptidoglycan mesh is loose.…”

Localization of Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Subunits during Intoxication of Live Cells

“…This technology has been successfully used to examine virus entry and trafficking in cells (26,27,36) and secretion of the cytosolic and periplasmic Xcp polar protein in Pseudomonas aeruginosa (35). Optimal biarsenical compound binding increased resistance to compound release by dithiol reducing agents, and increased fluorescence quantum yield was achieved by engineering the tetracysteine motif, Cys-Cys-ProGly-Cys-Cys, and a four-amino-acid spacer (Gly-Ala-Gly-Gly) into each subunit protein (24) (Invitrogen).…”

Transcriptional Profiling of Rice Leaves Undergoing a Hypersensitive Response Like Reaction Induced by Xanthomonas oryzae pv. oryzae Cellulase

“…oryzae is going to be much higher than that used in this experiment. Besides the above, there is evidence that secretion by T2S of V. cholerae and P. aeruginosa occurs in a polar manner because the T2S apparatus is localized to one pole of the cell, possibly at the point of attachment to host surface (Scott et al 2001;Senf et al 2008). If the T2S of X. oryzae pv.…”