Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition.

Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).

Membrane association and multimerization of secreton component pulC.

The PulC component of the Klebsiella oxytoca pullulanase secretion machinery (the secreton) was found by subcellular fractionation to be associated with both the cytoplasmic (inner) and outer membranes. Association with the outer membrane was independent of other secreton components, including the outer membrane protein PulD (secretin). The association of PulC with the inner membrane is mediated by the signal anchor sequence located close to its N terminus. These results suggest that PulC forms a bridge between the two membranes that is disrupted when bacteria are broken open for fractionation. Neither the signal anchor sequence nor the cytoplasmic N-terminal region that precedes it was found to be required for PulC function, indicating that PulC does not undergo sequence-specific interactions with other cytoplasmic membrane proteins. Cross-linking of whole cells resulted in the formation of a ca. 110-kDa band that reacted with PulC-specific serum and whose detection depended on the presence of PulD. However, antibodies against PulD failed to react with this band, suggesting that it could be a homo-PulC trimer whose formation requires PulD. The data are discussed in terms of the possible role of PulC in energy transduction for exoprotein secretion.

Structure-function analysis of XcpP, a component involved in general secretory pathway-dependent protein secretion in Pseudomonas aeruginosa.

The general secretory pathway of Pseudomonas aeruginosa is required for the transport of signal peptide-containing exoproteins across the cell envelope. After completion of the Sec-dependent translocation of exoproteins across the inner membrane and cleavage of the signal peptide, the Xcp machinery mediates translocation across the outer membrane. This machinery consists of 12 components, of which XcpQ (GspD) is the sole outer membrane protein. XcpQ forms a multimeric ring-shaped structure, with a central opening through which exoproteins could pass to reach the medium. Surprisingly, all of the other Xcp proteins are located in or are associated with the cytoplasmic membrane. This study is focused on the characteristics of one such cytoplasmic membrane protein, XcpP. An xcpP mutant demonstrated that the product of this gene is indeed an essential element of the P. aeruginosa secretion machinery. Construction and analysis of truncated forms of XcpP made it possible to define essential domains for the function of the protein. Some of these domains, such as the N-terminal transmembrane domain and a coiled-coil structure identified at the C terminus of XcpP, may be involved in protein-protein interaction during the assembly of the secretory apparatus.