Specific interaction between OutD, an Erwinia chrysanthemi outer membrane protein of the general secretory pathway, and secreted proteins

Abstract

OutD is an outer membrane component of the main terminal branch of the general secretory pathway (GSP) in Erwinia chrysanthemi. We analyzed the interactions of OutD with other components of the GSP (Out proteins) and with secreted proteins (PelB, EGZ and PemA). OutD is stabilized by its interaction with another GSP component, OutS. The 62 C‐terminal amino acids of OutD are necessary for this interaction. In vivo formation of OutD multimers, up to tetramers, was proved after the dissociation in mild conditions of the OutD aggregates formed in the outer membrane. Thus, OutD could form a channel‐like structure in the outer membrane. We showed that OutD is stabilized in vivo when co‐expressed with Out‐secreted proteins. This stabilization results from the formation of complexes that were detected in experiments of co‐immunoprecipitation and co‐sedimentation in sucrose density gradients. The presence of the N‐terminal part of OutD is required for this interaction. The interaction between OutD and the secreted protein PelB was confirmed in vitro, suggesting that no other component of the GSP is required for this recognition. No interaction was observed between the E.carotovora PelC and the E.chrysanthemi OutD. Thus, the interaction between GspD and the secreted proteins present in the periplasm could be the key to the specificity of the secretion machinery and a trigger for that process.

Translocation of Group 1 Capsular Polysaccharide in Escherichia coli Serotype K30

“…Secretins are multimeric OM proteins in which a conserved C-terminal ␤-barrel rich domain is implicated in forming the multimeric OM channel (39 -42). Typically, 6 -14 identical subunits form the ringlike structure of the secretin (39,(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52). Despite similarities in overall architecture of the secretin and Wza multimers, the respective monomers share no primary sequence similarity.…”

“…Non-acylated secretins typically require an additional OM lipoprotein ("secretin pilot") that associates with the secretin (61) and is required for formation of a stable multimer as well as, in some cases, localization of the multimer. Examples are found in type II (48,50,62,63) and III (45,46,64,65) protein secretion systems from various bacteria.…”

Cysteine Scanning Mutagenesis and Disulfide Mapping Analysis of Arrangement of GspC and GspD Protomers within the Type 2 Secretion System

“…Secretins form pore-like toroidal structures composed of 12-14 protomers, through which secretion substrates can be translocated (16, 18 -21). The poreforming activity has been attributed to the conserved C-terminal portion of secretins, whereas their variable N-terminal part is thought to span the periplasm and to be involved in the recognition of the secretion substrate (9,(22)(23)(24)(25). In the secretin GspD, this region consists of four domains, N0-N3.…”

Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD

“…Dodecameric PulD forms a translocation channel in the outer membrane (8). Dodecamer formation is essential for secreton function and does not require the N-terminal half of the protein (PulD-N) (9), for which a role in substrate recognition has been proposed (10,11). We generated a library of Ϸ3 ϫ 10 12 variants of Sac7d and selected those that bind to PulD-N in vitro by ribosome display (12).…”