PilY1 and minor pilins form a complex priming the type IVa pilus in Myxococcus xanthus

Abstract

Type IVa pili are ubiquitous and versatile bacterial cell surface filaments that undergo cycles of extension, adhesion and retraction powered by the cell-envelope spanning type IVa pilus machine (T4aPM). The overall architecture of the T4aPM and the location of 10 conserved core proteins within this architecture have been elucidated. Here, using genetics, cell biology, proteomics and cryo-electron tomography, we demonstrate that the PilY1 protein and four minor pilins, which are widely conserved in T4aP systems, are essential for pilus extension in Myxococcus xanthus and form a complex that is an integral part of the T4aPM. Moreover, these proteins are part of the extended pilus. Our data support a model whereby the PilY1/minor pilin complex functions as a priming complex in T4aPM for pilus extension, a tip complex in the extended pilus for adhesion, and a cork for terminating retraction to maintain a priming complex for the next round of extension.

The structure and mechanism of the bacterial type II secretion system

“…A similarly positioned plug density was observed in the cryo‐electron tomography (cryo‐ET) reconstruction of the Legionella pneumophila T2SS (Ghosal et al, 2019). Intriguingly, in the type 4 pilus system (T4PS), the N‐terminal von Willebrand factor A domain of PilY1 constitutes a secretin plug while its C‐terminus locates with the minor pilins at the pilus tip to form a priming complex (Treuner‐Lange et al, 2020).…”

“…Recently, cryo‐ET provided the first glimpse of a T2SS assembled in situ (Ghosal et al, 2019). Combined with cryo‐ET on the T4PS (Chang et al, 2017; Chang et al, 2016; Gold et al, 2015; Treuner‐Lange et al, 2020), we now have a topology map for the position of most components for both the T2SS and T4PS. In these systems, the secretin was observed in the outer membrane with its N‐terminal domains protruding into the periplasm.…”

“…Between the secretin base and the inner membrane, densities consistent with a ring‐like structure were observed and assigned to the periplasmic domains of GspL and GspM in the T2SS, or PilN and PilO in the T4PS. A central stem density was positioned within the GspLM ring, which likely relates to the pseudopilus tip proteins GspHIJK or their equivalent in the T4PS (Chang et al, 2016; Treuner‐Lange et al, 2020). Densities for the cytoplasmic domains of GspL or T4PS PilM were nestled on the inner membrane surface and were consistent with another ring‐like structure.…”

Going virtual: a report from the sixth Young Microbiologists Symposium on ‘Microbe Signalling, Organisation and Pathogenesis’

“…The core components of the machine were previously mapped by a highly innovative imaging approach combining cryo-electron tomography with systematic green fluorescent protein (GFP)-tagging of the machine components to precisely locate them in the complex and thereby infer how they contributed to the machine's overall function [10]. This lecture presented new structure-function data specifically on the hitherto unknown role of the minor pilins and the periplasmic protein PilY1 in the complex [11], combining biochemical and genetic analyses with similarly elegant imaging approaches to reveal their location and function at the pilus tip.…”

Multidisciplinary Interrogation of a Crucial Protein Interface in the Type II Secretion System

“…Minor pseudopilin subunits could not be resolved in the L. pneumophila T2SS cryo electron tomograms 23 . In a recent T4P cryo electron tomography study, minor pilins were attributed to density blobs but no conclusions could be drawn about which protein belonged to which density nor about the molecular nature of their interactions 34…”