CryoEM structure of the type IVa pilus secretin required for natural competence in Vibrio cholerae

Abstract

Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, a type IVa pilus (T4aP) is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. Here, we use a functional tagged allele of VcPilQ purified from native V. cholerae cells to determine the cryoEM structure of the VcPilQ secretin in amphipol to ~2.7 Å. We use bioinformatics to examine the domain architecture and gene neighborhood of T4aP secretins in Proteobacteria in comparison with VcPilQ. This structure highlights differences in the architecture of the T4aP secretin from the type II and type III secretion system secretins. Based on our cryoEM structure, we design a series of mutants to reversibly regulate VcPilQ gate dynamics. These experiments support the idea of VcPilQ as a potential druggable target and provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation.

Structure of the molecular bushing of the bacterial flagellar motor

“…The flagellum and injectisome are both classified as the type III secretion system, but FlgH and FlgI are not homologous to the components of injectisomes 26 , such as InvG of the type III secretion system 27 , as well as those of other secretion systems: GspD of the type II secretion system 28 , and PilQ of the type IVa pilus 29 . However, all of these proteins have similar cylindrical β barrel structures, except that the long β strands of FlgH shows different orientation (Supplementary Fig.…”

CryoEM map of Pseudomonas aeruginosa PilQ enables structural characterization of TsaP

“…For example, PilQ adopts distinct open and closed states depending on whether the pilus fiber occupies the central channel of the T4aP (Chang et al, 2016). Compared with T4aP maps obtained by electron cryotomography in which the pilus fiber does not occupy the T4aP, the outer membrane (OM) in the piliated state has moved 20 Å away from PilP and the peptidoglycan (Chang et al, 2016;Weaver et al, 2020). Although the secretin domain is embedded in the OM, the AMI_N and N0 domains of PilQ bind peptidoglycan and PilP, respectively (Berry et al, 2012).…”

“…PilQ secretins from Proteobacteria, structurally characterized at low resolution, show radial spikes emerging from a central barrel (Chang et al, 2016;Collins et al, 2004;Koo et al, 2016;Siewering et al, 2014). Models of PilQ from Thermus thermophilus (PilQ Tt ) and Vibrio cholerae (PilQ Vc ) competence pilus systems, which are divergent from Proteobacterial T4aP secretins, were recently built into 7.0and 2.7-Å resolution electron cryomicroscopy (cryoEM) maps, respectively (D'Imprima et al, 2017; Weaver et al, 2020). These competence secretins lack the lip subdomain and the radial spikes found in T4aP secretins.…”

The structure and mechanism of the bacterial type II secretion system

“…The N0 domain consists of two helices flanked on each side by β‐sheets that pack laterally to form the secretin base (Figure 1a,e). Recently, the structure of the PilQ secretin from the Vibrio cholerae type IV competence pilus was determined by cryo‐EM (Weaver et al, 2020). Although PilQ does not share N1‐N3 domains with the T2SS, it does have an N0 domain.…”