CryoEM structure of the Vibrio cholerae Type IV competence pilus secretin PilQ

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, the Type IV competence pilus 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. A lack of structural information has hindered our understanding of this process, however. Here, we solved the first ever high-resolution structure of a Type IV competence pilus secretin. A functional tagged allele of VcPilQ purified from native V. cholerae cells was used to determine the cryoEM structure of the PilQ secretin in amphipol to ~2.7 Å. This structure highlights for the first time key differences in the architecture of the Type IV competence pilus secretin from the Type II and Type III Secretin System secretins. Based on our cryoEM structure, we designed a series of mutants to interrogate the mechanism of PilQ. These experiments provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation. We prove that it is possible to reduce pilus biogenesis and natural transformation by sealing the gate, suggesting VcPilQ as a new drug target

CryoEM structure of the Vibrio cholerae Type IV competence pilus secretin PilQ

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, the Type IV competence pilus 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. A lack of structural information has hindered our understanding of this process, however. Here, we solved the first ever high-resolution structure of a Type IV competence pilus secretin. A functional tagged allele of VcPilQ purified from native V. cholerae cells was used to determine the cryoEM structure of the PilQ secretin in amphipol to ~2.7 Å. This structure highlights for the first time key differences in the architecture of the Type IV competence pilus secretin from the Type II and Type III Secretin System secretins. Based on our cryoEM structure, we designed a series of mutants to interrogate the mechanism of PilQ. These experiments provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation. We prove that it is possible to reduce pilus biogenesis and natural transformation by sealing the gate, suggesting VcPilQ as a new drug target

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

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

The quorum sensing transcription factor AphA directly regulates natural competence in Vibrio cholerae

Many bacteria use population density to control gene expression via quorum sensing. In Vibrio cholerae, quorum sensing coordinates virulence, biofilm formation, and DNA uptake by natural competence. The transcription factors AphA and HapR, expressed at low and high cell density respectively, play a key role. In particular, AphA triggers the entire virulence cascade upon host colonisation. In this work we have mapped genome-wide DNA binding by AphA. We show that AphA is versatile, exhibiting distinct modes of DNA binding and promoter regulation. Unexpectedly, whilst HapR is known to induce natural competence, we demonstrate that AphA also intervenes. Most notably, AphA is a direct repressor of tfoX, the master activator of competence. Hence, production of AphA markedly suppressed DNA uptake; an effect largely circumvented by ectopic expression of tfoX. Our observations suggest dual regulation of competence. At low cell density AphA is a master repressor whilst HapR activates the process at high cell density. Thus, we provide deep mechanistic insight into the role of AphA and highlight how V. cholerae utilises this regulator for diverse purposes

Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae

Natural transformation is a broadly conserved mechanism of horizontal gene transfer in bacterial species that can shape evolution and foster the spread of antibiotic resistance determinants, promote antigenic variation, and lead to the acquisition of novel virulence factors. Surface appendages called competence pili promote DNA uptake during the first step of natural transformation1 , however, their mechanism of action has remained unclear due to an absence of methods to visualize these structures in live cells. Here, using the model naturally transformable species Vibrio cholerae and a pilus labeling method, we define the mechanism for type IV competence pilus-mediated DNA uptake during natural transformation. First, we show that type IV competence pili bind to extracellular doublestranded DNA via their tip and demonstrate that this binding is critical for DNA uptake. Next, we show that type IV competence pili are dynamic structures and that pilus retraction brings tip-bound DNA to the cell surface. Finally, we show that pilus retraction is spatiotemporally coupled to DNA internalization and that sterically obstructing pilus retraction prevents DNA uptake. Together, these results indicate that type IV competence pili directly bind DNA via their tip and mediate DNA internalization through retraction during this conserved mechanism of horizontal gene transfe

Multiplex genome editing for synthetic biology in Vibrio natriegens

Vibrio natriegens has recently emerged as an alternative to Escherichia coli for molecular biology and biotechnology, but low-efficiency genetic tools hamper its development. Here, we uncover how to induce natural competence in V. natriegens and describe methods for multiplex genome editing by natural transformation (MuGENT). MuGENT promotes integration of multiple genome edits at high-efficiency on unprecedented timescales. Also, this method allows for generating highly complex mutant populations, which can be exploited for metabolic engineering efforts. As a proof-of-concept, we attempted to enhance production of the value added chemical poly-β-hydroxybutyrate (PHB) in V. natriegens by targeting the expression of nine genes involved in PHB biosynthesis via MuGENT. Within 1 week, we isolated edited strains that produced ~100 times more PHB than the parent isolate and ~3.3 times more than a rationally designed strain. Thus, the methods described here should extend the utility of this species for diverse academic and industrial applications

CryoEM Structure of the Vibrio cholerae Type IV Pilus Secretin PilQ

Natural competence is the process by which bacteria take up genetic material from their environment and integrate it into their genome using homologous recombination. In Vibrio cholerae, the Type IV pilus (T4P) is thought to mediate DNA uptake by binding DNA and retracting back toward the cell. How the DNA enters the periplasm is unclear. One hypothesis suggests that the DNA-bound T4P retracts completely so that the DNA would pass through the outer membrane secretin pore (PilQ). PilQ is a 870 kDa outer membrane pore with C14 symmetry. Here, we purify the V. cholerae PilQ secretin from V. cholerae cells in amphipols for single particle cryogenic electron microscopy (cryoEM). We solve the structure to 3.5 Å and provide insight on the channel DNA may traverse through during uptake

CryoEM Structure of the Vibrio cholerae Type IV Pilus Secretin PilQ

Natural competence is the process by which bacteria take up genetic material from their environment and integrate it into their genome using homologous recombination. In Vibrio cholerae, the Type IV pilus (T4P) is thought to mediate DNA uptake by binding DNA and retracting back toward the cell. How the DNA enters the periplasm is unclear. One hypothesis suggests that the DNA-bound T4P retracts completely so that the DNA would pass through the outer membrane secretin pore (PilQ). PilQ is a 870 kDa outer membrane pore with C14 symmetry. Here, we purify the V. cholerae PilQ secretin from V. cholerae cells in amphipols for single particle cryogenic electron microscopy (cryoEM). We solve the structure to 3.5 Å and provide insight on the channel DNA may traverse through during uptake