#Structural bases for F #plasmid #conjugation and F pilus #biogenesis in #Escherichia coli (Proc Natl Acad Sci USA, abstract)

[Source: Proceedings of the National Academy of Sciences of the United States of America, full page: (LINK). Abstract, edited.]

Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli

Bo Hu, Pratick Khara, and Peter J. Christie

PNAS first published June 25, 2019 / DOI: https://doi.org/10.1073/pnas.1904428116

Edited by Scott J. Hultgren, Washington University School of Medicine, St. Louis, MO, and approved June 5, 2019 (received for review March 14, 2019)



Bacterial “sex,” or conjugation, is a central mechanism underlying the proliferation of antibiotic resistance. Despite the discovery of conjugation by F plasmids in Escherichia coliover seven decades ago, we have only now visualized the F-encoded transfer channel and F pilus-associated platforms in the E. coli cell envelope by cryoelectron tomography. The channel supports plasmid transfer or assembly of F pili, which remarkably upon synthesis are deposited onto alternative basal structures around the cell surface. The F plasmid transfer system is a paradigm for the bacterial type IV secretion system (T4SS) superfamily. Consequently, the F-encoded structures are broadly informative of mechanisms underlying the biogenesis and function of type IV secretion machines and associated conjugative pili.



Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in the Enterobacteriaceae was first reported in the 1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or “mating” channel, the channel–pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs.

cryoelectron tomography – DNA conjugation – type IV secretion – pilus – protein transport



1 To whom correspondence may be addressed. Email: bo.hu@uth.tmc.edu or peter.j.christie@uth.tmc.edu.

Author contributions: B.H. and P.J.C. designed research; B.H. and P.K. performed research; B.H., P.K., and P.J.C. analyzed data; and P.J.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Density maps and coordinate data that support the F-encoded channel structures determined by cryoelectron tomography have been deposited in The Electron Microscopy Data Bank (EMDB), https://www.ebi.ac.uk/pdbe/emdb (entry nos. EMD-9344 and EMD-9347).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1904428116/-/DCSupplemental.

Published under the PNAS license.

Keywords: Enterobacteriaceae; Plasmids.



#mcr-9, an inducible #gene encoding an acquired phosphoethanolamine transferase in #Escherichia coli, and its origin (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

mcr-9, an inducible gene encoding an acquired phosphoethanolamine transferase in Escherichia coli, and its origin

Nicolas Kieffer, Guilhem Royer, Jean-Winoc Decousser, Anne-Sophie Bourrel, Mattia Palmieri, Jose-Manuel Ortiz De La Rosa, Hervé Jacquier, Erick Denamur, Patrice Nordmann,Laurent Poirel

DOI: 10.1128/AAC.00965-19



The plasmid-located mcr-9 gene encoding a putative phosphoethanolamine transferase was identified in a colistin-resistant human fecal Escherichia coli belonging to a very rare phylogroup D-ST69-O15:H6 clone. This MCR-9 protein shares 33-65% identity with the other plasmid-encoded MCR-type enzymes identified (MCR-1- to -8) that have been found as sources of acquired resistance to polymyxins in Enterobacteriaceae. Analysis of the lipopolysaccharide of the MCR-9-producing isolate revealed a similar function as MCR-1 by adding a phosphoethanolamine group to the lipid A and subsequently modifying the structure of the lipopolysaccharide. However, a minor impact on susceptibility to polymyxins was noticed once cloned and produced in an E. coli K-12 derived strain. Nevertheless, we showed here that sub-inhibitory concentrations of colistin induced the expression of the mcr-9 gene, leading to increased MIC levels. This inducible expression was mediated by a two-component regulatory system encoded by the qseC and qseB genes located downstream of mcr-9. Genetic analysis showed that the mcr-9 gene was carried by an IncHI2 plasmid. In silico analysis revealed that the plasmid-encoded MCR-9 shared significant amino acid identity (ca. 80%) with the chromosomally-encoded MCR-like proteins from Buttiauxella spp. In particular, Buttiauxella gaviniae was found to harbor a gene encoding MCR-BG, sharing 84% identity with MCR-9. That gene was neither expressed nor inducible in its original host, which was fully susceptible to polymyxins. This work showed that mcr genes may circulate quite silently and remaining undetected unless induced by colistin.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Antibiotics; Drugs Resistance; MCR9; Colistin; E. Coli.


IncI1 ST3 and IncI1 ST7 #plasmids from CTX-M-1-producing #Escherichia coli obtained from #patients with #bloodstream infections are closely related to plasmids from E. coli of #animal origin (J Antimicrob Chemother., abstract)

[Source: Journal of Antimicrobial Chemotherapy, full page: (LINK). Abstract, edited.]

IncI1 ST3 and IncI1 ST7 plasmids from CTX-M-1-producing Escherichia coli obtained from patients with bloodstream infections are closely related to plasmids from E. coli of animal origin

Adam Valcek, Louise Roer, Søren Overballe-Petersen, Frank Hansen, Valeria Bortolaia, Pimlapas Leekitcharoenphon, Helle B Korsgaard, Anne Mette Seyfarth, Rene S Hendriksen, Henrik Hasman, Anette M Hammerum

Journal of Antimicrobial Chemotherapy, dkz199, https://doi.org/10.1093/jac/dkz199

Published: 14 May 2019




Fully sequenced IncI1 plasmids obtained from CTX-M-1-producing Escherichia coli of human and animal origin were compared.


Twelve E. coli isolates sharing identical ESBL genes and plasmid multilocus STs sequenced on Illumina and MinION platforms were obtained from the Danish antimicrobial resistance surveillance programme, DANMAP. After de novoassembly, the sequences of plasmids harbouring blaCTX-M-1 were manually curated and ORFs annotated. Within-group comparisons were performed separately for the IncI1 ST3 plasmid type and the IncI1 ST7 plasmid type. The IncI1 ST3 plasmid group was obtained from 10 E. coli isolates (2 from patients with bloodstream infections, 6 from food and 2 from animals). The IncI1 ST7 plasmids originated from E. coli isolates obtained from a patient with bloodstream infection and from a pig. Sequences of IncI1 ST3 and IncI1 ST7 plasmids harbouring blaCTX-M-1 with determined origin were retrieved from GenBank and used for comparison within the respective group.


The 10 IncI1 ST3 blaCTX-M-1 plasmids were highly similar in structure and organization with only minor plasmid rearrangements and differences in the variable region. The IncI1 ST7 blaCTX-M-1 plasmids also showed high similarity in structure and organization. The high level of similarity was also observed when including plasmids from E. coli of animal origin from Australia, Switzerland, the Netherlands and France.


This study shows broad spread of a very successful CTX-M-1-producing IncI1 type plasmid among E. coli of both human and animal origin.

Topic: plasmids – drug resistance, microbial – food – genes – ichthyosis, x-linked – sequence tagged sites – escherichia coli – sodium thiosulfate – bloodstream infections – genbank


© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Keywords: Antibiotics; Drugs Resistance; E. Coli; Bacteremia; Pigs; Human; Plasmids.


Functional characterization of a Miniature Inverted Transposable Element at the origin of #mcr-5 gene acquisition in #Escherichia coli (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Functional characterization of a Miniature Inverted Transposable Element at the origin of mcr-5 gene acquisition in Escherichia coli

Nicolas Kieffer, Patrice Nordmann, Yves Millemann, Laurent Poirel

DOI: 10.1128/AAC.00559-19



Plasmid-mediated colistin resistance of the MCR type is a growing concern in Enterobacteriaceae since it has been described worldwide either in humans and in animals. Here we identified a series of MCR-producing Escherichia coli isolates, corresponding to two different clones (respectively represented by isolates PS1 and PS8b) producing MCR-1 and MCR-5, respectively, from pig fecal samples in France. Plasmid analysis showed that the plasmid carrying the mcr-1 gene (pPS1) possesses an IncHI2 backbone whereas the mcr-5gene was carried onto a 6,268 bp non-typeable, non self-conjugative plasmid (pPS8b). Detailed analysis of plasmid pPS8b revealed a 3,803 bp-long cassette containing the mcr-5 gene that was bracketed by two inverted-repeat sequences (IRs) with 5-bp long direct repeats at each extremity, similarly to an insertion sequence, but with the exception that no transposase gene was identified within this cassette. By performing in-vitro transposition experiments, we showed that the mcr-5 cassette could be mobilized by the TnAs1 transposase provided in-trans, displaying a similar mobilization mechanism as miniature inverted repeat transposable elements (MITEs).

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Antibiotics; Drugs Resistance; Colistin; E. Coli; MCR5; Plasmids.


Identification of Novel Mobilized #Colistin #Resistance #Gene #mcr9 in a #MDR, Colistin-Susceptible #Salmonella enterica Serotype #Typhimurium Isolate (mBio, abstract)

[Source: mBio, full page: (LINK). Abstract, edited.]

Identification of Novel Mobilized Colistin Resistance Gene mcr-9 in a Multidrug-Resistant, Colistin-Susceptible Salmonella enterica Serotype Typhimurium Isolate

Laura M. Carroll, Ahmed Gaballa, Claudia Guldimann, Genevieve Sullivan, Lory O. Henderson, Martin Wiedmann

Mark S. Turner, Editor

DOI: 10.1128/mBio.00853-19



Mobilized colistin resistance (mcr) genes are plasmid-borne genes that confer resistance to colistin, an antibiotic used to treat severe bacterial infections. To date, eight known mcrhomologues have been described (mcr-1 to -8). Here, we describe mcr-9, a novel mcrhomologue detected during routine in silico screening of sequenced Salmonella genomes for antimicrobial resistance genes. The amino acid sequence of mcr-9, detected in a multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium (S. Typhimurium) strain isolated from a human patient in Washington State in 2010, most closely resembled mcr-3, aligning with 64.5% amino acid identity and 99.5% coverage using Translated Nucleotide BLAST (tblastn). The S. Typhimurium strain was tested for phenotypic resistance to colistin and was found to be sensitive at the 2-mg/liter European Committee on Antimicrobial Susceptibility Testing breakpoint under the tested conditions. mcr-9 was cloned in colistin-susceptible Escherichia coliNEB5α under an IPTG (isopropyl-β-d-thiogalactopyranoside)-induced promoter to determine whether it was capable of conferring resistance to colistin when expressed in a heterologous host. Expression of mcr-9 conferred resistance to colistin in E. coli NEB5α at 1, 2, and 2.5 mg/liter colistin, albeit at a lower level than mcr-3. Pairwise comparisons of the predicted protein structures associated with all nine mcr homologues (Mcr-1 to -9) revealed that Mcr-9, Mcr-3, Mcr-4, and Mcr-7 share a high degree of similarity at the structural level. Our results indicate that mcr-9 is capable of conferring phenotypic resistance to colistin in Enterobacteriaceae and should be immediately considered when monitoring plasmid-mediated colistin resistance.



Colistin is a last-resort antibiotic that is used to treat severe infections caused by MDR and extensively drug-resistant (XDR) bacteria. The World Health Organization (WHO) has designated colistin as a “highest priority critically important antimicrobial for human medicine” (WHO, Critically Important Antimicrobials for Human Medicine, 5th revision, 2017, https://www.who.int/foodsafety/publications/antimicrobials-fifth/en/), as it is often one of the only therapies available for treating serious bacterial infections in critically ill patients. Plasmid-borne mcr genes that confer resistance to colistin pose a threat to public health at an international scale, as they can be transmitted via horizontal gene transfer and have the potential to spread globally. Therefore, the establishment of a complete reference of mcr genes that can be used to screen for plasmid-mediated colistin resistance is essential for developing effective control strategies.

Keywords: Antibiotics; Drugs Resistance; Enterobacteriaceae; Salmonella enterica; MCR9; MCR3; Colistin; USA; Plasmids.


#Plasmid carrying #blaCTX-M-2 and blaGES-1 in #XDR #Pseudomonas aeruginosa from #CSF (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Plasmid carrying blaCTX-M-2 and blaGES-1 in extensively drug-resistant Pseudomonas aeruginosa from cerebrospinal fluid

Anelise Stella Ballaben, Renata Galetti, Leonardo Neves Andrade, Joseane Cristina Ferreira, Doroti de Oliveira Garcia, Paulo da Silva, Yohei Doi, Ana Lucia Costa Darini

DOI: 10.1128/AAC.00186-19



Extended-spectrum β-lactamases (ESBL) are spread worldwide in Order Enterobacterales (1, 2) but are less common in Pseudomonas aeruginosa, consequently little is known regarding genetic environment and plasmid carrying blaESBL genes in this species (3).…


Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Antibiotics; Drugs Resistance; Beta-lactams; Pseudomonas aeruginosa; Plasmids.


Identification of a #Carbapenemase-Producing #Hypervirulent #Klebsiella pneumoniae Isolate, #USA (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Identification of a Carbapenemase-Producing Hypervirulent Klebsiella pneumoniae Isolate, United States

Maria Karlsson, Richard A. Stanton, Uzma Ansari, Gillian McAllister, Monica Y. Chan, Erisa Sula, Julian E. Grass, Nadezhda Duffy, Melissa L. Anacker, Medora L. Witwer, J. Kamile Rasheed,Christopher A. Elkins, Alison Laufer Halpin

DOI: 10.1128/AAC.00519-19



We report on a carbapenemase-producing hypervirulent Klebsiella pneumoniae (CP-hvKP) collected from a U.S. patient at an outpatient clinic. The isolate was identified as K. pneumoniae serotype K1, sequence type 23 and included both a hypervirulence (with rmpA, rmpA2 iroBCDN, peg-344 and iucABCD-iutA genes) and a carbapenemase-encoding (blaKPC-2) plasmid. The emergence of CP-hvKP underscores the importance of clinical awareness of this pathotype and the need for continued monitoring of CP-hvKP in the United States.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Keywords: Antibiotics; Drugs Resistance; Carbapenen; Klebsiella pneumoniae; Plasmids; USA.