The Type IV Pilus of Plasmid TP114 Displays Adhesins Conferring Conjugation Specificity and Is Important for DNA Transfer in the Mouse Gut Microbiota

Allard, Nancy; Neil, Kevin; Grenier, Frédéric; Rodrigue, Sébastien

doi:10.1128/spectrum.02303-21

Cited by 7 publications

(7 citation statements)

References 65 publications

(145 reference statements)

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“…Our data suggest that target-adapted conjugative systems are abundant in the human gut metagenome. Interestingly, we observed a higher frequency of conjugation of pCitro-15a to C. rodentium than to other bacteria, agreeing with studies that showed specificity in the conjugation process. , A number of groups have utilized conjugative systems with broad host ranges in an attempt to access a significant fraction of the targeted microbiome for editing. − While promising, low conjugation rates to diverse bacteria in the targeted microbiome and the stability of conjugative plasmids with resident plasmids are confounding factors. Bacterial resistance to Cas9-killing has also been documented and could be minimized by multiplexing sgRNAs to different targets and tighter regulation of Cas9 expression. , In many cases, conjugative systems required optimization by in vitro evolution, gene minimization, or other manipulations to enhance conjugation to desired recipients .…”

Section: Discussionsupporting

confidence: 83%

De Novo Synthesis of a Conjugative System from Human Gut Metagenomic Data for Targeted Delivery of Cas9 Antimicrobials

Hamilton,

Joris,

Shrestha

et al. 2023

ACS Synth. Biol.

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Metagenomic sequences represent an untapped source of genetic novelty, particularly for conjugative systems that could be used for plasmid-based delivery of Cas9-derived antimicrobial agents. However, unlocking the functional potential of conjugative systems purely from metagenomic sequences requires the identification of suitable candidate systems as starting scaffolds for de novo DNA synthesis. Here, we developed a bioinformatics approach that searches through the metagenomic “trash bin” for genes associated with conjugative systems present on contigs that are typically excluded from common metagenomic analysis pipelines. Using a human metagenomic gut data set representing 2805 taxonomically distinct units, we identified 1598 contigs containing conjugation genes with a differential distribution in human cohorts. We synthesized de novo an entire Citrobacter spp. conjugative system of 54 kb containing at least 47 genes and assembled it into a plasmid, pCitro. We found that pCitro conjugates from Escherichia coli to Citrobacter rodentium with a 30-fold higher frequency than to E. coli , and is compatible with Citrobacter resident plasmids. Mutations in the traV and traY conjugation components of pCitro inhibited conjugation. We showed that pCitro can be repurposed as an antimicrobial delivery agent by programming it with the TevCas9 nuclease and Citrobacter -specific sgRNAs to kill C. rodentium . Our study reveals a trove of uncharacterized conjugative systems in metagenomic data and describes an experimental framework to animate these large genetic systems as novel target-adapted delivery vectors for Cas9-based editing of bacterial genomes.

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Section: Discussionsupporting

confidence: 83%

De Novo Synthesis of a Conjugative System from Human Gut Metagenomic Data for Targeted Delivery of Cas9 Antimicrobials

Hamilton,

Joris,

Shrestha

et al. 2023

ACS Synth. Biol.

Self Cite

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“…It is possible that a virion having tail fibers of different host-specificities would exhibit impaired infectivity. As bacteria were observed to be connected through multiple pili ( 55 ), similar reasoning might lead to an analogous hypothesis for the shufflon PilV protein, which was shown to determine conjugation specificity ( 21 ).…”

Section: Discussionmentioning

confidence: 91%

“…Additionally, some phage tail genes are also targeted by programmed inversions, conferring different host specificity ( 22 ). Finally, shufflon programmed inversions were shown to provide different conjugative pilus specificities ( 21 ).…”

Section: Discussionmentioning

confidence: 99%

“…These variants were also observed to differ in opacity ( 10 ), virulence in mice ( 10 ), as well as human cell infection efficiency and survival in human blood ( 11 ), presumably resulting from Type I RM phase variants inducing differential methylation of host DNA ( 20 ). Other interesting phase variation systems include a programmed inversion targeting the pilV gene (shufflon), leading to phase variants differing in the specificity of their conjugative pili ( 21 ), and a programmed inversion targeting phage tail genes, conferring different host specificity ( 22 ).…”

Section: Introductionmentioning

confidence: 99%

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Systematic identification of gene-altering programmed inversions across the bacterial domain

Milman

Yelin

Kishony

2023

Nucleic Acids Research

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Programmed chromosomal inversions allow bacteria to generate intra-population genotypic and functional heterogeneity, a bet-hedging strategy important in changing environments. Some programmed inversions modify coding sequences, producing different alleles in several gene families, most notably in specificity-determining genes such as Type I restriction-modification systems, where systematic searches revealed cross phylum abundance. Yet, a broad, gene-independent, systematic search for gene-altering programmed inversions has been absent, and little is known about their genomic sequence attributes and prevalence across gene families. Here, identifying intra-species variation in genomes of over 35 000 species, we develop a predictive model of gene-altering inversions, revealing key attributes of their genomic sequence attributes, including gene-pseudogene size asymmetry and orientation bias. The model predicted over 11,000 gene-altering loci covering known targeted gene families, as well as novel targeted families including Type II restriction-modification systems, a protein of unknown function, and a fusion-protein containing conjugative-pilus and phage tail domains. Publicly available long-read sequencing datasets validated representatives of these newly predicted inversion-targeted gene families, confirming intra-population genetic heterogeneity. Together, these results reveal gene-altering programmed inversions as a key strategy adopted across the bacterial domain, and highlight programmed inversions that modify Type II restriction-modification systems as a possible new mechanism for maintaining intra-population heterogeneity.

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“…Transfer rates are however low for most recipients, and most conjugative systems work poorly in the gut environment 17 . High transfer efficiencies can be achieved in the animal gut, but rely on the stabilization of the mating pair through specific interactions between donor pili and receptors on the recipient surface 18 . Efficient conjugative delivery to different strains and species will thus likely require different specialized systems.…”

Section: Introductionmentioning

confidence: 99%

In situtargeted mutagenesis of gut bacteria

Brödel

Charpenay

Galtier

et al. 2022

Preprint

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Microbiome research is revealing a growing number of bacterial genes that impact our health. While CRISPR-derived tools have shown great success in editing disease-driving genes in human cells, we currently lack the tools to achieve comparable success for bacterial targets. Here we engineer a phage-derived particle to deliver a base editor and modify E. coli colonizing the mouse gut. This was achieved using a non-replicative DNA payload, preventing maintenance and dissemination of the payload, while allowing for an editing efficiency of up to 99.7% of the target bacterial population. The editing of a β-lactamase gene resulted in the stable maintenance of edited bacteria in the mouse gut at least 42 days after treatment. By enabling the in situ modification of bacteria directly in the gut, our approach offers a novel avenue to investigate the function of bacterial genes and provides an opportunity to develop novel microbiome-targeted therapies.

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The Type IV Pilus of Plasmid TP114 Displays Adhesins Conferring Conjugation Specificity and Is Important for DNA Transfer in the Mouse Gut Microbiota

Cited by 7 publications

References 65 publications

De Novo Synthesis of a Conjugative System from Human Gut Metagenomic Data for Targeted Delivery of Cas9 Antimicrobials

De Novo Synthesis of a Conjugative System from Human Gut Metagenomic Data for Targeted Delivery of Cas9 Antimicrobials

Systematic identification of gene-altering programmed inversions across the bacterial domain

In situtargeted mutagenesis of gut bacteria

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