Fine-tuning of a CRISPRi screen in the seventh pandemic Vibrio cholerae DOI
Kevin Debatisse, Théophile Niault,

Sarah Peeters

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: July 3, 2024

ABSTRACT Vibrio cholerae O1 El Tor , the etiological agent responsible for last cholera pandemic, has become a well-established model organism which some genetic tools exist. While CRISPRi been applied in V. improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing this bacterium. In study, we introduce genome wide library construction specifically optimized strain, characterized minimal cytotoxicity streamlined experimental setup. This allows depletion of 3, 674 (98.9%) annotated genes from genome. To confirm its effectiveness, screened essential during exponential growth rich medium identified 368 guides significantly depleted (log2FC < - 2). Remarkably, 82% these had previously described as hypothetical or closely related bacterium, natriegens . We thus validated robustness accuracy our CRISPRi-based approach assessing gene fitness given condition. Our findings highlight efficacy developed platform powerful tool functional genomics studies GRAPHICAL

Language: Английский

Integrons are anti-phage defence libraries in Vibrio parahaemolyticus DOI
Landon J. Getz,

Sam R Fairburn,

Yundan Liu

et al.

Nature Microbiology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 27, 2025

Language: Английский

Citations

1
Belt and braces: Two escape ways to maintain the cassette reservoir of large chromosomal integrons DOI Creative Commons
Egill Richard, Baptiste Darracq, Eloi Littner

et al.

PLoS Genetics, Journal Year: 2024, Volume and Issue: 20(4), P. e1011231 - e1011231

Published: April 5, 2024

Integrons are adaptive devices that capture, stockpile, shuffle and express gene cassettes thereby sampling combinatorial phenotypic diversity. Some integrons called sedentary chromosomal (SCIs) can be massive structures containing hundreds of cassettes. Since most these non-expressed, it is not clear how they remain stable over long evolutionary timescales. Recently, was found the experimental inversion SCI Vibrio cholerae led to a dramatic increase cassette excision rate associated with fitness defect. Here, we question sustainability this apparently counter selected genetic context. Through evolution, find integrase rapidly inactivated inverted recover its original orientation by homologous recombination between two insertion sequences (ISs) present in array. These outcomes restore normal growth prevent loss cassettes, enabling SCIs retain their roles as reservoirs functions. results illustrate nice interplay orientation, genome rearrangement, bacterial demonstrate benefit from embedded ISs.

Language: Английский

Citations

1
Chromosomal integrons are genetically and functionally isolated units of genomes DOI Creative Commons
Paula Blanco, Filipa Trigo da Roza, Laura Toribio-Celestino

et al.

Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 52(20), P. 12565 - 12581

Published: Sept. 23, 2024

Abstract Integrons are genetic elements that increase the evolvability of bacteria by capturing new genes and stockpiling them in arrays. Sedentary chromosomal integrons (SCIs) can be massive highly stabilized structures encoding hundreds genes, whose function remains generally unknown. SCIs have co-evolved with host for aeons intertwined their physiology from a mechanistic point view. But, paradoxically, other aspects, like variable content location within genome, suggest high functional independence. In this work, we explored connection to genome using as model Superintegron (SI), 179-cassette long SCI Vibrio cholerae N16961. We relocated deleted SI SeqDelTA, novel method allows counteract strong stabilization conferred toxin–antitoxin systems array. characterized depth impact V. cholerae’s physiology, measuring fitness, chromosome replication dynamics, persistence, transcriptomics, phenomics, natural competence, virulence resistance against protist grazing. The deletion did not produce detectable effects any condition, proving that—despite millions years co-evolution—SCIs genetically functionally isolated units genomes.

Language: Английский

Citations

1
Toxin-antitoxin systems reflect community interactions through horizontal gene transfer DOI Creative Commons
Jonathan H. Bethke, Jeffrey A. Kimbrel, Yongqin Jiao

et al.

Molecular Biology and Evolution, Journal Year: 2024, Volume and Issue: 41(10)

Published: Oct. 1, 2024

Abstract Bacterial evolution through horizontal gene transfer (HGT) reflects their community interactions. In this way, HGT networks do well at mapping interactions, but offer little toward controlling them—an important step in the translation of synthetic strains into natural contexts. Toxin–antitoxin (TA) systems serve as ubiquitous and diverse agents selection; however, utility is limited by erratic distribution hosts. Here we examine heterogeneous TAs a consequence mobility. By systematically TA across 10,000 plasmid network, find communities have unique predictable signatures. We propose these signatures arise from competition further potential to signal degree which plasmids, hosts, phage interact. To emphasize relationships, construct an network based solely on similarity, framing specific selection markers broader context bacterial communities. This work both clarifies unlocks common framework for manipulating interactions compatibility.

Language: Английский

Citations

1
Fine-tuning of a CRISPRi screen in the seventh pandemic Vibrio cholerae DOI Creative Commons
Kevin Debatisse, Théophile Niault,

Sarah Peeters

et al.

BMC Genomics, Journal Year: 2024, Volume and Issue: 25(1)

Published: Oct. 21, 2024

Vibrio cholerae O1 El Tor, the etiological agent responsible for last cholera pandemic, has become a well-established model organism which some genetic tools are available. While CRISPRi technology been applied to V. cholerae, improvements were necessary upscale it and enable pooled screening by high-throughput sequencing in this bacterium. In study, we present genome-wide CRISPR-dCas9 screen specifically optimized N16961 Tor strain of cholerae. This approach is characterized tight control dCas9 expression activity, as well streamlined experimental setup. Our library allows depletion 3,674 (98.9%) annotated genes from genome. To confirm its effectiveness, screened that essential during exponential growth rich medium identified 369 guides significantly depleted (log2FC < -2). Remarkably, 82% these had previously described hypothetical or closely related bacterium, natriegens. We thus validated robustness accuracy our CRISPRi-based assessing gene fitness given condition. findings highlight efficacy developed platform powerful tool functional genomics studies

Language: Английский

Citations

1
Controlling and controlled elements: highlights of the year in mobile DNA research DOI Creative Commons
Irina R. Arkhipova, Kathleen H. Burns, Pascale Lesage

et al.

Mobile DNA, Journal Year: 2024, Volume and Issue: 15(1)

Published: Dec. 31, 2024

Language: Английский

Citations

0
Fine-tuning of a CRISPRi screen in the seventh pandemic Vibrio cholerae DOI
Kevin Debatisse, Théophile Niault,

Sarah Peeters

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: July 3, 2024

ABSTRACT Vibrio cholerae O1 El Tor , the etiological agent responsible for last cholera pandemic, has become a well-established model organism which some genetic tools exist. While CRISPRi been applied in V. improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing this bacterium. In study, we introduce genome wide library construction specifically optimized strain, characterized minimal cytotoxicity streamlined experimental setup. This allows depletion of 3, 674 (98.9%) annotated genes from genome. To confirm its effectiveness, screened essential during exponential growth rich medium identified 368 guides significantly depleted (log2FC < - 2). Remarkably, 82% these had previously described as hypothetical or closely related bacterium, natriegens . We thus validated robustness accuracy our CRISPRi-based approach assessing gene fitness given condition. Our findings highlight efficacy developed platform powerful tool functional genomics studies GRAPHICAL

Language: Английский

Citations

0