Erasing the superintegron to understand the role of chromosomal integrons in bacterial evolution

Project Information

KryptonInt

Grant agreement ID: 803375

DOI

10.3030/803375

Project closed

EC signature date 9 November 2018

Start date 1 January 2019

End date 30 April 2025

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 499 516,00

EU contribution

€ 1 499 516,00

Coordinated by

UNIVERSIDAD COMPLUTENSE DE MADRID
Spain

Periodic Reporting for period 4 - KryptonInt (Erasing the superintegron to understand the role of chromosomal integrons in bacterial evolution)

Reporting period: 2023-07-01 to 2025-04-30

Summary of the context and overall objectives of the project

Antibiotic resistance is an urgent challenge for modern medicine. Bacteria adapt rapidly often using a genetic system called the integron. Integrons acquire and express new genes —including resistance genes—encoded in mobile units called "cassettes". While mobile integrons have been studied extensively, we know very little about the adaptive value of sedentary chromosomal integrons (SCI), which are present in many environmental and pathogenic bacteria. KRYPTONINT aimed to uncover their role by deleting the “Superintegron” (SI) of Vibrio cholerae. The SI is the paradigm of SCI and is highly stabilized by 19 toxin-antitoxin systems. We developed a novel deletion method (SeqDelTA), successfully erased the SI, and studied the impact in growth, transcriptomics, and virulence. We found no major differences with the wild type, showing that SCIs are genetically isolated and do not interfere with core cell functions. This deepens our understanding of integrons as modular genetic units. We discovered a new class of non-coding cassettes acting as internal promoters along arrays, changing our perception that SCIs are mainly silent structures. We have also shown that integrons encode phage defence systems, enlarging our functional and ecological perspective on these structures. Additionally, we have revealed that integrase expression is variable among cells—a form of phenotypic heterogeneity that could help bacteria pre-adapt to stress. Our findings provide new insight into how bacteria evolve and adapt, and deliver novel tools to study integrons and gene flow in microbial communities.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Over the course of the project, we achieved several scientific and technological milestones:

- We deleted the SI of V. cholerae for the 1st time, using our patented method SeqDelTA (ES2969666B2, ES2970040B2), generating a ΔSI strain free of off-target mutations.
- We show that this does not impact the host, suggesting that integrons are modular and functionally isolated (published in Nucleic Acids Research (2024)).
- We have explored the expression model of integrons and have found that the distance-to-Pc model that we have assumed for decades does not explain our observations. Instead, genes exert polar effects that modulate the expression of others downstream in a manner dependent on translation rates. The new “translation-driven polarity model” was published in Nature communications in 2024 and are developing further now.
- We developed the I3C tool: a bacterial platform that captures integron cassettes from environmental DNA, without knowing their sequence or function. It allowed us to discover novel antibiotic and phage resistance genes. The tool is protected by two patents and is the basis of a planned startup project. A dedicated publication is in preparation.
- We discovered that integrase expression varies between cells, a rare phenomenon known as phenotypic heterogeneity, which may lead to genetic diversity through cassette reshuffling. A dedicated publication is in preparation.
- We found that mobile integrons encode phage defence systems, revealing a second function for integrons and alerting to potential resistance to phage therapy. This breakthrough was published in Science (2025).

In total, the project generated five major publications in top-tier journals, three patents, and several public outreach actions. The PI will move to the National Centre for Biotechnology (CSIC), where the team will continue its research with international collaborations and funding prospects.
In addition to the main results described above, the KryptonInt project has supported several other peer-reviewed publications that acknowledge the ERC grant. These works, developed in the scientific context and infrastructure enabled by the project, further strengthen the impact of KryptonInt on related areas such as integron activity, gene expression, and antimicrobial resistance:
• Souque et al., eLife (2020) – on integron-mediated acceleration of antibiotic resistance evolution.
• Escudero et al., eLife (2021) – on coexisting primary and promiscuous functions during integron cassette recombination.
• Hipólito et al., npj Antimicrobials and Resistance (2023) – on integron prevalence and resistance profile..
• Papkou et al., Science (2023) – on fitness landscapes and gene function in bacteria.
• Hipólito et al. Nucleic Acids Research (2022), on the regulation of gene expression and the presence of riboswitches in integrons.
• Ortiz-Miravalles et al. Science of the Total Environment (2024), on the function of AMR genes in integrons in anaerobic conditions.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

KRYPTONINT has delivered multiple advances that push the boundaries of current knowledge in microbial genetics, integron biology, and biotechnology:
- Development of SeqDelTA, the first method to successfully delete highly stabilized SCIs such as the Superintegron of V. cholerae, overcoming the challenge posed by 19 toxin-antitoxin systems. This enables the study of SCIs in their native context and is protected by international patents (ES2969666B2, ES2970040B2, PCT/ES2024/070711).
- Erasing the Superintegron without altering host physiology, revealing that SCIs are genetically and functionally isolated from the core genome. This contradicts long-held assumptions and positions SCIs as modular, accessory units. This insight was published in Nucleic Acids Research (2024).
- Discovery of gene-less cassettes acting as internal promoters, changing our view of transcriptional regulation within long arrays. These elements modulate expression beyond the proximity to the main promoter, with consequences for co-selection and resistance dynamics. This is complemented by our finding on polar effects and the new translation-driven polarity model of expression of integrons. Together, both findings shift the paradigm of expression of the integron (Nature Communications, 2024; Nucleic Acids Research, 2024).
- Identification of integrase expression heterogeneity, a novel finding showing that integrase is expressed in a subset of cells even without stress. This phenotypic heterogeneity leads to genotypic diversification, unveiling a mechanism of bacterial pre-adaptation with strong evolutionary implications (manuscript in preparation, expected 2025).
- Discovery that mobile integrons encode phage defence systems, revealing a second major function for integrons beyond antibiotic resistance. This breakthrough has major implications for the future of phage therapy and was published in Science (2025).
- Creation of the I3C tool, a cassette capture platform that is agnostic to cassette sequence or function. It allows direct capture of cassettes from DNA samples and has led to the discovery of new antibiotic and phage resistance genes. This tool is protected by additional patents (ES2991744A1, PCT pending), with commercial interest from private equity funds and plans for future exploitation via an ERC Proof of Concept.
- Cross-disciplinary integration of microbiology, molecular genetics, single-cell analysis and synthetic biology, with high throughput and resolution.
By the end of the project, KRYPTONINT has not only resolved key fundamental questions about integron biology but has also delivered innovative tools and conceptual frameworks that redefine the field. The outputs of the project have high potential for translation into diagnostics, synthetic biology, and antimicrobial resistance surveillance.

The Superintegron