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

Periodic Reporting for period 2 - KryptonInt (Erasing the superintegron to understand the role of chromosomal integrons in bacterial evolution)

Reporting period: 2020-07-01 to 2021-12-31

Bacteria have demonstrated an unexpected evolvability that jeopardizes our efforts to control infections with antibiotics. An important element used by bacteria to evolve and adapt to clinical settings are integrons, a unique type of genetic platform that provides the bacterial host with novel functions through the recruitment of new genes encoded in cassettes. These elements play a major role in multidrug resistance in clinically relevant bacteria, since they are often found on plasmids, carrying a cargo of cassettes devoted to produce resistance to multiple antibiotics. But integrons are also found in the chromosomes of environmental bacteria harboring long arrays of cassettes of unknown function. In this project we want to understand the adaptive value of integrons in both environments. Yet the study of chromosomal integrons has been blocked since their discovery, because the native integron will interfere with our tools to study it, biasing our results. To overcome these limitations we propose to delete the Superintegron, a massive structure encoded in the chromosome of Vibrio cholerae, the bacteria that causes Cholera. We propose a novel approach to delete the structure and deliver a bacterial strain in which studies can be delivered. We will then use it to start answering major questions that have been out of reach. We will investigate the functions encoded in chromosomal integrons of environmental bacteria, including those of pathogenic Vibrio. We will deliver a method to unveil the genetic basis of how cassettes are created; and we will study the ecology of resistance genes in all fields of the One Health concept through the design of a biotechnological tool that captures cassettes from DNA samples. Understanding the origin and function of cassettes, and the circulation of resistance genes in cassettes will unveil important aspects of a genetic element that represents today a major threat to modern medicine.
We have performed an important amount of work since the beginning of the project. We have advanced in all different objectives proposed, producing high quality results. We have obtained a ∆SI strain that we have corrected to repair additional mutations appeared during the process. We are producing phenomic data on this strain to unveil the functions encoded in cassettes. We are also setting up a blind cassette capture system to explore cassette content in DNA samples. We have produced two publications in high impact journals on the evolutionary origin of integrons (Escudero et al. eLife 2019) and the proof of the Adaptation on demand hypothesis (Souque et al. eLife, 2020), and we are preparing another one on regulatory aspects of resistance genes (Hipólito et al. in preparation).
We have made several progresses beyond the state of the art in the field, such as:
-Developing a methodology to delete chromosomal integrons
-unblocking the field of study of chromosomal integrons.
-linking the Superintegron to novel virulence phenotypes
-Mixing methodologies from different fields to increase the throughput of recombination experiments.
-Building a complete library of resistance cassettes to analyze their evolutionary dynamics in a comprehensive, systematic and comparable manner.
The Superintegron