Biology subjects

Escudero, J. A.

Publications and source records attributed to Escudero, J. A..

10 recordsLinked to original sources

bioRxiv2026

Large-scale recovery of integron cassettes for gene discovery screens

Integrons capture and stockpile adaptive genes encoded in modular mobile genetic elements called integron cassettes (ICs). Present in 17% of bacterial genomes, integrons can harbor hundreds of cassettes, representing a hotspot of genetic variability. Some ICs encode antimicrobial or phage resistance genes, but most remain functionally uncharacterized, representing an untapped source of genes of biotechnological interest. Here, we present two tools, the cassette gatherer and hunter, that allow the swift establishment of libraries of genes either from genetically tractable strains or directly from DNA. By re-engineering a class 1 integron, these platforms capture single cassettes in a sequence and function-independent manner. When applied to Vibrio genomes, they recovered hundreds of single cassettes per assay with >99% specificity. To validate the usability of our tools for the discovery of new genes, we subjected these libraries to screens against phages ICP2 and T4, and identified nine phage-defense systems, including five previously undescribed. Hence, these tools provide a fast and simple method to recover thousands of ICs, in a way amenable to gene-discovery screens.

microbiology↗

bioRxiv2026

Fitness effects of antimicrobial resistance genes in changing environments

The evolutionary success of antimicrobial resistance (AMR) genes is generally seen as a trade-off between their function in the presence and their cost in the absence of antibiotics. Mobile integrons are genetic elements that recruit and disseminate dozens of AMR genes among Gram-negative pathogens. Here, we have measured the fitness effects of 136 integron genes conferring resistance against several antibiotic families. We have found a significant proportion having neutral and positive effects in the absence of antibiotics. We confirmed this using a mouse model, where we also observed cases of changes in the sign of fitness effects. This led us to unveil that oxygen availability modulates the cost of AMR genes. Using a stochastic model, we show that fluctuating aerobic/anaerobic conditions can rescue AMR genes in the absence of selective pressure. Here we provide a comprehensive analysis of the cost of AMR at the gene level challenging the traditional fitness-resistance trade-off hypothesis.

microbiology↗

bioRxiv2024

Mobile Integrons Encode Phage Defence Systems

Integrons are bacterial genetic elements that capture, stockpile and modulate the expression of genes encoded in integron cassettes. Mobile Integrons (MI) are borne on plasmids, acting as a vehicle for hundreds of antimicrobial resistance genes among key pathogens. These elements also carry gene cassettes of unknown function (gcus) whose role and adaptive value remains unexplored. Here we show that gcus encode phage resistance systems, many of which are novel. Bacteriophage resistance integron cassettes (BRiCs) can be combined and mixed with resistance cassettes to produce multiphage or drug/phage-resistance. The fitness costs of BRiCs are variable, dependent on the genetic context, and can be modulated by changing the order of cassettes in the array. Hence, MIs act as highly mobile, low-cost defense islands. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/601719v4_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@965766org.highwire.dtl.DTLVardef@442763org.highwire.dtl.DTLVardef@7600d4org.highwire.dtl.DTLVardef@13068ca_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOSummary FigureC_FLOATNO Novel phage defense systems identified in Mobile Integrons. We confronted genes of unknown function from mobile integrons against a panel of phage. We characterized 13 Bacteriophage Resistance integron Cassettes (BRiCs) and confirmed their function in Klebsiella pneumoniae and Pseudomonas aeruginosa. Combined with other cassettes, BRiCs produce multi-phage/antibiotic resistance. Additionally, their cost can be reduced in an array. C_FIG

microbiology↗

bioRxiv2024

Anaerobiosis modulates the performance of antimicrobial resistance genes in Enterobacteriaceae

Bacteria must face and adapt to a variety of physicochemical conditions in the environment and during infection. A key condition is the concentration of dissolved oxygen, proportional to the partial pressure of oxygen (PO2), which is extremely variable among environmental biogeographical areas and also compartments of the human and animal body. Here, we sought to understand if the phenotype of resistance determinants commonly found in Enterobacterales can be influenced by oxygen pressure. To do so, we have compared the MIC in aerobic and anaerobic conditions of isogenic Escherichia coli strains containing 136 different resistance genes against 9 antibiotic families. Our results show a complex landscape of changes in the performance of resistance genes in anaerobiosis. Certain changes are especially relevant for their intensity and the importance of the antibiotic family, like the large decreases in resistance observed against ertapenem and fosfomycin among blaVIM {beta}-lactamases and certain fos genes, respectively; however, the blaOXA-48 {beta}-lactamase from the clinically relevant pOXA-48 plasmid conferred 4-fold higher ertapenem resistance in anaerobiosis. Strong changes in resistance patterns in anaerobiosis were also conserved in Klebsiella pneumoniae. Our results suggest that anaerobiosis is a relevant aspect that can affect the action and selective power of antibiotics for specific AMRs in different environments.

microbiology↗

bioRxiv2024

The expression of integron arrays is shaped by the translation rate of cassettes

Integrons are key elements in the rise and spread of multidrug resistance in Gram-negative bacteria. These genetic platforms capture cassettes containing promoterless genes and stockpile them in arrays of variable length. In the current integron model, expression of cassettes is granted by the Pc promoter in the platform and is assumed to decrease as a function of its distance. Here we explored this model using a large collection of 136 antibiotic resistance cassettes and show that the effect of distance is in fact negligible. Instead, cassettes have a strong impact in the expression of downstream genes because their translation rate affects the stability of the whole polycistronic mRNA molecule. Hence, poorly translated cassettes decrease the expression and resistance phenotype of cassettes downstream. Our data puts forward a novel integron model in which expression is contingent on the translation of cassettes upstream, rather than on the distance to the Pc.

microbiology↗

bioRxiv2023

IDENTIFICATION OF PROMOTER ACTIVITY IN GENE-LESS CASSETTES FROM VIBRIONACEAE SUPERINTEGRONS

Integrons are genetic platforms that acquire new genes encoded in integron cassettes (ICs), building arrays of adaptive functions. ICs generally encode promoterless genes, whose expression relies on the platform-associated Pc promoter, with the cassette array functioning as an operon-like structure regulated by the distance to the Pc. This is relevant in large sedentary chromosomal integrons (SCIs) carrying hundreds of ICs, like those in Vibrio species. We selected 29 gene-less cassettes in four Vibrio SCIs, and explored whether their function could be related to the transcription regulation of adjacent ICs. We show that most gene-less cassettes have promoter activity on the sense strand, enhancing the expression of downstream cassettes. Additionally, we identified the transcription start sites of gene-less ICs through 5-RACE. Accordingly, we found that most of the superintegron in Vibrio cholerae is not silent. These promoter cassettes can trigger the expression of a silent dfrB9 cassette downstream, increasing trimethoprim resistance >512-fold in V. cholerae and Escherichia coli. Furthermore, one cassette with an antisense promoter can reduce trimethoprim resistance when cloned downstream. Our findings highlight the regulatory role of gene-less cassettes in the expression of adjacent cassettes, emphasizing their significance in SCIs and their clinical importance if captured by mobile integrons.

microbiology↗

bioRxiv2023

Chromosomal Integrons are Genetically and Functionally Isolated Units of Genomes

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

microbiology↗

bioRxiv2023

A rugged yet easily navigable fitness landscape of antibiotic resistance

A fitness landscape is a biological analogue of a physical landscape, in which each genotype occupies a location whose elevation corresponds to fitness. Theoretical models predict that rugged fitness landscapes with multiple peaks should impair Darwinian evolution, because natural selection prevents evolving populations from traversing the valleys that lie between peaks. Experimental tests of this prediction are very limited. Here we combine CRISPR-Cas9 genome editing and deep sequencing to map the fitness landscape of more than 260000 genotypes of the E. coli folA gene in an environment harboring the antibiotic trimethoprim. The folA gene encodes the key metabolic enzyme dihydrofolate reductase (DHFR), which is also a target of this antibiotic. With 514 mostly low fitness peaks, the DHFR fitness landscape is rugged. Despite this ruggedness, its highest fitness peaks are easily accessible to evolving populations. Fitness-increasing paths to high fitness peaks are abundant, and individual peaks have large basins of attractions. The basins of different peaks overlap, which renders the outcome of adaptive evolution highly contingent on chance events. In sum, ruggedness need not be an obstacle to Darwinian evolution but can reduce its predictability. If true in general, evolutionary biology and other fields of sciences in which landscapes play an important role may have to re-appraise the complexity of optimization problems on realistic landscapes.

evolutionary biology↗

bioRxiv2022

Off-target integron activity leads to rapid plasmid compensatory evolution in response to antibiotic selection pressure

Integrons are mobile genetic elements that have played an important role in the dissemination of antibiotic resistance. Under stress, the integron can generate combinatorial variation in resistance cassette expression by cassette re-shuffling, accelerating the evolution of resistance. However, the flexibility of the integron integrase site recognition motif hints at potential off-target effects of the integrase on the rest of the genome that may have important evolutionary consequences. Here we test this hypothesis by selecting for increased piperacillin resistance populations of P.aeruginosa with a mobile integron containing a hard-to-mobilise beta-lactamase cassette to minimize the potential for adaptive cassette re-shuffling. We found that integron activity can both decrease overall survival rate but also improve the fitness of the surviving populations. Off-target inversions mediated by the integron accelerated plasmid adaptation by disrupting costly conjugative genes otherwise mutated in control populations lacking a functional integrase. Plasmids containing integron-mediated inversions were associated with lower plasmid costs and higher stability than plasmids carrying mutations, albeit at a cost of reduced conjugative ability. These findings highlight the potential for integrons to create structural variation that can drive bacterial evolution, and they provide an interesting example showing how antibiotic pressure can drive the loss of conjugative genes.

evolutionary biology↗

bioRxiv2020

Integron activity accelerates the evolution of antibiotic resistance

Mobile integrons are widespread genetic platforms that allow bacteria to modulate the expression of antibiotic resistance cassettes by shuffling their position from a common promoter. Antibiotic stress induces the expression of an integrase that excises and integrates cassettes, and this unique recombination and expression system is thought to allow bacteria to evolve on demand in response to antibiotic pressure. To test this hypothesis, we inserted a custom three cassette integron into P. aeruginosa, and used experimental evolution to measure the impact of integrase activity on adaptation to gentamicin. Crucially, integrase activity accelerated evolution by increasing the expression of a gentamicin resistance cassette through duplications and by eliminating redundant cassettes. Importantly, we found no evidence of deleterious off-target effects of integrase activity. In summary, integrons accelerate resistance evolution by rapidly generating combinatorial variation in cassette composition while maintaining genomic integrity.

microbiology↗