Biology subjects

Redrejo-Rodriguez, M.

Publications and source records attributed to Redrejo-Rodriguez, M..

6 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↗

bioRxiv2025

Tn-seq of Thermus thermophilus genome reveals unexpected tolerance to insertions in bacterial common essential genes.

A large library based on a Tn5 minitransposon carrying a thermostable kanamycin resistance gene was prepared using Thermus thermophilus HB27 genomic DNA as target. To increase the yield of transformants, DNA from the in vitro transposition reaction was amplified using isothermal multiple displacement amplification. The resulting product was first transformed into the high-transformation efficiency{Delta} addAB/ppol strain and then into a wild-type HB27 strain. Tn-seq analysis of the libraries showed that almost all genes contained insertions and the distribution of the number of insertions per gene was unimodal, unlike the bimodal distribution reported in most Tn-seq analyses, thus hindering the discrimination of required or essential genes. Upon comparing the Tn-seq results with gene conservation in pangenomic analysis from Thermus thermophilus to Deiococcota levels, as well as with available HB27 RNA-seq data, we observed a very low correlation between core genes or gene transcription levels and Tn-seq insertion frequency. Notably, many genes largely deemed part of the essential bacterial core, supporting critical cellular pathways, showed relatively high transposon insertion numbers. In the case of DNA repair routes, which are essential but somewhat redundant, our results align well with previously published essentiality data, indicating that many genes are dispensable and permissive to insertions. The analysis of these striking results in the context of Thermus biology suggests that the polyploidy of the Thermus genome and the differential stability of proteins may explain the apparent non-essentiality of key genes. ImportanceThe Tn-seq technique, random insertion of transposons into an organisms genome followed by deep sequencing of the resulting population, has enabled precise identification of genes necessary for that organism to live under specified conditions. We have performed Tn-seq on the extremophilic bacterium Thermus thermophilus to gain information on the genes required for thriving at high temperature. Unexpectedly, we detected transposon insertions in the practical totality of the genes of this organism, hindering a clear discrimination of essential genes. Furthermore, a comparison with core genes derived from a pangenome analysis, spanning from the Thermus genus up to the phylum level, failed to establish a correlation between gene conservation and Tn-seq results. The polyploidy of the genome of this organism, along with potential regulatory mechanisms, may explain why commonly essential bacterial genes tolerate transposon insertions, a characteristic that appears particular for this organism.

microbiology↗

bioRxiv2022

ExplorePipolin: reconstruction and annotation of bacterial mobile elements from draft genomes

MotivationDetailed and accurate analysis of mobile genetic elements (MGEs) in bacteria is essential to deal with the current threat of multiresistant microbes. The overwhelming use of draft, contig-based genomes hinder the delineation of the genetic structure of these plastic and variable genomic stretches, as in the case of pipolins, a superfamily of MGEs that spans diverse integrative and plasmidic elements, characterized by the presence of a primer-independent DNA polymerase. ResultsExplorePipolin is a Python-based pipeline that screens for the presence of the element and performs its reconstruction and annotation. The pipeline can be used on virtually any genome from diverse organisms and of diverse quality, obtaining the highest-scored possible structure, and reconstructed out of different contigs if necessary. Then, predicted pipolin boundaries and pipolin encoded genes are subsequently annotated using a custom database, returning the standard file formats suitable for comparative genomics of this mobile element. AvailabilityAll code is available and can be accessed here: github.com/pipolinlab/ExplorePipolin Contactmodesto.redrejo@uam.es

bioinformatics↗

bioRxiv2022

Multiple Ciliary Localization Signals Control INPP5E Ciliary Targeting

Primary cilia are sensory membrane protrusions whose dysfunction causes diseases named ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, such as cilium stability, trafficking, signaling, or exovesicle release. Despite its key ciliary roles, how INPP5E accumulates in cilia remains poorly understood. Herein, we show that INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four ciliary localization signals (CLSs), the first two of which we newly discover: LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial redundancy between CLS1 and CLS4 explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 on the catalytic domain surface clarifies why CLS3 and CLS4 are together insufficient for ciliary accumulation. Furthermore, we reveal that some Joubert syndrome mutations in INPP5E catalytic domain affect its ciliary targeting, and shed light on the mechanisms of action of each CLS. Thus, we find that CLS2 and CLS3 promote interaction with TULP3 and ARL13B, while downregulating CEP164 binding. On the other hand, CLS4 recruits PDE6D, RPGR and ARL13B, and cooperates with CLS1 in ATG16L1 binding. Lastly, we show INPP5E immune synapse targeting is CLS-independent. Altogether, we reveal unusual complexity in INPP5E ciliary targeting mechanisms, likely reflecting its multiple key roles in ciliary biology.

cell biology↗

bioRxiv2021

UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions

Reverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs. In this work, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential.

evolutionary biology↗

bioRxiv2020

The fully resolved genome of Bacillus thuringiensis HER1410 reveals a cry-containing chromosome, two megaplasmids & an integrative plasmidial prophage

Bacillus thuringiensis is the most used biopesticide in agriculture. Its entomopathogenic capacity stems from the possession of plasmid-borne insecticidal crystal genes (cry), traditionally used as discriminant taxonomic feature for that species. As such, crystal and plasmid identification are key to the characterization of this species. To date, about 600 B. thuringiensis genomes have been reported, but less than 5% have been resolved, while the other draft genomes are incomplete, precluding plasmid delineation. Here we present the complete genome of Bacillus thuringiensis HER1410, a strain closely related to B. thuringiensis entomocidus and a known host for a variety of Bacillus phages. The combination of short and long-reads techniques allowed fully resolving the genome and delineation of three plasmids. This enabled the accurate detection of an unusual location of a unique cry gene, cry1Ba4, located in a genomic island near the chromosome replication origin. Two megaplasmids, pLUSID1 and pLUSID2 could be delineated: pLUSID1 (368kb), a likely conjugative plasmid involved in virulence, and pLUSID2 potentially related to the sporulation process. A smaller plasmidial prophage pLUSID3, with a dual lifestyle whose integration within the chromosome, causes the disruption of a flagellar key component. Finally, phylogenetic analysis located this strain within a clade comprising members from the B. thuringiensis serovar thuringiensis and other serovars and with B.cereus s. s. This highlights the intermingled taxonomy of B. cereus sensu lato group, where genomics alone does not support the present taxonomy between B. cereus s. s. and B. thuringiensis as species designation currently relies solely on the presence of entomocidal genes. ImportanceBacillus cereus group species have been extensively studied due to their economical and clinical relevance. This importance originally set the basis for B. cereus group members classification which are commonly based on phenotypical criteria. Sequencing era has shed light about genomic characterization of these species, showing their chromosomal genomic similarity and highlighting the role of mobile genetic elements, especially megaplasmids, in the classification and characterization of this group. However, only the 5% of the sequenced B. thuringiensis genomes have been fully resolved. Thus, here we addressed efficiently the study B. thuringiensis HER1410 genomic features by the use of a combination of short and long-reads sequencing. This methodology resulted in the high-quality assembly, which led to the identification of an uncommon location of a cry gene close to the chromosomal origin, as well as three fully resolved extrachromosomal elements, two megaplasmids and an integrative plasmidial prophage.

microbiology↗