Biology subjects

Garcillan-Barcia, M. P.

Publications and source records attributed to Garcillan-Barcia, M. P..

8 recordsLinked to original sources

bioRxiv2026

Plasmid-plasmid interactions reshape extracellular vesicle cargo and gene transfer potential

Plasmids are key drivers of horizontal gene transfer, yet their dissemination is not limited to conjugation. Extracellular vesicles (EVs) can transport plasmid DNA, but the factors governing plasmid incorporation into EVs remain poorly understood. Here, we tested whether principles of conjugative plasmid transfer, including plasmid mobility type and plasmid-plasmid interactions, extend to EV-mediated export. Using a conjugative plasmid (pKJK5) and a mobilizable plasmid (RSF1010) in two Gram-negative hosts, we quantified plasmid incorporation into EVs under single- and dual-plasmid conditions. When present individually, the conjugative plasmid was preferentially incorporated into EVs, exceeding RSF1010 by 10-23-fold despite its lower intracellular abundance. Under co-residence, this pattern reversed: RSF1010 became enriched by 13-39-fold, while pKJK5 was reduced by 2-7-fold. Consequently, EV-associated plasmid cargo shifted to RSF1010 dominance, deviating strongly from the expected 10-fold higher pKJK5 cargo if a stochastic model based on intracellular abundance and single-plasmid conditions were applicable. We propose that mobilizable plasmids under coexistence exploit conjugative plasmid transfer machinery to access membrane-associated sites, increasing their likelihood of incorporation into EVs. Our findings demonstrate that plasmid-plasmid interactions reshape EV cargo and identify a previously unrecognized mechanism that may influence extracellular gene transfer potential in microbial communities.

microbiology↗

bioRxiv2025

Plasmids drive the dissemination and diversification of Type VI Secretion Systems

The Type VI secretion system (T6SS) is a major determinant of bacterial competition, yet its dissemination across lineages remains unclear. Analyzing 43,213 plasmids and 29,161 chromosomes, we reveal plasmids as an underestimated reservoir and vehicle for T6SS diversification. We identified 405 complete plasmidencoded T6SSs and 929 orphan islands containing hcp, vgrG, and/or PAAR genes, often independent of full systems. Plasmidencoded T6SSs are biased toward large replicons, frequently megaplasmids, with distinct stability and mobility traits: orphan island plasmids are enriched in conjugation modules, whereas complete systems rely on partition and toxin-antitoxin maintenance. Phylogenomic analyses show some plasmid lineages stably integrating T6SSs as core traits, while others undergo recurrent acquisition and diversification. Comparative and ancestral analyses indicate pervasive bidirectional transfers between plasmids and chromosomes, mediated by insertion sequences. The presence of nearidentical homologs across compartments underscores the capacity of plasmids to transcend phylogenetic barriers and propagate these nanoweapons. Together, our results identify plasmids as dual evolutionary actors in T6SS ecology--short-term vectors that enable rapid horizontal spread, and long-term reservoirs that foster stabilization and adaptive diversification.

genomics↗

bioRxiv2025

Jaccard Index Network Analysis for pangenome analysis

ii.Summary/AbstractJaccard Index Network Analysis (JINA) is a comprehensive workflow designed to explore bacterial genome relationships through an integrated network-based approach. This workflow combines existing tools such as Jaccard Index (1), Gephi (2) and Pangraph (3). By integrating these methodologies into a unified framework, JINA enables efficient visualization and stratification of genomic data, facilitating the identification of meaningful patterns, groups, and associations within bacterial populations. The use of JINA ensures precision in capturing genomic variation including single nucleotide polymorphisms, insertions and deletions. While JINA does not implement Gephi, BLAST, and PanGraph directly in a single software, it guides their coordinated use to analyze and interpret genomic data effectively.

bioinformatics↗

bioRxiv2025

Exploring Clinical Class 1 Integrons as Valuable Targets for the Re-sensitization of Multidrug Resistant Pathogenic Bacteria Using CRISPR-Cas

The increasing prevalence of bacteria resistant to many or all types of antibiotics poses a major health crisis. Novel classes of antibiotics are only slowly being developed and alternative strategies are needed to tackle the issue. Mobile genetic elements and class 1 integrons are important facilitators for antibiotic resistance genes, with the latter being highly conserved in human pathogens. The growing prevalence of multidrug-resistant bacteria and the paucity in the development of new antibiotics underscore the urgent need for innovative approaches in the treatment of pathogens. Among these, CRISPR-Cas nucleases can be used to cleave acquired resistance genes, leading to either plasmid curing or cell death if the target is on a chromosome. In this study, we investigate the feasibility of using class 1 integrons as a target for Cas9-based cleavage leading to re-sensitizing antibiotic-resistant bacteria. We analyze the conserved and widespread integrase gene intI1 and conclude that it is a suitable target for Cas-based re-sensitization due to its high sequence conservation and its occurrence largely limited to human pathogens, alleviating the risk of targeting benign bacteria. We developed a broad host range conjugative plasmid encoding a class 1 integron-targeting Cas9 system that leads to removal of resistance plasmids in target bacteria with subsequent re-sensitization towards antibiotics. We find that 290 distinct ARGs co-occur on int1-harboring plasmids, showing the potential for re-sensitization towards a very broad range of antibiotics.

microbiology↗

bioRxiv2025

The Type IV Secretion System of Patescibacteria is homologous to the bacterial monoderm conjugation machinery

The Candidate Phyla Radiation, also known as Patescibacteria, represents a vast and diverse division of bacteria that has come to light via culture-independent "omics" technologies. Their limited biosynthetic capacity, along with evidence of their growth as obligate epibionts on other bacteria, suggests a broad reliance on host organisms for their survival. Nevertheless, our understanding of the molecular mechanisms governing their metabolism and lifestyle remains limited. The Type IV Secretion System (T4SS) represents a superfamily of translocation systems with a wide range of functional roles. T4SS genes have been identified in the CPR group Saccharibacteria as essential for their epibiotic growth. In this study, we used a comprehensive bioinformatics approach to investigate the diversity and distribution of T4SS within the Patescibacteria lineage. The phylogenetic analysis of the T4SS signature protein VirB4 suggests that most of these proteins cluster into a distinct monophyletic group with a shared ancestry to the MPFFATA class of T4SS. This class is found in the conjugative elements of Firmicutes, Actinobacteria, Tenericutes, and Archaea, indicating a possible horizontal gene transfer from these monoderm microorganisms to CPR bacteria. We identified additional T4SS components near virB4, particularly those associated with the MPFFATA class, as well as homologs of other T4SS classes, such as VirB2-like pilins, and observed their varied arrangements across different CPR phyla. The absence of a relaxase in most of these T4SS clusters suggests that the system has been co-opted for other functions in CPR bacteria. The proximity of T4SS components to the origin of replication (gene dnaA) in some CPR suggests a potential mechanism for increased expression. The broad ubiquity of a phylogenetically distinct T4SS in CPR, combined with its chromosomal location, underscores the significance of T4SS in the biology of Patescibacteria. Impact statementThe Candidate Phyla Radiation (CPR), or Patescibacteria, represents a highly diverse bacterial group that constitutes a significant fraction of the microbial dark matter. Known for their minimal biosynthetic capabilities and dependence on host bacteria, CPR bacteria exemplify fascinating yet poorly understood lifestyles. Our research sheds light on the molecular strategies underlying their survival by focusing on the Type IV Secretion System (T4SS), a versatile bacterial machinery typically involved in DNA transfer and effector molecule delivery. We revealed that T4SS is not only widespread in CPR but also forms a distinct evolutionary lineage, likely acquired via horizontal gene transfer from Gram-positive bacteria, and we illustrated its gene organization across different CPR phyla. Unlike canonical systems, CPR T4SS lacks key components for conjugation, hinting at a novel functional adaptation. Remarkably, T4SS genes in CPR are often located near the replication origin, suggesting a regulatory role linked to their expression. This research suggests the repurposing of T4SS in CPR to thrive under extreme reliance on other organisms. By unraveling the genomic and evolutionary peculiarities of T4SS in CPR, our work provides valuable insights into their unique biology, broadens our understanding of microbial diversity and innovation, and highlights this system as a strong candidate to sustain their parasitic episymbiotic lifestyle. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

genomics↗

bioRxiv2024

Specificities and commonalities of the Planctomycetes plasmidome

Although plasmids play a crucial role in antibiotic resistance and contemporary biotechnology, our comprehension of their natural ecological dynamics remains restricted to a few bacterial groups. Planctomycetes is a bacterial phylum with unusual molecular and cellular biology for which little is known about its plasmidome. This study provides the first comprehensive description of the diversity of the endogenous plasmids found in Planctomycetes, which may be used as starting points to generate different genetic tools for research on this phylum. Plasmids from Planctomycetes encode a wide variety of biological functions, although for a large portion of the encoded genes a function cannot be assigned, which is usual in Planctomycetes. They seemed to have largely coevolved with the genome of their hosts, with which they share many homologs. We also detected recent transfer events of insertion sequences between co-habiting chromosomes and plasmids. 60% of the plasmid genes are distantly related to chromosomally-encoded genes and 40% have homologs in plasmids from other bacterial groups, while 36% of the proteins composing the planctomycetal plasmidome are exclusive. Most planctomycetal plasmids encode a replication initiation protein of the RPA family in the proximity of a putative iteron-containing replication origin, as well as active type I partition systems. One conjugative and three mobilizable plasmids were identified, suggesting horizontal gene transfer via conjugation in this phylum.

microbiology↗

bioRxiv2023

Harnessing the pangenome for genomic surveillance: Salmonella enterica serotype Typhi as a paradigm

Public health genomic surveillance systems typically measure genome relatedness and infer molecular epidemiological relationships using chromosomal loci alone - an approximation of vertical evolution, or homology-by-descent. The accessory genome, composed of plasmids and other mobile genetic elements, reflects horizontal gene transfer and serves as an important mechanism of bacterial evolution, enabling rapid adaptation. Measuring homology in the accessory genome - homology-by-admixture - could offer important molecular epidemiological information for public health application. We applied Jaccard Index and a novel genome length distance metric to compute pangenome relatedness for the globally-important pathogen Salmonella enterica serotype Typhi (Typhi), and graphically express both homology-by-descent and homology-by-admixture in a reticulate network. Jaccard Index Network Analysis revealed structure in the Typhi pangenome that can be harnessed to enhance discriminatory power for surveillance, track antimicrobial resistance, and refine our understanding of homology for outbreak management and prevention. This offers a more intricate, multidimensional framework for understanding pathogen evolution. Significance StatementBacterial relatedness is often measured and visualized using chromosomal comparison and phylogenetic trees. While valuable, this approach captures only the vertical evolutionary dimension and excludes genetic material acquired or lost through horizontal gene transfer. We present an approach for measuring and visualizing bacterial relatedness using all core and accessory genetic material and discuss the interpretation of resulting reticulate networks of bacterial genomes. In application to Salmonella Typhi, Jaccard Index Network Analysis revealed structure in populations of this pathogen that may be harnessed for public health applications. This approach captures both vertical and horizontal evolutionary dimensions, offering an intricate genetic framework for exploring pathogen evolution.

genomics↗

bioRxiv2019

Comparative analysis of MOBQ4 plasmids demonstrates that MOBQ is a cis-acting-enriched relaxase protein family

A group of small mobilizable plasmids is increasingly being reported in epidemiology surveys of enterobacteria. Some of them encode colicins, while others are cryptic. All of them encode a relaxase belonging to a previously non-described group of the MOBQ class, MOBQ4. While highly similar in their mobilization module, two families with unrelated replicons can be distinguished, MOBQ41 and MOBQ42. Members of both groups were compatible between them and stably maintained in E. coli. MOBQ4 plasmids were mobilized by conjugation. They contain two transfer genes, mobA coding for the MOBQ4 relaxase and mobC, which was non-essential but enhanced the plasmid mobilization frequency. The origin of transfer was located between these two divergently transcribed mob genes. MPFI conjugative plasmids were the most efficient helpers for MOBQ4 conjugative transmission. No interference in mobilization was observed when both MOBQ41 and MOBQ42 were present in the same donor cell. Remarkably, MOBQ4 relaxases exhibited a cis-acting preference for their oriTs, a feature already observed in other MOBQ plasmids. These findings indicate that MOBQ4 plasmids can efficiently spread among enterobacteria aided by coresident IncI1, IncK and IncL/M plasmids, while ensuring their self-dissemination over highly-related elements.\n\nIMPORTANCEPlasmids are key vehicles of horizontal gene transfer and contribute greatly to bacterial genome plasticity. A group of plasmids, called mobilizable, is able to disseminate aided by helper conjugative plasmids. Here, we studied a group of phylogenetically-related mobilizable plasmids, MOBQ4, commonly found in clinically-relevant enterobacteria, uncovering the helper plasmids responsible for their dissemination. We found that the two plasmid species encompassed in the MOBQ4 group can coexist and transfer orthogonally, despite origin-of-transfer cross-recognition by their relaxases. Specific discrimination among their highly similar oriT sequences is guaranteed by the preferential cis activity of the MOBQ4 relaxases. Such strategy would be biologically relevant in a scenario of co-residence of non-divergent elements to favor self-dissemination.

microbiology↗