Cis-Acting Relaxases Guarantee Independent Mobilization of MOBQ4 Plasmids

Plasmids are key vehicles of horizontal gene transfer and contribute greatly to bacterial genome plasticity. In this work, we studied a group of plasmids from enterobacteria that encode phylogenetically related mobilization functions that populate the previously non-described MOBQ4 relaxase family. These plasmids encode two transfer genes: mobA coding for the MOBQ4 relaxase; and mobC, which is non-essential but enhances the plasmid mobilization frequency. The origin of transfer is located between these two divergently transcribed mob genes. We found that MPFI conjugative plasmids were the most efficient helpers for MOBQ4 conjugative dissemination among clinically relevant enterobacteria. While highly similar in their mobilization module, two sub-groups with unrelated replicons (Rep_3 and ColE2) can be distinguished in this plasmid family. These subgroups can stably coexist (are compatible) and transfer independently, 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 a strategy would be biologically relevant in a scenario of co-residence of non-divergent elements to favor self-dissemination.Funding: This work was supported by the Spanish Ministry of Economy and Competitiveness (BFU2017-86378-P, AEI/FEDER, UE, to FC) and Consejo Superior de Investigaciones Científicas (201820I143 to MG-B). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

ArdC, a ssDNA-binding protein with a metalloprotease domain, overpasses the recipient hsdRMS restriction system broadening conjugation host range

Plasmids, when transferred by conjugation in natural environments, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida. Expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. ardC was also required if the hsdRMS system was present in E. coli recipient cells. Thus, ArdC has antirestriction activity against the HsdRMS system and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and presence of Mn2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity was not needed for the antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggered an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies and settle into new hosts.This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness [BFU2014-55534-C2 to FdlC and GM] and by the Spanish Ministry of Education, Culture and Sports [FPU014/06013 to LG-M]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Plasmid conjugation from Proteobacteria as evidence for the origin of xenologous genes in Cyanobacteria

Comparative genomics showed that 5% of Synechococcus elongatus PCC 7942 genes are of probable proteobacterial origin. To investigate the role of inter-phylum conjugation in cyanobacterial gene acquisition, we tested the ability of a set of prototype proteobacterial conjugative plasmids (RP4, pKM101, R388, R64 and F) to transfer DNA from E. coli to S. elongatus. A series of BioBrick-compatible, mobilizable shuttle vectors was developed. These vectors were based on the putative origin of replication of the Synechococcus resident plasmid pANL. Not only broad-host range plasmids, such as RP4 and R388, but also narrower host-range plasmids, such as pKM101, all encoding MPFT-type IV secretion systems, were able to transfer plasmid DNA from E. coli to S. elongatus by conjugation. Neither MPFF, nor MPFI could be used as interphylum DNA delivery agents. Reciprocally, pANL-derived cointegrates could be introduced in E. coli by electroporation, where they conferred a functional phenotype. These results suggest the existence of potentially ample channels of gene flow between Proteobacteria and Cyanobacteria and point to MPFT-based inter-phylum conjugation as a potential mechanism to explain the proteobacterial origin of a majority of S. elongatus xenologous genes

Key components of the eight classes of type IV secretion systems involved in bacterial conjugation or protein secretion

Conjugation of DNA through a type IV secretion system (T4SS) drives horizontal gene transfer. Yet little is known on the diversity of these nanomachines. We previously found that T4SS can be divided in eight classes based on the phylogeny of the only ubiquitous protein of T4SS (VirB4). Here, we use an ab initio approach to identify protein families systematically and specifically associated with VirB4 in each class. We built profiles for these proteins and used them to scan 2262 genomes for the presence of T4SS. Our analysis led to the identification of thousands of occurrences of 116 protein families for a total of 1623 T4SS. Importantly, we could identify almost always in our profiles the essential genes of well-studied T4SS. This allowed us to build a database with the largest number of T4SS described to date. Using profile-profile alignments, we reveal many new cases of homology between components of distant classes of T4SS. We mapped these similarities on the T4SS phylogenetic tree and thus obtained the patterns of acquisition and loss of these protein families in the history of T4SS. The identification of the key VirB4-associated proteins paves the way toward experimental analysis of poorly characterized T4SS classes.Funding. Spanish Ministry of Economy [BFU2011-26608]; European Seventh Framework Program [282004/FP7-HEALTH.2011, 612146/FP7-ICT-2013]; European Research Council Grant [EVOMOBILOME no. 281605]. Source of open access funding: European Research Council grant to the PI

White Paper 4: Challenges In Biomedicine & Health

Publicado en Madrid, 231 p. ; 17 cm.A lesson that we have learned from the pandemia caused by coronavirus is that solutions in health require coordinated actions. Beside this and other emerging and re-emerging infectious diseases, millions of Europeans are suffering a plethora of disorders that are currently acquiring epidemic dimensions, including cancer, rare diseases, pain and food allergies, among others. New tools for prevention, diagnosis and treatment need to be urgently designed and implemented using new holistic and multidisciplinary approaches at three different levels (basic research, translational/clinical and public/social levels) and involving researchers, clinicians, industry and all stakeholders in the health system. The CSIC is excellently positioned to lead and coordinate these challenges in Biomedicine and Health.Peer reviewe

Type VI Secretion Systems encoded by plasmids

Trabajo presentado en la conferencia "Plasmids around the Globe", celebrada en modalidad virtual el 06 de mayo de 2021

Sistemas de Secreción Tipo VI como armas móviles

Resumen del trabajo presentado en el XXVIII Congreso de la Sociedad Española de Microbiología, celebrado en modalidad virtual del 28 de junio al 02 de julio de 2021.El Sistema de Secreción Tipo VI (T6SS) es un complejo multiproteico presente en bacterias Gramnegativas que permite inyectar toxinas de una manera dependiente de contacto a otras células, tanto procariotas como eucariotas. Su papel es crítico en la competición en comunidades microbianas y en la patogenicidad. La presencia de este sistema en plataformas móviles tales como plásmidos, parece anecdótica ya que, hasta ahora, la mayoría de T6SSs funcionales descritos son cromosómicos. Sin embargo, nuestro análisis de la base de datos plasmídica NCBI RefSeq identificó una cantidad significativa de plásmidos que codifican T6SSs. La mayoría de estos plásmidos codifican, además, la maquinaria necesaria para ser transmitidos mediante conjugación, incorporando esta arma al proceso de transferencia genética horizontal. Hemos comprobado la actividad antibacteriana de uno de estos T6SSs codificado en un plásmido conjugativo. Esto plantea la pregunta de cómo influye esta actividad antibacteriana en la transferencia del propio plásmido y viceversa, pero sobre todo, cómo los plásmidos conjugativos que codifican para este T6SS son capaces de decidir entre matar a una célula vecina o utilizarla como receptora.Ayuda predoctoral: Ayuda para Contratos Predoctorales de la Universidad de Cantabria (BOC No 187 29/09/2020

Are Type VI Secretion System-encoding plasmids vehicles for antimicrobial resistances?

Trabajo presentado en Workshop on Plasmids as Vehicles of AMR Spread, celebrado en Trieste (Italia) del 18 al 22 de septiembre de 2023.Peer reviewe

Distribución de sistemas de secreción tipo VI en plásmidos

Trabajo presentado en el II Basque Microbiology Meeting MikrobioGUNE, celebrado en Bilbao (España) el 13 de diciembre de 2022

Genomic inventory of mobile type VI secretion systems

Trabajo presentado en el International Symposium on Plasmid Biology, celebrado en Toulouse (Francia) del 18 al 23 de septiembre de 2022