Project description:Tn insertion library was used for recipient for conjugative transfer of pESBL, F, and R388 plasmids. For both recipient and the resulting exconjugant libraries, Tn insertion sites were determined by illumina sequencing
Project description:Rhizobia are gram-negative bacteria able to establish a symbiotic interaction with leguminous plants. Due to their nitrogen fixing capacity, the study of these microorganisms has acquired great relevance for the agriculture. Rhizobia usually harbor many plasmids in their genome which can be transferred to other organisms by conjugation. Two main mechanisms of regulation of rhizobial plasmid transfer have been described: Quorum sensing (QS) and rctA/rctB system. Nevertheless, new genes and molecules that modulate conjugative transfer have been recently described, demonstrating that new actors can tightly regulate the process. In this work, by means of bioinformatics tools and molecular biology approaches, two hypothetical genes are identified as playing key roles in conjugative transfer. These genes are located between conjugative genes of plasmid pLPU83a from Rhizobium favelukesii LPU83, a plasmid that showed a conjugative transfer behavior depending on the genomic background. One of the two mentioned genes, rcgA, is essential for conjugation, while the other, rcgR, acts as an inhibitor of the process. In addition to introducing this new regulatory mechanism, we show evidence of the functions of these genes in different genomic backgrounds, and confirmed that homologous proteins from non-closely related organisms play the same function. These findings set up a cornerstone for a new molecular circuit of conjugative transfer of plasmids.
Project description:We analyzed gene expression during conjugative transfer of plasmid RP4. Pairs of rifampicin-susceptible (RifS) and -resistance (RifR) strains of Pseudomonas putida KT2440 were conjugated for 10 minute on filter membrane in the presence of rifampicin to discriminate the expression changes in the donor and recipient cells.
Project description:Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution. Transfer of conjugative plasmids is a complex process that needs to be synchronized with the physiological state of the bacterial host. While several host transcription factors are known to control the plasmid-borne transfer control genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. Here, we describe a post-transcriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two different seed pairing domains to recognize and activate the mRNAs of both the sigma-factor S and RicI, a cytoplasmic membrane protein. The latter is a hitherto unknown conjugation inhibitor whose transcription requires S. Together, RprA and S constitute a feed-forward loop with AND-gate logic which tightly controls RicI synthesis for selective suppression of plasmid conjugation under membrane stress. This study reports the first sRNA-controlled feed-forward loop based on double target activation and an unexpected function for a core-genome encoded small RNA in controlling extrachromosomal DNA transfer.
Project description:Integrative and conjugative elements (ICEs), a.k.a., conjugative transposons, are mobile genetic elements involved in many biological processes, including the spread of antibiotic resistance. Unlike conjugative plasmids that are extra-chromosomal and replicate autonomously, ICEs are integrated in the chromosome and replicate passively during chromosomal replication. It is generally thought that ICEs do not replicate autonomously. We found that when induced, Bacillus subtilis ICEBs1 replicates as a plasmid. The ICEBs1 origin of transfer (oriT) served as the origin of replication and the conjugal DNA relaxase served as the replication initiation protein. Autonomous replication of ICEBs1 conferred genetic stability to the excised element, but was not required for mating. The B. subtilis helicase PcrA that mediates unwinding and replication of Gram-positive rolling circle replicating plasmids was required for ICEBs1 replication and mating. Nicking of oriT by the relaxase and unwinding by PcrA likely directs transfer of a single-strand of ICEBs1 into recipient cells. This SuperSeries is composed of the SubSeries listed below.
Project description:M. ciceri WSM1271 carries a novel integrative and conjugative element (ICE) termed a tripartite ICE (ICE3) that exists as thee separated DNA regions in the chromosome. Three site-specific recombinases acting on three distinct pairs of attachment sites catalyse recombination between the three ICE3 DNA regions leading to their excision from the chromosome to form a single plasmid-like entity for horizontal conjugative transfer. RNASeq was used to probe for differentially expressed ICE3-encoded genes in WSM1271 wild-type cells, and engineered WSM1271 cells carrying cloned copies of two quorum-sensing loci that induce ICE3 excision and transfer. Overall, 187 significantly differentially expressed genes (adjusted P-value < 0.05) were identified and although ICEMcSym1271 comprised only ~7.6% of the chromosome, 15.5% (29) of the differentially expressed genes were located on ICEMcSym1271. Genes likely involved in activation of excision and conjugation including rdfS, rlxS, msi172-msi171 and the type-IV conjugative pilus gene cluster msi031-trbBCDEJLFGI-msi021 were all significantly induced. The RNA-Seq libraries generated in this study have strongly aided the development of our model detailing the regulation of excision and transfer of ICE3s.
Project description:we examined the three different mature biofilms and searched the genes which promoted the rapid biofilm formation when their population hosing the plasmids. We investigate the global transcriptional differences between the non-conjugative or conjugative plasmid-carrying and plasmid-free strains.
Project description:Antibiotic resistance is exacerbated by the exchange of antibiotic resistance genes (ARGs) between microbes from diverse habitats. Plasmids are important ARGs mobile elements and are spread by horizontal gene transfer (HGT). In this study, we demonstrated the presence of multi-resistant plasmids from inhalable particulate matter (PM) and its effect on gene horizontal transfer. Three transferable multi-resistant plasmids were identified from PM in a hospital, using conjugative mating assays and nanopore sequencing. pTAir-3 contained 26 horizontal transfer elements and 10 ARGs. Importantly pTAir-5 harbored carbapenem resistance gene (blaOXA) which shows homology to plasmids from human and pig commensal bacteria, thus indicating that PM is a media for antibiotic resistant plasmid spread. In addition, 125 μg/mL PM2.5 and PM10 significantly increased the conjugative transfer rate by 110% and 30%, respectively, and augmented reactive oxygen species (ROS) levels. Underlying mechanisms were revealed by identifying the upregulated expressional levels of genes related to ROS, SOS, cell membranes, pilus generation, and transposition via genome-wide RNA sequencing. The study highlights the airborne spread of multi-resistant plasmids and the impact of inhalable PM on the horizontal transfer of antibiotic resistance.