Tnseq to identify recipient factor to modulate conjugative plasmid transfer
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Escherichia coli K-12
SUBMITTER:
PROVIDER: E-MTAB-11704 | biostudies-arrayexpress |
SECONDARY ACCESSION(S): E-MTAB-9713
REPOSITORIES: biostudies-arrayexpress
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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.
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Project description:Study of the possible existence of a replication fork trap in Vibrio cholerae. 1- FX85: EPV50(WT) grown in M9 minimal medium supplemented with 0.4 % fructose to exponential phase (0.2 OD 650 nm). 2- FX86: EPV50(WT) grown in M9 minimal medium supplemented with 0.4 % fructose to stationary phase (long overnight). 3- FX288: EGV140 (oriL3) grown in M9 minimal medium supplemented with 0.4 % fructose to exponential phase (0.2 OD 650 nm). 4- FX289:EGV140 (oriL3) grown in M9 minimal medium supplemented with 0.4 % fructose to stationary phase (long overnight). 5- FX290: EGV111 (oriR4) grown in M9 minimal medium supplemented with 0.4 % fructose to exponential phase (0.2 OD 650 nm). 6- FX291:EGV111 (oriR4) grown in M9 minimal medium supplemented with 0.4 % fructose to stationary phase (long overnight). 7- FX355:EPV50 (WT) grown in LB medium to exponential phase (0.2 OD 650nm) 8- FX356:EPV50 (WT) grown in LB medium to stationary phase (overnight) 9- FX286:EGV140 (oriL3) grown in LB medium to exponential phase (0.2 OD 650nm) 10- FX287:EGV140 (oriL3) grown in LB medium to stationary phase (overnight) 11- FX292:EGV111 (oriR4) grown in LB medium to exponential phase (0.2 OD 650nm) 12- FX49: MCH1 (WT monochromosome) grown in M9 minimal medium supplemented with 0.4 % fructose to exponential phase (0.2 OD 650 nm). 13- FX48: MCH1 (WT monochromosome) grown in M9 minimal medium supplemented with 0.4 % fructose to stationary phase (long overnight). 14- FX11: EGV369 (oriL3 monochromosome) grown in M9 minimal medium supplemented with 0.4 % fructose to exponential phase (0.2 OD 650 nm). 15- FX12: EGV366 (oriR4 monochromosome) grown in M9 minimal medium supplemented with 0.4 % fructose to exponential phase (0.2 OD 650 nm). 16- FX296 EPV50 M9 Exp 17- FX294 EPV50 M9 Stat 18-FX316 EGV140 M9 Exp 19- FX315 EGV111 M9 Exp 20-FX318 MCH1 M9 Exp 21- FX317 MCH1 M9 Stat 22- FX320 EGV369 M9 Exp 23- FX319 EGV366 M9 Exp Chromosomal DNA was extracted using the Sigma GenElute bacterial genomic DNA kit. 5 μg of DNA were used to generate a genomic library according to Illumina's protocol. The libraries and the sequencing were performed by the High-throughput Sequencing facility of the I2BC (http://www.i2bc.paris-saclay.fr/spip.php?article399〈=en,CNRS, Gif-sur-Yvette, France). Genomic DNA libraries were made with the ‘Nextera DNA library preparation kit’ (Illumina) following the manufacturer’s recommendations. Library quality was assessed on an Agilent Bioanalyzer 2100, using an Agilent High Sensitivity DNA Kit (Agilent technologies). Libraries were pooled in equimolar proportions. 75 bp single reads were generated on an Illumina MiSeq instrument, using a MiSeq Reagent kit V2 (500 cycles) (Illumina), with an expected depth of 217X. Reads were aligned on the in silico reconstituted genome of the cognate strain using BWA software. An in-lab written MATLAB-based script was used to perform marker frequency analysis. Data were normalized by dividing uniquely mapping sequence reads by the total number of reads. Enrichment of uniquely mapping sequence reads in 1 kb non-overlapping windows were calculated and plotted against the chromosomal coordinates.
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Project description:Tnseq to identify recipient factor to modulate conjugative plasmid transfer
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Project description:Integrative and conjugative elements (ICEs) are found in many bacterial species and are mediators of horizontal gene transfer. Tn916, an ICE found in several Gram-positive species, can integrate into many sites in the host chromosome, in contrast to the many ICEs that preferentially integrate into a single site. The consensus integration motif for Tn916, based on analyses of approximately 200 independent insertions, is an ~16 bp AT-rich sequence. Here, we describe the identification and mapping of approximately 105 independent Tn916 insertions in the Bacillus subtilis chromosome. The insertions were distributed between 1554 chromosomal sites, and approximately 99% of the insertions were in 303 sites and 65% were in only ten sites. One region, between ykuC and ykyB (kre), was a 'hot spot' for integration with ~22% of the insertions in that single location. In almost all of the top 99% of sites, Tn916 was found with similar frequencies in both orientations relative to the chromosome and relative to the direction of transcription, with a few notable exceptions. Using the sequences of all insertion regions, we determined a consensus motif which is similar to that previously identified for Clostridium difficile. The insertion sites are largely AT-rich, and some sites overlap with regions bound by the nucleoid-associated protein Rok, a functional analog of H-NS of Gram-negative bacteria. Rok functions as a negative regulator of at least some horizontally acquired genes. We found that the presence or absence of Rok had little or no effect on insertion site specificity of Tn916.
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