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Transcriptome Analysis of Zygotic Induction During Conjugative Transfer of Plasmid RP4.

ABSTRACT: Conjugative transfer of bacterial plasmid is one of the major mechanisms of horizontal gene transfer, which is mediated by direct contact between donor and recipient cells. Gene expression of a conjugative plasmid is tightly regulated mostly by plasmid-encoded transcriptional regulators, but it remains obscure how differently plasmid genes are expressed in each cell during the conjugation event. Here, we report a comprehensive analysis of gene expression during conjugative transfer of plasmid RP4, which is transferred between isogenic strains of Pseudomonas putida KT2440 at very high frequency. To discriminate the expression changes in the donor and recipient cells, we took advantage of conjugation in the presence of rifampicin (Rif). Within 10 min of mating, we successfully detected transient transcription of plasmid genes in the resultant transconjugant cells. This phenomenon known as zygotic induction is likely attributed to derepression of multiple RP4-encoded repressors. Interestingly, we also observed that the traJIH operon encoding relaxase and its auxiliary proteins were upregulated specifically in the donor cells. Identification of the 5' end of the zygotically induced traJ mRNA confirmed that the transcription start site of traJ was located 24-nt upstream of the nick site in the origin of transfer (oriT) as previously reported. Since the traJ promoter is encoded on the region to be transferred first, the relaxase may be expressed in the donor cell after regeneration of the oriT-flanking region, which in itself is likely to displace the autogenous repressors around oriT. This study provides new insights into the regulation of plasmid transfer processes.

SUBMITTER: Miyakoshi M

PROVIDER: S-EPMC7314908 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Transcriptome Analysis of Zygotic Induction During Conjugative Transfer of Plasmid RP4.

Miyakoshi Masatoshi M Ohtsubo Yoshiyuki Y Nagata Yuji Y Tsuda Masataka M

Frontiers in microbiology 20200618

Conjugative transfer of bacterial plasmid is one of the major mechanisms of horizontal gene transfer, which is mediated by direct contact between donor and recipient cells. Gene expression of a conjugative plasmid is tightly regulated mostly by plasmid-encoded transcriptional regulators, but it remains obscure how differently plasmid genes are expressed in each cell during the conjugation event. Here, we report a comprehensive analysis of gene expression during conjugative transfer of plasmid RP ...[more]

PMID: 32625173

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Project description:Multiwalled carbon nanotubes (MWCNTs) regularly enter aquatic environments due to their ubiquity in consumer products and engineering applications. However, the effects of MWCNT pollution on the environmental microbiome are poorly understood. Here, we evaluated whether these carbon nanoparticles can elevate the spread of antimicrobial resistance by promoting bacterial plasmid transfer, which has previously been observed for copper nanomaterials with antimicrobial properties as well as for microplastics. Through a combination of experimental liquid mating assays between Pseudomonas putida donor and recipient strains with plasmid pKJK5::gfpmut3b and mathematical modeling, we here demonstrate that the presence of MWCNTs leads to increased plasmid transfer rates in a concentration-dependent manner. The percentage of transconjugants per recipient significantly increased from 0.21 ± 0.04% in absence to 0.41 ± 0.09% at 10 mg L-1 MWCNTs. Similar trends were observed when using an Escherichia coli donor hosting plasmid pB10. The identified mechanism underlying the observed dynamics was the agglomeration of MWCNTs. A significantly increased number of particles with >6 μm diameter was detected in the presence of MWCNTs, which can in turn provide novel surfaces for bacterial interactions between donor and recipient cells after colonization. Fluorescence microscopy confirmed that MWCNT agglomerates were indeed covered in biofilms that contained donor bacteria as well as elevated numbers of green fluorescent transconjugant cells containing the plasmid. Consequently, MWCNTs provide bacteria with novel surfaces for intense cell-to-cell interactions in biofilms and can promote bacterial plasmid transfer, hence potentially elevating the spread of antimicrobial resistance. IMPORTANCE In recent decades, the use of carbon nanoparticles, especially multiwalled carbon nanotubes (MWCNTs), in a variety of products and engineering applications has been growing exponentially. As a result, MWCNT pollution into environmental compartments has been increasing. We here demonstrate that the exposure to MWCNTs can affect bacterial plasmid transfer rates in aquatic environments, an important process connected to the spread of antimicrobial resistance genes in microbial communities. This is mechanistically explained by the ability of MWCNTs to form bigger agglomerates, hence providing novel surfaces for bacterial interactions. Consequently, increasing pollution with MWCNTs has the potential to elevate the ongoing spread of antimicrobial resistance, a major threat to human health in the 21st century.

Dataset Information

Transcriptome Analysis of Zygotic Induction During Conjugative Transfer of Plasmid RP4.

Publications

Transcriptome Analysis of Zygotic Induction During Conjugative Transfer of Plasmid RP4.

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