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RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli

Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and trans...

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Autores principales: Zhang, Yanmei, Shi, Chunyu, Yu, Jiafei, Ren, Jingjing, Sun, Dongchang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306417/
https://www.ncbi.nlm.nih.gov/pubmed/22438941
http://dx.doi.org/10.1371/journal.pone.0033514
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author Zhang, Yanmei
Shi, Chunyu
Yu, Jiafei
Ren, Jingjing
Sun, Dongchang
author_facet Zhang, Yanmei
Shi, Chunyu
Yu, Jiafei
Ren, Jingjing
Sun, Dongchang
author_sort Zhang, Yanmei
collection PubMed
description Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and transformation regulation remain unclear in this new type of plasmid transformation. In this study, we developed our previous plasmid transformation system and induced competence at early stationary phase. Despite of inoculum size, the development of competence was determined by optical cell density. DNase I interruption experiment showed that DNA was taken up exponentially within the initial 2 minutes and most transforming DNA entered E. coli cells within 10 minutes on LB-agar plates. A half-order kinetics between recipient cells and transformants was identified when cell density was high on plates. To determine whether the stationary phase master regulator RpoS plays roles in plasmid transformation, we investigated the effects of inactivating and over-expressing its encoding gene rpoS on plasmid transformation. The inactivation of rpoS systematically reduced transformation frequency, while over-expressing rpoS increased plasmid transformation. Normally, RpoS recognizes promoters by its lysine 173 (K173). We found that the K173E mutation caused RpoS unable to promote plasmid transformation, further confirming a role of RpoS in regulating plasmid transformation. In classical transformation, DNA was transferred across membranes by DNA uptake proteins and integrated by DNA processing proteins. At stationary growth phase, RpoS regulates some genes encoding membrane/periplasmic proteins and DNA processing proteins. We quantified transcription of 22 of them and found that transcription of only 4 genes (osmC, yqjC, ygiW and ugpC) encoding membrane/periplasmic proteins showed significant differential expression when wildtype RpoS and RpoS(K173E) mutant were expressed. Further investigation showed that inactivation of any one of these genes did not significantly reduce transformation, suggesting that RpoS may regulate plasmid transformation through other/multiple target genes.
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spelling pubmed-33064172012-03-21 RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli Zhang, Yanmei Shi, Chunyu Yu, Jiafei Ren, Jingjing Sun, Dongchang PLoS One Research Article Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and transformation regulation remain unclear in this new type of plasmid transformation. In this study, we developed our previous plasmid transformation system and induced competence at early stationary phase. Despite of inoculum size, the development of competence was determined by optical cell density. DNase I interruption experiment showed that DNA was taken up exponentially within the initial 2 minutes and most transforming DNA entered E. coli cells within 10 minutes on LB-agar plates. A half-order kinetics between recipient cells and transformants was identified when cell density was high on plates. To determine whether the stationary phase master regulator RpoS plays roles in plasmid transformation, we investigated the effects of inactivating and over-expressing its encoding gene rpoS on plasmid transformation. The inactivation of rpoS systematically reduced transformation frequency, while over-expressing rpoS increased plasmid transformation. Normally, RpoS recognizes promoters by its lysine 173 (K173). We found that the K173E mutation caused RpoS unable to promote plasmid transformation, further confirming a role of RpoS in regulating plasmid transformation. In classical transformation, DNA was transferred across membranes by DNA uptake proteins and integrated by DNA processing proteins. At stationary growth phase, RpoS regulates some genes encoding membrane/periplasmic proteins and DNA processing proteins. We quantified transcription of 22 of them and found that transcription of only 4 genes (osmC, yqjC, ygiW and ugpC) encoding membrane/periplasmic proteins showed significant differential expression when wildtype RpoS and RpoS(K173E) mutant were expressed. Further investigation showed that inactivation of any one of these genes did not significantly reduce transformation, suggesting that RpoS may regulate plasmid transformation through other/multiple target genes. Public Library of Science 2012-03-16 /pmc/articles/PMC3306417/ /pubmed/22438941 http://dx.doi.org/10.1371/journal.pone.0033514 Text en Zhang et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Zhang, Yanmei
Shi, Chunyu
Yu, Jiafei
Ren, Jingjing
Sun, Dongchang
RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli
title RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli
title_full RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli
title_fullStr RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli
title_full_unstemmed RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli
title_short RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli
title_sort rpos regulates a novel type of plasmid dna transfer in escherichia coli
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306417/
https://www.ncbi.nlm.nih.gov/pubmed/22438941
http://dx.doi.org/10.1371/journal.pone.0033514
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