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Plasmid maintenance

Plasmid maintenance


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I have obtained some plasmids used as integration vectors, this question may apply to all plasmids. I would like to have a somewhat continuos source for these plasmids, let's say that the origin of replication is e-coli specific and can i then just transform the e-coli with the plasmid, let it grow to some density and then do miniprep? or can i also create glycerol stocks out of the bacteria that contain the plasmids and then harvest more plasmids as needed? i realize that both things are possible, but is this a good reasoning to follow?

Thanks


What I would usually do is transform E. coli with the plasmid, grow an overnight culture and mini prep the plasmid in the morning. Before you do the miniprep save a glycerol stock. Thus, you would have some plasmid to work with in the next couple of weeks, and also a glycerol stock to come back to. You can also keep the plate with your transformants, as you can go back to it within a month.


Plasmid partition system

A plasmid partition system is a mechanism that ensures the stable inheritance of plasmids during bacterial cell division. Each plasmid has its independent replication system which controls the number of copies of the plasmid in a cell. The higher the copy number is, the more likely the two daughter cells will contain the plasmid. Generally, each molecule of plasmid diffuses randomly, so the probability of having a plasmid-less daughter cell is 2 1−N , where N is the number of copies. For instance, if there are 2 copies of a plasmid in a cell, there is a 50% chance of having one plasmid-less daughter cell. However, high-copy number plasmids have a cost for the hosting cell. This metabolic burden is lower for low-copy plasmids, but those have a higher probability of plasmid loss after a few generations. To control vertical transmission of plasmids, in addition to controlled-replication systems, bacterial plasmids use different maintenance strategies, such as multimer resolution systems, post-segregational killing systems (addiction modules), and partition systems. [1]


Purification and properties of the plasmid maintenance proteins from the Borrelia burgdorferi linear plasmid lp17

The Lyme disease spirochete Borrelia burgdorferi carries more plasmids than any other bacterium, many of which are linear with covalently closed hairpin ends. These plasmids have also been referred to as mini-chromosomes and essential genetic elements and are integral components of its segmented genome. We have investigated two plasmid maintenance proteins, BBD14 (the replication initiator) and BBD21 (a presumptive ParA orthologue), encoded by the linear plasmid lp17 these proteins are representatives of paralogous families 62 and 32, respectively. We have purified recombinant 6-his-BBD21 and shown it possesses an ATPase activity. 6-his-BBD14 initially could not be overexpressed in Escherichia coli by itself. It was only effectively overproduced in recombinant form through coexpression with other B. burgdorferi proteins and codon optimization. Although the mechanism for increased production through coexpression is not clear, this method holds promise for expression and purification of other B. burgdorferi proteins, a number of which have remained recalcitrant to purification from E. coli. Finally, we present evidence for the physical interaction of BBD14 and BBD21, a feature suggesting that BBD21 and the paralogous family 32 proteins are more likely involved in DNA replication than functioning as simple ParA orthologues as previously surmised based upon sequence homology. Such a role would not preclude a function in plasmid partitioning through interaction with the replication initiator.

Figures

Putative B. burgdorferi plasmid replication…

Putative B. burgdorferi plasmid replication proteins. The 12 linear and 10 circular plasmids…

Alignment of BBD21 with two…

Alignment of BBD21 with two related protein families. The proposed ATPase domain of…

Induction and purification of 6-his-BBD21.…

Induction and purification of 6-his-BBD21. A Coomassie blue-stained 15% SDS-polyacrylamide gel is shown.…

Glycerol gradient purification and ATPase…

Glycerol gradient purification and ATPase activity of 6-his-BBD21. (A) Glycerol gradient purification of…

Construct for coexpression of BBD21…

Construct for coexpression of BBD21 and 6-his-BBD14. A map of the coexpression plasmid…

Coelution of BBD21 from Ni-NTA…

Coelution of BBD21 from Ni-NTA with His-tagged BBD14. (A) Wild-type BBD21 and His-tagged…


Plasmid Biology

Aims: To isolate and characterize multiple antibiotic resistance plasmids found in swine manure and test for plasmid-associated genetic markers in soil following manure application to an agricultural field.

Methods and Results: Plasmids were isolated from an erythromycin enrichment culture that used liquid swine manure as an inoculant. Plasmids were transformed into Escherichia coli DH10β for subsequent characterization. We isolated and DNA sequenced a 22 102-bp plasmid (pMC2) that confers macrolide, and tetracycline resistances, and carries genes predicted to code for mercury and chromium resistance. Conjugation experiments using an pRP4 derivative as a helper plasmid confirm that pMC2 has a functional mobilization unit. PCR was used to detect genetic elements found on pMC2 in DNA extracted from manure amended soil.

Conclusions: The pMC2 plasmid has a tetracycline-resistant core and has acquired additional resistance genes by insertion of an accessory region (12 762 bp) containing macrolide, mercury and chromium resistance genes, which was inserted between the truncated DDE motifs within the Tn903/IS102 mobile element.

Significance and Impact of the Study: Liquid swine manure used for manure spreading contains multiple antibiotic resistance plasmids that can be detected in soil following manure application.


An evolutionary perspective on plasmid lifestyle modes

Plasmid evolution reviewed from the perspective of plasmids.

Mobility, stability, and indispensability largely determine plasmid lifestyles.

Changes in plasmid traits facilitate transitions into new lifestyle modes.

The intensity of traits and their interaction defines the plasmid lifestyle mode.

Plasmids are extra-chromosomal genetic elements whose ecology and evolution depend on their genetic repertoire and interaction with the host. We review the events that lead to transitions between plasmid lifestyle modes – invasion, host range, plasmid persistence and adaptation – from a plasmid perspective. Plasmid lifestyle is determined by various traits, including mobility, stability and indispensability that vary in their magnitude. Transitions between the plasmid lifestyles, invasion, host range, plasmid persistence and adpatation, are caused by the interplay between plasmid traits and host biology. Mobility and indispensability are important in plasmid ecology, whereas plasmid stability is more relevant for long-term plasmid evolution. In transitioning into additional chromosomes plasmids loose their independence and enter the host lineage. Though plasmids are confined to their hosts, their evolution may be independent of prokaryotic chromosomes.


International Society for Plasmid Biology

Sunday 18th September: Arrivals

Evening: Welcome Reception, Clare College

Monday 19th September

Keynote address – Kenn Gerdes (University of Copenhagen, Denmark) : From plasmid maintenance to bacterial multi-drug tolerance.

Craig McLean (University of Oxford, UK) : Natural selection, gene expression and the emergence of plasmid stability in bacterial populations.

Alessandra Carattoli (Instituto Superiore di Sanita, Rome, Italy ) : The impact of plasmid classification on the epidemiology of antimicrobial resistance.

Neville Firth (University of Sydney, Australia) : Replication of pSK41-like conjugative plasmids.

Igor Konieczny (University of Gdansk, Poland) : Rep monomers and dimers, chaperones and proteases in control of iteron-containing plasmid replication.

Rafael Giraldo (Centro de Investigaciones Biológicas CSIC, Madrid, Spain) : Oligomeric Amyloid Assemblies of an Origin-Bound RepA Initiator Inhibit Plasmid DNA Replication.

Bruno Gonzalez-Zorn (Universidad Complutense de Madrid, Spain) : ColE1 plasmids reloaded: Novel aspects of host adaptation and coexistence.

Steven Hancock (University of Queensland, Australia) : Identification of IncA/C Replication and Maintenance Genes and Development of a Plasmid Multi-Locus Typing Scheme.

Jacques Oberto (Université Paris-Sud, France): Plasmid-driven evolution of Archaea Thermococcales genomes.

Franck Pasta (Paul Sabatier University, Toulouse, France) : Coordination and self-control for maintaining the four replicons of Burkholderia cenocepacia.

Francois Cornet (Paul Sabatier University, Toulouse, France ): Domestication of Xer recombination is an early step in chromosome genesis from plasmids ancestors.

Didier Mazel (Institut Pasteur, Paris) : Replication of the plasmid-related chromosome 2 is subservient to the replication of chromosome 1 by a novel mechanism in Vibrio cholerae.

Sally Partridge (University of Sydney, Australia) : Annotation and analysis of plasmid sequences.

Alan Grossman (Massachusetts Institute of Technology, USA) : Plasmid-like properties of integrative and conjugative elements.

Darek Bartosik (University of Warsaw, Poland): Essential extrachromosomal replicons of Paracoccus spp. (Alphaproteobacteria).

Ethan Wyrsch (University of Technology, Sydney, Australia) : WGS analysis of Australian porcine E. coli.

Tuesday 20th September

Michael Brockhurst (University of York, UK) : Solving the plasmid paradox.

Eva Top (University of Idaho, USA) : The effect of biofilm growth on the evolution of plasmid persistence and permissiveness.

Jan Kreft (University of Birmingham, UK) : Evolution of broad v narrow host range plasmids.

Martin Werisch (Technische Universität Dresden, Germany) : Adaptation and its relevance for plasmid maintenance.

Tatiana Dimitriu (University of Exeter, UK): Selection of plasmid transfer through indirect public goods benefits.

Daniela Barilla (University of York, UK) : Borrowing building blocks from bacteria and eukarya: a three-component DNA segregation machine in archaea.

Jean-Yves Bouet (Paul Sabatier University, Toulouse, France) : Mechanism of plasmid F segregation: what we learn from super-resolution microscopy?

Juan Alonso (National Centre for Biotechnology, CSIC, Madrid, Spain) : Molecular anatomy of the ParA-ParB partition complex within the nucleoid.

Punting on the Cam the Fitzwilliam Art Gallery/Museum the Botanical Gardens

Barbara Funnell ( University of Toronto , Canada): Timing is key: kinetics of ParB-ParA-DNA interactions during plasmid partition.

Yong Wang (University of Arkansas, USA ): To Cluster or Not to Cluster: New Insight into the Segregation Mechanism of High-Copy Bacterial Plasmids using Quantitative Localization Microscopy.

Alexander Harms (University of Copenhagen, Denmark) : Interbacterial effector proteins as an evolutionary missing link between toxin-antitoxin modules, conjugative DNA transfer, and host-targeting type IV secretion.

Wednesday 21st September

Fernando de la Cruz Calahorra (University of Cantabria, Santander, Spain) : Extent and control of plasmid conjugation: dynamics, interactions and barriers.

Gabriel Waksman (Birkbeck and University College London, UK) : Structural and molecular biology of bacterial secretion systems

Elizabeth Grohmann (Beuth University of Applied Sciences, Berlin, Germany) : Key players of an Enterococcus Type IV Secretion System.

Jacques Mahillon (Université Catholique de Louvain, Belgium) : Deciphering pXO16 complex biology .

Lina Thoma (University of Tubingen, Germany) : Intramycelial plasmid spreading during conjugative DNA transfer in Streptomyces.

Julian Rood (Monash University, Australia) : Structural and functional analysis of the conjugation system from Clostridium perfringens.

Guenther Koraimann (University of Graz, Austria) : A non-canonical RNAP drives F-like tra-operon expression.

Walter Keller (University of Graz, Austria ): TraN, a regulator of conjugative DNA-transfer

Christophe Merlin (University of Lorraine, France) : Exploring the effect of antibiotics at sub-inhibitory concentrations on the activity of promoters involved in the mobility of mobile genetic elements.

Susanna Brom (Centre for Genome Science, Cuernavaca, Mexico) : Does the nodulation process provide an adequate environment for rhizobial conjugation?

Matxalen Llosa (Universidad de Cantabria, Spain) : The conjugative relaxase TrwC delivers DNA into human cells and promotes its integration in the human genome.

Robert Moran (University of Sydney, Australia): A cryptic, RC plasmid from a commensal Escherichia coli has oriT sites and is mobilised by a B/O plasmid.

Hideaki Nojiri (University of Tokyo, Japan): Plasmid-borne nucleoid associated proteins, key factors determining host cell physiology and fitness.

Konny Smalla (Julius Kühn-Institut Federal Research Centre, Braunschweig, Germany) : Plant species dependent enrichment of IncP-1 in the rhizosphere.

Ben Raymond (Imperial College, London, UK) : Why here? Why there? Ecological and genetic determinants of plasmid distribution in Escherichia coli and Bacillus cereus s.l.

Thibault Stalder (University of Idaho, USA) : Spread of mobile genetic elements in agricultural soil as affected by fertilization practices.

ISPB Biannual General Meeting

Thursday 22nd September

Liz Wellington (University of Warwick, UK) : Plasmids and antibiotic resistance: how both persist in the environment.

Bart Smets (Technical University of Denmark, Lyngby, Denmark) : Measuring community-wide conjugal plasmid permissiveness.

Hannah Jordt (University of Idaho, USA ) Use it AND lose it: Alternating selection promotes horizontal gene transfer

Yohann Lacotte (University of Limoges, France) : Fitness cost of class 1 integrons in E. coli.

Ellie Harrison (University of York, UK) : Plasmid carriage can limit antagonistic bacteria – phage coevolution.

George Chaconas (University of Calgary, Canada) : Plasmid-encoded antigenic variation system of the Lyme disease spirochete.

Alvaro San Milan (Ramón y Cajal University Hospital, Madrid, Spain) : Multicopy plasmids potentiate the evolution of antibiotic resistance genes in bacteria.

Anna Shepherd (University of Oxford, UK) : Tn4401 mobility leads to dynamic plasmid structures in a hospital outbreak.

Mark Toleman (University of Cardiff, UK) : Integrative Conjugative Elements and antibiotic resistance. (Title to be confirmed, TBC)

Steve Djordjevic (University of Technology, Sydney, Australia ): Whole Genome Sequence analysis of multiple antibiotic resistant Escherichia coli from humans and food animals.

Piklu Roy Chowdhury (University of Technology, Sydney, Australia) : Inducing IS26 mediated gene-shuffling in complex multi-drug resistance loci.

Makoto Kuroda (National Institute of Infectious Diseases, Japan) : GenEpid-J: an integrated database of pathogen genomics and epidemiology focused on plasmids involving in the antimicrobial resistance.

Jon Iredell (University of Sydney, Australia) : In vivo plasmid husbandry – a bedside perspective.

Chris Thomas (University of Birmingham, UK) : IncP plasmids as vectors for plasmid curing.

Keynote address – Dhruba Chattoraj (NIH, Bethesda, USA): Random vs. cell cycle-regulated replication initiation in bacteria: Insights from studying a plasmid-like replicon in Vibrio cholerae.

Evening: Conference Dinner, Gonville and Caius College.

Friday 23rd September: Departures


  • Statistics and Probability
  • Modeling and Simulation
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Agricultural and Biological Sciences(all)
  • Applied Mathematics
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In: Mathematical Biosciences , Vol. 193, No. 2, 01.02.2005, p. 183-204.

Research output : Contribution to journal › Article › peer-review

T1 - Biofilms and the plasmid maintenance question

N2 - Can a conjugative plasmid encoding enhanced biofilm forming abilities for its bacterial host facilitate the persistence of the plasmid in a bacterial population despite conferring diminished growth rate and segregative plasmid loss on its bearers? We construct a mathematical model in a chemostat and in a plug flow environment to answer this question. Explicit conditions for an affirmative answer are derived. Numerical simulations support the conclusion.

AB - Can a conjugative plasmid encoding enhanced biofilm forming abilities for its bacterial host facilitate the persistence of the plasmid in a bacterial population despite conferring diminished growth rate and segregative plasmid loss on its bearers? We construct a mathematical model in a chemostat and in a plug flow environment to answer this question. Explicit conditions for an affirmative answer are derived. Numerical simulations support the conclusion.


Acknowledgements

We thank Kees Veldman en Ben Wit for providing the resistant strains and Sarah Duxbury for assistance in identifying appropriate proper analysis programs and all three for stimulating discussions. The MSc students Lisa Daniels, Sidra Kashif, Selin Alpagot and Eva Kozanli performed plasmid isolations and transfer experiments as part of their degree requirements. We thank Ali Farmand Kabir Noori for programming software to circularize plasmids and assistance with data analysis using AI methodologies.


Plasmid maintenance - Biology

International Symposium on Plasmid Biology 2022

We are pleased to announce you that new dates have been released for the symposium.

The Plasmid Biology 2022 will take place on September 18 to 23, 2022 at the University of Toulouse III- Paul Sabatier, France. The conference is the biennial International Symposium of the International Society for Plasmid Biology and other Mobile Genetic Elements (ISPB).

IMPORTANT ANNOUNCEMENT RELATIVE TO COVID-19

In the context of the Covid-19 worldwide expansion, the organizers and the Plasmid Society board have decided to postpone the ISPB2020 meeting. The Plasmid Biology 2022 will take place on September 18 to 23, 2022. We will keep you informed about any other change. We wish you all the best during this epidemic period.

The conference will cover all aspects of plasmids and mobile genetic elements (MGEs), including replication and maintenance, horizontal transfer, genomics and systems biology, synthetic biology, industrial, agricultural and medical biotechnology, evolution, ecology, epidemiology, and the role of plasmids/MGEs in bacterial pathogenesis.

The conference will start on Sunday evening, September 18 th with a keynote lecture on &ldquoBacterial secretion systems&rdquo given by Eric Cascales from the CNRS, LISM in Marseille, France. The closing lecture on September 23 rd will be given by the President of the ISPB, Barbara Funnell, from the University of Toronto.

We will showcase diverse topics and presenters. Graduate students, postdoctoral fellows and early career scientists are particularly encouraged to attend and contribute to the meeting. Oral sessions will feature invited talks by leading international researchers and presentations chosen from submitted abstracts. The poster sessions will give all attendees an opportunity to present their latest findings, with ample time for informal discussions, socializing, and networking

We look forward to welcoming you to Toulouse in September 2022: come to learn about exciting science related to plasmids and other mobile elements and to explore the beautiful city of Toulouse.

The conference is organised by a local organising committee of the "Laboratoire de Microbiologie et Génétique Moléculaires" (LMGM) - Centre for Integrative Biology (CBI) of Toulouse: Dr Jean-Yves Bouet and Dr François Cornet, PI at the LMGM and Dr Isabelle Saves, head of international Cooperation at the CBI-Toulouse. Contact: [email protected]

The Plasmid Biology 2020 s cientific committee is composed of Pr Daniella Barilla (University of York, UK), Pr Marie-Cécile Ploy (University of Limoges, France), Pr Fernando de la Cruz (University of Santander, Spain) and Pr Didier Mazel (Pasteur Institute, Paris, France).


Plasmid maintenance - Biology

We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin's mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, a short half-life of the protein toxin is also beneficial to the function of the toxin-antitoxin system. In addition, we study a therapeutic scenario in which a competitor mRNA is introduced to sequester the sRNA antitoxin, causing the toxic protein to be expressed.