Measuring the Plasmid Copy Number of Single E. Coli by Digital PCR: Are Plasmids Partitioned in Clusters during Cell Division?

AbstractPathogenic Yersinia spp. depend on the activity of a potent virulence plasmid-encoded ysc/yop type 3 secretion system (T3SS) to colonize hosts and cause disease. It was recently shown that Y. pseudotuberculosis up-regulates the virulence plasmid copy number (PCN) during infection and the resulting elevated gene dose of plasmid-encoded T3SS genes is essential for virulence. When and how this novel regulatory mechanism is deployed and regulates the replication of the virulence plasmid during infection is unknown. In the current study, we applied droplet digital PCR (ddPCR) to investigate the dynamics of Y. pseudotuberculosis virulence PCN variations and growth rates in infected mouse organs. We demonstrated that both PCN and growth varied in different tissues and over time throughout the course of infection, indicating that the bacteria adapted to discrete microenvironments during infection. The PCN was highest in Peyer’s Patches and caecum during the clonal invasive phase of the infection, while the fastest growth rates were found in the draining mesenteric lymph nodes. In deeper, systemic organs, the PCN was lower and more modest growth rates were recorded. Our study indicates that increased gene dosage of the plasmid-encoded T3SS genes is most important early in the infection during invasion of the host. The described ddPCR approach will greatly simplify analyses of PCN, growth dynamics, and bacterial loads in infected tissues, and will be readily applicable to other infection models.ImportanceStudying pathogenic bacteria proliferating inside infected hosts is challenging using traditional methods, especially the transit and reversible genetic events. The bacteria are effectively diluted by the overwhelming number of host cells present in infected tissues. Using an innovative droplet digital PCR (ddPCR) approach, we have determined the virulence plasmid copy number (PCN) variations and growth rates of Yersinia during the course of infection in a mouse model. Here, we show that both the virulence plasmid copy number and bacterial growth rates display spatiotemporal variations in mice during infection. We demonstrate that the peak-to-trough ratio can be used as a proxy for determining the growth rate of invasive bacterial pathogen during infection, and ddPCR as the method of choice for quantifying DNA in host-pathogen interaction context. This proof-of-concept ddPCR approach can be easily applied for any bacterial pathogens and any infection models, for analysis of PCN, growth dynamics and bacterial loads.

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Spatiotemporal Variations in Growth Rate and Virulence Plasmid Copy Number during Yersinia pseudotuberculosis Infection

Infection and Immunity ◽

10.1128/iai.00710-20 ◽

2021 ◽

Author(s):

Stephan Schneiders ◽

Tifaine Hechard ◽

Tomas Edgren ◽

Kemal Avican ◽

Maria Fällman ◽

...

Keyword(s):

Growth Rates ◽

Copy Number ◽

Yersinia Pseudotuberculosis ◽

Gene Dosage ◽

Growth Dynamics ◽

Digital Pcr ◽

Plasmid Copy Number ◽

Virulence Plasmid ◽

Mesenteric Lymph Nodes ◽

Plasmid Copy

Pathogenic Yersinia spp. depend on the activity of a potent virulence plasmid-encoded ysc/yop type 3 secretion system (T3SS) to colonize hosts and cause disease. It was recently shown that Y. pseudotuberculosis up-regulates the virulence plasmid copy number (PCN) during infection and the resulting elevated gene dose of plasmid-encoded T3SS genes is essential for virulence. When and how this novel regulatory mechanism is deployed and regulates the replication of the virulence plasmid during infection is unknown. In the current study, we applied droplet digital PCR (ddPCR) to investigate the dynamics of Y. pseudotuberculosis virulence PCN variations and growth rates in infected mouse organs. We demonstrated that both PCN and growth varied in different tissues and over time throughout the course of infection, indicating that the bacteria adapted to discrete microenvironments during infection. The PCN was highest in Peyer’s Patches and caecum during the clonal invasive phase of the infection, while the fastest growth rates were found in the draining mesenteric lymph nodes. In deeper, systemic organs, the PCN was lower and more modest growth rates were recorded. Our study indicates that increased gene dosage of the plasmid-encoded T3SS genes is most important early in the infection during invasion of the host. The described ddPCR approach will greatly simplify analyses of PCN, growth dynamics, and bacterial loads in infected tissues, and will be readily applicable to other infection models.

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Restoration of wild-type motility to flagellin-knockout Escherichia coli

10.1101/700492 ◽

2019 ◽

Author(s):

Nicholas M. Thomson ◽

Mark J. Pallen

Keyword(s):

Escherichia Coli ◽

Copy Number ◽

Plasmid Copy Number ◽

Major Constituent ◽

Strongly Correlated ◽

Wild Type ◽

Plasmid Copy ◽

Inducible Promoters ◽

E Coli ◽

Flagellar Filament

AbstractFlagellin is the major constituent of the flagellar filament and faithful restoration of wild-type motility to flagellin mutants may be beneficial for studies of flagellar biology and biotechnological exploitation of the flagellar system. Therefore, we explored the restoration of motility by flagellin expressed from a variety of combinations of promoter, plasmid copy number and induction strength. Motility was only partially restored using the tightly regulated rhamnose promoter, but wild-type motility was achieved with the T5 promoter, which, although leaky, allowed titration of induction strength. Motility was little affected by plasmid copy number when dependent on inducible promoters. However, plasmid copy number was important when expression was controlled by the native E. coli flagellin promoter. Motility was poorly correlated with flagellin transcription levels, but strongly correlated with the amount of flagellin associated with the flagellar filament, suggesting that excess monomers are either not exported or not assembled into filaments. This study provides a useful reference for further studies of flagellar function and a simple blueprint for similar studies with other proteins.

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Accurate Determination of Plasmid Copy Number of Flow-Sorted Cells using Droplet Digital PCR

Analytical Chemistry ◽

10.1021/ac501118v ◽

2014 ◽

Vol 86 (12) ◽

pp. 5969-5976 ◽

Cited By ~ 32

Author(s):

Michael Jahn ◽

Carsten Vorpahl ◽

Dominique Türkowsky ◽

Martin Lindmeyer ◽

Bruno Bühler ◽

...

Keyword(s):

Copy Number ◽

Accurate Determination ◽

Digital Pcr ◽

Plasmid Copy Number ◽

Droplet Digital Pcr ◽

Plasmid Copy

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Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

PLoS ONE ◽

10.1371/journal.pone.0169846 ◽

2017 ◽

Vol 12 (1) ◽

pp. e0169846 ◽

Cited By ~ 8

Author(s):

Magdalena Plotka ◽

Mateusz Wozniak ◽

Tadeusz Kaczorowski

Keyword(s):

Copy Number ◽

Digital Pcr ◽

Plasmid Copy Number ◽

Droplet Digital Pcr ◽

Plasmid Copy ◽

Single Colour

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Segregational drift and the interplay between plasmid copy number and evolvability

10.1101/369579 ◽

2018 ◽

Cited By ~ 1

Author(s):

Judith Ilhan ◽

Anne Kupczok ◽

Christian Woehle ◽

Tanita Wein ◽

Nils F. Hülter ◽

...

Keyword(s):

Cell Division ◽

Copy Number ◽

Plasmid Copy Number ◽

Fixation Time ◽

Multicopy Plasmid ◽

Plasmid Copy ◽

Plasmid Evolution ◽

Host Chromosome ◽

Daughter Cells ◽

Multiple Copies

AbstractThe ubiquity of plasmids in all prokaryotic phyla and habitats and their ability to transfer between cells marks them as prominent constituents of prokaryotic genomes. Many plasmids are found in their host cell in multiple copies. This leads to an increased mutational supply of plasmid-encoded genes and genetically heterogeneous plasmid genomes. Nonetheless, the segregation of plasmid copies into daughter cells during cell division is considered to occur in the absence of selection on the plasmid alleles. We investigate the implications of random genetic drift of multicopy plasmids during cell division – termed here segregational drift – to plasmid evolution. Performing experimental evolution of low- and high-copy non-mobile plasmids in Escherichia coli, we find that the evolutionary rate of multicopy plasmids does not reflect the increased mutational supply expected according to their copy number. In addition, simulated evolution of multicopy plasmid alleles demonstrates that segregational drift leads to increased loss frequency and extended fixation time of plasmid mutations in comparison to haploid chromosomes. Furthermore, an examination of the experimentally evolved hosts reveals a significant impact of the plasmid type on the host chromosome evolution. Our study demonstrates that segregational drift of multicopy plasmids interferes with the retention and fixation of novel plasmid variants. Depending on the selection pressure on newly emerging variants, plasmid genomes may evolve slower than haploid chromosomes, regardless of their higher mutational supply. We suggest that plasmid copy number is an important determinant of plasmid evolvability due to the manifestation of segregational drift.

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A model for predicting plasmid copy number and genetic stability in E. coli

Trends in Biotechnology ◽

10.1016/0167-7799(87)90097-7 ◽

1987 ◽

Vol 5 (9) ◽

pp. 245

Keyword(s):

Genetic Stability ◽

Copy Number ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

E Coli

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Escherichia coli O157:H7 Strains Isolated from High-Event Period Beef Contamination Have Strong Biofilm-Forming Ability and Low Sanitizer Susceptibility, Which Are Associated with High pO157 Plasmid Copy Number

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-113 ◽

2016 ◽

Vol 79 (11) ◽

pp. 1875-1883 ◽

Cited By ~ 8

Author(s):

RONG WANG ◽

BRANDON E. LUEDTKE ◽

JOSEPH M. BOSILEVAC ◽

JOHN W. SCHMIDT ◽

NORASAK KALCHAYANAND ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Biofilm Formation ◽

Copy Number ◽

Plasmid Copy Number ◽

Bacterial Biofilm ◽

Escherichia Coli O157 ◽

Plasmid Copy ◽

E Coli ◽

High Event

ABSTRACT In the meat industry, a high-event period (HEP) is defined as a time period when beef processing establishments experience an increased occurrence of product contamination by Escherichia coli O157:H7. Our previous studies suggested that bacterial biofilm formation and sanitizer resistance might contribute to HEPs. We conducted the present study to further characterize E. coli O157:H7 strains isolated during HEPs for their potential to cause contamination and to investigate the genetic basis for their strong biofilm-forming ability and high sanitizer resistance. Our results show that, compared with the E. coli O157:H7 diversity control panel strains, the HEP strains had a significantly higher biofilm-forming ability on contact surfaces and a lower susceptibility to common sanitizers. No difference in the presence of disinfectant-resistant genes or the prevalence of antibiotic resistance was observed between the HEP and control strains. However, the HEP strains retained significantly higher copy numbers of the pO157 plasmid. A positive correlation was observed among a strain's high plasmid copy number, strong biofilm-forming ability, low sanitizer susceptibility, and high survival and recovery capability after sanitization, suggesting that these specific phenotypes could be either directly correlated to gene expression on the pO157 plasmid or indirectly regulated via chromosomal gene expression influenced by the presence of the plasmid. Our data highlight the potential risk of biofilm formation and sanitizer resistance in HEP contamination by E. coli O157:H7, and our results call for increased attention to proper and effective sanitization practices in meat processing facilities.

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