scholarly journals Piscirickettsia salmonis Cryptic Plasmids: Source of Mobile DNA and Virulence Factors

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 269
Author(s):  
Javiera Ortiz-Severín ◽  
Dante Travisany ◽  
Alejandro Maass ◽  
Francisco P. Chávez ◽  
Verónica Cambiazo
Keyword(s):  
Virulence Factors ◽  
Sequence Similarity ◽  
Functional Characterization ◽  
Plasmid Copy Number ◽  
Mobile Dna ◽  
Plasmid Copy ◽  
Infection Models ◽  
Bacterial Fitness ◽  
Piscirickettsia Salmonis ◽  
Cryptic Plasmids

Four large cryptic plasmids were identified in the salmon pathogen Piscirickettsia salmonis reference strain LF-89. These plasmids appeared highly novel, with less than 7% nucleotidic identity to the nr plasmid database. Plasmid copy number analysis revealed that they are harbored in chromosome equivalent ratios. In addition to plasmid-related genes (plasmidial autonomous replication, partitioning, maintenance, and mobilization genes), mobile genetic elements such as transposases, integrases, and prophage sequences were also identified in P. salmonis plasmids. However, bacterial lysis was not observed upon the induction of prophages. A total of twelve putative virulence factors (VFs) were identified, in addition to two global transcriptional regulators, the widely conserved CsrA protein and the regulator Crp/Fnr. Eleven of the putative VFs were overexpressed during infection in two salmon-derived cellular infection models, supporting their role as VFs. The ubiquity of these plasmids was also confirmed by sequence similarity in the genomes of other P. salmonis strains. The ontology of P. salmonis plasmids suggests a role in bacterial fitness and adaptation to the environment as they encode proteins related to mobilization, nutrient transport and utilization, and bacterial virulence. Further functional characterization of P. salmonis plasmids may improve our knowledge regarding virulence and mobile elements in this intracellular pathogen.

scholarly journals Cloning and functional characterization of Komagataella phaffii centromeres by a color-based plasmid stability assay

2018 ◽  
Author(s):  
Luiza Cesca Piva ◽  
Janice Lisboa De Marco ◽  
Lidia Maria Pepe de Moraes ◽  
Viviane Castelo Branco Reis ◽  
Fernando Araripe Gonçalves Torres
Keyword(s):  
Copy Number ◽  
Plasmid Stability ◽  
Functional Characterization ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Komagataella Phaffii ◽  
High Transformation ◽  
Colony Color ◽  
Stable Vectors

AbstractThe yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work we designed a color-based genetic assay to investigate genetic stability in K. phaffii. Plasmids bearing K. phaffii centromeres and the ADE3 marker were evaluated in terms of mitotic stability in an ade2/ade3 auxotrophic strain which allows plasmid screening through colony color. Plasmid copy number was verified through qPCR. Our results confirmed that the centromeric plasmids were maintained at low copy number as a result of typical chromosome-like segregation during cell division. These features, combined with high transformation efficiency and in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.

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

2020 ◽  
Author(s):  
Stephan Schneiders ◽  
Tifaine Hechard ◽  
Tomas Edgren ◽  
Kemal Avican ◽  
Maria Fällman ◽  
...  
Keyword(s):  
Growth Rates ◽  
Copy Number ◽  
Growth Dynamics ◽  
Digital Pcr ◽  
Plasmid Copy Number ◽  
Droplet Digital Pcr ◽  
Virulence Plasmid ◽  
Spatiotemporal Variations ◽  
Plasmid Copy ◽  
Infection Models

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.

Bacterial fitness and plasmid loss: the importance of culture conditions and plasmid size

1998 ◽  
Vol 44 (4) ◽  
pp. 351-355 ◽  
Author(s):  
M Alex Smith ◽  
Michael J Bidochka
Keyword(s):  
Copy Number ◽  
Luria Broth ◽  
Plasmid Copy Number ◽  
Culture Conditions ◽  
Lag Phase ◽  
Multicopy Plasmid ◽  
Plasmid Copy ◽  
Plasmid Loss ◽  
Bacterial Fitness ◽  
Plasmid Size

Several pBluescript-derived plasmids of various sizes were constructed to study the effects of multicopy plasmid size on bacterial fitness and plasmid loss. Transformed and untransformed bacterial clones were grown in media with or without ampicillin. Bacterial fitness (measured by growth rate), plasmid presence or absence, and plasmid copy number were assessed during successive subculturings. In selective media (minimal medium or Luria Broth plus ampicillin), the clone transformed with the largest plasmid (pBluescript with a 9000-bp insert) had a significantly longer lag phase than all other clones. In nonselective media the rate of plasmid loss during successive subculturings was greatest in the clone with the largest insert. The clone with the largest insert displayed a lower plasmid copy number than clones with a small insert or no insert at all. Plasmid loss in the form of segregational instability and plasmid copy number reduction in nonselective environments are important to the understanding of the evolution of the bacteria-plasmid associations and the appreciation of the potential for altering the genetic properties of a clone maintained or subcultured on a standard medium.Key words: pBluescript, plasmid, stress, fitness, starvation.

scholarly journals Small-Plasmid-Mediated Antibiotic Resistance Is Enhanced by Increases in Plasmid Copy Number and Bacterial Fitness

2015 ◽  
Vol 59 (6) ◽  
pp. 3335-3341 ◽  
Author(s):  
Alvaro San Millan ◽  
Alfonso Santos-Lopez ◽  
Rafael Ortega-Huedo ◽  
Cristina Bernabe-Balas ◽  
Sean P. Kennedy ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Bacterial Host ◽  
Plasmid Copy ◽  
Small Plasmid ◽  
Content Type ◽  
Competitive Disadvantage ◽  
Bacterial Fitness ◽  
Horizontal Spread

ABSTRACTPlasmids play a key role in the horizontal spread of antibiotic resistance determinants among bacterial pathogens. When an antibiotic resistance plasmid arrives in a new bacterial host, it produces a fitness cost, causing a competitive disadvantage for the plasmid-bearing bacterium in the absence of antibiotics. On the other hand, in the presence of antibiotics, the plasmid promotes the survival of the clone. The adaptations experienced by plasmid and bacterium in the presence of antibiotics during the first generations of coexistence will be crucial for the progress of the infection and the maintenance of plasmid-mediated resistance once the treatment is over. Here we developed a model system using the human pathogenHaemophilus influenzaecarrying the small plasmid pB1000 conferring resistance to β-lactam antibiotics to investigate host and plasmid adaptations in the course of a simulated ampicillin therapy. Our results proved that plasmid-bearing clones compensated for the fitness disadvantage during the first 100 generations of plasmid-host adaptation. In addition, ampicillin treatment was associated with an increase in pB1000 copy number. The augmentation in both bacterial fitness and plasmid copy number gave rise toH. influenzaepopulations with higher ampicillin resistance levels. In conclusion, we show here that the modulations in bacterial fitness and plasmid copy number help a plasmid-bearing bacterium to adapt during antibiotic therapy, promoting both the survival of the host and the spread of the plasmid.

scholarly journals Chlamydia trachomatis intra-bacterial and total plasmid copy number in clinical urogenital samples

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
J. A. M. C. Dirks ◽  
K. Janssen ◽  
C. J. P. A. Hoebe ◽  
T. H. B. Geelen ◽  
M. Lucchesi ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Diagnostic Value ◽  
Plasmid Copy Number ◽  
Extracellular Dna ◽  
Chromosomal Dna ◽  
Plasmid Copy ◽  
Clinical Sensitivity ◽  
Copy Numbers ◽  
Vaginal Swabs ◽  
And Gender

AbstractChlamydia trachomatis (CT) increases its plasmid numbers when stressed, as occurs in clinical trachoma samples. Most CT tests target the plasmid to increase the test sensitivity, but some only target the chromosome. We investigated clinical urogenital samples for total plasmid copy numbers to assess its diagnostic value and intra-bacterial plasmid copy numbers to assess its natural variation. Both plasmid and chromosome copies were quantified using qPCR, and the plasmid:chromosome ratio (PCr) calculated in two cohorts: (1) 383 urogenital samples for the total PCR (tPCr), and (2) 42 vaginal swabs, with one half treated with propium-monoazide (PMA) to prevent the quantification of extracellular DNA and the other half untreated to allow for both tPCr and intra-bacterial PCr (iPCr) quantification. Mann–Whitney U tests compared PCr between samples, in relation to age and gender. Cohort 1: tPCr varied greatly (1–677, median 16). Median tPCr was significantly higher in urines than vaginal swabs (32 vs. 11, p < 0.001). Cohort 2: iPCr was more stable than tPCr (range 0.1–3 vs. 1–11). To conclude, tPCr in urogenital samples was much more variable than previously described. Transport time and temperature influences DNA degradation, impacting chromosomal DNA more than plasmids and urine more than vaginal samples. Data supports a plasmid target in CT screening assays to increase clinical sensitivity.

scholarly journals Plasmid Replicons for the Production of Pharmaceutical-Grade pDNA, Proteins and Antigens by Lactococcus lactis Cell Factories

2021 ◽  
Vol 22 (3) ◽  
pp. 1379
Author(s):  
Sofia O.D. Duarte ◽  
Gabriel A. Monteiro
Keyword(s):  
Lactococcus Lactis ◽  
Recombinant Proteins ◽  
Plasmid Copy Number ◽  
Fermented Food ◽  
Added Value ◽  
Regulatory Sequences ◽  
Plasmid Copy ◽  
Mucosal Vaccination ◽  
Cell Factories ◽  
Plasmid Replicons

The Lactococcus lactis bacterium found in different natural environments is traditionally associated with the fermented food industry. But recently, its applications have been spreading to the pharmaceutical industry, which has exploited its probiotic characteristics and is moving towards its use as cell factories for the production of added-value recombinant proteins and plasmid DNA (pDNA) for DNA vaccination, as a safer and industrially profitable alternative to the traditional Escherichia coli host. Additionally, due to its food-grade and generally recognized safe status, there have been an increasing number of studies about its use in live mucosal vaccination. In this review, we critically systematize the plasmid replicons available for the production of pharmaceutical-grade pDNA and recombinant proteins by L. lactis. A plasmid vector is an easily customized component when the goal is to engineer bacteria in order to produce a heterologous compound in industrially significant amounts, as an alternative to genomic DNA modifications. The additional burden to the cell depends on plasmid copy number and on the expression level, targeting location and type of protein expressed. For live mucosal vaccination applications, besides the presence of the necessary regulatory sequences, it is imperative that cells produce the antigen of interest in sufficient yields. The cell wall anchored antigens had shown more promising results in live mucosal vaccination studies, when compared with intracellular or secreted antigens. On the other side, engineering L. lactis to express membrane proteins, especially if they have a eukaryotic background, increases the overall cellular burden. The different alternative replicons for live mucosal vaccination, using L. lactis as the DNA vaccine carrier or the antigen producer, are critically reviewed, as a starting platform to choose or engineer the best vector for each application.

scholarly journals Segregational Drift and the Interplay between Plasmid Copy Number and Evolvability

2018 ◽  
Vol 36 (3) ◽  
pp. 472-486 ◽  
Author(s):  
Judith Ilhan ◽  
Anne Kupczok ◽  
Christian Woehle ◽  
Tanita Wein ◽  
Nils F Hülter ◽  
...  
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy

scholarly journals RNA polyadenylation and its consequences in prokaryotes

2018 ◽  
Vol 373 (1762) ◽  
pp. 20180166 ◽  
Author(s):  
Eliane Hajnsdorf ◽  
Vladimir R. Kaberdin
Keyword(s):  
Gene Expression ◽  
State Level ◽  
Rna Degradation ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Protein Coding ◽  
Mrna Metabolism ◽  
Isolation And Characterization ◽  
Polymerase I ◽  
The Impact

Post-transcriptional addition of poly(A) tails to the 3′ end of RNA is one of the fundamental events controlling the functionality and fate of RNA in all kingdoms of life. Although an enzyme with poly(A)-adding activity was discovered in Escherichia coli more than 50 years ago, its existence and role in prokaryotic RNA metabolism were neglected for many years. As a result, it was not until 1992 that E. coli poly(A) polymerase I was purified to homogeneity and its gene was finally identified. Further work revealed that, similar to its role in surveillance of aberrant nuclear RNAs of eukaryotes, the addition of poly(A) tails often destabilizes prokaryotic RNAs and their decay intermediates, thus facilitating RNA turnover. Moreover, numerous studies carried out over the last three decades have shown that polyadenylation greatly contributes to the control of prokaryotic gene expression by affecting the steady-state level of diverse protein-coding and non-coding transcripts including antisense RNAs involved in plasmid copy number control, expression of toxin–antitoxin systems and bacteriophage development. Here, we review the main findings related to the discovery of polyadenylation in prokaryotes, isolation, and characterization and regulation of bacterial poly(A)-adding activities, and discuss the impact of polyadenylation on prokaryotic mRNA metabolism and gene expression. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

scholarly journals Plasmid copy number noise in monoclonal populations of bacteria

2010 ◽  
Vol 81 (1) ◽  
Author(s):  
Jérôme Wong Ng ◽  
Didier Chatenay ◽  
Jérôme Robert ◽  
Michael Guy Poirier
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy
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