Genomic Characterization and Copy Number Variation ofBacillus anthracisPlasmids pXO1 and pXO2 in a Historical Collection of 412 Strains

ABSTRACTBacillus anthracisplasmids pXO1 and pXO2 carry the main virulence factors responsible for anthrax. However, the extent of copy number variation within the species and how the plasmids are related to pXO1/pXO2-like plasmids in other species of theBacillus cereussensu latogroup remain unclear. To gain new insights into these issues, we sequenced 412B. anthracisstrains representing the total phylogenetic and ecological diversity of the species. Our results revealed thatB. anthracisgenomes carried, on average, 3.86 and 2.29 copies of pXO1 and pXO2, respectively, and also revealed a positive linear correlation between the copy numbers of pXO1 and pXO2. No correlation between the plasmid copy number and the phylogenetic relatedness of the strains was observed. However, genomes of strains isolated from animal tissues generally maintained a higher plasmid copy number than genomes of strains from environmental sources (P< 0.05 [Welch two-sample t test]). Comparisons againstB. cereusgenomes carrying complete or partial pXO1-like and pXO2-like plasmids showed that the plasmid-based phylogeny recapitulated that of the main chromosome, indicating limited plasmid horizontal transfer between or within these species. Comparisons of gene content revealed a closed pXO1 and pXO2 pangenome; e.g., plasmids encode <8 unique genes, on average, and a single large fragment deletion of pXO1 in oneB. anthracisstrain (2000031682) was detected. Collectively, our results provide a more complete view of the genomic diversity ofB. anthracisplasmids, their copy number variation, and the virulence potential of otherBacillusspecies carrying pXO1/pXO2-like plasmids.IMPORTANCEBacillus anthracismicroorganisms are of historical and epidemiological importance and are among the most homogenous bacterial groups known, even though theB. anthracisgenome is rich in mobile elements. Mobile elements can trigger the diversification of lineages; therefore, characterizing the extent of genomic variation in a large collection of strains is critical for a complete understanding of the diversity and evolution of the species. Here, we sequenced a large collection ofB. anthracisstrains (>400) that were recovered from human, animal, and environmental sources around the world. Our results confirmed the remarkable stability of gene content and synteny of the anthrax plasmids and revealed no signal of plasmid exchange betweenB. anthracisand pathogenicB. cereusisolates but rather predominantly vertical descent. These findings advance our understanding of the biology and pathogenomic evolution ofB. anthracisand its plasmids.

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Plasmid copy number variation of a modular vector set in Shewanella oneidensis MR-1

2020 42nd Annual International Conference of the IEEE Engineering in Medicine & Biology Society (EMBC) ◽

10.1109/embc44109.2020.9176029 ◽

2020 ◽

Author(s):

Meghdad Hajimorad ◽

Jeffrey A. Gralnick

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Shewanella Oneidensis ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Number Variation

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Noise in a minimal regulatory network: plasmid copy number control

Quarterly Reviews of Biophysics ◽

10.1017/s0033583501003663 ◽

2001 ◽

Vol 34 (1) ◽

pp. 1-59 ◽

Cited By ~ 111

Author(s):

Johan Paulsson ◽

Måns Ehrenberg

Keyword(s):

Copy Number Variation ◽

Rate Constants ◽

Copy Number ◽

Time Delays ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Copy Number Control ◽

Plasmid R1 ◽

Number Variation ◽

Control Curves

1. Introduction 22. Plasmid biology 32.1 What are plasmids? 32.2 Evolution of CNC: cost and benefit 42.3 Plasmids are semi-complete regulatory networks 62.4 The molecular mechanisms of CNC for plasmids ColE1 and R1 62.4.1 ColE1 72.4.2 R1 72.5 General simplifying assumptions and values of rate constants 93. Macroscopic analysis 113.1 Regulatory logic of inhibitor-dilution CNC 113.2 Sensitivity amplification 123.3 Plasmid control curves 133.4 Multistep control of plasmid ColE1: exponential control curves 143.5 Multistep control of plasmid R1: hyperbolic control curves 163.6 Time-delays, oscillations and critical damping 184. Mesoscopic analysis 204.1 The master equation approach 204.2 A random walker in a potential well 234.3 CNC as a stochastic process 244.4 Sensitivity amplification 264.4.1 Single-step hyperbolic control 264.4.2 ColE1 multistep control can eliminate plasmid copy number variation 284.4.3 Replication backup systems – the Rom protein of ColE1 and CopB of R1 294.5 Time-delays 304.5.1 Limited rate of inhibitor degradation 304.5.2 Precise delays – does unlimited sensitivity amplification always reduce plasmid losses? 324.6 Order and disorder in CNC 334.6.1 Disordered CNC 344.6.2 Ordered CNC: R1 multistep control gives narrowly distributed interreplication times 344.7 Noisy signalling – disorder and sensitivity amplification 374.7.1 Eliminating a fast but noisy variable 384.7.2 Conditional inhibitor distribution: Poisson 394.7.3 Increasing inhibitor variation I: transcription in bursts 404.7.4 Increasing inhibitor variation II: duplex formation 414.7.5 Exploiting fluctuations for sensitivity amplification: stochastic focusing 444.7.6 A kinetic uncertainty principle 454.7.7 Disorder and stochastic focusing 464.7.8 Do plasmids really use stochastic focusing? 474.8 Metabolic burdens and values of in vivo rate constants 485. Previous models of copy number control 495.1 General models of CNC 495.2 Modelling plasmid ColE1 CNC 495.3 Modelling plasmid R1 CNC 526. Summary and outlook: the plasmid paradigm 537. Acknowledgements 568. References 56This work is a theoretical analysis of random fluctuations and regulatory efficiency in genetic networks. As a model system we use inhibitor-dilution copy number control (CNC) of the bacterial plasmids ColE1 and R1. We chose these systems because they are simple and well-characterised but also because plasmids seem to be under an evolutionary pressure to reduce both average copy numbers and statistical copy number variation: internal noise.

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Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02010-12 ◽

2013 ◽

Vol 57 (4) ◽

pp. 1850-1856 ◽

Cited By ~ 17

Author(s):

L. C. Cook ◽

G. M. Dunny

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Copy Number ◽

Antibiotic Resistance Genes ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Rapid Reduction ◽

Biofilm Growth ◽

Content Type

ABSTRACTBiofilm growth causes increased average plasmid copy number as well as increased copy number heterogeneity inEnterococcus faecaliscells carrying plasmid pCF10. In this study, we examined whether biofilm growth affected the copy number and expression of antibiotic resistance determinants for several plasmids with diverse replication systems. Four differentE. faecalisplasmids, unrelated to pCF10, demonstrated increased copy number in biofilm cells. In biofilm cells, we also observed increased transcription of antibiotic resistance genes present on these plasmids. The increase in plasmid copy number correlated with increased plating efficiency on high concentrations of antibiotics. Single-cell analysis of strains carrying two different plasmids suggested that the increase in plasmid copy number associated with biofilm growth was restricted to a subpopulation of biofilm cells. Regrowth of harvested biofilm cells in liquid culture resulted in a rapid reduction of plasmid copy number to that observed in the planktonic state. These results suggest a possible mechanism by which biofilm growth could reduce susceptibility to antibiotics in clinical settings.

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Pervasive gene content variation and copy number variation in maize and its undomesticated progenitor

Genome Research ◽

10.1101/gr.109165.110 ◽

2010 ◽

Vol 20 (12) ◽

pp. 1689-1699 ◽

Cited By ~ 208

Author(s):

R. A. Swanson-Wagner ◽

S. R. Eichten ◽

S. Kumari ◽

P. Tiffin ◽

J. C. Stein ◽

...

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Gene Content ◽

Content Variation ◽

Number Variation

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Industrial Relevance of Chromosomal Copy Number Variation in Saccharomyces Yeasts

Applied and Environmental Microbiology ◽

10.1128/aem.03206-16 ◽

2017 ◽

Vol 83 (11) ◽

Cited By ~ 26

Author(s):

Arthur R. Gorter de Vries ◽

Jack T. Pronk ◽

Jean-Marc G. Daran

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Evolutionary Significance ◽

Evolutionary Engineering ◽

Content Type ◽

Recent Developments ◽

Number Variation ◽

Chromosomal Copy Number ◽

Chromosomal Copy ◽

Chromosomal Copy Number Variation

ABSTRACT Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyces strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes.

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Modified Antibiotic Adjuvant Ratios Can Slow and Steer the Evolution of Resistance: Co-amoxiclav as a Case Study

mBio ◽

10.1128/mbio.01831-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 3

Author(s):

Richard C. Allen ◽

Sam P. Brown

Keyword(s):

Antibiotic Resistance ◽

Treatment Efficacy ◽

Copy Number ◽

Resistance Mechanisms ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Content Type ◽

Resistance Evolution ◽

Evolution Of Resistance ◽

Lactamase Inhibitor

ABSTRACT As antibiotic resistance spreads, developing sustainable methods to restore the efficacy of existing antibiotics is increasingly important. One widespread method is to combine antibiotics with synergistically acting adjuvants that inhibit resistance mechanisms, allowing drug killing. Here we use co-amoxiclav (a clinically important combination of the β-lactam antibiotic amoxicillin and the β-lactamase inhibitor clavulanate) to ask whether treatment efficacy and resistance evolution can be decoupled via component dosing modifications. A simple mathematical model predicts that different ratios of these two drug components can produce distinct evolutionary responses irrespective of the initial efficacy. We test this hypothesis by selecting Escherichia coli with a plasmid-encoded β-lactamase (CTX-M-14), against different concentrations of amoxicillin and clavulanate. Consistent with our theory, we found that while resistance evolved under all conditions, the component ratio influenced both the rate and mechanism of resistance evolution. Specifically, we found that the current clinical practice of high amoxicillin-to-clavulanate ratios resulted in the most rapid adaptation to antibiotics via gene dosing responses. Increased plasmid copy number allowed E. coli to increase β-lactamase dosing and effectively titrate out low quantities of clavulanate, restoring amoxicillin resistance. In contrast, high clavulanate ratios were more robust—plasmid copy number did not increase, although porin or efflux resistance mechanisms were found, as for all drug ratios. Our results indicate that by changing the ratio of adjuvant to antibiotic we can slow and steer the path of resistance evolution. We therefore suggest using increased adjuvant dosing regimens to slow the rate of resistance evolution. IMPORTANCE As antibiotic resistance spreads, a promising approach is to restore the effectiveness of existing drugs via coadministration with adjuvants that inhibit resistance. However, as for monotherapy, antibiotic-adjuvant therapies can select for a variety of resistance mechanisms, so it is imperative that adjuvants be used in a sustainable manner. We test whether the rate of resistance evolution can be decoupled from treatment efficacy using co-amoxiclav, a clinically important combination of the β-lactam amoxicillin and β-lactamase inhibitor clavulanate. Using experimental evolution and a simple theoretical model, we show that the current co-amoxiclav formulation with a high proportion of amoxicillin rapidly selects for resistance via increased β-lactamase production. On the other hand, formulations with more clavulanate and less amoxicillin have similar efficacies yet prevent the selective benefit of increased β-lactamase. We suggest that by blocking common paths to resistance, treatment combinations with the adjuvant in excess can slow the evolution of resistance.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00235-15 ◽

2015 ◽

Vol 59 (6) ◽

pp. 3335-3341 ◽

Cited By ~ 34

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.

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Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains

Applied and Environmental Microbiology ◽

10.1128/aem.01263-15 ◽

2015 ◽

Vol 81 (18) ◽

pp. 6253-6267 ◽

Cited By ~ 43

Author(s):

M. van den Broek ◽

I. Bolat ◽

J. F. Nijkamp ◽

E. Ramos ◽

M. A. H. Luttik ◽

...

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Phenotypic Traits ◽

Genome Sequences ◽

Content Type ◽

Chromosome Copy Number ◽

Group I ◽

Saccharomyces Pastorianus ◽

Karyotypic Variability ◽

Number Variation

ABSTRACTLager brewing strains ofSaccharomyces pastorianusare natural interspecific hybrids originating from the spontaneous hybridization ofSaccharomyces cerevisiaeandSaccharomyces eubayanus. Over the past 500 years,S. pastorianushas been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics ofS. pastorianusgenomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed thatS. pastorianusstrain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants inS. pastorianusCBS1483. Analysis of eight additionalS. pastorianusstrains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes.

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