A Naturally Occurring Single Nucleotide Polymorphism in a Multicopy Plasmid Produces a Reversible Increase in Antibiotic Resistance

Santos-López, Alfonso; Bernabe-Balas, Cristina; Ares-Arroyo, Manuel; Ortega-Huedo, Rafael; Hoefer, Andreas; Millán, Álvaro San; González‐Zorn, Bruno

doi:10.1128/aac.01735-16

Cited by 42 publications

(45 citation statements)

References 25 publications

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“…In others words they will be heteroplasmic to a different degree. Recent data from another ColE1 plasmid (pB1000 in Haemophilus influenzae ) shows heteroplasmic cells to have higher overall number of plasmid copies compared to cells that are monomorphic for the mutated plasmid (here, like in our case, the mutation leads to copy number increase) 58 . Thus the differences in the extent of heteroplasmy (a mutant-to-wild-type ratio within a cell) will lead to different plasmid copy numbers within the daughter cells.…”

Section: Plasmid Heteroplasmy Likely Prevents Alterations In Copy Numbersupporting

confidence: 51%

A high resolution view of adaptive events

Han

Arbeithuber

Cremoina

et al. 2018

Preprint

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Coadaptation between bacterial hosts and plasmids often involves a small number of highly reproducible mutations. Yet little is known about the underlying complex dynamics that leads to such a single "correct" solution. Observing mutations in fine detail along the adaptation trajectory is necessary for understanding this phenomenon. We studied coadaptation between E. coli and a common artificial plasmid, pBR322, in a continuous turbidostat culture. To obtain a high resolution picture of early adaptive events, we used a highly sensitive duplex sequencing strategy to directly observe and track mutations with frequencies undetectable with conventional methods. The observed highly reproducible trajectories are governed by clonal interference and show rapid increases in the frequencies of beneficial mutations controlling plasmid replication followed by a profound diversity crash corresponding to the emergence of chromosomal variants. To the best of our knowledge our study represents the first comprehensive assessment of adaptive processes at a very fine level of resolution. Our work highlights the hidden complexity of coadaptation, and provides an experimental and theoretical foundation for future studies.2

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Section: Plasmid Heteroplasmy Likely Prevents Alterations In Copy Numbersupporting

confidence: 51%

A high resolution view of adaptive events

Han

Arbeithuber

Cremoina

et al. 2018

Preprint

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“…Previous reports showed that the biological cost of a single plasmid, including a ColE1 plasmid (18, 35), is proportional to its copy number in the host cell (19, 36). We measured plasmid copy number (PCN) of pB1000, pB1005 and pB1006 in all the strains using quantitative PCR (qPCR) (Table 1, Figure 3).…”

Section: Resultsmentioning

confidence: 99%

“…These plasmids replicate via two RNAs (16). Natural SNPs in these RNAs allow different ColE1-like plasmids to stably cohabit within the same cell (17, 18). If the plasmids bear antibiotic resistance genes, this cohabitation confers antibiotic multiresistance to the host bacteria (9).…”

Section: Introductionmentioning

confidence: 99%

Compensatory evolution facilitates the acquisition of multiple plasmids in bacteria

Santos-López

Bernabe-Balas

Millán

et al. 2017

Preprint

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17The coexistence of multicopy plasmids is a common phenomenon. However, the 18 evolutionary forces promoting these genotypes are poorly understood. In this study, we 19 have analyzed multiple ColE1 plasmids (pB1000, pB1005 and pB1006) coexisting 20 within Haemophilus influenzae RdKW20 in all possible combinations. When 21 transformed into the naïve host, each plasmid type presented a particular copy number 22 and produced a specific resistance profile and biological cost, whether alone or 23 coexisting with the other plasmids. Therefore, there was no fitness advantage associated 24 with plasmid coexistence that could explain these common plasmid associations in 25 nature. Using experimental evolution, we showed how H. influenzae Rd was able to 26 completely compensate the fitness cost produced by any of these plasmids. Crucially, 27 once the bacterium has compensated for a first plasmid, the acquisition of new 28 multicopy plasmid(s) did not produced any extra biological cost. We argue therefore 29 that compensatory adaptation pave the way for the acquisition of multiple coexisting 30 ColE1 plasmids. 31 Importance 32Antibiotic resistance is a major concern for human and animal health. Plasmids play a 33 major role in the acquisition and dissemination of antimicrobial resistance genes. In this 34 report we investigate, for the first time, how plasmids are capable to cohabit stably in 35 populations. This coexistence of plasmids is driven by compensatory evolution 36 alleviating the cost of a first plasmid, which potentiates the acquisition of further 37 plasmids at no extra cost. This phenomenon explains the high prevalence of plasmids 38 coexistance in wild type bacteria, which generates multiresistant clones and contributes 39 to the maintenance and spread of antibiotic resistance genes within bacterial 40 populations. 41 42Antibiotic resistance is a serious problem in animal and human health and bacterial 43 plasmids play an essential role in the dissemination of resistance (1). In last years, 44 numerous works have described the importance of small ColE1-like plasmids in the 45 dissemination of resistance genes (2-15). These plasmids replicate via two RNAs (16). 46Natural SNPs in these RNAs allow different ColE1-like plasmids to stably cohabit 47 within the same cell (17, 18). If the plasmids bear antibiotic resistance genes, this 48 cohabitation confers antibiotic multiresistance to the host bacteria (9). 49The acquisition of plasmids usually entails a biological cost to the host bacterium that 50 will generate a selection against plasmid-bearing clones (19, 20). Thus, it is reasonable 51 to assume that the accumulation of various plasmids will decrease the fitness of bacteria 52 and therefore clones bearing several plasmids will be outcompeted in bacterial 53 populations. Notwithstanding, ColE1 plasmids cohabitation is a common phenomenon 54 in nature (9, 11, 13, 14, 21). In this study we test two hypotheses that could explain the 55 high prevalence of plasmid coexistence in nature: i) Positive epi...

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“…Secondly, while we examined rescue via single mutations, if multiple mutations with different dominance were available, populations at different ploidy levels may tend to evolve via different pathways (43). Furthermore, while we exclusively considered chromosomal mutations, mutations on plasmids, particularly those with high copy number (44), should show similar effects, although segregation patterns and hence time to achieve homozygosity are likely to differ. Finally, models developed thus far have assumed constant ploidy, whereas future modeling efforts could incorporate the dynamically changing and environment-dependent nature of bacterial ploidy.…”

Section: Discussionmentioning

confidence: 99%

Effective polyploidy causes phenotypic delay and influences bacterial evolvability

Sun

Alexander

Bogos

et al. 2017

Preprint

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Whether mutations in bacteria exhibit a noticeable delay before expressing their corresponding mutant phenotype was discussed intensively in the 1940s-50s, but the discussion eventually waned for lack of supportive evidence and perceived incompatibility with observed mutant distributions in fluctuation tests. Phenotypic delay in bacteria is widely assumed to be negligible, despite lack of direct evidence. Here we revisited the question using recombineering to introduce antibiotic resistance mutations into E. coli at defined time points and then tracking expression of the corresponding mutant phenotype over time. Contrary to previous assumptions, we found a substantial median phenotypic delay of 3-4 generations. We provided evidence that the primary source of this delay is multifork replication causing cells to be effectively polyploid, whereby wild-type gene copies transiently mask the phenotype of recessive mutant gene copies in the same cell. Using modeling and simulation methods, we explored the consequences of effective polyploidy for mutation rate estimation by fluctuation tests and sequencing-based methods. For recessive mutations, despite the substantial phenotypic delay, the per-copy or per-genome mutation rate is accurately estimated. However, the per-cell rate cannot be estimated by existing methods. Finally, with a mathematical model, we showed that effective polyploidy increases the frequency of costly recessive mutations in the standing genetic variation, and thus their potential contribution to evolutionary adaptation, while drastically reducing the chance that de novo recessive mutations can rescue populations facing a harsh environmental change such as antibiotic treatment. Overall, we have identified phenotypic delay and effective polyploidy as previously overlooked but essential components in bacterial evolvability, including antibiotic resistance evolution. Author summaryWhat is the time delay between the occurrence of a genetic mutation in a bacterial cell and manifestation of its phenotypic effect? We show that antibiotic resistance mutations in E.coli show a remarkably long phenotypic delay of 3-4 bacterial generations. The primary underlying mechanism of this delay is effective polyploidy. In a polyploid cell with multiple chromosomes, once a mutation arises on one of the chromosomes, the presence of non-mutated, wild-type gene copies on other chromosomes may mask the . CC-BY 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/226654 doi: bioRxiv preprint first posted online Nov. 29, 2017; 2 phenotype of the mutation. One implication of this finding is that conventional methods to determine the mutation rates of bacteria do not detect polyploidy and thus underestimate their potential for adaptation. More generally, the effect that a new mutation may become useful only in the "grand-children of the grand-children" suggests that pre-existing mutations are more importan...

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A Naturally Occurring Single Nucleotide Polymorphism in a Multicopy Plasmid Produces a Reversible Increase in Antibiotic Resistance

Cited by 42 publications

References 25 publications

A high resolution view of adaptive events

A high resolution view of adaptive events

Compensatory evolution facilitates the acquisition of multiple plasmids in bacteria

Effective polyploidy causes phenotypic delay and influences bacterial evolvability

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