Seventy Years on from the Luria and Delbrûck Fluctuation Analysis: A Comparison of three Methods for Estimating Mutation Rate

2015 ◽  
Vol 6 ◽  
pp. 50-58
Author(s):  
I W Nyinoh
Keyword(s):  
Mutation Rate ◽  
Resistance Mutation ◽  
Model Organism ◽  
Relative Fitness ◽  
Mutation Rates ◽  
Fluctuation Analysis ◽  
Cellular Processes ◽  
Scientific Disciplines ◽  
Rate Fluctuation ◽  
Contemporary Interpretation

Seventy years ago, Luria and Delbrûck discovered fluctuation assay for estimating mutation rates. While this method is slightly dated, it is one of the few methods for estimating mutation rates in batch culture. Mutation rates when determined expose information on cellular processes and fundamental mutagenic mechanisms. Formerly, inferences drawn from fluctuation assay were sufficient to answer a specific question inbacterial genetics. However, contemporary interpretation of results goes far beyond the motive originally intended. As the fluctuation assay has gained popularity in various scientific disciplines, analyses of results obtained are not same. This study aims to compare the estimation of mutation rates using the Poison distribution (Po) method with, the Ma-Sarka Sandri maximum likelihood estimator and the Lea-Coulson median estimator. Mycobacterium smegmatismc 2 155was used as a model organism for Mycobacterium tuberculosis, and spontaneous mutations that arose in stationary phase cells exposed to antibiotic stress were investigated. Ten to twenty-four parallel cultures were tested with various anti-tuberculosis drugs; isoniazid, kanamycin, rifampicin and streptomycin. Minimum Inhibitory Concentration (MIC) of the drugs were also determinedto be; 8 ìg/mL, 0.24 ìg/mL, 16 ìg/mL and 0.5 ìg/mL for isoniazid, kanamycin, rifampicin and streptomycin respectively. The mutation rates obtained with the methods were very similar. To improve the power of deductions drawn from fluctuation assay, efforts should be made to experimentally determine the relative fitness of wild-type to mutant bacteria.This comparison is only a guide providing evidence regarding the authenticity of some of the methods currently available to researchers interested in estimating bacterial mutation rates.Keywords: antibiotic resistance, mutation rate, fluctuation assay, fluctuation analysis calculator.

scholarly journals Localized hypermutation drives the evolution of unstable colistin resistance in small populations of Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Natalia Kapel ◽  
Julio Felix Diaz Caballero ◽  
Craig MacLean
Keyword(s):  
Antimicrobial Peptides ◽  
Mutation Rate ◽  
Resistance Mutation ◽  
Host Immunity ◽  
Mutation Rates ◽  
High Dose ◽  
Small Populations ◽  
Colistin Resistance ◽  
Two Component System ◽  
Antibiotic Resistant

Colistin has emerged as an important last line of defence for the treatment of infections caused by antibiotic resistant Gram-negative pathogens. Here we investigate the responses of ≈1,000 small populations (≈104 CFU) of an MDR strain of P. aeruginosa to a high dose of colistin (2 mg/L). Colistin exposure resulted in rapid cell death, but a sub-set of populations eventually recovered due to the outgrowth of heteroresistant cells. Genome sequencing revealed that heteroresistance was primarily driven by mutations in the PmrAB two-component system that occurred at a rate (≈2x10-5 per cell division) that was 103-104 fold higher than typical resistance mutation rates. Crucially, this elevated mutation rate was only found in pmrB, demonstrating that hypermutability is localized to this gene. PmrAB provides resistance to antimicrobial peptides that are involved in host immunity, suggesting that this pathogen may have evolved a high mutation rate as an adaption to generate mutants that are resistant to host antimicrobial peptides that are secreted during infection. Interestingly, we found no mutations in some heteroresister populations, suggesting that a small sub-set of P. aeruginosa cells are intrinsically resistant to colistin as a result of phenotypic plasticity.

scholarly journals Spontaneous mutations conferring antibiotic resistance to antitubercular drugs at a range of concentrations in Mycobacterium smegmatis

2018 ◽  
Author(s):  
Iveren W Nyinoh ◽  
Johnjoe McFadden
Keyword(s):  
Antibiotic Resistance ◽  
Mutation Rate ◽  
Mycobacterium Smegmatis ◽  
Future Generations ◽  
Mutation Rates ◽  
Fluctuation Analysis ◽  
Spontaneous Mutations ◽  
Likelihood Estimator ◽  
Antitubercular Drugs

AbstractMycobacteria population can undergo mutations in their DNA sequence during replication, which if not repaired, would be transferred to future generations. In this study, in vitro spontaneous mutations in Mycobacterium smegmatis mc2155 (Msm) conferring resistance to isoniazid (INHr), rifampicin (RIFr), kanamycin (KANr) and streptomycin (STRr) were determined at several concentrations in a fluctuation assay. Mutation rate was estimated using the P₀ method, and estimates were then compared with the Lea-Coulson method of the median and Ma-Sandri-Sarkar Maximum Likelihood Estimator (MSS-MLE) method available on the Fluctuation analysis calculator (FALCOR). The mutation rates of RIFr ranged from 9.24 × 10-8 - 2.18 × 10-10, INHr 1.2 × 10-7 - 1.20×10-9, STRr 2.77 × 10-8 - 5.31 × 10-8 and KANr 1.7 × 10-8 mutations per cell division. This study provides mutation rate estimates to key antitubercular drugs at a range of concentrations.

scholarly journals Population persistence under high mutation rate: from evolutionary rescue to lethal mutagenesis

2019 ◽  
Author(s):  
Yoann Anciaux ◽  
Amaury Lambert ◽  
Ophelie Ronce ◽  
Lionel Roques ◽  
Guillaume Martin
Keyword(s):  
Mutation Rate ◽  
Geometric Model ◽  
Resistance Mutation ◽  
Theoretical Work ◽  
Mutation Rates ◽  
Moderate Increase ◽  
Asexual Population ◽  
Lethal Mutagenesis ◽  
Recent Theoretical Work ◽  
Evolutionary Rescue

AbstractPopulations may genetically adapt to severe stress that would otherwise cause their extirpation. Recent theoretical work, combining stochastic demography with Fisher’s geometric model of adaptation, has shown how evolutionary rescue becomes unlikely beyond some critical intensity of stress. Increasing mutation rates may however allow adaptation to more intense stress, raising concerns about the effectiveness of treatments against pathogens. This previous work assumes that populations are rescued by the rise of a single resistance mutation. However, even in asexual organisms, rescue can also stem from the accumulation of multiple mutations in a single genome. Here, we extend previous work to study the rescue process in an asexual population where the mutation rate is sufficiently high so that such events may be common. We predict both the ultimate extinction probability of the population and the distribution of extinction times. We compare the accuracy of different approximations covering a large range of mutation rates. Moderate increase in mutation rates favors evolutionary rescue. However, larger increase leads to extinction by the accumulation of a large mutation load, a process called lethal mutagenesis. We discuss how these results could help design “evolution-proof” anti-pathogen treatments that even highly mutable strains could not overcome.

scholarly journals Identifying Mutator Phenotypes among Fluoroquinolone-Resistant Strains of Streptococcus pneumoniae Using Fluctuation Analysis

2007 ◽  
Vol 51 (9) ◽  
pp. 3225-3229 ◽  
Author(s):  
Carolyn V. Gould ◽  
Paul D. Sniegowski ◽  
Mikhail Shchepetov ◽  
Joshua P. Metlay ◽  
Jeffrey N. Weiser
Keyword(s):  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Mismatch Repair ◽  
Mutation Rate ◽  
In Vitro Selection ◽  
Clinical Isolates ◽  
Mutation Rates ◽  
Fluctuation Analysis ◽  
Missense Mutations ◽  
Resistant Strains

ABSTRACT The occurrence of mutator phenotypes among laboratory-generated and clinical levofloxacin-resistant strains of Streptococcus pneumoniae was determined using fluctuation analysis. The in vitro selection for levofloxacin-resistant mutants of strain D39, each with point mutations in both gyrA and parC or parE, was not associated with a significant change in the mutation rate. Two of eight clinical isolates resistant to levofloxacin (MIC, >8 μg/ml) had estimated mutation rates of 1.2 × 10−7 and 9.4 × 10−8 mutations per cell division, indicating potential mutator phenotypes, compared to strain D39, which had an estimated mutation rate of 1.4 × 10−8 mutations per cell division. The levofloxacin-resistant isolates with the highest mutation rates showed evidence of dysfunctional mismatch repair and contained missense mutations in mut genes at otherwise highly conserved sites. The association of hypermutability in levofloxacin-resistant S. pneumoniae clinical isolates with mutations in DNA mismatch repair genes provides further evidence that mismatch repair mutants may have a selective advantage in the setting of antibiotic pressure, facilitating the development of further antibiotic resistance.

scholarly journals Fluctuation analysis: the probability distribution of the number of mutants under different conditions.

Genetics ◽  
1990 ◽  
Vol 124 (1) ◽  
pp. 175-185 ◽  
Author(s):  
F M Stewart ◽  
D M Gordon ◽  
B R Levin
Keyword(s):  
Growth Rate ◽  
Probability Distribution ◽  
Mutation Rate ◽  
Point Of View ◽  
Mutation Rates ◽  
Fluctuation Analysis ◽  
Constant Multiple ◽  
Single Culture ◽  
Biological Reality ◽  
Fluctuation Experiment

Abstract In the 47 years since fluctuation analysis was introduced by Luria and Delbrück, it has been widely used to calculate mutation rates. Up to now, in spite of the importance of such calculations, the probability distribution of the number of mutants that will appear in a fluctuation experiment has been known only under the restrictive, and possibly unrealistic, assumptions: (1) that the mutation rate is exactly proportional to the growth rate and (2) that all mutants grow at a rate that is a constant multiple of the growth rate of the original cells. In this paper, we approach the distribution of the number of mutants from a new point of view that will enable researchers to calculate the distribution to be expected using assumptions that they believe to be closer to biological reality. The new idea is to classify mutations according to the number of observable mutants that derive from the mutation when the culture is selectively plated. This approach also simplifies the calculations in situations where two, or many, kinds of mutation may occur in a single culture.

scholarly journals Learning the distribution of single-cell chromosome conformations in bacteria reveals emergent order across genomic scales

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joris J. B. Messelink ◽  
Muriel C. F. van Teeseling ◽  
Jacqueline Janssen ◽  
Martin Thanbichler ◽  
Chase P. Broedersz
Keyword(s):  
Single Cell ◽  
Caulobacter Crescentus ◽  
Model Organism ◽  
Super Resolution ◽  
Chromosome Organization ◽  
General Strategy ◽  
Cell Axis ◽  
Cellular Processes ◽  
Emergent Order ◽  
Genomic Clusters

AbstractThe order and variability of bacterial chromosome organization, contained within the distribution of chromosome conformations, are unclear. Here, we develop a fully data-driven maximum entropy approach to extract single-cell 3D chromosome conformations from Hi–C experiments on the model organism Caulobacter crescentus. The predictive power of our model is validated by independent experiments. We find that on large genomic scales, organizational features are predominantly present along the long cell axis: chromosomal loci exhibit striking long-ranged two-point axial correlations, indicating emergent order. This organization is associated with large genomic clusters we term Super Domains (SuDs), whose existence we support with super-resolution microscopy. On smaller genomic scales, our model reveals chromosome extensions that correlate with transcriptional and loop extrusion activity. Finally, we quantify the information contained in chromosome organization that may guide cellular processes. Our approach can be extended to other species, providing a general strategy to resolve variability in single-cell chromosomal organization.

scholarly journals Mutation rate analysis via parent–progeny sequencing of the perennial peach. II. No evidence for recombination-associated mutation

2016 ◽  
Vol 283 (1841) ◽  
pp. 20161785 ◽  
Author(s):  
Long Wang ◽  
Yanchun Zhang ◽  
Chao Qin ◽  
Dacheng Tian ◽  
Sihai Yang ◽  
...  
Keyword(s):  
Mutation Rate ◽  
Recombination Rate ◽  
Mutation Rates ◽  
Recombination Rates ◽  
Rate Analysis ◽  
A Genome ◽  
Break Points ◽  
New Mutations

Mutation rates and recombination rates vary between species and between regions within a genome. What are the determinants of these forms of variation? Prior evidence has suggested that the recombination might be mutagenic with an excess of new mutations in the vicinity of recombination break points. As it is conjectured that domesticated taxa have higher recombination rates than wild ones, we expect domesticated taxa to have raised mutation rates. Here, we use parent–offspring sequencing in domesticated and wild peach to ask (i) whether recombination is mutagenic, and (ii) whether domesticated peach has a higher recombination rate than wild peach. We find no evidence that domesticated peach has an increased recombination rate, nor an increased mutation rate near recombination events. If recombination is mutagenic in this taxa, the effect is too weak to be detected by our analysis. While an absence of recombination-associated mutation might explain an absence of a recombination–heterozygozity correlation in peach, we caution against such an interpretation.

Evolution of mutation rate and virulence among human retroviruses

1994 ◽  
Vol 346 (1317) ◽  
pp. 333-343 ◽  
Keyword(s):  
Immune System ◽  
Mutation Rate ◽  
Hiv Infections ◽  
Molecular Data ◽  
Mutation Rates ◽  
Rapid Progression ◽  
Human T Cell ◽  
Large Numbers ◽  
The Individual ◽  
Multicellular Organisms

High mutation rates are generally considered to be detrimental to the fitness of multicellular organisms because mutations untune finely tuned biological machinery. However, high mutation rates may be favoured by a need to evade an immune system that has been strongly stimulated to recognize those variants that reproduced earlier during the infection, hiv infections conform to this situation because they are characterized by large numbers of viruses that are continually breaking latency and large numbers that are actively replicating throughout a long period of infection. To be transmitted, HIVS are thus generally exposed to an immune system that has been activated to destroy them in response to prior viral replication in the individual. Increases in sexual contact should contribute to this predicament by favouring evolution toward relatively high rates of replication early during infection. Because rapid replication and high mutation rate probably contribute to rapid progression of infections to aids, the interplay of sexual activity, replication rate, and mutation rate helps explain why HIV-1 has only recently caused a lethal pandemic, even though molecular data suggest that it may have been present in humans for more than a century. This interplay also offers an explanation for geographic differences in progression to cancer found among infections due to the other major group of human retroviruses, human T-cell lymphotropic viruses (HTLV). Finally, it suggests ways in which we can use natural selection as a tool to control the aids pandemic and prevent similar pandemics from arising in the future.

scholarly journals Transfer RNA genes experience exceptionally elevated mutation rates

2018 ◽  
Vol 115 (36) ◽  
pp. 8996-9001 ◽  
Author(s):  
Bryan P. Thornlow ◽  
Josh Hough ◽  
Jacquelyn M. Roger ◽  
Henry Gong ◽  
Todd M. Lowe ◽  
...  
Keyword(s):  
Mutation Rate ◽  
Trna Gene ◽  
Gene Evolution ◽  
Purifying Selection ◽  
Mutation Rates ◽  
Model Organisms ◽  
Biological Synthesis ◽  
Human Populations ◽  
Trna Genes ◽  
Simple Method

Transfer RNAs (tRNAs) are a central component for the biological synthesis of proteins, and they are among the most highly conserved and frequently transcribed genes in all living things. Despite their clear significance for fundamental cellular processes, the forces governing tRNA evolution are poorly understood. We present evidence that transcription-associated mutagenesis and strong purifying selection are key determinants of patterns of sequence variation within and surrounding tRNA genes in humans and diverse model organisms. Remarkably, the mutation rate at broadly expressed cytosolic tRNA loci is likely between 7 and 10 times greater than the nuclear genome average. Furthermore, evolutionary analyses provide strong evidence that tRNA genes, but not their flanking sequences, experience strong purifying selection acting against this elevated mutation rate. We also find a strong correlation between tRNA expression levels and the mutation rates in their immediate flanking regions, suggesting a simple method for estimating individual tRNA gene activity. Collectively, this study illuminates the extreme competing forces in tRNA gene evolution and indicates that mutations at tRNA loci contribute disproportionately to mutational load and have unexplored fitness consequences in human populations.

Y-chromosomal microsatellite mutation rates: Differences in mutation rate between and within loci

2004 ◽  
Vol 23 (2) ◽  
pp. 117-124 ◽  
Author(s):  
B. Myhre Dupuy ◽  
M. Stenersen ◽  
T. Egeland ◽  
B. Olaisen
Keyword(s):  
Mutation Rate ◽  
Mutation Rates ◽  
Microsatellite Mutation
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