Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx

ABSTRACT The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase (dinP) and a DNA damage response regulator (umuDAb [the umuD gene of A. baylyi]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting “clean-genome” ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits.

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First Estimation of the Spontaneous Mutation Rate in Diatoms

Genome Biology and Evolution ◽

10.1093/gbe/evz130 ◽

2019 ◽

Vol 11 (7) ◽

pp. 1829-1837 ◽

Cited By ~ 11

Author(s):

Marc Krasovec ◽

Sophie Sanchez-Brosseau ◽

Gwenael Piganeau

Keyword(s):

Mutation Rate ◽

Spontaneous Mutation ◽

Mutation Accumulation ◽

Mutation Rates ◽

Model Organisms ◽

Base Substitution ◽

Unicellular Eukaryote ◽

Fundamental Parameter ◽

Secondary Endosymbiosis ◽

Spontaneous Mutation Rate

Abstract Mutations are the origin of genetic diversity, and the mutation rate is a fundamental parameter to understand all aspects of molecular evolution. The combination of mutation–accumulation experiments and high-throughput sequencing enabled the estimation of mutation rates in most model organisms, but several major eukaryotic lineages remain unexplored. Here, we report the first estimation of the spontaneous mutation rate in a model unicellular eukaryote from the Stramenopile kingdom, the diatom Phaeodactylum tricornutum (strain RCC2967). We sequenced 36 mutation accumulation lines for an average of 181 generations per line and identified 156 de novo mutations. The base substitution mutation rate per site per generation is μbs = 4.77 × 10−10 and the insertion–deletion mutation rate is μid = 1.58 × 10−11. The mutation rate varies as a function of the nucleotide context and is biased toward an excess of mutations from GC to AT, consistent with previous observations in other species. Interestingly, the mutation rates between the genomes of organelles and the nucleus differ, with a significantly higher mutation rate in the mitochondria. This confirms previous claims based on indirect estimations of the mutation rate in mitochondria of photosynthetic eukaryotes that acquired their plastid through a secondary endosymbiosis. This novel estimate enables us to infer the effective population size of P. tricornutum to be Ne∼8.72 × 106.

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Functional Analysis of the NucS/EndoMS of the Hyperthermophilic Archaeon Sulfolobus islandicus REY15A

Frontiers in Microbiology ◽

10.3389/fmicb.2020.607431 ◽

2020 ◽

Vol 11 ◽

Author(s):

Sohail Ahmad ◽

Qihong Huang ◽

Jinfeng Ni ◽

Yuanxi Xiao ◽

Yunfeng Yang ◽

...

Keyword(s):

Mismatch Repair ◽

Mutation Rate ◽

Spontaneous Mutation ◽

Repair Pathway ◽

Wild Type ◽

Spontaneous Mutation Rate ◽

Hyperthermophilic Archaeon ◽

Cleavage Activity ◽

Sulfolobus Islandicus ◽

Mismatch Repair Pathway

EndoMS is a recently identified mismatch specific endonuclease in Thermococcales of Archaea and Mycobacteria of Bacteria. The homologs of EndoMS are conserved in Archaea and Actinobacteria, where classic MutS-MutL-mediated DNA mismatch repair pathway is absent or non-functional. Here, we report a study on the in vitro mismatch cleavage activity and in vivo function of an EndoMS homolog (SisEndoMS) from Sulfolobus islandicus REY15A, the model archaeon belonging to Crenarchaeota. SisEndoMS is highly active on duplex DNA containing G/T, G/G, and T/T mismatches. Interestingly, the cleavage activity of SisEndoMS is stimulated by the heterotrimeric PCNAs, and when Mn2+ was used as the co-factor instead of Mg2+, SisEndoMS was also active on DNA substrates containing C/T or A/G mismatches, suggesting that the endonuclease activity can be regulated by ion co-factors and accessory proteins. We compared the spontaneous mutation rate of the wild type strain REY15A and ∆endoMS by counter selection against 5-fluoroorotic acid (5-FOA). The endoMS knockout mutant had much higher spontaneous mutation rate (5.06 × 10−3) than that of the wild type (4.6 × 10−6). A mutation accumulation analysis also showed that the deletion mutant had a higher mutation occurrence than the wild type, with transition mutation being the dominant, suggesting that SisEndoMS is responsible for mutation avoidance in this hyperthermophilic archaeon. Overexpression of the wild type SisEndoMS in S. islandicus resulted in retarded growth and abnormal cell morphology, similar to strains overexpressing Hje and Hjc, the Holliday junction endonucleases. Transcriptomic analysis revealed that SisEndoMS overexpression led to upregulation of distinct gene including the CRISPR-Cas IIIB system, methyltransferases, and glycosyltransferases, which are mainly localized to specific regions in the chromosome. Collectively, our results support that EndoMS proteins represent a noncanonical DNA repair pathway in Archaea. The mechanism of the mismatch repair pathway in Sulfolobus which have a single chromosome is discussed.

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Spontaneous mutation rate estimates for the principal malaria vectors Anopheles coluzzii and Anopheles stephensi

Scientific Reports ◽

10.1038/s41598-021-03943-z ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Iliyas Rashid ◽

Melina Campos ◽

Travis Collier ◽

Marc Crepeau ◽

Allison Weakley ◽

...

Keyword(s):

Confidence Interval ◽

Mutation Rate ◽

Anopheles Stephensi ◽

Spontaneous Mutation ◽

False Negative ◽

False Negative Rate ◽

Mutation Rates ◽

Malaria Vectors ◽

Base Substitution ◽

Anopheles Coluzzii

AbstractUsing high-depth whole genome sequencing of F0 mating pairs and multiple individual F1 offspring, we estimated the nuclear mutation rate per generation in the malaria vectors Anopheles coluzzii and Anopheles stephensi by detecting de novo genetic mutations. A purpose-built computer program was employed to filter actual mutations from a deep background of superficially similar artifacts resulting from read misalignment. Performance of filtering parameters was determined using software-simulated mutations, and the resulting estimate of false negative rate was used to correct final mutation rate estimates. Spontaneous mutation rates by base substitution were estimated at 1.00 × 10−9 (95% confidence interval, 2.06 × 10−10—2.91 × 10−9) and 1.36 × 10−9 (95% confidence interval, 4.42 × 10−10—3.18 × 10−9) per site per generation in A. coluzzii and A. stephensi respectively. Although similar studies have been performed on other insect species including dipterans, this is the first study to empirically measure mutation rates in the important genus Anopheles, and thus provides an estimate of µ that will be of utility for comparative evolutionary genomics, as well as for population genetic analysis of malaria vector mosquito species.

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Overexpression of RamA, Which Regulates Production of the Multidrug Resistance Efflux Pump AcrAB-TolC, Increases Mutation Rate and Influences Drug Resistance Phenotype

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02460-19 ◽

2020 ◽

Vol 64 (4) ◽

Cited By ~ 2

Author(s):

Elizabeth M. Grimsey ◽

Natasha Weston ◽

Vito Ricci ◽

Jack W. Stone ◽

Laura J. V. Piddock

Keyword(s):

Multidrug Resistance ◽

Mutation Rate ◽

Selective Pressure ◽

Efflux Pump ◽

Transcriptional Regulators ◽

Multidrug Resistant ◽

Minor Role ◽

Wild Type ◽

Content Type ◽

Serovar Typhimurium

ABSTRACT In Enterobacteriales, the AcrAB-TolC efflux pump exports substrates, including antimicrobials, from the cell. Overexpression of AcrAB-TolC can occur after exposure to fluoroquinolones, leading to multidrug resistance. The expression of AcrAB-TolC in Salmonella is primarily regulated by the transcriptional activator RamA. However, other transcriptional activators, such as MarA, SoxRS, and Rob, can influence AcrAB-TolC expression. This study determined whether the overproduction or absence of RamA influences the mutation rate or the phenotype of mutants selected in Salmonella enterica serovar Typhimurium SL1344 after ciprofloxacin exposure. The absence of RamA (SL1344 ramA::aph) resulted in mutation frequencies/rates similar to those of wild-type Salmonella Typhimurium SL1344. However, the overproduction of RamA (SL1344 ramR::aph) and, consequently, AcrB resulted in a significantly higher mutation frequency and rate than for wild-type Salmonella Typhimurium SL1344. Whole-genome sequencing revealed that in addition to selecting gyrA mutants resistant to quinolones, SL1344 and SL1344 ramA::aph also produced multidrug-resistant (MDR) mutants, associated with mutations in soxR. Conversely, mutations in SL1344 ramR::aph occurred in gyrA only. Although transcriptional regulators such as SoxRS are believed to play a minor role in AcrAB-TolC regulation under antibiotic selective pressure, we show that soxR mutants can be selected after exposure to ciprofloxacin, including when RamA is absent. This demonstrates that under selective pressure, Salmonella can respond to increased efflux pump expression by mutating other AcrAB-TolC regulatory genes, allowing for the evolution of MDR. Understanding how Salmonella responds to antibiotic pressure in the absence/overproduction of RamA is important if targeting transcriptional regulators to alter efflux is to be considered an avenue for future drug discovery.

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A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens

mBio ◽

10.1128/mbio.00120-19 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 6

Author(s):

Erika Shor ◽

Jessica Schuyler ◽

David S. Perlin

Keyword(s):

Drug Resistance ◽

Mutation Rate ◽

Fungal Pathogen ◽

Candida Glabrata ◽

Drug Tolerance ◽

Clinical Isolates ◽

Mutation Rates ◽

Mismatch Repair Gene ◽

Repair Gene ◽

Content Type

ABSTRACT All evolutionary processes are underpinned by a cellular capacity to mutate DNA. To identify factors affecting mutagenesis, it is necessary to compare mutation rates between different strains and conditions. Drug resistance-based mutation reporters are used extensively to measure mutation rates, but they are suitable only when the compared strains have identical drug tolerance levels—a condition that is not satisfied under many “real-world” circumstances, e.g., when comparing mutation rates among a series of environmental or clinical isolates. Candida glabrata is a fungal pathogen that shows a high degree of genetic diversity and fast emergence of antifungal drug resistance. To enable meaningful comparisons of mutation rates among C. glabrata clinical isolates, we developed a novel fluorescence-activated cell sorting-based approach to measure the mutation rate of a chromosomally integrated GFP gene. We found that in Saccharomyces cerevisiae this approach recapitulated the reported mutation rate of a wild-type strain and the mutator phenotype of a shu1Δ mutant. In C. glabrata, the GFP reporter captured the mutation rate increases caused either by a genotoxic agent or by deletion of DNA mismatch repair gene MSH2, as well as the specific mutational signature associated with msh2Δ. Finally, the reporter was used to measure the mutation rates of C. glabrata clinical isolates carrying different alleles of MSH2. Together, these results show that fluorescence-based mutation reporters can be used to measure mutation rates in microbes under conditions of unequal drug susceptibility to reveal new insights about drivers of mutagenesis. IMPORTANCE Measurements of mutation rates—i.e., how often proliferating cells acquire mutations in their DNA—are essential for understanding cellular processes that maintain genome stability. Many traditional mutation rate measurement assays are based on detecting mutations that cause resistance to a particular drug. Such assays typically work well for laboratory strains but have significant limitations when comparing clinical or environmental isolates that have various intrinsic levels of drug tolerance, which confounds the interpretation of results. Here we report the development and validation of a novel method of measuring mutation rates, which detects mutations that cause loss of fluorescence rather than acquisition of drug resistance. Using this method, we measured the mutation rates of clinical isolates of fungal pathogen Candida glabrata. This assay can be adapted to other organisms and used to compare mutation rates in contexts where unequal drug sensitivity is anticipated.

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Vibrio fischeriDarR Directs Responses to D-Aspartate and Represents a Group of Similar LysR-Type Transcriptional Regulators

Journal of Bacteriology ◽

10.1128/jb.00773-17 ◽

2018 ◽

Vol 200 (15) ◽

Cited By ~ 2

Author(s):

Richard M. Jones ◽

David L. Popham ◽

Alicia L. Schmidt ◽

Ellen L. Neidle ◽

Eric V. Stabb

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Transcriptional Control ◽

Vibrio Fischeri ◽

Transcriptional Response ◽

Control Mechanisms ◽

Wild Type ◽

Acinetobacter Baylyi ◽

Transcriptional Responses ◽

Content Type

ABSTRACTMounting evidence suggests thatd-amino acids play previously underappreciated roles in diverse organisms. In bacteria, evend-amino acids that are absent from canonical peptidoglycan (PG) may act as growth substrates, as signals, or in other functions. Given these proposed roles and the ubiquity ofd-amino acids, the paucity of knownd-amino-acid-responsive transcriptional control mechanisms in bacteria suggests that such regulation awaits discovery. We found that DarR, a LysR-type transcriptional regulator (LTTR), activates transcription in response tod-Asp. Thed-Glu auxotrophy of aVibrio fischerimurI::Tn mutant was suppressed, with the wild-type PG structure maintained, by a point mutation indarR. ThisdarRmutation resulted in the overexpression of an adjacent operon encoding a putative aspartate racemase, RacD, which compensated for the loss of the glutamate racemase encoded bymurI. Using transcriptional reporters, we found that wild-type DarR activatedracDtranscription in response to exogenousd-Asp but not upon the addition ofl-Asp,l-Glu, ord-Glu. A DNA sequence typical of LTTR-binding sites was identified betweendarRand the divergently orientedracDoperon, and scrambling this sequence eliminated activation of the reporter in response tod-Asp. In several proteobacteria, genes encoding LTTRs similar to DarR are linked to genes with predicted roles ind- and/orl-Asp metabolism. To test the functional similarities in another bacterium,darRandracDmutants were also generated inAcinetobacter baylyi. InV. fischeriandA. baylyi, growth ond-Asp required the presence of bothdarRandracD. Our results suggest that multiple bacteria have the ability to sense and respond tod-Asp.IMPORTANCEd-Amino acids are prevalent in the environment and are generated by organisms from all domains of life. Although some biological roles ford-amino acids are understood, in other cases, their functions remain uncertain. Given the ubiquity ofd-amino acids, it seems likely that bacteria will initiate transcriptional responses to them. Elucidatingd-amino acid-responsive regulators along with the genes they control will help uncover bacterial uses ofd-amino acids. Here, we report the discovery of DarR, a novel LTTR inV. fischerithat mediates a transcriptional response to environmentald-Asp and underpins the catabolism ofd-Asp. DarR represents the founding member of a group of bacterial homologs that we hypothesize control aspects of aspartate metabolism in response tod-Asp and/or tod-Asp-containing peptides.

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MUTATION RATES FOR ENZYME AND MORPHOLOGICAL LOCI IN BARLEY (HORDEUM VULGARE L.)

Genetics ◽

10.1093/genetics/106.4.729 ◽

1984 ◽

Vol 106 (4) ◽

pp. 729-734

Author(s):

A L Kahler ◽

R W Allard ◽

R D Miller

Keyword(s):

Mutation Rate ◽

Spontaneous Mutation ◽

Mutation Rates ◽

Isogenic Line ◽

Probability Level ◽

Short Term ◽

Hordeum Vulgare L ◽

Spontaneous Mutation Rate ◽

Enzyme Loci ◽

Morphological Variants

ABSTRACT Spontaneous mutation rates were estimated by assaying 84,126 seedlings of a highly homozygous barley line (isogenic line 2025) for five enzyme loci. No mutants were observed in 841,260 allele replications. This result excludes, at probability level 0.95, a spontaneous mutation rate larger than 3.56 x 10-6/locus/gamete/generation for these enzyme loci. Isogenic line 2025 also was scored for mutants at four loci governing morphological variants. No mutants were observed in 3,386,850 allele replications which indicates that the upper bound for the mutation rate for these loci is 8.85 x 10-7. It was concluded that, even though spontaneous mutation has been important in creating variability in the barley species at the loci scored, the rate is too low to have much affect on the short-term dynamics of barley populations.

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Fitness landscape analysis reveals that the wild type allele is sub-optimal and mutationally robust

10.1101/2021.09.27.461914 ◽

2021 ◽

Author(s):

Tzahi Gabzi ◽

Yitzhak Tzachi Pilpel ◽

Tamar Friedlander

Keyword(s):

Mutation Rate ◽

Evolutionary Biology ◽

Evolutionary Dynamics ◽

Fitness Landscape ◽

Mutation Rates ◽

Wild Type Allele ◽

Wild Type ◽

Type Allele ◽

Landscape Mapping ◽

Evolutionary Trajectories

Fitness landscape mapping and the prediction of evolutionary trajectories on these landscapes are major tasks in evolutionary biology research. Evolutionary dynamics is tightly linked to the landscape topography, but this relation is not straightforward. Models predict different evolutionary outcomes depending on mutation rates: high-fitness genotypes should dominate the population under low mutation rates and lower-fitness, mutationally robust (also called 'flat') genotypes - at higher mutation rates. Yet, so far, flat genotypes have been demonstrated in very few cases, particularly in viruses. The quantitative conditions for their emergence were studied only in simplified single-locus, two-peak landscapes. In particular, it is unclear whether within the same genome some genes can be flat while the remaining ones are fit. Here, we analyze a previously measured fitness landscape of a yeast tRNA gene. We found that the wild type allele is sub-optimal, but is mutationally robust ('flat'). Using computer simulations, we estimated the critical mutation rate in which transition from fit to flat allele should occur for a gene with such characteristics. We then used a scaling argument to extrapolate this critical mutation rate for a full genome, assuming the same mutation rate for all genes. Finally, we propose that while the majority of genes are still selected to be fittest, there are a few mutation hot-spots like the tRNA, for which the mutationally robust flat allele is favored by selection.

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THE ORIGIN OF SPONTANEOUS MUTATION IN SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/96.4.819 ◽

1980 ◽

Vol 96 (4) ◽

pp. 819-839 ◽

Cited By ~ 1

Author(s):

Siew-Keen Quah ◽

R C von Borstel ◽

P J Hastings

Keyword(s):

Mutation Rate ◽

Spontaneous Mutation ◽

Repair System ◽

Wild Type ◽

Spontaneous Mutation Rate ◽

Double Mutant Strain ◽

In The Wild ◽

Radiation Resistant ◽

Almost All

ABSTRACT Characterization of two antimutator loci in yeast shows that both are members of the same mutagenic repair system known to be responsible for almost all induced mutation (Lawrence and Christensen 1976, 1979a,b; Prakash 1976). One of the these newly isolated antimutator mutations is an allele of rev3 (Lemontt 1971b). Two other alleles of rev3 were tested and were also found to be antimutators. Double mutants carrying rev3 and mutator mutations of rad3, rad51 or rad18 are like rev3 single mutants with respect to spontaneous mutation rate, supporting the hypothesis (Hastings, Quah and von Borstel 1976) that many mutators in yeast act by channelling spontaneous lesions from accurate to mutagenic repair. However, the enhanced mutation rate seen in a radiation-resistant mutator mutant mut1 is not dependent on REV3, but is dependent on another gene designated ANT1. An additive effect on the reduction in spontaneous mutation, seen in the ant1 rev3 double-mutant strain, leads to the conclusion that at least 90% of spontaneous mutations seen in the wild type are caused by mutagenic repair of spontaneous lesions.

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Spontaneous Mutation Rate of Measles Virus: Direct Estimation Based on Mutations Conferring Monoclonal Antibody Resistance

Journal of Virology ◽

10.1128/jvi.73.1.51-54.1999 ◽

1999 ◽

Vol 73 (1) ◽

pp. 51-54 ◽

Cited By ~ 64

Author(s):

Stephanie J. Schrag ◽

Paul A. Rota ◽

William J. Bellini

Keyword(s):

Monoclonal Antibody ◽

Mutation Rate ◽

Measles Virus ◽

Spontaneous Mutation ◽

Single Point ◽

Test Method ◽

Mutation Rates ◽

Viral Population ◽

Single Point Mutation ◽

Direct Estimate

ABSTRACT High mutation rates typical of RNA viruses often generate a unique viral population structure consisting of a large number of genetic microvariants. In the case of viral pathogens, this can result in rapid evolution of antiviral resistance or vaccine-escape mutants. We determined a direct estimate of the mutation rate of measles virus, the next likely target for global elimination following poliovirus. In a laboratory tissue culture system, we used the fluctuation test method of estimating mutation rate, which involves screening a large number of independent populations initiated by a small number of viruses each for the presence or absence of a particular single point mutation. The mutation we focused on, which can be screened for phenotypically, confers resistance to a monoclonal antibody (MAb 80-III-B2). The entire H gene of a subset of mutants was sequenced to verify that the resistance phenotype was associated with single point mutations. The epitope conferring MAb resistance was further characterized by Western blot analysis. Based on this approach, measles virus was estimated to have a mutation rate of 9 × 10−5 per base per replication and a genomic mutation rate of 1.43 per replication. The mutation rates we estimated for measles virus are comparable to recent in vitro estimates for both poliovirus and vesicular stomatitis virus. In the field, however, measles virus shows marked genetic stability. We briefly discuss the evolutionary implications of these results.

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