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Gravity and land plant evolution: experimental induction of lignification by simulated hypergravity and water stress.

The ability to lignify cell walls has long been associated with the upright condition and sometimes massive body of the land plant, and gravitational regulation of lignification has been demonstrated by clinostat/centrifuge techniques. Previous centrifuge experiments were conducted using plants in the early stages of growth, hence already programmed for immediate lignification. Elodea, an aquatic Angiosperm long believed to be incapable of lignification synthesizes lignin or lignin-like phenolics during 6 days incubation at 80 g . Yields in some cases exceeded stationary controls by as much as 300%. Supporting data were obtained using bean cotyledons. Exposure to 80 g induced lignification in extra-vascular tissue along with vascular lignin deposits. The loss of a buoyant medium also deprived early land plants of unlimited water: the lignin content in cucumber seedlings grown in water is reduced 50% or more relative to pot grown plants but water stress elevates lignin content as much as 100%. This suggests that the water status of the land environment may have provided a "backup" or "reinforcement" signal for an adaptive axial support system.

Biological Evolution↗

Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution.

We improved the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Thermus thermophilus, as a host cell. The leuB gene encoding B. subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a leuB-deficient strain of T. thermophilus. The resulting transformant showed a leucine-autotrophy at 56 degrees C but not at 61 degrees C and above. Phenotypically thermostabilized strains that can grow at 61 degrees C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70 degrees C and mutants that showed a leucine-autotrophic growth at 70 degrees C were obtained. DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine. The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme. Furthermore, the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type enzyme.

3-Isopropylmalate Dehydrogenase↗

Experimental evolution of a dense cluster of residues in tyrosyl-tRNA synthetase: quantitative effects on activity, stability and dimerization.

A dense cluster of eight residues was identified at the crossing of two alpha-helices in tyrosyl-tRNA synthetase (TyrRS) from the thermophile Bacillus stearothermophilus. Its mechanism of evolution was characterized. Four residues of this cluster are not conserved in TyrRS from the mesophile Escherichia coli. The corresponding mutations were constructed in TyrRS(Delta1), a derivative of TyrRS from B. stearothermophilus in which the anticodon binding domain is deleted. Mutations I52L (i.e. Ile52 into Leu), M55L and L105V did not affect the activity of TyrRS(Delta1) in the pyrophosphate exchange reaction whereas T51P increased it. The kinetic stabilities of TyrRS(Delta1) and its mutant derivatives at 68.5 degreesC were determined from experiments of irreversible thermal precipitation. They were in the order L105V<I52L<T51P<Wild Type</=M55L; mutation I52L partially compensated L105V in these experiments whereas M55L was coupled neither to I52L nor to L105V. Mutations I52L and L105V affected the stability of the dimeric TyrRS(Delta1) at different steps of its unfolding by urea, monitored under equilibrium conditions by spectrofluorometry or size exclusion chromatography. I52L destabilized the association between the subunits even though residue Ile52 is more than 20 A away from the subunit interface. L105V destabilized the monomeric intermediate of unfolding. The two mutational pathways, going from the wild-type TyrRS(Delta1) to the I52L-L105V double mutant through each of the single mutants were not equivalent for the stability of the monomeric intermediate and for the total stability of the dimer. One pathway contained two neutral steps whereas the other pathway contained a destabilizing step followed by a stabilizing step. Mutation I52L allowed L105V along the first pathway and compensated it along the second pathway. Thus, the effects of I52L and L105V on stability depended on the structural context. The gain in activity due to T51P was at the expense of a slight destabilization.

Amino Acid Substitution↗

Experimental evolution of a novel pathway for glycerol dissimilation in Escherichia coli.

Wild-type Escherichia coli utilizes glycerol aerobically through an inducible pathway mediated by an ATP-dependent kinase and a glycerol 3-phosphate dehydrogenase which is a flavoprotein. A mutant, strain ECL424, employing a novel pathway for glycerol utilization was isolated. The novel pathway is mediated by an NAD-linked dehydrogenase and a dihydroxyacetone specific enzyme II of the phosphoenolpyruvate phosphotransferase system. This study describes the selection from strain ECL424, a derivative which grows more rapidly on glycerol. The derivative, strain ECL428, produces twice the parental levels of both the dehydrogenase and the enzyme II during growth on glycerol. The function of the dehydrogenase in wild-type cells is unknown, although hydroxyacetone (acetol), 3-hydroxy-2-butanone (acetoin), and 1-amino-2-propanone are gratuitous inducers. The induction can be prevented by glucose whose effect can be cancelled by external cyclic AMP. The effects of hydroxyacetone, glucose, and cyclic AMP are attenuated in the two mutants in which the dehydrogenase is produced at high basal levels. The dihydroxyacetone specific enzyme II is inducible by the substrate in both wild-type and mutant strains and serves for growth on the triose.

Aerobiosis↗

Modeling the impact of periodic bottlenecks, unidirectional mutation, and observational error in experimental evolution.

Antibiotic resistant bacteria are a constant threat in the battle against infectious diseases. One strategy for reducing their effect is to temporarily discontinue the use of certain antibiotics in the hope that in the absence of the antibiotic the resistant strains will be replaced by the sensitive strains. An experiment where this strategy is employed in vitro produces data which showed a slow accumulation of sensitive mutants. Here we propose a mathematical model and statistical analysis to explain this data. The stochastic model elucidates the trend and error structure of the data. It provides a guide for developing future sampling strategies, and provides a framework for long term predictions of the effects of discontinuing specific antibiotics on the dynamics of resistant bacterial populations.

Bacteria↗

Experimental evolution of mating discrimination in budding yeast.

Assortative mating, when individuals of similar phenotypes mate, likely plays a key role in preventing gene flow during speciation. Reinforcement occurs when two previously geographically separated (allopatric) groups meet after having evolved partial postzygotic isolation; they are selected to evolve or enhance assortative mating to prevent costly intergroup matings that produce only maladaptive or sterile hybrids. Studies in Drosophila have shown that the genetic architectures of mating discrimination could differ significantly with or without reinforcement, suggesting that the evolution of assortative mating may be more complicated than expected. To study the evolution of assortative mating, we evolved mating discrimination in populations of the budding yeast, Saccharomyces cerevisiae. After 36 cycles of selection, these cells are five times more likely to mate with each other than to their ancestors, despite detectable one-way gene flow between the selected and reference populations. Several individual cultures evolved mating discrimination by changing their mating kinetics, with some mating more rapidly and others more slowly than the ancestral population. Genetic analysis indicates that multiple mutations have accumulated to produce the altered mating preference. Our results show that subtle details of mating behavior can play an important role in the evolution of reproductive isolation.

Biological Evolution↗

The catalytic consequences of experimental evolution. Transition-state structure during catalysis by the evolved beta-galactosidases of Escherichia coli (ebg enzymes) changed by a single mutational event.

1. The first chemical step in the hydrolysis of galactosylpyridinium ions by the evolvant ebg enzyme is less sensitive to leaving-group acidity than in the case of the wild-type ebg enzyme, implying less glycone-aglycone-bond fission at the transition state. 2. The first chemical step in the hydrolysis of aryl galactosides by ebg enzyme is probably less sensitive to leaving-group acidity than in the case of ebg enzyme, possibly as a consequence of resulting in more effective proton donation to the leaving aglycone. 3. alpha-Deuterium kinetic isotope effects of 1.1(0) and beta-deuterium kinetic isotope effects of 1.0(0) were measured for the hydrolysis of galactosyl-enzyme intermediates derived from ebg and ebg enzymes: these effects are not compatible with reaction of the sugar ring through a 4C1-like conformation, or with an ionic glycosyl-enzyme intermediate. 4. The variation with pH of steady-state kinetic parameters for hydrolysis of p-nitrophenyl galactoside by ebg and ebg enzymes and of 3-methylphenyl beta-galactoside, 3,4-dinitrophenyl beta-galactoside and beta-galactosyl-3-bromopyridinium ion by ebg enzyme was measured. The steep, non-classical, fall in activity against p-nitrophenyl galactoside at low pH observed with ebg and ebg enzymes is not observed with ebg enzymes.

Catalysis↗

Large changes of transition-state structure during experimental evolution of an enzyme.

The question of whether, during the evolution of an enzyme, the transition state of the catalysed reaction is largely unchanged, or whether transition state and protein change together, was examined using the egb beta-galactosidases of Escherichia coli. Charge development at the first chemical state was assumed [Konstantinidis and Sinnott (1991) Biochem. J. 279, 587-593] to be proportional to delta delta G++, the ratio of second-order rate constants for the hydrolysis of beta-D-galactopyranosyl fluoride and 1-fluoro-D-galactopyranosyl fluoride, expressed as a free-energy difference. delta delta G++ (kJ.mol-1) falls from 10.4 for wild-type enzyme to 6.8 and 7.2 as a consequence of two different single amino-acid changes (which arise from single evolutionary events), to 6.3 as a consequence of the two amino-acid changes together, and then increases slightly to 7.3 as a consequence of a third single evolutionary change involving three further amino-acid changes.

Amino Acid Sequence↗

Characteristic genome rearrangements in experimental evolution of Saccharomyces cerevisiae.

Genome rearrangements, especially amplifications and deletions, have regularly been observed as responses to sustained application of the same strong selective pressure in microbial populations growing in continuous culture. We studied eight strains of budding yeast (Saccharomyces cerevisiae) isolated after 100-500 generations of growth in glucose-limited chemostats. Changes in DNA copy number were assessed at single-gene resolution by using DNA microarray-based comparative genomic hybridization. Six of these evolved strains were aneuploid as the result of gross chromosomal rearrangements. Most of the aneuploid regions were the result of translocations, including three instances of a shared breakpoint on chromosome 14 immediately adjacent to CIT1, which encodes the citrate synthase that performs a key regulated step in the tricarboxylic acid cycle. Three strains had amplifications in a region of chromosome 4 that includes the high-affinity hexose transporters; one of these also had the aforementioned chromosome 14 break. Three strains had extensive overlapping deletions of the right arm of chromosome 15. Further analysis showed that each of these genome rearrangements was bounded by transposon-related sequences at the breakpoints. The observation of repeated, independent, but nevertheless very similar, chromosomal rearrangements in response to persistent selection of growing cells parallels the genome rearrangements that characteristically accompany tumor progression.

Aneuploidy↗

Experimental evolution of aging, growth, and reproduction in fruitflies.

We report in this paper an evolutionary experiment on Drosophila that tested life-history theory and the evolutionary theory of aging. As theory predicts, higher extrinsic mortality rates did lead to the evolution of higher intrinsic mortality rates, to shorter lifespans, and to decreased age and size at eclosion; peak fecundity also shifted earlier in life. These results confirm the key role of extrinsic mortality rates in the evolution of growth, maturation, reproduction, and aging, and they do so with a selection regime that maintained selection on fertility throughout life while holding population densities constant.

Aging↗

Long-term experimental evolution in Escherichia coli. VI. Environmental constraints on adaptation and divergence.

The effect of environment on adaptation and divergence was examined in two sets of populations of Escherichia coli selected for 1000 generations in either maltose-or glucose-limited media. Twelve replicate populations selected in maltose-limited medium improved in fitness in the selected environment, by an average of 22.5%. Statistically significant among-population genetic variation for fitness was observed during the course of the propagation, but this variation was small relative to the fitness improvement. Mean fitness in a novel nutrient environment, glucose-limited medium, improved to the same extent as in the selected environment, with no statistically significant among-population genetic variation. In contrast, 12 replicate populations previously selected for 1000 generations in glucose-limited medium showed no improvement, as a group, in fitness in maltose-limited medium and substantial genetic variation. This asymmetric pattern of correlated responses suggests that small changes in the environment can have profound effects on adaptation and divergence.

Adaptation, Physiological↗

Long-term experimental evolution in Escherichia coli. IX. Characterization of insertion sequence-mediated mutations and rearrangements.

As part of a long-term evolution experiment, two populations of Escherichia coli B adapted to a glucose minimal medium for 10,000 generations. In both populations, multiple IS-associated mutations arose that then went to fixation. We identify the affected genetic loci and characterize the molecular events that produced nine of these mutations. All nine were IS-mediated events, including simple insertions as well as recombination between homologous elements that generated inversions and deletions. Sequencing DNA adjacent to the insertions indicates that the affected genes are involved in central metabolism (knockouts of pykF and nadR), cell wall synthesis (adjacent to the promoter of pbpA-rodA), and ill-defined functions (knockouts of hokB-sokB and yfcU). These genes are candidates for manipulation and competition experiments to determine whether the mutations were beneficial or merely hitchhiked to fixation.

Biological Evolution↗

Mode of selection and experimental evolution of antifungal drug resistance in Saccharomyces cerevisiae.

We show that mode of selection, degree of dominance of mutations, and ploidy are determining factors in the evolution of resistance to the antifungal drug fluconazole in yeast. In experiment 1, yeast populations were subjected to a stepwise increase in fluconazole concentration over 400 generations. Under this regimen, two mutations in the same two chromosomal regions rose to high frequency in parallel in three replicate populations. These mutations were semidominant and additive in their effect on resistance. The first of these mutations mapped to PDR1 and resulted in the overexpression of the ABC transporter genes PDR5 and SNQ2. These mutations had an unexpected pleiotropic effect of reducing the residual ability of the wild type to reproduce at the highest concentrations of fluconazole. In experiment 2, yeast populations were subjected to a single high concentration of fluconazole. Under this regimen, a single recessive mutation appeared in each of three replicate populations. In a genome-wide screen of approximately 4700 viable deletion strains, 13 were classified as resistant to fluconazole (ERG3, ERG6, YMR102C, YMR099C, YPL056C, ERG28, OSH1, SCS2, CKA2, SML1, YBR147W, YGR283C, and YLR407W). The mutations in experiment 2 all mapped to ERG3 and resulted in the overexpression of the gene encoding the drug target ERG11, but not PDR5 and SNQ2. Diploid hybrids from experiments 1 and 2 were less fit than the parents in the presence of fluconazole. In a variation of experiment 2, haploids showed a higher frequency of resistance than diploids, suggesting that degree of dominance and ploidy are important factors in the evolution of antifungal drug resistance.

Biological Evolution↗

Experimental evolution of a new enzymatic function. II. Evolution of multiple functions for ebg enzyme in E. coli.

The evolution of ebgo enzyme of Escherichia coli, an enzyme which is unable to hydrolyze lactose, lactulose, lactobionate, or galactose-arabinoside effectively, has been directed in successive steps so that the evolved enzyme is able to hydrolyze these galactosides effectively. I show that in order for a strain of E. coli with a lacZ deletion to evolve the ability to use lactobionate as a carbon source, a series of mutations must occur in the ebg genes, and that these mutations must be selected in a particular order. The ordered series of mutations constitutes an obligatory evolutionary pathway for the acquisition of a new function for ebgo enzyme. A comparison of newly evolved strains with parental strains shows that when ebg enzyme acquires a new function, its old functions often suffer; but that in several cases old functions are either unaffected or are improved. I conclude that divergence of functions catalyzed by an enzyme need not require gene duplication.

Biological Evolution↗

A cold-adapted protease engineered by experimental evolution system.

A new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which we developed for the screening of mutant subtilisins with increased activity at low temperature. The m-51 mutant showed 70% higher catalytic efficiency, expressed by the k(cat)/K(m) value, than the wild-type at 10 degrees C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate. This cold-adaptation was achieved mainly by the increase in the k(cat) value in a temperature-dependent manner. Genetic analysis revealed that m-51 had three mutations, Ala-->Thr at position -31 (A-31T) in the prodomain, Ala-->Val at position 88 (A88V), and Ala-->Thr at position 98 (A98T). From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V. The A-31T mutation severely constrained the autoprocessing-mediated maturation of the pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis. No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51. In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points. In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred. This mutual effect may be related to the enhanced activity of the double mutant.

Base Sequence↗