Nonlethal Selection Research Articles

Effect of Non-Lethal Selection on Spontaneous Revertants of Frameshift Mutations: The Escherichia coli hisF Case

Microorganisms possess the potential to adapt to fluctuations in environmental parameters, and their evolution is driven by the continuous generation of mutations. The reversion of auxotrophic mutations has been widely studied; however, little is known about the reversion of frameshift mutations resulting in amino acid auxotrophy and on the structure and functioning of the protein encoded by the revertant mutated gene. The aims of this work were to analyze the appearance of reverse mutations over time and under different selective pressures and to investigate revertant enzymes’ three-dimensional structures and their correlation with a different growth ability. Escherichia coli FB182 strain, carrying the hisF892 single nucleotide deletion resulting in histidine auxotrophy, was subjected to different selective pressures, and revertant mutants were isolated and characterized. The obtained results allowed us to identify different indels of different lengths located in different positions in the hisF gene, and relations with the incubation time and the selective pressure applied were observed. Moreover, the structure of the different mutant proteins was consistent with the respective revertant ability to grow in absence of histidine, highlighting a correlation between the mutations and the catalytic activity of the mutated HisF enzyme.

The SNAP hypothesis: Chromosomal rearrangements could emerge from positive Selection during Niche Adaptation.

The relative linear order of most genes on bacterial chromosomes is not conserved over evolutionary timescales. One explanation is that selection is weak, allowing recombination to randomize gene order by genetic drift. However, most chromosomal rearrangements are deleterious to fitness. In contrast, we propose the hypothesis that rearrangements in gene order are more likely the result of selection during niche adaptation (SNAP). Partial chromosomal duplications occur very frequently by recombination between direct repeat sequences. Duplicated regions may contain tens to hundreds of genes and segregate quickly unless maintained by selection. Bacteria exposed to non-lethal selections (for example, a requirement to grow on a poor nutrient) can adapt by maintaining a duplication that includes a gene that improves relative fitness. Further improvements in fitness result from the loss or inactivation of non-selected genes within each copy of the duplication. When genes that are essential in single copy are lost from different copies of the duplication, segregation is prevented even if the original selection is lifted. Functional gene loss continues until a new genetic equilibrium is reached. The outcome is a rearranged gene order. Mathematical modelling shows that this process of positive selection to adapt to a new niche can rapidly drive rearrangements in gene order to fixation. Signature features (duplication formation and divergence) of the SNAP model were identified in natural isolates from multiple species showing that the initial two steps in the SNAP process can occur with a remarkably high frequency. Further bioinformatic and experimental analyses are required to test if and to which extend the SNAP process acts on bacterial genomes.

Evolution of antibiotic resistance at non-lethal drug concentrations

Human use of antimicrobials in the clinic, community and agricultural systems has driven selection for resistance in bacteria. Resistance can be selected at antibiotic concentrations that are either lethal or non-lethal, and here we argue that selection and enrichment for antibiotic resistant bacteria is often a consequence of weak, non-lethal selective pressures - caused by low levels of antibiotics - that operates on small differences in relative bacterial fitness. Such conditions may occur during antibiotic therapy or in anthropogenically drug-polluted natural environments. Non-lethal selection increases rates of mutant appearance and promotes enrichment of highly fit mutants and stable mutators.

The Origin of Mutants under Selection: Interactions of Mutation, Growth, and Selection.

The classical experiments of Luria and Delbrück showed convincingly that mutations exist before selection and do not contribute to the creation of mutations when selection is lethal. In contrast, when nonlethal selections are used,measuring mutation rates and separating the effects of mutation and selection are difficult and require methods to fully exclude growth after selection has been applied. Although many claims of stress-induced mutagenesis have been made, it is difficult to exclude the influence of growth under nonlethal selection conditions in accounting for the observed increases in mutant frequency. Instead, for many of the studied experimental systems the increase in mutant frequency can be explainedbetter by the ability of selection to detect small differences in growth rate caused by common small effect mutations. A verycommon mutant class,found in response to many different types of selective regimensin which increased gene dosage can resolve the problem, is gene amplification. In the well-studiedlac system of Cairns and Foster, the apparent increase in Lac+revertants can be explained by high-level amplification of the lac operon and the increased probability for a reversion mutation to occur in any one of the amplified copies. The associated increase in general mutation rate observed in revertant cells in that system is an artifact caused by the coincidental co-amplification of the nearby dinB gene (encoding the error-prone DNA polymerase IV) on the particular plasmid used for these experiments. Apart from the lac system, similar gene amplification processes have been described for adaptation to toxic drugs, growth in host cells, and various nutrient limitations.

Adaptive mutation: how growth under selection stimulates Lac(+) reversion by increasing target copy number.

From the time of Darwin until about 1950, a controversy continued over whether selective stress induces mutations or only affects the relative reproductive success of organisms with different genotypes (30). The controversy was resolved by the classic experiments of Luria and Delbruck (27) and of Leder- berg and Lederberg (25), who showed that some bacterial mutants arise prior to application of the selection that allows their detection and thus could not have been caused by selec- tive conditions. However, these experiments used lethal selec- tions and therefore did not eliminate the possibility that an- other fraction of total mutations might be formed in response to stress and be detected only by nonlethal selection. Shapiro and Cairns et al. reopened the controversy by pointing out this caveat and presenting data that seemed to support stress-in- duced mutation (7, 45). Because very few genetic systems behave in ways that sug- gest stress-induced mutation, the rare cases that seem to ex- hibit such behavior have attracted close attention. In one case, mutants were later shown to preexist selection (14, 28, 29, 44). For the system devised by Cairns and Foster (5), we suggest that reversion occurs by a multistep process initiated prior to selection and the appearance of stress-induced mutagenesis results from growth under strong selection.

Development of a non-lethal selection system by using the aadA marker gene for efficient recovery of transgenic rice (Oryza sativa L.).

The application of aminoglycoside-3"-adenyltransferase ( aadA) gene-mediated streptomycin resistance for non-lethal selection of transgenic rice resulted in plant regeneration frequencies under selection pressure as high as those in non-transformed controls without selection. Since streptomycin does not kill non-transgenic cells, and allows plant regeneration from them, a selection procedure was developed that made the visual identification of transgenic calli and regenerants possible. For callus-level selection, a vital pH indicator-Chlorophenol Red-was applied together with streptomycin, making use of the phenomenon that fast-growing cell lines lower the pH in the culture medium. Transgenic plants were selected according to their main distinctive features; their green colour (photomixotrophic assimilation), and more intense growth. At the same time, non-transgenic regenerants were bleached (heterotrophic assimilation), and growth was retarded in the presence of streptomycin and sucrose. The final efficiency of genetic transformation based on streptomycin resistance was found to be double that of transformations where the selective agent was l-phosphinothricin, and nearly three times more compared to transformations resulting in hygromycin-resistant regenerants. To the best of our knowledge, this is the first report on producing nuclear transformed rice plants by using a non-lethal selection strategy based on the chimaeric aadA gene.

Persistence of unselected transgenic DNA during a plastid transformation and segregation approach to herbicide resistance.

The use of a nonlethal selection scheme, most often using the aadA gene that confers resistance to spectinomycin and streptomycin, has been considered critical for recovery of plastid transformation events. In this study, the plastid-lethal markers, glyphosate or phosphinothricin herbicides, were used to develop a selection scheme for plastids that circumvents the need for integration of an antibiotic resistance marker. The effect of selective agents on tobacco (Nicotiana tabacum) mesophyll chloroplasts was first examined by transmission electron microscopy. We found that at concentrations typically used for selection of nuclear transformants, herbicides caused rapid disintegration of plastid membranes, whereas antibiotics had no apparent effect. To overcome this apparent herbicide lethality to plastids, a "transformation segregation" scheme was developed that used two independent transformation vectors for a cotransformation approach and two different selective agents in a phased selection scheme. One transformation vector carried an antibiotic resistance (aadA) marker used for early nonlethal selection, and the other transformation vector carried the herbicide (CP4 or bar) resistance marker for use in a subsequent lethal selection phase. Because the two markers were carried on separate plasmids and were targeted to different locations on the plastid genome, we reasoned that segregation of the two markers in some transplastomic lines could occur. We report here a plastid cotransformation frequency of 50% to 64%, with a high frequency (20%) of these giving rise to transformation segregants containing exclusively the initially nonselected herbicide resistance marker. Our studies indicate a high degree of persistence of unselected transforming DNA, providing useful insights into plastid chromosome dynamics.

The effect of genomic position on reversion of a lac frameshift mutation (lacIZ33) during non-lethal selection (adaptive mutation).

In a system described by Cairns and Foster, starvation of a particular leaky lac mutant (lacIZ33) in the presence of lactose appears to direct mutation in non-growing cells to sites that allow growth (adaptive mutation). This behaviour requires that the lac operon be located on an F' plasmid. This position effect was investigated by placing the mutant lac operon at many sites in the genome of Salmonella enterica (Typhimurium; LT2) and testing reversion behaviour. Genomic position did not affect reversion during non-selective growth. When lac was at any of 550 chromosomal sites, starvation caused little or no enhancement of reversion. In the 28 strains with the lac on Salmonella's conjugative plasmid (pSLT), selection enhanced reversion strongly, just as seen for strains with lac on an F' plasmid. In 46 strains, the lac operon was inserted within a small chromosomal duplication, and selection stimulated RecA-dependent partial reversion by simple amplification (about 8x) of the mutant lac region. The position of lac on a conjugative plasmid is important to reversion because it allows more frequent gene duplication and amplification. These events are central to growth and reversion under selection because they increase the number of replicating lac alleles within each developing revertant clone.

A new experimental system for study on adaptive mutations

A super-repressed mutant of purR (purR(S)), which encodes a repressor protein controlling expression of purine biosynthetic genes inSalmonella typhimurium, grew very slowly on NCE medium with 10 mug/mL Ade and lactose as sole carbon source (cannot form colonies). However, a phenomenon of late-arising mutations was observed when purR(S) mutants were spread on NCE+lactose plates and subjected to a prolonged non-lethal selection. The reconstruction experiments of revertants showed that the late-arising "lac(+)" mutants are not slow growing mutants. Statistical analysis indicated that the distribution of late-arising mutants is Poisson distribution, showing that reversion occurred after plating. The result of co-transductional analysis preliminarily showed that late-arising mutation occurred at selected genepurR or 16 bp PUR box,cis element of structural genepurD. The above results suggest that the phenomenon of late-arising mutation observed by our system is a result of adaptive mutations which are different from random mutations. This is the first time to extend target genes at which adaptive mutations could occur from structural genes involved in carbon metabolism and amino acid biosynthesis totrans regulatory gene coding repressor protein. Our results have provided not only a new proof for generality of adaptive mutations but also a new system for study on adaptive mutations.

Adaptive mutation: implications for evolution.

Adaptive mutation is defined as a process that, during nonlethal selections, produces mutations that relieve the selective pressure whether or not other, nonselected mutations are also produced. Examples of adaptive mutation or related phenomena have been reported in bacteria and yeast but not yet outside of microorganisms. A decade of research on adaptive mutation has revealed mechanisms that may increase mutation rates under adverse conditions. This article focuses on mechanisms that produce adaptive mutations in one strain of Escherichia coli, FC40. These mechanisms include recombination-induced DNA replication, the placement of genes on a conjugal plasmid, and a transient mutator state. The implications of these various phenomena for adaptive evolution in microorganisms are discussed.

Transposon stability and a role for conjugational transfer in adaptive mutability.

Lac(+) revertants of Escherichia coli that occur after prolonged nonlethal selection display a high frequency of transposon loss when the transposon Tn10 and the reverting lacI33 allele are linked on an F'128 episome. As many as 20% of the Lac(+) revertants are sensitive to tetracycline, about half because of transposon loss, nearly all by precise excision, and the remainder because of amplification of both the transposon and the linked lac allele. Lethality of the amplified products in the presence of tetracycline is a peculiarity of the tetA gene at high gene dosage. The selective conditions on lactose medium result in 10% transposon-free revertants, whether or not a requirement for conjugal DNA transfer is imposed. In addition, a similar fraction, about 5% of Lac(-) unreverted colonies that are products of transfer between cells experiencing nonlethal selection are also tetracycline-sensitive, and all are attributable to loss of the Tn10 transposon. These results suggest the possibility that the high frequency of transposon loss is a consequence of conjugal transfer, making this loss a marker for that transfer. We suggest that conjugal DNA transfer may be a prominent feature in the mutability process that occurs during nonlethal selection and that the subset of bacteria displaying hypermutability are those that experience such transfer.

Some features of the mutability of bacteria during nonlethal selection.

We describe the mutability of the Trp(-) chromosomal +1 frameshift mutation trpE7999 during nonlethal selection, finding that the appearance of Trp(+) revertants behaves similarly to that of episomal Lac(+) revertants. In addition, we show that a feature of the Lac(+) and Trp(+) mutability is the accumulation of Trp(+) and Lac(+) revertants with additional unselected mutations, most of which are not due to heritable mutators. The cells undergoing nonlethal selection apparently experience an epigenetic change resulting in a subset of bacteria with elevated mutability that often remain hypermutable for the duration of selection. The epigenetic change provoked by nonlethal selection appears to be mediated by a unique function provided by the F'128 episome.

Adaptive mutation in Escherichia coli.

We usually think of spontaneous mutations as the result of replication errors made while cells are growing exponentially. That mutations do arise during cell proliferation and without regard to selection was, of course, shown by Luria and Delbruck (1943). Several years ago, John Cairns and his collaborators (Cairns et al. 1988) demonstrated that mutations can also arise in nonproliferating cells when they are subjected to a nonlethal selection. These experiments extended earlier observations made by Francis Ryan (Ryan 1955; Ryan et al. 1961) and James Shapiro (Shapiro 1984). Luria and Delbruck’s proof that mutations arise at random in a growing population was based on large fluctuations in mutant numbers among parallel cultures of bacteria. These large fluctuations occur because clones of different sizes are produced by mutants that appear at different points during the growth of the cultures. Cairns et al. (1988) used the absence of large fluctuations in mutant numbers among parallel cultures to demonstrate that mutations also arise after selection had been imposed. Because on solid selective medium each mutant produces a colony of progeny cells, and so is counted as one, the distribution of mutant numbers is Poisson with a variation equal to its mean. Luria and Delbruck could not have seen this Poisson component; the selections they used were lethal, so no mutants could have arisen after selection was imposed, as Delbruck himself pointed out at the 1946 Cold Spring Harbor Symposium (see Lwoff 1946, p. 154). Although the occurrence of mutations in nonproliferating cells is an interesting and potentially important phenomenon, what was startling about the results of Cairns et al. (1988) was that mutations appeared to be “directed” by the selective conditions; i.e., while selected mutations were accumulating, deleterious or neutral mutations were not. As Cairns put it “populations of bacteria, in stationary phase, have some way of producing (or selectively retaining) only the most appropriate mutations” (Cairns et al. 1988). Some of the more dramatic cases of “directed” mutation have now been shown to have other causes, so at this point we are left with rather few examples of apparent directedness that are still unexplained (for review, see Foster 1999b). The phenomenon has come to be called “adaptive mutation” by which is meant a process that during nonlethal selection produces mutations that relieve the selected pressure, whether or not other nonselected mutations are also occurring (Foster 1999b). Work in my laboratory has focused on a strain of Escherichia coli, FC40, that cannot utilize lactose (Lac−) but that readily reverts to lactose utilization (Lac+) when plated on medium with lactose as the sole carbon and energy source (Cairns and Foster 1991). These Lac+ mutations are not “directed” in the original sense because non-selected mutations in another gene closely linked to lac also accumulate in the Lac− population during lactose selection (Foster 1997). Nonetheless, research on FC40 has provided insights into mechanisms by which mutations can occur in nonproliferating cells. In the first part of this paper, I review the characteristics of adaptive Lac+ mutation in FC40 and present a model for how the mutations are produced. Included are new data indicating that three of E. coli’s DNA polymerases are active in this process. In the second part of the paper, I focus on the occurrence of transient mutators in populations of cells under selection and discuss possible mechanisms by which mutation rates can be temporarily increased.

The role of transient hypermutators in adaptive mutation in Escherichia coli.

Microbial populations under nonlethal selection can give rise to mutations that relieve the selective pressure, a phenomenon that has come to be called "adaptive mutation." One explanation for adaptive mutation is that a small proportion of the cells experience a period of transient hypermutation, and that these hypermutators account for the mutations that appear. The experiments reported here investigated the contribution that hypermutators make to the mutations occurring in a Lac- strain of Escherichia coli during selection for lactose utilization. A broad mutational screen, loss of motility, was used to compare the frequency of nonselected mutations in starved Lac- cells, in Lac+ revertants, and in Lac+ revertants carrying yet another nonselected mutation. These frequencies allowed us to calculate that the hypermutating subpopulation makes up approximately 0.06% of the population and that its mutation rate is elevated approximately 200-fold. From these numbers we conclude that the hypermutators are responsible for nearly all multiple mutations but produce only approximately 10% of the adaptive Lac+ mutations.

Mechanisms of mutation in nondividing cells. Insights from the study of adaptive mutation in Escherichia coli.

When populations of cells are subjected to nonlethal selection, mutations arise in the absence of cell division, a phenomenon that has been called "adaptive mutation." In a strain of Escherichia coli that cannot metabolize lactose (Lac-) but that reverts to lactose utilization (Lac+) when lactose is its sole energy and carbon source, the mutational process consists of two components. (1) A highly efficient, recombination-dependent mechanism giving rise to mutations on the F' episome that carries the Lac- allele; and (2) a less efficient, unknown mechanism giving rise to mutations elsewhere in the genome. Both selected and nonselected mutations arise in the Lac- population, but nonselected mutations are enriched in Lac+ mutants, suggesting that some Lac+ cells have passed though a transient period of increased mutation. These results have several evolutionary implications. (1) DNA synthesis initiated by recombination could be an important source of spontaneous mutation, particularly in cells that are not undergoing genomic replication. (2) The highly active mutational mechanism on the episome could be important in the horizontal transfer of variant alleles among species that carry and exchange conjugal plasmids. (3) A sub-population of cells in a state of transient mutation could be a source of multiple variant alleles and could provide a mechanism for rapid adaptive evolution under adverse conditions.

Hypermutation in stationary-phaseE. coli: tales from thelac operon

Escherichia coli cells are capable of complex regulatory responses to environmental conditions and stresses. In some circumstances, the response includes an increase in the mutation rate, effectively mutagenizing the genome. The classic example is the SOS response to DNA damage. Recent work indicates that other environmental stresses can also result in mutation of the genome. Modulation of mutation rate may be a more prevalent stress response than previously thought. In this review we focus on genome-wide mutation inE. coli cells subjected to a nonlethal genetic selection for reversion of alac frameshift allele. Reversion of thelac frameshift allele occurs via a novel mechanism that requires homologous recombination functions, and is enhanced by transiently diminished postsynthesis mismatch repair. A model for recombination-dependent stationary-phase mutation will be presented and its relevance for genome-wide mutation discussed.

Comparison of spontaneous and adaptive mutation spectra in yeast

Adaptive mutations occur in nongrowing populations of cells to overcome strong, nonlethal selection conditions. Several models have been proposed for the molecular mechanism(s) for this phenomenon inEscherichia coli, but the mechanisms involved in adaptive mutagenesis in the yeastSaccharomyces cerevisiae are largely unknown. We present here a comparison of the mutational spectra of spontaneous and adaptive frameshift reversion events in yeast. In contrast to results fromE. coli, we find that the mutational spectrum of adaptive mutations inS. cerevisiae is not similar to that seen in mismatch repair defective cells, but rather resembles the spontaneous mutational events that occur during normal growth.

Adaptive mutagenesis at ebgR is regulated by PhoPQ.

Adaptive mutations are mutations that occur in nondividing or very slowly dividing microbial cells during prolonged nonlethal selection and that are specific to the challenge of the selection in the sense that the only mutations that can be detected are those that provide a growth advantage to the cell. The phoPQ genes encode a two-component positively acting regulatory system that controls expression of at least 25 to 30 genes in Escherichia coli and Salmonella typhimurium. PhoPQ responds to a variety of environmental stress signals including Mg2+ starvation and nutritional deprivation. Here I show that disruption of phoP or phoQ by Tn10dCam significantly reduces the adaptive mutation rate to ebgR, indicating that the adaptive mutagenesis machinery is regulated, directly or indirectly, by phoPQ. The finding that it is regulated implies that adaptive mutagenesis does not simply result from a failure of various error correction mechanisms during prolonged starvation.

On the specificity of adaptive mutations.

Adaptive mutations are mutations that occur in nondividing or slowly dividing cells during prolonged nonlethal selection, and that appear to be specific to the challenge of the selection in the sense that the only mutations that arise are those that provide a growth advantage to the cell. The issue of the specificity has been controversial because it violates our most basic assumptions about the randomness of mutations with respect to their effect on the cell. Although a variety of experiments in several systems in both bacteria and yeast have claimed to demonstrate that specificity, those experiments have been subjected to a variety of technical criticisms suggesting that the specificity may not be real. Here I use the ebg system to provide evidence that when selection is applied to one specific nucleotide site within a gene, mutation occurs at that site but not at an alternative and equally mutable site within the same gene.

Green fluorescent protein expressed in living mosquitoes--without the requirement of transformation.

Mosquitoes transmit viruses, protozoa and nematodes that are major causes of morbidity and mortality in humans. Details of arthropod anatomy and development, and the replication and development of pathogens in the arthropod vector, have relied upon examination of dissected or histologically processed material. We constructed a double-subgenomic Sindbis (dsSIN) virus expressing green fluorescent protein to demonstrate the potential of this protein for studying pathogen development in living arthropods. We were able to observe dissemination of virus, and furthermore, it was possible to observe components of the nervous system of mosquito larvae in extraordinary detail and record this on video tape. Although green fluorescent protein has been used as a reporter gene in a number of organisms, expression has relied upon transformation of cells or embryos. Transformation technology has limited applicability, thus we have described an alternative system that, due to the broad host range and viral tropisms of dsSIN viruses, may be useful to scientists in a range of disciplines. Green fluorescent protein may also provide a non-lethal selection method for use in transgenic arthropod research.