Quantifying the risks of invasion by genetically engineered organisms.
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Gene Frequency↗
Biomedical subjects
D E Dykhuizen
Publications and source records attributed to D E Dykhuizen.
Recombination in Escherichia coli and the definition of biological species.
The DNA sequence of part of the gnd (6-phosphogluconate dehydrogenase) gene was determined for eight wild strains of Escherichia coli and for Salmonella typhimurium. Since a region of the trp (tryptophan) operon and the phoA (alkaline phosphatase) gene have been sequenced from the same strains, the gene trees for these three regions were determined and compared. Gene trees are different from species or strain trees in that a gene tree is derived from a particular segment of DNA, whereas a species or strain tree should be derived from many such segments and is the tree that best represents the phylogenetic relationship of the species or strains. If there were no recombination in E. coli, the gene trees for different genes would not be statistically different from the strain tree or from each other. But, if the gene trees are significantly different, there must have been recombination. Methods are proposed that show these gene trees to be statistically different. Since the gene trees are different, we conclude that recombination is important in natural populations of E. coli. Finally, we suggest that gene trees can be used to create an operational means of defining bacterial species by using the biological species definition.
Alleles↗
Evolution. Mountaineering with microbes.
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Adaptation, Physiological↗
Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli.
Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.
Blotting, Southern↗
Genetic exchange among natural isolates of bacteria: recombination within the phoA gene of Escherichia coli.
An 1871-nucleotide region including the phoA gene (the structural gene encoding alkaline phosphatase, EC 3.1.3.1) was cloned and sequenced from eight naturally occurring strains of Escherichia coli. Alignment with the sequence from E. coli K-12 made apparent that there were 87 polymorphic nucleotide sites, of which 42 were informative for phylogenetic analysis. Maximum parsimony analysis revealed six equally parsimonious trees with a consistency index of 0.80. Of the 42 informative sites, 22 were inconsistent with each of the maximum parsimony trees. The spatial distribution of the inconsistent sites was highly nonrandom in a manner implying that intragenic recombination has played a major role in determining the evolutionary history of the nine alleles. The implication is that different segments of the phoA gene have different phylogenetic histories.
Alkaline Phosphatase↗
Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli.
Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey-lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.
Alleles↗
Fitness effects of a deletion mutation increasing transcription of the 6-phosphogluconate dehydrogenase gene in Escherichia coli.
Directed evolution in microbial organisms provides an experimental approach to molecular evolution in which selective forces can be controlled and favorable mutations analyzed at the molecular level. Here we present an analysis of a mutation selected in Escherichia coli in response to growth in a chemostat in which the limiting nutrient was gluconate. The selectively favored mutation, designated gnd+ (862), occurred in the gene gnd coding for 6-phosphogluconate dehydrogenase, used in gluconate metabolism. Although the allele is strongly favored in chemostats in which the limiting nutrient is gluconate, the selective effects of gnd+ (862) are highly dependent on growth conditions. In chemostats in which growth is limited by a mixture of gluconate and either ribose, glucose, or succinate, the gnd+ (862) allele is favored, disfavored, or neutral according to the relative concentrations of the substrates. The gnd+ (862) allele results from a deletion of 385 nucleotide pairs in the region 5' to the promoter of gnd, and one endpoint of the deletion is contiguous with the terminus of an IS5 insertion sequence located near gnd in E. coli K12. The gnd+ (862) allele shows a marked increase in transcription that accounts for most or all of the increased enzyme activity.
Alleles↗
Confidence interval for the number of selectively neutral amino acid polymorphisms.
A statistical approach to the analysis of DNA sequences has been developed, which provides a confidence interval estimate for the proportion of naturally occurring amino acid polymorphisms that are selectively neutral. When applied to the gnd gene coding for 6-phosphogluconate dehydrogenase in a sample of seven natural isolates of Escherichia coli, the method indicates that the proportion of observed amino acid polymorphisms that are selectively neutral is unlikely to be greater than 49% (upper 95% confidence limit). On the other hand, the observations are also consistent with a model in which all of the observed amino acid substitutions are mildly deleterious with an average selection coefficient approximating 1.6 X 10(-7). Various models for the distribution of configurations at silent sites are also investigated.
Amino Acids↗
Metabolic flux and fitness.
Studies of Escherichia coli under competition for lactose in chemostat cultures have been used to determine the selective effects of variation in the level of the beta-galactoside permease and the beta-galactosidase enzyme. The results determine the adaptive topography of these gene products relative to growth in limiting lactose and enable predictions concerning the selective effects of genetic variants found in natural populations. In the terms of metabolic control theory, the beta-galactosidase enzyme at wild-type-induced levels has a small control coefficient with respect to fitness (C = 0.018), and hence genetic variants resulting in minor changes in enzyme activity have disproportionately small effects on fitness. However, the apparent control coefficient of the beta-galactoside permease at wild-type-induced levels is large (C = 0.551), and hence even minor changes in activity affect fitness. Therefore, we predict that genetic polymorphisms in the lacZ gene are subject to less effective selection in natural populations than are those in the lacY gene. The beta-galactoside permease is also less efficient than might be expected, and possible forces resulting in selection for an intermediate optimum level of permease activity are considered. The selective forces that maintain the lactose operon in a regulated state in natural populations are also discussed.
Bacterial Proteins↗
Distribution and abundance of insertion sequences among natural isolates of Escherichia coli.
A reference collection of 71 natural isolates of Escherichia coli (the ECOR collection) has been studied with respect to the distribution and abundance of transposable insertion sequences using DNA hybridization. The data include 1173 occurrences of six unrelated insertion sequences (IS1, IS2, IS3, IS4, IS5 and IS30). The number of insertion elements per strain, and the sizes of DNA restriction fragments containing them, is highly variable and can be used to discriminate even among closely related strains. The occurrence and abundance of pairs of unrelated insertion sequences are apparently statistically independent, but significant correlations result from stratifications in the reference collection. However, there is a highly significant positive association among the insertion sequences considered in the aggregate. Nine branching process models, which differ in assumptions regarding the regulation of transposition and the effect of copy number on fitness, have been evaluated with regard to their fit of the observed distributions. No single model fits all copy number distributions. The best models incorporate no regulation of transposition and a moderate to strong decrease in fitness with increasing copy number for IS1 and IS5, strong regulation of transposition and a negligible to weak decrease in fitness with increasing copy number for IS3, and less than strong regulation of transposition for IS2, IS4 and IS30.
Base Sequence↗
The evolution of DNA sequences in Escherichia coli.
It is proposed that certain families of transposable elements originally evolved in plasmids and functioned in forming replicon fusions to aid in the horizontal transmission of non-conjugational plasmids. This hypothesis is supported by the finding that the transposable elements Tn3 and gamma delta are found almost exclusively in plasmids, and also by the distribution of the unrelated insertion sequences IS4 and IS5 among a reference collection of 67 natural isolates of Escherichia coli. Each insertion sequence was found to be present in only about one-third of the strains. Among the ten strains found to contain both insertion sequences, the number of copies of the elements was negatively correlated. With respect to IS5, approximately half of the strains containing a chromosomal copy of the insertion element also contained copies within the plasmid complement of the strain.
Base Sequence↗
Fitness as a function of beta-galactosidase activity in Escherichia coli.
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Escherichia coli↗
Joint distribution of insertion elements IS4 and IS5 in natural isolates of Escherichia coli.
A reference collection of natural isolates of Escherichia coli has been studied in order to determine the distribution, abundance and joint occurrence of DNA insertion elements IS4 and IS5. Among these isolates, 36% were found to contain IS4 and 30% were found to contain IS5. Among strains containing IS4 the mean number of copies per strain was 4.4 +/- 0.8; the comparable figure for IS5 was 3.7 +/- 1.0. Although the presence of the elements among the isolates was independent, among those isolates containing both IS4 and IS5, there was a significant negative correlation in the number of copies of the elements. The reference collection was also studied for the presence of the DNA sequences flanking the single copy of IS4 in the chromosome of E. coli K12. Homologous sequences were found in only 26% of the isolates. The sequences flanking the IS4 invariably occur together, and their presence is significantly correlated with the presence of IS4. In eight of the strains that carry these flanking sequences, an IS4 is located between them, and the sequences are present at the homologous position as in the K12 strain. We suggest that IS4 and its flanking sequences share a common mechanism of dissemination, such as plasmids, and we present evidence that they are included in a much larger transposable element.
Base Composition↗
Limits of adaptation: the evolution of selective neutrality.
Many enzymes in intermediary metabolism manifest saturation kinetics in which flux is a concave function of enzyme activity and often of the Michaelis-Menten form. The result is that, when natural selection favors increased enzyme activity so as to maximize flux, a point of diminishing returns will be attained in which any increase in flux results in a disproportionately small increase in fitness. Enzyme activity ultimately will reach a level at which the favorable effect of an increase in activity is of the order 1/(4Ne) or smaller, where Ne is the effective population number. At this point, many mutations that result in small changes in activity will result in negligible changes in fitness and will be selectively nearly neutral. We propose that this process is a mechanism whereby conditions for the occurrence of nearly neutral mutations and gene substitutions can be brought about by the long-continued action of natural selection. Evidence for the hypothesis derives from metabolic theory, direct studies of flux, studies of null and other types of alleles in Drosophila melanogaster and chemostat studies in Escherichia coli. Limitations and complications of the theory include changes in environment or genetic background, enzymes with sharply defined optima of activity, overdominance, pleiotropy, multifunctional enzymes and branched metabolic pathways. We conclude that the theory is a useful synthesis that unites many seemingly unrelated observations. The principal theoretical conclusion is that the conditions for the occurrence of neutral evolution can be brought about as an indirect result of the action of natural selection.
Adaptation, Physiological↗