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A central assumption of models of molecular evolution, that each site in a sequence evolves independently of all other sites, lacks empirical support. We investigated the extent to which sites evolve codependently in triosephosphate isomerase (TIM), a ubiquitous glycolytic enzyme conserved in both structure and function. Codependencies among sites, or concomitantly variable codons (covarions), are evident from the reduced function and misfolding of hybrid TIM proteins. Although they exist, we find covarions are relatively rare, and closely related proteins are unlikely to have developed them. However, the potential for covarions increases with genetic distance so that highly divergent proteins may have evolved codependencies between many sites. The evolution of covarions undermines a key assumption in phylogenetics and calls into question our ability to disentangle ancient relationships among major taxonomic groups. covarion | phylogeny | hybrid incompatibilities

The mechanism by which protein functional diversity expands is an important evolutionary issue. Studies of recently evolved chimeric genes permit direct investigation of the origin of new protein functions before they become obscured by subsequent evolution. Found in several African Drosophila species, jingwei (jgw), a recently evolved gene with a domain derived from the still extant short-chain alcohol dehydrogenase (ADH) through retroposition, provides an opportunity to examine this previously undescribed process directly. We expressed JGW proteins in a microbial expression system and, after purification, investigated their enzymatic properties. We found that, unexpectedly, positive Darwinian selection for amino acid replacements outside the active site of JGW produced a novel dehydrogenase with altered substrate specificity compared with the ancestral ADH. Instead of detoxifying and assimilating ethanol like its Adh parental gene, we observe that JGW efficiently utilizes long-chain primary alcohols found in hormone and pheromone metabolism. These data suggest that protein functional diversity can expand rapidly under the joint forces of exon shuffling, gene duplication, and natural selection.

We used a combination of genomic techniques to monitor chromosomal evolution across hundreds of generations as Escherichia coli adapted to growth-limiting concentrations of either lactulose, methyl-galactoside, or a 72:28 mixture of the two. DNA microarrays identified 8 unique duplications and 16 unique deletions among 42 evolvants from 23 chemostat experiments. Each mutation was confirmed by sequencing PCR-amplified flanking genomic DNA and, except for one deletion, an insertion sequence was found at the break point, vPCR of insertion sequences identified these same mutations and 16 additional insertions (all confirmed by sequencing). The pattern of genomic evolution is highly reproducible. Statistical analyses show that duplications at lac and mutations in mgl are adaptations specific to lactulose and to methyl-galactoside, respectively. Adaptation to mixed sugars is characterized by similar mutations, but lac duplications and mgl mutations usually arise in different backgrounds, producing ecological specialists for each sugar. This suggests that an antagonistic pleiotropic tradeoff between duplications at lac and mutations in mgl retards the evolution of generalists. Other mutations that repeatedly appear in replicate experiments are adaptations to the chemostat environment and are not specific to one or the other sugar.

Gene regulation is commonly assumed to have evolved in response to environmental variability. Although tightly regulated in Escherichia coli strain K12, transcriptional control of arginine biosynthesis is deregulated in strain B. Caused by a single amino acid replacement in the arginine repressor, these contrasting regulatory strategies result in a fitness tradeoff. The K12 repressor is selectively favored in the presence of arginine and disfavored in its absence. In environments that cycle between high and low arginine, short seasons favor the K12 allele, whereas long seasons favor the B allele. Unexpectedly then, deregulated expression is adaptive in some seasonal habitats.

The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD (calculated as the ratio of [k.sub.cat]/[K.sub.m). Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to introduce six substitutions in the adenosine binding pocket that systematically shift coenzyme preference toward NAD. The engineered enzyme displays an 850-fold preference for NAD over NADP, which exceeds the 140-fold preference displayed by a homologous NAD-dependent enzyme. Of the six mutations introduced, only one is identical in all related NAD-dependent enzyme sequences - strict adherence to homology as a criterion for replacing these amino acids impairs function. Two additional mutations at remote sites improve performance further, resulting in a final mutant enzyme with kinetic characteristics and coenzyme preference comparable to naturally occurring homologous NAD-dependent enzymes.

Existing theory predicts competitors (species or genetic clones) cannot coexist in a fluctuating environment unless relative fitness is negatively frequency-dependent (relative fitness declines as the frequency of a competitor increases). We develop simple theory to show coexistence does not require frequency-dependent selection, and we confirm this prediction by direct experiment. The conditions for coexistence in a fluctuating environment are precisely the same as those for coexistence in a spatially variable environment, conditions that arise naturally whenever population abundances are bounded. Simulations show the likelihood of coexistence increases with environmental uncertainty. The capacity of temporally variable environments to maintain biological diversity is far broader than generally envisaged. [ABSTRACT FROM AUTHOR]

We used a combination of genomic techniques to monitor chromosomal evolution across hundreds of generations as Escherkhia coil adapted to growth-limiting concentrations of either lactulose, methyl-galactoside, or a 72:28 mixture of the two. DNA microarrays identified 8 unique duplications and 16 unique deletions among 42 evolvants from 23 chemostat experiments. Each mutation was confirmed by sequencing PCR-amplified flanking genomic DNA and, except for one deletion, an insertion sequence was found at the break point. vPCR of insertion sequences identified these same mutations and 16 additional insertions (all confirmed by sequencing). The pattern of genomic evolution is highly reproducible. Statistical analyses show that duplications at lac and mutations in mgl are adaptations specific to lactulose and to methyl-galactoside, respectively. Adaptation to mixed sugars is characterized by similar mutations, but lac duplications and mgl mutations usually arise in different backgrounds, producing ecological specialists for each sugar. This suggests that an antagonistic pleiotropic tradeoff between duplications at lac and mutations in mgl retards the evolution of generalists. Other mutations that repeatedly appear in replicate experiments are adaptations to the chemostat environment and are not specific to one or the other sugar. [ABSTRACT FROM AUTHOR]

Describes the identification of amino acids responsible for differing coenzyme specificities. X-ray crystallographic structures of Escherichia coli isocitrate dehydrogenase and Thermus thermophilus NAD-dependent isopropylmalate dehydrogenase; Niche expansion during growth on acetate; Site-directed mutagenesis.

Existing theory predicts competitors (species or genetic clones) cannot coexist in a fluctuating environment unless relative fitness is negatively frequency-dependent (relative fitness declines as the frequency of a competitor increases). We develop simple theory to show coexistence does not require frequency-dependent selection, and we confirm this prediction by direct experiment. The conditions for coexistence in a fluctuating environment are precisely the same as those for coexistence in a spatially variable environment, conditions that arise naturally whenever population abundances are bounded. Simulations show the likelihood of coexistence increases with environmental uncertainty. The capacity of temporally variable environments to maintain biological diversity is far broader than generally envisaged.