Genome Sequences of Escherichia coli B strains REL606 and BL21(DE3)

Escherichia coli K-12 and B have been the subjects of classical experiments from which much of our understanding of molecular genetics has emerged. We present here complete genome sequences of two E. coli B strains, REL606, used in a long-term evolution experiment, and BL21(DE3), widely used to express recombinant proteins. The two genomes differ in length by 72,304 bp and have 426 single base pair differences, a seemingly large difference for laboratory strains having a common ancestor within the last 67 years. Transpositions by IS1 and IS150 have occurred in both lineages. Integration of the DE3 prophage in BL21(DE3) apparently displaced a defective prophage in the λ attachment site of B. As might have been anticipated from the many genetic and biochemical experiments comparing B and K-12 over the years, the B genomes are similar in size and organization to the genome of E. coli K-12 MG1655 and have > 99% sequence identity over ∼ 92% of their genomes. E. coli B and K-12 differ considerably in distribution of IS elements and in location and composition of larger mobile elements. An unexpected difference is the absence of a large cluster of flagella genes in B, due to a 41 kbp IS1-mediated deletion. Gene clusters that specify the LPS core, O antigen, and restriction enzymes differ substantially, presumably because of horizontal transfer. Comparative analysis of 32 independently isolated E. coli and Shigella genomes, both commensals and pathogenic strains, identifies a minimal set of genes in common plus many strain-specific genes that constitute a large E. coli pan-genome.     

Rare missense and synonymous variants in UBE1 are associated with X-linked infantile spinal muscular atrophy

X-linked infantile spinal muscular atrophy (XL-SMA) is an X-linked disorder presenting with the clinical features hypotonia, areflexia, and multiple congenital contractures (arthrogryposis) associated with loss of anterior horn cells and infantile death. To identify the XL-SMA disease gene, we performed large-scale mutation analysis in genes located between markers DXS8080 and DXS7132 (Xp11.3–Xq11.1). This resulted in detection of three rare novel variants in exon 15 of UBE1 that segregate with disease: two missense mutations (c.1617 G→T, p.Met539Ile; c.1639 A→G, p.Ser547Gly) present each in one XL-SMA family, and one synonymous C→T substitution (c.1731 C→T, p.Asn577Asn) identified in another three unrelated families. Absence of the missense mutations was demonstrated for 3550 and absence of the synonymous mutation was shown in 7914 control X chromosomes; therefore, these results yielded statistical significant evidence for the association of the synonymous substitution and the two missense mutations with XL-SMA (p = 2.416 × 10⁻¹⁰, p = 0.001815). We also demonstrated that the synonymous C→T substitution leads to significant reduction of UBE1 expression and alters the methylation pattern of exon 15, implying a plausible role of this DNA element in developmental UBE1 expression in humans. Our observations indicate first that XL-SMA is part of a growing list of neurodegenerative disorders associated with defects in the ubiquitin-proteasome pathway and second that synonymous C→T transitions might have the potential to affect gene expression.     

Genomic divergence of Escherichia coli strains: evidence for horizontal transfer and variation in mutation rates.

This report describes the sequencing in the Escherichia coli B genome of 36 randomly chosen regions that are present in most or all of the fully sequenced E. coli genomes. The phylogenetic relationships among E. coli strains were examined, and evidence for the horizontal gene transfer and variation in mutation rates was determined. The overall phylogenetic tree indicated that E. coli B and K-12 are the most closely related strains, with E. coli O157:H7 being more distantly related, Shigella flexneri 2a even more, and E. coli CFT073 the most distant strain. Within the B, K-12, and O157:H7 clusters, several regions supported alternative topologies. While horizontal transfer may explain these phylogenetic incongruities, faster evolution at synonymous sites along the O157:H7 lineage was also identified. Further interpretation of these results is confounded by an association among genes showing more rapid evolution and results supporting horizontal transfer. Using genes supporting the B and K-12 clusters, an estimate of the genomic mutation rate from a long-term experiment with E. coli B, and an estimate of 200 generations per year, it was estimated that B and K-12 diverged several hundred thousand years ago, while O157:H7 split off from their common ancestor about 1.5-2 million years ago.     

Genetic analysis of a plasmid-encoded, host genotype-specific enhancement of bacterial fitness.

In the absence of antibiotics, carriage of pACYC184 reduces the competitive fitness of an Escherichia coli B genotype that was not previously selected for plasmid carriage, relative to that of an isogenic plasmid-free competitor. However, a host genotype propagated with the plasmid for 500 generations evolved an unexpected competitive advantage from plasmid carriage, relative to its own isogenic plasmid-free segregant. We manipulated the pACYC184 genome in order to identify the plasmid-encoded function that was required for the enhancement of the coevolved host genotype's competitive fitness. Inactivation of the plasmid-encoded tetracycline resistance gene, by deletion of either the promoter region or the entire gene, eliminated the beneficial effect of plasmid carriage for the coevolved host. This beneficial effect for the coevolved host was also manifest with pBR322, which contains a tetracycline resistance gene identical to that of pACYC184 but is otherwise heterologous.     

Long-term experimental evolution in Escherichia coli. XII. DNA topology as a key target of selection

The genetic bases of adaptation are being investigated in 12 populations of Escherichia coli, founded from a common ancestor and serially propagated for 20,000 generations, during which time they achieved substantial fitness gains. Each day, populations alternated between active growth and nutrient exhaustion. DNA supercoiling in bacteria is influenced by nutritional state, and DNA topology helps coordinate the overall pattern of gene expression in response to environmental changes. We therefore examined whether the genetic controls over supercoiling might have changed during the evolution experiment. Parallel changes in topology occurred in most populations, with the level of DNA supercoiling increasing, usually in the first 2000 generations. Two mutations in the topA and fis genes that control supercoiling were discovered in a population that served as the focus for further investigation. Moving the mutations, alone and in combination, into the ancestral background had an additive effect on supercoiling, and together they reproduced the net change in DNA topology observed in this population. Moreover, both mutations were beneficial in competition experiments. Clonal interference involving other beneficial DNA topology mutations was also detected. These findings define a new class of fitness-enhancing mutations and indicate that the control of DNA supercoiling can be a key target of selection in evolving bacterial populations.     

Pleiotropic effects of beneficial mutations in Escherichia coli

Micromutational models of adaptation have placed considerable weight on antagonistic pleiotropy as a mechanism that prevents mutations of large effect from achieving fixation. However, there are few empirical studies of the distribution of pleiotropic effects, and no studies that have examined this distribution for a large number of adaptive mutations. Here we examine the form and extent of pleiotropy associated with beneficial mutations in Escherichia coli. To do so, we used a collection of independently evolved genotypes, each of which contains a beneficial mutation that confers increased fitness in a glucose-limited environment. To determine the pleiotropic effects of these mutations, we examined the fitnesses of the mutants in five novel resource environments. Our results show that the majority of mutations had significant fitness effects in alternative resources, such that pleiotropy was common. The predominant form of this pleiotropy was positive--that is, most mutations that conferred increased fitness in glucose also conferred increased fitness in novel resources. We did detect some deleterious pleiotropic effects, but they were primarily limited to one of the five resources, and within this resource, to only a subset of mutants. Although pleiotropic effects were generally positive, fitness levels were lower and more variable on resources that differed most in their mechanisms of uptake and catabolism from that of glucose. Positive pleiotropic effects were strongly correlated in magnitude with their direct effects, but no such correlation was found among mutants with deleterious pleiotropic effects. Whereas previous studies of populations evolved on glucose for longer periods of time showed consistent declines on some of the resources used here, our results suggest that deleterious pleiotropic effects were limited to only a subset of the beneficial mutations available.     

Long-Term Experimental Evolution in Escherichia coli. VIII. Dynamics of a Balanced Polymorphism

We describe the short- and long-term dynamics of a phenotypic polymorphism that arose in a population of Escherichia coli while it was serially propagated for almost 20,000 generations in a glucose-limited minimal medium. The two types, designated L and S, differ conspicuously in the size of the colonies they form on agar plates as well as the size of their individual cells, and these differences are heritable. The S type reached a detectable frequency (>1%) at generation 6,000, and it remained above that frequency throughout the subsequent generations. In addition to morphological differences, L and S diverged in important ecological properties. With clones isolated at 18,000 generations, L has a maximal growth rate in fresh medium that is approximately 20% higher than that of S. However, experiments with conditioned media demonstrate that L and S secrete one or more metabolites that promote the growth of S but not of L. The death rate of L during stationary phase also increases when S is abundant, which suggests that S may either secrete a metabolite that is toxic to L or remove some factor that enables the survival of L. One-day competition experiments with the clones isolated at generation 18,000 show that their relative fitness is frequency dependent, with each type having an advantage when rare. When these two types are grown together for a period of several weeks, they converge on an equilibrium frequency that is consistent with the 1-d competition experiments. Over the entire 14,000-generation period of coexistence, however, the frequency of the S type fluctuated between approximately 10% and 85%. We offer several hypotheses that may explain the fluctuations in this balanced polymorphism, including the possibility of coevolution between the two types.