Jeffrey E. Barrick

Genome evolution and adaptation in a long-term experiment with Escherichia coli

The relationship between rates of genomic evolution and organismal adaptation remains uncertain, despite considerable interest. The feasibility of obtaining genome sequences from experimentally evolving populations offers the opportunity to investigate this relationship with new precision. Here we sequence genomes sampled through 40,000 generations from a laboratory population of Escherichia coli. Although adaptation decelerated sharply, genomic evolution was nearly constant for 20,000 generations. Such clock-like regularity is usually viewed as the signature of neutral evolution, but several lines of evidence indicate that almost all of these mutations were beneficial. This same population later evolved an elevated mutation rate and accumulated hundreds of additional mutations dominated by a neutral signature. Thus, the coupling between genomic and adaptive evolution is complex and can be counterintuitive even in a constant environment. In particular, beneficial substitutions were surprisingly uniform over time, whereas neutral substitutions were highly variable.

Riboswitches Control Fundamental Biochemical Pathways in Bacillus subtilis and Other Bacteria

Riboswitches are metabolite binding domains within certain messenger RNAs that serve as precision sensors for their corresponding targets. Allosteric rearrangement of mRNA structure is mediated by ligand binding, and this results in modulation of gene expression. We have identified a class of riboswitches that selectively recognizes guanine and becomes saturated at concentrations as low as 5 nM. In Bacillus subtilis, this mRNA motif is located on at least five separate transcriptional units that together encode 17 genes that are mostly involved in purine transport and purine nucleotide biosynthesis. Our findings provide further examples of mRNAs that sense metabolites and that control gene expression without the need for protein factors. Furthermore, it is now apparent that riboswitches contribute to the regulation of numerous fundamental metabolic pathways in certain bacteria.

An mRNA structure that controls gene expression by binding S-adenosylmethionine.

Riboswitches are metabolite-binding RNA structures that serve as genetic control elements for certain messenger RNAs. These RNA switches have been identified in all three kingdoms of life and are typically responsible for the control of genes whose protein products are involved in the biosynthesis, transport or utilization of the target metabolite. Herein, we report that a highly conserved RNA domain found in bacteria serves as a riboswitch that responds to the coenzyme S-adenosylmethionine (SAM) with remarkably high affinity and specificity. SAM riboswitches undergo structural reorganization upon introduction of SAM, and these allosteric changes regulate the expression of 26 genes in Bacillus subtilis. This and related findings indicate that direct interaction between small metabolites and allosteric mRNAs is an important and widespread form of genetic regulation in bacteria.

The distributions, mechanisms, and structures of metabolite-binding riboswitches

BackgroundRiboswitches are noncoding RNA structures that appropriately regulate genes in response to changing cellular conditions. The expression of many proteins involved in fundamental metabolic processes is controlled by riboswitches that sense relevant small molecule ligands. Metabolite-binding riboswitches that recognize adenosylcobalamin (AdoCbl), thiamin pyrophosphate (TPP), lysine, glycine, flavin mononucleotide (FMN), guanine, adenine, glucosamine-6-phosphate (GlcN6P), 7-aminoethyl 7-deazaguanine (preQ1), and S-adenosylmethionine (SAM) have been reported.ResultsWe have used covariance model searches to identify examples of ten widespread riboswitch classes in the genomes of organisms from all three domains of life. This data set rigorously defines the phylogenetic distributions of these riboswitch classes and reveals how their gene control mechanisms vary across different microbial groups. By examining the expanded aptamer sequence alignments resulting from these searches, we have also re-evaluated and refined their consensus secondary structures. Updated riboswitch structure models highlight additional RNA structure motifs, including an unusual double T-loop arrangement common to AdoCbl and FMN riboswitch aptamers, and incorporate new, sometimes noncanonical, base-base interactions predicted by a mutual information analysis.ConclusionRiboswitches are vital components of many genomes. The additional riboswitch variants and updated aptamer structure models reported here will improve future efforts to annotate these widespread regulatory RNAs in genomic sequences and inform ongoing structural biology efforts. There remain significant questions about what physiological and evolutionary forces influence the distributions and mechanisms of riboswitches and about what forms of regulation substitute for riboswitches that appear to be missing in certain lineages.

Genomic analysis of a key innovation in an experimental Escherichia coli population

Evolutionary novelties have been important in the history of life, but their origins are usually difficult to examine in detail. We previously described the evolution of a novel trait, aerobic citrate utilization (Cit+), in an experimental population of Escherichia coli. Here we analyse genome sequences to investigate the history and genetic basis of this trait. At least three distinct clades coexisted for more than 10,000 generations before its emergence. The Cit+ trait originated in one clade by a tandem duplication that captured an aerobically expressed promoter for the expression of a previously silent citrate transporter. The clades varied in their propensity to evolve this novel trait, although genotypes able to do so existed in all three clades, implying that multiple potentiating mutations arose during the population’s history. Our findings illustrate the importance of promoter capture and altered gene regulation in mediating the exaptation events that often underlie evolutionary innovations.

Second-order selection for evolvability in a large Escherichia coli population

Descendants of bacterial lineages that retained adaptation potential outcompeted competitors of higher fitness. In theory, competition between asexual lineages can lead to second-order selection for greater evolutionary potential. To test this hypothesis, we revived a frozen population of Escherichia coli from a long-term evolution experiment and compared the fitness and ultimate fates of four genetically distinct clones. Surprisingly, two clones with beneficial mutations that would eventually take over the population had significantly lower competitive fitness than two clones with mutations that later went extinct. By replaying evolution many times from these clones, we showed that the eventual winners likely prevailed because they had greater potential for further adaptation. Genetic interactions that reduce the benefit of certain regulatory mutations in the eventual losers appear to explain, at least in part, why they were outcompeted.

Repeatability and Contingency in the Evolution of a Key Innovation in Phage Lambda

Natural Selection Caught in the Act Understanding how new functions evolve has been of long-standing interest. However, the number of mutations needed to evolve a key innovation is rarely known, or whether other sets of mutations would also suffice, whether the intermediate steps are driven by natural selection, or how contingent the outcome is on steps along the way. Meyer et al. (p. 428; see the Perspective by Thompson) answer these questions for a case in which phage lambda evolved the ability to infect its host Escherichia coli through a novel receptor. This shift required four mutations, which accumulated under natural selection in concert with coevolution of the host. However, when Tenaillon et al. (p. 457) exposed 115 lines of E. coli to high temperature and sequenced them, adaptation occurred through many different genetic paths, showing parallelism at the level of genes and interacting protein complexes, but only rarely at the nucleotide level. Thus, epistasis—nonadditive genetic interaction—is likely to play an important part in the process of adaptation to this environment. A receptor shift required four mutations that accumulated by natural selection and with the host’s coevolution. The processes responsible for the evolution of key innovations, whereby lineages acquire qualitatively new functions that expand their ecological opportunities, remain poorly understood. We examined how a virus, bacteriophage λ, evolved to infect its host, Escherichia coli, through a novel pathway. Natural selection promoted the fixation of mutations in the virus’s host-recognition protein, J, that improved fitness on the original receptor, LamB, and set the stage for other mutations that allowed infection through a new receptor, OmpF. These viral mutations arose after the host evolved reduced expression of LamB, whereas certain other host mutations prevented the phage from evolving the new function. This study shows the complex interplay between genomic processes and ecological conditions that favor the emergence of evolutionary innovations.

Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq.

Next-generation DNA sequencing (NGS) can be used to reconstruct eco-evolutionary population dynamics and to identify the genetic basis of adaptation in laboratory evolution experiments. Here, we describe how to run the open-source breseq computational pipeline to identify and annotate genetic differences found in whole-genome and whole-population NGS data from haploid microbes where a high-quality reference genome is available. These methods can also be used to analyze mutants isolated in genetic screens and to detect unintended mutations that may occur during strain construction and genome editing.

A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain.

A previous bioinformatics-based search for riboswitches yielded several candidate motifs in eubacteria. One of these motifs commonly resides in the 5′ untranslated regions of genes involved in the biosynthesis of queuosine (Q), a hypermodified nucleoside occupying the anticodon wobble position of certain transfer RNAs. Here we show that this structured RNA is part of a riboswitch selective for 7-aminomethyl-7-deazaguanine (preQ1), an intermediate in queuosine biosynthesis. Compared with other natural metabolite-binding RNAs, the preQ1 aptamer appears to have a simple structure, consisting of a single stem-loop and a short tail sequence that together are formed from as few as 34 nucleotides. Despite its small size, this aptamer is highly selective for its cognate ligand in vitro and has an affinity for preQ1 in the low nanomolar range. Relatively compact RNA structures can therefore serve effectively as metabolite receptors to regulate gene expression.

Balancing robustness and evolvability.

Can a single unifying mathematical framework help to explain robustness - the ability of organisms to persist in the face of changing conditions - at all biological scales, from biochemical to ecological?