DNA superhelicity is one of the most important regulatory networks in bacteria [1], involved in many vital processes, from chromosome replication and segregation, transcription, translation, recombination, to responses to environmental conditions. In particular, it has been suggested that DNA supercoiling can act as both a sensor of external stimuli and a transmitter to the global expression profile of the cells. Hence, DNA superhelicity has a dynamic structural organization, transiently modified during all environmental changes tested so far, like nutritional conditions [2,3], oxidative stress [4], osmotic stress [5], thermal stress [6], pH stress [7], presence or absence of oxygen [8], or intracellular growth of pathogens [9]. Upon different stresses, transient alterations of the DNA superhelicity level generate transient modifications of global transcriptional patterns, including activation of genes involved in stress survival, leading to phenotypic acclimation of bacteria to their environment [4,5]. Changes in DNA superhelicity are known to affect transcription of at least 10% of the Escherichia coli genes [10]. The DNA superhelicity control of gene transcription involves two levels : one local whereupon it directly and dynamically alters the physical properties of promoter sequences, for example by influencing the opening of DNA duplex [11], and one global. Hence, the bacterial chromosome is organized first into fluid topological domains of 10kb on average [12,13], and second into large macrodomains of several hundreds of kilobase pairs [14] which may lead to expression coupling of spatially separated genes involved in phenotypic responses to particular stresses.
These different levels of chromosomal organization correlate with the existence of spatial patterns of transcription in the E. coli genome [15], which can be altered by modulation of the DNA superhelicity level. Structural organization of the chromosome, DNA superhelicity and global transcription patterns are therefore highly interconnected processes allowing cells to cope with fluctuating environmental conditions.
The DNA superhelicity level is precisely adjusted by the combined activities of topoisomerases [16] and histone-like proteins [17]. Topoisomerases are enzymes that introduce or remove supercoils by cutting, transferring and religating DNA strands. In E. coli, three major topoisomerases maintain the appropriate level of DNA supercoiling : topoisomerases I, encoded by topA [18] and IV, encoded by parC and parE [19] relax DNA, whereas DNA gyrase, encoded by gyrA and gyrB is the only known topoisomerase able to introduce negative supercoils into the bacterial chromosome [20]. Binding of histone-like
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barriers for the formation of topological domains [22]. Most histone-like proteins have also been shown to be global regulators of gene transcription in bacteria, like Fis [23], H-NS [24,25], HU [26] or IHF [27]. They are expressed at various levels during the bacterial cell cycle, generating yet another level of highly dynamic interplay between environmental signals, DNA superhelicity and global transcription profiles.
Of primary importance among these histone-like proteins connecting DNA superhelicity and global transcription to environmental signals is the Fis protein [28]. After binding DNA, Fis leads to the extrusion of DNA loops [29]. This constitutes the common mechanism by which Fis can regulate gene expression by local binding to high-affinity sites [28], organize the chromosome at the global level into topological domains by binding non-specifically to DNA [22], or facilitate site-specific recombination events catalysed by serine invertase and tyrosine integrase recombinases [30]. Several lines of evidence suggest that Fis plays a crucial role in the adaptation of bacteria to the nutritional state of the environment by sensing nutrient upshifts and deficiencies, modifying DNA superhelicity at both local and global levels, thereby adjusting global gene expression profiles to provide bacteria with the appropriate phenotypic response: i) Fis exerts a homeostatic control on DNA superhelicity, allowing a physiologically relevant supercoiling level in vivo. Fis represses transcription of gyrA and gyrB [31] and in turn, fis transcription is strongly dependent on the level of DNA supercoiling [32]; ii) Nutrient upshifts lead to high negative superhelicity, which triggers high Fis levels (50,000 molecules per cell) [33], which in turn activates transcription of ribosomal RNA operons and other genes necessary to cell growth. Transition to stationary phase is accompanied by DNA relaxation, leading to a dramatic decrease in Fis levels (100 molecules per cell), as well as in transcription of rRNA operons. This favours transcription of genes by the σS sigma factor of RNA polymerase, specific to the stationary phase [34–36]. Besides being a sensor of the nutritional state of the environment, Fis also helps bacterial cells to cope with certain stresses, in particular oxidative stress, again through its ability of adjusting the DNA superhelicity level [4]; iii) After binding to numerous sites on the chromosome, Fis acts as a global regulator of gene transcription [23,37], regulating ribosomal RNA operons [38], genes involved in cellular metabolism [39], and virulence genes necessary to intracellular bacterial growth [9].
The last two decades have witnessed the development of experimental evolution strategies, which consist in propagating living organisms for hundreds or thousands of
generations in a defined environment [40], representing a powerful tool to study the dynamics and genetic bases of adaptation. Using the longest-running evolution experiment, we recently further extended the crucial role of DNA supercoiling in cellular life by showing that it can be involved in evolutionary processes [41]. Hence, DNA supercoiling was shown to be one of the major targets of natural selection. In particular, we demonstrated that both a topoisomerase and a histone-like protein could be directly involved in adaptive evolution. In this long-term evolution experiment, Lenski and colleagues have propagated 12 populations of E. coli by serial daily transfers for more than 40,000 generations at 37°C in a minimal medium supplemented with limiting amounts of glucose [42–44]. Six of the 12 populations, called Ara-1 to Ara-6, were founded from an E. coli B ancestor clone unable to use arabinose as a carbon source (Ara-) [45], while the 6 others (Ara+1 to Ara+6) were founded from an Ara+
revertant of the ancestor clone. The arabinose utilization phenotype was used as an internal neutral marker in competition experiments to assess the fitness of evolved clones compared to the ancestor [46]. All replicate populations achieved parallel gains in competitive fitness, indicative of substantial adaptation. The fitness increase reaches about 70% in all 12 populations after 20,000 generations of evolution [43]. Interestingly, four populations evolved defects in their DNA repair pathways, leading to a hypermutable phenotype [47,48]. During 40,000 generations, evolution is characterized by a strong level of phenotypic parallelism.
Hence, several phenotypic traits in addition to fitness evolved in parallel in most populations, including cell size [49], growth parameters [50], catabolic functions [43,51], global gene expression [51,52] and DNA topology [41]. Various genetic studies, specifically aimed to understand these parallel phenotypic changes, revealed that the same genes were reproducibly altered by mutations in most populations : rbs, the ribose utilization operon [53]; spoT, one of the genes involved in (p)ppGpp metabolism during the stringent response [51,52] and malT, encoding the transcriptional activator of the maltose utilization operon [51]. These genetic changes were demonstrated to be beneficial under the environmental conditions of the evolution experiment. We investigated the observed parallel changes in DNA supercoiling levels only in one model population, where two successive increases in DNA superhelicity were detected [41]. We found two mutations, one in topA, which encodes topoisomerase I, and one in fis, encoding the histone-like protein. By genetically manipulating these mutations, we could demonstrate that both were beneficial in these conditions. However, and this constitutes one of the objectives of this manuscript, this study was not extended in the other 11 populations to analyse the level of genetic parallelism linked to these parallel increases of DNA supercoiling.
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These mutations affect nadR involved in NAD metabolism, pykF encoding pyruvate kinase I, pbpA-rodA involved in cell wall biosynthesis and hokB-sokB encoding a plasmid maintenance module [55,56]. Sequencing these 4 loci in all 12 populations revealed mutations in most populations [56]. Statistical tests were used to compare these results to a set of 36 randomly chosen genes that were sequenced in evolved clones isolated at 20,000 generations from all 12 populations. Almost no mutations were detected [57]. These statistical tests of the pattern of molecular evolution all indicate that the parallel genetic changes in the 4 loci were driven through adaptation by natural selection [56], although no experimental proof confirmed these conclusions.
Phenotypic and genetic parallelism occurring in several lineages is a hallmark of this evolution experiment. In the conditions prevailing in this experiment, all possible genetic changes are potentially possible, given the predicted mutation rate (about 2.10-10 mutations/bp/generation), the genome and population sizes and the number of generations [44,57]. Therefore, parallel evolution provides strong evidence of adaptation by natural selection for the parallel evolved traits. Parallel evolution, both at the phenotypic and genetic levels, is widespread in nature. Similar phenotypes evolved repeatedly when independent populations colonize similar environments, including the morphology of lizards and fishes [58,59], the wing length in Drosophila [60]. Parallel genomic changes have been described for viral and bacterial pathogens [61,62]. Linkage between environmental parameters and evolution of organismal phenotypes unlikely arises by random genetic drift and strongly involves natural selection. Despite numerous cases of parallel evolution, the only way to precisely quantify parallelism and the level it can reach during evolutionary processes is to measure phenotypic repeatability in independent populations during experimental evolution strategies where the common ancestor is available to experimental and statistical tests. This would however be crucial to understand the underlying molecular bases of adaptation and to use evolutionary biology as a predicting tool. The DNA supercoiling target of natural selection constitutes an ideal candidate to analyse in deep details the level of parallelism of evolutionary processes, since more than 20 genes (encoding topoisomerases, histone-like proteins and gene expression regulators) are involved in its regulation in E. coli.
Here, we sought to gain further insights into both the level of parallelism characterizing evolutionary processes and the underlying molecular and ecological mechanisms by analyzing
the relationships between DNA supercoiling and adaptation by natural selection. We raised three different types of questions. We first asked whether the observed parallel changes in DNA supercoiling were due to an underlying genetic parallelism, by sequencing 9 topology- related loci (including topA and fis) in both the ancestral and evolved clones isolated from all 12 populations. On one extreme, mutations in various genes involved in DNA superhelicity control could explain the observed parallel phenotypic increases in DNA supercoiling. On the other extreme, only a small number of genes may be repeatedly targets of natural selection in the 12 independently evolving populations. Second, as we detected genetic parallelism at the level of the gene, but genetic divergence at the level of mutation alleles, we asked how this is related to the level of molecular gene regulation. Third, only few of the 12 populations did not show any increase in the DNA superhelicity level [41]. We asked whether the absence of changes was linked to any molecular explanation or was simply due to the random processes of mutation fixation within the evolving populations. Our results revealed an exceptional high level of genetic and molecular parallelism. During the course of our work, we also discovered a new beneficial mutation.