Analysis of the evolution of resistance to multiple antibiotics enables prediction of the Escherichia coli phenotype-based fitness landscape.

The fitness landscape represents the complex relationship between genotype or phenotype and fitness under a given environment, the structure of which allows the explanation and prediction of evolutionary trajectories. Although previous studies have constructed fitness landscapes by comprehensively studying the mutations in specific genes, the high dimensionality of genotypic changes prevents us from developing a fitness landscape capable of predicting evolution for the whole cell. Herein, we address this problem by inferring the phenotype-based fitness landscape for antibiotic resistance evolution by quantifying the multidimensional phenotypic changes, i.e., time-series data of resistance for eight different drugs. We show that different peaks of the landscape correspond to different drug resistance mechanisms, thus supporting the validity of the inferred phenotype-fitness landscape. We further discuss how inferred phenotype-fitness landscapes could contribute to the prediction and control of evolution. This approach bridges the gap between phenotypic/genotypic changes and fitness while contributing to a better understanding of drug resistance evolution.

Experimental evidence for the correlation between RNA structural fluctuations and the frequency of beneficial mutations.

RNA has been used as a model molecule to understand the adaptive evolution process owing to the simple relationship between the structure (i.e., phenotype) and sequence (i.e., genotype). RNA usually forms multiple substructures with similar thermodynamic stabilities, called structural fluctuations. Ancel and Fontana theoretically proposed that structural fluctuation is directly related to the ease of change in structures by mutations and thus works as a source of adaptive evolution; however, experimental verification is limited. Here, we analyzed 76 RNA genotypes that appeared in our previous in vitro evolution to examine whether (i) RNA fluctuation decreases as adaptive evolution proceeds and (ii) RNAs that have larger fluctuations tend to have higher frequencies of beneficial mutations. We first computationally estimated the structural fluctuations of all RNAs and observed that they tended to decrease as their fitness increased. We next measured the frequency of beneficial mutations for 10 RNA genotypes and observed that the total number of beneficial mutations was correlated with the size of the structural fluctuations. These results consistently support the idea that the structural fluctuation of RNA, at least those evaluated in our study, works as a source of adaptive evolution.

Purifying selection enduringly acts on the sequence evolution of highly expressed proteins in Escherichia coli.

The evolutionary speed of a protein sequence is constrained by its expression level, with highly expressed proteins evolving relatively slowly. This negative correlation between expression levels and evolutionary rates (known as the E-R anticorrelation) has already been widely observed in past macroevolution between species from bacteria to animals. However, it remains unclear whether this seemingly general law also governs recent evolution, including past and de novo, within a species. However, the advent of genomic sequencing and high-throughput phenotyping, particularly for bacteria, has revealed fundamental gaps between the 2 evolutionary processes and has provided empirical data opposing the possible underlying mechanisms which are widely believed. These conflicts raise questions about the generalization of the E-R anticorrelation and the relevance of plausible mechanisms. To explore the ubiquitous impact of expression levels on molecular evolution and test the relevance of the possible underlying mechanisms, we analyzed the genome sequences of 99 strains of Escherichia coli for evolution within species in nature. We also analyzed genomic mutations accumulated under laboratory conditions as a model of de novo evolution within species. Here, we show that E-R anticorrelation is significant in both past and de novo evolution within species in E. coli. Our data also confirmed ongoing purifying selection on highly expressed genes. Ongoing selection included codon-level purifying selection, supporting the relevance of the underlying mechanisms. However, the impact of codon-level purifying selection on the constraints in evolution within species might be smaller than previously expected from evolution between species.

Development of a device that generates a temperature gradient in a microtiter plate for microbial culture.

Although temperature is a fundamental parameter in biology, testing various temperature conditions simultaneously is often difficult. In the present study, we developed a device for generating a temperature gradient in arrays of wells on a microtiter plate. This device consists of a pair of Peltier elements and temperature sensors placed on both ends of a flat aluminum bar to generate a linear temperature gradient. The device loads a microtiter plate at the center of the aluminum bar and transfers the temperature gradient to the bottom of the wells in the plate. This device successfully maintained a temperature gradient of 38.2 to 43.1°C on the horizontal axis of a 96-well microtiter plate in an incubator at 31°C. Furthermore, using this device, we demonstrated a laboratory evolution experiment of Escherichia coli, which was selected on the basis of its ability to grow at high temperatures. The developed device also facilitates a two-dimensional assay to determine the effects of temperature and drug concentrations on cellular growth.

Acceleration of target production in co-culture by enhancing intermediate consumption through adaptive laboratory evolution.

Co-culture is a promising way to alleviate metabolic burden by dividing the metabolic pathways into several modules and sharing the conversion processes with multiple strains. Since an intermediate is passed from the donor to the recipient via the extracellular environment, it is inevitably diluted. Therefore, enhancing the intermediate consumption rate is important for increasing target productivity. In the present study, we demonstrated the enhancement of mevalonate consumption in Escherichia coli by adaptive laboratory evolution and applied the evolved strain to isoprenol production in an E. coli (upstream: glucose to mevalonate)-E. coli (downstream: mevalonate to isoprenol) co-culture. An engineered mevalonate auxotroph strain was repeatedly sub-cultured in a synthetic medium supplemented with mevalonate, where the mevalonate concentration was decreased stepwise from 100 to 20 µM. In five parallel evolution experiments, all growth rates gradually increased, resulting in five evolved strains. Whole-genome re-sequencing and reverse engineering identified three mutations involved in enhancing mevalonate consumption. After introducing nudF gene for producing isoprenol, the isoprenol-producing parental and evolved strains were respectively co-cultured with a mevalonate-producing strain. At an inoculation ratio of 1:3 (upstream:downstream), isoprenol production using the evolved strain was 3.3 times higher than that using the parental strain.

Mutational property of newly identified mutagen l-glutamic acid γ-hydrazide in Escherichia coli.

We previously found that an l-glutamine analog l-glutamic acid γ-hydrazide has high mutagenic activity through the high-throughput laboratory evolution of Escherichia coli. In this study, mutagenicity and mutational property of l-glutamic acid γ-hydrazide were examined by the Ames test and mutation accumulation experiments using E. coli. The Ames test revealed that l-glutamic acid γ-hydrazide showed higher mutagenic activity without metabolic activation than known mutagens 2-aminoanthracene, and cobalt(II) acetate tetrahydrate. This result indicates that l-glutamic acid γ-hydrazide does not require metabolic activation for mutagenic activity in E. coli. Mutation accumulation experiments and whole-genome sequencing analysis revealed the number and spectrum of the accumulated mutations with or without l-glutamic acid γ-hydrazide. In the presence of l-glutamic acid γ-hydrazide, MDS42 strain accumulated 392.3 ± 116.2 point mutations during 30 passages corresponding to 777 generations, while MDS42 strain accumulated 1.5 ± 2.5 point mutations without l-glutamic acid γ-hydrazide during 50 passages corresponding to 1341 generations. The mutational spectrum of l-glutamic acid γ-hydrazide was G/C to A/T transition (82.2 ± 4.3 %) and A/T to G/C transition (17.4 ± 4.3 %). These results indicated that l-glutamic acid γ-hydrazide has a strong mutagenic activity.

Complete Genome Sequence of a Radioresistant Bacterial Strain, Deinococcus grandis ATCC 43672.

Deinococcus grandis is a radioresistant bacterial species isolated from freshwater fish. In this article, we report the complete genome sequence of D. grandis strain ATCC 43672. This sequence is useful for comparative genomics to understand the traits of Deinococcus species and can be used as a reference in experimental genetics.

Complete Genome Sequences of Three Star-Shaped Bacteria, Stella humosa, Stella vacuolata, and Stella Species ATCC 35155.

Stella species are unique star-shaped alphaproteobacteria found in various environments. We report the complete genome sequences of three Stella strains, Stella humosa ATCC 43930, Stella vacuolata ATCC 43931, and Stella species ATCC 35155. These are the first complete genome sequences of members of the genus Stella.

Development of an Automated UV Irradiation Device for Microbial Cell Culture.

Ultraviolet (UV) mutagenesis is a widely used technique to increase bacterial mutation rates in laboratory experiments. UV mutagenesis requires fine regulation of UV dose, because the number of dead cells increases exponentially as the dose increases. Ignoring this hazard can cause extinction of UV-exposed populations. Therefore, an automated system that cooperatively conducts both growth measurement and UV irradiation is needed for efficient UV mutagenesis experiments. To address this task, we constructed an automated UV irradiation device for microbial cell culture. This device can measure cell density and irradiate the bacterial cells with UV light automatically according to the state of cell growth. We demonstrated that this growth feedback control avoided extinction and enabled accumulation of mutations in bacterial genomes at a rapid rate for a long period. Whole-genome sequencing revealed the high accumulation rate, neutrality, and spectrum of UV-induced mutations. These characteristics were all consistent with those obtained by manual UV irradiation. These results indicate that our automated device is useful in accelerating mutation accumulation over a long duration.

Enhancement of K-strategy evolution in histidine utilization using a container with compartments.

Evolutionary strategies in growth improvement can be classified into r- or K-strategies. The former strategy corresponds to an evolutionary increase in growth rate, whereas the latter corresponds to an increase in the maximum amount of organisms or carrying capacity. What determines the strategies to be adopted during evolution? Spatial structures that compartmentalize the population into small patches are key to inducing the K-strategy. Interestingly, previous evolution experiments using Escherichia coli in a glucose-limited batch culture showed that carrying capacity could improve evolutionally even in the absence of spatial structures. However, it is unclear if the lack of spatial structures can direct evolution toward high carrying capacity for utilization of other resources. To address this question, we established a simplified evolution experiment using histidine-requiring E. coli grown under histidine limitation in a container with compartments. We confirmed the importance of spatial structures in K-strategy evolution in histidine utilization. Whole genome sequencing of the K-adapted strains showed functional variety of the mutated genes during the fitness-increasing period. These results validate the importance of spatial structures and imply that restriction of K-strategy evolution on a sort of nutrients is attributable to a paucity of appropriate selection rather than a paucity of causal mutation.

Attraction of posture and motion-trajectory elements of conspecific biological motion in medaka fish.

Visual recognition of conspecifics is necessary for a wide range of social behaviours in many animals. Medaka (Japanese rice fish), a commonly used model organism, are known to be attracted by the biological motion of conspecifics. However, biological motion is a composite of both body-shape motion and entire-field motion trajectory (i.e., posture or motion-trajectory elements, respectively), and it has not been revealed which element mediates the attractiveness. Here, we show that either posture or motion-trajectory elements alone can attract medaka. We decomposed biological motion of the medaka into the two elements and synthesized visual stimuli that contain both, either, or none of the two elements. We found that medaka were attracted by visual stimuli that contain at least one of the two elements. In the context of other known static visual information regarding the medaka, the potential multiplicity of information regarding conspecific recognition has further accumulated. Our strategy of decomposing biological motion into these partial elements is applicable to other animals, and further studies using this technique will enhance the basic understanding of visual recognition of conspecifics.

Mutation accumulation under UV radiation in Escherichia coli.

Mutations are induced by not only intrinsic factors such as inherent molecular errors but also by extrinsic mutagenic factors such as UV radiation. Therefore, identifying the mutational properties for both factors is necessary to achieve a comprehensive understanding of evolutionary processes both in nature and in artificial situations. Although there have been extensive studies on intrinsic factors, the mutational profiles of extrinsic factors are poorly understood on a genomic scale. Here, we explored the mutation profiles of UV radiation, a ubiquitous mutagen, in Escherichia coli on the genomic scale. We performed an evolution experiment under periodic UV radiation for 28 days. The accumulation speed of the mutations was found to increase so that it exceeded that of a typical mutator strain with deficient mismatch repair processes. The huge contribution of the extrinsic factors to all mutations consequently increased the risk of the destruction of inherent error correction systems. The spectrum of the UV-induced mutations was broader than that of the spontaneous mutations in the mutator. The broad spectrum and high upper limit of the frequency of occurrence suggested ubiquitous roles for UV radiation in accelerating the evolutionary process.

Genomic confirmation of nutrient-dependent mutability of mutators in Escherichia coli.

Mutators with increased mutation rates are prevalent in various environments and have important roles in accelerating adaptive evolution. Previous studies on mutator strains of microorganisms have shown that some mutators have constant mutation rates, whereas others exhibit switchable mutation rates depending on nutritional conditions. This suggests that the contributions of mutators on evolution vary with fluctuating nutritional conditions. However, such conditional mutability has been unclear at the genomic level. In addition, it is still unknown why mutation rates change with nutritional condition. Here, we used two mutator strains of Escherichia coli to explore the nutrient dependence of mutation rates at the genomic level. These strains were transferred repeatedly under different nutritional conditions for hundreds of generations to accumulate mutations. Whole-genome sequencing of the offspring showed that the nutrient dependence of the mutation rates was pervasive at the genomic scale. Neutrality in the mutation accumulation processes and constancy in the mutational bias suggested that nutrient dependence was not derived from conditional selective purges or from shifts of mutational bias. Some mutators could simply switch their mutation rates for both transitions and transversions in response to nutritional shifts.