Transcriptome profile of subsynaptic myonuclei at the neuromuscular junction in embryogenesis.

Skeletal muscle fiber is a large syncytium with multiple and evenly distributed nuclei. Adult subsynaptic myonuclei beneath the neuromuscular junction (NMJ) express specific genes, the products of which coordinately function in the maintenance of the pre- and post-synaptic regions. However, the gene expression profiles that promote the NMJ formation during embryogenesis remain largely unexplored. We performed single-nucleus RNA sequencing (snRNA-seq) analysis of embryonic and neonatal mouse diaphragms, and found that each myonucleus had a distinct transcriptome pattern during the NMJ formation. Among the previously reported NMJ-constituting genes, Dok7, Chrna1, and Chrnd are specifically expressed in subsynaptic myonuclei at E18.5. In the E18.5 diaphragm, ca. 10.7% of the myonuclei express genes for the NMJ formation (Dok7, Chrna1, and Chrnd) together with four representative ß-catenin regulators (Amotl2, Ptprk, Fam53b, and Tcf7l2). Additionally, the temporal gene expression patterns of these seven genes are synchronized in differentiating C2C12 myoblasts. Amotl2 and Ptprk are expressed in the sarcoplasm, where ß-catenin serves as a structural protein to organize the membrane-anchored NMJ structure. In contrast, Fam53b and Tcf7l2 are expressed in the myonucleus, where ß-catenin serves as a transcriptional coactivator in Wnt/ß-catenin signaling at the NMJ. In C2C12 myotubes, knockdown of Amotl2 or Ptprk markedly, and that of Fam53b and Tcf7l2 less efficiently, impair the clustering of acetylcholine receptors. In contrast, knockdown of Fam53b and Tcf7l2, but not of Amotl2 or Ptprk, impairs the gene expression of Slit2 encoding an axonal attractant for motor neurons, which is required for the maturation of motor nerve terminal. Thus, Amotl2 and Ptprk exert different roles at the NM compared to Fam53b and Tcf7l2. Additionally, Wnt ligands originating from the spinal motor neurons and the perichondrium/chondrocyte are likely to work remotely on the subsynaptic nuclei and the myotendinous junctional nuclei, respectively. We conclude that snRNA-seq analysis of embryonic/neonatal diaphragms reveal a novel coordinated expression profile especially in the Wnt/ß-catenin signaling that regulate the formation of the embryonic NMJ.

Metagenomic Thermometer.

Various microorganisms exist in environments, and each of them has its optimal growth temperature (OGT). The relationship between genomic information and OGT of each species has long been studied, and one such study revealed that OGT of prokaryotes can be accurately predicted based on the fraction of seven amino acids (IVYWREL) among all encoded amino-acid sequences in its genome. Extending this discovery, we developed a 'Metagenomic Thermometer' as a means of predicting environmental temperature based on metagenomic sequences. Temperature prediction of diverse environments using publicly available metagenomic data revealed that the Metagenomic Thermometer can predict environmental temperatures with small temperature changes and little influx of microorganisms from other environments. The accuracy of the Metagenomic Thermometer was also confirmed by a demonstration experiment using an artificial hot water canal. The Metagenomic Thermometer was also applied to human gut metagenomic samples, yielding a reasonably accurate value for human body temperature. The result further suggests that deep body temperature determines the dominant lineage of the gut community. Metagenomic Thermometer provides a new insight into temperature-driven community assembly based on amino-acid composition rather than microbial taxa.

Experimental Evolution Expands the Breadth of Adaptation to an Environmental Gradient Correlated With Genome Reduction.

Whether and how adaptive evolution adjusts the breadth of adaptation in coordination with the genome are essential issues for connecting evolution with ecology. To address these questions, experimental evolution in five Escherichia coli strains carrying either the wild-type genome or a reduced genome was performed in a defined minimal medium (C0). The ancestral and evolved populations were subsequently subjected to fitness and chemical niche analyses across an environmental gradient with 29 combinations of eight chemical components of the minimal medium. The results showed that adaptation was achieved not only specific to the evolutionary condition (C0), but also generally, to the environmental gradient; that is, the breadth of adaptation to the eight chemical niches was expanded. The magnitudes of the adaptive improvement and the breadth increase were both correlated with genome reduction and were highly significant in two out of eight niches (i.e., glucose and sulfate). The direct adaptation-induced correlated adaptation to the environmental gradient was determined by only a few genome mutations. An additive increase in fitness associated with the stepwise fixation of mutations was consistently observed in the reduced genomes. In summary, this preliminary survey demonstrated that evolution finely tuned the breadth of adaptation correlated with genome reduction.

Primordial mimicry induces morphological change in Escherichia coli.

The morphology of primitive cells has been the subject of extensive research. A spherical form was commonly presumed in prebiotic studies but lacked experimental evidence in living cells. Whether and how the shape of living cells changed are unclear. Here we exposed the rod-shaped bacterium Escherichia coli to a resource utilization regime mimicking a primordial environment. Oleate was given as an easy-to-use model prebiotic nutrient, as fatty acid vesicles were likely present on the prebiotic Earth and might have been used as an energy resource. Six evolutionary lineages were generated under glucose-free but oleic acid vesicle (OAV)-rich conditions. Intriguingly, fitness increase was commonly associated with the morphological change from rod to sphere and the decreases in both the size and the area-to-volume ratio of the cell. The changed cell shape was conserved in either OAVs or glucose, regardless of the trade-offs in carbon utilization and protein abundance. Highly differentiated mutations present in the genome revealed two distinct strategies of adaption to OAV-rich conditions, i.e., either directly targeting the cell wall or not. The change in cell morphology of Escherichia coli for adapting to fatty acid availability supports the assumption of the primitive spherical form.

Correlation between the spatial distribution and colony size was common for monogenetic bacteria in laboratory conditions.

BACKGROUND: Geographically separated population growth of microbes is a common phenomenon in microbial ecology. Colonies are representative of the morphological characteristics of this structured population growth. Pattern formation by single colonies has been intensively studied, whereas the spatial distribution of colonies is poorly investigated. RESULTS: The present study describes a first trial to address the questions of whether and how the spatial distribution of colonies determines the final colony size using the model microorganism Escherichia coli, colonies of which can be grown under well-controlled laboratory conditions. A computational tool for image processing was developed to evaluate colony density, colony size and size variation, and the Voronoi diagram was applied for spatial analysis of colonies with identical space resources. A positive correlation between the final colony size and the Voronoi area was commonly identified, independent of genomic and nutritional differences, which disturbed the colony size and size variation. CONCLUSIONS: This novel finding of a universal correlation between the spatial distribution and colony size not only indicated the fair distribution of spatial resources for monogenetic colonies growing with identical space resources but also indicated that the initial localization of the microbial colonies decided by chance determined the fate of the subsequent population growth. This study provides a valuable example for quantitative analysis of the complex microbial ecosystems by means of experimental ecology.

Correlated chromosomal periodicities according to the growth rate and gene expression.

Linking genetic information to population fitness is crucial to understanding living organisms. Despite the abundant knowledge of the genetic contribution to growth, the overall patterns/features connecting genes, their expression, and growth remain unclear. To reveal the quantitative and direct connections, systematic growth assays of single-gene knockout Escherichia coli strains under both rich and poor nutritional conditions were performed; subsequently, the resultant growth rates were associated with the original expression levels of the knockout genes in the parental genome. Comparative analysis of growth and the transcriptome identified not only the nutritionally differentiated fitness cost genes but also a significant correlation between the growth rates of the single-gene knockout strains and the original expression levels of these knockout genes in the parental strain, regardless of the nutritional variation. In addition, the coordinated chromosomal periodicities of the wild-type transcriptome and the growth rates of the strains lacking the corresponding genes were observed. The common six-period periodicity was somehow attributed to the essential genes, although the underlying mechanism remains to be addressed. The correlated chromosomal periodicities associated with the gene expression-growth dataset were highly valuable for bacterial growth prediction and discovering the working principles governing minimal genetic information.

The highly conserved chromosomal periodicity of transcriptomes and the correlation of its amplitude with the growth rate in Escherichia coli.

The growth rate, representing the fitness of a bacterial population, is determined by the transcriptome. Chromosomal periodicity, which is known as the periodic spatial pattern of a preferred chromosomal distance in microbial genomes, is a representative overall feature of the transcriptome; however, whether and how it is associated with the bacterial growth rate are unknown. To address these questions, we analysed a total of 213 transcriptomes of multiple Escherichia coli strains growing in an assortment of culture conditions varying in terms of temperature, nutrition level and osmotic pressure. Intriguingly, Fourier transform analyses of the transcriptome identified a common chromosomal periodicity of transcriptomes, which was independent of the variation in genomes and environments. In addition, fitting of the data to a theoretical model, we found that the amplitudes of the periodic transcriptomes were significantly correlated with the growth rates. These results indicated that the amplitude of periodic transcriptomes is a parameter representing the global pattern of gene expression in correlation with the bacterial growth rate. Thus, our study provides a novel parameter for evaluating the adaptiveness of a growing bacterial population and quantitatively predicting the growth dynamics according to the global expression pattern.

Experimental Challenges for Reduced Genomes: The Cell Model Escherichia coli.

Genome reduction, as a top-down approach to obtain the minimal genetic information essential for a living organism, has been conducted with bacterial cells for decades. The most popular and well-studied cell models for genome reduction are Escherichia coli strains. As the previous literature intensively introduced the genetic construction and application of the genome-reduced Escherichia coli strains, the present review focuses the design principles and compares the reduced genome collections from the specific viewpoint of growth, which represents a fundamental property of living cells and is an important feature for their biotechnological application. For the extended simplification of the genomic sequences, the approach of experimental evolution and concern for medium optimization are newly proposed. The combination of the current techniques of genomic construction and the newly proposed methodologies could allow us to acquire growing Escherichia coli cells carrying the extensively reduced genome and to address the question of what the minimal genome essential for life is.

A decay effect of the growth rate associated with genome reduction in Escherichia coli.

BACKGROUND: Bacterial growth is an important topic in microbiology and of crucial importance to better understand living cells. Bacterial growth dynamics are quantitatively examined using various methods to determine the physical, chemical or biological features of growing populations. Due to methodological differences, the exponential growth rate, which is a parameter that is representative of growth dynamics, should be differentiated. Ignoring such differentiation in the growth analysis might overlook somehow slight but significant changes in cellular features of the growing population. Both experimental and theoretical investigations are required to address these issues. RESULTS: This study experimentally verified the differentiation in growth rates attributed to different methodologies, and demonstrated that the most popular method, optical turbidity, led to the determination of a lower growth rate in comparison to the methods based on colony formation and cellular adenosine triphosphate, due to a decay effect of reading OD600 during a population increase. Accordingly, the logistic model, which is commonly applied to the high-throughput growth data reading the OD600, was revised by introducing a new parameter: the decay rate, to compensate for the lowered estimation in growth rates. An improved goodness of fit in comparison to the original model was acquired due to this revision. Applying the modified logistic model to hundreds of growth data acquired from an assortment of Escherichia coli strains carrying the reduced genomes led to an intriguing finding of a correlation between the decay rate and the genome size. The decay effect seemed to be partially attributed to the decrease in cell size accompanied by a population increase and was medium dependent. CONCLUSIONS: The present study provides not only an improved theoretical tool for the high-throughput studies on bacterial growth dynamics linking with optical turbidity to biological meaning, but also a novel insight of the genome reduction correlated decay effect, which potentially reflects the changing cellular features during population increase. It is valuable for understanding the genome evolution and the fitness increase in microbial life.

Precise, High-throughput Analysis of Bacterial Growth.

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Coordinated Changes in Mutation and Growth Rates Induced by Genome Reduction.

Genome size is determined during evolution, but it can also be altered by genetic engineering in laboratories. The systematic characterization of reduced genomes provides valuable insights into the cellular properties that are quantitatively described by the global parameters related to the dynamics of growth and mutation. In the present study, we analyzed a small collection of W3110 Escherichia coli derivatives containing either the wild-type genome or reduced genomes of various lengths to examine whether the mutation rate, a global parameter representing genomic plasticity, was affected by genome reduction. We found that the mutation rates of these cells increased with genome reduction. The correlation between genome length and mutation rate, which has been reported for the evolution of bacteria, was also identified, intriguingly, for genome reduction. Gene function enrichment analysis indicated that the deletion of many of the genes encoding membrane and transport proteins play a role in the mutation rate changes mediated by genome reduction. Furthermore, the increase in the mutation rate with genome reduction was highly associated with a decrease in the growth rate in a nutrition-dependent manner; thus, poorer media showed a larger change that was of higher significance. This negative correlation was strongly supported by experimental evidence that the serial transfer of the reduced genome improved the growth rate and reduced the mutation rate to a large extent. Taken together, the global parameters corresponding to the genome, growth, and mutation showed a coordinated relationship, which might be an essential working principle for balancing the cellular dynamics appropriate to the environment.IMPORTANCE Genome reduction is a powerful approach for investigating the fundamental rules for living systems. Whether genetically disturbed genomes have any specific properties that are different from or similar to those of natively evolved genomes has been under investigation. In the present study, we found that Escherichia coli cells with reduced genomes showed accelerated nucleotide substitution errors (mutation rates), although these cells retained the normal DNA mismatch repair systems. Intriguingly, this finding of correlation between reduced genome size and a higher mutation rate was consistent with the reported evolution of mutation rates. Furthermore, the increased mutation rate was quantitatively associated with a decreased growth rate, indicating that the global parameters related to the genome, growth, and mutation, which represent the amount of genetic information, the efficiency of propagation, and the fidelity of replication, respectively, are dynamically coordinated.

Correlation between genome reduction and bacterial growth.

Genome reduction by removing dispensable genomic sequences in bacteria is commonly used in both fundamental and applied studies to determine the minimal genetic requirements for a living system or to develop highly efficient bioreactors. Nevertheless, whether and how the accumulative loss of dispensable genomic sequences disturbs bacterial growth remains unclear. To investigate the relationship between genome reduction and growth, a series of Escherichia coli strains carrying genomes reduced in a stepwise manner were used. Intensive growth analyses revealed that the accumulation of multiple genomic deletions caused decreases in the exponential growth rate and the saturated cell density in a deletion-length-dependent manner as well as gradual changes in the patterns of growth dynamics, regardless of the growth media. Accordingly, a perspective growth model linking genome evolution to genome engineering was proposed. This study provides the first demonstration of a quantitative connection between genomic sequence and bacterial growth, indicating that growth rate is potentially associated with dispensable genomic sequences.