Saburo Tsuru

Noisy cell growth rate leads to fluctuating protein concentration in bacteria

The present study discusses a prime cause of fluctuating protein concentrations, which play a significant role in generating phenotypic diversity in bacteria. A genetic circuit integrated in a bacterial genome was used to evaluate the cell-to-cell variation in protein concentration. A simple dynamic model, comprising terms for synthesis and dilution, was used to elucidate the contributions of distinct noises to the fluctuation in cell protein concentration. Experimental and theoretical results demonstrated that noise in the rate of increase in cell volume (cell growth rate) serves as a source of extrinsic noise that accounts for dozens of percent of the total noise, whereas intrinsic noise in protein synthesis makes only a moderate contribution to the fluctuation in protein concentration. This suggests that such external noise in the cell growth rate has a global effect on cellular components, resulting in a large fluctuation in protein concentration in bacterial cells.

Construction of Escherichia coli gene expression level perturbation collection.

We generated 61 strains of Escherichia coli in which the expression level of a specific single gene can be changed continuously over a physiologically significant range. In each strain, one auxotrophic gene was deleted from its original position and reinserted at a specific position on the chromosome under the control of the tetA promoter. Therefore, the level of expression of the target gene can be controlled easily by altering the concentrations of inducers, e.g., anhydrotetracycline and doxycycline, in the medium. Protein and mRNA levels and changes in proliferation rate were examined in some of the strains in our collection to determine the ability to control the level of target gene expression over a physiologically significant range. These strains will be useful for extracting omics data sets and for the construction of genome-scale mathematical models, because causality between perturbations in gene expression level and their consequences can be clearly determined.

Stochasticity in Gene Expression in a Cell-Sized Compartment

The gene expression in a clonal cell population fluctuates significantly, and its relevance to various cellular functions is under intensive debate. A fundamental question is whether the fluctuation is a consequence of the complexity and redundancy in living cells or an inevitable attribute of the minute microreactor nature of cells. To answer this question, we constructed an artificial cell, which consists of only necessary components for the gene expression (in vitro transcription and translation system) and its boundary as a microreactor (cell-sized lipid vesicle), and investigated the gene expression noise. The variation in the expression of two fluorescent proteins was decomposed into the components that were correlated and uncorrelated between the two proteins using a method similar to the one used by Elowitz and co-workers to analyze the expression noise in E. coli. The observed fluctuation was compared with a theoretical model that expresses the amplitude of noise as a function of the average number of intermediate molecules and products. With the assumption that the transcripts are partly active, the theoretical model was able to well describe the noise in the artificial system. Furthermore, the same measurement for E. coli cells harboring an identical plasmid revealed that the E. coli exhibited a similar level of expression noise. Our results demonstrated that the level of fluctuation found in bacterial cells is mostly an intrinsic property that arises even in a primitive form of the cell.

Adaptation by stochastic switching of a monostable genetic circuit in Escherichia coli

Stochastic switching is considered as a cost‐saving strategy for adaptation to environmental challenges. We show here that stochastic switching of a monostable circuit can mediate the adaptation of the engineered OSU12‐hisC Escherichia coli strain to histidine starvation. In this strain, the hisC gene was deleted from the His operon and placed under the control of a monostable foreign promoter. In response to histidine depletion, the OSU12‐hisC population shifted to a higher HisC expression level, which is beneficial under starving conditions but is not favoured by the monostable circuit. The population shift was accompanied by growth recovery and was reversible upon histidine addition. A weak directionality in stochastic switching of hisC was observed in growing microcolonies under histidine‐free conditions. Directionality and fate decision were in part dependent on the initial cellular status. Finally, microarray analysis indicated that OSU12‐hisC reorganized its transcriptome to reach the appropriate physiological state upon starvation. These findings suggest that bacteria do not necessarily need to evolve signalling mechanisms to control gene expression appropriately, even for essential genes.

Growth rate-coordinated transcriptome reorganization in bacteria

BackgroundCell growth rate reflects an organism’s physiological state and largely relies on the ability of gene expression to respond to the environment. The relationship between cellular growth rate and gene expression remains unknown.ResultsGrowth rate-coordinated changes in gene expression were discovered by analyzing exponentially growing Escherichia coli cells cultured under multiple defined environments, in which osmotic pressure, temperature and starvation status were varied. Gene expression analyses showed that all 3,740 genes in the genome could be simply divided into three clusters (C1, C2 and C3), which were accompanied by a generic trend in the growth rate that was coordinated with transcriptional changes. The direction of transcriptional change in C1 indicated environmental specificity, whereas those in C2 and C3 were correlated negatively and positively with growth rates, respectively. The three clusters exhibited differentiated gene functions and gene regulation task division.ConclusionsWe identified three gene clusters, exhibiting differential gene functions and distinct directions in their correlations with growth rates. Reverses in the direction of the growth rate correlated transcriptional changes and the distinguished duties of the three clusters indicated how transcriptome homeostasis is maintained to balance the total expression cost for sustaining life in new habitats.

Stochastic Switching Induced Adaptation in a Starved Escherichia coli Population

Population adaptation can be determined by stochastic switching in living cells. To examine how stochastic switching contributes to the fate decision for a population under severe stress, we constructed an Escherichia coli strain crucially dependent on the expression of a rewired gene. The gene essential for tryptophan biosynthesis, trpC, was removed from the native regulatory unit, the Trp operon, and placed under the extraneous control of the lactose utilisation network. Bistability of the network provided the cells two discrete phenotypes: the induced and suppressed level of trpC. The two phenotypes permitted the cells to grow or not, respectively, under conditions of tryptophan depletion. We found that stochastic switching between the two states allowed the initially suppressed cells to form a new population with induced trpC in response to tryptophan starvation. However, the frequency of the transition from suppressed to induced state dropped off dramatically in the starved population, in comparison to that in the nourished population. This reduced switching rate was compensated by increasing the initial population size, which probably provided the cell population more chances to wait for the rarely appearing fit cells from the unfit cells. Taken together, adaptation of a starved bacterial population because of stochasticity in the gene rewired from the ancient regulon was experimentally confirmed, and the nutritional status and the population size played a great role in stochastic adaptation.

Global coordination in adaptation to gene rewiring

Gene rewiring is a common evolutionary phenomenon in nature that may lead to extinction for living organisms. Recent studies on synthetic biology demonstrate that cells can survive genetic rewiring. This survival (adaptation) is often linked to the stochastic expression of rewired genes with random transcriptional changes. However, the probability of adaptation and the underlying common principles are not clear. We performed a systematic survey of an assortment of gene-rewired Escherichia coli strains to address these questions. Three different cell fates, designated good survivors, poor survivors and failures, were observed when the strains starved. Large fluctuations in the expression of the rewired gene were commonly observed with increasing cell size, but these changes were insufficient for adaptation. Cooperative reorganizations in the corresponding operon and genome-wide gene expression largely contributed to the final success. Transcriptome reorganizations that generally showed high-dimensional dynamic changes were restricted within a one-dimensional trajectory for adaptation to gene rewiring, indicating a general path directed toward cellular plasticity for a successful cell fate. This finding of global coordination supports a mechanism of stochastic adaptation and provides novel insights into the design and application of complex genetic or metabolic networks.

A reduced genome decreases the host carrying capacity for foreign DNA

BackgroundHost-plasmid interactions have been discussed largely in terms of the influences of plasmids, whereas the contributions of variations in host genomes to host interactions with foreign DNA remain unclear. A strain with a so-called “clean genome” (i.e., MDS42) of reduced genome size has recently been generated from the wild-type strain MG1655, a commonly used host strain. A quantitative evaluation of the influence of plasmid burdens in these two Escherichia coli strains can not only provide an understanding of how a reduced genome responds to foreign DNA but also offer insights into the proper application of these strains.ResultsThe decreases in growth caused by the cost of carrying foreign DNA were similar for the wild-type and clean-genome strains. A negative correlation between the growth rate and the total amount of exogenous DNA was observed in both strains, but a better theoretical fit with a higher statistical significance was found for the strain with the clean genome. Compared to the wild-type strain, the clean-genome strain exhibited a reduced carrying capacity for exogenous DNA, which was largely attributed to its ability to restrict the replication of foreign DNA. A tendency to allocate energy and resources toward gene expression, but not DNA replication, was observed in the strain with the clean genome.ConclusionsThe possession of a clean genome constrained the plasmid copy number to a wild-type-equivalent load. The results indicate that the wild-type strain possesses a greater tolerance for foreign DNA, as in endosymbiosis, and that the use of strains with clean genomes will be favorable in the applications that require precise control and theoretical prediction.

Directed evolution of cell size in Escherichia coli.

BackgroundIn bacteria, cell size affects chromosome replication, the assembly of division machinery, cell wall synthesis, membrane synthesis and ultimately growth rate. In addition, cell size can also be a target for Darwinian evolution for protection from predators. This strong coupling of cell size and growth, however, could lead to the introduction of growth defects after size evolution. An important question remains: can bacterial cell size change and/or evolve without imposing a growth burden?ResultsThe directed evolution of particular cell sizes, without a growth burden, was tested with a laboratory Escherichia coli strain. Cells of defined size ranges were collected by a cell sorter and were subsequently cultured. This selection-propagation cycle was repeated, and significant changes in cell size were detected within 400 generations. In addition, the width of the size distribution was altered. The changes in cell size were unaccompanied by a growth burden. Whole genome sequencing revealed that only a few mutations in genes related to membrane synthesis conferred the size evolution.ConclusionsIn conclusion, bacterial cell size could evolve, through a few mutations, without growth reduction. The size evolution without growth reduction suggests a rapid evolutionary change to diverse cell sizes in bacterial survival strategies.

Density-Dependent Recycling Promotes the Long-Term Survival of Bacterial Populations during Periods of Starvation

ABSTRACT The amount of natural resources in the Earth’s environment is in flux, which can trigger catastrophic collapses of ecosystems. How populations survive under nutrient-poor conditions is a central question in ecology. Curiously, some bacteria persist for a long time in nutrient-poor environments. Although this survival may be accomplished through cell death and the recycling of dead cells, the importance of these processes and the mechanisms underlying the survival of the populations have not been quantitated. Here, we use microbial laboratory experiments and mathematical models to demonstrate that death and recycling are essential activities for the maintenance of cell survival. We also show that the behavior of the survivors is governed by population density feedback, wherein growth is limited not only by the available resources but also by the population density. The numerical simulations suggest that population density-dependent recycling could be an advantageous behavior under starvation conditions. IMPORTANCE How organisms survive after exhaustion of resources is a central question in ecology. Starving Escherichia coli constitute a model system to understand survival mechanisms during long-term starvation. Although death and the recycling of dead cells might play a key role in the maintenance of long-term survival, their mechanisms and importance have not been quantitated. Here, we verified the significance of social recycling of dead cells for long-term survival. We also show that the survivors restrained their recycling and did not use all available nutrients released from dead cells, which may be advantageous under starvation conditions. These results indicate that not only the utilization of dead cells but also restrained recycling coordinate the effective utilization of limited resources for long-term survival under starvation. IMPORTANCE How organisms survive after exhaustion of resources is a central question in ecology. Starving Escherichia coli constitute a model system to understand survival mechanisms during long-term starvation. Although death and the recycling of dead cells might play a key role in the maintenance of long-term survival, their mechanisms and importance have not been quantitated. Here, we verified the significance of social recycling of dead cells for long-term survival. We also show that the survivors restrained their recycling and did not use all available nutrients released from dead cells, which may be advantageous under starvation conditions. These results indicate that not only the utilization of dead cells but also restrained recycling coordinate the effective utilization of limited resources for long-term survival under starvation.