Scaffold size-dependent effect on the enhanced uptake of antibiotics and other compounds by Escherichia coli and Pseudomonas aeruginosa.

The outer membrane of Gram-negative bacteria functions as an impermeable barrier to foreign compounds. Thus, modulating membrane transport can contribute to improving susceptibility to antibiotics and efficiency of bioproduction reactions. In this study, the cellular uptake of hydrophobic and large-scaffold antibiotics and other compounds in Gram-negative bacteria was investigated by modulating the homolog expression of bamB encoding an outer membrane lipoprotein and tolC encoding an outer membrane efflux protein via gene deletion and gene silencing. The potential of deletion mutants for biotechnological applications, such as drug screening and bioproduction, was also demonstrated. Instead of being subjected to gene deletion, wild-type bacterial cells were treated with cell-penetrating peptide conjugates of a peptide nucleic acid (CPP-PNA) against bamB and tolC homologs as antisense agents. Results revealed that the single deletion of bamB and tolC in Escherichia coli increased the uptake of large- and small-scaffold hydrophobic compounds, respectively. A bamB-and-tolC double deletion mutant had a higher uptake efficiency for certain antibiotics and other compounds with high hydrophobicity than each single deletion mutant. The CPP-PNA treated E. coli and Pseudomonas aeruginosa cells showed high sensitivity to various antibiotics. Therefore, these gene deletion and silencing approaches can be utilized in therapeutic and biotechnological fields.

MazF activation causes ACA sequence-independent and selective alterations in RNA levels in Escherichia coli.

Escherichia coli MazF is a toxin protein that cleaves RNA at ACA sequences. Its activation has been thought to cause growth inhibition, primarily through indiscriminate cleavage of RNA. To investigate responses following MazF activation, transcriptomic profiles of mazF-overexpressing and non-overexpressing E. coli K12 cells were compared. Analyses of differentially expressed genes demonstrated that the presence and the number of ACA trimers in RNA was unrelated to cellular RNA levels. Mapping differentially expressed genes onto the chromosome identified two chromosomal segments in which upregulated genes formed clusters, and these segments were absent in the chromosomes of E. coli strains other than K12. These results suggest that MazF regulates selective, rather than indiscriminate, categories of genes, and is involved in the regulation of horizontally acquired genes. We conclude that the primary role of MazF is not only cleaving RNA indiscriminately but also generating a specific cellular state.

Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits.

Reporter genes have contributed to advancements in molecular biology. Binding of an upstream regulatory protein to a downstream reporter promoter allows quantification of the activity of the upstream protein produced from the corresponding gene. In studies of synthetic biology, analyses of reporter gene activities ensure control of the cell with synthetic genetic circuits, as achieved using a combination of in silico and in vivo experiments. However, unexpected effects of downstream reporter genes on upstream regulatory genes may interfere with in vivo observations. This phenomenon is termed as retroactivity. Using in silico and in vivo experiments, we found that a different copy number of regulatory protein-binding sites in a downstream gene altered the upstream dynamics, suggesting retroactivity of reporters in this synthetic genetic oscillator. Furthermore, by separating the two sources of retroactivity (titration of the component and competition for degradation), we showed that, in the dual-feedback oscillator, the level of the fluorescent protein reporter competing for degradation with the circuits' components is important for the stability of the oscillations. Altogether, our results indicate that the selection of reporter promoters using a combination of in silico and in vivo experiments is essential for the advanced design of genetic circuits.

A Highly Bioactive Lys-Deficient IFN Leads to a Site-Specific Di-PEGylated IFN with Equivalent Bioactivity to That of Unmodified IFN-α2b.

Although conjugation with polyethylene glycol (PEGylation) improves the pharmacokinetics of therapeutic proteins, it drastically decreases their bioactivity. Site-specific PEGylation counters the reduction in bioactivity, but developing PEGylated proteins with equivalent bioactivity to that of their unmodified counterparts remains challenging. This study aimed to generate PEGylated proteins with equivalent bioactivity to that of unmodified counterparts. Using interferon (IFN) as a model protein, a highly bioactive Lys-deficient protein variant generated using our unique directed evolution methods enables the design of a site-specific di-PEGylated protein. Antiviral activity of our di-PEGylated IFN was similar to that of unmodified IFN-α2b. The di-PEGylated IFN exhibited 3.0-fold greater antiviral activity than that of a commercial PEGylated IFN. Moreover, our di-PEGylated IFN showed higher in vitro and in vivo stability than those of unmodified IFN-α2b. Hence, we propose that highly bioactive Lys-deficient proteins solve the limitation of conventional PEGylation with respect to the reduction in bioactivity of PEGylated proteins.

Effect of light wavelength on hot spring microbial mat biodiversity.

Hot spring associated phototrophic microbial mats are purely microbial communities, in which phototrophic bacteria function as primary producers and thus shape the community. The microbial mats at Nakabusa hot springs in Japan harbor diverse photosynthetic bacteria, mainly Thermosynechococcus, Chloroflexus, and Roseiflexus, which use light of different wavelength for energy conversion. The aim of this study was to investigate the effect of the phototrophs on biodiversity and community composition in hot spring microbial mats. For this, we specifically activated the different phototrophs by irradiating the mats with different wavelengths in situ. We used 625, 730, and 890 nm wavelength LEDs alone or in combination and confirmed the hypothesized increase in relative abundance of different phototrophs by 16S rRNA gene sequencing. In addition to the increase of the targeted phototrophs, we studied the effect of the different treatments on chemotrophic members. The specific activation of Thermosynechococcus led to increased abundance of several other bacteria, whereas wavelengths specific to Chloroflexus and Roseiflexus induced a decrease in >50% of the community members as compared to the dark conditions. This suggests that the growth of Thermosynechococcus at the surface layer benefits many community members, whereas less benefit is obtained from an increase in filamentous anoxygenic phototrophs Chloroflexus and Roseiflexus. The increases in relative abundance of chemotrophs under different light conditions suggest a relationship between the two groups. Aerobic chemoheterotrophs such as Thermus sp. and Meiothermus sp. are thought to benefit from aerobic conditions and organic carbon in the form of photosynthates by Thermosynechococcus, while the oxidation of sulfide and production of elemental sulfur by filamentous anoxygenic phototrophs benefit the sulfur-disproportionating Caldimicrobium thiodismutans. In this study, we used an experimental approach under controlled environmental conditions for the analysis of natural microbial communities, which proved to be a powerful tool to study interspecies relationships in the microbiome.

A Bacterial Continuous Culture System Based on a Microfluidic Droplet Open Reactor.

Recently, micrometer-sized bacterial culture systems have attracted attention as useful tools for synthetic biology studies. Here, we present the development of a bacterial continuous culture system based on a microdroplet open reactor consisting of two types of water-in-oil microdroplets with diameters of several hundred micrometers. A continuous culture was realized the through supply of nutrient substrates and the removal of waste and excess bacterial cells based on repeated fusion and fission of droplets. The growth dynamics was controlled by the interval of fusion. We constructed a microfluidic system and quantitatively assessed the dynamics of the bacterial growth using a mathematical model. This system will facilitate the study of synthetic biology and metabolic engineering in the future.

An observation method for autonomous signaling-mediated synthetic diversification in Escherichia coli.

Phenotypic diversification of cells in development and regeneration is conceptually modeled by the motion of marbles rolling down valleys on the Waddington landscape, the main feature of which is bifurcations of the valleys. We have experimentally shown that this feature is sufficient to achieve phenotypic diversification by the construction of a synthetic phenotypic diversification system in Escherichia coli. Cells containing the synthetic phenotypic diversification system were diversified into two distinct cell states, high and low, through autonomous signaling-mediated bifurcation, when all cells were initialized to the low state. In this chapter, we illustrate the detailed experimental procedures involved in the initialization of cells and the observation of the phenotypic diversification.

An aptazyme-based molecular device that converts a small-molecule input into an RNA output.

We describe the construction of an aptazyme-based molecular device that converts, through a cascade of reactions, a small-molecule input into output RNA strands. This device is applicable as an interface between a small molecule and a molecular system that accepts only nucleic acid input.

RTRACS: a modularized RNA-dependent RNA transcription system with high programmability.

Creating artificial biological systems is an important research endeavor. Each success contributes to synthetic biology and adds to our understanding of the functioning of the biomachinery of life. In the construction of large, complex systems, a modular approach simplifies the design process: a multilayered system can be prepared by integrating simple modules. With the concept of modularity, a variety of synthetic biological systems have been constructed, both in vivo and in vitro. But to properly develop systems with desired functions that integrate multiple modules, researchers need accurate mathematical models. In this Account, we review the development of a modularized artificial biological system known as RTRACS (reverse transcription and transcription-based autonomous computing system). In addition to modularity, model-guided predictability is an important feature of RTRACS. RTRACS has been developed as an in vitro artificial biological system through the assembly of RNA, DNA, and enzymes. A fundamental module of RTRACS receives an input RNA with a specific sequence and returns an output RNA with another specific sequence programmed in the main body, which is composed of DNA and enzymes. The conversion of the input RNA to the output RNA is achieved through a series of programmed reactions performed by the components assembled in the module. Through the substitution of a subset of components, a module that performs the AND operation was constructed. Other logical operations could be constructed with RTRACS modules. An integration of RTRACS modules has allowed the theoretical design of more complex functions, such as oscillation. The operations of these RTRACS modules were readily predicted with a numerical simulation based on a mathematical model using realistic parameters. RTRACS has the potential to model highly complex systems that function like a living cell. RTRACS was designed to be integrated with other molecules or molecular devices, for example, aptazymes, cell-free expression systems, and liposomes. For the integration of these new modules, the quantitative controls of each module based on the numerical simulation will be instructive. The capabilities of RTRACS promise to provide models of complex biomolecular systems that are able to detect the environment, assess the situation, and react to overcome the situation. Such a smart biomolecular system could be useful in many applications, such as drug delivery systems.

Tunable synthetic phenotypic diversification on Waddington's landscape through autonomous signaling.

Phenotypic diversification of cells is crucial for developmental and regenerative processes in multicellular organisms. The diversification concept is described as the motion of marbles rolling down Waddington's landscape, in which the number of stable states changes as development proceeds. In contrast to this simple concept, the complexity of natural biomolecular processes prevents comprehension of their design principles. We have constructed, in Escherichia coli, a synthetic circuit with just four genes, which programs cells to autonomously diversify as the motion on the landscape through cell-cell communication. The circuit design was based on the combination of a bistable toggle switch with an intercellular signaling system. The cells with the circuit diversified into two distinct cell states, "high" and "low," in vivo and in silico, when all of the cells started from the low state. The synthetic diversification was affected by not only the shape of the landscape determined by the circuit design, which includes the synthesis rate of the signaling molecule, but also the number of cells in the experiments. This cell-number dependency is reminiscent of the "community effect": The fates of developing cells are determined by their number. Our synthetic circuit could be a model system for studying diversification and differentiation in higher organisms. Prospectively, further integrations of our circuit with different cellular functions will provide unique tools for directing cell fates on the population level in tissue engineering.

Construction of a genetic AND gate under a new standard for assembly of genetic parts.

BACKGROUND: Appropriate regulation of respective gene expressions is a bottleneck for the realization of artificial biological systems inside living cells. The modification of several promoter sequences is required to achieve appropriate regulation of the systems. However, a time-consuming process is required for the insertion of an operator, a binding site of a protein for gene expression, to the gene regulatory region of a plasmid. Thus, a standardized method for integrating operator sequences to the regulatory region of a plasmid is required. RESULTS: We developed a standardized method for integrating operator sequences to the regulatory region of a plasmid and constructed a synthetic promoter that functions as a genetic AND gate. By standardizing the regulatory region of a plasmid and the operator parts, we established a platform for modular assembly of the operator parts. Moreover, by assembling two different operator parts on the regulatory region, we constructed a regulatory device with an AND gate function. CONCLUSIONS: We implemented a new standard to assemble operator parts for construction of functional genetic logic gates. The logic gates at the molecular scale have important implications for reprogramming cellular behavior.