群体感应系统在合成生物学中的应用*

群体感应(quorum sensing, QS)是一种广泛存在于多种微生物中的胞间通信系统,细菌产生的自诱导物随着种群密度的增加而积累,诱导细菌对种群密度的响应,调节生物膜的形成或特定基因的表达。近年来,随着群体感应系统原理与关键元件的逐渐清晰,应用合成生物学手段进行多技术联合以及多系统间正交性设计具有极大的发展潜力,群体感应系统已成为合成生物学家动态调控胞间通信常用的重要手段之一。在群体感应是细胞-细胞间通信系统的基础上,对多种群体感应系统的联合设计在生物基化学品生产中自动化调控的研究进展进行综述;并针对群体感应系统在生物电化学转化领域实现双向生物信息交流的应用进行总结;同时归纳了医学领域中群体感应系统的动态调控功能与多种疾病诊断及治疗结合的研究进展,讨论了群体感应系统在多细胞通信和实际应用等方面的发展前景。     

Cell population dynamics (apoptosis, mitosis, and cell-cell communication) during disruption of homeostasis

The sequence of events involved in maintenance of homeostasis must encompass mechanisms within single cells as well as interactions between cells within a population. To investigate the interaction among these inter- and intracellular mechanisms, disruption of homeostasis by serum deprivation was performed in WB-F344, a normal diploid epithelial cell line. Changes in cell-cell communication (gap junction function) at the population level and in individual cells were monitored using the scrape load/dye transfer and fluorescence redistribution after photobleaching assays. Apoptosis and mitosis were measured using internucleosomal DNA ladder assays and fluorescence-activated cell sorting. The results indicate that a common element in early apoptosis and early mitosis is sustained gap junction function. As cell life (mitosis) and cell death (apoptosis) progressed, a common process of change in gap junction function occurred. A transient stimulation of mitosis concomitant with increased apoptosis was also observed during serum deprivation. Gap junctions may play a regulatory role during initiation of these opposite yet equally important mechanisms of maintaining homeostasis. This model system is useful for further studies on the relationships among inter- and intracellular mechanisms of homeostasis.     

Modular construction of multi-subunit protein complexes using engineered tags and microbial transglutaminase

Motivations for the hierarchical assembly of protein complexes are diverse spanning biosensing, biomedical and bioreactor applications. The assembly processes should be simple, scalable, versatile, and biologically benign to minimize loss of component parts. A “plug and play” methodology comprising a generic linking apparatus may enable rapid design and optimization. One application that desires these qualities is metabolon construction wherein multiple enzymes are organized in defined pathways to mediate biochemical flux. Here, we propose a modular design by incorporation of crosslinking-compliant amino acid tags comprised of lysine or glutamine residues at the N- or C-termini of the to-be-assembled proteins. These amino acid tags enable covalent crosslinking using microbial transglutaminase (mTG). Modularity is demonstrated where stoichiometries and relative positions of enzymes and other functional proteins are altered. Construction of multifunctional complexes is demonstrated by crosslinking domains of different function and origin. Namely, we built a two-subunit quorum sensing (QS) biosynthetic metabolon on solid supports and altered stoichiometries of the limiting constituents to increase the overall rate of reaction. To display functionality beyond biosynthesis, we constructed a molecular communication ‘device’ (antibody binding Protein G–QS complex) to target bacterial cells and demonstrated tailored QS responses among targeted bacteria. We propose that this approach, solid phase mTG-mediated linkage of biological components, can be used for assembly within many environments including microreactors or lab-on-a-chip systems. Because the methodology is general, we envision construction of multi-functional protein complexes in a ‘plug and play’ fashion for a variety of biosensing and synthetic biology applications.     

Quorum-sensing linked RNA interference for dynamic metabolic pathway control in Saccharomyces cerevisiae

Some of the most productive metabolic engineering strategies involve genetic modifications that cause severe metabolic burden on the host cell. Growth-limiting genetic modifications can be more effective if they are ‘switched on’ after a population growth phase has been completed. To address this problem we have engineered dynamic regulation using a previously developed synthetic quorum sensing circuit in Saccharomyces cerevisiae. The circuit autonomously triggers gene expression at a high population density, and was linked with an RNA interference module to enable target gene silencing. As a demonstration the circuit was used to control flux through the shikimate pathway for the production of para-hydroxybenzoic acid (PHBA). Dynamic RNA repression allowed gene knock-downs which were identified by elementary flux mode analysis as highly productive but with low biomass formation to be implemented after a population growth phase, resulting in the highest published PHBA titer in yeast (1.1 mM).     

Bioelectricity enhancement via overexpression of quorum sensing system in Pseudomonas aeruginosa-inoculated microbial fuel cells

Low electron transfer efficiency from bacteria to electrodes remains one of the major bottlenecks that limit industrial applications of microbial fuel cells (MFCs). Elucidating biological mechanism of the electron transfer processes is of great help in improving the efficiency of MFCs. Here, we reported that Pseudomonas aeruginosa could use different electron shuttles in a MFC under different quorum sensing (QS) expression patterns. An electron shuttle (rather than phenazines) with a high mid-point potential of 0.20 V (vs. Ag/AgCl–KCl saturated electrode) was found to be the dominating shuttle in a wild-type P. aeruginosa strain. Strikingly, upon genetic overexpression of rhl QS system in this wild-type strain, the electron shuttle was substituted by phenazines (pyocyanin and phenazine-1-carboxylate, with a low mid-point potential of −0.17 V and −0.28 V, respectively), which directly resulted in an increase of about 1.6 times of the maximum current of the rhl overexpressed strain over the wild-type strain. Our result implied that manipulating electron transfer pathways to improve MFCs’ efficiency could be achieved by rewiring gene regulatory circuits, thus synthetic biology strategies would be adopted.     

Modeling population dynamics in a microbial consortium under control of a synthetic pheromone‐mediated communication system

Microbial consortia can be used to catalyze complex biotransformations. Tools to control the behavior of these consortia in a technical environment are currently lacking. In the present study, a synthetic biology approach was used to build a model consortium of two Saccharomyces cerevisiae strains where growth and expression of the fluorescent marker protein EGFP by the receiver strain is controlled by the concentration of α‐factor pheromone, which is produced by the emitter strain. We have developed a quantitative experimental and theoretical framework to describe population dynamics in the model consortium. We measured biomass growth and metabolite production in controlled bioreactor experiments, and used flow cytometry to monitor changes of the subpopulations and protein expression under different cultivation conditions. This dataset was used to parameterize a segregated mathematical model, which took into account fundamental growth processes, pheromone‐induced growth arrest and EGFP production, as well as pheromone desensitization after extended exposure. The model was able to predict the growth dynamics of single‐strain cultures and the consortium quantitatively and provides a basis for using this approach in actual biotransformations.     

Sensitivity of the quorum sensing system is achieved by low pass filtering

Autoinducer sensing, also known as quorum sensing, is the communication of bacteria by autoinducer (small signaling molecules). Cells respond on extremely low concentrations of autoinducer: only one or two molecules per cell are sufficient. At this signal level a high degree of noise is inherent. We ask for the mechanism that is able to overcome the stochasticity of the signal. By means of a model and parameter fitting we show that the sensing module acts as a low pass filter, representing the biochemical equivalent of a moving average. It is shown that the system works most sensitive in the range of 0-50 nM autoinducer. Moreover, the time scale of the reaction depends on the signal strength in a crucial manner. Nonlinear feedback is able to further enhance the sensitivity. The biological implications of the low pass filter property are discussed.     

A modular autoinduction device for control of gene expression in Bacillus subtilis

Intense synthesis of proteins and chemicals in engineered microbes impose metabolic burden, frequently leading to reduced growth and heterogeneous cell population. Thus, the correct balance between growth and production is important. Such balance can be engineered through dynamic control of pathways, but few broadly applicable tools are available to achieve this. We present an autonomous control of gene expression mediated by quorum sensing in Bacillus subtilis, able to self-monitor and induce expression without human supervision. Two variations of the induction module and seven of the response module were engineered generating a range of induction folds and strengths for gene expression control. Our strongest response promoter is 2.5 and 3.2 times stronger than the well-characterized promoters P_srfA and P_veg, respectively. We applied our strongest autoinduction device for the production of the vitamin B₂. This study presents a toolbox of autoinduction modules for B. subtilis that is modular and tunable.     

Developing next generation antimicrobials by intercepting AI-2 mediated quorum sensing

Bacteria have been evolving antibiotic resistance since their discovery in the early twentieth century. Most new antibiotics are derivatives of older generations and there are now bacteria that are virtually resistant to almost all antibiotics. This poses a global threat to human health and has been classified as a clinical “super-challenge”, which has necessitated research into new antimicrobials that inhibit bacterial virulence while minimizing selective pressures that lead to the emergence of resistant strains. Quorum sensing (QS), the process of population dependent bacterial cell-cell signaling, can accelerate bacterial virulence and is an increasingly interesting target for developing next generation antimicrobials. Most QS inhibitors target species-specific signals, such as acylhomoserine lactones (AHLs) and oligopeptides. Methodologies for intercepting the cross-species signal, autoinducer-2 (AI-2), have only recently emerged. We review these strategies to prevent the relay of the AI-2 signal amongst pathogens, including Escherichia coli, Salmonella enterica serovar Typhimurium, Vibrio cholerae and Pseudomonas aeruginosa. Inhibition mechanisms are categorized based on the target (i.e., enzymes for signal generation, the signal molecule itself, or the various components of the signal transduction process). The universal nature of the AI-2 signal imparts on its inhibitors the potential for broad spectrum use.     

Exosomes and nanotubes: Control of immune cell communication

Cell–cell communication is critical to coordinate the activity and behavior of a multicellular organism. The cells of the immune system not only must communicate with similar cells, but also with many other cell types in the body. Therefore, the cells of the immune system have evolved multiple ways to communicate. Exosomes and tunneling nanotubes (TNTs) are two means of communication used by immune cells that contribute to immune functions. Exosomes are small membrane vesicles secreted by most cell types that can mediate intercellular communication and in the immune system they are proposed to play a role in antigen presentation and modulation of gene expression. TNTs are membranous structures that mediate direct cell-cell contact over several cell diameters in length (and possibly longer) and facilitate the interaction and/or the transfer of signals, material and other cellular organelles between connected cells. Recent studies have revealed additional, but sometimes conflicting, structural and functional features of both exosomes and TNTs. Despite the new and exciting information in exosome and TNT composition, origin and in vitro function, biologically significant functions are still being investigated and determined. In this review, we discuss the current field regarding exosomes and TNTs in immune cells providing evaluation and perspectives of the current literature.     

Bacterial communication through membrane vesicles

ABSTRACT

Bacteria can communicate through diffusible signaling molecules that are perceived by cognate receptors. It is now well established that bacterial communication regulates hundreds of genes. Hydrophobic molecules which do not diffuse in aqueous environments alone have been identified in bacterial communication, that raised the question on how these molecules are transported between cells and trigger gene expressions. Recent studies show that these hydrophobic signaling molecules, including a long-chain N-acyl homoserine lactone signal produced in Paracoccus denitrificans, are carried by membrane vesicles (MVs). MVs were thought to be formed only through the blebbing of the cell membrane, but new findings in Pseudomonas aeruginosa and Bacillus subtilis revealed that different types of MVs can be formed through explosive cell lysis or bubbling cell death, which findings have certain implications on our view of bacterial interactions.     

Characterization of molecules mediating cell-cell communication in human cardiac valve interstitial cells

Cell-cell interactions and adhesion determine cellular architectural organization, proliferation, signaling, differentiation, and death. We have identified the molecular components of different cell-cell junctions in human valve interstitial cells (ICs) both in situ and in culture. ICs were isolated, cultured, and phenotyped for cell surface and cytoplasmic markers by flow cytometry and immunocytochemistry. Western blotting was used to identify and quantify the molecular components of these cell-cell junctions in human valve ICs and compared with expression in smooth muscle and fibroblast cell types. N-cadherin and desmoglein were weakly detected on a low percentage of ICs, and the other classical cadherins were not detected. α- and β-catenin, but not γ-catenin, were expressed at equivalent levels by all valve ICs. Valve ICs did not express connexin-32 and-40; however, connexin-26 and-43 were equally expressed by a low percentage of ICs, demonstrating cell surface and cytoplasmic expression, and connexin-45 was weakly expressed. The other cell types also expressed N-cadherin, α- and β-catenin, desmoglein and connexin-43. The expression of these junctional molecules was predominantly by valve ICs on the inflow side of the valves. Human valve ICs have the ability to communicate with other valve ICs and mediate cell-cell adhesion via N-cadherin, connexin-26 and-43, and desmoglein. The junctions between valve ICs could support an interconnecting and coordinated cellular unit capable of controlling the functionality of the valve.     

Involvement of protein kinases in self-organization of the rhythm of protein synthesis by direct cell-cell communication

Primary cultures of rat hepatocytes grown on slides were studied in serum-free medium. Ultradian protein synthesis rhythm was used as a marker of synchronization of individual oscillations, resulting in the formation of a common rhythm of the cell population, i.e. cell-cell self-organization. Dense synchronous and sparse non-synchronous cultures were used to estimate effect of protein kinase activity on the kinetics of protein synthesis. Treatment of dense cultures with the inhibitors H7 (40 microM) or H8 (25 microM) resulted in a loss of the protein synthesis rhythm, a suppression of the cell-cell self-organization. Stimulation of protein kinase activity with either 0.5 or 1.0 microM phorbol 12-miristate-13-acetate (PMA) or 10 microM forskolin caused the appearance of the synthetic rhythm in non-synchronous sparse cultures under otherwise normal conditions. Inhibition of protein kinase activity with H7 resulted in signal factors, such as gangliosides and phenylephrine, failing to initiate this rhythm in sparse cultures. Activation of protein kinase activity with PMA shifted the phase pattern of the protein synthesis rhythm. Thus, according to our previous and the new data, protein kinase activity and consequently protein phosphorylation is the crucial step of sequence of processes resulting in synchronization during self-organization of cells in producing a common rhythm in the population. The general pathway can be presented as follows: signaling of gangliosides or other calcium agonists-->efflux of calcium ion from intracellular stores, with elevation of calcium concentration in the cytoplasm-->activation of protein kinases-->protein phosphorylation-->synchronization of individual oscillations in protein synthesis rates-->induction of a common rhythm throughout this population. The data have been discussed concerning similarity of the direct cell-cell communication and the cell self-organization in cultures and in organism.     

Towards one sample per second for mass spectrometric screening of engineered microbial strains

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LRRC8 proteins share a common ancestor with pannexins, and may form hexameric channels involved in cell-cell communication

Leucine-rich repeat-containing 8 (LRRC8) proteins are composed of four transmembrane helices and 17 leucine-rich repeats (LRR). Although LRRC8 proteins have been associated with important processes, like maturation of B cells or adipocyte differentiation, their biology and molecular function are largely unknown. We found that LRRC8 proteins originated from the combination of a pannexin and an LRR domain (most likely related to the SHOC2, LAP, RSU1 and LRRIQ4 protein families) before the diversification of chordates. We propose that, like pannexins, LRRC8 proteins form hexameric channels, which participate in cell-cell communication processes. According to the inferred topological model, and contrary to what was previously assumed, the six LRR domains are located in the cytoplasm, and could participate in the organisation of signalling cascades. By compiling available proteomics and gene expression data, and on the basis of the LRRC8 proposed hexameric channel structure, we present clues to the function of this family.