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.     

革兰氏阴性细菌LuxR/I型群体感应抑制剂研究进展

摘要：细菌群体感应（Quorum sensing, QS）被视为对抗细菌感染与解决细菌耐药性问题的新靶点。以AHLs为信号分子的LuxR/I型群体感应系统广泛存在于革兰氏阴性菌包括多种临床致病菌中,因此寻找LuxR/I型群体感应抑制剂（Quorum sensing inhibitors, QSIs）是研发抗革兰氏阴性致病菌药物的重要途径。迄今为止,已知的LuxR/I型小分子QSIs来源包括化学合成、天然产物与已知药物库的化合物,大分子则包括群体感应淬灭酶与群体感应淬灭抗体。本文总结了近年来LuxR/I型QSIs研究进展,为新型抗菌药物研发提供理论依据。     

Programming microbial population dynamics by engineered cell-cell communication

A major aim of synthetic biology is to program novel cellular behavior using engineered gene circuits. Early endeavors focused on building simple circuits that fulfill simple functions, such as logic gates, bistable toggle switches, and oscillators. These gene circuits have primarily focused on single-cell behaviors since they operate intracellularly. Thus, they are often susceptible to cell-cell variations due to stochastic gene expression. Cell-cell communication offers an efficient strategy to coordinate cellular behavior at the population level. To this end, we review recent advances in engineering cell-cell communication to achieve reliable population dynamics, spanning from communication within single species to multispecies, from one-way sender-receiver communication to two-way communication in synthetic microbial ecosystems. These engineered systems serve as well-defined model systems to better understand design principles of their naturally occurring counterparts and to facilitate novel biotechnology applications.     

Regulatory dynamics of synthetic gene networks with positive feedback

Biological processes are governed by complex networks ranging from gene regulation to signal transduction. Positive feedback is a key element in such networks. The regulation enables cells to adopt multiple internal expression states in response to a single external input signal. However, past works lacked a dynamical aspect of this system. To address the dynamical property of the positive feedback system, we employ synthetic gene circuits in Escherichia coli to measure the rise-time of both the no-feedback system and the positive feedback system. We show that the kinetics of gene expression is slowed down if the gene regulatory system includes positive feedback. We also report that the transition of gene switching behaviors from the hysteretic one to the graded one occurs. A mathematical model based on the chemical reactions shows that the response delay is an inherited property of the positive feedback system. Furthermore, with the aid of the phase diagram, we demonstrate the decline of the feedback activation causes the transition of switching behaviors. Our findings provide a further understanding of a positive feedback system in a living cell from a dynamical point of view.     

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。     

Phytopathogenic bacteria utilize host glucose as a signal to stimulate virulence through LuxR homologues

Chemical signal-mediated biological communication is common within bacteria and between bacteria and their hosts. Many plant-associated bacteria respond to unknown plant compounds to regulate bacterial gene expression. However, the nature of the plant compounds that mediate such interkingdom communication and the underlying mechanisms remain poorly characterized. Xanthomonas campestris pv. campestris (Xcc) causes black rot disease on brassica vegetables. Xcc contains an orphan LuxR regulator (XccR) which senses a plant signal that was validated to be glucose by HPLC-MS. The glucose concentration increases in apoplast fluid after Xcc infection, which is caused by the enhanced activity of plant sugar transporters translocating sugar and cell-wall invertases releasing glucose from sucrose. XccR recruits glucose, but not fructose, sucrose, glucose 6-phosphate, and UDP-glucose, to activate pip expression. Deletion of the bacterial glucose transporter gene sglT impaired pathogen virulence and pip expression. Structural prediction showed that the N-terminal domain of XccR forms an alternative pocket neighbouring the AHL-binding pocket for glucose docking. Substitution of three residues affecting structural stability abolished the ability of XccR to bind to the luxXc box in the pip promoter. Several other XccR homologues from plant-associated bacteria can also form stable complexes with glucose, indicating that glucose may function as a common signal molecule for pathogen–plant interactions. The conservation of a glucose/XccR/pip-like system in plant-associated bacteria suggests that some phytopathogens have evolved the ability to utilize host compounds as virulence signals, indicating that LuxRs mediate an interkingdom signalling circuit.     

Modeling direct positive feedback between predators and prey

Predators can have positive impacts on their prey through such mechanisms as nutrient mineralization and prey transport. These positive feedbacks have the potential to change predictions based on food web theory, such as the assertion that enrichment is destabilizing. We present a model of a simple food web, consisting of a resource, a consumer, and its predator. We assume that the predator has a direct positive effect on the consumer, by increasing the rate at which the consumer acquires resources. We consider two cases: the feedback strength is a saturating function of predator density, or it is proportional to the encounter rate between predators and prey. In both cases, the positive feedback is stabilizing, delaying or preventing the onset of oscillations due to enrichment. Positive feedback can introduce an Allee effect for the predator population, yielding multiple stable equilibria. Strong positive feedback can yield counterintuitive results such as a transient increase in consumer density following the introduction of predators, and a decrease in the resource pool following enrichment.     

Binding mode exploration of LuxR-thiazolidinedione analogues,e-pharmacophore-based virtual screening in the designing of LuxR inhibitors and its biological evaluation

Master quorum sensing (QS) regulator LuxR of Vibrio harveyi is a unique member of the TetR protein superfamily. Recent studies have demonstrated the contribution of thiazolidinedione analogues in blocking QS by decreasing the DNA-binding ability of LuxR. However, the precise mechanism of thiazolidinedione analogues binding to LuxR is still unclear. In the present study, molecular docking combined with molecular dynamics (MD) simulations was performed to understand the mechanism of ligand binding to the protein. The binding pattern of thiazolidinedione analogues showed strong hydrogen bonding interactions with the amine group (NH) of polar amino acid residue Asn133 and carbonyl (C=O) interaction with negatively charged amino acid residue Gln137 in the binding site of LuxR. The stability of the protein–ligand complexes was confirmed by running 50 ns of MD simulations. Further, the four-featured pharmacophore hypothesis (AHHD) consists of one acceptor (A), two hydrophobic regions (HH) and one donor (D) group was used to screen compounds from ChemBridge database. The identified hit molecules were shown to have excellent pharmacokinetic properties under the acceptable range. Based on the computational studies, ChemBridge_5343641 was selected for in vitro assays. The 1-(4-chlorophenoxy)-3-[(4,6-dimethyl-2-pyrimidinyl)thio]-2-propanol (ChemBridge_5343641) showed significant reduction in bioluminescence in a dose-dependent manner. In addition, ChemBridge_5343641 inhibits biofilm formation and motility in V. harveyi. The result from the study suggests that ChemBridge_5343641 could serve as an anti-QS molecule.     

Considerations for using integral feedback control to construct a perfectly adapting synthetic gene network

It has long been known to control theorists and engineers that integral feedback control leads to, and is necessary for, “perfect” adaptation to step input perturbations in most systems. Consequently, implementation of this robust control strategy in a synthetic gene network is an attractive prospect. However, the nature of genetic regulatory networks (density-dependent kinetics and molecular signals that easily reach saturation) implies that the design and construction of such a device is not straightforward. In this study, we propose a generic two-promoter genetic regulatory network for the purpose of exhibiting perfect adaptation; our treatment highlights the challenges inherent in the implementation of a genetic integral controller. We also present a numerical case study for a specific realization of this two-promoter network, “constructed” using commonly available parts from the bacterium Escherichia coli. We illustrate the possibility of optimizing this network's transient response via analogy to a linear, free-damped harmonic oscillator. Finally, we discuss extensions of this two-promoter network to a proportional-integral controller and to a three-promoter network capable of perfect adaptation under conditions where first-order protein removal effects would otherwise disrupt the adaptation.     

Equilibria and stability of a class of positive feedback loops

Positive feedback loops are common regulatory elements in metabolic and protein signalling pathways. The length of such feedback loops determines stability and sensitivity to network perturbations. Here we provide a mathematical analysis of arbitrary length positive feedback loops with protein production and degradation. These loops serve as an abstraction of typical regulation patterns in protein signalling pathways. We first perform a steady state analysis and, independently of the chain length, identify exactly two steady states that represent either biological activity or inactivity. We thereby provide two formulas for the steady state protein concentrations as a function of feedback length, strength of feedback, as well as protein production and degradation rates. Using a control theory approach, analysing the frequency response of the linearisation of the system and exploiting the Small Gain Theorem, we provide conditions for local stability for both steady states. Our results demonstrate that, under some parameter relationships, once a biological meaningful on steady state arises, it is stable, while the off steady state, where all proteins are inactive, becomes unstable. We apply our results to a three-tier feedback of caspase activation in apoptosis and demonstrate how an intermediary protein in such a loop may be used as a signal amplifier within the cascade. Our results provide a rigorous mathematical analysis of positive feedback chains of arbitrary length, thereby relating pathway structure and stability.     

群体感应信号分子及其抑制剂快速检测方法的建立

细菌能自发产生、释放一些特定的信号分子,并能感知其浓度变化,调节微生物的群体行为,这一调控系统称为群体感应。细菌群体感应参与包括人类、动植物病原菌致病力在内的多种生物学功能的调节,群体感应抑制剂成为抗感染药物开发的靶点。利用紫色色杆菌(Chromobacterium violaceum)和根癌农杆菌(Agrobacterium tumefaciens)作为指示菌,建立检测高丝氨酸内酯(AHLs)及其抑制剂的简便方法。结果表明,通过平板交叉划线接种,使用指示菌能够有效地检测AHLs,并且通过薄层层析(TLC)与细菌生物感应器相结合的方法可以快速、方便地鉴定AHLs的种类;通过双层平板法观察指示菌色素产生情况,能够有效地检测群体感应信号分子AHLs抑制剂,且该方法简单易行。     

AKT as locus of cancer positive feedback loops and extreme robustness

A positive feedback loops induce extreme robustness in metastatic cancer, relapsed leukemia, myeloma or lymphoma. The loops are generated by the signaling interactome networks of autocrine and paracrine elements from cancer hypoxic microenvironment. The elements of the networks are signaling proteins synthesized in hypoxic microenvironment such as the vascular endothelial growth factor, HIF‐1α, hepatocyte growth factor, and molecules nitric oxide and H₂O₂. The signals from upstream or rebound downstream pathways are amplified by the short or wide positive feedback loops, hyperstimulating AKT‐inducing cancer extreme robustness. Targeting the phosphorylated AKT locus by an oxidant/antioxidant modulation induces collapse of positive feedback loops and establishment of negative feedback loops leading to stability of the system and disappearance of cancer extreme robustness. This is a new principle for the conversion of cancer positive loops into negative feedback loops by the locus chemotherapy. J. Cell. Physiol. 228: 522–524, 2013. © 2012 Wiley Periodicals, Inc.