Yes / Pantoea stewartii strain M073a is a Gram-negative bacterium isolated from a tropical waterfall. This strain exhibits quorum-sensing activity. Here, the assembly and annotation of its genome are presented. / High Impact Research Grants from the University of Malaya (UM.C/625/1/HIR/MOHE/CHAN/01, grant no. A-000001-50001 and UM-MOHE HIR Grant UM.C/625/1/HIR/MOHE/ CHAN/14/1, no. H-50001-A000027)

Natural cellular networks are very good at processing diverse inputs, generating complicated responses, and confounding researchers with their complexities. As an alternative to traditional cellular engineering approaches, the field of synthetic biology attempts to avoid the complexities of natural systems by focusing on the bottom-up construction of artificial cellular circuits. By rationally building up circuit complexity, synthetic biologists hope to both create novel systems capable of programming unique cellular responses, and gain insights into the design principles of natural systems. Circuits that allow for the programming of intercellular responses are of particular interest, and researchers have focused on the use of bacterial communication mechanisms (quorum sensing) to construct such circuits. At their most basic, quorum-sensing systems are composed of three main components, making them amenable to genetic manipulation. These components, however, have properties that have been finely tuned through evolution to function in very specific ways, and repurposing them for our own uses requires methods to overcome their naturally evolved properties. This thesis details our work in the construction and engineering of synthetic circuits based on components of the LuxI-LuxR quorum-sensing system. Using these components, we demonstrate methods for altering both the sensitivity and the form of the quorum-sensing response through the creation of three unique systems: an ultrasensitive positive feedback loop, a logical AND gate, and a coupled feedback loop oscillator. Construction and tuning of each circuit's properties were achieved through a mixture of rational and evolutionary approaches, with particular emphasis on the directed evolution of the LuxR transcriptional activator. Mathematical modeling was also used during the construction of the more complex circuits to predict the properties that were essential to their functionalities. With the construction and characterization of these circuits, we have provided both well-defined modules that can be used in the construction of more complex systems, and developed methods that will allow for the creation and engineering of additional synthetic circuits.

No description available.

Work over the past two decades has revealed that non-coding RNAs (ncRNAs) are prevalent in all kingdoms of life. Using RNA-seq we discovered hundreds of ncRNAs in the antibiotic-producing genus of bacteria, Streptomyces. These included trans-encoded small RNAS (sRNAs), cis-antisense RNAs, and a new type of antisense RNA we termed cutoRNAs (convergent untranslated overlapping RNAs) that arise when transcription termination does not occur in the intergenic region between two convergently arranged genes. Many of these ncRNAs feature prominently in the specialized metabolite biosynthetic clusters (e.g. antibiotics, anticancer agents, immunosuppressants). Hence, it is likely that understanding the functions of these RNAs will be important for new molecule discovery. We found that one highly expressed antisense RNA (ScbN) was expressed opposite the -butyrolactone synthase scbA in the model streptomycete Streptomyces coelicolor. However, ScbN had no detectible impact on the expression of scbA. Instead, the transcription terminator of scbN, which also forms a hairpin within the coding sequence of scbA, was found to reduce expression of scbA more than 10-fold. This led us to bioinformatically search for similar coding-sequence hairpins throughout all bacteria, leading to the discovery of many stable RNA structures with conserved locations throughout very divergent bacteria (e.g. Streptomyces, Escherichia coli, Bacillus subtilis). / Thesis / Doctor of Philosophy (PhD) / The flow of genetic information, from DNA to RNA to proteins, often portrays RNA as a mere intermediary molecule. An alternative, and perhaps more accurate, way to view RNA is that it is central to all cellular processes. Many RNAs are not translated into proteins and instead act as regulatory molecules, impacting the expression of other genes. In this work we found many examples of these regulatory RNAs in a group of bacteria known to produce many of the world’s antibiotics. Understanding the roles these regulatory RNAs play in impacting gene expression will be important for the discovery of new molecules, such as antibiotics. In addition to distinct regulatory RNAs mentioned above, we found that RNA structures within the coding sequences of mRNAs that are translated into proteins have dramatic regulatory consequences. We describe the characterization of one such RNA structure in a gene involved in bacterial communication, and develop a bioinformatic tool to hunt for other such structures conserved throughout bacteria.

Swimming bacteria are ubiquitous in aqueous environments ranging from oceans to fluidic environments within a living host. Furthermore, engineered bacteria are being increasingly utilized for a host of applications including environmental bioremediation, biosensing, and for the treatment of diseases. Often driven by chemotaxis (i.e. biased migration in response to gradients of chemical effectors) and quorum sensing (i.e. number density dependent regulation of gene expression), bacterial population dynamics and emergent behavior play a key role in regulating their own life and their impact on their immediate environment. Computational models that accurately and robustly describe bacterial population behavior and response to environmental stimuli are crucial to both understanding the dynamics of microbial communities and efficiently utilizing engineered microbes in practice. Many existing computational frameworks are finely-detailed at the cellular level, leading to extended computational time requirements, or are strictly population scale models, which do not permit population heterogeneities or spatiotemporal variability in the environment. To bridge this gap, we have created and experimentally validated a scalable, computationally-efficient, agent-based model of bacterial chemotaxis and quorum sensing (QS) which robustly simulates the stochastic behavior of each cell across a wide range of bacterial populations, ranging from a few to several hundred cells. We quantitatively and accurately capture emergent behavior in both isogenic QS populations and the altered QS response in a mixed QS and quorum quenching (QQ) microbial community. Finally, we show that the model can be used to predictively design synthetic genetic components towards programmed microbial behavior. / Master of Science

The LuxR protein family is a class of quorum-sensing regulated bacterial transcription factors that alter gene expression as a function of ligand detection. This coincides with a high population density and/or a low rate of signal ligand diffusion. The majority of LuxR proteins are activated only in the presence of the signal ligand, an acyl-homoserine lactone (AHL). EsaR, from the corn pathogen Pantoea stewartii, represents a subset of LuxR homologues that are active in the absence of AHL and deactivated by its presence. The mechanism by which EsaR responds to AHL in a manner opposite to that of the majority of LuxR homologues remains elusive. Unlike the majority of LuxR homologues, which require AHL for purification, EsaR can be purified and biochemically investigated in the absence and presence of AHL. This work sought to answer questions regarding the structure-function relationship of the LuxR homologue, EsaR. Fluorescence anisotropy was used to determine the relative DNA-binding affinity of wild type EsaR and three AHL-independent EsaR variants in the presence and absence of AHL. This enabled for quantitative analysis of the relative binding affinities of these AHL-independent variants for the EsaR binding site, the esa box. The results demonstrate that one AHL-independent EsaR variant has a slightly higher affinity for the esa box in the presence, rather than the absence of AHL. The affinity of the other two for the DNA is not impacted by AHL, potentially due to an inability to transduce the signal of ligand detection to the DNA binding domain. Constructs containing only the EsaR N-terminal domain (NTD) were also developed. These constructs circumvented solubility issues associated with the full-length protein, allowing for additional biochemical analysis. It was determined that the EsaR NTD alone is sufficient for multimerization and ligand binding. Additionally, preliminary X-ray crystallography efforts have established some of the early parameters required to solve the crystal structure of the EsaR ligand binding domain in both the presence and absence of AHL. If pursued, these structures would be the first solved of a LuxR homologue ligand binding domain in both the presence and absence of the native AHL, potentially demonstrating the conformational change that occurs as a result of ligand binding. Collectively, these findings have established some of the groundwork required to resolve the question of what sort of conformational changes occur in EsaR as a result of ligand binding. / Master of Science

The quorum sensing signaling system based on intercellular exchange of N-acyl-homoserine lactones is used by many proteobacteria to regulate the transcription of essential genes in a signal density-dependent manner. It is involved in a number of processes including the development of highly organized bacterial communities, e.g., biofilms, the regulation of expression of virulence factors, production of antibiotics, and bioluminescence. The extensive genetic and biochemical data available on the quorum sensing system in Vibrio fischeri allows the development of a systems biology approach to undertake a spatial and dynamical analysis of the regulation throughout the population. The quorum sensing regulated lux genes are organized in two divergent transcriptional units: luxR and luxICDABEG. The latter contains the genes required for luminescence and the luxI gene necessary for synthesis of an N-acyl-homoserine lactone commonly called autoinducer (AI). The luxR gene codes for a transcriptional regulatory protein that activates the transcription of both operons at a threshold concentration of AI. The positive feedback loop induces a rapid increase of transcription level of the lux genes when a critical population density is reached (reflected by the concentration of AI in the environment). With a combination of molecular biology tools, physiological analysis, and mathematical modeling we identified critical characteristics of the system and expect to assign parameter values in order to achieve a comprehensive understanding of the dynamics. An ordinary differential equation mathematical model is used to investigate the dynamics of the system and derive parameter values. In parallel a novel microfluidic cell culture experimental set-up is used to carefully control environmental parameters as well as to achieve chemostatic conditions for high-density cell populations. An unstable variant of the green fluorescent protein was used as a reporter to follow the time response at a single cell level. Thus spatial organization and noise across the population can be analyzed. Plasmids carrying different genetic constructs were transformed in a recombinant Escherichia coli strain to specifically identify genetic and biochemical elements involved in the regulation of the lux genes under diverse conditions. Then the quantitative data extracted from batch culture and microfluidic assays were used to assign parameter values in the models. The particular question being investigated first is the nature of the regulation to increasing concentration of the signal. The hypothesis tested is that the regulation of the production of the signal by individual cells is biphasic and, therefore, quorum sensing should be robust to global and local variations in cell density. / Master of Science

Bio-hybrid approaches have recently provided a possible solution to address the challenge of on-board actuation, control and communication modules for micro/nanoscale cargo-carrying vehicles by integrating live prokaryotic or eukaryotic cells with synthetic objects. More specifically, because micro/nanoparticles are able to transport cargos efficiently and bacteria can play the role of targeted and selective delivery agents, a hybrid of these two can advance the current strategies for environmental monitoring, drug delivery and medical imaging. The main goal of this dissertation was to fabricate, assemble, and characterize different components of a mobile network of bacteria-based bio-hybrid systems for long-term applications in drug delivery and biosensing. First, a new library of bacteria-enabled delivery systems was developed by coupling live engineered bacteria with non-spherical particles and the transport of these bacteria-based systems was investigated in the absence and presence of chemical cues using microfluidic platforms. Next, a quorum-sensing (QS) based bacterial cell-cell communication network was characterized in a high-throughput manner in order to understand the coordinated behavior of the bacterial species ferrying the cargoes. Lastly, the QS behavior of a chemotactic population of the bacterial species in response to the endogenously produced signaling molecules was studied. The work presented in this dissertation lays the foundation for a well-characterized generation of bacteria-assisted cargo delivery devices with enhanced transport properties and capable of executing pre-programmed multi-agent coordinated tasks upon their arrival at the target site. / Ph. D.

The LuxR family of proteins serves as quorum-sensing transcriptional regulators in proteobacteria. At high population densities, a small acyl-homoserine lactone (AHL) molecule, produced by a LuxI homologue, accumulates in the environment. The LuxR proteins bind to their respective AHL when the ligand accumulates to sufficient levels. Once bound to AHL, the holoproteins usually become functional as transcriptional activators. However, there is a subset of LuxR homologues, the EsaR subfamily, which is active without the AHL ligand and becomes inactivated once bound to it. EsaR is the best understood member of this subfamily. It controls virulence in the corn pathogen Pantoea stewartii ssp. stewartii. Solubility issues have previously limited structural studies of LuxR homologues as the proteins could not be purified without the AHL ligand. A soluble recombinant EsaR protein, HMGE, is biologically active and can be purified in the absence and presence of AHL, unlike most other LuxR homologues. Using HMGE, amino acid substitutions and Förster resonance energy transfer (FRET), experimental methods were designed for determining the dimerization interface of EsaR and for testing the hypothesis that EsaR undergoes a conformational shift when presented with the AHL ligand. To identify residues of the dimerization interface, heterodimerization assays were designed, involving either coexpression or coincubation of wild-type EsaR and variant HMGE proteins. In this assay, the inability of the proteins to copurify by nickel affinity chromatography would indicate that the modified residue(s) are important for dimerization of EsaR. To determine the conformational change that EsaR undergoes when bound to the AHL ligand, a FRET assay was developed to estimate the distances between amino acid residues in the absence and presence of AHL. Future work will have to include a few modifications to the methods and/or control experiments. This study provides the basis upon which the present methods can be further developed and later used for structural studies of EsaR. / Master of Science

Quorum sensing is a bacterial signaling system that controls gene expression in a population density-dependent manner. In Gram-negative proteobacteria, the cell density control of luminescence was first observed in the symbiotic marine bacterium Vibrio fischeri and this system is one of the best studied quorum sensing systems. Two-dimensional sodium dodecyl sulfate-polyacrylamide (2D-SDS) gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). A lacZ reporter was used to show that the promoters for qsrP, acfA, and ribB were directly activated via LuxR-3-oxo-C6-HSL in recombinant Escherichia coli. The sites of transcription initiation were established via primer extension analysis. Based on the position of the lux box-binding site near position â 40, all three promoters appear to have a class II-type promoter structure. Real-time reverse transcription-PCR was used to study the temporal expression of qsrP, acfA, and ribB during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities of LuxR to the promoters under investigation. In order to fully characterize the LuxR regulon in V. fischeri ES114, microarray analysis was performed in the Greenberg lab (University of Washington) and 18 LuxR-3-oxo-C6-HSL regulated promoters were found including 2 genes (qsrP and acfA) identified previously in MJ-100 in addition to the well-studied lux operon. In collaboration with them, full-length purified LuxR protein was used to show direct interaction between the LuxR protein and 7 genes/operons newly identified out of 13 genes/operons examined. The binding affinity between LuxR proteins and those genes was also measured. Based on the sequence of the lux boxes of the known genes regulated by LuxR and LitR, a position specific weight matrix (PSWM) was created and used to search through the intergenic regions of the V. fischeri ES114 genome. Some potential LuxR and LitR-regulated genes with high score were tested experimently to confirm direct activation. For the LuxR regulon, these possible LuxR-regulated promoters were cloned into a lacZ reporter and tested for their LuxR dependence. Beyond the genes found in microarray, the promoter of the intergenic region VFA0658-0659 was found to be activated by LuxR and 3-oxo-C6-HSL. For the LitR regulon, two LitR-regulated genes found in the microarray were also identified using PSWM and confirmed by real-time PCR to be dependent on LitR for expression. EMSA experiments showed that LitR can specifically bind to the litR boxes of LitR-regulated genes, litR and VF0170 which confirmed that the regulation is direct. / Ph. D.