In order to save energy consumption in idle states, low duty-cycled operation is widely used in Wireless Sensor Networks (WSNs), where each node periodically switches between sleeping mode and awake mode. Although efficient toward saving energy, duty-cycling causes many challenges, such as difficulty in neighbor discovery due to asynchronous wakeup/sleep scheduling, time-varying transmission latencies due to varying neighbor discovery latencies, and difficulty on multihop broadcasting due to non-simultaneous wakeup in neighborhood. This dissertation focuses on this problem space. Specifically, we focus on three co-related problems in duty-cycled WSNs: wakeup scheduling, routing and broadcasting. We propose an asynchronous quorum-based wakeup scheduling scheme, which optimizes heterogenous energy saving ratio and achieves bounded neighbor discovery latency, without requiring time synchronization. Our solution is based on quorum system design. We propose two designs: cyclic quorum system pair (cqs-pair) and grid quorum system pair (gqs-pair). We also present fast offline construction algorithms for such designs. Our analytical and experimental results show that cqs-pair and gqs-pair achieve better trade-off between the average discovery delay and energy consumption ratio. We also study asymmetric quorum-based wakeup scheduling for two-tiered network topologies for further improving energy efficiency. Heterogenous duty-cycling causes transmission latencies to be time-varying. Hence, the routing problem becomes more complex when the time domain must be considered for data delivery in duty-cycled WSNs. We formulate the routing problem as time-dependent Bellman-Ford problem, and use vector representation for time-varying link costs and end-to-end (E2E) distances. We present efficient algorithms for route construction and maintenance, which have bounded time and message complexities in the worst case by ameliorating with beta-synchronizer. Multihop broadcast is complex in duty-cycled WSNs due to non simultaneous wakeup in neighborhoods. We present Hybrid-cast, an asynchronous multihop broadcast protocol, which can be applied to low duty-cycling or quorum-based duty-cycling schedules, where nodes send out a beacon message at the beginning of wakeup slots. Hybrid-cast achieves better tradeoff between broadcast latency and broadcast count compared to previous broadcast solutions. It adopts opportunistic data delivery in order to reduce the broadcast latency. Meanwhile, it reduces redundant transmission via delivery deferring and online forwarder selection. We analytically establish the upper bound of broadcast count and the broadcast latency under Hybrid-cast. To verify the feasibility, effectiveness, and performance of our solutions for asynchronous wakeup scheduling, we developed a prototype implementation using Telosb and TinyOS 2.0 WSN platforms. We integrated our algorithms with the existing protocol stack in TinyOS, and compared them with the CSMA mechanism. Our implementation measurements illustrate the feasibility, performance trade-off, and effectiveness of the proposed solutions for low duty-cycled WSNs. / Ph. D.

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.

The goal of this project is to decipher the quorum sensing (cell-to-cell signaling) abilities of Shewanella oneidensis MR-1, a Gram-negative bacterium well known for its ability to use geologic substrates, such as Fe and Mn oxides, for respiratory purposes. Overall our results show that S. oneidensis cannot utilize either an acyl-homoserine lactone (AHL) or AI-2 quorum sensing signal, despite previous work that indicated that it produced an AHL that would enhance it ability to growth in certain anaerobic environments. Using a variety of quorum sensing signal sensors, no evidence could be found that S. oneidensis has a typical AHL signal. An in silco analysis of the genome also produced little evidence that S. oneidensis has the genes to accept or relay an AHL signal. S. oneidensis can produce a luminescence response in the AI-2 reporter strain, Vibrio harveyi MM32. This luminescence response is abolished upon deletion of luxS, the gene responsible for catalyzing AI-2. Deletion of luxS also affected biofilm formation. Within 16 hours of growth in a biofilm flow-through reactor, the luxS mutant had an inhibited ability to initiate biofilm formation. After 48 hours of growth, the mutant's biofilm had developed similarly to wild-type. The addition of synthetic AI-2 did not restore the mutant's ability to initiation biofilm formation, which led to the conclusion that AI-2 is not likely used as a quorum sensing signal in S. oneidensis for this phenotype. Because of the involvement of LuxS in the activated methyl cycle (AMC) in other organisms, growth on various sulfur sources was examined. A mutation in luxS produced a reduced ability to growth with methionine as the sole sulfur source. Methionine is a key metabolite used in the AMC to produce a methyl source in the cell and homocysteine. This data suggests that LuxS is important in metabolizing methionine and the AMC in S. oneidensis. / Ph. D.

Properly representing the principle physical interactions of complex biological systems is paramount for building powerful, yet simple models. As an in depth look into different biological systems at different scales, multiple models are presented. At the molecular scale, an analytical solution to the (linearized) Poisson-Boltzmann equation for the electrostatic potential of any size biomolecule is derived using spherical geometry. The solution is tested both on an ideal sphere relative to an exact solution and on a multitude of biomolecules relative to a numerical solution. In all cases, the bulk of the error is within thermal noise. The computational power of the solution is demonstrated by finding the electrostatic potential at the surface of a viral capsid that is nearly half a million atoms in size. Next, a model of the nucleosome using simplified geometry is presented. This system is a complex of protein and DNA and acts as the first level of DNA compaction inside the nucleus of eukaryotes. The analytical model reveals a mechanism for controlling the stability of the nucleosome via changes to the total charge of the protein globular core. The analytical model is verified by a computational study on the stability change when the charge of individual residues is altered. Finally, a multiple model approach is taken to study bacteria that are capable of different responses depending on the size of their surrounding colony. The first model is capable of determining how the system propagates the information about the colony size to those specific genes that control the concentration of a master regulatory protein. A second model is used to analyze the direct RNA interference mechanism the cell employs to tune the available gene transcripts of the master regulatory protein, i.e. small RNA - messenger RNA regulation. This model provides a possible explanation for puzzling experimentally measured phenotypic responses. / Ph. D.

Pantoea stewartii subsp. stewarti is the causative agent of Stewart's wilt disease in maize. Disease symptoms develop after the bacteria grow to high cell densities in the plant xylem and secrete an abundance of exopolysaccharide (EPS). EPS production is regulated by quorum sensing. Two regulatory proteins are key to the process of quorum sensing, the LuxI and LuxR homologues EsaI and EsaR. Most LuxR homologues function as activators of transcription in the presence of their cognate acylated homoserine lactone signal (AHL). EsaR utilizes an AHL-response opposite of the majority of the LuxR homologues. EsaR represses EPS production at low cell densities. However, at high cell densities when high concentrations of AHL are present, EsaR is inactivated and derepression of EPS production occurs. The mechanism that enables EsaR to respond to AHL in a manner opposite to that of most LuxR homologues remains elusive. A comparative study of EsaR and the well characterized quorum-sensing regulators LuxR from Vibrio fischeri and TraR from Agrobacterium tumefaciens was initiated. Previous studies demonstrated that in the absence of AHL, EsaR retains the ability to function as a weak activator of the lux operon in recombinant Escherichia coli. This thesis research further characterized the role of EsaR as an activator. Variant forms of EsaR with deletions or single residue substitutions were generated and their ability to regulate transcription was examined in vivo. Furthermore, a native EsaR-activated promoter has been identified, which controls expression of a putative regulatory sRNA in P. stewartii. It is apparent that EsaR functions as a transcription factor at low concentrations of AHL as demonstrated by its ability to inhibit EPS production. At high concentrations, the AHL appears to bind and cause a conformational shift in the protein leading to its inactivation. The second goal of this study was to further elucidate the mechanism by which AHL regulates EsaR. Pulse-chase experiments demonstrated that EsaR is resistant to proteases with or without AHL in vivo. Limited proteolytic digestions in vitro suggest that the protein does undergo conformational changes in response to AHL. Gel filtration chromatography, sucrose gradient ultracentrifugation, and cross-linking experiments proved that this conformational change does not impact the multimeric state of EsaR. To better understand the mechanism of regulation by AHL, the final goal of this project was to examine the interactions which result in EsaR-responsiveness to AHL. Several individual amino acid substitutions were identified that cause EsaR to function in an AHL-independent manner, by which variants retain the ability to bind and block gene expression in the presence of AHL. These residues have been mapped onto a homology model of EsaR and their role has been examined in vitro. The ability of these EsaR* variants to bind AHL and an analysis of the effects individual mutations have on the overall conformation of the protein was performed. Overall this study has revealed several unique aspects of the quorum-sensing system in P. stewartii whereby gene expression is regulated at both low and high cell density. Studies were also initiated to examine the mechanism of AHL-responsiveness of EsaR. The mechanism by which AHL modulates most LuxR homologues remains elusive. The ability to purify EsaR +/- its cognate AHL may prove critical in elucidating this mechanism. / Ph. D.

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

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)