The objectives of this work are: 1) to agarose-stabilize fragile biofilms for quantitative structure analysis; 2) to understand the influences of LuxS on biofilm formation; 3) to improve teacher quality by preparing Georgia’s middle school science teachers to integrate inquiry-based, hands-on research modules in the classroom. Quantitative digital image analysis demonstrated the effectiveness of the agarose stabilization technique for generating reproducible measurements of three dimensional biofilm structure. The described method will also benefit researchers who transport their flow cell-cultivated biofilms to a core facility for imaging. AI-2-dependent and independent effects of LuxS on biofilm-related phenotypes were revealed, suggesting that LuxS is a versatile enzyme, possessing multiple functions in E. coli ecology that could assist E. coli in adapting to diverse conditions. Overall, the work presented in this dissertation supported the concept that quorum sensing, biofilm formation, and cell adhesion are largely related. Additionally, through this project, teachers enhanced content knowledge and confidence levels, mastered innovative teaching strategies and integrated inquiry-based, inter-disciplinary, hands-on activities in the classroom. As a result, student learning was enhanced, and teachers are better equipped to give Georgia’s students a solid foundation in the sciences.

Regularity is a shared memory consistency condition that has received considerable attention, notably in connection with quorum-based shared memory. Lamport's original definition of regularity assumed a single-writer model, however, and is not well defined when each shared variable may have multiple writers. In this thesis, we address this need by formally extending the notion of regularity to a multi-writer model. We have shown that the extension is not trivial. While there exist various ways to extend the single-writer definition, the resulting definitions will have different strengths. Specifically, we give several possible definitions of regularity in the presence of multiple writers. We then present a quorum-based algorithm to implement each of the proposed definitions and prove them correct. We study the relationships between these definitions and a number of other well-known consistency conditions, and give a partial order describing the relative strengths of these consistency conditions. Finally, we provide a practical context for our results by studying the correctness of two well-known algorithms for mutual exclusion under each of our proposed consistency conditions.

Regularity is a shared memory consistency condition that has received considerable attention, notably in connection with quorum-based shared memory. Lamport's original definition of regularity assumed a single-writer model, however, and is not well defined when each shared variable may have multiple writers. In this thesis, we address this need by formally extending the notion of regularity to a multi-writer model. We have shown that the extension is not trivial. While there exist various ways to extend the single-writer definition, the resulting definitions will have different strengths. Specifically, we give several possible definitions of regularity in the presence of multiple writers. We then present a quorum-based algorithm to implement each of the proposed definitions and prove them correct. We study the relationships between these definitions and a number of other well-known consistency conditions, and give a partial order describing the relative strengths of these consistency conditions. Finally, we provide a practical context for our results by studying the correctness of two well-known algorithms for mutual exclusion under each of our proposed consistency conditions.

This dissertation describes the development and application of an approach for creating multiphoton crosslinked protein microchambers to characterize bacterial group behaviors in small populations (~10¹ - 10⁵ cells). Porous protein cavities of desired size and geometry are made with sub-micrometer three-dimensional (3D) resolution using a dynamic mask-based multiphoton lithography (MPL) technique previously developed in the Shear Group. One aspect of this dissertation focuses on basic characterizations of properties of these materials key to their utility in studying entrapped bacteria. Studies are presented on the mass transport across microcavity walls (important for growth and signaling), and the temperature- and light-induced volume response (used to open/close microchamber apertures for cell entry/exit). Fabrication parameters are optimized to trap and manipulate small populations under in vitro conditions that are relevant to in vivo environments. The ability to culture bacteria at physiologic growth rates within protein microstructures has provided a unique platform to study the group behaviors of quorum sensing (QS) and antibiotic resistance in biologically relevant population sizes, a platform I have exploited to study group behaviors in the opportunistic pathogen, Pseudomonas aeruginosa. This work presents the first experimental evidence supporting the efficiency sensing QS model by showing that QS-dependent gene expression is affected by both the population size and density, as well the external flow rate in the surrounding environment. The onset of antibiotic resistance is observed in as few as ~150 P. aeruginosa cells, and is shown to increase with cell density. Lastly, the development of a gelatin-based MPL approach that is demonstrated in situ to create confined populations of non-motile cells, free-floating 3D cultures, nested colonies, and spatially patterned polymicrobial communities of P. aeruginosa and Staphylococcus aureus. / text

The coral holobiont contains diverse communities of bacteria that play a role in the maintenance of coral ecosystems, however little is known about the structure and conservation of the host-bacterial relationship. Declines in coral ecosystems have been partly attributed to outbreaks of disease in tropical and sub-tropical regions, which have been linked to increasing temperatures. Bacteria are thought to play a role in some of these diseases, however little is understood about the mechanisms behind disease progression or the series of events involved in the shifts of coral-associated bacteria from conserved, potentially beneficial communities to those including potential pathogens. Investigations into a cold-water gorgonian coral, Eunicella verrucosa, have shown similar bacterial communities to those present in tropical and sub-tropical regions, with high proportions of Spongiobacter and Endozoicomonas genera, suggesting an important role for these associates in the coral holobiont irrespective of location or the presence of zooxanthellae. A shift in bacterial community with disease was also shown, with suggestions that sedimentation and depth may affect the extent of bacterial community alteration. With the increasing knowledge that bacteria exhibit elaborate systems of intercellular communication (quorum sensing; QS) to allow a population response and to control the expression of genes for pathogenesis, antibiotic production and biofilm formation, the present study showed the presence, stability and species-specific nature of N-acyl-homoserine lactones (AHLs; most prevalent type of QS) in situ in a number of coral species. This finding and a high proportion of coral-associated bacteria found producing AHLs suggests an important role for QS in the coral holobiont. Further, AHL signals have been shown to break down in Stylophora pistillata kept at 30 °C, which coincided with a drop in bacterial numbers and a changing bacterial community which included more quorum quenching (QQ; AHL-degrading) bacteria. Temperature was shown to affect AHL-QS in a strain-dependent manner in E. verrucosa isolates, suggesting that the decline seen in S. pistillata is not primarily an effect of temperature. Further experiments with three species of soft coral (Sinularia sp., Discosoma sp. and a gorgonian) showed no such decline in AHLs at 30 °C and instead show a coral-specific response to temperature, including the ability of coral extracts to inhibit putative pathogens. A decline in the ability of crude coral extract to degrade AHLs in the Discosoma sp. and the high QQ activity in crude extract from all three species suggests a role for QQ in the coral holobiont, confirmed by the high percentage of QQ found in coral-associated bacterial isolates; again suggesting a role in the maintenance of bacterial communities. Further investigations attempted to link QS and QQ to antagonism and susceptibility in coral associated bacteria; however these results were inconclusive. The thesis concludes that priority should be given to further research of QS and QQ in the coral holobiont, which will reveal important knowledge that may lead to future mitigation of some forms of coral disease.

Gram-negative bacteria use N-acyl homoserine lactones (AHLs) to sense population density and regulate gene expression, including virulent phenotypes. The quorum-quenching AHL lactonase from Bacillus thuringiensis cleaves the lactone ring of AHLs, disabling this mode of gene regulation. Despite the potential applications of this enzyme as an antibacterial weapon, little was known about it's lactone ring-opening mechanism. As a member of the metallo-beta-lactamase superfamily, AHL lactonase requires two divalent metal ions for catalysis. NMR experiments confirm that these metal ions are also involved in proper enzyme folding. The chemical mechanism of ring opening was explored using isotope incorporation studies, and hydrolysis was determined to proceed via a nucleophilic attack by a solvent-derived hydroxide at the carbonyl of the lactone ring. A transient, kinetically significant metal-leaving group interaction was detected in steady-state kinetic assays with AHL lactonase containing alternative divalent metal ions hydrolyzing a sulfur-containing substrate. High-resolution crystal structures implicated two residues in substrate binding and hydrolysis, Tyr194 and Asp108. Site-directed mutagenesis of these residues followed by steady-state kinetic studies with wild-type and mutant enzymes hydrolyzing a spectrum of AHL substrates revealed that mutations Y194F and D108N significantly affect catalysis. Combining these results allows the proposal of a detailed hydrolytic mechanism. The binding site for the N-acyl hydrophobic moiety was probed using steady-state kinetics with a variety of naturally occurring and non-natural AHL substrates, and these studies indicate that AHL lactonase will accept a broad range of homoserine lactone containing substrates. Crystal structures with AHL substrates and non-hydrolyzable analogs reveal two distinct binding sites for this N-acyl group. Based on the ability of this enzyme to accommodate a variety of substrates, AHL lactonase was shown to have the ability to quench quorum sensing regulated by a newly discovered class of homoserine lactone signal molecules possessing an N-aryl group using a bioassay. Steady-state kinetic studies confirm that this class of signal molecules are indeed substrates for AHL lactonase. / text

No description available.

Aeromonas spp. are important biofilm-forming fish pathogens causing great economic loss in aquaculture. Bacterial cells within biofilms communicate with each other via the production of quorum sensing (QS) signalling molecules called acyl-homoserine lactones (AHLs), which influence biofilm development and production of virulence factors. QS together with efflux pumps, extracellular polymeric substances (EPS) and eDNA are associated with resistance of bacteria to antimicrobial agents. These mechanisms provide a target for different control strategies. The objectives of this study were to: (i) determine effective antimicrobial agents and exposure concentrations against aeromonad biofilms; (ii) ascertain whether Aeromonas spp. produce QS molecules or display efflux pump phenotypes, and (iii) investigate the effect of antimicrobial agents, lytic enzymes, efflux pump inhibitors and QS inhibitors on biofilm formation by Aeromonas spp. isolates.signalling MICs of azithromycin, ciprofloxacin, ceftazidime, and tetracycline ranged between 0.064-64 μg/ml. Gentamicin had the lowest MICs which ranged between 0.0048-32 μg/ml.The highest MBIC at which antimicrobial agents exhibited inhibition was 4096 μg/ml. Majority of the isolates displayed MIC levels ranging from 2-32 μg/ml, and thus a ≥ 128-fold increase was observed for MBICs. Of the sub-MIC, MIC and supra-MIC exposures tested, MIC exposure of biofilms was the most effective. Gentamicin MIC exposures inhibited initial attachment of 100% (28/28) of isolates tested, while azithromycin MIC exposure detached 82.1% (23/28) of isolates. Carbonyl cyanide 3-chlorophenylhydrazone completely inhibited efflux of cefpodoximeby 14.8% of isolates. However, 1-(1-naphthylmethyl)-piperazinewas more effective, decreasing adherence of 98.1% (53/54) of isolates and increasing detachment of 100% (54/54) of isolates. DNase I was more effective against the mature biofilm,where it increased biofilm detachment of 64.8% of isolates. Of the 48 Aeromonas spp. and six Plesiomonas spp. isolates used, only a single isolate induced the production of violacein by the C. violaceum CV026 biosensor, while all isolates induced the utilization of X-gal to produce a visible blue colour with the A.tumefaciens A136 biosensor. Based on the reaction to the two biosensors, aeromonads appeared to produce long-chain acylhomoserine lactones. By blocking QS, S-adenosyl homoserinewas more effective in inhibiting both initial attachment (72.2% of isolates) and pre-formed biofilms (detached 74.1% of isolates). The investigated strategies are promising for Aeromonas spp. biofilm inhibition. Thesecould be explored aspotential therapeutic measures in aquaculture systems to limit aeromonad pathogenicity and overcome antimicrobial resistance. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

Quorum sensing is a cell density-dependent bacterial gene regulatory mechanism used for the expression of colonization-related genes. The symbiotic relationship between the luminescent bacterium Vibrio fischeri and the Hawaiian bobtail squid Euprymna scolopes serves as a model system to study the molecular processes underlying bacterial colonization. This system is especially well-suited for the investigation of the impact of quorum sensing on colonization because (i) it is an easily accessible, natural, two-species colonization model, and (ii) quorum sensing regulates luminescence expression in V. fischeri, which allows the non-invasive detection of quorum-sensing activity both in culture and in symbiosis. While the impact of one of V. fischeri's quorum-sensing systems, lux, on luminescence expression and symbiotic competence has been extensively studied, little was known about other putative systems. The results of this study demonstrate that the V. fischeri ain system is essential for both maximal luminescence expression and symbiotic competence. The ain system predominantly induces luminescence expression at intermediate cell densities, which occur in culture, while the lux system is responsible for luminescence expression at the high cell densities found in symbiosis, suggesting the sequential induction of luminescence gene expression by these two systems. Furthermore, the ain quorum sensing system is important for the processes underlying colonization initiation, while the impact of the lux system is apparent only in later stages of the symbiosis, indicating distinct functions of these two systems during the colonization process. A global transcriptome. analysis of quorum-sensing mutants revealed that ain quorum sensing represses motility gene expression, providing a likely explanation for the initiation defect. Although it has been known that many bacterial species possess multiple quorum-sensing systems, this is the first study demonstrating that two quorum-sensing systems are employed to specifically regulate functions important at distinct cell densities occurring during the colonization process.

Nano-sized materials are being used to address some of humanities greatest challenges— cancer therapy, food and water security, and environmental remediation. While extremely promising for these applications, the production, use, and disposal of nanomaterials have resulted in their release into environmental compartments. One major concern of any novel contaminant is how it interacts with bacteria. Bacteria play essential roles in human health, engineered systems, and ecological functioning. Bacteria are capable of macro-scale influence because they have evolved communication systems that enable coordinated behaviors. Communication among cells involves chemical signals that enter the environment, where they are subjected to its biogeochemistry, which now includes novel nanomaterials. The overall goal of this thesis was to improve understanding of the relationship between nanoparticles and cell-to-cell signaling behavior in bacteria focusing on two population-level behaviors: autolysis and quorum sensing. Specifically, this project sought to: (1) improve our understanding of how metal-oxide nanoparticles affect the autolytic process in Bacillus subtilis, by elucidating the biological response of the interactions between titanium dioxide nanoparticles and biomolecules; (2) reveal the interactions between quorum sensing signaling molecules and metal cations commonly used in antimicrobial nanomaterials, silver and copper; and (3) demonstrate the potential of quorum sensing-regulated cyanide production to affect oxidation and dissolution of gold nanoparticles in an environmentally relevant system. By addressing these objectives, the work demonstrated that: 1. TiO2 nanoparticles disrupt the autolytic process by delaying the onset of autolysis, and intercepting released autolytic enzymes, preventing the enzymes from degrading peptidoglycan in neighboring cells. 2. Quorum sensing signaling molecules form complexes with Ag+ and Cu2+, removing the most bioavailable form (free HHL, Ag+, and Cu2+) from the cells’ environment. 3. Quorum sensing-regulated cyanide production induces oxidative dissolution in Au nanoparticles, which were previously assumed to be inert in environmental systems. Taken together, this body of work highlights the relationship between nanoparticles and population-level behavior in bacteria. The presence of nanoparticles can have significant effects on population-level behaviors, and the activity of population-level behaviors can have significant effects on nanoparticles behavior. This inter-connected relationship, where the nanoparticles are both acted on and act upon their environment, must be considered in nanoparticle-based studies and applications.