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.

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.

abstract: The portability of genetic tools from one organism to another is a cornerstone of synthetic biology. The shared biological language of DNA-to-RNA-to-protein allows for expression of polypeptide chains in phylogenetically distant organisms with little modification. The tools and contexts are diverse, ranging from catalytic RNAs in cell-free systems to bacterial proteins expressed in human cell lines, yet they exhibit an organizing principle: that genes and proteins may be treated as modular units that can be moved from their native organism to a novel one. However, protein behavior is always unpredictable; drop-in functionality is not guaranteed. My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli. 1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin. 2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2017

Individual cells in a genetically identical population, exposed to the same environment, can show great variation in their protein expression levels. This is due to noise, which is inherent in many biological processes, due in part to the low molecule numbers and probabilistic interactions which lead to stochasticity. Much of the work in the field of noise and its propagation in gene expression networks, whether it is experimental, modelling or theoretical, has been conducted on networks/systems that occur within a single cell. However, cells do not exist solely in isolation and understanding how cells are able to coordinate their behaviour despite this noise is an interesting area of expansion for the field. In this study, a synthetic intercellular communication system was designed that allows the investigation of how noise is propagated in intercellular communication. The communication system consists of separate sender and receiver cells incorporating components of the Lux quorum sensing system of Vibrio fischeri. The sender cell was designed so that the production of the signalling molecule, 3-oxohexanoyl homoserine lactone, is able to be controlled by addition of isopropyl-β-D-thio-galactoside (IPTG) and monitored via a reporter gene. The receiver cell was designed with a dual reporter system to enable the response of the cell to the signalling molecule to be monitored and the intrinsic and extrinsic noise contributions to the total noise to be calculated. Sender and the receiver cells were engineered in Escherichia coli. The functionality of the receiver cells was tested in the presence of known concentrations of the signalling molecule. The population response and the noise characteristics of the receiver cells in the homogeneous environment were determined from single cell measurements. The functionality of the sender cells was tested in the presence of a range of IPTG concentrations and the induction of expression from the LacI-repressible promoter was monitored. Mathematical models of the system were developed. Stochastic simulations of the models were used to investigate any unexplained behaviour seen in the characterisation of the cells. The full functionality of the intercellular communication system was then tested by growing the receiver in the collected media of the induced sender cells. The response of the receiver cells to the signalling molecule in the media was again characterised using single cell measurements of the reporter expression levels. The analysis of mixed populations of the sender and receiver cells was hampered by the technical limitations of the instruments used for the single cell measurements. Difficulties were encountered in simultaneous and specific measurement of the three reporter genes. Two methods for overcoming this issue were proposed using microscopy, and one of these methods was shown to have potential in overcoming the issue.

The scope of this project was to implement a quorum sensing model capable of synchronised oscillations from the article ”A synchronized quorum of genetic clocks” [1] in the software framework URDME [2]. The model consists of a system of partial differential equations describing time delayed and coupled biochemical reactions. In URDME, the time delay system was formed using a cascade of reactions in which the rate of each reaction was set so that the expected total time for all reactions in the cascade corresponds to a certain delay time. One reason for this cascade delay model is that it might better capture the inherently stochastic nature of the delay mechanism in the quorum sensing network, as opposed to a model using explicit delays.Another reason is simplicity of implementation, as delays are not explicitly supported in URDME. After initial tests suggested that the cascade delay model gave satisfying results, it was incorporated into the quorum sensing model from the article, which was implemented by rewriting the differential equations as a system of biochemical reactions. Simulations in one and two dimensions were then done, with both stochastic and deterministic solution methods. The one dimensional and two dimensional simulations yielded distinct synchronised oscillations with a cascade delay containing five sub-reactions. Several results from the simulations of the original article could be reproduced. From the results, it was concluded that the proposed cascade delay model was successful in modelling the delayed reactions in the quorum sensing network. In future studies, it is suggested that the individual cells, in which most of the reactions in the quorum sensing network happen, are modelled with greater resolution.

No description available.

Polymicrobial communities of bacterial and fungal species are present on the skin and mucosal surfaces of the body. Invasive infections caused by Candida species are commonly seen in immunocompromised individuals (HIV, transplants, cancer) and ranks as the third leading cause of infection in hospitalized patients. C. albicans is a polymorphic opportunistic fungus that infects critically ill patients and has the ability to change its morphology from yeast to hyphal form.The morphogenesis of C. albicans is a major aspect of its virulence and is regulated by quorum sensing (QS) molecules they produce, as well as the presence of neighboring microbes.In this study, we examined two transporter mutants, cdr4 and ssu1, for their ability to form biofilms in the presence of cyclic-di-GMP and 3-oxo-12-homoserine lactone. To quantify biomass, wild type and mutant cells were grown overnight at 30˚C in YPD. The cells were washed, counted and diluted to a desired density of 106 cells/ml in medium 199, pH7.5. Cells were added to 96-well plates pre-incubated with 5% fetal bovine serum at densities of 105, 104, and 103 cells/well and allowed to adhere at 37˚C for one hour. The wells were then covered with fresh M199 media containing the QS molecule and monitored for 48 hours at 37˚C. After this time, the media and planktonic cells were removed. The biofilms were fixed with methanol, dried, then stained with 0.05% crystal violet. Bulk biomass was assessed by spectrophotometry. We did observe a difference in biofilm density when incubated in the presence of cyclic-diGMP. We noted that for the wild type and ssu1 strain their biofilms biomass increased by as much as 10% at 104 and 103 cell densities when compared with the control. While the cdr4 strain had a slight reduction in biofilm density when cyclic-diGMP was present. This result also indicates a potentially positive role in which cyclic-diGMP can help C. albicans develop denser biofilms, potentially in the presence of bacteria like P. aeruginsa, which secrete cyclic di-GMP, but kill hyphal forms of C. albicans. Additionally, it has been shown that C. albicans mutants lacking the hybrid histidine kinase, Chk1p, are refractory to the effects of farnesol, a QS molecule that inhibits morphogenesis.Given that mutations in CDR4 and SSU1 impact the QS response in C. albicans, we investigated whether these genes were regulated through two-component signaling by Chk1. To assess CDR4 and SSU1 expression, wild type and mutant strains were grown overnight in YPD media at 30˚C. Cells were then harvested and RNA was obtained by acid phenol extraction. Using RT-PCR, we determined both CDR4 and SSU1 expression is reduced or highly repressed in the chk1, ypd1, and skn7 null strains. These results suggest the two genes are downstream targets in a pathway regulated by Chk1p. The finding that QS proceeds through a two-component pathway can be exploited in antifungal drug development. Given that two-component signaling is absent in mammalian cells, development of novel compounds that interfere with this pathway may be a useful alternative for treating patients with candidiasis.