A molecular basis for polyketide biosynthesis in marine dinoflagellates with emphasis on Karenia brevis

Polyketides derived from dinoflagellates are among the most complex and unique structures identified to date. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). No studies of the biosynthesis of dinoflagellate derived polyketides at the genomic level have been reported to date. Nine strains representing seven different species of dinoflagellates were screened for the presence of type I and type II polyketide synthases (PKS) by PCR and RT-PCR. Seven of the nine strains yielded products that were homologous with known and putative type I polyketide synthases. In each case, the presence of a PKS gene was correlated with the presence of bacteria in the cultures as identified by amplification of the bacterial 16S rRNA gene. However, residual phylogenetic signals, resistance to methylation sensitive restriction enzymes and the lack of hybridization to bacterial isolates support a dinoflagellate origin for most of these genes. ^ A more detailed analysis of Karenia brevis, a toxic marine dinoflagellate endemic to the Gulf of Mexico, also supports the hypothesis that dinoflagellates have polyketide synthase genes. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. PKS encoding genes amplified from K. brevis culture were found to be similar to PKS genes from the closely related protist, Cryptosporidium parvum. This suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. This dissertation reports the localization of these PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate. ^

Identification and localization of polyketide synthase genes from cultures of diarrheic shellfish poisoning toxin producing dinoflagellates of the genus Prorocentrum

Aquatic toxins are responsible for a number of acute and chronic diseases in humans. Okadaic acid (OA) and other dinoflagellate derived polyketide toxins pose serious health risks on a global scale. Ingestion of OA contaminated shellfish causes diarrheic shellfish poisoning (DSP). Some evidence also suggests tumor promotion in the liver by OA. Microcystin-LR (MC-LR) is produced by cyanobacteria and is believed to be the most common freshwater toxin in the US. Humans may be exposed to this acute hepatotoxin through drinking or recreational use of contaminated waters. ^ OA producing dinoflagellates have not been cultured axenically. The presence of associated bacteria raises questions about the ultimate source of OA. Identification of the toxin-producing organism(s) is the first step in identifying the biosynthetic pathways involved in toxin production. Polyketide synthase (PKS) genes of toxic and non-toxic species were surveyed by construction of clonal libraries from PCR amplicons of various toxic and non-toxic species of Prorocentrum in an effort to identify genes, which may be part of the biosynthetic pathway of OA. Analysis of the PKS sequences revealed that toxic species shared identical PKS genes not present in non-toxic species. Interestingly, the same PKS genes were identified in a library constructed from associated bacteria. ^ Subsequent bacterial small subunit RNA (16S) clonal libraries identified several common bacterial species. The most frequent 16S sequences found were identified as species of the genus Roseobacter which has previously been implicated in the production of OA. Attempts to culture commonly occurring bacteria resulted in the isolation of Oceanicaulis alexandrii , a novel marine bacterium previously isolated from the dinoflagellate Alexandrium tamarense, from both P. lima, and P. hoffmanianum. ^ Metabolic studies of microcystin-LR, were conducted to probe the activity of the major human liver cytochromes (CYP) towards the toxin. CYPs may provide alternate routes of detoxification of toxins when the usual routes have been inhibited. For example, some research indicates that cyanobacterial xenobiotics, in particular, lipopolysaccharides may inhibit glutathione S-transferases allowing the toxin to persist long enough to be acted upon by other enzymes. These studies found that at least one human liver CYP was capable of metabolizing the toxin. ^

Leveraging symbiotic relationships for emulation of computer networks

The lack of analytical models that can accurately describe large-scale networked systems makes empirical experimentation indispensable for understanding complex behaviors. Research on network testbeds for testing network protocols and distributed services, including physical, emulated, and federated testbeds, has made steady progress. Although the success of these testbeds is undeniable, they fail to provide: 1) scalability, for handling large-scale networks with hundreds or thousands of hosts and routers organized in different scenarios, 2) flexibility, for testing new protocols or applications in diverse settings, and 3) inter-operability, for combining simulated and real network entities in experiments. This dissertation tackles these issues in three different dimensions. First, we present SVEET, a system that enables inter-operability between real and simulated hosts. In order to increase the scalability of networks under study, SVEET enables time-dilated synchronization between real hosts and the discrete-event simulator. Realistic TCP congestion control algorithms are implemented in the simulator to allow seamless interactions between real and simulated hosts. SVEET is validated via extensive experiments and its capabilities are assessed through case studies involving real applications. Second, we present PrimoGENI, a system that allows a distributed discrete-event simulator, running in real-time, to interact with real network entities in a federated environment. PrimoGENI greatly enhances the flexibility of network experiments, through which a great variety of network conditions can be reproduced to examine what-if questions. Furthermore, PrimoGENI performs resource management functions, on behalf of the user, for instantiating network experiments on shared infrastructures. Finally, to further increase the scalability of network testbeds to handle large-scale high-capacity networks, we present a novel symbiotic simulation approach. We present SymbioSim, a testbed for large-scale network experimentation where a high-performance simulation system closely cooperates with an emulation system in a mutually beneficial way. On the one hand, the simulation system benefits from incorporating the traffic metadata from real applications in the emulation system to reproduce the realistic traffic conditions. On the other hand, the emulation system benefits from receiving the continuous updates from the simulation system to calibrate the traffic between real applications. Specific techniques that support the symbiotic approach include: 1) a model downscaling scheme that can significantly reduce the complexity of the large-scale simulation model, resulting in an efficient emulation system for modulating the high-capacity network traffic between real applications; 2) a queuing network model for the downscaled emulation system to accurately represent the network effects of the simulated traffic; and 3) techniques for reducing the synchronization overhead between the simulation and emulation systems.

Understanding the role of Dimethylsulfoniopropionate as a potential chemotactic cue for coral-associated bacteria

Most reef-building corals are known to engage in non-pathogenic symbiosis not only with unicellular dinoflagellates from the genus Symbiodinium, but also with other microscopic organisms such as bacteria, fungi, and viruses. The functional details of these highly complex associations remain largely unclear. The impetus of this study is to gain a better understanding of the symbiotic interaction between marine bacteria and their coral host. Studies have shown that certain bacterial orders associate with specific certain coral species, thus making the symbiotic synergy a non-random consortium. Consequently both corals and bacteria may be capable of emitting chemical cues that enable both parties to find one another and thus generate the symbiosis. The production of these cues by the symbionts may be the result of environmental stimuli such as elevated ocean temperatures, increased water acidity, and even predation. One potential chemical cue could be the compound DMSP (Dimethylsulfoniopropionate) and its sulphur derivatives. Reef-building corals are believed to be the major producers of the DMSP during times of stress. Marine bacteria utilize DMSP as a source of sulfur and carbon. As a result corals could potentially attract their bacterial consortium depending on their DMSP production. This would enable them to adapt to fluctuating environmental conditions by changing their bacterial communities to that which may aid in survival. To test the hypothesis that coral-produced DMSP plays a role in attracting symbiotic bacteria, this study utilized the advent of high-throughput sequencing paired with chemotactic assays to determine the response of coral-associated bacterial isolates towards the DMSP compound at differing concentrations. Chemotaxis assays revealed that some isolates responded positively towards the DMSP compound. This finding adds to existing evidence suggesting that coral-associated pathogens utilize chemotaxis as a host colonization and detection mechanism. Thus the symbiotic bacteria that make up the coral microbiome may also employ this process. Furthermore this study demonstrates that bacterial motility may be a strong contributing factor in the response to the chemotactic cue. Swarming motility may be better suited for bacteria that need to respond to a chemical gradient on the surface of the coral. Therefore the isolates that were able to swarm seemed to respond more strongly to the DMSP.

Understanding the role of Dimethylsulfoniopropionate as a Potential Chemotactic Cue for Coral-Associated Bacteria

Most reef-building corals are known to engage in symbiosis not only with unicellular dinoflagellates from the genus, Symbiodinium, but they also sustain highly complex symbiotic associations with other microscopic organisms such as bacteria, fungi, and viruses. The details of these non-pathogenic interactions remain largely unclear. The impetus of this study is to gain a better understanding of the symbiotic interaction between marine bacteria and a variety of coral species representative of differing morphologies. Studies have shown that certain bacterial orders associate specifically with certain coral species, thus making the symbiotic synergy a non-random consortium. Consequently both corals and bacteria may be capable of emitting chemical cues that enables both parties to find one another and thus creating the symbiosis. One potential chemical cue could be the compound DMSP (Dimethylsulfoniopropionate) and its sulphur derivatives. Reef-building corals are believed to be the major producers of the DMSP and its derivatives during times of stress. As a result corals could potentially attract their bacterial consortium depending on their DMSP production. Corals may be able to adapt to fluctuating environmental conditions by changing their bacterial communities to that which may aid in survival. The cause of this attraction may stem from the capability of a variety of marine bacteria to catabolize DMSP into different metabolically significant pathways, which may be necessary for the survival of these mutualistic interactions. To test the hypothesis that coral-produced DMSP play a role in attracting symbiotic bacteria, this study utilized the advent of high-through sequencing paired with bacterial isolation techniques to properly characterize the microbial community in the stony coral Porites astreoides. We conducted DMSP swarming and chemotaxis assays to determine the response of these coral-associated bacterial isolates towards the DMSP compound at differing concentrations. Preliminary data from this study suggests that six out of the ten bacterial isolates are capable of conducting unidirectional motility; these six isolates are also capable of conducting swarming motility in the direction of an increasing DMSP concentration gradient. This would indicate that there is a form of positive chemotaxis on behalf of the bacteria towards the DMSP compound. By obtaining a better understanding of the dynamics that drive the associations between bacterial communities and corals, we can further aid in the protection and conservation processes for corals. Also this study would further elucidate the significance of the DMSP compound in the survival of corals under times of stress.

Understanding the role of Dimethylsulfoniopropionate as a Potential Chemotactic Cue for Coral-Associated Bacteria

Most reef-building corals are known to engage in symbiosis not only with unicellular dinoflagellates from the genus, Symbiodinium, but they also sustain highly complex symbiotic associations with other microscopic organisms such as bacteria, fungi, and viruses. The details of these non-pathogenic interactions remain largely unclear. The impetus of this study is to gain a better understanding of the symbiotic interaction between marine bacteria and a variety of coral species representative of differing morphologies. Studies have shown that certain bacterial orders associate specifically with certain coral species, thus making the symbiotic synergy a non-random consortium. Consequently both corals and bacteria may be capable of emitting chemical cues that enables both parties to find one another and thus creating the symbiosis. One potential chemical cue could be the compound DMSP (Dimethylsulfoniopropionate) and its sulphur derivatives. Reef-building corals are believed to be the major producers of the DMSP and its derivatives during times of stress. As a result corals could potentially attract their bacterial consortium depending on their DMSP production. Corals may be able to adapt to fluctuating environmental conditions by changing their bacterial communities to that which may aid in survival. The cause of this attraction may stem from the capability of a variety of marine bacteria to catabolize DMSP into different metabolically significant pathways, which may be necessary for the survival of these mutualistic interactions. To test the hypothesis that coral-produced DMSP play a role in attracting symbiotic bacteria, this study utilized the advent of high-through sequencing paired with bacterial isolation techniques to properly characterize the microbial community in the stony coral Porites astreoides. We conducted DMSP swarming and chemotaxis assays to determine the response of these coral-associated bacterial isolates towards the DMSP compound at differing concentrations. Preliminary data from this study suggests that six out of the ten bacterial isolates are capable of conducting unidirectional motility; these six isolates are also capable of conducting swarming motility in the direction of an increasing DMSP concentration gradient. This would indicate that there is a form of positive chemotaxis on behalf of the bacteria towards the DMSP compound. By obtaining a better understanding of the dynamics that drive the associations between bacterial communities and corals, we can further aid in the protection and conservation processes for corals. Also this study would further elucidate the significance of the DMSP compound in the survival of corals under times of stress.

Leveraging Symbiotic Relationships for Emulation of Computer Networks

The lack of analytical models that can accurately describe large-scale networked systems makes empirical experimentation indispensable for understanding complex behaviors. Research on network testbeds for testing network protocols and distributed services, including physical, emulated, and federated testbeds, has made steady progress. Although the success of these testbeds is undeniable, they fail to provide: 1) scalability, for handling large-scale networks with hundreds or thousands of hosts and routers organized in different scenarios, 2) flexibility, for testing new protocols or applications in diverse settings, and 3) inter-operability, for combining simulated and real network entities in experiments. This dissertation tackles these issues in three different dimensions. First, we present SVEET, a system that enables inter-operability between real and simulated hosts. In order to increase the scalability of networks under study, SVEET enables time-dilated synchronization between real hosts and the discrete-event simulator. Realistic TCP congestion control algorithms are implemented in the simulator to allow seamless interactions between real and simulated hosts. SVEET is validated via extensive experiments and its capabilities are assessed through case studies involving real applications. Second, we present PrimoGENI, a system that allows a distributed discrete-event simulator, running in real-time, to interact with real network entities in a federated environment. PrimoGENI greatly enhances the flexibility of network experiments, through which a great variety of network conditions can be reproduced to examine what-if questions. Furthermore, PrimoGENI performs resource management functions, on behalf of the user, for instantiating network experiments on shared infrastructures. Finally, to further increase the scalability of network testbeds to handle large-scale high-capacity networks, we present a novel symbiotic simulation approach. We present SymbioSim, a testbed for large-scale network experimentation where a high-performance simulation system closely cooperates with an emulation system in a mutually beneficial way. On the one hand, the simulation system benefits from incorporating the traffic metadata from real applications in the emulation system to reproduce the realistic traffic conditions. On the other hand, the emulation system benefits from receiving the continuous updates from the simulation system to calibrate the traffic between real applications. Specific techniques that support the symbiotic approach include: 1) a model downscaling scheme that can significantly reduce the complexity of the large-scale simulation model, resulting in an efficient emulation system for modulating the high-capacity network traffic between real applications; 2) a queuing network model for the downscaled emulation system to accurately represent the network effects of the simulated traffic; and 3) techniques for reducing the synchronization overhead between the simulation and emulation systems.