Ambientes dulcícolas ocupam uma pequena parcela da superfície do planeta comparada aos ambientes marinhos e terrestres, porém são de suma importância para diversas atividades humanas e essenciais à manutenção da vida. Por estarem localizadas próximas a áreas urbanizadas passam a servir como corpos receptores de efluentes domésticos e até industriais. A cidade de Cubatão, SP, abriga um importante pólo industrial do país, sendo o rio Cubatão o corpo receptor principal de efluentes domésticos e industriais. O rio Cubatão é o principal da região e abastece quase toda a Baixada Santista juntamente com o rio Pilões. A intenção deste presente trabalho é evidenciar as fontes de contaminação do Rio Cubatão, por meio da literatura e de resultados de efeitos ecotoxicológicos (crônico e agudo) determinados em águas superficiais, utilizando os organismos Ceriodaphnia dubia e Vibro fischeri, respectivamente. Forte esforço foi aplicado a fim de quantificar os surfactantes (princípio ativo de detergentes) e outros contaminantes de interesse que permitam estabelecer a relação causa-efeito da toxicidade. Para as análises químicas e biológicas foram coletadas águas superficiais em cinco pontos do rio Cubatão, 2 pontos nos canais de Fuga da Usina Henry Borden e um ponto nos rios Pilões e Perequê. Os ensaios realizados com V. fischeri possibilitaram identificar toxicidade em todos os pontos amostrais, entre as cinco campanhas, cujos valores variaram entre CE(I)50 25,98% e 61%. Os resultados obtidos com C. dubia, tratados pelo Teste t por bioequivalência, mostraram toxicidade nos pontos P0; P1; P3; P4; P5 e P6 na terceira campanha. Na segunda e terceira campanha, os valores médios para a reprodução de C.dubia variaram entre 2,9 a 12,6 neonatas, possível efeito observado para a espécie, já que estes valores estiveram abaixo de 15 organismos, número mínimo recomendado pela ABNT NBR 13373:2004 para C.dubia. Com relação à condutividade das águas, o valor médio de 774,18 μS/cm-1 foi determinado para o P5, sendo esta média significativamente maior em relação aos demais pontos, possivelmente associado a proximidade das salinas da Carbocloro S.A. Indústrias Químicas. O P5 apresentou valor médio de 74,68 mg/L para cloretos.Os valores de compostos fenólicos, na segunda e terceira campanha, variaram entre 0,05 e 3,62 mg/L, concentrações essas acima do estabelecido pelo CONAMA 357:2005, para rios de classe 1 e 2 (0,003 mg/L). Os valores médios de surfactantes aniônicos estiveram entre 0,01 e 0,04 mg/L. Embora duas substâncias tenham sido empregadas na calibração do método (LAS) e (DSS), os resultados mostraram que não há diferença significativa na quantificação de surfactantes, pelo método (MBAS methylene blue active substance - APHA, 2005) (p ≤ 0,01). Em análise qualitativa foram identificados compostos aromáticos como tolueno, xileno, benzeno, etilbenzeno e diclorobenzeno, nos pontos amostrados em fevereiro de 2011. / Freshwater environments occupy a small portion of the surface compared to marine and terrestrial environments, but are critical for many activities essential to maintaining human life. By being located close to urban areas they serve as receptors bodies and even industrial effluents. The city of Cubatão, SP, houses an important industrial center in the country. Cubatão river is an important receptor of sewage and industrial effluents. It is the principal river of the region and supplies almost all of Baixada Santista with the Pilões river. The intention of this paper is to highlight the sources of contamination for Cubatão, through literature and results of ecotoxicological effects (chronic and acute) to be determined in surface water bodies using Ceriodaphnia dubia and Vibro fischeri, respectively. Strong effort was applied to quantify surfactant (active ingredient of detergents) and other contaminants of interest for determining the cause-effect relationship of toxicity. For chemical and biological analysis surface waters of the Cubatão river were collected in five points, two points in the channels of Henry Borden Plant, one on the Pilões and Perequê river. Through tests using V. fischeri toxicity was identified in all nine sampling sites, whose values ranged from EC(I)50 25.98% up to 60%. The results obtained from C. dubia , using \"t for bioequivalence\" showed toxic effects at P0, P1, P3, P4, P5 and P6 , third campaign. Dring the second and third campaign, it was obtained 2.9 to 12.6 neonates of C.dubia , numbers interpreted as chronic effects once they were quite below 15 neonates, number recommended by Brazilian environmental agencies (ABNT NBR 13373:2004). Water conductivity was much higher at P5, 774.18 μS/cm-1, mean value, probably related to the salt source from Carbocloro SA Chemical Industries. Chlorides at P5 was 74.68 mg/L, confirming the last statemente. Phenolic compounds varied from 0.05 to 3.62 mg/L, which is above the recommended by CONAMA 357:2005 (0,003 mg/L). Anionic surfactants varied from 0,01 to 0,04 mg/L. Although using two substances for internal method calibration (LAS) e (DSS), no statistically diference was obtained (p ≤ 0,01) (MBAS methylene blue active substance - APHA, 2005). Quantitative analysis reveled the following aromatic substances: toluene, xilene, benzene, ethylbenzene and dichlorobenzene, during February 2011 samplings.

Ambientes dulcícolas ocupam uma pequena parcela da superfície do planeta comparada aos ambientes marinhos e terrestres, porém são de suma importância para diversas atividades humanas e essenciais à manutenção da vida. Por estarem localizadas próximas a áreas urbanizadas passam a servir como corpos receptores de efluentes domésticos e até industriais. A cidade de Cubatão, SP, abriga um importante pólo industrial do país, sendo o rio Cubatão o corpo receptor principal de efluentes domésticos e industriais. O rio Cubatão é o principal da região e abastece quase toda a Baixada Santista juntamente com o rio Pilões. A intenção deste presente trabalho é evidenciar as fontes de contaminação do Rio Cubatão, por meio da literatura e de resultados de efeitos ecotoxicológicos (crônico e agudo) determinados em águas superficiais, utilizando os organismos Ceriodaphnia dubia e Vibro fischeri, respectivamente. Forte esforço foi aplicado a fim de quantificar os surfactantes (princípio ativo de detergentes) e outros contaminantes de interesse que permitam estabelecer a relação causa-efeito da toxicidade. Para as análises químicas e biológicas foram coletadas águas superficiais em cinco pontos do rio Cubatão, 2 pontos nos canais de Fuga da Usina Henry Borden e um ponto nos rios Pilões e Perequê. Os ensaios realizados com V. fischeri possibilitaram identificar toxicidade em todos os pontos amostrais, entre as cinco campanhas, cujos valores variaram entre CE(I)50 25,98% e 61%. Os resultados obtidos com C. dubia, tratados pelo Teste t por bioequivalência, mostraram toxicidade nos pontos P0; P1; P3; P4; P5 e P6 na terceira campanha. Na segunda e terceira campanha, os valores médios para a reprodução de C.dubia variaram entre 2,9 a 12,6 neonatas, possível efeito observado para a espécie, já que estes valores estiveram abaixo de 15 organismos, número mínimo recomendado pela ABNT NBR 13373:2004 para C.dubia. Com relação à condutividade das águas, o valor médio de 774,18 μS/cm-1 foi determinado para o P5, sendo esta média significativamente maior em relação aos demais pontos, possivelmente associado a proximidade das salinas da Carbocloro S.A. Indústrias Químicas. O P5 apresentou valor médio de 74,68 mg/L para cloretos.Os valores de compostos fenólicos, na segunda e terceira campanha, variaram entre 0,05 e 3,62 mg/L, concentrações essas acima do estabelecido pelo CONAMA 357:2005, para rios de classe 1 e 2 (0,003 mg/L). Os valores médios de surfactantes aniônicos estiveram entre 0,01 e 0,04 mg/L. Embora duas substâncias tenham sido empregadas na calibração do método (LAS) e (DSS), os resultados mostraram que não há diferença significativa na quantificação de surfactantes, pelo método (MBAS methylene blue active substance - APHA, 2005) (p ≤ 0,01). Em análise qualitativa foram identificados compostos aromáticos como tolueno, xileno, benzeno, etilbenzeno e diclorobenzeno, nos pontos amostrados em fevereiro de 2011. / Freshwater environments occupy a small portion of the surface compared to marine and terrestrial environments, but are critical for many activities essential to maintaining human life. By being located close to urban areas they serve as receptors bodies and even industrial effluents. The city of Cubatão, SP, houses an important industrial center in the country. Cubatão river is an important receptor of sewage and industrial effluents. It is the principal river of the region and supplies almost all of Baixada Santista with the Pilões river. The intention of this paper is to highlight the sources of contamination for Cubatão, through literature and results of ecotoxicological effects (chronic and acute) to be determined in surface water bodies using Ceriodaphnia dubia and Vibro fischeri, respectively. Strong effort was applied to quantify surfactant (active ingredient of detergents) and other contaminants of interest for determining the cause-effect relationship of toxicity. For chemical and biological analysis surface waters of the Cubatão river were collected in five points, two points in the channels of Henry Borden Plant, one on the Pilões and Perequê river. Through tests using V. fischeri toxicity was identified in all nine sampling sites, whose values ranged from EC(I)50 25.98% up to 60%. The results obtained from C. dubia , using \"t for bioequivalence\" showed toxic effects at P0, P1, P3, P4, P5 and P6 , third campaign. Dring the second and third campaign, it was obtained 2.9 to 12.6 neonates of C.dubia , numbers interpreted as chronic effects once they were quite below 15 neonates, number recommended by Brazilian environmental agencies (ABNT NBR 13373:2004). Water conductivity was much higher at P5, 774.18 μS/cm-1, mean value, probably related to the salt source from Carbocloro SA Chemical Industries. Chlorides at P5 was 74.68 mg/L, confirming the last statemente. Phenolic compounds varied from 0.05 to 3.62 mg/L, which is above the recommended by CONAMA 357:2005 (0,003 mg/L). Anionic surfactants varied from 0,01 to 0,04 mg/L. Although using two substances for internal method calibration (LAS) e (DSS), no statistically diference was obtained (p ≤ 0,01) (MBAS methylene blue active substance - APHA, 2005). Quantitative analysis reveled the following aromatic substances: toluene, xilene, benzene, ethylbenzene and dichlorobenzene, during February 2011 samplings.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 118-142).

Made available in DSpace on 2018-08-24T22:53:03Z (GMT). No. of bitstreams: 1 tese_3679_dissert_vesaofinal.pdf: 2234737 bytes, checksum: 7b99be2ff949fbc75aaf953220288ab6 (MD5) Previous issue date: 2009-08-19 / Os manguezais são constantemente atingidos por derramamentos de hidrocarbonetos de petróleo, provenientes das crescentes indústrias petroquímicas ou embarcações que transportam estes minerais brutos, ou como produtos derivados, dentre eles os diversos tipos de combustíveis. Por isso, estes ambientes são considerados ecossistemas vulneráveis e os mais sensíveis a derramamento de hidrocarbonetos de petróleo, devido à facilidade de o óleo aderir ao sedimento causando efeitos agudos e crônicos a toda biodiversidade. Sendo assim, uma das formas mais precisas de monitoramento biológico aos impactos antrópicos nos ecossistemas pode ser feitos através dos ensaios ecotoxicológicos agudos e ou crônicos, com organismos que sejam representativos ao ambiente. Com base nisso, para uma avaliação detalhada de um impacto por derrame de petróleo e derivados no manguezal, o presente trabalho buscou desenvolver uma metodologia de monitoramento do sedimento com estes contaminantes em etapa preliminar ocorrida em um mês e definitiva ocorrida em três meses, ambas com diferentes tipos de unidades experimentais (em recipientes Vaso e Lata/PET, respectivamente) fora do seu ambiente natural, por meio de ensaios ecotoxicológicos agudos, com a bactéria Vibrio fischeri. Os contaminantes testados foram o petróleo (16,9 ºAPI, pesado) e três frações provenientes deste mesmo hidrocarboneto, com densidades diferentes comparadas a alguns tipos de combustíveis, uma similar ao querosene (fração 2, 40,6 ºAPI, leve), outra similar ao óleo diesel (fração 6, 28 ºAPI, média) e a última similar ao óleo lubrificante (fração 12, 19,4 ºAPI, pesada). A toxicidade aguda apresentou relação com a densidade do óleo, pois foi sempre mais acentuada ao contaminante de grau API mais alto, onde também foi observada uma atenuação maior nos períodos experimentais, principalmente nas primeiras análises. Nos sedimentos contendo a fração 2 como contaminante apresentaram maior toxicidade aguda inicial, tanto para os experimentos preliminares quanto para os experimentos definitivos, de 81,12% e 75,46%, respectivamente.

A poluição dos rios brasileiros é, atualmente, um grande problema para as autoridades governamentais já que a demanda dos recursos hídricos vem crescendo nos últimos anos. Os rios Tietê, Pinheiros e Tamanduateí foram de grande importância para a cidade de São Paulo em seu período de industrialização, contudo o próprio desenvolvimento industrial impactou permanentemente os mesmos. Preocupados com a saúde ambiental, órgãos fiscalizadores estaduais iniciaram forte fiscalização para coibir e responsabilizar os principais culpados. Esta política pública resultou em uma diminuição das concentrações de poluentes orgânicos e inorgânicos, porém muito pouco foi efetivamente feito com relação ao esgoto doméstico, e, como consequência, a toxicidade das águas dos rios permanece elevada. Os estudos de Avaliação e identificação da toxicidade englobam diversas metodologias reunidas em três documentos pela USEPA - Agência de Proteção Ambiental dos Estados Unidos, onde método físico-químico e testes toxicológicos são usados para identificar substâncias tóxicas, ou grupo de substâncias tóxicas que contribuem efetivamente para a toxicidade total de uma amostra. A abordagem rotineiramente utilizada baseia-se no fracionamento da amostra de acordo com suas propriedades físico-químicas: volatilidade, solubilidade e capacidade de formar complexos, ou de ser reduzida, oxidada, ionizada, entre outras. Em conjunto com os ensaios químicos, os ensaios toxicológicos, em especial o Sistema Microtox®, contribuem na investigação da toxicidade para os rios metropolitanos Tietê, Pinheiros e Tamanduateí, que se mostrou eficaz na determinação dos grupos de compostos, sendo os compostos orgânicos apolares, orgânicos voláteis, tensoativos e as partículas, as principais fontes da toxicidade para esses rios. Embora muitas informações sobre a toxicidade tenham sido descobertas, a identificação dos compostos só pode ser realizada por análises químicas mais robustas uma vez que um grande número de substâncias, metabólitos e subprodutos de degradação são facilmente encontrados neste tipo de amostra. Mesmo sofrendo algumas modificações, deve-se incentivar o uso desta metodologia para outros rios a fim de determinar os principais toxicantes responsáveis pela toxicidade. / Pollution of Brazilian rivers is currently a major problem for government authorities as demand for water resources has been increasing in recent years. Tietê, Pinheiros and Tamanduateí rivers were of great importance for the city of São Paulo during its industrialization period, however, the industrial development itself permanently impacted them. Concerned about environmental health, state supervisory bodies have begun strong enforcement to curb and hold the main culprits accountable. This public policy has resulted in a decrease in concentrations of organic and inorganic chemicals, but very little has been done in relation to domestic sewage, and as a consequence, the toxicity of the rivers remains high. The Toxicology Assessment and Identification studies encompass various methodologies gathered in three documents by the USEPA, where physico-chemical method and toxicological tests are used to identify toxic substances, or group of toxic substances that effectively contribute to the total toxicity of a sample. The routinely used approach is based on fractionation of the sample according to its physicochemical properties: volatility, solubility and chemical properties such as the ability to form complexes, or to be reduced, oxidized, ionized, among others. In conjunction with the chemical tests, the toxicological tests in particular the Microtox® System contribute to the investigation of the toxicity to the metropolitan rivers, Tietê, Pinheiros and Tamanduateí, which proved to be effective in the determination of the groups of compounds, the organic compounds being apolar, organic volatiles, surfactants and particulates, the main sources of toxicity for the studied rivers. Although much information on toxicity has been discovered, the identification of compounds can only be performed by more robust chemical analyzes since a large number of substances, metabolites, degradation byproducts are readily found in this type of sample. Even with some modifications, it is necessary to encourage the use of this methodology for other rivers in order to determine the main xenobiotics responsible for toxicity.

A poluição dos rios brasileiros é, atualmente, um grande problema para as autoridades governamentais já que a demanda dos recursos hídricos vem crescendo nos últimos anos. Os rios Tietê, Pinheiros e Tamanduateí foram de grande importância para a cidade de São Paulo em seu período de industrialização, contudo o próprio desenvolvimento industrial impactou permanentemente os mesmos. Preocupados com a saúde ambiental, órgãos fiscalizadores estaduais iniciaram forte fiscalização para coibir e responsabilizar os principais culpados. Esta política pública resultou em uma diminuição das concentrações de poluentes orgânicos e inorgânicos, porém muito pouco foi efetivamente feito com relação ao esgoto doméstico, e, como consequência, a toxicidade das águas dos rios permanece elevada. Os estudos de Avaliação e identificação da toxicidade englobam diversas metodologias reunidas em três documentos pela USEPA - Agência de Proteção Ambiental dos Estados Unidos, onde método físico-químico e testes toxicológicos são usados para identificar substâncias tóxicas, ou grupo de substâncias tóxicas que contribuem efetivamente para a toxicidade total de uma amostra. A abordagem rotineiramente utilizada baseia-se no fracionamento da amostra de acordo com suas propriedades físico-químicas: volatilidade, solubilidade e capacidade de formar complexos, ou de ser reduzida, oxidada, ionizada, entre outras. Em conjunto com os ensaios químicos, os ensaios toxicológicos, em especial o Sistema Microtox®, contribuem na investigação da toxicidade para os rios metropolitanos Tietê, Pinheiros e Tamanduateí, que se mostrou eficaz na determinação dos grupos de compostos, sendo os compostos orgânicos apolares, orgânicos voláteis, tensoativos e as partículas, as principais fontes da toxicidade para esses rios. Embora muitas informações sobre a toxicidade tenham sido descobertas, a identificação dos compostos só pode ser realizada por análises químicas mais robustas uma vez que um grande número de substâncias, metabólitos e subprodutos de degradação são facilmente encontrados neste tipo de amostra. Mesmo sofrendo algumas modificações, deve-se incentivar o uso desta metodologia para outros rios a fim de determinar os principais toxicantes responsáveis pela toxicidade. / Pollution of Brazilian rivers is currently a major problem for government authorities as demand for water resources has been increasing in recent years. Tietê, Pinheiros and Tamanduateí rivers were of great importance for the city of São Paulo during its industrialization period, however, the industrial development itself permanently impacted them. Concerned about environmental health, state supervisory bodies have begun strong enforcement to curb and hold the main culprits accountable. This public policy has resulted in a decrease in concentrations of organic and inorganic chemicals, but very little has been done in relation to domestic sewage, and as a consequence, the toxicity of the rivers remains high. The Toxicology Assessment and Identification studies encompass various methodologies gathered in three documents by the USEPA, where physico-chemical method and toxicological tests are used to identify toxic substances, or group of toxic substances that effectively contribute to the total toxicity of a sample. The routinely used approach is based on fractionation of the sample according to its physicochemical properties: volatility, solubility and chemical properties such as the ability to form complexes, or to be reduced, oxidized, ionized, among others. In conjunction with the chemical tests, the toxicological tests in particular the Microtox® System contribute to the investigation of the toxicity to the metropolitan rivers, Tietê, Pinheiros and Tamanduateí, which proved to be effective in the determination of the groups of compounds, the organic compounds being apolar, organic volatiles, surfactants and particulates, the main sources of toxicity for the studied rivers. Although much information on toxicity has been discovered, the identification of compounds can only be performed by more robust chemical analyzes since a large number of substances, metabolites, degradation byproducts are readily found in this type of sample. Even with some modifications, it is necessary to encourage the use of this methodology for other rivers in order to determine the main xenobiotics responsible for toxicity.

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.

Bioassays for baseline toxicity, which measure toxicants’ non-specific effects, have been shown in previous studies to effectively correlate with the increased presence of pharmaceuticals, personal care products, endocrine-disrupting compounds, and other synthetic organics in treated sewage effluent. This study investigated how the baseline toxicity of anthropogenic compounds-spiked wastewater changed during the treatment of biofiltration and ozone oxidation, as measured by the bioluminescence inhibition of the Vibrio fischeri bacterium. The water quality parameters of dissolved organic carbon, seven common anions, and fluorescence spectroscopy were used to corroborate and collate with the toxicity results. Water quality was evaluated on two bench-scale soil filtration columns, which were configured for pre-ozonation and post-ozonation. Both systems’ soil aerobically removed similar amounts of dissolved organic carbon, and the reduction ranged between 57.7% and 62.1% for the post-ozonation and pre-ozonation systems, respectively. Biological removal of DOC, protein-like, humic-like, and soluble microbial product-like material was highest in the first 28.5 cm of each 114 cm-long system. While bioluminescence inhibition showed that ozonation was effective at lowering baseline toxicity, this study’s bioassay procedure was a very poor indicator of soil filtration treatment; both system’s effluents were significantly more toxic than their non-ozonated influents.

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

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.