Quorum sensing in Gram-negative bacteria is best understood in the bioluminescent marine microorganism, <i>Vibrio fischeri</i>. In <i>V. fischeri</i>, the luminescence or <i>lux</i> genes are regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule (3-oxo-hexanoyl homoserine lactone). LuxR, which binds to the <i>lux</i> operon promoter at position -42.5, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the <font face = "symbol">a</font>CTD of RNAP in LuxR-dependent transcriptional activation of the <i>lux</i> operon promoter has been investigated. The effect of seventy alanine substitution variants of the <font face = "symbol">a</font> subunit was determined <i>in vivo</i> by measuring the rate of transcription of the <i>lux</i> operon via luciferase assays in recombinant <i>Escherichia coli</i>. The mutant RNAPs from strains exhibiting at least two fold increased or decreased activity in comparison to the wild-type were further examined by <i>in vitro</i> assays. Since full-length LuxR has not been purified to date, an autoinducer-independent N-terminal truncated form of LuxR, LuxR<font face = "symbol">D</font>N, was used for <i>in vitro</i> studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxR<font face = "symbol">D</font>N, and fourteen residues in the <font face = "symbol">a</font>CTD were identified as having negative effects on the rate of transcription from the <i>lux</i> operon promoter. Five of these fourteen residues were also involved in the mechanism of both LuxR and LuxR<font face = "symbol">D</font>N-dependent activation <i>in vivo</i> and were chosen for further analysis by DNA mobility shift assays. Results from these assays indicate that while the wild-type <font face = "symbol">a</font>CTD is capable of interacting with the <i>lux</i> DNA fragment tested, all five of the variant forms of the <font face = "symbol">a</font>CTD tested appear to be deficient in their ability to recognize and bind the DNA. These findings suggest that <font face = "symbol">a</font>CTD-DNA interactions may play a role in LuxR-dependent transcriptional activation of the <i>lux</i> operon during quorum sensing. / Master of Science

The mechanism of gene regulation used by Gram-negative bacteria during quorum sensing is well understood in the bioluminescent marine bacterium Vibrio fischeri. The cell-density dependent activation of the luminescence (lux) genes of V. fischeri relies on the formation of a complex between the autoinducer molecule, N-(3-oxohexanoyl) homoserine lactone, and the autoinducer-dependent transcriptional activator LuxR. LuxR, a 250 amino acid polypeptide, binds to a site known as the lux box centered at position -42.5 relative to the luxI transcriptional start site. During transcriptional activation of the lux operon, LuxR is thought to function as an ambidextrous activator capable of making multiple contacts with RNA polymerase (RNAP). The specific role of region 4 of the Escherichia coli sigma 70 subunit of RNAP in LuxR-dependent transcriptional activation of the luxI promoter has been investigated. Rich in basic amino acids, this conserved portion of sigma 70 is likely to be surface-exposed and available to interact with transcription factors bound near the -35 element. The effect of 16 single and 2 triple alanine substitution variants of sigma 70 between amino acid residues 590 and 613, was determined in vivo by measuring the rate of transcription from a luxI-lacZ translational fusion via b-galactosidase assays in recombinant E. coli. In vitro work was performed with LuxRDN, the autoinducer-independent C-terminal domain (amino acids 157 to 250) of LuxR because purified, full length LuxR is unavailable. Single-round transcription assays were performed in the presence of LuxRDN and 19 variant RNAPs, one of which contained a C-terminally truncated sigma 70 subunit devoid of region 4. Results indicate that region 4 is essential for LuxRDN-dependent luxI transcription with two specific amino acid residues, E591 and K597, having negative effects on the rate of LuxRDN-dependent luxI transcription in vivo and in vitro. None of the residues tested were identified as having any effect on LuxR-dependent luxI transcription in vivo. These findings suggest that region 4.2 is most likely to be in close proximity to LuxR when bound to the luxI promoter. However, unlike the situation found for other ambidextrous activators, no single residue within region 4.2 of sigma 70 may be critical by itself for LuxR-dependent during transcriptional activation. / Master of Science

<I>Vibrio fischeri</I>, a symbiotic bioluminescent bacterium, serves as one of the best understood model systems for a mechanism of cell-density dependent bacterial gene regulation known as quorum sensing. During quorum sensing in <I>V. fischeri</I>, an acylated homoserine chemical signal (autoinducer) is synthesized by the bacteria and used to sense their own species in a given environment. As the autoinducer levels rise, complexes form between the autoinducer and the N-terminal domain of a regulatory protein, LuxR. In response to autoinducer binding, LuxR is believed to undergo a conformational change that allows the C-terminal domain to activate transcription of the luminescence or <I>lux</I> operon. To further understand the mechanism of LuxR-dependent transcriptional activation of the <I>lux</I> operon, PCR-based site-directed mutagenesis procedures have been used to generate alanine-substitution mutants in the C-terminal forty-one amino acid residues of LuxR, a region that has been hypothesized to play a critical role in the activation process. An <I>in vivo</I> luminescence assay was first used to test the effects of the mutations on LuxR-dependent activation of the <I>lux</I> operon in recombinant <I>Escherichia coli</I>. Luciferase levels present in cell extracts obtained from these strains were also quantified and found to correlate with the luminescence results. Eight strains encoding altered forms of LuxR exhibited a "dark" phenotype with luminescence output less than 50% and luciferase levels less than 50% of the wildtype control strain. Western immunoblotting analysis with cell extracts from the luminescence and luciferase assays verified that the altered forms of LuxR were expressed at levels approximately equal to wildtype. Therefor, Low luminescence and luciferase levels could be the result of a mutation that either affects the ability of LuxR to recognize and bind its DNA target (the <I>lux</I> box) or to establish associations with RNA polymerase (RNAP) at the <I>lux</I> operon promoter necessary for transcriptional initiation. A third <I>in vivo </I>assay was used to test the ability of the altered forms of LuxR to bind to the <I>lux</I> box (DNA binding assay/repression). All of the LuxR variants exhibiting the "dark" phenotype in the luminescence and luciferase assay were also found to be unable to bind to the <I>lux</I> box in the<I> </I>DNA binding assay. Therefore, it can be concluded that the alanine substitutions made at these positions affect the ability of LuxR to bind to the <I>lux</I> box in the presence and absence of RNA polymerase. Another class of mutants exhibited wildtype phenotypes in the luminescence and luciferase assays but were unable to bind to the <I>lux</I> box in the DNA binding assay. The alanine substitutions made at these amino acid residues may be making contacts with RNAP that are important for maintaining the stability of the DNA binding region of LuxR. Alanine substitutions made at these positions have a defect in DNA binding at the promoter of the <I>lux</I> operon only in the absence of RNAP. None of the alanine substitutions made in the C-terminal forty-one amino acids of LuxR were found to affect activation of transcription of the <I>lux</I> operon without also affecting DNA binding. Taken together, these results support the conclusion that the C-terminal forty-one amino acids of LuxR are important for DNA recognition and binding of the <I>lux</I> box rather than positive control of the process of transcription initiation. / Master of Science

The primary contributor to Vibrio fischeri aminopeptidase activity is aminopeptidase N, PepN. Colonization assays revealed the pepN mutant strain to be deficient at forming dense aggregates and populating the host’s light organ compared to wildtype within the first 12 hours of colonization; however the mutant competed normally at 24 hours. To address the role of PepN in colonization initiation and establish additional phenotypes for the pepN mutant strain, stress response and other physiological assays were employed. Marked differences were found between pepN mutant and wildtype strain in response to salinity, acidity, and antibiotic tolerance. This study has provided a foundation for future work on identifying a putative role for V. fischeri PepN in regulating stress response.

En aquest estudi, la toxicitat de diversos metalls pesants i l'arsènic va ser analitzada utilitzant diferents models biològics.En la primera part d'aquest treball, el bioassaig de toxicitat Microtox, el qual està basat en la variació de l'emissió lumínica del bacteri luminiscent Vibrio fischeri, va ser utilitzat per establir les corbes dosi-resposta de diferents elements tòxics com el Zn(II), Pb(II), Cu(II), Hg(II), Ag(I), Co(II), Cd(II), Cr(VI), As(V) i As(III) en solucions aquoses. Els experiments es varen portar a terme a pH 6.0 i 7.0 per tal de mostrar que el pH pot influir en la toxicitat final mesurada d'alguns metalls degut als canvis relacionats amb la seva especiació química. Es varen trobar diferents tipus de corbes dosi-resposta depenent del metall analitzat i el pH del medi. En el cas de l'arsènic, l'efecte del pH en la toxicitat de l'arsenat i l'arsenit es va investigar utilitzant l'assaig Microtox en un rang de pHs comprès entre pH 5.0 i 9.0. Els valors d'EC50 determinats per l'As(V) disminueixen, reflectint un augment de la toxicitat, a mesura que el pH de la solució augmenta mentre que, en el cas de l'As(III), els valors d'EC50 quasi bé no varien entre pH 6.0 i 8.0 i només disminueixen a pH 9.0. HAsO42- i H2AsO3- es varen definir com les espècies més tòxiques. Així mateix, una anàlisi estadística va revelar un efecte antagònic entre les espècies químiques d'arsenat que es troben conjuntament a pH 6.0 i 7.0.D'altra banda, els resultats de dos mètodes estadístics per predir la toxicitat i les possibles interaccions entre el Co(II), Cd(II), Cu(II), Zn(II) i Pb(II) en mescles binàries equitòxiques es varen comparar amb la toxicitat observada sobre el bacteri Vibrio fischeri. L'efecte combinat d'aquests metalls va resultar ser antagònic per les mescles de Co(II)-Cd(II), Cd(II)-Zn(II), Cd(II)-Pb(II) i Cu(II)-Pb(II), sinèrgic per Co(II)-Cu(II) i Zn(II)-Pb(II) i additiu en els altres casos, revelant un patró complex de possibles interaccions. L'efecte sinèrgic de la combinació Co(II)-Cu(II) i la forta disminució de la toxicitat del Pb(II) quan es troba en presència de Cd(II) hauria de merèixer més atenció quan s'estableixen les normatives de seguretat ambiental.La sensibilitat de l'assaig Microtox també va ser determinada. Els valors d'EC20, els quals representen la toxicitat llindar mesurable, varen ser determinats per cada element individualment i es va veure que augmenten de la següent manera: Pb(II) < Ag(I) < Hg(II) &#61627; Cu(II) < Zn(II) < As(V) < Cd(II) &#61627; Co(II) < As(III) < Cr(VI). Aquests valors es varen comparar amb les concentracions permeses en aigues residuals industrials establertes per la normativa oficial de Catalunya (Espanya). L'assaig Microtox va resultar ser suficientment sensible per detectar els elements assajats respecte a les normes oficials referents al control de la contaminació, excepte en el cas del cadmi, mercuri, arsenat, arsenit i cromat. En la segona part d'aquest treball, com a resultats complementaris dels resultats previs obtinguts utilitzant l'assaig de toxicitat aguda Microtox, els efectes crònics del Cd(II), Cr(VI) i As(V) es varen analitzar sobre la taxa de creixement i la viabilitat en el mateix model biològic. Sorprenentment, aquests productes químics nocius varen resultar ser poc tòxics per aquest bacteri quan es mesura el seu efecte després de temps d'exposició llargs. Tot i això, en el cas del Cr(VI), l'assaig d'inhibició de la viabilitat va resultar ser més sensible que l'assaig de toxicitat aguda Microtox. Així mateix, també va ser possible observar un clar fenomen d'hormesis, especialment en el cas del Cd(II), quan s'utilitza l'assaig d'inhibició de la viabilitat. A més a més, diversos experiments es varen portar a terme per intentar explicar la manca de toxicitat de Cr(VI) mostrada pel bacteri Vibrio fischeri. La resistència mostrada per aquest bacteri podria ser atribuïda a la capacitat d'aquest bacteri de convertir el Cr(VI) a la forma menys tòxica de Cr(III). Es va trobar que aquesta capacitat de reducció depèn de la composició del medi de cultiu, de la concentració inicial de Cr(VI), del temps d'incubació i de la presència d'una font de carboni. En la tercera part d'aquest treball, la línia cel·lular humana HT29 i cultius primaris de cèl·lules sanguínies de Sparus sarba es varen utilitzar in vitro per detectar la toxicitat llindar de metalls mesurant la sobreexpressió de proteines d'estrès. Extractes de fangs precedents de diverses plantes de tractament d'aigues residuals i diferents metalls, individualment o en combinació, es varen analitzar sobre cultius cel·lulars humans per avaluar el seu efecte sobre la taxa de creixement i la capacitat d'induir la síntesi de les proteïnes Hsp72 relacionades amb l'estrès cel·lular. No es varen trobar efectes adversos significatius quan els components s'analitzen individualment. Nogensmenys, quan es troben conjuntament, es produeix un afecte advers sobre tan la taxa de creixement com en l'expressió de proteins d'estrès. D'altra banda, cèl·lules sanguínies procedents de Sparus sarba es varen exposar in vitro a diferents concentracions de cadmi, plom i crom. La proteïna d'estrès HSP70 es va sobreexpressar significativament després de l'exposició a concentracions tan febles com 0.1 &#61549;M. Sota les nostres condicions de treball, no es va evidenciar una sobreexpressió de metal·lotioneïnes. Nogensmenys, les cèl·lules sanguínies de peix varen resultar ser un model biològic interessant per a ser utilitzat en anàlisis de toxicitat. Ambdós models biològics varen resultar ser molt adequats per a detectar acuradament la toxicitat produïda per metalls. En general, l'avaluació de la toxicitat basada en l'anàlisi de la sobreexpressió de proteïnes d'estrès és més sensible que l'avaluació de la toxicitat realitzada a nivell d'organisme.A partir dels resultats obtinguts, podem concloure que una bateria de bioassaigs és realment necessària per avaluar acuradament la toxicitat de metalls ja que existeixen grans variacions entre els valors de toxicitat obtinguts emprant diferents organismes i molts factors ambientals poden influir i modificar els resultats obtinguts. / In this study, the toxicity of some metals and arsenic was investigated using three different biological models. In the first part of this work, the Microtox® bioassay, which is based on variation in light emission by Vibrio fischeri luminescent bacteria, was used to establish dose-response curves for several toxic elements, namely, Zn(II), Pb(II), Cu(II), Hg(II), Ag(I), Co(II), Cd(II), Cr(VI), As(V), and As(III), in aqueous solutions. Experiments were carried out at either pH 6.0 or pH 7.0 to indicate that pH may influence the measured toxicity of some elements due to pH-related changes in their chemical speciation. Different types of dose-response curves were found depending on the analyzed metal and pH. In the case of arsenic, effect of pH on either arsenate or arsenite toxicity, was investigated using the Microtox® bioassay within a 5.0 - 9.0 pH range. EC50 values for As(V) were found to decrease, reflecting an increase in toxicity, as pH became basic, whereas in the case of As(III), EC50 values were almost unchanged within a 6.0 - 8.0 pH range and lowered at pH 9.0 only. HAsO42- and H2AsO3- were found to be the most toxic species. A statistical approach revealed an antagonistic effect between the arsenate chemical species found in combination at pH 6.0 or 7.0.On the other hand, results from two mathematical approaches to predict the toxicity of all the possible binary equitoxic mixtures of Co(II), Cd(II), Cu(II), Zn(II), and Pb(II) were compared to the observed toxicity of these mixtures to Vibrio fischeri bacteria. Combined effect of the metals was found to be antagonistic for Co(II)-Cd(II), Cd(II)-Zn(II), Cd(II)-Pb(II), and Cu(II)-Pb(II), synergistic for Co(II)-Cu(II) and Zn(II)-Pb(II) and merely additive in other cases, revealing a complex pattern of possible interactions. The synergistic effect of the Co(II)-Cu(II) combination and the strong decrease of Pb(II) toxicity when in the presence of Cd(II) should deserve much attention when establishing environmental safety regulations. Microtox bioassay sensitivity was also analyzed. EC20 values, which represent a measurable threshold of toxicity, were determined for each element individually and were found to rank as Pb(II) < Ag(I) < Hg(II) &#61627; Cu(II) < Zn(II) < As(V) < Cd(II) &#61627; Co(II) < As(III) < Cr(VI). These values were compared to the concentration levels allowed in industrial wastewater according to the official regulations in Catalonia (Spain). It appears that the Microtox® test is sensitive enough to detect the tested elements with respect to official regulations dealing with pollution control, with the exception of cadmium, mercury, arsenate, arsenite and chromate.In the second part of this work, as a complement to previous results obtained using the standard Microtox® acute toxicity test, the long-term effects of Cd(II), Cr(VI), and As(V) were studied on growth rate and viability of the same biological model. Surprisingly, these poisonous chemicals were found not to be very toxic to these bacteria when measuring their effect on viability or growth after long periods of exposure. Nevertheless, in the case of Cr(VI), the inhibition viability assay resulted to be more sensitive than the Microtox acute toxicity test was. Interestingly, it was possible to observe a clear hormesis phenomenon, especially for Cd(II), under the conditions of the viability assay. In addition, several experiments were performed as an attempt to explain the lack of Cr(VI) toxicity shown by Vibrio fischeri bacteria. The resistance shown by Vibrio fischeri bacteria could be attributed to the capacity of the bacteria to convert Cr(VI) ions into less toxic Cr(III) ions. This capacity of reduction was found to depend on culture medium composition, initial concentration of chromium, incubation time, and the presence of a carbon source. In the third part of this work, the HT29 human cell line and primary cultures of Sparus sarba blood cells were used in vitro to detect metal toxicity thresholds by measuring the overexpression of stress proteins. Sludge extracts from several wastewater treatment plants and metals, individually or in combination, were tested on human cultured cells for evaluating their ability to affect the growth rate and trigger a synthesis of the stress-related HSP72i proteins. No significant adverse effects were found when given individually. When given in combination, they were however found to affect both cell growth and stress proteins expression. On the other hand, blood cells freshly collected from Sparus sarba were exposed in vitro to different concentrations of cadmium, lead or chromium(VI). HSP70 stress protein was significantly overexpressed after exposure to a metal concentration as low as 0.1 µM. Under our experimental conditions, no overexpression of metallothioneins was evidenced. Nevertheless, fish blood cells appear as an interesting biological model for experimental toxicology.Both biological models were found convenient to detect toxicity produced by metals. In general, evaluation of toxicity based on stress proteins overexpression was found to be more sensitive than evaluation of toxicity performed at the organism level.Based on the results, it can be concluded that a battery of bioassays is necessary to accurately evaluate toxicity of metals since important variations between different organisms can be found and a lot of environmental factors may influence as well as modify the obtained results.

Made available in DSpace on 2016-06-02T19:31:43Z (GMT). No. of bitstreams: 1 2493.pdf: 1820155 bytes, checksum: c4fbdc0a3fdb7a9316ba5c6c6a6905c8 (MD5) Previous issue date: 2009-05-15 / Universidade Federal de Sao Carlos / The objective of the present dissertation was to make a toxicity assessment of the application of calcium nitrate solution as a remediation procedure for sediments of a eutrophic aquatic ecosystem. The study was carried out using microcosms with superficial sediments and water from sediment-water interface of the Ibirité Reservoir located in the metropolitan area of Belo Horizonte (Minas Gerais, SE Brazil). The experiment lasted 135 days and the following treatment or incubation periods were applied: t=0, t=5, t=10, t=25, t=50, t=85 and t=135 days. In each period, one controlmicrocosm and three treatment-microcosms were disassembled and, chemically and ecotoxicologically analyzed. The organisms Ceriodaphnia silvestrii and Vibrio fischeri (Microtox® System) were used for the acute toxicity assessment of the water from sediment-water interface and the pore water of sediments, whereas the organism Chironomus xanthus was used for the toxicity assessment of bulk sediment. The toxicity tests were run in parallel with chemical analyzes of dissolved inorganic nitrogen species (nitrate, nitrate and ammonium), sulfate, and metals in the interface sediment-water and interstitial water samples. Acid volatile sulfide (AVS), simultaneously extracted metal (SEM) and potentially bioavailable metals were analyzed in bulk sediment. The overall results indicate that nitrate whose concentration reached 1,200 mg N-NO3 - L-1 in sediment pore water samples from treatment-microcosms is the most probable compound causing toxicity to the tests organisms. For Chironomus xanthus sediments were deleterious to the exposed organisms in all microcosm run, except in the period of t= 135 days. For the experimental conditions of this work, the application of calcium nitrate as a remediation procedure for sediments from Ibirité Reservoir indicated to be inadequate from the ecotoxicological pint of view. / O presente trabalho visou à avaliação da toxicidade da aplicação de solução de nitrato de cálcio, como procedimento de intervenção para remediação de sedimentos de um ambiente aquático eutrofizado. O estudo foi realizado através de microcosmos com sedimento e água de interface sedimento-água da Represa Ibirité, situada na região metropolitana de Belo Horizonte (Minas Gerais, Brasil). Os experimentos tiveram a duração total de 135 dias, divididos nos tempos de tratamento ou incubação de: t=0, t=5; t=10; t=25; t=50; t=85 e t=135 dias. Em cada tempo de tratamento foram analisados um microcosmo-controle e três microcosmostratamento. Os organismos Ceriodaphnia silvestrii e Vibrio fischeri (Sistema Microtox®) foram utilizados para avaliação da toxicidade aguda das águas de interface sedimento-água e intersticial dos sedimentos, enquanto o organismo Chironomus xanthus para avaliação do sedimento integral. Em paralelo aos testes de toxicidade foram realizadas análises químicas da série nitrogenada (nitrato, nitrito e amônia), sulfato, e metais nas amostras de água de interface sedimento-água e intersticial dos sedimentos, sulfetos volatilizáveis por acidificação (SVA), metais extraídos simultaneamente (MES) e metais potencialmente biodisponíveis nos sedimentos. Os resultados mostraram que o nitrato, chegando a concentração superior a 1.200 mg N-NO3 - L-1 nas amostras de água intersticial dos sedimentos dos microcosmos-tratamentos, foi considerado o causador mais provável da toxicidade das amostras dos microcosmos-tratamento para os organismos-teste empregados. Para o organismo Chironomus xanthus, os sedimentos em tratamento foram deletérios aos organismos expostos em todos os tempos de incubação, exceto no tempo t=135 dias. Estritamente do ponto de vista ecotoxicológico e para as condições experimentais deste trabalho, a aplicação do nitrato como forma de intervenção para remediação dos sedimentos da Represa Ibirité não se mostrou adequada.

To determine the ecotoxicity of analgetics, first the individual ecotoxicity values of individual analgetics are determined and then a mixture of two analgetics is tested. To determine the toxicity, both standard and alternative toxicity tests are used (daphnia magna, sinapsis alba, scenedesmus subspicatum, vibrio fischeri, thamnotoxkit FTM a daphnotoxkit FTM magna). The analgetics being whish tested are the commonly used medicines ibuprofen, ASA, diclofenac and paracetamol, which are the most frequently used medicines in the Czech Republic and whole Europe. To determine the ecotoxicity of the polymers, I‘m using an indirect method of examination, in which I determine the antagonistic or synergistic effects of the mixture of monomers from which the polymer is prepared and into which it slowly decomposes in nature. For the determination both standard and alternative toxicity tests are used. The polymers the toxicity of which is being determined are the habitually used polymer PET and the formaldehyde resine known as bakelite in Eastern Europe.