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This study provided evidence of cell-density dependent signalling interactions between the autotrophic ammonia-oxidising bacterium Nitrosomonas europaea and a variety of terrestrial heterotrophic bacteria. The autoinducer signal molecule N-(3-hexanoyl)-L-homoserine lactone (HHL) extended the lag phase of N. europaea recovering from starvation while N-(3-oxooctanoyl)-L-homoserine lactone (OOHL) reduced the lag phase. However, the autoinducers N-(3-oxohexanoyl)-L-homoserine lactone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), N-(3-oxobutanoyl)-L-homoserine lactone(OBHL) and N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) did not exert an effect. Spent cell-free medium from early stationary phase cultures of Agrobacterium tumefaciens, Pseudomonas fluorescens and Micrococcus luteus reduced the lag phase of N. europaea recovering from starvation. Supplementing solid medium with spent cell-free medium from the heterotrophs A. tumefaciens, P. fluorescens and M. luteus reduced the incubation period required for the development of colonies of the ammonia-oxidiser. For example, the presence of spent cell-free medium from A. tumefaciens reduced the required incubation period from 20 weeks to 2 weeks. Spent cell-free medium from the heterotrophs Comomonas testosteroni, Erwinia carotovora and Rhizobium leguminosarum had no effect on the growth of N. europaea. A. tumefaciens produces the N-acyl autoinducer OOHL which reduces the lag phase of N. europaea. A mutant unable to produce this autoinducer was generated and spent cell-free medium from this organism had no effect on the recovery of N. europaea from starvation or the incubation period required for the development of colonies of N. europaea on solid medium. Enrichment cultures of ammonia-oxidising bacteria were established from soil and the heterotrophs present in the final stages of enrichment were isolated and identified. Spent cell-free medium from these organisms also reduced the lag phase of N. europaea recovering from starvation and reduced the incubation period required for the development of colonies of N. europaea on solid medium.

Listeria monocytogenes é um micro-organismo Gram-positivo que está comumente associado a doenças de origem alimentar. Possui a capacidade de sobreviver a condições adversas e de formar biofilme em diferentes superfícies abióticas, tornando-se um problema constante para a indústria de alimentos, pois pode comprometer a sanitização e aumentar o risco de contaminação pós-processamento. A formação de biofilme pode ser regulada por um mecanismo denominado quorum sensing, no qual ocorre intensa comunicação célula-célula, mediada por moléculas químicas, chamadas de autoindutoras. Pouco se sabe sobre a ocorrência de interação entre bactérias Gram- positivas e negativas na formação de biofilmes, sendo mais frequentes estudos entre bactérias do mesmo grupo. A fim de avaliar a ocorrência de interação entre Escherichia coli e L. monocytogenes (Lm), desenvolveu-se esta pesquisa com os seguintes objetivos: i) verificar a capacidade de Lm sorotipo 1/2a selvagem e sua mutante isogênica (ΔprfA ΔsigB) formar biofilme em presença de Escherichia coli, avaliando-se a importância dos reguladores de virulência, prfA e sigB, no processo; e ii) verificar a produção e interferência de moléculas autoindutoras de E. coli E2348/69 na formação de biofilme por Lm. Os ensaios de formação de biofilme foram realizados utilizando-se lâminas de aço-inoxidável AISI 304 #4 imersas em caldo infusão de cérebro e coração (BHI) e em meio pré-condicionado (MPC) por E. coli, com incubação a 25 ºC. Foram testadas duas concentrações iniciais de Lm (102 e 106 UFC.mL-1) e amostragens em diferentes tempos de incubação. Utilizou-se um método de quantificação indireto com coloração do biofilme por cristal violeta e posterior leitura da absorbância. Observou-se que Lm 1/2a selvagem e sua mutante isogênica (ΔprfA ΔsigB) são capaz de formar biofilme na presença de Escherichia coli e que uma maior quantidade de biofilme foi formada por Lm selvagem quando comparada à sua mutante, em meio não pré-condicionado (controle), indicando que prfA e sigB estão envolvidos no processo de formação de biofilme. Quando em MPC, o biofilme formado pela cepa selvagem foi menor que no meio controle (BHI), indicando que E. coli E2348/69, utilizada no pré-condicionamento do meio, produz moléculas capazes de interferir no processo de formação e na quantidade de biofilme formado por Lm; e para o biofilme formado pela cepa mutante, houve uma maior quantificação em MPC em comparação ao meio controle, o que sugere que os genes deletados possam estar envolvidos no reconhecimento das moléculas autoindutoras. Assim, os dados obtidos permitem concluir que há interação e interferência por parte de E. coli na formação de biofilme por Lm mediante produção de moléculas autoindutoras. / Listeria monocytogenes (Lm) is a Gram-positive microorganism commonly associated with foodborne diseases. Due to its ability to survive under adverse environmental conditions and to form biofilm in different abiotic surfaces, this bacterium is a concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Biofilm formation can be regulated by a quorum sensing mechanism, in which there is intense cell-cell communication mediated by chemical molecules, called autoinducers. Little is known about the occurrence of interaction between Gram-positive and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate the occurrence of interaction between Escherichia coli and Lm, this study was developed including the following objectives: i) to evaluate the ability of Lm 1/2a and its isogenic mutant strain (ΔprfAΔsigB) to form biofilm on the presence of Escherichia coli, assessing the importance of virulence regulators, prfA and sigB, in this process; and ii) to verify the production and interference autoinducers of E. coli E2348/69 on biofilm formation by Lm. Biofilm formation assays were conducted using stainless steel AISI 304 #4 immersed into broth brain heart infusion (BHI) and into preconditioned medium (MPC) by E. coli, following incubation at 25 °C. Lm at two initial concentrations (102 and 106 CFU.mL-1) and under different incubation time was tested. An indirect method for quantification of cells was applied, using crystal violet to color the biofilm, followed by optical density measurement. It was observed that Lm 1/2a and its isogenic mutant (ΔprfA ΔsigB) are able to form biofilm in the presence of Escherichia coli and a larger amount of biofilm was formed by wild strain Lm compared to its mutant, in a non-preconditioned medium (control), indicating that prfA and sigB are involved in biofilm formation. For MPC, the biofilm formation by the wild strain was lower than in the control (BHI), indicating that E. coli E2348/69, used in the preconditioned medium, produces molecules that can affect the formation process and the amount of biofilm formed by Lm; and in the biofilm formed by the mutant strain, there was a higher quantification of MPC compared to the control, suggesting that the deleted genes may be involved in recognition the of autoinducers. These results suggest that there is an interaction and interference of E. coli on biofilm formation by Lm due the production of autoinducers.

Listeria monocytogenes é um micro-organismo Gram-positivo que está comumente associado a doenças de origem alimentar. Possui a capacidade de sobreviver a condições adversas e de formar biofilme em diferentes superfícies abióticas, tornando-se um problema constante para a indústria de alimentos, pois pode comprometer a sanitização e aumentar o risco de contaminação pós-processamento. A formação de biofilme pode ser regulada por um mecanismo denominado quorum sensing, no qual ocorre intensa comunicação célula-célula, mediada por moléculas químicas, chamadas de autoindutoras. Pouco se sabe sobre a ocorrência de interação entre bactérias Gram- positivas e negativas na formação de biofilmes, sendo mais frequentes estudos entre bactérias do mesmo grupo. A fim de avaliar a ocorrência de interação entre Escherichia coli e L. monocytogenes (Lm), desenvolveu-se esta pesquisa com os seguintes objetivos: i) verificar a capacidade de Lm sorotipo 1/2a selvagem e sua mutante isogênica (ΔprfA ΔsigB) formar biofilme em presença de Escherichia coli, avaliando-se a importância dos reguladores de virulência, prfA e sigB, no processo; e ii) verificar a produção e interferência de moléculas autoindutoras de E. coli E2348/69 na formação de biofilme por Lm. Os ensaios de formação de biofilme foram realizados utilizando-se lâminas de aço-inoxidável AISI 304 #4 imersas em caldo infusão de cérebro e coração (BHI) e em meio pré-condicionado (MPC) por E. coli, com incubação a 25 ºC. Foram testadas duas concentrações iniciais de Lm (102 e 106 UFC.mL-1) e amostragens em diferentes tempos de incubação. Utilizou-se um método de quantificação indireto com coloração do biofilme por cristal violeta e posterior leitura da absorbância. Observou-se que Lm 1/2a selvagem e sua mutante isogênica (ΔprfA ΔsigB) são capaz de formar biofilme na presença de Escherichia coli e que uma maior quantidade de biofilme foi formada por Lm selvagem quando comparada à sua mutante, em meio não pré-condicionado (controle), indicando que prfA e sigB estão envolvidos no processo de formação de biofilme. Quando em MPC, o biofilme formado pela cepa selvagem foi menor que no meio controle (BHI), indicando que E. coli E2348/69, utilizada no pré-condicionamento do meio, produz moléculas capazes de interferir no processo de formação e na quantidade de biofilme formado por Lm; e para o biofilme formado pela cepa mutante, houve uma maior quantificação em MPC em comparação ao meio controle, o que sugere que os genes deletados possam estar envolvidos no reconhecimento das moléculas autoindutoras. Assim, os dados obtidos permitem concluir que há interação e interferência por parte de E. coli na formação de biofilme por Lm mediante produção de moléculas autoindutoras. / Listeria monocytogenes (Lm) is a Gram-positive microorganism commonly associated with foodborne diseases. Due to its ability to survive under adverse environmental conditions and to form biofilm in different abiotic surfaces, this bacterium is a concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Biofilm formation can be regulated by a quorum sensing mechanism, in which there is intense cell-cell communication mediated by chemical molecules, called autoinducers. Little is known about the occurrence of interaction between Gram-positive and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate the occurrence of interaction between Escherichia coli and Lm, this study was developed including the following objectives: i) to evaluate the ability of Lm 1/2a and its isogenic mutant strain (ΔprfAΔsigB) to form biofilm on the presence of Escherichia coli, assessing the importance of virulence regulators, prfA and sigB, in this process; and ii) to verify the production and interference autoinducers of E. coli E2348/69 on biofilm formation by Lm. Biofilm formation assays were conducted using stainless steel AISI 304 #4 immersed into broth brain heart infusion (BHI) and into preconditioned medium (MPC) by E. coli, following incubation at 25 °C. Lm at two initial concentrations (102 and 106 CFU.mL-1) and under different incubation time was tested. An indirect method for quantification of cells was applied, using crystal violet to color the biofilm, followed by optical density measurement. It was observed that Lm 1/2a and its isogenic mutant (ΔprfA ΔsigB) are able to form biofilm in the presence of Escherichia coli and a larger amount of biofilm was formed by wild strain Lm compared to its mutant, in a non-preconditioned medium (control), indicating that prfA and sigB are involved in biofilm formation. For MPC, the biofilm formation by the wild strain was lower than in the control (BHI), indicating that E. coli E2348/69, used in the preconditioned medium, produces molecules that can affect the formation process and the amount of biofilm formed by Lm; and in the biofilm formed by the mutant strain, there was a higher quantification of MPC compared to the control, suggesting that the deleted genes may be involved in recognition the of autoinducers. These results suggest that there is an interaction and interference of E. coli on biofilm formation by Lm due the production of autoinducers.

Made available in DSpace on 2014-08-20T13:32:45Z (GMT). No. of bitstreams: 1 tese_rita_de_cassia_dos_santos_da_conceicao.pdf: 1008237 bytes, checksum: 314712b961ce0f1c183271a96a57f4ea (MD5) Previous issue date: 2012-12-21 / Salmonella spp. is the most commonly bacterium transmitted through contamined food. The foods of animal are the most responsible for the worldwide distribution of this bacterium. Salmonella spp. is able to induce a variety of diseases, ranging from an enteric fever, septicemia, gastroenteritis and infections focused. This is due to the bacteria produce different pathogenicity factors and these factors, the flagellum was the target of our study and it is one of the pathogenicity factors induced by Quorum Sensing. Quorum Sensing is a bacterial signaling system that operates through the so-called autoinducer (AI). Gram-negative bacteria produce three auto-inducers. Studies find that catecholamines are able to use the same pathway the autoinducer 3 (AI-3) to active certain genes. Epinephrine and norepinephrine are catecholamines that are present in the mammalian gastrointestinal tract can modulate bacterial gene expression. This work had as objectives to evaluate the Quorum Sensing signaling system on motility, on the growth and flagellar assembly gene expression of Salmonella enterica serovar Typhimurium (ST) and do a review about pathogenicity factors of Salmonella spp. induced by Quorum Sensing. ST was exposed to different concentrations of epinephrine and conditioned medium and its association. The combination of 500 μM epinephrine with 50 % conditioned medium increased ST bacterial motility, growth and increased the expression of the fliC, motA, motB and fliA genes. These results suggest that epinephrine in association with conditioned medium increases ST growth and motility, by modulating the expression of genes involved in flagellum assembly. These observations provide valuable insight into the association between catecholamine and autoinducer and their role in the outcome of Salmonella spp. infection. / Salmonella spp. é um dos principais patógenos transmitidos por alimentos. Os produtos de origem animal são os maiores responsáveis pela distribuição mundial desta bactéria. Salmonella spp. é capaz de induzir uma série de doenças, que vão desde uma febre entérica, septicemia, gastroenterite e infecções focalizadas. Isto é decorrente da bactéria produzir diferentes fatores de patogenicidade e dentre estes fatores, o flagelo foi o alvo no nosso estudo e é um dos fatores de patogenicidade induzidos por Quorum Sensing. Este é um de sistema de sinalização entre as bactérias, através de substâncias denominadas de auto-indutores (AI). As bactérias Gram-negativas produzem três auto-indutores. Estudos verificaram que catecolaminas são capazes de usar a mesma via de sinalização do auto-indutor 3 (AI-3) para ativar determinados genes. Adrenalina e noradrenalina são catecolaminas presentes no trato gastrointestinal de humanos e animais que são capazes de modular a expressão de gênica de bactérias. O presente trabalho teve por objetivos avaliar o sistema de sinalização Quorum Sensing (AI-3) na motilidade, no crescimento celular e na expressão de genes envolvidos na montagem do flagelo de Salmonella enterica sorovar Typhimurium (ST) e fazer uma revisão bibliográfica de fatores de patogenicidade de Salmonella spp. induzidos por Quorum Sensing. ST foi exposta a diferentes concentrações de adrenalina e esta associada ao meio condicionado. A combinação de 500 μM de adrenalina + 50% de meio condicionado aumentou a motilidade de ST, o crescimento celular e induziu a expressão dos genes motA, motB, fliA e fliC. Estes resultados sugerem que a maior motilidade de ST foi decorrente de uma maior expressão de genes envolvidos com a montagem do flagelo. Estas observações fornecem dados valiosos sobre a associação da adrenalina com os auto-indutores e seu papel na infecção causada por Salmonella spp..

Les bactéries sont capables de communiquer entre elles afin de coordonner l’expression de certains gènes en fonction de leur densité de population. Ce système de communication utilise de petites molécules appelées autoinducteurs (AIs) comme messagers chimiques et est connu sous le nom de Quorum Sensing (QS). Chez les bactéries pathogènes, les gènes régulés sont, en particulier, ceux codant pour la production des facteurs de virulence et la structuration des biofilms. Ainsi des inhibiteurs du QS chez ces bactéries pourraient être de nouveaux agents anti-bactériens alternatifs aux antibiotiques actuels. Chez les bactéries à Gram-négatif, les AIs sont très majoritairement des acyl-homosérine lactones (AHLs). Utilisant deux approches, la conception rationnelle et le criblage virtuel, nous avons découvert cinq nouvelles familles d’antagonistes des AHLs, ainsi qu’un certain nombre d’agonistes. Nous avons également préparé des analogues d’antagonistes naturels de la famille des bromo-furanones, afin d’établir une étude structure-activité de ce type de composés. / The bacteria are able to communicate with each other for coordinating gene expression depending on their population density. This communication system use small molecules called autoinducers (AIs) as chemical messengers and is referred to as quorum sensing (QS). In pathogenic bacteria, the regulated genes are, in particular, those coding for the production of virulence factors and biofilms formation. Thus, inhibitors of bacterial QS could be used as new anti-bacterial agents providing an alternative to current antibiotics. In Gram-negative bacteria, the main AIs are acyl-homoserine lactones (AHLs). Using two approaches, rational design and virtual screening, we have discovered five new families of AHLs antagonists, and some agonists. We have also prepared analogues of natural bromo-furanones antagonists, in order to establish a structure-activity study of this type of compounds.

The individual behaviour of prokaryotic organisms such as bacteria often gives rise to complexity that is commonly associated with multicellular behaviour. The transition from unicellular to multicellular behaviour occurs in response to chemical signals, called autoinducers, which bacteria generate and receive internally within a given population. These autoinducers control the gene expression necessary for the emergence of group-behaviour-phenotype. This phenomenon is called quorum sensing (QS). An example of the quorum sensing control of gene regulation has been the luminescence (lux) operon in Vibrio fischeri. The luxI and ainS quorum signalling systems work in conjunction to regulate luminescence in V. fischeri. LuxI and AinS are acyl-synthases that catalyse the production of the autoinducers C6-HSL and C8-HSL respectively. These autoinducers bind to LuxR, a transcriptional activator of the lux operon, which activates expression of the lux genes causing an increase in luminescence. It was shown that quorum signalling also affects motility and biofilm formation in bacteria. However, the evidence with respect to these phenotypes is conflicting and inconclusive, the reason being the state of quorum is ambiguously defined. It is not properly known whether the observed collective behaviour is purely a result of physical crowding of bacteria, or that both chemical signalling and crowding contribute to this phenomenon. This work attempts to address these issues by studying luminescence, motility, and biofilm, a diverse set of behaviours, yet closely linked to each other in V. fischeri-squid symbiosis. We studied the luminescence response of V. fischeri to both endogenous and externally added signals at per-cell and population level. Experiments with ES114, a wild-type strain of V. fischeri, and ainS mutant showed that (i) luminescence per cell does not mutually correlate with the cell-density, indicating that bacteria do not show greater response to the signal at higher densities; (ii) the activity of the lux signalling circuit shows a strong dependence on the growth stage, (iii) the cells do not show enhanced growth, i.e., they do not derive fitness benefits at higher densities in response to the signal. We anticipated that the culture with a higher cell-density should exhibit greater per-cell-luminescence. However, we found that the luminescence curve of the culture with lower density crosses that of the cultures with higher densities during the exponential phase. Kinetic modelling of the luxI mRNA expression showed that the expression profile qualitatively agrees with the luminescence trend observed in the cultures, supporting the observation that growth-phase plays a major role in regulating the luminescence gene expression. We also studied the effect of autoinducers on motility of V. fischeri. V. fischeri uses flagella to move into the inner crypts of the light organ of the squid. The bacterium secretes autoinducers, encounters secretions of the light organ, and slows down during the final stage of colonization process. Studies have shown that flagellar elaboration is repressed as a consequence of ainS signalling. However, those studies were soft-agar migration assays and carried out with the mutant strain of ainS. We measured real-time planktonic motility of ES114 and the signalling mutant strains of V. fischeri in response to autoinducers added exogenously at different concentrations. We found that the autoinducers do not affect the motility of the strains. We also showed that reduction in motility is purely a consequence of physical crowding of bacteria, and chemical signalling may not be involved in the process. It was shown that reduction in motility leads to biofilm formation. Motile bacteria must lose flagella in order to form biofilm, and signalling controls biofilm formation in many species. Our study on motility showed that reduction in motility occurs because of physical crowding in V. fischeri. Hence, we explored the possibility that physical crowding might lead to formation of biofilm rather than signalling in this species. We quantified exopolysaccharide production by crystal violet assay, which revealed that planktonic cells produce exopolysaccharides, in addition to biofilm cells. The study revealed that V. fischeri cells always produce exopolysaccharides irrespective of their physiological state. We examined the effect of signalling on biofilm in ES114 and the mutant strains using gene-expression analysis. We quantified the expression of various genes involved in biofilm formation and found that both ES114 and the mutants expressed rscS and sypP indicating that exopolysaccharide production is not under the control of autoinducers. Therefore, we hypothesized that biofilm formation in V. fischeri may be a result of physical agglomeration of cells. Our observations indicate that the state of quorum is inadequately defined and there is no direct measure of the underlying process. Multicellular behaviour in V. fischeri is regulated by a complex interplay of cell-density, signalling, and other factors such as the growth phase of the culture, indicating that the state of quorum employs different mechanisms to regulate various phenotypes. Our study reveals that QS is an intricate process, and the accepted mechanisms for QS are incomplete at best.