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Temperature up-shift and UV-A radiation effects on growth, lipid damage, fatty acid (FA) composition and expression of desaturase genes desA and desB were investigated in the cyanobacteria Microcystis aeruginosa. Although UV-A damaging effect has been well documented, reports on the interactive effects of UV radiation exposure and warming on cyanobacteria are scarce. Temperature and UV-A doses were selected based on the physiological responses previously obtained by studies with the same M. aeruginosa strain used in this study. Cells pre-grown at 26 °C were incubated at the same temperature or 29 °C and exposed to UV-A + PAR and only PAR for 9 days. Growth rate was significantly affected by UV-A radiation independently of the temperature throughout the experiment. High temperature produced lipid damage significantly higher throughout the experiment, decreasing at day 9 as compared to 26 °C. In addition, the cells grown at 29 °C under UV-A displayed a decrease in polyunsaturated FA (PUFA) levels, with ω3 PUFA being mostly affected at the end of exposure. Previously, we reported that UV-A-induced lipid damage affects differentially ω3 and ω6 PUFAs. We report that UV-A radiation leads to an upregulation of desA, possibly due to lipid damage. In addition, the temperature up-shift upregulates desA and desB regardless of the radiation. The lack of lipid damage for UV-A on ω3 could explain the lack of transcription induction of desB. The significant ω6 decrease at 26 °C in cells exposed to UV-A could be due to the lack of upregulation of desA.
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Ácido Graso Desaturasas , Ácidos Grasos , Microcystis , Temperatura , Rayos Ultravioleta , Microcystis/efectos de la radiación , Ácidos Grasos/metabolismo , Ácido Graso Desaturasas/metabolismo , Ácido Graso Desaturasas/genética , Aclimatación , Estrés FisiológicoRESUMEN
Extracellular vesicles (EVs) are biomolecule carriers for intercellular communication in health and disease. Nef is a HIV virulence factor that is released from cells within EVs and is present in plasma EVs of HIV-1 infected individuals. We performed a quantitative proteomic analysis to fully characterize the Nef-induced changes in protein composition of T cell-derived EVs and identify novel host targets of HIV. Several proteins with well-described roles in infection or not previously associated with HIV pathogenesis were specifically modulated by Nef in EVs. Among the downregulated proteins are the interferon-induced transmembrane 1, 2, and 3 (IFITM1-3) proteins, broad-spectrum antiviral factors known to be cell-to-cell transferable by EVs. We demonstrate that Nef depletes IFITM1-3 from EVs by excluding these proteins from the plasma membrane and lipid rafts, which are sites of EVs biogenesis in T cells. Our data establish Nef as a modulator of EVs' global protein content and as an HIV factor that antagonizes IFITMs.
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Vesículas Extracelulares , Infecciones por VIH , VIH-1 , Humanos , Linfocitos T , Proteoma/metabolismo , Proteómica , Vesículas Extracelulares/metabolismo , Interferones/metabolismo , Infecciones por VIH/metabolismo , Antivirales/metabolismoRESUMEN
Pseudomonas aeruginosa is a versatile bacterium capable of adapting to a wide range of stress factors, including solar UVA radiation (400-315 nm). High UVA doses produce lethal effects due to the action of reactive oxygen species. Sublethal UVA doses also induces oxidative damage, but, in addition, it triggers a variety of adaptive responses, including the overexpression of pelA and pslA genes in P. aeruginosa. These genes encode the synthesis of Pel and Psl, which are essential polysaccharides in biofilm formation. The present study analysed the role of Pel and Psl in the adaptive responses generated by exposure to low UVA doses, and their importance in the response to lethal doses of UVA, hydrogen peroxide (H2O2), and sodium hypochlorite, in both planktonic cells and submerged and air-liquid interface (ALI) biofilms. It also studied the roles of Pel and Psl in P. aeruginosa-Staphylococcus aureus interaction. The results demonstrate that the capacity of sublethal UVA exposure to increase cell hydrophobicity and cell attachment and generate cross-protection phenomena in P. aeruginosa depends on the presence of Pel and Psl. The study also shows that Pel and Psl have a key role in the tolerance to lethal doses of UVA radiation, sodium hypochlorite and H2O2, in both biofilms and planktonic cells. Finally, co-culture assays showed total inhibition of S. aureus growth in presence of P. aeruginosa. This phenomenon depends, at least in part, on the simultaneous presence of Pel and Psl in planktonic cells and biofilms, suggesting a relevant role of these polysaccharides in the interaction between these species.
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Peróxido de Hidrógeno , Hipoclorito de Sodio , Peróxido de Hidrógeno/farmacología , Hipoclorito de Sodio/farmacología , Pseudomonas aeruginosa/fisiología , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Polisacáridos Bacterianos/metabolismo , Biopelículas , Estrés OxidativoRESUMEN
Pseudomonas aeruginosa is an extremely versatile microorganism that survives in a wide variety of niches. It is capable to respond rapidly to changes in the environment by producing secondary metabolites and virulence factors, including alginate. Alginate is an extracellular polysaccharide that protects the bacteria from antibiotics and oxidative agents, and enhances cell adhesion to solid surfaces in the process of biofilm formation. In the present study, we analyzed the role of alginate in the response of P. aeruginosa to lethal doses of ultraviolet-A (UVA) radiation, the major fraction of solar UV radiation reaching the Earth's surface. We also studied the role of alginate in the context of the adaptive responses generated when P. aeruginosa is exposed to sublethal doses of UVA radiation. The survival studies demonstrated that alginate has a key role in the resistance of P. aeruginosa to the oxidative stress generated by lethal UVA doses, both in planktonic cells and in static biofilms. In addition, the presence of alginate proved to be essential in the occurrence of adaptive responses such as induction of biofilm formation and cross-protection against hydrogen peroxide and sodium hypochlorite, both generated by exposure to low UVA doses. Finally, we demonstrated that the increase of biofilm formation is accompanied by an increase in alginate concentration in the biofilm matrix, possibly through the ppGpp-dependent induction of genes related to alginate regulation (algR and algU) and biosynthesis (algD operon). Given the importance of alginate in biofilm formation and its protective roles, better understanding of the mechanisms associated to its functions and synthesis is relevant, given the normal exposure of P. aeruginosa to UVA radiation and other types of oxidative stresses.
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Plancton , Pseudomonas aeruginosa , Alginatos/metabolismo , Alginatos/farmacología , Biopelículas , Peróxido de Hidrógeno/farmacología , Pseudomonas aeruginosa/fisiologíaRESUMEN
In bacteria, exposure to changes in environmental conditions can alter membrane fluidity, thereby affecting its essential functions in cell physiology. To adapt to these changes, bacteria maintain appropriate fluidity by varying the composition of the fatty acids of membrane phospholipids, a phenomenon known as homeophasic adaptation. In Pseudomonas aeruginosa, this response is achieved mainly by two mechanisms of fatty acid desaturation: the FabA-FabB and DesA-DesB systems. This study analyzed the effect of ultraviolet-A (UVA) radiation-the major fraction of solar UV radiation reaching the Earth's surface-on the homeophasic process. The prototypical strain PAO1 was grown under sublethal UVA doses or in the dark, and the profiles of membrane fatty acids were compared at early logarithmic, logarithmic and stationary growth phases. In the logarithmic growth phase, it was observed that growth under sublethal UVA doses induced the expression of the desaturase-encoding genes desA and desB and increased the proportion of unsaturated fatty acids; in addition, membrane fluidity could also increase, as suggested by the indices used as indicators of this parameter. The opposite effect was observed in the stationary growth phase. These results demonstrate the relevant role of UVA on the homeophasic response at transcriptional level.
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Ácidos Grasos , Pseudomonas aeruginosa , Adaptación Fisiológica/genética , Fosfolípidos , Rayos UltravioletaRESUMEN
Pseudomonas aeruginosa, a versatile bacterium present in terrestrial and aquatic environments and a relevant opportunistic human pathogen, is largely known for the production of robust biofilms. The unique properties of these structures complicate biofilm eradication, because they make the biofilms very resistant to diverse antibacterial agents. Biofilm development and establishment is a complex process regulated by multiple regulatory genetic systems, among them is quorum sensing (QS), a mechanism employed by bacteria to regulate gene transcription in response to population density. In addition, environmental factors such as UVA radiation (400-315 nm) have been linked to biofilm formation. In this work, we further investigate the mechanism underlying the induction of biofilm formation by UVA, analysing the role of QS in this phenomenon. We demonstrate that UVA induces key genes of the Las and Rhl QS systems at the transcriptional level. We also report that pelA and pslA genes, which are essential for biofilm formation and whose transcription depends in part on QS, are significantly induced under UVA exposure. Finally, the results demonstrate that in a relA strain (impaired for ppGpp production), the UVA treatment does not induce biofilm formation or QS genes, suggesting that the increase of biofilm formation due to exposure to UVA in P. aeruginosa could rely on a ppGpp-dependent QS induction.
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Biopelículas/efectos de la radiación , Pseudomonas aeruginosa/fisiología , Percepción de Quorum/fisiología , 4-Butirolactona/análogos & derivados , 4-Butirolactona/genética , 4-Butirolactona/metabolismo , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Genes Bacterianos/genética , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Mutación , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/metabolismo , Pseudomonas aeruginosa/efectos de la radiación , Percepción de Quorum/genética , Percepción de Quorum/efectos de la radiación , Transcripción Genética/efectos de la radiación , Rayos UltravioletaRESUMEN
Activation of photocatalytic titania by ultraviolet-A (UVA) radiation has been proposed as a good approach for combating bacteria. Titania powder, in solution or immobilized on a surface, has excellent UVA-assisted killing properties on several microorganisms. However, these properties could not be demonstrated in biofilms of Pseudomonas aeruginosa, a resistant opportunistic human pathogen that can cause severe complications in patients who are immunocompromised or have burn wounds or cystic fibrosis. P. aeruginosa biofilms have detrimental effects on health and industry, causing serious economic damage. In this study, the effect of titania photocatalysis for controlling P. aeruginosa biofilms was investigated by employing different coatings obtained through sol-gel and evaporation-induced self-assembly. Biofilms were grown on non-mesoporous and mesoporous titania surfaces with different pore sizes, which were achieved based on the use of surfactants Brij-58 and Pluronics-F127. In addition, two structural forms of titania were assayed: amorphous and anatase. As well as inhibiting biofilm formation, these coatings significantly enhanced the bactericidal effect of UVA on P. aeruginosa biofilms. The most efficient surface with regard to total antibacterial effect was the mesoporous Brij-58-templated anatase film, which, compared to control biofilms, decreased the number of viable bacteria by about 5 orders, demonstrating the efficacy of this methodology as a disinfection system.
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Biopelículas/efectos de los fármacos , Nanopartículas del Metal/toxicidad , Pseudomonas aeruginosa/fisiología , Titanio/química , Rayos Ultravioleta , Biopelículas/efectos de la radiación , Catálisis , Nanopartículas del Metal/química , Porosidad , Tensoactivos/químicaRESUMEN
The HIV-1 accessory protein Nef downregulates the cell surface expression of major histocompatibility complex class I (MHC-I) molecules to facilitate virus spreading. The Nef-induced downregulation of MHC-I molecules such as HLA-A requires the clathrin adaptor protein 1 (AP-1) complex. The cooperative interaction of Nef, AP-1, and the cytosolic tail (CT) of HLA-A leads to a redirection of HLA-A targeting from the trans-Golgi network (TGN) to lysosomes for degradation. Although the γ-adaptin subunit of AP-1 has two distinct isoforms (γ1 and γ2), which may form two AP-1 complex variants, so far, only the importance of AP-1γ1 in MHC-I downregulation by Nef has been investigated. Here, we report that the AP-1γ2 isoform also participates in this process. We found that AP-1γ2 forms a complex with Nef and HLA-A2_CT and that this interaction depends on the Y320 residue in HLA-A2_CT and Nef expression. Moreover, Nef targets AP-1γ1 and AP-1γ2 to different compartments in T cells, and the depletion of either AP-1 variant impairs the Nef-mediated reduction of total endogenous HLA-A levels and rescues HLA-A levels on the cell surface. Finally, immunofluorescence and immunoelectron microscopy analyses reveal that the depletion of γ2 in T cells compromises both the Nef-mediated retention of HLA-A molecules in the TGN and targeting to multivesicular bodies/late endosomes. Altogether, these results show that in addition to AP-1γ1, Nef also requires the AP-1γ2 variant for efficient MHC-I downregulation.IMPORTANCE HIV-1 Nef mediates evasion of the host immune system by inhibiting MHC-I surface presentation of viral antigens. To achieve this goal, Nef modifies the intracellular trafficking of MHC-I molecules in several ways. Despite being the subject of intense study, the molecular details underlying these modifications are not yet fully understood. Adaptor protein 1 (AP-1) plays an essential role in the Nef-mediated downregulation of MHC-I molecules such as HLA-A in different cell types. However, AP-1 has two functionally distinct variants composed of either γ1 or γ2 subunit isoforms. Because previous studies on the role of AP-1 in MHC-I downregulation by Nef focused on AP-1γ1, an important open question is the participation of AP-1γ2 in this process. Here, we show that AP-1γ2 is also essential for Nef-mediated depletion of surface HLA-A molecules in T cells. Our results indicate that Nef hijacks AP-1γ2 to modify HLA-A intracellular transport, redirecting these proteins to lysosomes for degradation.
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Regulación hacia Abajo , Regulación de la Expresión Génica , Antígeno HLA-A2/metabolismo , Factor de Transcripción AP-1/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Endosomas/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisosomas/metabolismo , Microscopía Inmunoelectrónica , Transporte de Proteínas , Linfocitos T/inmunología , Linfocitos T/virología , Red trans-Golgi/metabolismoRESUMEN
Pseudomonas aeruginosa is a human pathogen capable to form robust biofilms. P. aeruginosa biofilms represent a serious problem because of the adverse effects on human health and industry, from sanitary and economic points of view. Typical strategies to break down biofilms have been long used, such as the use of disinfectants or antibiotics, but also, according to their high resistance to standard antimicrobial approaches, alternative strategies employing photocatalysis or control of biofilm formation by modifying surfaces, have been proposed. Colony forming units (cfu) counting and live/dead staining, two classic techniques used for biofilm quantification, are detailed in this work. Both methods assess cell viability, a key factor to analyze the microbial susceptibility to given treatment, then, they represent a good approach for evaluation of an antibiofilm strategy.
RESUMEN
Pseudomonas extremaustralis is an Antarctic bacterium with high stress resistance, able to grow under cold conditions. It is capable to produce polyhydroxyalkanoates (PHAs) mainly as polyhydroxybutyrate (PHB) and, to a lesser extent, medium-chain length polyhydroxyalkanoates (mclPHAs). In this work, we analyzed the role of PHAs and cold adaptation in the survival of P. extremaustralis after lethal UVA exposure. P. extremaustralis presented higher radiation resistance under polymer accumulation conditions. This result was also observed in the derivative mutant strain PHA-, deficient for mclPHAs production. On the contrary, the PHB- derivative mutant, deficient for PHB production, showed high sensitivity to UVA exposure. Complementation of the PHB- strain restored the wild-type resistance level, indicating that the UVA-sensitive phenotype is due to the lack of PHB. All strains exhibited high sensitivity to radiation when cultured under PHAs non-accumulation conditions. A slight decrease in PHB content was observed after UVA exposure in association with increased survival. The scattering of UVA radiation by intracellular PHAs granules could also result in bacterial cell protection. In addition, cold conditions improved UVA tolerance, probably depending on PHB mobilization. Results showed that PHB accumulation is crucial in the resistance to UVA in P. extremaustralis. Mechanisms involved probably entail depolymerization and light scattering acting as a screen, both conferring protection against oxidative stress.
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Pseudomonas , Regiones Antárticas , Polihidroxialcanoatos , Factores Protectores , Rayos UltravioletaRESUMEN
Cells from all kingdoms of life can release membrane-enclosed vesicles to the extracellular milieu. These extracellular vesicles (EVs) may function as mediators of intercellular communication, allowing the transfer of biologically active molecules between cells and organisms. It has become clear that HIV particles and certain types of EVs, such as exosomes, share many similarities regarding morphology, composition, and biogenesis. This review presents a summary of the literature describing the intricate relationship between HIV and EVs biogenesis. Also, we discuss the latest progress toward understanding the mechanisms by which EVs influence HIV pathogenesis, as well as, how HIV modulates EVs composition in infected cells to facilitate viral spread.
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The establishment of bacterial biofilms on abiotic surfaces is a complex process regulated by multiple genetic regulators and environmental factors which are able to modulate the passage of planktonic cells to a sessile state. Solar ultraviolet-A radiation (UVA, 315-400) is one of the main environmental stress factors that bacteria must face at the Earth´s surface. The deleterious effects of UVA are mainly due to oxidative damage. This paper reports that exposure to low UVA doses promotes biofilm formation in three prototypical strains of Pseudomonas aeruginosa, a relevant opportunistic human pathogen. It demonstrates that exposure of planktonic cells to sublethal doses of UVA can increase cell surface hydrophobicity and swimming motility, two parameters known to favor cell adhesion. These results suggest that UVA radiation acts, at least in part, by promoting the first stages of biofilm development.
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Biopelículas/efectos de la radiación , Pseudomonas aeruginosa/efectos de los fármacos , Rayos Ultravioleta , Biopelículas/crecimiento & desarrollo , Pseudomonas aeruginosa/fisiologíaRESUMEN
Salmonella enterica serovar Typhimurium (S. typhimurium) can cause food- and water-borne illness with diverse clinical manifestations. One key factor for S. typhimurium pathogenesis is the alternative sigma factor σE, which is encoded by the rpoE gene and controls the transcription of genes required for outer-membrane integrity in response to alterations in the bacterial envelope. The canonical pathway for σE activation involves proteolysis of the antisigma factor RseA, which is triggered by unfolded outer-membrane porins (OMPs) and lipopolysaccharides (LPS) that have accumulated in the periplasm. This study reports new stress factors that are able to activate σE expression. We demonstrate that UVA radiation induces σE activity in a pathway that is dependent on the stringent response regulator ppGpp. Survival assays revealed that rpoE has a role in the defence against lethal UVA doses that is mediated by functions that are dependent on and independent of the alternative sigma factor RpoS. We also report that the envelope stress generated by phage infection requires a functional rpoE gene for optimal bacterial tolerance and that it is able to induce σE activity in an RseA-dependent fashion. σE activity is also induced by hypo-osmotic shock in the absence of osmoregulated periplasmic glucans (OPGs). It is known that the rpoE gene is not essential in S. typhimurium. However, we report here two cases of the conditional lethality of rpoE mutations in this micro-organism. We demonstrate that rpoE mutations are not tolerated in the absence of OPGs (at low to moderate osmolarity) or LPS O-antigen. The latter case resembles that of the prototypic Escherichia coli strain K12, which neither synthesizes a complete LPS nor tolerates null rpoE mutations.
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Regulación Bacteriana de la Expresión Génica , Salmonella typhimurium/fisiología , Factor sigma/genética , Factor sigma/metabolismo , Estrés Fisiológico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriófago P22/fisiología , Glucanos/metabolismo , Guanosina Tetrafosfato/metabolismo , Viabilidad Microbiana , Mutación , Antígenos O/metabolismo , Presión Osmótica , Salmonella typhimurium/efectos de la radiación , Salmonella typhimurium/virología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Rayos UltravioletaRESUMEN
Bacteria in nature and as pathogens commonly face oxidative stress which causes damage to proteins, lipids and DNA. This damage is produced by the action of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), singlet oxygen, superoxide anion and hydroxyl radical. ROS are generated by antimicrobials, environmental factors (e.g., ultraviolet radiation, osmotic stress), aerobic respiration, and host phagocytes during infective processes. Pseudomonas aeruginosa, a versatile bacterium, is a prevalent opportunistic human pathogen which possesses several defense strategies against ROS. Among them, two catalases (KatA and KatB) have been well characterized by their role on the defense against multiple types of stress. In this protocol, KatA and KatB activities are detected by polyacrylamide gel electrophoresis (PAGE). It is also suggested that the detection of KatB is elusive.
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Bacteria attached to solid surfaces and encased in a self-synthesized matrix, so-called biofilms, are highly difficult to eradicate and present negative impact on industry and human health. The ability of supramolecularly templated mesoporous silica coatings to inhibit biofilm formation in Pseudomonas aeruginosa is shown here. Assays employing submerged and air-liquid interface biofilms demonstrated that mesoporous coatings with tuned pore size significantly reduce the number of attached bacteria and matrix production. Given its versatility, scalability, robustness and low cost, our proposal is attractive for the production of transparent, inert and permanent antibiofilm coatings that could be applied on multiple surfaces.
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Antibacterianos/farmacología , Dióxido de Silicio/farmacología , Bacterias , Biopelículas , Porosidad , Pseudomonas aeruginosaRESUMEN
Solar UVA radiation is one of the main environmental stress factors for Pseudomonas aeruginosa. Exposure to high UVA doses produces lethal effects by the action of the reactive oxygen species (ROS) it generates. P. aeruginosa has several enzymes, including KatA and KatB catalases, which provide detoxification of ROS. We have previously demonstrated that KatA is essential in defending P. aeruginosa against high UVA doses. In order to analyse the mechanisms involved in the adaptation of this micro-organism to UVA, we investigated the effect of exposure to low UVA doses on KatA and KatB activities, and the physiological consequences. Exposure to UVA induced total catalase activity; assays with non-denaturing polyacrylamide gels showed that both KatA and KatB activities were increased by radiation. This regulation occurred at the transcriptional level and depended, at least partly, on the increase in H2O2 levels. We demonstrated that exposure to low UVA produced a protective effect against subsequent lethal doses of UVA, sodium hypochlorite and H2O2. Protection against lethal UVA depends on katA, whilst protection against sodium hypochlorite depends on katB, demonstrating that different mechanisms are involved in the defence against these oxidative agents, although both genes can be involved in the global cellular response. Conversely, protection against lethal doses of H2O2 could depend on induction of both genes and/or (an)other defensive factor(s). A better understanding of the adaptive response of P. aeruginosa to UVA is relevant from an ecological standpoint and for improving disinfection strategies that employ UVA or solar irradiation.
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Adaptación Fisiológica/fisiología , Catalasa/metabolismo , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Estrés Oxidativo/efectos de la radiación , Pseudomonas aeruginosa/efectos de la radiación , Hipoclorito de Sodio/farmacología , Adaptación Fisiológica/genética , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción/efectos de la radiación , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Rayos UltravioletaRESUMEN
One of the main stress factors that bacteria face in the environment is solar ultraviolet-A (UVA) radiation, which leads to lethal effects through oxidative damage. The aim of this work was to investigate the role of 2-heptyl-3-hydroxi-4-quinolone (the Pseudomonas quinolone signal or PQS) in the response of Pseudomonas aeruginosa to UVA radiation. PQS is an intercellular quorum sensing signal associated to membrane vesicles which, among other functions, regulates genes related to iron acquisition, forms stable complexes with iron and participates in oxidative phenomena. UVA exposure of the wild-type PAO1 strain and a pqsA mutant unable to produce PQS revealed a sensitising role for this signal. Research into the mechanism involved in this phenomenon revealed that catalase, an essential factor in the UVA defence, is not related to PQS-mediated UVA sensitivity. Absorption of UVA by PQS produced its own photo-degradation, oxidation of the probe 2',7'- dichlorodihydrofluorescein and generation of singlet oxygen and superoxide anion, suggesting that this signal could be acting as an endogenous photosensitiser. The results presented in this study could explain the high sensitivity to UVA of P. aeruginosa when compared to enteric bacteria.
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Pseudomonas aeruginosa/fisiología , Percepción de Quorum/efectos de la radiación , Rayos Ultravioleta , Catalasa/metabolismo , Hierro/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de la radiación , Fotólisis/efectos de la radiación , Quinolonas/química , Quinolonas/metabolismo , Oxígeno Singlete/química , Oxígeno Singlete/metabolismo , Superóxidos/química , Superóxidos/metabolismoRESUMEN
One of the more stressful factors that Pseudomonas aeruginosa must face in nature is solar UVA radiation. In this study, the protective role of KatA catalase in both planktonic cells and biofilms of P. aeruginosa against UVA radiation was determined by using the wild-type (PAO1) and an isogenic catalase deficient strain (katA). The katA strain was more sensitive than the wild-type, especially in the case of biofilms. Moreover, the wild-type biofilm was more resistant than its planktonic counterpart, but this was not observed in the katA strain. Striking KatA activity was detected in the matrix of katA(+) strains, and to our knowledge, this is the first report of this activity in the matrix of P. aeruginosa biofilms. Provision of bovine catalase or KatA to the matrix of a katA biofilm significantly increased its UVA tolerance, demonstrating that extracellular KatA is essential to optimal defense against UVA in P. aeruginosa biofilms. Efficiency of photocatalytic treatments using TiO2 and UVA was lower in biofilms than in planktonic cells, but KatA and KatB catalases seem not to be responsible for the higher resistance of the sessile cells to this treatment.
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Catalasa/metabolismo , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/efectos de la radiación , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/efectos de la radiación , Catalasa/genética , Bovinos , Espacio Extracelular/enzimología , Prueba de Complementación Genética , Plancton , Pseudomonas aeruginosa/enzimología , Rayos UltravioletaRESUMEN
The exposure of Pseudomonas aeruginosa cells to very low UVA fluences induces a growth delay, a phenomenon proposed in Escherichia coli as an adaptive mechanism related to protection against lethal and mutagenic effects of UVA. This paper reports that the treatment with low UVA irradiation fluences protects P. aeruginosa PAO1 strain from a subsequent lethal exposure. This phenomenon depends on the relA gene, coding for the main (p)ppGpp synthetase, and is unrelated to the induction of quorum sensing or catalase activity, two essential factors involved in the response of P. aeruginosa to UVA. Cross-protection between osmotic stress and UVA is observed when a great protective response to lethal UVA is caused by the induction of resistance to osmotic stress. The increase in resistance to osmotic shock observed in the pre-irradiated PAO1 strain but not in its relA derivative, unable to show photo-protection, leads us to hypothesize that the photo-protection could be attributed to an adaptive response to osmotic stress. It is concluded that the exposure of P. aeruginosa to low UVA doses induces a relA-dependent adaptive response that protects against cell death induced by high doses and causes an increase in the resistance to osmotic stress.
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Ligasas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Catalasa/metabolismo , Relación Dosis-Respuesta en la Radiación , Ligasas/metabolismo , Presión Osmótica/efectos de la radiación , Pseudomonas aeruginosa/citología , Pseudomonas aeruginosa/fisiología , Percepción de Quorum/efectos de la radiación , Estrés Fisiológico/efectos de la radiaciónRESUMEN
The aim of this study was to compare the photoprotective effect of carotenoids in phylogentically related bacteria, which synthesize structurally different pigments. Two organisms were isolated from the same environment. Their 16S rDNA sequences and phenotypic characteristics identified them as members of the family Micrococcaceae. Reverse phase HPLC and absorption spectroscopy revealed that one of them, designated RMB40, synthesized 3 carotenoids with 9 conjugated double bonds, whilst the other, designated RMB42, synthesized a single and more hydrophobic pigment carrying 11 conjugated double bonds. Survival curves were obtained during sunlight exposure for both organisms and for carotenoid deficient mutants derived from them. Increased sunlight sensitivity was found in the carotenoidless mutant derived from RMB42. In contrast, pigment depletion had no appreciable effect on the sunlight response of RMB40. It is concluded that the structure of bacterial carotenoid probably exert an important influence on the effectiveness of these compounds to provide photoprotection in vivo.