Effects of temperature up-shift and UV-A radiation on fatty acids content and expression of desaturase genes in cyanobacteria Microcystis aeruginosa: stress tolerance and acclimation responses.

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.

Role of Pel and Psl polysaccharides in the response of Pseudomonas aeruginosa to environmental challenges: oxidative stress agents (UVA, H2O2, sodium hypochlorite) and its competitor Staphylococcus aureus.

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.

UVA as environmental signal for alginate production in Pseudomonas aeruginosa: role of this polysaccharide in the protection of planktonic cells and biofilms against lethal UVA doses.

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.

Homeophasic Adaptation in Response to UVA Radiation in Pseudomonas aeruginosa: Changes of Membrane Fatty Acid Composition and Induction of desA and desB Expression.

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.

Determination of Antibacterial Activity of Film Coatings Against Four Clinically Relevant Bacterial Strains.

Antibacterial coatings have currently gained great importance in biomedical technology investigations. Because of the spatial arrangement of the film coatings, evaluation of antibacterial activity presents a new challenge regarding traditional bacterial counting methods. In this protocol, four clinically relevant pathogens, Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were incubated on titania mesostructured thin film coatings for 24 h. Then, cell viability was studied considering three methods: counting of the number of colony forming units (CFU), live/dead staining, and quantification of extracellular DNA in suspension. Firstly, bacterial count was determined by the standard plate-count technique. Secondly, bacteria membrane integrity was evaluated by utilization of two fluorescent dyes, which allow distinction between live (membrane intact) and dead (membrane disrupted) bacteria. Lastly, extracellular DNA was quantified by spectrophotometry. In this manner, the three aforementioned techniques enabled the study of bacterial viability by qualitative and quantitative analyses.

Effect of temperature on microcystin-LR removal and lysis activity on Microcystis aeruginosa (cyanobacteria) by an indigenous bacterium belonging to the genus Achromobacter.

Microcystis is a frequent cyanobacterium bloom-forming with cosmopolitan distribution which can produce a hepatotoxin group called microcystins (MCs). These MCs are resistant to the traditional processes employed in the water treatment plants and they are often detected after conventional treatments. Because of this, the bio-removal studies have obtained a great interest in the last decades. In this work, a bacterial strain namely LG1 with the ability to remove microcystin-LR (MC-LR) under laboratory conditions was isolated from Rio de la Plata River and it was identified as Achromobacter spp. This ubiquitous bacterium was able to remove 79.5% MC-LR in 7 days with average removal time of 3.33 ± 0.08, 3.06 ± 0.05, and 2.77 ± 0.05 days at 28, 32, and 36 ± 1 °C, being higher at high temperature (36 °C) with an activation energy = 16.79 ± 1.99 kJ mol-1. LG1 grew better at higher temperature (from 28 to 36 ± 1 °C) increasing the specific growth rate (µ) and reducing 2-fold the lag phase duration (LPD) without significant differences (p > 0.05) between maximum population density (MPD). In addition, LG1 showed a lysis activity on two M. aeruginosa native strains in 7 days measured as chlorophyll a (Chl-a) concentration. The lysis activity increased around 2-fold when increasing the temperature from 28 to 36 ± 1 °C. This is the first report of an indigenous bacterium belonging to the genus Achromobacter spp. isolated from the Rio de la Plata River with the capacity to remove MC-LR and lysis activity on M. aeruginosa.

Role of quorum sensing in UVA-induced biofilm formation in Pseudomonas aeruginosa.

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.

Antibiofilm effect of mesoporous titania coatings on Pseudomonas aeruginosa biofilms.

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.

Evaluation of Viable Cells in Pseudomonas aeruginosa Biofilmsby Colony Count and Live/Dead Staining.

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.

Response to lethal UVA radiation in the Antarctic bacterium Pseudomonas extremaustralis: polyhydroxybutyrate and cold adaptation as protective factors.

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.

Transforming an inert nanopolymer into broad-spectrum bactericidal by superstructure tuning.

Poloxamer block copolymers (also known as Pluronic®) are particularly useful for drug delivery and self-assembly techniques. These nanopolymers are generally considered to be biologically inert and they were used to generate only bacteria repellent surfaces but keeps bacteria alive and as a latent threat. However, the inherent capabilities of these nanopolymers to kill bacteria have been largely overlooked. Here, we report that Pluronic shaped as superstructures (self-organized array of micelles) in fact possess a broad-spectrum bactericidal activity (capability of killing bacteria) similar to that shown for some antibiotics. This further represents the first report that shows that appropriate control of superstructured mesophase architecture is a key parameter for bactericidal efficacy. Based on this finding, we have developed a highly bactericidal coating (>99.9% kill) against all tested Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Salmonella typhimurium LT2, Escherichia coli K12 and Pseudomonas aeruginosa PAO1) bacteria which moreover allows the adhesion and proliferation of mammalian cells. The inexpensiveness and ease of production make these versatile nanopolymer structures a powerful tool for the development of a new generation of highly effective antimicrobial coatings.

Exposure to low doses of UVA increases biofilm formation in Pseudomonas aeruginosa.

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.

New envelope stress factors involved in σE activation and conditional lethality of rpoE mutations in Salmonella enterica.

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.

Detection of Catalase Activity by Polyacrylamide Gel Electrophoresis (PAGE)in Cell Extracts from Pseudomonas aeruginosa.

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.

Antibiofilm effect of supramolecularly templated mesoporous silica coatings.

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.

Exposure to low UVA doses increases KatA and KatB catalase activities, and confers cross-protection against subsequent oxidative injuries in Pseudomonas aeruginosa.

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.

Role of the Pseudomonas quinolone signal (PQS) in sensitising Pseudomonas aeruginosa to UVA radiation.

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.

Protective role of extracellular catalase (KatA) against UVA radiation in Pseudomonas aeruginosa biofilms.

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.

Protective effect of low UVA irradiation against the action of lethal UVA on Pseudomonas aeruginosa: role of the relA gene.

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.

The relationship between carotenoids and sunlight response in members of the family Micrococcaceae.

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.