RESUMO
The two-component system GacS/A and the posttranscriptional control system Rsm constitute a genetic regulation pathway in Gammaproteobacteria; in some species of Pseudomonas, this pathway is part of a multikinase network (MKN) that regulates the activity of the Rsm system. In this network, the activity of GacS is controlled by other kinases. One of the most studied MKNs is the MKN-GacS of Pseudomonas aeruginosa, where GacS is controlled by the kinases RetS and LadS; RetS decreases the kinase activity of GacS, whereas LadS stimulates the activity of the central kinase GacS. Outside of the Pseudomonas genus, the network has been studied only in Azotobacter vinelandii. In this work, we report the study of the RetS kinase of A. vinelandii; as expected, the phenotypes affected in gacS mutants, such as production of alginates, polyhydroxybutyrate, and alkylresorcinols and swimming motility, were also affected in retS mutants. Interestingly, our data indicated that RetS in A. vinelandii acts as a positive regulator of GacA activity. Consistent with this finding, mutation in retS also negatively affected the expression of small regulatory RNAs belonging to the Rsm family. We also confirmed the interaction of RetS with GacS, as well as with the phosphotransfer protein HptB.
Assuntos
Alginatos , Azotobacter vinelandii , Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Azotobacter vinelandii/genética , Azotobacter vinelandii/enzimologia , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Alginatos/metabolismo , Resorcinóis/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Poliésteres/metabolismo , Hidroxibutiratos/metabolismoRESUMO
Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.
Assuntos
Antibacterianos , Biofilmes , Paenibacillus , Polissacarídeo-Liases , Pseudomonas aeruginosa , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Polissacarídeo-Liases/metabolismo , Polissacarídeo-Liases/genética , Antibacterianos/farmacologia , Paenibacillus/genética , Paenibacillus/enzimologia , Paenibacillus/efeitos dos fármacos , Gentamicinas/farmacologia , Amicacina/farmacologia , Fermentação , Testes de Sensibilidade Microbiana , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Alginatos/metabolismoRESUMO
We conducted two experiments. The first aimed to obtain and characterize microparticles of slow-release urea (SRU) using calcium alginate as the encapsulating agent. The second experiment evaluated their inclusion in sheep diets. In the first experiment, four treatments from a completely randomized design were employed to develop an SRU through the ionic gelification technique testing two drying methods (oven and lyophilizer) and addition or no of sulfur (S): SRU oven-dried with sulfur (MUSO) and without sulfur (MUO), SRU freeze-dried/lyophilized with (MUSL), and without sulfur (MUL). MUO exhibited better yield and encapsulation efficiency among these formulations than the others. Therefore, the second experiment was conducted to compare free urea (U) as control and three proportions (1%, 1.5%, and 2% of total dry matter) of MUO in the diet of sheep. Twenty-four non-castrated male Santa Ines lambs, with an average body weight of 22 ± 3.0 kg, were used and distributed in a completely randomized design with four treatments and six replications. The inclusion of 1% alginate-encapsulated urea (MUO1%) resulted in higher dry matter (DM) intake than free urea (p ≤ 0.05). MUO2% inclusion promoted higher NDF digestibility than U and MUO1%. MUO1% showed higher DM than MUO2% and higher NFC digestibility than U and MUO2% (p ≤ 0.05). Sheep fed MUO1.5% and MUO2% exhibited similar nutrient intake and digestibility. Sheep receiving MUO1% had higher N-intake, N-urinary, N-excretion total, N-digested, and N-retained compared to U. Sheep fed MUO1% showed greater N-retained (as % ingested and digested), microbial protein production, and efficiency when compared to other treatments (p ≤ 0.05). MUO2% addition (SRU) promoted the lowest microbial protein production and efficiency in sheep. MUO dietary inclusion increased feeding time and reduced idleness time compared to U, regardless of the MUO level (p ≤ 0.05). Adding MUO1% improved the intake efficiency of DM and NDF and resulted in more feed boli than the other MUO levels (p ≤ 0.05). Sheep receiving U had (4 h after fending) higher NH3-N, pH, and blood urea nitrogen (BUN) and lower TGL serum compared to sheep fed MUO (p ≤ 0.05), without significant difference among MUO levels (p > 0.05), except NH3-N was higher in MUO1.5% and MUO2% compared to MUO1.0%. The external ionic gelation technique proved suitable for urea microencapsulation in calcium alginate (3%), demonstrating high quality, efficiency, and yield. MUO represents a promising slow-release urea for ruminants and is recommended for sheep diets at an inclusion level of 1.0%. This inclusion level improves intake efficiency and nutrient digestibility, increases rumen nitrogen retention, and reduces BUN without compromising sheep health.
Assuntos
Digestão , Ureia , Animais , Masculino , Alginatos/metabolismo , Alginatos/farmacologia , Ração Animal/análise , Dieta/veterinária , Nitrogênio/metabolismo , Rúmen/metabolismo , Ovinos , Enxofre , Ureia/metabolismoRESUMO
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.
Assuntos
Plâncton , Pseudomonas aeruginosa , Alginatos/metabolismo , Alginatos/farmacologia , Biofilmes , Peróxido de Hidrogênio/farmacologia , Pseudomonas aeruginosa/fisiologiaRESUMO
BACKGROUND: Alginates are polysaccharides used in a wide range of industrial applications, with their functional properties depending on their molecular weight. In this study, alginate production and the expression of genes involved in polymerization and depolymerization in batch cultures of Azotobacter vinelandii were evaluated under controlled and noncontrolled oxygen transfer rate (OTR) conditions. RESULTS: Using an oxygen transfer rate (OTR) control system, a constant OTR (20.3 ± 1.3 mmol L 1 h 1 ) was maintained during cell growth and stationary phases. In cultures subjected to a controlled OTR, alginate concentrations were higher (5.5 ± 0.2 g L 1 ) than in cultures under noncontrolled OTR. The molecular weight of alginate decreased from 475 to 325 kDa at the beginning of the growth phase and remained constant until the end of the cultivation period. The expression level of alyA1, which encodes an alginate lyase, was more affected by OTR control than those of other genes involved in alginate biosynthesis. The decrease in alginate molecular weight can be explained by a higher relative expression level of alyA1 under the controlled OTR condition. CONCLUSIONS: This report describes the first time that alginate production and alginate lyase (alyA1) expression levels have been evaluated in A. vinelandii cultures subjected to a controlled OTR. The results show that automatic control of OTR may be a suitable strategy for improving alginate production while maintaining a constant molecular weight.
Assuntos
Polissacarídeo-Liases/metabolismo , Transferência de Oxigênio , Azotobacter vinelandii/metabolismo , Oxigênio/metabolismo , Expressão Gênica , Reação em Cadeia da Polimerase , Azotobacter vinelandii/genética , Alginatos/metabolismo , Fermentação , Peso MolecularRESUMO
Alginates can be used to elaborate hydrogels, and their properties depend on the molecular weight (MW) and the guluronic (G) and mannuronic (M) composition. In this study, the MW and G/M ratio were evaluated in cultures of Azotobacter vinelandii to 3 and 30 L scales at different oxygen transfer rates (OTRs) under diazotrophic conditions. An increase in the maximum OTR (OTRmax) improved the alginate production, reaching 3.3 ± 0.2 g L-1. In the cultures conducted to an OTR of 10.4 mmol L-1 h-1 (500 rpm), the G/M increased during the cell growth phase and decreased during the stationary phase; whereas, in the cultures at 19.2 mmol L-1 h-1 was constant throughout the cultivation. A higher alginate MW (520 ± 43 kDa) and G/M ratio (0.86 ± 0.01) were obtained in the cultures conducted at 10.4 mmol L-1 h-1. The OTR as a criterion to scale up alginate production allowed to replicate the concentration and the alginate production rate; however, it was not possible reproduce the MW and G/M ratio. Under a similar specific oxygen uptake rate (qO2) (approximately 65 mmol g-1 h-1) the alginate MW was similar (approximately 365 kDa) in both scales. The evidences revealed that the qO2 can be a parameter adequate to produce alginate MW similar in two bioreactor scales. Overall, the results have shown that the alginate composition could be affected by cellular respiration, and from a technological perspective the evidences contribute to the design process based on oxygen consumption to produce alginates defined.
Assuntos
Alginatos , Azotobacter vinelandii/crescimento & desenvolvimento , Reatores Biológicos , Ácidos Hexurônicos , Alginatos/análise , Alginatos/química , Alginatos/metabolismo , Ácidos Hexurônicos/análise , Ácidos Hexurônicos/química , Ácidos Hexurônicos/metabolismo , Peso MolecularRESUMO
The engineering and microbiological aspects involved in the production of alginate-like exopolysaccharides (ALE) and tryptophan (TRY) in aerobic granular sludge systems were evaluated. The inclusion of short anoxic phase (A/O/A cycle-anaerobic, oxic, and anoxic phase) and the control of sludge retention time (SRT ≈ 10 days) proved to be an important strategy to increase the content of these bioproducts in granules. The substrate concentration also has a relevant impact on the production of ALE and TRY. The results of the microbiological analysis showed that slow-growing heterotrophic microbial groups (i.e., PAOs and GAOs) might be associated with the production of ALE, and the EPS-producing fermentative bacteria might be associated with the TRY production. The preliminary economic evaluation indicated the potential of ALE recovery in AGS systems in decreasing the OPEX (operational expenditure) of the treatment, especially for larger sewage treatment plants or industrial wastewaters with a high organic load.
Assuntos
Alginatos/metabolismo , Reatores Biológicos , Esgotos/microbiologia , Triptofano/biossíntese , AerobioseRESUMO
Nanotechnology is a very promising technological tool to combat health problems associated with the loss of effectiveness of currently used antibiotics. Previously, we developed a formulation consisting of a chitosan and tween 80-decorated alginate nanocarrier that encapsulates rifampicin and the antioxidant ascorbic acid (RIF/ASC), intended for the treatment of respiratory intracellular infections. Here, we investigated the effects of RIF/ASC-loaded NPs on the respiratory mucus and the pulmonary surfactant. In addition, we evaluated their cytotoxicity for lung cells in vitro, and their biodistribution on rat lungs in vivo after their intratracheal administration. Findings herein demonstrated that RIF/ASC-loaded NPs display a favorable lung biocompatibility profile and a uniform distribution throughout lung lobules. RIF/ASC-loaded NPs were mainly uptaken by lung macrophages, their primary target. In summary, findings show that our novel designed RIF/ASC NPs could be a suitable system for antibiotic lung administration with promising perspectives for the treatment of pulmonary intracellular infections.
Assuntos
Alginatos/química , Ácido Ascórbico/química , Pneumopatias/tratamento farmacológico , Pneumopatias/metabolismo , Nanopartículas/química , Rifampina/metabolismo , Rifampina/toxicidade , Células A549 , Alginatos/metabolismo , Alginatos/toxicidade , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Antioxidantes/toxicidade , Ácido Ascórbico/metabolismo , Ácido Ascórbico/toxicidade , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Quitosana/metabolismo , Quitosana/toxicidade , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/toxicidade , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Masculino , Nanopartículas/metabolismo , Nanopartículas/toxicidade , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/toxicidade , Polímeros/metabolismo , Polímeros/toxicidade , Ratos , Ratos Wistar , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Rifampina/farmacologia , Suínos , Distribuição TecidualRESUMO
Azotobacter vinelandii produces the linear exopolysaccharide alginate, a compound of significant biotechnological importance. The biosynthesis of alginate in A. vinelandii and Pseudomonas aeruginosa has several similarities but is regulated somewhat differently in the two microbes. Here, we show that the second messenger cyclic dimeric GMP (c-di-GMP) regulates the production and the molecular mass of alginate in A. vinelandii The hybrid protein MucG, containing conserved GGDEF and EAL domains and N-terminal HAMP and PAS domains, behaved as a c-di-GMP phosphodiesterase (PDE). This activity was found to negatively affect the amount and molecular mass of the polysaccharide formed. On the other hand, among the diguanylate cyclases (DGCs) present in A. vinelandii, AvGReg, a globin-coupled sensor (GCS) DGC that directly binds to oxygen, was identified as the main c-di-GMP-synthesizing contributor to alginate production. Overproduction of AvGReg in the parental strain phenocopied a ΔmucG strain with regard to alginate production and the molecular mass of the polymer. MucG was previously shown to prevent the synthesis of high-molecular-mass alginates in response to reduced oxygen transfer rates (OTRs). In this work, we show that cultures exposed to reduced OTRs accumulated higher levels of c-di-GMP; this finding strongly suggests that at least one of the molecular mechanisms involved in modulation of alginate production and molecular mass by oxygen depends on a c-di-GMP signaling module that includes the PAS domain-containing PDE MucG and the GCS DGC AvGReg.IMPORTANCE c-di-GMP has been widely recognized for its essential role in the production of exopolysaccharides in bacteria, such as alginate produced by Pseudomonas and Azotobacter spp. This study reveals that the levels of c-di-GMP also affect the physical properties of alginate, favoring the production of high-molecular-mass alginates in response to lower OTRs. This finding opens up new alternatives for the design of tailor-made alginates for biotechnological applications.
Assuntos
Alginatos/metabolismo , Azotobacter vinelandii/metabolismo , GMP Cíclico/análogos & derivados , Polissacarídeos Bacterianos/biossíntese , Alginatos/química , Azotobacter vinelandii/enzimologia , Azotobacter vinelandii/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Peso Molecular , Oxigênio/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Polissacarídeos Bacterianos/químicaRESUMO
The genus Azotobacter, belonging to the Pseudomonadaceae family, is characterized by the formation of cysts, which are metabolically dormant cells produced under adverse conditions and able to resist desiccation. Although this developmental process has served as a model for the study of cell differentiation in Gram-negative bacteria, the molecular basis of its regulation is still poorly understood. Here, we report that the ubiquitous second messenger cyclic dimeric GMP (c-di-GMP) is critical for the formation of cysts in Azotobacter vinelandii Upon encystment induction, the levels of c-di-GMP increased, reaching a peak within the first 6 h. In the absence of the diguanylate cyclase MucR, however, the levels of this second messenger remained low throughout the developmental process. A. vinelandii cysts are surrounded by two alginate layers with variable proportions of guluronic residues, which are introduced into the final alginate chain by extracellular mannuronic C-5 epimerases of the AlgE1 to AlgE7 family. Unlike in Pseudomonas aeruginosa, MucR was not required for alginate polymerization in A. vinelandii Conversely, MucR was necessary for the expression of extracellular alginate C-5 epimerases; therefore, the MucR-deficient strain produced cyst-like structures devoid of the alginate capsule and unable to resist desiccation. Expression of mucR was partially dependent on the response regulator AlgR, which binds to two sites in the mucR promoter, enhancing mucR transcription. Together, these results indicate that the developmental process of A. vinelandii is controlled through a signaling module that involves activation by the response regulator AlgR and c-di-GMP accumulation that depends on MucR.IMPORTANCEA. vinelandii has served as an experimental model for the study of the differentiation processes to form metabolically dormant cells in Gram-negative bacteria. This work identifies c-di-GMP as a critical regulator for the production of alginates with specific contents of guluronic residues that are able to structure the rigid laminated layers of the cyst envelope. Although allosteric activation of the alginate polymerase complex Alg8-Alg44 by c-di-GMP has long been recognized, our results show a previously unidentified role during the polymer modification step, controlling the expression of extracellular alginate epimerases. Our results also highlight the importance of c-di-GMP in the control of the physical properties of alginate, which ultimately determine the desiccation resistance of the differentiated cell.