RESUMEN
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.
Asunto(s)
Alginatos , Azotobacter vinelandii , Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Azotobacter vinelandii/genética , Azotobacter vinelandii/enzimología , Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Alginatos/metabolismo , Resorcinoles/metabolismo , Histidina Quinasa/genética , Histidina Quinasa/metabolismo , Poliésteres/metabolismo , Hidroxibutiratos/metabolismoRESUMEN
Non-symbiotic N2-fixation would greatly increase the versatility of N-biofertilizers for sustainable agriculture. Genetic modification of diazotrophic bacteria has successfully enhanced NH4+ release. In this study, we compared the competitive fitness of A. vinelandii mutant strains, which allowed us to analyze the burden of NH4+ release under a broad dynamic range. Long-term competition assays under regular culture conditions confirmed a large burden for NH4+ release, exclusion by the wt strain, phenotypic instability, and loss of the ability to release NH4+. In contrast, co-inoculation in mild autoclaved soil showed a much longer co-existence with the wt strain and a stable NH4+ release phenotype. All genetically modified strains increased the N content and changed its chemical speciation in the soil. This study contributes one step forward towards bridging a knowledge gap between molecular biology laboratory research and the incorporation of N from the air into the soil in a molecular species suitable for plant nutrition, a crucial requirement for developing improved bacterial inoculants for economic and environmentally sustainable agriculture. KEY POINTS: ⢠Genetic engineering for NH4+ excretion imposes a fitness burden on the culture medium ⢠Large phenotypic instability for NH4+-excreting bacteria in culture medium ⢠Lower fitness burden and phenotypic instability for NH4+-excreting bacteria in soil.
Asunto(s)
Compuestos de Amonio , Azotobacter vinelandii , Microbiología del Suelo , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Compuestos de Amonio/metabolismo , Fijación del Nitrógeno , Nitrógeno/metabolismo , Aptitud Genética , Fenotipo , Suelo/química , Medios de Cultivo/química , Ingeniería GenéticaRESUMEN
In several Gram-negative bacteria, the general stress response is mediated by the alternative sigma factor RpoS, a subunit of RNA polymerase that confers promoter specificity. In Escherichia coli, regulation of protein levels of RpoS involves the adaptor protein RssB, which binds RpoS for presenting it to the ClpXP protease for its degradation. However, in species from the Pseudomonadaceae family, RpoS is also degraded by ClpXP, but an adaptor has not been experimentally demonstrated. Here, we investigated the role of an E. coli RssB-like protein in two representative Pseudomonadaceae species such as Azotobacter vinelandii and Pseudomonas aeruginosa. In these bacteria, inactivation of the rssB gene increased the levels and stability of RpoS during exponential growth. Downstream of rssB lies a gene that encodes a protein annotated as an anti-sigma factor antagonist (rssC). However, inactivation of rssC in both A. vinelandii and P. aeruginosa also increased the RpoS protein levels, suggesting that RssB and RssC work together to control RpoS degradation. Furthermore, we identified an in vivo interaction between RssB and RpoS only in the presence of RssC using a bacterial three-hybrid system. We propose that both RssB and RssC are necessary for the ClpXP-dependent RpoS degradation during exponential growth in two species of the Pseudomonadaceae family.
Asunto(s)
Azotobacter vinelandii , Proteínas de Escherichia coli , Factor sigma/genética , Factor sigma/metabolismo , Factores de Transcripción/metabolismo , Escherichia coli/metabolismo , Proteínas de Unión al ADN/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas de Escherichia coli/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
Encapsulation is an immobilization method characterized by restricting microbial cells to a delimited area while preserving their metabolic viability. This technique represents an alternative to improve the adaptive capacity of bacteria in the face of interactions with native microorganisms and environmental factors that limit their inoculation. This study aimed to evaluate the effect of Azotobacter vinelandii ATCC 12837 encapsulated in alginate-Na beads as an inoculant of tomato (Solanum Lycopersicum L) seedlings. Two inoculation treatments were carried out: liquid and encapsulated, and the control without microorganisms. Physiological variables, microbial viability, and the presence of A. vinelandii were determined by qPCR. Inoculation with A. vinelandii in liquid and encapsulated form favored seedling growth. Plants with the encapsulated inoculum significantly increased germination percentage (20%), stem diameter (38%), seedling height (34%), root length (69%), NO3 concentration (41%), and Na (30%); compared to the control. Encapsulation of A. vinelandii in alginate-Na macrocapsules allowed its establishment in the rhizosphere and was corroborated by viable count and molecular methods. The viability of the bacteria was maintained for 28 days using both inoculation methods, and not detected in the control treatment.
Asunto(s)
Azotobacter vinelandii , Solanum lycopersicum , Alginatos , Azotobacter vinelandii/genética , Rizosfera , PlantonesRESUMEN
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.
Asunto(s)
Polisacárido Liasas/metabolismo , Transferencia de Oxígeno , Azotobacter vinelandii/metabolismo , Oxígeno/metabolismo , Expresión Génica , Reacción en Cadena de la Polimerasa , Azotobacter vinelandii/genética , Alginatos/metabolismo , Fermentación , Peso MolecularRESUMEN
Poly-3-hydroxybutyrate (P3HB) is a biopolymer, which presents characteristics similar to those of plastics derived from the petrochemical industry. The thermomechanical properties and biodegradability of P3HB are influenced by its molecular weight (MW). The aim of the present study was to evaluate the changes of the molecular weight of P3HB as a function of oxygen transfer rate (OTR) in the cultures using two strains of Azotobacter vinelandii, a wild-type strain OP, and PhbZ1 mutant with a P3HB depolymerase inactivated. Both strains were grown in a bioreactor under different OTR conditions. An inverse relationship was found between the average molecular weight of P3HB and the OTRmax, obtaining a polymer with a maximal MW (8000-10,000 kDa) from the cultures developed at OTRmax of 5 mmol L-1 h-1 using both strains, with respect to the cultures conducted at 8 and 11 mmol L-1 h-1, which produced a P3HB between 4000 and 5000 kDa. The increase in MW of P3HB was related to the activity of enzymes involved in the synthesis and depolymerization. Overall, our results show that it is possible to modulate the average molecular weight of P3HB by manipulating oxygen transfer conditions with both strains (OP and PhbZ1 mutant) of A. vinelandii.
Asunto(s)
Azotobacter vinelandii , Reactores Biológicos , Hidroxibutiratos/metabolismo , Mutación , Poliésteres/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/crecimiento & desarrollo , Peso MolecularRESUMEN
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.
Asunto(s)
Alginatos/metabolismo , Azotobacter vinelandii/metabolismo , GMP Cíclico/análogos & derivados , Polisacáridos Bacterianos/biosíntesis , Alginatos/química , Azotobacter vinelandii/enzimología , Azotobacter vinelandii/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Peso Molecular , Oxígeno/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , Polisacáridos Bacterianos/químicaRESUMEN
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.
Asunto(s)
Azotobacter vinelandii/enzimología , Proteínas Bacterianas/metabolismo , Carbohidrato Epimerasas/metabolismo , GMP Cíclico/análogos & derivados , Alginatos/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/crecimiento & desarrollo , Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/genética , Carbohidrato Epimerasas/genética , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMEN
Azotobacter vineladii is a Gram-negative bacterium that produces alginate and poly-hydroxybutyrate (PHB), two polymers of biotechnological interest. This bacterium has the ability to form desiccation-resistant cysts. In the cyst the membrane phospholipids are replaced with a family of phenolic lipids called alkylresorcinols (ARs). The alginate, PHB, and ARs are controlled by the GacS/A two-component system and the small regulatory RNA (sRNA) RsmZ1, belonging to the Rsm (Csr) regulatory system. The Rsm (Csr) systems usually possess two or more sRNAs, in this regard A. vinelandii is the bacterium with the highest number of rsm-sRNAs. Originally, the presence of two sRNAs of the RsmY family (RsmY1 and RsmY2) was reported, but in a subsequent work it was suggested that they conformed to a single sRNA. In this work we provide genetic evidence confirming that rsmY1 and rsmY2 constitute a single gene. Also, it was established that rsmY mutation decreased alginate and ARs production, but did not affect the PHB synthesis. Transcriptional studies showed that rsmY has its higher expression during the stationary growth phase, and in the absence of RsmZ1, rsmY increases its transcription. Interestingly, rsmY expression was influenced by the carbon source, but its expression did not correlate with alginate production.
Asunto(s)
Alginatos/metabolismo , Azotobacter vinelandii/metabolismo , ARN Bacteriano/metabolismo , Resorcinoles/metabolismo , Azotobacter vinelandii/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Hidroxibutiratos/metabolismo , Mutación , ARN Bacteriano/genéticaRESUMEN
Pseudomonas aeruginosa is a metabolically versatile bacterium and also an important opportunistic pathogen. It has a remarkable genomic structure since the genetic information encoding its pathogenicity-related traits belongs to its core-genome while both environmental and clinical isolates are part of the same population with a highly conserved genomic sequence. Unexpectedly, considering the high level of sequence identity and homologue gene number shared between different P. aeruginosa isolates, the presence of specific essential genes of the two type strains PAO1 and PA14 has been reported to be highly variable. Here we report the detailed bioinformatics analysis of the essential genes of P. aeruginosa PAO1 and PA14 that have been previously experimentally identified and show that the reported gene variability was owed to sequencing and annotation inconsistencies, but that in fact they are highly conserved. This bioinformatics analysis led us to the definition of 348 P. aeruginosa general essential genes. In addition we show that 342 of these 348 essential genes are conserved in Azotobacter vinelandii, a nitrogen-fixing, cyst-forming, soil bacterium. These results support the hypothesis of A. vinelandii having a polyphyletic origin with a Pseudomonads genomic backbone, and are a challenge to the accepted theory of bacterial evolution.
Asunto(s)
Azotobacter vinelandii/genética , Bacterias/genética , Evolución Biológica , Genes Esenciales , Pseudomonas aeruginosa/genética , Azotobacter vinelandii/patogenicidad , Bacterias/clasificación , Biología Computacional/métodos , Secuencia Conservada , Evolución Molecular , Genes Bacterianos , Genoma Bacteriano , Pseudomonas aeruginosa/patogenicidadRESUMEN
In bacteria, the 5'-end-dependent RNA degradation is triggered by the RNA pyrophosphohydrolase RppH converting tri/diphosphate to monophosphate transcripts. This study shows that in the soil bacterium Azotobacter vinelandii, inactivation of rppH gene negatively affected the production of bioplastic poly-ß-hydroxybutyrate (PHB) by reducing the expression at the translational level of PhbR, the specific transcriptional activator of the phbBAC biosynthetic operon. The effect of RppH on the translation of phbR seemed to be exerted through the translational repressor RsmA, as the inactivation of rsmA in the rppH mutant restored the phbR expression. Interestingly, in Escherichia coli inactivation of rppH also affected the expression of CsrA, the RsmA homolog. The level of the csrA transcript was higher and more stable in the E. coli rppH mutant than in the wild type strain. Additionally, and in contrast to the csrA mutants that are known to have a defective swimming phenotype, the E. coli rppH mutant showed a hyper-swimming phenotype that was suppressed by a csrA mutation, and the AvRppH restored to wild type level the swimming phenotype to the E. coli rppH mutant. We propose that in both A. vinelandii and E. coli, RppH activity plays a role in the expression of the translational regulator protein RsmA/CsrA.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Unión al ARN/biosíntesis , Proteínas Represoras/biosíntesis , Eliminación de Gen , Biosíntesis de ProteínasRESUMEN
Alginate is a linear polysaccharide that can be used for different applications in the food and pharmaceutical industries. These polysaccharides have a chemical structure composed of subunits of (1-4)-ß-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G). The monomer composition and molecular weight of alginates are known to have effects on their properties. Currently, these polysaccharides are commercially extracted from seaweed but can also be produced by Azotobacter vinelandii and Pseudomonas spp. as an extracellular polymer. One strategy to produce alginates with different molecular weights and with reproducible physicochemical characteristics is through the manipulation of the culture conditions during fermentation. This mini-review provides a comparative analysis of the metabolic pathways and molecular mechanisms involved in alginate polymerization from A. vinelandii and Pseudomonas spp. Different fermentation strategies used to produce alginates at a bioreactor laboratory scale are described.
Asunto(s)
Alginatos/metabolismo , Azotobacter vinelandii/crecimiento & desarrollo , Pseudomonas/crecimiento & desarrollo , Alginatos/química , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Reactores Biológicos , Fermentación , Ácido Glucurónico/química , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/química , Ácidos Hexurónicos/metabolismo , Redes y Vías Metabólicas , Peso Molecular , Pseudomonas/genética , Pseudomonas/metabolismoRESUMEN
Alginate production and gene expression of genes involved in alginate biosynthesis were evaluated in continuous cultures under dissolved oxygen tension (DOT) controlled conditions. Chemostat at 8% DOT showed an increase in the specific oxygen uptake rate [Formula: see text] from 10.9 to 45.3 mmol g-1 h-1 by changes in the dilution rate (D) from 0.06 to 0.10 h-1, whereas under 1% DOT the [Formula: see text] was not affected. Alginate molecular weight was not affected by DOT. However, chemostat at 1% DOT showed a downregulation up to 20-fold in genes encoding both the alginate polymerase (alg8, alg44), alginate acetylases (algV, algI) and alginate lyase AlgL. alyA1 and algE7 lyases gene expressions presented an opposite behavior by changing the DOT, suggesting that A. vinelandii can use specific depolymerases depending on the oxygen level. Overall, the DOT level have a differential effect on genes involved in alginate synthesis, thus a gene expression equilibrium determines the production of alginates of similar molecular weight under DOT controlled.
Asunto(s)
Azotobacter vinelandii/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Polisacárido Liasas/metabolismo , Acetilación , Alginatos , Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/genética , Medios de Cultivo/química , Fermentación , Ácido Glucurónico/biosíntesis , Ácidos Hexurónicos , Peso Molecular , Oxígeno/metabolismo , Polisacárido Liasas/genéticaRESUMEN
In this study, the respiratory activity and carbon usage of the mutant strain of A. vinelandii AT6, impaired in poly-ß-hydroxybutyrate (PHB) production, and their relationship with the synthesis of alginate were evaluated. The alginate yield and the specific oxygen uptake rate were higher (2.5-fold and 62 %, respectively) for the AT6 strain, compared to the control strain (ATCC 9046), both in shake flasks cultures and in bioreactor, under fixed dissolved oxygen tension (1 %). In contrast, the degree of acetylation was similar in both strains. These results, together with the analysis of carbon usage (% C-mol), suggest that in the case of the AT6 strain, the flux of acetyl-CoA (precursor molecule for PHB biosynthesis and alginate acetylation) was diverted to the respiratory chain passing through the tricarboxylic acids cycle, and an important % C-mol was directed through alginate biosynthesis, up to 25.9 % and to a lesser extent, to biomass production (19.7 %).
Asunto(s)
Azotobacter vinelandii/metabolismo , Carbono/metabolismo , Acetilcoenzima A/metabolismo , Alginatos , Azotobacter vinelandii/genética , Ácido Glucurónico/biosíntesis , Ácidos Hexurónicos , Hidroxibutiratos/metabolismo , Mutación , Oxígeno/metabolismo , Poliésteres/metabolismoRESUMEN
Azotobacter vinelandii is a soil bacterium that forms desiccation-resistant cysts, and the exopolysaccharide alginate is essential for this process. A. vinelandii also produces alginate under vegetative growth conditions, and this production has biotechnological significance. Poly-ß-hydroxybutyrate (PHB) is another polymer synthetized by A. vinelandii that is of biotechnological interest. The GacS/A two-component signal transduction system plays an important role in regulating alginate production, PHB synthesis, and encystment. GacS/A in turn controls other important regulators such as RpoS and the ncRNAs that belong to the Rsm family. In A. vinelandii, RpoS is necessary for resisting oxidative stress as a result of its control over the expression of the catalase Kat1. In this work, we characterized a new ncRNA in A. vinelandii that is homologous to the P16/RsgA reported in Pseudomonas. We found that the expression of rgsA is regulated by GacA and RpoS and that it was essential for oxidative stress resistance. However, the activity of the catalase Kat1 is unaffected in rgsA mutants. Unlike those reported in Pseudomonas, RgsA in A. vinelandii regulates biofilm formation but not polymer synthesis or the encystment process.
Asunto(s)
Azotobacter vinelandii/fisiología , Biopelículas , Estrés Oxidativo , ARN Bacteriano/metabolismo , ARN Largo no Codificante/metabolismo , Azotobacter vinelandii/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Bacteriano/genética , ARN Largo no Codificante/genética , Factor sigma/genética , Factor sigma/metabolismoRESUMEN
Alginates are polysaccharides used as food additives and encapsulation agents in biotechnology, and their functional properties depend on its molecular weight. In this study, different steady-states in continuous cultures of A. vinelandii were established to determine the effect of the dilution rate (D) and the agitation rate on alginate production and expression of genes involved in alginate polymerization and depolymerization. Both, the agitation and dilution rates, determined the partitioning of the carbon utilization from sucrose into alginate and CO2 under oxygen-limiting conditions. A low D (0.07 h(-1)) and 500 rpm resulted in the highest carbon utilization into alginate (25%). Quantitative real-time polymerase chain reaction was used to determine the transcription level of six genes involved in alginate polymerization and depolymerization. In chemostat cultures at 0.07 h(-1), the gene expression was affected by changes in the agitation rate. By increasing the agitation rate from 400 to 600 rpm, the algE7 gene expression decreased tenfold, whereas alyA1, algL and alyA2 gene expression increased between 1.5 and 2.8 times under similar conditions evaluated. Chemostat at 0.07 h(-1) showed a highest alginate molecular weight (580 kDa) at 500 rpm whereas similar molecular weights (480 kDa) were obtained at 400 and 600 rpm. The highest molecular weight was not explained by changes in the expression of alg8 and alg44 (genes involved in alginate polymerization). Nonetheless, a different expression pattern observed for lyases could explain the highest alginate molecular weight obtained. Overall, the results suggest that the control of alginate molecular weight in A. vinelandii cells growing in continuous mode is determined by a balance between the gene expression of intracellular and extracellular lyases in response to oxygen availability. These findings better our understanding of the biosynthesis of bacterial alginate and help us progress toward obtain tailor-made alginates.
Asunto(s)
Azotobacter vinelandii/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Microbiología Industrial , Oxígeno/metabolismo , Alginatos , Azotobacter vinelandii/genética , Biomasa , Carbono/metabolismo , Medios de Cultivo/química , Fermentación , Ácido Glucurónico/biosíntesis , Ácidos Hexurónicos , Peso Molecular , Sacarosa/metabolismoRESUMEN
The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. In this study we compared the effect of controlling the maximum activation state of the Azotobacter vinelandii glutamine synthase by a point mutation at the active site (D49S mutation) and impairing the ammonium-dependent homeostatic control of nitrogen-fixation genes expression by the ΔnifL mutation on ammonium release by the cells. Strains bearing the single D49S mutation were more efficient ammonium producers under carbon/energy limiting conditions and sustained microalgae growth at the expense of atmospheric N2 in synthetic microalgae-bacteria consortia. Ammonium delivery by the different strains had implications for the microalga׳s cell-size distribution. It was uncovered an extensive cross regulation between nitrogen fixation and assimilation that extends current knowledge on this key metabolic pathway and might represent valuable hints for further improvements of versatile N2-fixing microbial-cell factories.
Asunto(s)
Amoníaco/metabolismo , Azotobacter vinelandii , Ingeniería Metabólica/métodos , Microalgas/crecimiento & desarrollo , Consorcios Microbianos , Fijación del Nitrógeno/genética , Azotobacter vinelandii/enzimología , Azotobacter vinelandii/genética , Dominio Catalítico , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Mutación PuntualRESUMEN
The biosynthesis of poly-3-hydroxybutyrate (P3HB), a biodegradable bio-plastic, requires acetyl-CoA as precursor and NADPH as cofactor. Escherichia coli has been used as a heterologous production model for P3HB, but metabolic pathway analysis shows a deficiency in maintaining high levels of NADPH and that the acetyl-CoA is mainly converted to acetic acid by native pathways. In this work the pool of NADPH was increased 1.7-fold in E. coli MG1655 through plasmid overexpression of the NADP(+)-dependent glyceraldehyde 3-phosphate dehydrogenase gene (gapN) from Streptococcus mutans (pTrcgapN). Additionally, by deleting the main acetate production pathway (ackA-pta), the acetic acid production was abolished, thus increasing the acetyl-CoA pool. The P3HB biosynthetic pathway was heterologously expressed in strain MG1655 Δack-pta/pTrcgapN, using an IPTG inducible vector with the P3HB operon from Azotobacter vinelandii (pPHB Av ). Cultures were performed in controlled fermentors using mineral medium with glucose as the carbon source. Accordingly, the mass yield of P3HB on glucose increased to 73 % of the maximum theoretical and was 30 % higher when compared to the progenitor strain (MG1655/pPHB Av ). In comparison with the wild type strain expressing pPHB Av , the specific accumulation of PHB (gPHB/gDCW) in MG1655 Δack-pta/pTrcgapN/pPHB Av increased twofold, indicating that as the availability of NADPH is raised and the production of acetate abolished, a P3HB intracellular accumulation of up to 84 % of the E. coli dry weight is attainable.
Asunto(s)
Acetilcoenzima A/metabolismo , Vías Biosintéticas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroxibutiratos/metabolismo , Ingeniería Metabólica , NADP/metabolismo , Poliésteres/metabolismo , Azotobacter vinelandii/enzimología , Azotobacter vinelandii/genética , Reactores Biológicos/microbiología , Medios de Cultivo/química , Escherichia coli/enzimología , Eliminación de Gen , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/genética , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/metabolismo , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus mutans/enzimología , Streptococcus mutans/genéticaRESUMEN
In Azotobacter vinelandii, a cyst-forming bacterium, the alternative sigma factor RpoS is essential to the formation of cysts resistant to desiccation and to synthesis of the cyst-specific lipids, alkylresorcinols. In this study, we carried out a proteome analysis of vegetative cells and cysts of A. vinelandii strain AEIV and its rpoS mutant derivative AErpoS. This analysis allowed us to identify a small heat-shock protein, Hsp20, as one of the most abundant proteins of cysts regulated by RpoS. Inactivation of hsp20 did not affect the synthesis of alkylresorcinols or the formation of cysts with WT morphology; however, the cysts formed by the hsp20 mutant strain were unable to resist desiccation. We also demonstrated that expression of hsp20 from an RpoS-independent promoter in the AErpoS mutant strain is not enough to restore the phenotype of resistance to desiccation. These results indicate that Hsp20 is essential for the resistance to desiccation of A. vinelandii cysts, probably by preventing the aggregation of proteins caused by the lack of water. To our knowledge, this is the first report of a small heat-shock protein that is essential for desiccation resistance in bacteria.
Asunto(s)
Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas del Choque Térmico HSP20/genética , Proteínas del Choque Térmico HSP20/metabolismo , Factor sigma/genética , Factor sigma/metabolismo , Secuencia de Bases , Desecación , Silenciador del Gen , Proteínas del Choque Térmico HSP20/química , Metabolismo de los Lípidos , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Proteoma , Proteómica , Procesamiento Postranscripcional del ARN , Transcripción GenéticaRESUMEN
The transcription of genes involved in alginate polymerization and depolymerization, as well as the alginase activity (extracellular and intracellular) under oxygen-limited and non oxygen-limited conditions in cultures of A. vinelandii, was studied. Two levels of dissolved oxygen tension (DOT) (1% and 5%, oxygen-limited and non-oxygen-limited, respectively) strictly controlled by gas blending, were evaluated in a wild type strain. In cultures at low DOT (1%), in which a high molecular weight alginate (1200 kDa) was synthesized, the transcription levels of alg8 and alg44 (genes encoding alginate polymerase complex), and algX (encoding a protein involved in polymer transport through periplasmic space) were considerably higher as compared to cultures conducted at 5% DOT, under which an alginate with a low MW (42 kDa) was produced. In the case of genes encoding for intracellular and extracellular alginases, the levels of these transcripts were higher at 1% DOT. However, intracellular and extracellular alginase activity were lower (0.017 and 0.01 U/mg protein, respectively) in cultures at 1% DOT, as compared with the activities measured at 5% DOT (0.027 and 0.052 U/mg protein for intracellular and extracellular maximum activity, respectively). The low alginase activity measured in cultures at 1% DOT and the high level of transcription of genes constituting alginate polymerase complex might be mechanisms by which oxygen regulates the production of alginates with a high MW.