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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.

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Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a polymer produced by Azotobacter vinelandii OP. In the bioreactor, PHBV production and its molar composition are affected by aeration rate. PHBV production by A. vinelandii OP was evaluated using extended batch cultures at different aeration rates, which determined different oxygen transfer rates (OTR) in the cultures. Under the conditions evaluated, PHBV with different 3-hydroxyvalerate (3HV) fractions were obtained. In the cultures with a low OTR (6.7 mmol L-1 h-1, at 0.3 vvm), a PHBV content of 38% w w-1 with 9.1 mol % 3HV was achieved. The maximum PHBV production (72% w w-1) was obtained at a high OTR (18.2 mmol L-1 h-1, at 1.0 vvm), both at 48 h. Thus, PHBV production increased in the bioreactor with an increased aeration rate, but not the 3HV fraction in the polymer chain. An OTR of 24.9 mmol L-1 h-1 (at 2.1 vvm) was the most suitable for improving the PHBV content (61% w w-1) and a high 3HV fraction of 20.8 mol % (at 48 h); and volumetric productivity (0.15 g L-1 h-1). The findings indicate that the extended batch culture at 2.1 vvm is the most adequate mode of cultivation to produce higher biomass and PHBV with a high 3HV fraction. Overall, the results have shown that the PHBV production and 3HV fraction could be affected by the aeration rate and the proposed approach could be applied to implement cultivation strategies to optimize PHBV production for different biotechnological applications.

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Late embryogenesis abundant (LEA) proteins are hydrophilic proteins that lack a well-ordered tertiary structure and accumulate to high levels in response to water deficit, in organisms such as plants, fungi, and bacteria. The mechanisms proposed to protect cellular structures and enzymes are water replacement, ion sequestering, and membrane stabilization. The activity of some proteins has a limited shelf-life due to instability that can be caused by their structure or the presence of a stress condition that limits their activity; several LEA proteins have been shown to behave as cryoprotectants in vitro. Here, we report a group1 LEA from Azotobacter vinelandii AvLEA1, capable of conferring protection to lactate dehydrogenase, catechol dioxygenase, and Baylase peroxidase against freeze-thaw treatments, desiccation, and oxidative damage, making AvLEA a promising biological stabilizer reagent. This is the first evidence of protection provided by this LEA on enzymes with biotechnological potential, such as dioxygenase and peroxidase under in vitro stress conditions. Our results suggest that AvLEA could act as a molecular chaperone, or a "molecular shield," preventing either dissociation or antiaggregation, or as a radical scavenger, thus preventing damage to these target enzymes during induced stress. KEY POINTS: • This work expands the basic knowledge of the less-known bacterial LEA proteins and their in vitro protection potential. • AvLEA is a bacterial protein that confers in vitro protection to three enzymes with different characteristics and oligomeric arrangement. • The use of AvLEA as a stabilizer agent could be further explored using dioxygenase and peroxidase in bioremediation treatments. AvLEA1 protects against freeze-thaw treatments, desiccation, and oxidative damage on three different enzymes with biotechnological potential.

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[This corrects the article DOI: 10.3389/fmicb.2022.845473.].

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Alginates are a family of polymers composed of guluronate and mannuronate monomers joined by ß (1-4) links. The different types of alginates have variations in their monomer content and molecular weight, which determine the rheological properties and their applications. In industry, alginates are commonly used as additives capable of viscosifying, stabilizing, emulsifying, and gelling aqueous solutions. Recently, additional specialized biomedical uses have been reported for this polymer. Currently, the production of alginates is based on the harvesting of seaweeds; however, the composition and structure of the extracts are highly variable. The production of alginates for specialized applications requires a precise composition of monomers and molecular weight, which could be achieved using bacterial production systems such as those based on Azotobacter vinelandii, a free-living, non-pathogenic bacterium. In this mini-review, we analyze the latest advances in the regulation of alginate synthesis in this model.

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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.

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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.

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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.

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Azotobacter vinelandii is a gram-negative soil bacterium that produces two biopolymers of biotechnological interest, alginate and poly(3-hydroxybutyrate), and it has been widely studied because of its capability to fix nitrogen even in the presence of oxygen. This bacterium is characterized by its high respiration rates, which are almost 10-fold higher than those of Escherichia coli and are a disadvantage for fermentation processes. On the other hand, several works have demonstrated that adequate control of the oxygen supply in A. vinelandii cultivations determines the yields and physicochemical characteristics of alginate and poly(3-hydroxybutyrate). Here, we summarize a review of the characteristics of A. vinelandii related to its respiration systems, as well as some of the most important findings on the oxygen consumption rates as a function of the cultivation parameters and biopolymer production.

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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.

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RESUMEN La batata (Ipomoea batatas L.) se cultiva en todo el mundo como fuente de carbohidratos, y su producción comercial requiere un alto aporte de fertilizantes químicos, lo cual eleva los costos de producción. Los inoculantes microbianos, se emplean como una fuente alternativa de nutrición vegetal. El objetivo de esta investigación fue evaluar el efecto de Pseudomonas denitrificans IBVS2 y Azotobacter vinelandii IBVS13 con diferentes niveles fertilización química nitrogenada en el cultivo de batata en la microrregión del Valle del Sinú en el Caribe Colombiano. Para los montajes de los experimentos se utilizó un diseño completamente aleatorizado, ocho tratamientos y tres repeticiones usando como material vegetal plántulas obtenidas in vitro endurecidas en invernadero. Los resultados demostraron que la cepa Azotobacter vinelandii IBVS13 con un 75% de fertilización nitrogenada (FN) mejoró la capacidad de acumulación de materia seca en los tubérculos de batata, generando incrementos de 6,65 t/ha respecto al testigo químico y 3,18 t/ha en relación con el testigo absoluto, garantizando un incremento del rendimiento. Así mismo, el contenido de proteína bruta aumentó 13,93% al realizar la inoculación de las plantas con esta cepa. En el mismo sentido, la cepa Pseudomonas denitrificans IBVS2+ fertilización nitrogenada 50% presentó aumentos en la variable de fibra cruda 31,75% respecto al testigo absoluto, contribuyendo de manera eficaz como bioestimulante microbiano en la agricultura.

ABSTRACT Sweet potatoes (Ipomoea batatas L.) are grown worldwide as a source of carbohydrates, and their commercial production requires a high contribution of chemical fertilizers, which increases production costs. Microbial inoculants are used as an alternative source of plant nutrition. The objective of this research was to evaluate the effect of Pseudomonas denitrificans IBVS2 and Azotobacter vinelandii IBVS13 with different levels of nitrogen chemical fertilization in the sweet potato crop in the microregion of the Sinú Valley in the Colombian Caribbean. A completely randomized design was used for the experiment development, eight treatments was evaluated and three repetitions were carried out. In vitro hardened seedlings was used as a plant material. The results showed that the Azotobacter vinelandii IBVS1 3 strain with 75% nitrogen fertilization (FM) improved the accumulation capacity of dry matter in sweet potato roots, generating increases of 6.65 t / ha compared to the chemical control and 3.18 t / ha in relation to the absolute control, guaranteeing an increase in yield. The crude protein content was increased in 13.93% when inoculating the plants with this strain. In the same way, with the inoculation of strain Pseudomonas denitrificans IBVS2 + 50% nitrogen fertilization the crude fiber variable was increased in 31.75% compared to the absolute control, contributing effectively as a microbial biostimulant in agriculture.

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Poly-(3-hydroxyalkanoates) (PHAs) are bacterial carbon and energy storage compounds. These polymers are synthesized under conditions of nutritional imbalance, where a nutrient is growth-limiting while there is still enough carbon source in the medium. On the other side, the accumulated polymer is mobilized under conditions of nutrient accessibility or by limitation of the carbon source. Thus, it is well known that the accumulation of PHAs is affected by the availability of nutritional resources and this knowledge has been used to establish culture conditions favoring high productivities. In addition to this effect of the metabolic status on PHAs accumulation, several genetic regulatory networks have been shown to drive PHAs metabolism, so the expression of the PHAs genes is under the influence of global or specific regulators. These regulators are thought to coordinate PHAs synthesis and mobilization with the rest of bacterial physiology. While the metabolic and biochemical knowledge related to the biosynthesis of these polymers has led to the development of processes in bioreactors for high-level production and also to the establishment of strategies for metabolic engineering for the synthesis of modified biopolymers, the use of knowledge related to the regulatory circuits controlling PHAs metabolism for strain improvement is scarce. A better understanding of the genetic control systems involved could serve as the foundation for new strategies for strain modification in order to increase PHAs production or to adjust the chemical structure of these biopolymers. In this review, the regulatory systems involved in the control of PHAs metabolism are examined, with emphasis on those acting at the level of expression of the enzymes involved and their potential modification for strain improvement, both for higher titers, or manipulation of polymer properties. The case of the PHAs producer Azotobacter vinelandii is taken as an example of the complexity and variety of systems controlling the accumulation of these interesting polymers in response to diverse situations, many of which could be engineered to improve PHAs production.

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In the present study, the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Azotobacter vinelandii was evaluated in shake flasks and bioreactors, utilizing different precursors and oxygen transfer rates (OTRs). In shake flask cultures, the highest PHBV yield from sucrose (0.16 g g-1) and 3-hydroxyvalerate (3HV) fraction in the PHA chain (27.4 mol%) were obtained with valerate (1.0 g L-1). In the bioreactor, the cultures were grown under oxygen-limited conditions, and the maximum OTR (OTRmax) was varied by adjusting the agitation rate. In the cultures grown at low OTRmax (4.3 mmol L-1 h-1), the intracellular content of PHBV (73% w w-1) was improved, whereas a maximum 3HV fraction (35 mol %) was obtained when a higher OTRmax (17.2 mmol L-1 h-1, to 600 rpm) was employed. The findings obtained suggest that the PHBV production and the content of 3HV incorporated into the polymer were affected by the OTR. Based on the evidence, it is possible to produce PHBV with a different composition by varying the OTR of the culture; thus, the approach in this study could be used to scale up PHBV production.

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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.

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Azotobacter vinelandii OP is a bacterium that produces poly(3-hydroxybutyrate) (PHB). PHB production in a stirred bioreactor, at different oxygen transfer strategies, was evaluated. By applying different oxygen contents in the inlet gas, the oxygen transfer rate (OTR) was changed under a constant agitation rate. Batch cultures were performed without dissolved oxygen tension (DOT) control (using 9% and 21% oxygen in the inlet gas) and under DOT control (4%) using gas blending. The cultures that developed without DOT control were limited by oxygen. As result of varying the oxygen content in the inlet gas, a lower OTR (4.6 mmol L-1 h-1) and specific oxygen uptake rate (11.6 mmol g-1 h-1) were obtained using 9% oxygen in the inlet gas. The use of 9% oxygen in the inlet gas was the most suitable for improving the intracellular PHB content (56 ± 6 w w-1). For the first time, PHB accumulation in A. vinelandii OP cultures, developed with different OTRs, was compared under homogeneous mixing conditions, demonstrating that bacterial respiration affects PHB synthesis. These results can be used to design new oxygen transfer strategies to produce PHB under productive conditions.

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BACKGROUND: Azotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); however, the role of NAD(P)H/NAD(P)+ ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown. The aim of this study was to evaluate the NAD(P)H/NAD(P) + ratios and the metabolic fluxes involved in alginate and P3HB biosynthesis, under oxygen-limiting and non-limiting oxygen conditions. RESULTS: The results reveal that changes in the oxygen availability have an important effect on the metabolic fluxes and intracellular NADPH/NADP+ ratio, showing that at the lowest OTR (2.4 mmol L-1 h-1), the flux through the tricarboxylic acid (TCA) cycle decreased 27.6-fold, but the flux through the P3HB biosynthesis increased 6.6-fold in contrast to the cultures without oxygen limitation (OTR = 14.6 mmol L-1 h-1). This was consistent with the increase in the level of transcription of phbB and the P3HB biosynthesis. In addition, under conditions without oxygen limitation, there was an increase in the carbon uptake rate (twofold), as well as in the flux through the pentose phosphate (PP) pathway (4.8-fold), compared to the condition of 2.4 mmol L-1 h-1. At the highest OTR condition, a decrease in the NADPH/NADP+ ratio of threefold was observed, probably as a response to the high respiration rate induced by the respiratory protection of the nitrogenase under diazotrophic conditions, correlating with a high expression of the uncoupled respiratory chain genes (ndhII and cydA) and induction of the expression of the genes encoding the nitrogenase complex (nifH). CONCLUSIONS: We have demonstrated that changes in oxygen availability affect the internal redox state of the cell and carbon metabolic fluxes. This also has a strong impact on the TCA cycle and PP pathway as well as on alginate and P3HB biosynthetic fluxes.

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ABSTRACT Using the classic biotechnological methods, the dependence of A. vinelandii D-05 culture alginate production from the media carbon and nitrogen content was investigated. The maximal alginate production was observed during cultivation bacterium in the medium with 2 to 4% of sucrose, but the maximal growth was found in the medium with 4% glucose. It was found that for the alginate production the optimal nitrogen contents could take from 0.05% yeast extract (carbon: nitrogen ratio 168:1). For the first time we demonstrated possibility the A. vinelandii growth during the cultivation in a medium with molasses (a by-product of sugar production) and the significant polysaccharide production (16.6 g/l) was obtained. It was established, that A. vinelandii culture broth could be used as a biological binder for obtaining the biocomposite materials.

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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.

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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 %).

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