The carbon source influences the energetic efficiency of the respiratory chain of N2-fixing Acetobacter diazotrophicus

Acetobacter diazotrophicus is a diazotrophic bacterium that colonizes sugarcane tissues. Glucose is oxidized to gluconate in the periplasm prior to uptake and metabolism. A membrane-bound glucose dehydrogenase quinoenzyme [which contains pyrroloquinoline quinone (PQQ) as the prosthetic group] is involved in that oxidation. Gluconate is oxidized further via the hexose monophosphate pathway and tricarboxylic acid cycle. A. diazotrophicus PAL3 was grown in a chemostat with atmospheric nitrogen as the sole N source provided that the dissolved oxygen was maintained at 1.0–2.0% air saturation. The biomass yields of A. diazotrophicus growing with glucose or gluconate with fixed N were very low compared with other heterotrophic bacteria. The biomass yields under N-fixing conditions were more than 30% less than with ammonium as the N source using gluconate as the carbon source but, surprisingly, were only about 14% less with glucose. The following scheme for the metabolism of A. diazotrophicus through the different pathways emerged: (1) the respiratory chain of this organism had a different efficiency of ATP production in the respiratory chain (P:O ratio) under different culture conditions; and (2) N fixation was one (but not the sole) condition under which a higher P:O ratio was observed. The other condition appears to be the expression of an active PQQ-linked glucose dehydrogenase.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Mineral phosphate solubilization activity of gluconacetobacter diazotrophicus under P-limitation and plant root environment

The ability to solubilize insoluble inorganic phosphate compounds by Gluconacetobacter diazotrophicus was studied using different culture approaches. Qualitative plate assays using tricalcium phosphate as the sole P-source showed that G. diazotrophicus produced solubilization only when aldoses were used as the C-source. Extracellular aldose oxidation via a pyrroloquinoline quinone-linked glucose dehydrogenase (PQQ-GDH) is the main pathway for glucose metabolism in G. diazotrophicus. In batch cultures with 5 g l-1 of hydroxyapatite as the P-source and glucose as the C-source, more than 98% of insoluble P was solubilized. No solubilization was observed neither using glycerol nor culturing a PQQ-GDH mutant of G. diazotrophicus. Solubilizaton was not affected by adding 100 mmol l-1 of MES buffer. Continuous cultures of G. diazotrophicus showed significant activities of PQQ-GDH either under C or P limitation. An intense acidification in the root environment of tomato and wheat seedlings inoculated with a G. diazotrophicus PAL5 was observed. Seedlings inoculated with a PQQ-GDH mutant strain of G. diazotrophicus showed no acidification. Our results suggest that G. diazotrophicus is an excellent candidate to be used as biofertilizer because in addition to the already described plant growth-promoting abilities of this organism, it shows a significant mineral phosphate solubilization capacity.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Colonization of sorghum and wheat by seed inoculation with <i>Gluconacetobacter diazotrophicus</i>

Colonization of sorghum and wheat after seed inoculation with Gluconacetobacter diazotrophicus strains PAL 5 and UAP 5541/pRGS561 (containing the marker gene gusA) was studied by colony counting and microscopic observation of plant tissues. Inoculum levels as low as 10² CFU per seed were enough for root colonization and further spreading in aerial tissues. Rhizoplane colonization was around 7 log CFU g⁻¹ (fresh weight). G. diazotrophicus was found inside sorghum and wheat roots with populations higher than 5 log CFU g⁻¹ (fresh weight). Stem colonization remained stable for 30 days post inoculation with endophyte concentrations from 4 to 5 log CFU g⁻¹ (fresh weight) (in both plants). Population in leaves decreased continuously being undetectable after 17 days post inoculation.Centro de Investigación y Desarrollo en Fermentaciones Industriale

MICROBIOTA BACTERIANA ASOCIADA AL CULTIVO DE TOMATE: CARACTERIZACIÓN Y SELECCIÓN DE BACTERIAS PROMOTORAS DEL CRECIMIENTO VEGETAL

El Cinturón Hortícola Platense concentra una de las mayores superficies del país con cultivos bajo cubierta. Las principales hortalizas cultivadas son en orden de importancia: tomate, apio, lechuga y pimiento. Su producción se caracteriza por su alta intensividad, lo que trae aparejado ciertos problemas como la aparición de plagas, enfermedades y degradación de los suelos, con un uso elevado de agroquímicos. En este contexto, resulta de importancia el empleo de tecnologías que favorezcan la producción hortícola y que reduzcan el impacto ambiental que producen los agroquímicos además de proteger la biodiversidad de estos agroecosistemas. Entre estas tecnologías sustentables, una de las más prometedoras son los inoculantes –productos tecnológicos cuyo principio activo es un microorganismo vivo– formulados a base de bacterias promotoras del crecimiento vegetal (PGPB, del inglés Plant Growth Promoting Bacteria), debido a su seguridad (recrean interacciones naturalmente existentes), su eficiencia y el menor costo de los mismos. Es por ello que el objetivo planteado en esta tesis doctoral es contribuir a la sustentabilidad del Cinturón Hortícola de La Plata mediante la caracterización de la microbiota bacteriana asociada a plantas de tomate cultivadas en la región. Particularmente se caracterizarán las comunidades bacterianas de suelo, rizosfera y endófitas –que se localizan en tejidos internos del vegetal– de raíz, tallo y fruto, diferenciando dos establecimientos: (i) un establecimiento con una larga historia productiva (+25 años de horticultura) y (ii) otro establecimiento en el cual se incorporó una superficie de pastizal natural y se realizó el primer cultivo de tomate. Esta caracterización de las comunidades bacterianas se abordará por un lado utilizando técnicas de biología molecular para realizar una caracterización más exhaustiva de las comunidades bacterianas asociadas a este cultivo (ya que comprende tanto a las bacterias cultivables y no cultivables), buscando realizar un análisis comparativo de ambos establecimientos para determinar el impacto de la acumulación de tantos años de actividad hortícola en las comunidades bacterianas asociadas al cultivo de tomate. Por otro lado, desde el enfoque de la microbiología clásica, se obtendrán aislamientos bacterianos en medios de cultivo nutritivos buscando generar una colección de cepas que serán caracterizadas y posteriormente seleccionadas en base a sus capacidades para promover el crecimiento y desarrollo vegetal, con la intención de obtener un microorganismo adaptado a las condiciones de nuestra región y que pueda ser empleado como un bioinsumo agrícola ya sea actuando como biofertilizante, fitoestimulante o biocontrolador

Energy generation by extracellular aldose oxidation in N2-fixing Gluconacetobacter diazotrophicus

Gluconacetobacter diazotrophicus PAL3 was grown in a chemostat with N2 and mixtures of xylose and gluconate. Xylose was oxidized to xylonate, which was accumulated in the culture supernatants. Biomass yields and carbon from gluconate incorporated into biomass increased with the rate of xylose oxidation. By using metabolic balances it is demonstrated that extracellular xylose oxidation led N2-fixing G. diazotrophicus cultures to increase the efficiency of energy generation.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Induction by Bradyrhizobium diazoefficiens of Different Pathways for Growth in D-mannitol or L-arabinose Leading to Pronounced Differences in CO2 Fixation, O2 Consumption, and Lateral-Flagellum Production

Bradyrhizobium diazoefficiens, a soybean N2-fixing symbiont, constitutes the basic input in one of the most prominent inoculant industries worldwide. This bacterium may be cultured with D-mannitol or L-arabinose as carbon-plus-energy source (C-source) with similar specific growth rates, but with higher biomass production with D-mannitol. To better understand the bacterium's carbon metabolism, we analyzed, by liquid chromatography and tandem mass spectrometry (MS), the whole set of proteins obtained from cells grown on each C-source. Among 3,334 proteins identified, 266 were overproduced in D-mannitol and 237 in L-arabinose, but among these, only 22% from D-mannitol cultures and 35% from L-arabinose cultures were annotated with well defined functions. In the D-mannitol-differential pool we found 19 enzymes of the pentose-phosphate and Calvin-Benson-Bassham pathways and accordingly observed increased extracellular-polysaccharide production by D-mannitol grown bacteria in a CO2-enriched atmosphere. Moreover, poly-3-hydroxybutyrate biosynthesis was increased, suggesting a surplus of reducing power. In contrast, the L-arabinose-differential pool contained 11 enzymes of the L-2-keto-3-deoxyarabonate pathway, 4 enzymes for the synthesis of nicotinamide-adenine dinucleotide from aspartate, with those cultures having a threefold higher O2-consumption rate than the D-mannitol cultures. The stoichiometric balances deduced from the modeled pathways, however, resulted in similar O2 consumptions and ATP productions per C-mole of substrate. These results suggested higher maintenance-energy demands in L-arabinose, which energy may be used partly for flagella-driven motility. Since B. diazoefficiens produces the lateral-flagella system in only L-arabinose, we calculated the O2-consumption rates of a lafR::Km mutant devoid of lateral flagella cultured in L-arabinose or D-mannitol. Contrary to that of the wild-type, the O2-consumption rate of this mutant was similar on both C-sources, and accordingly outcompeted the wild-type in coculture, suggesting that the lateral flagella behaved as parasitic structures under these conditions. Proteomic data are available via ProteomeXchange with identifier PXD008263.Instituto de Biotecnologia y Biologia MolecularCentro de Investigación y Desarrollo en Fermentaciones Industriale

Mineral phosphate solubilization activity of gluconacetobacter diazotrophicus under P-limitation and plant root environment

The ability to solubilize insoluble inorganic phosphate compounds by Gluconacetobacter diazotrophicus was studied using different culture approaches. Qualitative plate assays using tricalcium phosphate as the sole P-source showed that G. diazotrophicus produced solubilization only when aldoses were used as the C-source. Extracellular aldose oxidation via a pyrroloquinoline quinone-linked glucose dehydrogenase (PQQ-GDH) is the main pathway for glucose metabolism in G. diazotrophicus. In batch cultures with 5 g l-1 of hydroxyapatite as the P-source and glucose as the C-source, more than 98% of insoluble P was solubilized. No solubilization was observed neither using glycerol nor culturing a PQQ-GDH mutant of G. diazotrophicus. Solubilizaton was not affected by adding 100 mmol l-1 of MES buffer. Continuous cultures of G. diazotrophicus showed significant activities of PQQ-GDH either under C or P limitation. An intense acidification in the root environment of tomato and wheat seedlings inoculated with a G. diazotrophicus PAL5 was observed. Seedlings inoculated with a PQQ-GDH mutant strain of G. diazotrophicus showed no acidification. Our results suggest that G. diazotrophicus is an excellent candidate to be used as biofertilizer because in addition to the already described plant growth-promoting abilities of this organism, it shows a significant mineral phosphate solubilization capacity.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Mineral Phosphate Solubilization in Burkholderia tropica Involves an Inducible PQQ-Glucose Dehydrogenase

Aims: The objective of this work was to provide knowledgement about the mechanism and regulation of the mineral phosphate solubilization in Burkholderia. tropica. To this end, the expression of the direct extracellular oxidative pathway in B. tropica was studied using different culture approaches.Study Design: Plate assays and batch cultures in flasks and bioreactor were carried out in this study with B. tropica Mto-293 like target organism. The experiments were achieved at least three times with two repetitions per time.Place and Duration of Study: Departamento de Química, Centro de Investigación y Desarrollo en Fermentaciones Industriales, UNLP, CCT-La Plata-CONICET, between November 2014-2015.Methodology: Qualitative plate assays with different Carbon sources were carried out for the evaluation of Mineral Phosphate Solubilization phenotype. Batch cultures in flasks were carried out with different Carbon, Phosphorus and Nitrogen sources to determine quantitatively soluble phosphorus, gluconic acid and other ketoacids in the supernatants, and also PQQ-linked glucose and gluconate dehydrogenase activities in whole cells. Cultures with some of the conditions mentioned before were carried out in bioreactor specifically to control pH.Results: This organism was able to produce significant amounts of gluconic acid via the expression of a PQQ-GDH and also showed a significant activity of GaDH. However, the direct oxidative pathway was only observed under conditions of Phosphorus starvation and/or Nitrogen fixation.Conclusion: The Mineral Phosphate Solubilization phenotype for B. tropica can be ascribed to the expression of the direct oxidative pathway which involves the expression of an active PQQ- linked glucose dehydrogenase. Nevertheless, this pathway is not expressed constitutively in this bacterium. Environmental conditions, like low P and N availability, led to an active extracellular glucose oxidation. Therefore, mineral phosphate solubilization in B. tropica involves an inducible pyrroloquinoline quinone-linked glucose dehydrogenase. These findings may contribute to the use of this bacterium as plant growth promoting bacteria reducing the dependence on chemical fertilizer.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Mineral Phosphate Solubilization in Burkholderia tropica Involves an Inducible PQQ-Glucose Dehydrogenase

Aims: The objective of this work was to provide knowledgement about the mechanism and regulation of the mineral phosphate solubilization in Burkholderia. tropica. To this end, the expression of the direct extracellular oxidative pathway in B. tropica was studied using different culture approaches.Study Design: Plate assays and batch cultures in flasks and bioreactor were carried out in this study with B. tropica Mto-293 like target organism. The experiments were achieved at least three times with two repetitions per time.Place and Duration of Study: Departamento de Química, Centro de Investigación y Desarrollo en Fermentaciones Industriales, UNLP, CCT-La Plata-CONICET, between November 2014-2015.Methodology: Qualitative plate assays with different Carbon sources were carried out for the evaluation of Mineral Phosphate Solubilization phenotype. Batch cultures in flasks were carried out with different Carbon, Phosphorus and Nitrogen sources to determine quantitatively soluble phosphorus, gluconic acid and other ketoacids in the supernatants, and also PQQ-linked glucose and gluconate dehydrogenase activities in whole cells. Cultures with some of the conditions mentioned before were carried out in bioreactor specifically to control pH.Results: This organism was able to produce significant amounts of gluconic acid via the expression of a PQQ-GDH and also showed a significant activity of GaDH. However, the direct oxidative pathway was only observed under conditions of Phosphorus starvation and/or Nitrogen fixation.Conclusion: The Mineral Phosphate Solubilization phenotype for B. tropica can be ascribed to the expression of the direct oxidative pathway which involves the expression of an active PQQ- linked glucose dehydrogenase. Nevertheless, this pathway is not expressed constitutively in this bacterium. Environmental conditions, like low P and N availability, led to an active extracellular glucose oxidation. Therefore, mineral phosphate solubilization in B. tropica involves an inducible pyrroloquinoline quinone-linked glucose dehydrogenase. These findings may contribute to the use of this bacterium as plant growth promoting bacteria reducing the dependence on chemical fertilizer.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Plant growth promotion activity of Keratinolytic fungi growing on a recalcitrant waste known as "hair waste"

Purpureocillium lilacinum (Thom) Samsom is one of the most studied fungi in the control of plant parasitic nematodes. However, there is not specific information on its ability to inhibit some pathogenic bacteria, fungi, or yeast. This work reports the production of several antifungal hydrolytic enzymes by a strain of P. lilacinum when it is grown in a medium containing hair waste. The growth of several plant-pathogenic fungi, Alternaria alternata, Aspergillus niger, and Fusarium culmorum, was considerably affected by the presence of P. lilacinum’s supernatant. Besides antifungal activity, P. lilacinum demonstrates the capability to produce indoleacetic acid and ammonia during time cultivation on hair waste medium. Plant growth-promoting activity by cell-free supernatant was evidenced through the increase of the percentage of tomato seed germination from 71 to 85% after 48 hours. A 21-day plant growth assay using tomato plants indicates that crude supernatant promotes the growth of the plants similar to a reference fertilizer ( > 0.05). These results suggest that both strain and the supernatant may have potential to be considered as a potent biocontrol agent with multiple plant growth-promoting properties. To our knowledge, this is the first report on the antifungal, IAA production and tomato growth enhancing compounds produced by P. lilacinum LPSC #876.Centro de Investigación y Desarrollo en Fermentaciones Industriale