Criptobiósis de plásmidos de Azotobacter Vinelandii

Tesis (Doctor en Ciencias con Especialidad en Microbiología) U.A.N.L.

A metodologia de superfície de resposta como ferramenta para a avaliação da produção de alginato e poli-hidroxibutirato pela Azotobacter vinelandii

The response surface methodology as a tool for assessing the production of alginate and polyhydroxybutirate by Azotobacter vinelandii. Alginate is a polysaccharide extracted from cell walls of brown algae and used in the food, pharmaceuticals and biotech industries. Production is concentrated on the cultivation of brown seaweed, but several bacteria of the genus Pseudomonas and Azotobacter produce alginate. The chemical structure of alginates produced by algae is similar to those synthesized by A. vinelandii. The bacteria also produce intracellular polymers such as polyhydroxybutyrate (PHB), known as bioplastic. This work studied the simultaneous alginate and PHB production by A. vinelandii using sucrose and different parameters of fermentation in an orbital shaker. The optimal values for the production of these compounds were determined by the MSR. The first experiment was a 2(6-2) factorial design. The second was based on significant variables of the first, resulting in a full 3(3-0) factorial design. From the first to the second, an increase was observed in the PHB productivity from 12 to 45 mg g(-1) cell h(-1) and alginate from 100 to 1,600 mg g(-1) of cell h(-1). The productivity of both compounds was in the maximum incubation temperature of 62 degrees C, in the shortest time of incubation (18h) and the sucrose concentration, 11 g L(-1). In both experiments the PHB extracted presented purity of 94%.

Produção otimizada de alginato e plástico biodegradável (poli-hidroxibutirato) por Azotobacter vinelandii

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Substratos alternativos para a produção de poli-hidroxibutirato e alginato por Azotobacter vinelandii

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cassava wastewater as carbon source for the polyhydroxybutyrate production by Azotobacter vinelandii CCT 2841

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Azotobacter vinelandii nitrogenase: “Kinetics of nif gene expression and insights into the roles of FdxN and NifQ in FeMo-co biosynthesis”

The Molybdenum-nitrogenase is responsible for most biological nitrogen fixation activity (BNF) in the biosphere. Due to its great agronomical importance, it has been the subject of profound genetic and biochemical studies. The Mo nitrogenase carries at its active site a unique iron-molybdenum cofactor (FeMoco) that consists of an inorganic 7 Fe, 1 Mo, 1 C, 9 S core coordinated to the organic acid homocitrate. Biosynthesis of FeMo-co occurs outside nitrogenase through a complex and highly regulated pathway involving proteins acting as molecular scaffolds, metallocluster carriers or enzymes that provide substrates in appropriate chemical forms. Specific expression regulatory factors tightly control the accumulation levels of all these other components. Insertion of FeMo-co into a P-cluster containing apo-NifDK polypeptide results in nitrogenase reconstitution. Investigation of FeMo-co biosynthesis has uncovered new radical chemistry reactions and new roles for Fe-S clusters in biology.

In Azotobacter vinelandii, the E1 subunit of the pyruvate dehydrogenase complex binds fpr promoter region DNA and ferredoxin I

In Azotobacter vinelandii, deletion of the fdxA gene that encodes a well characterized seven-iron ferredoxin (FdI) is known to lead to overexpression of the FdI redox partner, NADPH:ferredoxin reductase (FPR). Previous studies have established that this is an oxidative stress response in which the fpr gene is transcriptionally activated to the same extent in response to either addition of the superoxide propagator paraquat to the cells or to fdxA deletion. In both cases, the activation occurs through a specific DNA sequence located upstream of the fpr gene. Here, we report the identification of the A. vinelandii protein that binds specifically to the paraquat activatable fpr promoter region as the E1 subunit of the pyruvate dehydrogenase complex (PDHE1), a central enzyme in aerobic respiration. Sequence analysis shows that PDHE1, which was not previously suspected to be a DNA-binding protein, has a helix–turn–helix motif. The data presented here further show that FdI binds specifically to the DNA-bound PDHE1.

Azotobacter vinelandii NIFL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox-sensitive switch.

The NIFL regulatory protein controls transcriptional activation of nitrogen fixation (nif) genes in Azotobacter vinelandii by direct interaction with the enhancer binding protein NIFA. Modulation of NIFA activity by NIFL, in vivo occurs in response to external oxygen concentration or the level of fixed nitrogen. Spectral features of purified NIFL and chromatographic analysis indicate that it is a flavoprotein with FAD as the prosthetic group, which undergoes reduction in the presence of sodium dithionite. Under anaerobic conditions, the oxidized form of NIFL inhibits transcriptional activation by NIFA in vitro, and this inhibition is reversed when NIFL is in the reduced form. Hence NIFL is a redox-sensitive regulatory protein and may represent a type of flavoprotein in which electron transfer is not coupled to an obvious catalytic activity. In addition to its ability to act as a redox sensor, the activity of NIFL is also responsive to adenosine nucleotides, particularly ADP. This response overrides the influence of redox status on NIFL and is also observed with refolded NIFL apoprotein, which lacks the flavin moiety. These observations suggest that both energy and redox status are important determinants of nif gene regulation in vivo.

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: cysteine ligation of the [4Fe-4S] cluster with protein rearrangement is preferred over serine ligation.

The [4Fe-4S] cluster of Azotobacter vinelandii ferredoxin I receives three of its four ligands from a Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys sequence at positions 39-45 while the fourth ligand, Cys20, is provided by a distal portion of the sequence. Previously we reported that the site-directed mutation of Cys20 to Ala (C20A protein) resulted in the formation of a new [4Fe-4S] cluster that obtained its fourth ligand from Cys24, a free cysteine in the native structure. That ligand exchange required significant protein rearrangement. Here we report the conversion of Cys20 to Ser (C20S protein), which gives the protein the opportunity either to retain the native structure and use the Ser20 O gamma as a ligand or to rearrange and use Cys24. X-ray crystallography demonstrates that the cluster does not use the Ser20 O gamma as a ligand; rather it rearranges to use Cys24. In the C20S protein the [4Fe-4S] cluster has altered stability and redox properties relative to either C20A or the native protein.

Homéostasie des cofacteurs métalliques de la nitrogénase (molybdène et vanadium) chez Azotobacter vinelandii en présence de matières organique et minérale

La fixation biologique d’azote (réduction du N2 atmosphérique, non biodisponible, en ammonium (NH3) bioassimilable) est catalysée par la métalloenzyme nitrogénase. Cette enzyme existe sous trois isoformes chez la bactérie du sol Azotobacter vinelandii: les nitrogénases au molybdène (Mo), au vanadium (V) et au fer (Fe). L’acquisition des métaux cofacteurs constitue un paramètre d’intérêt majeur car Mo et V sont fortement complexés à la matrice (matière organique et oxydes) ce qui peut limiter leur disponibilité. Ces travaux ont montré que la présence d’acide tannique et d’oxydes de fer entraîne des changements majeurs dans la gestion des métaux cofacteurs (Mo et V) chez A. vinelandii. Les stratégies d’acquisition des métaux cofacteurs sont fortement modifiées en présence de ces complexants avec (i) un changement important de la quantité des metallophores produits ainsi que (ii) une acquisition simultanée de Mo et V dans des conditions traditionnellement considérées comme non limitantes en Mo. Ceci se traduit par un changement important dans l’utilisation des nitrogénases; les niveaux de transcrits élevés des gènes nifD et vnfD, spécifiques des nitrogénases au Mo et au V respectivement, suggèrent une utilisation simultanée de ces isoenzymes pour assurer la fixation d’azote. Ce projet a permis de mettre en évidence que face à un stress métallique, l’utilisation des isoformes de la nitrogénase par A. vinelandii est un processus plus versatile que précédemment décrit et que le coût d’acquisition des métaux dans ces conditions constitue un facteur important de la régulation de l’activité des nitrogénases. Ceci suggère que les nitrogénases alternatives pourraient contribuer à la fixation d’azote de manière plus importante que présentement admis.

Azotobacter genomes: the genome of Azotobacter chroococcum NCIMB 8003 (ATCC 4412)

The genome of the soil-dwelling heterotrophic N2-fixing Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 (ATCC 4412) (Ac-8003) has been determined. It consists of 7 circular replicons totalling 5,192,291 bp comprising a circular chromosome of 4,591,803 bp and six plasmids pAcX50a, b, c, d, e, f of 10,435 bp, 13,852, 62,783, 69,713, 132,724, and 311,724 bp respectively. The chromosome has a G+C content of 66.27% and the six plasmids have G+C contents of 58.1, 55.3, 56.7, 59.2, 61.9, and 62.6% respectively. The methylome has also been determined and 5 methylation motifs have been identified. The genome also contains a very high number of transposase/inactivated transposase genes from at least 12 of the 17 recognised insertion sequence families. The Ac-8003 genome has been compared with that of Azotobacter vinelandii ATCC BAA-1303 (Av-DJ), a derivative of strain O, the only other member of the Azotobacteraceae determined so far which has a single chromosome of 5,365,318 bp and no plasmids. The chromosomes show significant stretches of synteny throughout but also reveal a history of many deletion/insertion events. The Ac-8003 genome encodes 4628 predicted protein-encoding genes of which 568 (12.2%) are plasmid borne. 3048 (65%) of these show > 85% identity to the 5050 protein-encoding genes identified in Av-DJ, and of these 99 are plasmid-borne. The core biosynthetic and metabolic pathways and macromolecular architectures and machineries of these organisms appear largely conserved including genes for CO-dehydrogenase, formate dehydrogenase and a soluble NiFe-hydrogenase. The genetic bases for many of the detailed phenotypic differences reported for these organisms have also been identified. Also many other potential phenotypic differences have been uncovered. Properties endowed by the plasmids are described including the presence of an entire aerobic corrin synthesis pathway in pAcX50f and the presence of genes for retro-conjugation in pAcX50c. All these findings are related to the potentially different environmental niches from which these organisms were isolated and to emerging theories about how microbes contribute to their communities.

Caracterização de dois novos tipos de centros ferro-enxofre em duas proteínas isoladas de bactérias redutoras de sulfato (desulfovibrio gigas NCIB 9332)

Tese apresentada para obtenção do grau de Doutor

Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0.

The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC ... ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

Alginato bacteriano: aspectos tecnológicos, características e produção

Alginate is a biopolymer used for a variety of industrial applications, for example, in the textiles, cosmetics, foods, agricultural and biotechnological industries. This biopolymer is traditionally extracted from some brown seaweeds (Phaeophyceae) and can be produced by bacteria isolated from soil, as Azotobacter vinelandii, like capsular polysaccharide using glucose, sucrose, among others as carbon sources. The main difference between the alginate of seaweed and the bacterial ones, is the biggest degree of acetylation of this last one, with great influence in the gel force. These chemical characteristics and production of bacterial alginate are presented in this work.

Process Improvement of Coir Pith Degradation Involving White Rot Fungi By Substitution of Urea With Nitrogen Fixing Bacteria and Application of Biodegraded Coir Pith as Plant Growth Medium

In this study, a novel improved technology could be developed to convert the recalcitrant coir pith into environmental friendly organic manure. The standard method of composting involves the substitution of urea with nitrogen fixing bacteria viz. Azotobacter vinelandii and Azospirillum brasilense leading to the development of an improved method of coir pith. The combined action of the microorganisms could enhance the biodegradation of coir pith. In the present study, Pleurotus sajor caju, an edible mushroom which has the ability to degrade coir pith, and the addition of nitrogen fixing bacteria like Azotobacter vinelandii and Azospirillum brasilense could accelerate the action of the fungi on coir pith. The use of these microorganisms brings about definite changes in the NPK, Ammonia, Organic Carbon and Lignin contents in coir pith. This study will encourage the use of biodegraded coir pith as organic manure for agri/horti purpose to get better yields and can serve as a better technology to solve the problem of accumulated coir pith in coir based industries