Orientador: Crispin Humberto Garcia-Cruz / Banca: Eleni Gomes / Banca: José Roberto Ernandes / Banca: Mário Antônio Alves da Cunha / Banca: Vanildo Luiz Del Bianchi / Resumo: Atualmente a destinação do lixo é uma das grandes preocupações da organização urbana e os problemas ambientais causados pela produção e acúmulo de materiais plásticos de origem petroquímica têm incentivado muitos países a realizarem estudos de gerenciamento do volume de lixo sólido, incluindo a diminuição de resíduos plásticos por meio do desenvolvimento de bioplásticos. Os bioplásticos possuem propriedades semelhantes às dos plásticos convencionais e apresentam a vantagem de serem facilmente degradados pela ação de microrganismos no ambiente, podendo citar como exemplo os poli-hidroxialcanoatos (PHA), dentre eles o poli-hidroxibutirato (PHB). Estes polímeros podem representar até 80% da massa seca total da célula, tendo como característica principal a biodegradabilidade em solos e a biocompatibilidade com o tecido animal. Entre os microrganismos produtores de PHAs, a bactéria Azotobacter vinelandii pode acumular grandes quantidades de PHB intracelular com a vantagem de utilizar durante seu crescimento uma ampla variedade de açúcares como os encontrados em melaço de cana-de-açúcar, beterraba e xarope de milho, além de resíduos da suinocultura, agroindustriais, etc. Além do PHB, a bactéria A. vinelandii é capaz de produzir alginato, composto muito empregado na área de análogos de frutas ou produtos tipo imitação como: fatias de pimentão para recheios de azeitonas, imitação de anéis de cebola, imitações de caviar, carne, pescados, produtos marinhos, etc. Tendo em vista que os principais fatores limitantes para a produção de biopolímeros estão associados, principalmente, com os custos dos substratos e ao fato de que muitos microrganismos são patogênicos dificultando a sua aceitação pela comunidade em geral, este trabalho teve como objetivo utilizar... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Currently, the waste disposal is a major concern of urban organization and the environmental problems caused by production and accumulation of petrochemical plastics have encouraged many countries to management studies of the solid waste volume, including the waste plastics reduction through the bioplastics development. Bioplastics have similar properties to conventional plastics and the advantage of being easily degraded by the microorganisms action in the environment, for example, poly-hydroxyalcanoatos (PHA), including poly-hydroxybutyrate (PHB). These polymers can represent up to 80% of total dry mass of the cell, having as main feature the biodegradability in soil and the biocompatibility with animal tissue. Among the microorganisms producing PHAs, the bacterium Azotobacter vinelandii can accumulate large amounts of intracellular PHB with the advantage that they grow a wide sugars variety like those found in molasses cane sugar, beet sugar and corn syrup, and swine waste, agribusiness, etc.. Besides the PHB, the bacterium A. vinelandii is able to produce alginate, a very useful compound in the similar area of type of fruit and imitation as sliced peppers for stuffing olives, onion rings imitation, caviar, meat, fish and marine products imitation, etc.. Given that the main limiting factors for the biopolymers production are mainly associated with the substrates costs and the fact that many microorganisms are pathogenic hindering its acceptance by the community in general, this study aimed to use the pollutant by-products environment (residual oil frying, glycerin, cassava wastewater - "manipueira", vinasse and wastewater industry carbonated beverages or soft drinks) as a substrate for the poly-hydroxybutyrate and alginate production by non-pathogenic bacterium Azotobacter vinelandii. Fermentations... (Complete abstract click electronic access below) / Doutor

Azotobacter vinelandii, grown with ammonium as a nitrogen source, was shown to possess an active transport system which could concentrate ammonium 44 to 58 fold. Ammonium uptake was inhibited by the glutamate analog methionine sulfone. The properties of the ammonium uptake system (transport and metabolism) were investigated using the ammonium analog methylammonium. The uptake of methylammonium was inhibited by arsenate indicating that phosphate bond energy was required. Methylammonium uptake was also inhibited by the electron transport inhibitor, cyanide, and the uncoupler, carbonyl cyanide- m-chlorophenyl hydrazone. However, it was shown that these agents served to deplete ATP pools in A. vinelandii. Uptake of methylammonium was sensitive to a Tris-Mg⁺⁺ shock treatment suggesting the possible involvement of a periplasmic binding protein, however, methylammonium-binding activity was not found in periplasmic extracts. A. vinelandii was shown to exhibit a positive chemotactic response toward ammonium as well as acetate, glucose and sucrose. Comparison of outer membrane proteins from nitrogen-fixing cells and ammonium-grown cells revealed the production of a 44,000 dalton protein in membranes from nitrogen-fixing cells. Inner membranes from nitrogen-fixing cells contained a 41,000 dalton protein which was present in low amounts in the membranes of ammonium-grown cells. It was shown that the outer membranes of ammonium-grown cells contained a major protein which was "heat modifiable" in that its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined by the temperature of solubilization prior to electrophoresis. Methylammonium was shown to be metabolized to N-methylglutamine. Strain JK301, an L-methionine-D,L-sulfoximine-resistant mutant of A. vinelandii, was unable to catalyse N-methylglutamine synthesis in vivo or in cell-free extracts and lacked detectable methylammonium uptake activity. Glutamine synthetase in cell-free extracts of JK301 had a Km for glutamate approximately three-fold higher and a Vmax approximately fourfold lower than enzyme from the wild type strain. It was concluded that methylammonium uptake reflects, in part, metabolism to N-methylglutamine by glutamine synthetase. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The work described in this thesis was undertaken to study the reasons why Azotobacter vinelandii ATCC 12837 after incubation in Burk's nitrogen-free liquid will not form as many colonies when plated on Difco Tryptic Soy Agar as when planted on Burk's nitrogen-free agar. The difference in growth of A. vinelandii on the two agars was established by performing viable cell-plate counts. The difference in growth was most apparent at 24-hours incubation of the Burk's liquid-media cultures. Phase contrast microscopic observations of Tryptic Soy media cultures of A. vinelandii disclosed the regular formation of fungoid cells at early stages of growth of the bacteria, 18 to 24 hours.

Azotobacter vinelandii contains an iron-regulatory small RNA ArrF whose expression is dependent upon the levels of iron and ferric uptake regulator. The deletion of ArrF-encoding gene resulted in a 300old increase in the production of polyhydroxybutyrate (PHB), a polymer of industrial importance. This ∆arrF mutant exhibited wild-type growth and growth-associated PHB production. Limited iron and aeration elevated the PHB production in the mutant as well as wild type. SDS-PAGE and MALDI-MS/MS revealed the overexpression of acetyl-CoA reductase, a phbBAC operon enzyme and the proteins that would alleviate the stress due to PHB accumulation in the ∆arrF mutant. Real-time RT-PCR revealed that phbR, phbB, phbA and phbC were upregulated in the mutant. Increased levels of activator PhbR in the mutant elevates the expression of phbB, phbA and phbC, resulting in the PHB overproduction. The proteins differentially expressed in the ∆arrF mutant were determined by gel-based proteomics and confirmed by real time RT-PCR. 6-phosphogluconolactonase that involve in the production of NADPH and acetyl-CoA, was upregulated, while the proteins involved in the TCA cycle that consumes acetyl-CoA were downregulated. Heat-shock proteins such as HSP20 and GroEL were overexpressed in the mutant. In addition, antioxidant proteins such as Fe-containing supeoxide dismutase (FeSOD), a putative oxidoreductase with unknown function, alkyl hydroperoxide reductase, flavorprotein WrbA and cysteine synthase were also upregulated, indicating that the PHB accumulation is highly stressful to the cells. Upregulated in the ∆arrF mutant were acetyl-CoA carboxylase, flagellin, and adenylate kinase. Among the genes upregulated in the ∆arrF mutant, sodB gene coding for Fe-superoxide dismutase and phbF gene encoding PHB synthesis regulator appears to be negatively regulated by small RNA ArrF in an antisense mechanism. However, all the TCA cycle genes were downregulated in the ∆arrF mutant. In addition to the TCA cycles enzyme, glutamate synthetase, elongation factor-Tu, iron ABC transporter, and major outer membrane porin OprF were downregulated in the ∆arrF mutant. Based on the results, it is concluded that several factors are responsible for the overproduction of PHB polymer in the ∆arrF mutant and one of which is the direct effect of small RNA ArrF on the expression of PhbF .

No description available.

A system was developed for the controlled expression of genes in Azotobacter vinelandii by using genomic fusions to the sucrose catabolic regulon. This system was used for the functional analysis of the A. vinelandii isc genes, whose products are involved in the maturation of [Fe-S] proteins. For this analysis the scrX gene, contained within the sucrose catabolic regulon, was replaced by the A. vinelandii iscS, iscU, iscA, hscB, hscA, fdx, iscX gene cluster, resulting in duplicate genomic copies of these genes, one whose expression is directed by the normal isc regulatory elements (Pisc) and the other whose expression is directed by the scrX promoter (PscrX). Functional analysis of [Fe-S] protein maturation components was achieved by placing a mutation within a particular Pisc-controlled gene with subsequent repression of the corresponding PscrX-controlled component by growth on glucose as the carbon source. This experimental strategy was used to show that IscS, IscU, HscBA and Fdx are essential in A. vinelandii and that their depletion results in a deficiency in the maturation of aconitase, an enzyme that requires a [4Fe-4S] cluster for its catalytic activity. Depletion of IscA results in null growth only when cells are cultured under conditions of elevated oxygen, marking the first null phenotype associated with the loss of a bacterial IscA-type protein. Furthermore, the null growth phenotype of cells depleted for HscBA could be partially reversed by culturing cells under conditions of low oxygen. These results are interpreted to indicate that HscBA and IscA could have functions related to the protection or repair of the primary IscS/IscU machinery when grown under aerobic conditions. Conserved amino acid residues within IscS, IscU, and IscA that are essential for their respective functions and/or display a partial or complete dominant-negative growth phenotype were also identified using this system. Inactivation of the IscR repressor protein resulted in a slow growth phenotype that could be specifically attributed to the elevated expression of an intact [Fe-S] cluster biosynthetic system. This system was also used to investigate the extent to which the two [Fe-S] biosynthetic systems in A. vinelandii, Nif and Isc, can perform overlapping functions. Under normal laboratory growth conditions, no cross-talk between the two systems could be detected. However, elevated expression of Isc components as a consequence of inactivation of the IscR repressor protein results in a modest ability of the Isc [Fe-S] protein maturation components to replace the function of Nif-specific [Fe-S] protein maturation components. Similarly, when expressed at very high levels the Nif-specific [Fe-S] protein maturation components could functionally replace the Isc components. Oxygen levels were also found to affect the ability of the Nif and Isc systems to perform common functions. Nevertheless, the lack of significant reciprocal cross-talk between the Nif and Isc systems when they are produced only at levels necessary to satisfy their respective physiological functions, indicates a high level of target specificity with respect to [Fe-S] protein maturation. / Ph. D.

Previous studies revealed that α-275-Cys provides an essential ligand to one of the Fe atoms on the FeMo-cofactor, and its substitutions resulted in inactive nitrogenase. In order to study the structural-functional relationship of the protein environment in this region with respect to the FeMo-cofactor, subtle changes were introduced through substitutions using a site-directed mutagenesis and gene-replacement method at α-274-His, α-276-Tyr and α-277-Arg in Azotobacter vinelandii nitrogenase. Characterization of mutants strains resulting from amino acid substitutions at residues, α-274-His, α-276-Tyr or α-277-Arg, using activity assays, resulted in mixed Nif phenotypes. Therefore, none of these residues is absolutely required for nitrogenase activity. However, the changed EPR spectra of the altered MoFe proteins from some strains with substitutions at either α-276-Tyr or α-277-Arg indicated that the FeMo-cofactor environment had been perturbed by these substitutions. Together with its changed EPR spectrum, substituting α-277-Arg with His showed some extraordinary catalytic features, such as its inability to reduce N₂ while retaining respectable C₂H₂- and H⁺-reduction activities. It was also found that this altered protein used a higher percentage of total electron flux for H₂ evolution under an C₂H₂/Ar atmosphere than did wild type. Further characterization of the purified α-277^his MoFe protein in parallel with its wild type counterpart revealed that the alteration in the α-277^his MoFe protein caused a lower affinity for C₂H₂ binding, whereas it did not affect the CO binding. Interestingly, CO-induced cooperativity during C₂H₂ reduction was observed in this altered MoFe protein clearly indicating two sites for C₂H₄ evolution, one of which might be in the vicinity of this residue. Furthermore, the α-277^his MoFe protein does not bind or reduce N₂ leading to the proposal of a nonexistent E₄ redox state in the MoFe protein catalytic cycle which was supported by stopped-flow spectrophotometric evidence. This altered α-277^his MoFe protein showed comparable physical stabilities to that of the wild-type protein, and its ATP hydrolysis rates remained constant under a number of substrates assayed. Therefore, the substitution has not affected the overall protein structure, rather, it has changed the local FeMo-cofactor environment. When we studied the purified α-276^his and α-274^gin/α-276^his MoFe proteins and compared the results with the data from the α-277^his and wild-type MoFe protein, we found that there is no direct correlation between the additional set of EPR signals observed in these altered MoFe proteins and their catalytic activities. The current understanding concerning the functionality of these residues is that they are involved in maintaining a proper environment for FeMoco to bind and in stabilizing the different redox states of the enzyme during catalysis. / Ph. D.

Os cofatores de ferro-enxofre [Fe-S] estão entre os mais versáteis e antigos cofatores enzimáticos encontrados na natureza. As células têm explorado as propriedades eletrônicas e estruturais destes cofatores inorgânicos para uma ampla variedade de atividades incluindo a transferência de elétrons, a catálise e a ativação de substratos. Um grande número de proteínas está envolvido na biogênese dos cofatores [Fe-S], e este processo pode ser dividido em três etapas principais: (i) formação do enxofre elementar, (ii) montagem do cofator [Fe-S], e (iii) inserção do cofator em apoproteínas. As plantas realizam fotossíntese e respiração, dois processos que requerem proteínas Fe-S, sendo os únicos organismos em que a biossíntese destas proteínas é compartimentalizada. Diversos fatores afetam o desenvolvimento das plantas, entre eles, a temperatura baixa, fator limitante à produtividade e à distribuição geográfica das plantas, incluindo Eucalyptus grandis, uma espécie com grande importância econômica. Devido a esse fato, foi realizada uma análise transcricional dos genes codificados pelo sistema ISC de biossíntese de cofatores [Fe-S] NFS1, ISA1 e ISU1 de E. grandis por meio de PCR quantitativa (RT-qPCR), após plântulas desta espécie serem submetidas ao tratamento de frio. O gene NFS1 teve sua expressão reprimida nas primeiras 48 horas de tratamento, porém, após esse período observa-se um aumento da expressão gênica em relação à condição controle. O genes ISU1 e ISA1 apresentaram maior expressão gênica nas primeiras duas horas de tratamento, diminuindo drasticamente logo após este período. Foi verificado um aumento na quantidade relativa de Fe e S nos nas plântulas submetidas ao tratamento de frio, indicando um possível aumento na quantidade de cofatores [Fe-S] requeridos para o reestabelecimento da homeostase celular. As bactérias, por sua vez, desenvolveram pelo menos três sistemas de biossíntese, altamente conservados, que estão envolvidos na formação dos cofatores [Fe-S], sendo estes NIF, ISC e SUF. Em muitas proteobactérias, a regulação da produção de cofatores [Fe-S] pelos sistemas ISC e SUF é controlada por uma única proteína, IscR, pertencente à família de reguladores Rrf2. A proteína IscR possui um domínio de ligação ao DNA na região N-terminal e um segundo domínio de ligação de cofatores com três resíduos de cisteínas (Cys) altamente conservados. A ligação de um cofator do tipo [2Fe-2S] reprime a transcrição do seu próprio promotor in vitro. O genoma de Azotobacter vinelandii não inclui um sistema SUF completo e, portanto, permite o estudo dos efeitos da regulação de IscR não relacionada a SUF. No presente trabalho, objetivamos analisar a expressão do operon isc em linhagens selvagens e mutantes para IscR de A. vinelandii por meio das técnicas de sequenciamento do transcritoma e RT-qPCR. As substituições das Cys92, Cys104, His107 e a deleção de 120 pb da região codificadora do segundo domínio de IscR levaram à indução de um aumento da expressão de todo o operon isc. Notou-se também uma diferença fenotípica clara no tamanho das colônias portadoras das substituições de Cys e His, sendo estas menores em relação à linhagem selvagem. As substituições das Cys98 e Cys111, ou ainda a dupla substituição Cys98/111 não levaram a alteração da expressão do operon. A ligação ou não do cofator [Fe-S] é, portanto, responsável pela regulação do operon isc em A. vinelandii, bem como, de outros operons codificadores de proteínas envolvidas em cadeias tranportadoras de elétrons. / The iron-sulfur clusters [Fe-S] are among the oldest and most versatile enzyme cofactors found in nature. The cells have explored the structural and electronic properties of these inorganic clusters for a wide variety of activities including electron transfer, catalysis and activation of substrates. A large number of proteins is involved in the biogenesis of the [Fe-S] clusters, and this process can be divided into three main steps: (i) formation of elemental sulfur, (ii) assembly of the [Fe-S] cluster and (iii) insertion into apoproteins. Plants perform photosynthesis and respiration, two processes that require Fe-S protein, and in these organisms the synthesis of these proteins is compartmentalized. Several factors affect the development of plants, among them, the low temperature is a limiting factor to productivity and geographical distribution of plants, including Eucalyptus grandis, a specie with great economic importance. Due to this fact, we performed a transcriptional analysis by quantitative PCR (RT-qPCR) of the genes encoded by the E. grandis [Fe-S] cluster ISC system NFS1, ISA1 and ISU1 after seedlings were submitted to the chilling treatment. The NFS1 gene expression is repressed in the first 48 hours of treatment, but after this period there was an increase in gene expression relating to the control condition. The genes ISU1 and ISA1 showed higher gene expression in the first two hours of treatment, followed by a sharp decrease. There was an increase in the relative amount of Fe and S in the seedlings subjected to cold treatment, indicating a possible increase in the amount of [Fe-S] clusters, required for the reestablishment of cellular homeostasis. Bacteria have developed at least three synthesis systems, highly conserved, which are involved in the formation of Fe-S proteins, NIF, ISC and SUF. In many proteobacteria, the regulation of clusters production by ISC and SUF is controlled by a single protein, IscR, belonging to the Rrf2 regulators family. The protein IscR has a DNA binding site at the N-terminal domain and second cofactors binding domain with three cysteine residues (Cys) highly conserved. The binding of a [2Fe-2S] cluster represses the transcription of its own promoter in vitro. The genome of Azotobacter vinelandii does not include a full SUF system and thus permits the study of the effects of IscR regulation unrelated to SUF. In this study, the aim was to analyze the expression of isc operon in wild type and mutant strains of A. vinelandii IscR by the techniques of the transcriptome sequencing and qRT-PCR. The replacement of Cys92, Cys104, His107 and a deletion of 120 bp region encoding the second IscR domain led to an increased expression of the whole isc operon. It also showed a clear phenotypic difference in colonies size in the strains carrying the substitutions of His and Cys, it was smaller compared to the wild type strain. The replacement of Cys98 and Cys111, or the double substitution Cys98/111 not led to an altered operon expression. The [Fe-S] cluster binding or not, is therefore responsible for the regulation of the isc operon in A. vinelandii as well as of other operons encoding proteins involved in electron tranport chains.

Os cofatores de ferro-enxofre [Fe-S] estão entre os mais versáteis e antigos cofatores enzimáticos encontrados na natureza. As células têm explorado as propriedades eletrônicas e estruturais destes cofatores inorgânicos para uma ampla variedade de atividades incluindo a transferência de elétrons, a catálise e a ativação de substratos. Um grande número de proteínas está envolvido na biogênese dos cofatores [Fe-S], e este processo pode ser dividido em três etapas principais: (i) formação do enxofre elementar, (ii) montagem do cofator [Fe-S], e (iii) inserção do cofator em apoproteínas. As plantas realizam fotossíntese e respiração, dois processos que requerem proteínas Fe-S, sendo os únicos organismos em que a biossíntese destas proteínas é compartimentalizada. Diversos fatores afetam o desenvolvimento das plantas, entre eles, a temperatura baixa, fator limitante à produtividade e à distribuição geográfica das plantas, incluindo Eucalyptus grandis, uma espécie com grande importância econômica. Devido a esse fato, foi realizada uma análise transcricional dos genes codificados pelo sistema ISC de biossíntese de cofatores [Fe-S] NFS1, ISA1 e ISU1 de E. grandis por meio de PCR quantitativa (RT-qPCR), após plântulas desta espécie serem submetidas ao tratamento de frio. O gene NFS1 teve sua expressão reprimida nas primeiras 48 horas de tratamento, porém, após esse período observa-se um aumento da expressão gênica em relação à condição controle. O genes ISU1 e ISA1 apresentaram maior expressão gênica nas primeiras duas horas de tratamento, diminuindo drasticamente logo após este período. Foi verificado um aumento na quantidade relativa de Fe e S nos nas plântulas submetidas ao tratamento de frio, indicando um possível aumento na quantidade de cofatores [Fe-S] requeridos para o reestabelecimento da homeostase celular. As bactérias, por sua vez, desenvolveram pelo menos três sistemas de biossíntese, altamente conservados, que estão envolvidos na formação dos cofatores [Fe-S], sendo estes NIF, ISC e SUF. Em muitas proteobactérias, a regulação da produção de cofatores [Fe-S] pelos sistemas ISC e SUF é controlada por uma única proteína, IscR, pertencente à família de reguladores Rrf2. A proteína IscR possui um domínio de ligação ao DNA na região N-terminal e um segundo domínio de ligação de cofatores com três resíduos de cisteínas (Cys) altamente conservados. A ligação de um cofator do tipo [2Fe-2S] reprime a transcrição do seu próprio promotor in vitro. O genoma de Azotobacter vinelandii não inclui um sistema SUF completo e, portanto, permite o estudo dos efeitos da regulação de IscR não relacionada a SUF. No presente trabalho, objetivamos analisar a expressão do operon isc em linhagens selvagens e mutantes para IscR de A. vinelandii por meio das técnicas de sequenciamento do transcritoma e RT-qPCR. As substituições das Cys92, Cys104, His107 e a deleção de 120 pb da região codificadora do segundo domínio de IscR levaram à indução de um aumento da expressão de todo o operon isc. Notou-se também uma diferença fenotípica clara no tamanho das colônias portadoras das substituições de Cys e His, sendo estas menores em relação à linhagem selvagem. As substituições das Cys98 e Cys111, ou ainda a dupla substituição Cys98/111 não levaram a alteração da expressão do operon. A ligação ou não do cofator [Fe-S] é, portanto, responsável pela regulação do operon isc em A. vinelandii, bem como, de outros operons codificadores de proteínas envolvidas em cadeias tranportadoras de elétrons. / The iron-sulfur clusters [Fe-S] are among the oldest and most versatile enzyme cofactors found in nature. The cells have explored the structural and electronic properties of these inorganic clusters for a wide variety of activities including electron transfer, catalysis and activation of substrates. A large number of proteins is involved in the biogenesis of the [Fe-S] clusters, and this process can be divided into three main steps: (i) formation of elemental sulfur, (ii) assembly of the [Fe-S] cluster and (iii) insertion into apoproteins. Plants perform photosynthesis and respiration, two processes that require Fe-S protein, and in these organisms the synthesis of these proteins is compartmentalized. Several factors affect the development of plants, among them, the low temperature is a limiting factor to productivity and geographical distribution of plants, including Eucalyptus grandis, a specie with great economic importance. Due to this fact, we performed a transcriptional analysis by quantitative PCR (RT-qPCR) of the genes encoded by the E. grandis [Fe-S] cluster ISC system NFS1, ISA1 and ISU1 after seedlings were submitted to the chilling treatment. The NFS1 gene expression is repressed in the first 48 hours of treatment, but after this period there was an increase in gene expression relating to the control condition. The genes ISU1 and ISA1 showed higher gene expression in the first two hours of treatment, followed by a sharp decrease. There was an increase in the relative amount of Fe and S in the seedlings subjected to cold treatment, indicating a possible increase in the amount of [Fe-S] clusters, required for the reestablishment of cellular homeostasis. Bacteria have developed at least three synthesis systems, highly conserved, which are involved in the formation of Fe-S proteins, NIF, ISC and SUF. In many proteobacteria, the regulation of clusters production by ISC and SUF is controlled by a single protein, IscR, belonging to the Rrf2 regulators family. The protein IscR has a DNA binding site at the N-terminal domain and second cofactors binding domain with three cysteine residues (Cys) highly conserved. The binding of a [2Fe-2S] cluster represses the transcription of its own promoter in vitro. The genome of Azotobacter vinelandii does not include a full SUF system and thus permits the study of the effects of IscR regulation unrelated to SUF. In this study, the aim was to analyze the expression of isc operon in wild type and mutant strains of A. vinelandii IscR by the techniques of the transcriptome sequencing and qRT-PCR. The replacement of Cys92, Cys104, His107 and a deletion of 120 bp region encoding the second IscR domain led to an increased expression of the whole isc operon. It also showed a clear phenotypic difference in colonies size in the strains carrying the substitutions of His and Cys, it was smaller compared to the wild type strain. The replacement of Cys98 and Cys111, or the double substitution Cys98/111 not led to an altered operon expression. The [Fe-S] cluster binding or not, is therefore responsible for the regulation of the isc operon in A. vinelandii as well as of other operons encoding proteins involved in electron tranport chains.

Os cofatores de ferro-enxofre [Fe-S] estão entre os mais versáteis e antigos cofatores enzimáticos encontrados na natureza. As células têm explorado as propriedades eletrônicas e estruturais destes cofatores inorgânicos para uma ampla variedade de atividades incluindo a transferência de elétrons, a catálise e a ativação de substratos. Um grande número de proteínas está envolvido na biogênese dos cofatores [Fe-S], e este processo pode ser dividido em três etapas principais: (i) formação do enxofre elementar, (ii) montagem do cofator [Fe-S], e (iii) inserção do cofator em apoproteínas. As plantas realizam fotossíntese e respiração, dois processos que requerem proteínas Fe-S, sendo os únicos organismos em que a biossíntese destas proteínas é compartimentalizada. Diversos fatores afetam o desenvolvimento das plantas, entre eles, a temperatura baixa, fator limitante à produtividade e à distribuição geográfica das plantas, incluindo Eucalyptus grandis, uma espécie com grande importância econômica. Devido a esse fato, foi realizada uma análise transcricional dos genes codificados pelo sistema ISC de biossíntese de cofatores [Fe-S] NFS1, ISA1 e ISU1 de E. grandis por meio de PCR quantitativa (RT-qPCR), após plântulas desta espécie serem submetidas ao tratamento de frio. O gene NFS1 teve sua expressão reprimida nas primeiras 48 horas de tratamento, porém, após esse período observa-se um aumento da expressão gênica em relação à condição controle. O genes ISU1 e ISA1 apresentaram maior expressão gênica nas primeiras duas horas de tratamento, diminuindo drasticamente logo após este período. Foi verificado um aumento na quantidade relativa de Fe e S nos nas plântulas submetidas ao tratamento de frio, indicando um possível aumento na quantidade de cofatores [Fe-S] requeridos para o reestabelecimento da homeostase celular. As bactérias, por sua vez, desenvolveram pelo menos três sistemas de biossíntese, altamente conservados, que estão envolvidos na formação dos cofatores [Fe-S], sendo estes NIF, ISC e SUF. Em muitas proteobactérias, a regulação da produção de cofatores [Fe-S] pelos sistemas ISC e SUF é controlada por uma única proteína, IscR, pertencente à família de reguladores Rrf2. A proteína IscR possui um domínio de ligação ao DNA na região N-terminal e um segundo domínio de ligação de cofatores com três resíduos de cisteínas (Cys) altamente conservados. A ligação de um cofator do tipo [2Fe-2S] reprime a transcrição do seu próprio promotor in vitro. O genoma de Azotobacter vinelandii não inclui um sistema SUF completo e, portanto, permite o estudo dos efeitos da regulação de IscR não relacionada a SUF. No presente trabalho, objetivamos analisar a expressão do operon isc em linhagens selvagens e mutantes para IscR de A. vinelandii por meio das técnicas de sequenciamento do transcritoma e RT-qPCR. As substituições das Cys92, Cys104, His107 e a deleção de 120 pb da região codificadora do segundo domínio de IscR levaram à indução de um aumento da expressão de todo o operon isc. Notou-se também uma diferença fenotípica clara no tamanho das colônias portadoras das substituições de Cys e His, sendo estas menores em relação à linhagem selvagem. As substituições das Cys98 e Cys111, ou ainda a dupla substituição Cys98/111 não levaram a alteração da expressão do operon. A ligação ou não do cofator [Fe-S] é, portanto, responsável pela regulação do operon isc em A. vinelandii, bem como, de outros operons codificadores de proteínas envolvidas em cadeias tranportadoras de elétrons. / The iron-sulfur clusters [Fe-S] are among the oldest and most versatile enzyme cofactors found in nature. The cells have explored the structural and electronic properties of these inorganic clusters for a wide variety of activities including electron transfer, catalysis and activation of substrates. A large number of proteins is involved in the biogenesis of the [Fe-S] clusters, and this process can be divided into three main steps: (i) formation of elemental sulfur, (ii) assembly of the [Fe-S] cluster and (iii) insertion into apoproteins. Plants perform photosynthesis and respiration, two processes that require Fe-S protein, and in these organisms the synthesis of these proteins is compartmentalized. Several factors affect the development of plants, among them, the low temperature is a limiting factor to productivity and geographical distribution of plants, including Eucalyptus grandis, a specie with great economic importance. Due to this fact, we performed a transcriptional analysis by quantitative PCR (RT-qPCR) of the genes encoded by the E. grandis [Fe-S] cluster ISC system NFS1, ISA1 and ISU1 after seedlings were submitted to the chilling treatment. The NFS1 gene expression is repressed in the first 48 hours of treatment, but after this period there was an increase in gene expression relating to the control condition. The genes ISU1 and ISA1 showed higher gene expression in the first two hours of treatment, followed by a sharp decrease. There was an increase in the relative amount of Fe and S in the seedlings subjected to cold treatment, indicating a possible increase in the amount of [Fe-S] clusters, required for the reestablishment of cellular homeostasis. Bacteria have developed at least three synthesis systems, highly conserved, which are involved in the formation of Fe-S proteins, NIF, ISC and SUF. In many proteobacteria, the regulation of clusters production by ISC and SUF is controlled by a single protein, IscR, belonging to the Rrf2 regulators family. The protein IscR has a DNA binding site at the N-terminal domain and second cofactors binding domain with three cysteine residues (Cys) highly conserved. The binding of a [2Fe-2S] cluster represses the transcription of its own promoter in vitro. The genome of Azotobacter vinelandii does not include a full SUF system and thus permits the study of the effects of IscR regulation unrelated to SUF. In this study, the aim was to analyze the expression of isc operon in wild type and mutant strains of A. vinelandii IscR by the techniques of the transcriptome sequencing and qRT-PCR. The replacement of Cys92, Cys104, His107 and a deletion of 120 bp region encoding the second IscR domain led to an increased expression of the whole isc operon. It also showed a clear phenotypic difference in colonies size in the strains carrying the substitutions of His and Cys, it was smaller compared to the wild type strain. The replacement of Cys98 and Cys111, or the double substitution Cys98/111 not led to an altered operon expression. The [Fe-S] cluster binding or not, is therefore responsible for the regulation of the isc operon in A. vinelandii as well as of other operons encoding proteins involved in electron tranport chains.