RESUMO
Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone.
Assuntos
Aldeído Desidrogenase/metabolismo , Citocromos b/metabolismo , Citocromos c/metabolismo , Gluconacetobacter/enzimologia , Cofator PQQ/química , Aldeído Desidrogenase/química , Aldeído Desidrogenase/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular , Citocromos b/química , Citocromos c/química , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , NADH NADPH Oxirredutases/metabolismo , OxirreduçãoRESUMO
AIMS: Gluconacetobacter xylinum is well known for its ability to produce large amounts of cellulose, however, little is known about its cell physiology. Our goal was to study the respiratory metabolism and components of the respiratory system of this bacterium in static cultures. To reach our goal, a medium formulation had to be designed to improve cell growth and cellulose production together with a novel method for the recovery of cells from cellulose pellicles. METHODS AND RESULTS: Successive modifications of a nutrient medium improved G. xylinum cell growth 4.5-fold under static culture conditions. A blender homogenization procedure for the releasing of cells from the cellulose matrix gave a high yield of cells recovered. Respiratory activities of purified cells were greatly stimulated by exogenous substrates and showed to be resistant to KCN. Unexpectedly, exogenous NADH was oxidized at high rates. Cytochromes a, b, c and d were identified after spectral analyses. CONCLUSIONS: Partial bioenergetic characterization of G. xylinum cells allowed us to propose a scheme for its respiratory system. In addition, the growth medium for biomass production and the procedure for the efficient recovery of cells from cellulose pellicles were significantly improved. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the first-ever bioenergetic characterization of G. xylinum grown in static cultures. In addition, a novel methodology to obtain purified cells in suitable quantities for biochemical research is described.
Assuntos
Celulose , Gluconacetobacter xylinus/fisiologia , Monóxido de Carbono/metabolismo , Meios de Cultura , Citocromos/metabolismo , Metabolismo Energético/fisiologia , Inibidores Enzimáticos/farmacologia , Gluconacetobacter xylinus/efeitos dos fármacos , Gluconacetobacter xylinus/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , NAD/metabolismo , Oxirredução , Cianeto de Potássio/farmacologiaRESUMO
Heterogeneous populations of mitochondria have been described in helminths. Mitochondria from different tissues have been isolated in adult organisms. However, in larvae, due to their small size, isolation from tissues has not been feasible. A method for the isolation of tegumental mitochondria from the larval stage of Taenia crassiceps is described. After solubilization of the plasma membrane with saponin, tegumental mitochondria were purified by a simple and rapid protocol of differential centrifugation, which allowed the retention of suitable quantities of well-preserved mitochondria, as judged by biochemical and ultrastructural parameters. Respiratory activity evoked by exogenous NADH was negligible, but its oxidation increased several-fold after sonication of intact mitochondria. Other substrates, e.g., succinate and malate-glutamate, were oxidized at high rate, leading to the formation of a H+ gradient across the inner mitochondrial membrane, which, in turn, supported oxidative phosphorylation. These results indicate that tegumental mitochondria carry out aerobic metabolism.
Assuntos
Cysticercus/ultraestrutura , Mitocôndrias/metabolismo , Animais , Fracionamento Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Cysticercus/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Malatos/metabolismo , Potenciais da Membrana , Camundongos , Microscopia Eletrônica , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , NAD/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio , Saponinas/farmacologia , Solubilidade , Succinato Desidrogenase/análise , Succinatos/metabolismoRESUMO
The cycHJKL gene locus was cloned from Rhizobium etli by the rescue of a Tn5mob insertion of a mutant (IFC01) which was affected in the production of c-type cytochromes. The cycH, cycJ, cycK and cycL genes are proposed to code for different subunits of a haem lyase complex involved in the attachment of haem to cytochrome c apoproteins. CycH of 365 aa shared 27, 36, 47 and 63% identity with CycH from Paracoccus denitrificans, Bradyrhizobium japonicum, R. meliloti, and R. leguminosarum, respectively. CycJ of 153 aa shared 52, 71, and 85% identity to the cycJ gene product of B. japonicum, R. meliloti, R. leguminosarum, respectively. CycK of 666 aa shared 62, 73, and 90% homology with CycK from B. japonicum, R. meliloti, and R. leguminosarum, respectively, while CycL of 151 aa shared 57, 67 and 86% hómology with CycL from the abovementioned species. The Tn5mob insertion present in the IFC01 strain was located in the cycH gene. This strain was able to infect bean plants, but unable to fix nitrogen during symbiosis. A previously described R. etli cytochrome c-deficient MuD1lac-induced mutant (CFN4202) that induced empty nodules on Phaseolus vulgaris, also have lesions in cycH. Complementation analysis suggested that the MuD1lac insertion of the CFN4202 strain was polar on expression of genes downstream of cycH in contrast with the Tn5mob insertion present in IFC01, which showed no polarity on cycJKL. Our data suggest that CycH may not be essential for the infection process, but is necessary for nitrogen fixation.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Grupo dos Citocromos c/biossíntese , Proteínas de Membrana , Rhizobium/genética , Proteínas de Bactérias/biossíntese , Mapeamento Cromossômico , Clonagem Molecular , Grupo dos Citocromos c/genética , Genes Bacterianos , Mutagênese Insercional , Óperon , Paracoccus denitrificans/genética , Proteínas Recombinantes/biossíntese , Rhizobium/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The biosynthetic pathway for myo-inositol consist of two enzymatic steps: first, the cycloaldolization of glucose-6P to L-myo-inositol-IP followed by its hydrolysis to form free myo-inositol. The former reaction is catalyzed by myo-inositol-IP synthase (MIPS) while, a phosphatase is responsible for the hydrolysis step. Depending on its degree of purification and storage age, MIPS activity us to be, from partial to fully, dependent on added NAD. Therefore, we decided to study the kinetic properties of the enzyme within the cell, specially its requirements for free NAD. To this purpose, a method was designed for the assay of MIPS-activity in situ, using toluene permeabilized mycelia. MIPS-activity "in situ" was fully displayed in the absence of added NAD; on the contrary, the purified enzyme showed only 33% of that activity displayed when NAD was included in the assay. Thus, it seems that the native enzyme contains tightly bound NAD, instrumental for its activity, and that during purification or storage, the coenzyme is progressively lost, rendering the NAD-dependent enzyme, as was previously envisage. In addition, the in situ assay method for MIP-Synthase was applied to several mutants of N. crassa having the inosphenotype. Our results showed that only in 3 of 14 cases analyzed the phenotype could be clearly associated to the lack of MIP-synthase activity. Indeed most of the mutants analyzed showed significant levels (from 5 to 21%) of MIP-synthase, when compared to the activity shown by the RL-21 WT strain. Finally, all the mutants and WT strains were zymographically analyzed for phosphatase activity and showed close to equal strong reaction levels.