RESUMO
The physiological role of the membrane-bound pyrophosphatase of Rhodospirillum rubrum was investigated by the characterization of a mutant strain. Comparisons of growth levels between the wild type and the mutant under different low-potential conditions and during transitions between different metabolisms indicate that this enzyme provides R. rubrum with an alternative energy source that is important for growth in low-energy states.
Assuntos
Pirofosfatase Inorgânica/fisiologia , Rhodospirillum rubrum/enzimologia , Trifosfato de Adenosina/metabolismo , Aerobiose , Metabolismo Energético , Luz , Fotossíntese , Rhodospirillum rubrum/crescimento & desenvolvimentoRESUMO
Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions.
Assuntos
Azospirillum brasilense/genética , Regulação Bacteriana da Expressão Gênica , N-Glicosil Hidrolases , Nitrogenase/biossíntese , Processamento de Proteína Pós-Traducional , Compostos de Amônio Quaternário/farmacologia , ADP Ribose Transferases , Azospirillum brasilense/efeitos dos fármacos , Azospirillum brasilense/enzimologia , Dinitrogenase Redutase/genética , Regulação Enzimológica da Expressão Gênica , Genes Bacterianos , Glicosídeo Hidrolases , Mutagênese Sítio-Dirigida , Fixação de Nitrogênio/genética , Rhodospirillum rubrum/enzimologia , Rhodospirillum rubrum/genética , Especificidade da EspécieRESUMO
Reversible ADP ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase activating glycohydrolase (DRAG) regulatory system, has been characterized in both Rhodospirillum rubrum and Azospirillum brasilense. Although the general functions of DRAT and DRAG are very similar in these two organisms, there are a number of interesting differences, e.g., in the timing and extent of the regulatory response to different stimuli. In this work, the basis of these differences has been studied by the heterologous expression of either draTG or nifH from A. brasilense in R. rubrum mutants that lack these genes, as well as the expression of draTG from R. rubrum in an A. brasilense draTG mutant. In general, these hybrid strains respond to stimuli in a manner similar to that of the wild-type parent of the recipient strain rather than the wild-type source of the introduced genes. These results suggest that the differences seen in the regulatory response in these organisms are not primarily a result of different properties of DRAT, DRAG, or dinitrogenase reductase. Instead, the differences are likely the result of different signal pathways that regulate DRAG and DRAT activities in these two organisms. Our results also suggest that draT and draG are cotranscribed in A. brasilense.
Assuntos
ADP Ribose Transferases/genética , Azospirillum brasilense/genética , Regulação Bacteriana da Expressão Gênica , Glicosídeo Hidrolases/genética , N-Glicosil Hidrolases , Rhodospirillum rubrum/genética , ADP Ribose Transferases/biossíntese , Adenosina Difosfato Ribose/metabolismo , Azospirillum brasilense/enzimologia , Azospirillum brasilense/efeitos da radiação , Escuridão , Dinitrogenase Redutase/metabolismo , Regulação Enzimológica da Expressão Gênica , Glicosídeo Hidrolases/biossíntese , Luz , Fixação de Nitrogênio/fisiologia , Nitrogenase/metabolismo , Compostos de Amônio Quaternário/farmacologia , Rhodospirillum rubrum/enzimologia , Rhodospirillum rubrum/efeitos da radiação , Especificidade da Espécie , Transcrição GênicaRESUMO
A comparative study of the citrate synthases purified from the facultatively photosynthetic bacterium Rhodospirillum rubrum (Gram negative) and the thermophile Bacillus stearothermophilus (Gram positive) was made. The citrate synthase from R. rubrum was activated by KCl (6-fold at 0.1 M KCl) and, less effectively, by NaCl and NH4Cl. Its molecular weight was about 300,000. The enzyme was strongly inhibited by NADH, and this inhibition was counteracted by AMP. The citrate synthase from B. stearothermophilus was little affected by KCl, NaCl and NH4Cl, all of which activated by about 25% at 0.1 M. Its molecular weight was ca 100,000. The enzyme was not affected by NADH or AMP. Both citrate synthases were insensitive to alpah-oxoglutarate concentrations up to 5 mM, and were inhibited by ATP; the B. stearothermophilus enzyme was more strongly inhibited than the R. rubrum enzyme. In both cases the ATP inhibition was strictly competitive towards acetyl-CoA and non-competitive towards oxaloacetate. Both enzymes, in spite of the peculiar physiological properties of their bacterial sources, followed the close correlation between the properties of the citrate synthase and the taxonomical position of the microorganism, proposed by Weitzman and his co-workers.