scholarly journals Adenine nucleotide levels in Rhodospirillum rubrum during switch-off of whole-cell nitrogenase activity

1984 ◽  
Vol 224 (3) ◽  
pp. 961-969 ◽  
Author(s):  
T D Paul ◽  
P W Ludden
Keyword(s):  
Protein Modification ◽  
Rhodospirillum Rubrum ◽  
Adenine Nucleotide ◽  
Nitrogenase Activity ◽  
Energy Charge ◽  
Whole Cell ◽  
Fe Protein ◽  
Nucleotide Pools ◽  
Phenazine Methosulphate

Adenine nucleotide pools were measured in Rhodospirillum rubrum cultures that contained nitrogenase. The average energy charge [([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP])] was found to be 0.66 and 0.62 in glutamate-grown and N-limited cultures respectively. Treatment of glutamate-grown cells with darkness, ammonia, glutamine, carbonyl cyanide m-chlorophenylhydrazone, or phenazine methosulphate resulted in perturbations in the adenine nucleotide pools, and led to loss of whole-cell nitrogenase activity and modification in vivo of the Fe protein. Treatment of N-limited cells resulted in similar changes in adenine nucleotide pools but not enzyme modification. No correlations were found between changes in adenine nucleotide pools or ratios of these pools and switch-off of nitrogenase activity by Fe protein modification in vivo. Phenazine methosulphate inhibited whole-cell activity at low concentrations. The effect on nitrogenase activity was apparently independent of Fe protein modification.

scholarly journals Characterization of the DraT/DraG System for Posttranslational Regulation of Nitrogenase in the Endophytic Betaproteobacterium Azoarcus sp. Strain BH72

2009 ◽  
Vol 191 (11) ◽  
pp. 3726-3735 ◽  
Author(s):  
Janina Oetjen ◽  
Barbara Reinhold-Hurek
Keyword(s):  
Blot Analysis ◽  
Protein Modification ◽  
Nitrogenase Activity ◽  
Posttranslational Regulation ◽  
Fe Protein ◽  
Grass Endophyte ◽  
Genes Encoding ◽  
Microaerobic Conditions ◽  
First Time

ABSTRACT DraT/DraG-mediated posttranslational regulation of the nitrogenase Fe protein by ADP-ribosylation has been described for a few diazotrophic bacteria belonging to the class Alphaproteobacteria. Here we present for the first time the DraT/DraG system of a betaproteobacterium, Azoarcus sp. strain BH72, a diazotrophic grass endophyte. Its genome harbors one draT ortholog and two physically unlinked genes coding for ADP-ribosylhydrolases. Northern blot analysis revealed cotranscription of draT with two genes encoding hypothetical proteins. Furthermore, draT and draG2 were expressed under all studied conditions, whereas draG1 expression was nitrogen regulated. By using Western blot analysis of deletion mutants and nitrogenase assays in vivo, we demonstrated that DraT is required for the nitrogenase Fe protein modification but not for the physiological inactivation of nitrogenase activity. A second mechanism responsible for nitrogenase inactivation must operate in this bacterium, which is independent of DraT. Fe protein demodification was dependent mainly on DraG1, corroborating the assumption from phylogenetic analysis that DraG2 might be mostly involved in processes other than the posttranslational regulation of nitrogenase. Nitrogenase in vivo reactivation was impaired in a draG1 mutant and a mutant lacking both draG alleles after anaerobiosis shifts and subsequent adjustment to microaerobic conditions, suggesting that modified dinitrogenase reductase was inactive. Our results demonstrate that the DraT/DraG system, despite some differences, is functionally conserved in diazotrophic proteobacteria.

Regulation of nitrogenase in the photosynthetic bacteriumRhodobacter sphaeroidescontainingdraTGandnifHDKgenes fromRhodobacter capsulatus

2001 ◽  
Vol 47 (3) ◽  
pp. 206-212 ◽  
Author(s):  
Alexander F Yakunin ◽  
Alexander S Fedorov ◽  
Tatyana V Laurinavichene ◽  
Vadim M Glaser ◽  
Nikolay S Egorov ◽  
...  
Keyword(s):  
Photosynthetic Bacteria ◽  
Rhodospirillum Rubrum ◽  
Rhodobacter Capsulatus ◽  
Nitrogenase Activity ◽  
Photosynthetic Bacterium ◽  
Adp Ribosylation ◽  
Fe Protein ◽  
Dinitrogenase Reductase ◽  
Different Strains

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.Key words: nitrogenase regulation, nitrogenase modification, photosynthetic bacteria.

Changes in nucleotide pools during conidial germination in Neurospora crassa

1981 ◽  
Vol 59 (11-12) ◽  
pp. 899-905 ◽  
Author(s):  
Richard L. Sabina ◽  
Paulette Dalke ◽  
Alan R. Hanks ◽  
Jane M. Magill ◽  
Clint W. Magill
Keyword(s):  
Neurospora Crassa ◽  
Adenine Nucleotide ◽  
Energy Charge ◽  
Fold Increase ◽  
Conidial Germination ◽  
Adenylate Energy Charge ◽  
Wild Type ◽  
Nucleotide Pools ◽  
Nucleotide Sugars ◽  
Purine Pathway

The acid-soluble nucleotide pools of wild type and several adenine auxotrophs of Neurospora crassa were studied immediately prior to and during conidial germination in the presence of adenine. A two- to four-fold increase in most nucleotide pools was observed after 6 h of germination at 33 °C indicating a general increase in nucleotide pools during this developmental period. The largest components of the acid-soluble nucleotide pools were uracil-containing nucleotide–sugars, which are precursors of chitin and glucan, the major constituents of cell walls. On removal of adenine, the UDP–sugar pools decreased significantly, in adenine auxotrophs, while the pools of UTP increased significantly. ATP levels increased approximately twofold in the first 6 h of germination. After 1 h without exogenous adenine, ATP dropped twofold or more in adenine auxotrophs but not in wild type. There was a net decrease in all adenine nucleotide pools during adenine starvation and a much greater decrease was seen in adenine auxotrophs than in wild type. The adenylate energy charge remained stable despite major changes in the adenylate pools.Accumulation of intermediates was observed in germinated conidia from purine auxotrophs blocked at various steps in the purine pathway. IMP accumulated in ungerminated and in starved conidia of the adenine-8 (ad-8) strain. Ungerminated conidia of the ad-5 strain contained a large pool of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) while the ad-1 strain had smaller amounts of AICAR, but significantly more than any other strain tested. The AICAR pools disappeared from ad-5 and ad-1 in the presence of 50 mg histidine/100 mL. Similarly the IMP pools in ad-8 decreased markedly in the presence of histidine, indicating that the contribution from the histidine biosynthetic pathway to purine nucleotide formation is significant.

Changes in the adenylate energy charge ofNematospiroides dubiusandTrichostrongylus colubriformisparalysed by levamisolein vivo

Parasitology ◽  
1980 ◽  
Vol 81 (3) ◽  
pp. 593-601 ◽  
Author(s):  
M. J. Sharpe
Keyword(s):  
Oxygen Tension ◽  
Adenine Nucleotide ◽  
Energy Charge ◽  
Adenylate Energy Charge ◽  
Laboratory Mice ◽  
Trichostrongylus Colubriformis ◽  
Nucleotide Content ◽  
Nematospiroides Dubius ◽  
Low Levels

SUMMARYThe adenine nucleotide content and adenylate energy charge ofNematospiroides dubiusfrom laboratory mice and ofTrichostrongylus colubriformisfrom lambs has been measured. Administration of the anthelmintic, levamisole, to infected hosts resulted in only a slight fall in the adenylate energy charge ofN. dubiusover a 3-h period but there was a greater fall in the adenylate energy charge ofT. colubriformisduring this period. In neither case did the energy charge fall quickly, nor did it fall to the low levels which would be expected if the levamisole were inhibiting synthesis of ATP. The changes in energy charge of the nematodes which occurred following administration of levamisole to their hosts was of the order which can be satisfactorily explained by changes in the environment of the nematodes, such as reduced oxygen tension. It is concluded that the maintenance of levamisole-induced paralysis of these two species of trichostrongylein vivodoes not rely on the inhibition of fumarate reductase.

scholarly journals Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

2001 ◽  
Vol 183 (1) ◽  
pp. 250-256 ◽  
Author(s):  
Yan Ma ◽  
Paul W. Ludden
Keyword(s):  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria.Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of thenifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding.

Hepatotrophic effects of insulin on glucose, glycogen, and adenine nucleotides in hepatocytes isolated from fed adult rats

1980 ◽  
Vol 58 (10) ◽  
pp. 1004-1011 ◽  
Author(s):  
Khursheed N. Jeejeebhoy ◽  
Joseph Ho ◽  
Rajni Mehra ◽  
Alan Bruce-Robertson
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Close Correlation ◽  
Adult Rats ◽  
Fed State ◽  
Adenine Nucleotide Pool ◽  
Intracellular Glucose

In vivo observations have suggested that there is an hepatotrophic effect of insulin. By contrast, subsequent in vitro work, using the isolated perfused liver system, showed no effect or indeterminate effects of insulin on the transport of glucose into the hepatocyte. However because this system may not have endured long enough to show such an influence we explored the transport of glucose using a 48-h suspension culture of hepatocytes isolated from young adult fed rats, the suspension being infused continuously with insulin at a rate approximating the maximum entering portal blood in the fed state. (In a separate study phloridzin was added after 2 h of incubation.) DNA, intracellular glucose and its inward transport, glycogen, and the adenine nucleotides were measured at intervals. By comparison with control or untreated cells, insulin-treated cells showed significantly more DNA and intracellular glucose, and the differences were abolished by phloridzin. Glucose transport rates fell to low values in untreated controls and still lower with insulin plus phloridzin. but the initial rate was maintained to the end (48 h) by insulin alone. Results for glycogen were similar to those for intracellular glucose. There was a close correlation (r = 0.96) between these two. The total adenine nucleotide pool and the concentration of ATP were maintained for about 24 h and fell to half their initial values by 48 h. Insulin had increased these concentrations significantly by 6 h. Although concentrations of ADP and AMP decreased gradually in all groups of cells, insulin enhanced the level of ADP by 12 h but had no measurable effect on that of AMP. The energy charge increased slightly throughout incubation but more so (by 6 h) in the presence of insulin. In conclusion the data support the concept that in the longer term (> 12 h) insulin in the portal circulation maintains the characteristic free permeability of the hepatocyte to glucose and this permits a variety of effects related to glucose entry into the hepatocyte.

Effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum in darkness

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1834-1840 ◽  
Author(s):  
Tiago Toscano Selao ◽  
Tomas Edgren ◽  
He Wang ◽  
Agneta Norén ◽  
Stefan Nordlund
Keyword(s):  
Nitrogen Source ◽  
Metabolic Regulation ◽  
Rhodospirillum Rubrum ◽  
De Novo ◽  
Nitrogenase Activity ◽  
Signal Transduction Pathway ◽  
Nitrogen Sources ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Fe Protein

Rhodospirillum rubrum, a photosynthetic diazotroph, is able to regulate nitrogenase activity in response to environmental factors such as ammonium ions or darkness, the so-called switch-off effect. This is due to reversible modification of the Fe-protein, one of the two components of nitrogenase. The signal transduction pathway(s) in this regulatory mechanism is not fully understood, especially not in response to darkness. We have previously shown that the switch-off response and metabolic state differ between cells grown with dinitrogen or glutamate as the nitrogen source, although both represent poor nitrogen sources. In this study we show that pyruvate affects the response to darkness in cultures grown with glutamate as nitrogen source, leading to a response similar to that in cultures grown with dinitrogen. The effects are related to PII protein uridylylation and glutamine synthetase activity. We also show that pyruvate induces de novo protein synthesis and that inhibition of pyruvate formate-lyase leads to loss of nitrogenase activity in the dark.

Expression of the activating enzyme and Fe protein of nitrogenase from Rhodospirillum rubrum

1982 ◽  
Vol 152 (2) ◽  
pp. 786-791
Author(s):  
E W Triplett ◽  
J D Wall ◽  
P W Ludden
Keyword(s):  
Carbon Source ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
N Sources ◽  
Fe Protein ◽  
N Source ◽  
In Vitro Activation ◽  
High Ammonia ◽  
Cells Cultured

Activating enzyme (AE) is responsible for the in vitro activation of inactive Fe protein of nitrogenase from Rhodospirillum rubrum cells cultured anaerobically with glutamate as the N source. The expression of Fe protein and AE was examined in R. rubrum cultured photosynthetically or aerobically on media containing malate as the carbon source. One of the following N sources was used in each culture: glutamate, glutamine, limiting ammonia, high ammonia, glutamate plus histidine, and high ammonia plus histidine. Chromatophores from every culture exhibited AE activity; activity was highest in glutamate-grown cells. Fe protein was observed by rocket immunoelectrophoresis in cultures with nitrogenase activity. Several Nif-, Gln-, and His- mutants of R. rubrum were assayed for AE activity, nitrogenase activity, and Fe protein. Every mutant expressed AE activity, and Fe protein was observed in those cultures with nitrogenase activity. AE from every preparation was O2 labile, and each O2-denatured AE preparation inhibited activation by active AE.

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