Regulation of nitrogenase activity in Rhodobacter capsulatus under dark microoxic conditions

Rhodobacter capsulatus modulates its in vivo nitrogenase activity in the light in response to the addition of NH4+ in a variety of ways: with ADP-ribosylation of the Fe-protein of nitrogenase, with a switch-off response that is independent of ADP-ribosylation, and with a "magnitude response." In the light, these responses are differentially shown by cultures that differ in the degree of their nitrogen limitation. Here we examined the response of these culture types to the addition of NH4+ under dark, microoxic conditions and found that all three responses can be observed under these conditions. However, the magnitude response was much more sensitive to the ammonium concentration, and the ADP-ribosylation response correlated only poorly with activity changes, similar to results obtained in the light. In contrast to previous reports, Fe-protein was not ADP-ribosylated in response to the presence of oxygen.     

Specific Interactions between Retrovirus Env and Gag Proteins in Rat Neurons

In this work we have studied the intracellular localization properties of the Gag and Env proteins of Moloney murine leukemia virus (MLV) and human immunodeficiency virus (HIV) in dorsal root ganglion (DRG) neurons of rat. These neurons form thick bundles of axons, which facilitates protein localization studies by immunofluorescence analyses. When such neuron cultures were infected with recombinant Semliki Forest virus particles carrying the gag genes of either retrovirus, the expressed Gag proteins were localized to both the somatic and the axonal regions of the DRG neurons. In contrast, the Env proteins were confined only to the somatic region. When the Gag and Env proteins were coexpressed, the Gag proteins were also excluded from the axons. This effect of the Env proteins was shown to be dependent on the concentration of the Gag proteins in the neuron and also to be specific for homologous pairs of retrovirus proteins. Therefore, the results suggest that there are specific interactions between the Env and the Gag proteins of MLV and HIV in the DRG neurons.     

Biochemical Analysis of Interactions between Outer Membrane Proteins That Contribute to Starch Utilization by Bacteroides thetaiotaomicron

An early step in the utilization of starch by Bacteroides thetaiotaomicron is the binding of starch to the bacterial surface. Four starch-associated outer membrane proteins of B. thetaiotaomicron that have no starch-degrading activity have been identified. Two of these, SusC and SusD, have been shown by genetic analysis to be required for starch binding. In this study, we provide the first biochemical evidence that these two proteins interact physically with each other. Both formaldehyde cross-linking and nondenaturing gel electrophoresis experiments showed that SusC and SusD interact to form a complex. Two other proteins encoded by genes in the same operon, SusE and SusF, proved not to be essential for starch utilization and actually decreased starch binding when they were present along with SusC and SusD. Consistent with this, nondenaturing gel analysis revealed that in a strain producing SusC, SusD, and SusE, the SusCD complex was partially destabilized. The strain producing SusC, SusD, and SusE also grew more slowly on starch than a strain producing SusC, SusD, SusE, and SusF (mu(max), 0.29 and 0.37/h, respectively). Thus, SusE appears to interact with the SusCD complex. SusE also interacts with SusF, because SusE was less susceptible to proteinase K digestion when SusF was present, and nondenaturing gel analysis detected a complex formed by these two proteins. Our results indicate that SusC, SusD, SusE, and SusF form a protein complex in the outer membrane but that SusE and SusF are dispensable members of this complex.     

Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates ammonia and other forms of reduced nitrogen either through the GS/GOGAT pathway or by the concerted action of l-alanine dehydrogenase and aminotransferases. These routes are light-independent and very responsive to the carbon and nitrogen sources used for cell growth. GS was most active in cells grown on nitrate or l-glutamate as nitrogen sources, whereas it was heavily adenylylated and siginificantly repressed by ammonium, glycine, l-alanine, l-aspartate, l-asparagine and l-glutamine, under which conditions specific aminotransferases were induced. GOGAT activity was kept at constitutive levels in cells grown on l-amino acids as nitrogen sources except on l-glutamine where it was significantly induced during the early phase of growth. In vitro, GOGAT activity was strongly inhibited by l-tyrosine and NADPH. In cells using l-asparagine or l-aspartate as nitrogen source, a concerted induction of l-aspartate aminotransferase and l-asparaginase was observed. Enzyme level enhancements in response to nitrogen source variation involved de novo protein synthesis and strongly correlated with the cell growth phase.Abbreviations ADH l-alanine dehydrogenase - AOAT l-alanine:2-oxoglutarate aminotransferase - Asnase l-asparaginase - GOAT Glycine: oxaloacetate aminotransferase - GOGAT Glutamate synthase - GOT l-aspartate: 2-oxoglutarate aminotransferase - GS Glutamine synthetase - HPLC High-Pressure Liquid Chromatography - MOPS 2-(N-morpholino)propanesulfonic acid - MSX l-methionine-d,l-sulfoximine     

Regulation of synthesis of pyruvate carboxylase in the photosynthetic bacterium Rhodobacter capsulatus.

The synthesis of pyruvate carboxylase (PC) was studied by using quantitative immunoblot analysis with an antibody raised against PC purified from Rhodobacter capsulatus and was found to vary 20-fold depending on the growth conditions. The PC content was high in cells grown on pyruvate or on carbon substrates metabolized via pyruvate (lactate, D-malate, glucose, or fructose) and low in cells grown on tricarboxylic acid (TCA) cycle intermediates or substrates metabolized without intermediate formation of pyruvate (acetate or glutamate). Under dark aerobic growth conditions with lactate as a carbon source, the PC content was approximately twofold higher than that found under light anaerobic growth conditions. The results of incubation experiments demonstrate that PC synthesis is induced by pyruvate and repressed by TCA cycle intermediates, with negative control dominating over positive control. The content of PC in R. capsulatus cells was also directly related to the growth rate in continuous cultures. The analysis of intracellular levels of pyruvate and TCA cycle intermediates in cells grown under different conditions demonstrated that the content of PC is directly proportional to the ratio between pyruvate and C4 dicarboxylates. These results suggest that the regulation of PC synthesis by oxygen and its direct correlation with growth rate may reflect effects on the balance of intracellular pyruvate and C4 dicarboxylates. Thus, this important enzyme is potentially regulated both allosterically and at the level of synthesis.     

Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus

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.     

Nucleotide transport in Rhodobacter capsulatus.

Cytoplasmic membrane vesicles isolated from the gram-negative photosynthetic bacterium Rhodobacter capsulatus catalyzed the transport of nucleotides. No transport occurred in the intact bacteria unless they were pretreated with EDTA. The transport rate was measured by incorporation of radioactive phosphate into externally added ADP or by incorporation of nonradioactive phosphate into added labeled ADP. The catalytic activities which utilized the added ADP were photosynthetic ATP synthesis, Pi-ADP exchange, and adenylate kinase. These activities were shown to occur on the cytoplasmic side of the internal membrane. The products were found in the outer medium. The rate of nucleotide transport across the membranes was comparable to the rate of photophosphorylation. These results indicated that nucleotides can be transported across the cytoplasmic membrane but not across the outer membrane of the native R. capsulatus cell. Therefore, by analogy to the mitochondrial ATP-ADP translocator, the exchange might function as an energy transfer system to the periplasm of these bacteria.     

The interaction between dimethylsulfoxide and nitrate reducing pathways in Rhodobacter capsulatus

Abstract The interaction between nitrate- and dimethyl-sulphoxide (DMSO)-reducing pathways was demonstrated in intact cells of Rhodobacter capsulatus AD2 removed from cultures grown under different conditions. The results provide evidence of competition between the DMSO and nitrate reductases for a common electron pool. Furthermore, strong inhibition was observed of the anaerobic dark DMSO-dependent growth of R. capsulatus by nitrate in the growth medium. This phenomenon is also discussed.     

Interactions Between Nitrate Assimilation and 2,4-Dinitrophenol Cometabolism in Rhodobacter capsulatus E1F1

The phototrophic, nitrate-photoassimilating bacterium Rhodobacter capsulatus E1F1 cometabolizes 2,4-dinitrophenol (DNP) by photoreducing it to 2-amino-4-nitrophenol under anaerobic conditions. DNP uptake and nitrate metabolism share some biochemical features, and in this article we show that both processes are influenced by each other. Thus, as was demonstrated for nitrate assimilation, DNP uptake requires a thermolabile periplasmic component. Nitrate assimilation is inhibited by DNP, which probably affects the nitrite reduction step because neither nitrate reductase activity nor the transport of nitrate or nitrite is inhibited. On the other hand, DNP uptake is competitively inhibited by nitrate, probably at the transport level, because the nitroreductase activity is not inhibited in vitro by nitrate, nitrite, or ammonium. In addition, the decrease in the intracellular DNP concentration in the presence of nitrate probably inactivates the nitroreductase. These results allow prediction of a negative environmental effect if nitrate and DNP are released together to natural habitats, because it may lead to a lower rate of DNP metabolism and to nitrite accumulation.     

Sulfide-quinone and sulfide-cytochrome reduction in Rhodobacter capsulatus

The reduction by sulfide of exogenous ubiquinone is compared to the reduction of cytochromes in chromatophores of Rhodobacter capsulatus. From titrations with sulfide values for V_max of 300 and 10 moles reduced/mg bacteriochlorophyll a·h, and for K_m of 5 and 3 M were estimated, for decyl-ubiquinone-and cytochrome c-reduction, respectively. Both reactions are sensitive to KCN, as has been found for sulfide-quinone reductase (SQR) in Oscillatoria limnetica, which is a flavoprotein. Effects of inhibitors interfering with quinone binding sites suggest that at least part of the electron transport from sulfide in R. capsulatus employs the cytochrome bc ₁-complex via the ubiquinone pool.Abbreviations BChl a bacteriochlorophyll a - DAD diaminodurene - decyl-UQ decyl-ubiquinone - LED light emitting diode - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - PQ-1 plastoquinone 1 - SQR sulfide-quinone reductase (E.C. 1.8.5.'.) - UQ ubiquinone 10 - Q_c the quinone reduction site on the cytochrome b ₆ f/bc ₁, complex (also termed Q_i or Q_r or Q_n) - Q_s the quinone reduction site on SQR - Q_z quinol oxidation site on the b ₆ f/bc ₁, complex (also termed Q_o or Q_p)     

Conservation and variation between Rhodobacter capsulatus and Escherichia coli Tat systems

The Tat system allows the translocation of folded and often cofactor-containing proteins across biological membranes. Here, we show by an interspecies transfer of a complete Tat translocon that Tat systems are largely, but not fully, interchangeable even between different classes of proteobacteria. The Tat apparatus from the alpha-proteobacterium Rhodobacter capsulatus was transferred to a Tat-deficient Escherichia coli strain, which is a gamma-proteobacterium. Similar to that of E. coli, the R. capsulatus Tat system consists of three components, rc-TatA, rc-TatB, and rc-TatC. A fourth gene (rc-tatF) is present in the rc-tatABCF operon which has no apparent relevance for translocation. The translational starts of rc-tatC and rc-tatF overlap in four nucleotides (ATGA) with the preceding tat genes, pointing to efficient translational coupling of rc-tatB, rc-tatC, and rc-tatF. We show by a variety of physiological and biochemical assays that the R. capsulatus Tat system functionally targets the E. coli Tat substrates TorA, AmiA, AmiC, and formate dehydrogenase. Even a Tat substrate from a third organism is accepted, demonstrating that usually Tat systems and Tat substrates from different proteobacteria are compatible with each other. Only one exceptional Tat substrate of E. coli, a membrane-anchored dimethyl sulfoxide (DMSO) reductase, was not targeted by the R. capsulatus Tat system, resulting in a DMSO respiration deficiency. Although the general features of Tat substrates and translocons are similar between species, the data indicate that details in the targeting pathways can vary considerably.     

Uncovering Specific Electrostatic Interactions in the Denatured States of Proteins

The stability and folding of proteins are modulated by energetically significant interactions in the denatured state that is in equilibrium with the native state. These interactions remain largely invisible to current experimental techniques, however, due to the sparse population and conformational heterogeneity of the denatured-state ensemble under folding conditions. Molecular dynamics simulations using physics-based force fields can in principle offer atomistic details of the denatured state. However, practical applications are plagued with the lack of rigorous means to validate microscopic information and deficiencies in force fields and solvent models. This study presents a method based on coupled titration and molecular dynamics sampling of the denatured state starting from the extended sequence under native conditions. The resulting denatured-state pK_as allow for the prediction of experimental observables such as pH- and mutation-induced stability changes. I show the capability and use of the method by investigating the electrostatic interactions in the denatured states of wild-type and K12M mutant of NTL9 protein. This study shows that the major errors in electrostatics can be identified by validating the titration properties of the fragment peptides derived from the sequence of the intact protein. Consistent with experimental evidence, our simulations show a significantly depressed pK_a for Asp⁸ in the denatured state of wild-type, which is due to a nonnative interaction between Asp⁸ and Lys¹². Interestingly, the simulation also shows a nonnative interaction between Asp⁸ and Glu⁴⁸ in the denatured state of the mutant. I believe the presented method is general and can be applied to extract and validate microscopic electrostatics of the entire folding energy landscape.     

Relations between the chemotype and the cell electrophoretic properties in Rhodobacter capsulatus strains

The cells of two Rhodobacter capsulatus strains, B10 and PG, and the LPS of their cell walls were studied by electrophysical and biochemical methods. Strain B10 was found to belong to the R chemotype, and strain PG, to the RS chemotype. A relation was revealed between the chemotype of the photosynthesizing bacteria Rhodobacter capsulatus and the electrophoretic properties of their cells.