Metabolism of L-Phenylalanine and L-Tyrosine by the Phototrophic Bacterium Rhodobacter capsulatus

The phototrophic bacterium Rhodobacter capsulatus utilizes the aromatic amino acids L-phenylalanine and L-tyrosine as nitrogen source. L-Phenylalanine is hydroxylated to L-tyrosine, which is further converted into p-hydroxyphenyl pyruvate (pHPP) by a transamination reaction. The bacterium is unable to grow at the expense of these amino acids as the sole carbon source, although it is able to degrade them to homogentisate, probably by unspecific hydroxylation reactions. Metabolization of L-phenylalanine or L-tyrosine as nitrogen source requires phototrophic growth conditions and does not produce free ammonium inside the cells. A low aminotransferase activity with 2-oxoglutarate and L-tyrosine as substrates can be detected in crude extracts of R. capsulatus. Uptake of both amino acids by R. capsulatus was completely inhibited by ammonium addition, which also prevents aminotransferase induction. Received: 21 July 1998 / Accepted: 19 August 1998     

Assimilation of D-Malate by Rhodobacter capsulatus E1F1

Rhodobacter capsulatus grew by using either L- or D-malate as carbon sources under light/anaerobic conditions. The cellular yields were the same with D- or L-malate. Both L-malate dehydrogenase and L-malic enzyme activities were detected in cell-free extracts from cells grown in both isomers. By contrast, a racemase activity converting D-malate into L-malate was induced only when D-malate was present in the culture medium. This racemase activity was Mn²⁺-dependent and was measured by coupling it either to the malate dehydrogenase or to the fumarase activities. The racemase activity was partially purified by anion-exchange chromatography. Received: 30 November 2000/Accepted: 10 January 2001     

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     

Characterization of a methionine sulfoximine resistant strain of Rhodobacter capsulatus E1F1

The transposon-loaded plasmid pAS8-121, incapable of autonomous replication in Gram-negative bacteria of non-enteric group, was transferred to Methylobacillus flagellatum KT wild type strain MFK1. The transconjugants arose at a frequency of 10(-7) per donor cell. The majority of the transconjugants tested exhibited the transfer of all selected chromosomal markers at rather high (10(-4)-10(-6) per donor cell) but similar frequencies. Only one of the obtained donors, designated MFK 64, was capable of mobilizing M. flagellatum KT chromosome in a polarized manner. The integrated nature of the plasmid in this and other MFK1 (pAS8-121) derivatives was supported by the results of DNA-DNA hybridization.     

Light-dependent degradation of nitrophenols by the phototrophic bacterium Rhodobacter capsulatus E1F1.

Rhodobacter capsulatus E1F1, a phototrophic purple nonsulfur bacterium capable of photoassimilating nitrate or nitrite, grew phototrophically in the presence of mono- and dinitrophenols with acetate as a carbon source, the highest growth levels being obtained under microaerobic conditions. Utilization of 2,4-dinitrophenol was strictly light dependent, was inhibited by O2 and by ammonium, and took place with the simultaneous and stoichiometric production of 2-amino-4-nitrophenol, which accumulated in the medium and was poorly used for further growth in anaerobiosis. Metabolism of mononitrophenols was also light dependent but was activated by O2 and by ammonium. Metabolism of nitrophenols seemed to depend on inducible systems which were repressed in nitrogen-starved cells. Induction of the in vivo 2,4-dinitrophenol reducing system was strongly inhibited by chloramphenicol.     

Light-dependent degradation of nitrophenols by the phototrophic bacterium Rhodobacter capsulatus E1F1.

Rhodobacter capsulatus E1F1, a phototrophic purple nonsulfur bacterium capable of photoassimilating nitrate or nitrite, grew phototrophically in the presence of mono- and dinitrophenols with acetate as a carbon source, the highest growth levels being obtained under microaerobic conditions. Utilization of 2,4-dinitrophenol was strictly light dependent, was inhibited by O2 and by ammonium, and took place with the simultaneous and stoichiometric production of 2-amino-4-nitrophenol, which accumulated in the medium and was poorly used for further growth in anaerobiosis. Metabolism of mononitrophenols was also light dependent but was activated by O2 and by ammonium. Metabolism of nitrophenols seemed to depend on inducible systems which were repressed in nitrogen-starved cells. Induction of the in vivo 2,4-dinitrophenol reducing system was strongly inhibited by chloramphenicol.     

Characterization of a nitrophenol reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1.

The phototrophic bacterium Rhodobacter capsulatus E1F1 photoreduced 2,4-dinitrophenol to 2-amino-4-nitrophenol by a nitrophenol reductase activity which was induced in the presence of nitrophenols and was repressed in ammonium-grown cells. The enzyme was located in the cytosol, required NAD(P)H as an electron donor, and used several nitrophenol derivatives as alternative substrates. The nitrophenol reductase was purified to electrophoretic homogeneity by a simple method. The enzyme was composed of two 27-kDa subunits, was inhibited by metal chelators, mercurial compounds, and Cu2+, and contained flavin mononucleotide and possibly nonheme iron as prosthetic groups. Purified enzyme also exhibited NAD(P)H diaphorase activity which used tetrazolium salt as an electron acceptor.     

Immunocytochemical localization of glutamine synthetase in Rhodobacter capsulatus E1F1 and Rhodopseudomonas acidophila

Intracellular localization of glutamine synthetase has been studied by immunochemical techniques with cryosections and London Resin sections of Rhodobacter capsulatus E1F1 and Rhodopseudomonas acidophila. For immunostaining, sections were sequentially incubated with monospecific anti-glutamine synthetase antibodies (R. capsulatus) and gold labelled goat anti-rabbit antibodies. Gold label was present in the cytoplasm but not in the cell walls. The antigen is not associated with the cell membrane or with photosynthetic vesicle whether these are round and randomly distributed (R. capsulatus) or flattened and organized in well defined stacks (R. acidophila). Our results also indicate that glutamine synthetase is absent from the central, nucleoid part of the cell. The enzyme is present in dense cytoplasmic patches, which appear to be RNA-ribosome-containing areas.Abbreviations GS glutamine synthetase - LR London Resin White     

Effects of the Metalloid Oxyanion Tellurite (TeO32−) on Growth Characteristics of the Phototrophic Bacterium Rhodobacter capsulatus

This work examines the effects of potassium tellurite (K₂TeO₃) on the cell viability of the facultative phototroph Rhodobacter capsulatus. There was a growth mode-dependent response in which cultures anaerobically grown in the light tolerate the presence of up to 250 to 300 μg of tellurite (TeO₃²⁻) per ml, while dark-grown aerobic cells were inhibited at tellurite levels as low as 2 μg/ml. The tellurite sensitivity of aerobic cultures was evident only for growth on minimal salt medium, whereas it was not seen during growth on complex medium. Notably, through the use of flow cytometry, we show that the cell membrane integrity was strongly affected by tellurite during the early growth phase (≤50% viable cells); however, at the end of the growth period and in parallel with massive tellurite intracellular accumulation as elemental Te⁰ crystallites, recovery of cytoplasmic membrane integrity was apparent (≥90% viable cells), which was supported by the development of a significant membrane potential (Δψ = 120 mV). These data are taken as evidence that in anaerobic aquatic habitats, the facultative phototroph R. capsulatus might act as a natural scavenger of the highly soluble and toxic oxyanion tellurite.     

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.     

Purification and properties of L-alanine dehydrogenase of the phototrophic bacterium Rhodobacter capsulatus E1F1.

In the phototrophic nonsulfur bacterium Rhodobacter capsulatus E1F1, L-alanine dehydrogenase aminating activity functions as an alternative route for ammonia assimilation when glutamine synthetase is inactivated. L-Alanine dehydrogenase deaminating activity participates in the supply of organic carbon to cells growing on L-alanine as the sole carbon source. L-Alanine dehydrogenase is induced in cells growing on pyruvate plus nitrate, pyruvate plus ammonia, or L-alanine under both light-anaerobic and dark-heterotrophic conditions. The enzyme has been purified to electrophoretic and immunological homogeneity by using affinity chromatography with Red-120 agarose. The native enzyme was an oligomeric protein of 246 kilodaltons (kDa) which consisted of six identical subunits of 42 kDa each, had a Stokes' radius of 5.8 nm, an s20.w of 10.1 S, a D20,w of 4.25 x 10(-11) m2 s-1, and a frictional quotient of 1.35. The aminating activity was absolutely specific for NADPH, whereas deaminating activity was strictly NAD dependent, with apparent Kms of 0.25 (NADPH), 0.15 (NAD+), 1.25 (L-alanine), 0.13 (pyruvate), and 16 (ammonium) mM. The enzyme was inhibited in vitro by pyruvate or L-alanine and had two sulfhydryl groups per subunit which were essential for both aminating and deaminating activities.     

Expression and characterization of the assimilatory NADH-nitrite reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1

A nas gene region from Rhodobacter capsulatus E1F1 containing the putative nasB gene for nitrite reductase was previously cloned. The recombinant His₆-NasB protein overproduced in E. coli showed nitrite reductase activity in vitro with both reduced methyl viologen and NADH as electron donors. The apparent K _m values for nitrite and NADH were 0.5 mM and 20 μM, respectively, at the pH and temperature optima (pH 9 and 30°C). The optical spectrum showed features that indicate the presence of FAD, iron-sulfur cluster and siroheme as prosthetic groups, and nitrite reductase activity was inhibited by sulfide and iron reagents. These results indicate that the phototrophic bacterium R. capsulatus E1F1 possesses an assimilatory NADH-nitrite reductase similar to that described in non-phototrophic organisms.     

Nitrate reductase activity in spheroplasts from Rhodobacter capsulatus E1F1 requires a periplasmic protein

Spheroplasts from Rhodobacter capsulatus E1F1 cells grown in nitrate maintained nitrate uptake and nitrate reductase activity only when they were illuminated under anaerobiosis in the presence of the periplasmic fraction and nitrate. The effects on nitrate uptake and nitrate reductase activity of spheroplasts were observed at low concentrations of periplasmic protein (about 50 x ml^-1). Periplasm from nitrate-grown cells was also required for nitrate reductase activity in spheroplasts isolated from ammonia-grown or diazotrophic cells which initially lacked this enzymatic activity. Both the maintenance of nitrate reductase in spheroplasts from nitrate-grown cells and the appearance of the activity in spheroplasts from diazotrophic cells were dependent on de novo protein synthesis. A periplasmic, 45-kDa protein which maintained the activity of nitrate reductase in spheroplasts was partially purified by gel filtration chromatography of periplasm obtained from nitrate-grown cells.Abbreviations NR nitrate reductase - CCCP carbonyl cyanide m-chlorophenylhydrazone - CAM chloramphenicol     

Role of glutamine as a direct co-repressor of glutamine synthetase in Rhodobacter capsulatus E1F1

High performance liquid chromatography (HPLC) has been used to determine the internal levels of amino acids in Rhodobacter capsulatus E1F1 cells, subjected to different treatments and nutritional conditions. Glutamine synthetase activity and enzyme concentration correlated negatively with the level of glutamine, suggesting that glutamine per se acts as a co-repressor in the enzyme synthesis. Moreover, addition of the specific inhibitor L-methionine-D,L-sulfoximine, that produced an increase in enzyme concentration, specifically promoted a depletion of intracellular glutamine.     

Effect of Selenite on Growth and Protein Synthesis in the Phototrophic Bacterium Rhodobacter sphaeroides

The effect of selenite on the growth rate and protein synthesis has been investigated in Rhodobacter sphaeroides. This photosynthetic bacterium efficiently reduces selenite with intracellular accumulation under both dark aerobic and anaerobic photosynthetic conditions. Addition of 1 mM selenite under these two growth conditions does not affect the final cell density, although a marked slowdown in growth rate is observed under aerobic growth. The proteome analysis of selenite response by two-dimensional gel electrophoresis shows an enhanced synthesis of some chaperones, an elongation factor, and enzymes associated to oxidative stress. The induction of these antioxidant proteins confirms that the major toxic effect of selenite is the formation of reactive oxygen species during its metabolism. In addition, we show that one mutant unable to precipitate selenite, selected from a transposon library, is affected in the smoK gene. This encodes a constituent of a putative ABC transporter implicated in the uptake of polyols. This mutant is less sensitive to selenite and does not express stress proteins identified in the wild type in response to selenite. This suggests that the entry of selenite into the cytoplasm is mediated by a polyol transporter in R. sphaeroides.     

The ATP Synthase atpHAGDC (F1) Operon from Rhodobacter capsulatus

The atpHAGDC operon of Rhodobacter capsulatus, containing the five genes coding for the F₁ sector of the ATP synthase, has been cloned and sequenced. The promoter region has been defined by primer extension analysis. It was not possible to obtain viable cells carrying atp deletions in the R. capsulatus chromosome, indicating that genes coding for ATP synthase are essential, at least under the growth conditions tested. We were able to circumvent this problem by combining gene transfer agent transduction with conjugation. This method represents an easy way to construct strains carrying mutations in indispensable genes.     

Regulation of isocitrate lyase inRhodobacter capsulatus E1F1

InRhodobacter capsulatus E1F1, isocitrate lyase (ICL) (EC 4.5.3.1) is a regulatory enzyme whose levels are increased in the presence of acetate as the sole carbon source. Acetate activated isocitrate lyase in a process dependent on energy supply and de novo protein synthesis. In contrast to isocitrate lyase, isocitrate dehydrogenase (ICDH) activity was independent of the carbon source used for growth and significantly increased in darkened cells. Pyruvate or yeast extract prevented in vivo activation of isocitrate lyase in cells growing on acetate. The enzyme was reversibly inactivated to a great extent in vitro by pyruvate and other oxoacids presumably involved in acetate metabolism. These results suggest that, inR. capsulatus E1F1, isocitrate lyase is regulated by both enzyme synthesis and oxoacid inactivation.     

Regulation of nitrate reductase inRhodobacter capsulatus E1F1

The photosynthetic purple non-sulfur nitrate-assimilating bacteriumRhodobacter capsulatus E1F1 has an adaptive nitrate reductase activity inducible by either nitrate or nitrite and molybdenum traces. Nitrate reductase induction by nitrate did not occur in media with nitrate and ammonium, which showed no effect if nitrite was the inductor instead of nitrate or in the presence ofl-methionine-dl-sulfoximine (MSX) plus nitrate. In vivo, tungstate inhibited nitrate reductase activity, and this was not recovered upon addition of molybdenum unless de novo protein synthesis took place. Nitrate reductase was also repressed in nitrogen-starved cells or after the addition of azaserine to cells growing phototrophically with nitrate. Moreover, higher rates of nitrate reductase induction and nitrite excretion were found in illuminated cells grown with nitrate under air than in those grown under argon.     

Characterization of urease from the phototrophic bacteriumRhodobacter capsulatus E1F1

Rhodobacter capsulatus E1F1 showed high cytosolic urease activity when growing on urea, purines, and purine metabolites as nitrogen source. Molecular mass ofR. capsulatus enzyme is similar to that of other bacteria and greatly differs from that of jack bean. Kinetic parameters of partially purifiedR. capsulatus enzyme resemble those described in other bacterial ureases. The activity was inhibited by metal-chelating agents and by mercurials. Urease fromR. capsulatus E1F1 was negligible in nitrogen-starved cells or in cells cultured with nitrate, ammonium, or amino acids. Moreover, ammonium inhibited both the urea uptake and the urease activity expression inR. capsulatus cells.