Nasser Gad'on

Phylogenetic and metabolic diversity of bacteria degrading aromatic compounds under denitrifying conditions, and description of Thauera phenylacetica sp. nov., Thauera aminoaromatica sp. nov., and Azoarcus buckelii sp. nov.

Abstract. Six strains of denitrifying bacteria isolated from various oxic and anoxic habitats on different monocyclic aromatic substrates were characterized by sequencing 16S rRNA genes, determining physiological and morphological traits, and DNA-DNA hybridization. According to these criteria, strains S100, SP and LG356 were identified as members of Thauera aromatica. Strains B5–1 and B5–2 were tentatively affiliated to the species Azoarcus tolulyticus. Strains B4P and S2 were only distantly related to each other and to other described Thauera species. These two strains are proposed as the type strains of two new species, Thauera phenylacetica sp. nov. and Thauera aminoaromatica sp. nov., respectively. By 16S rRNA gene analysis, strain U120 was highly related to the type strains of Azoarcus evansii and Azoarcusanaerobius, whereas corresponding DNA-DNA reassociation values indicated only a low degree of genomic relatedness. Based upon a low DNA similarity value and the presence of distinguishing physiological properties, strain U120 is proposed as the type strain of a new species, Azoarcus buckelii sp. nov. Almost all of the new isolates were obtained with different substrates. The highly varied substrate spectra of the isolates indicates that an even higher diversity of denitrifying bacteria degrading aromatic compounds would be discovered in the different habitats by using a larger spectrum of aromatic substrates for enrichment and isolation.

Autotrophic CO(2) fixation by Chloroflexus aurantiacus: study of glyoxylate formation and assimilation via the 3-hydroxypropionate cycle.

In the facultative autotrophic organism Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, the Calvin cycle does not appear to be operative in autotrophic carbon assimilation. An alternative cyclic pathway, the 3-hydroxypropionate cycle, has been proposed. In this pathway, acetyl coenzyme A (acetyl-CoA) is assumed to be converted to malate, and two CO(2) molecules are thereby fixed. Malyl-CoA is supposed to be cleaved to acetyl-CoA, the starting molecule, and glyoxylate, the carbon fixation product. Malyl-CoA cleavage is shown here to be catalyzed by malyl-CoA lyase; this enzyme activity is induced severalfold in autotrophically grown cells. Malate is converted to malyl-CoA via an inducible CoA transferase with succinyl-CoA as a CoA donor. Some enzyme activities involved in the conversion of malonyl-CoA via 3-hydroxypropionate to propionyl-CoA are also induced under autotrophic growth conditions. So far, no clue as to the first step in glyoxylate assimilation has been obtained. One possibility for the assimilation of glyoxylate involves the conversion of glyoxylate to glycine and the subsequent assimilation of glycine. However, such a pathway does not occur, as shown by labeling of whole cells with [1,2-(13)C(2)]glycine. Glycine carbon was incorporated only into glycine, serine, and compounds that contained C(1) units derived therefrom and not into other cell compounds.

Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica

Abstract Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica. This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) the central benzoyl-CoA pathway comprising ring-reduction of benzoyl-CoA and subsequent β-oxidation to 3-hydroxypimelyl-CoA, and (3) the pathway of β-oxidation of 3-hydroxypimelyl-CoA to three acetyl-CoA and CO2. Regulation was studied by three methods. 1. Determination of protein patterns of cells grown on different substrates. This revealed several strongly substrate-induced polypeptides that were missing in cells grown on benzoate or other intermediates of the respective metabolic pathways. 2. Measurement of activities of known enzymes involved in this metabolism in cells grown on different substrates. The enzyme pattern found is consistent with the regulatory pattern deduced from simultaneous adaptation of cells to utilisation of other aromatic substrates. 3. Immunological detection of catabolic enzymes in cells grown on different substrates. Benzoate-CoA ligase and 4-hydroxybenzoate-CoA ligase were detected only in cells yielding the respective enzyme activity. However, presence of the subunits of benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase was also recorded in some cell batches lacking enzyme activity. This possibly indicates an additional level of regulation on protein level for these two reductases.

Crystallization of the photosynthetic light‐harvesting pigment‐protein complex B800‐850 of Rhodopseudomonas capsulata

The B800‐850 light‐harvesting complex of Rhodopseudomonas capsulata was crystallized in the presence of detergents. The crystals were obtained by a vapour diffusion technique, using polyethylene glycol as a precipitant. Crystals grew to a size of 0.5 × 0.5 × 0.3 mm within two weeks. Two different crystal forms were obtained; one is supposed to be triclinic, space group PI, the other orthorhombic, space group C2221. Both crystal forms diffract to approximately 1.0 nm. Absorption spectra and polyacrylamide gel electrophoresis demonstrate that all expected components, i.e. three polypeptides of apparent M r,8000, 10000 and 14000, bacteriochlorophyll a and carotenoids, are present and in native conformation.

Phenylphosphate Synthase: a New Phosphotransferase Catalyzing the First Step in Anaerobic Phenol Metabolism in Thauera aromatica

The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via para-carboxylation of phenol (biological Kolbe-Schmitt carboxylation). In the first step, phenol is converted to phenylphosphate which is then carboxylated to 4-hydroxybenzoate in the second step. Phenylphosphate formation is catalyzed by the novel enzyme phenylphosphate synthase, which was studied. Phenylphosphate synthase consists of three proteins whose genes are located adjacent to each other on the phenol operon and were overproduced in Escherichia coli. The promoter region and operon structure of the phenol gene cluster were investigated. Protein 1 (70 kDa) resembles the central part of classical phosphoenolpyruvate synthase which contains a conserved histidine residue. It catalyzes the exchange of free [(14)C]phenol and the phenol moiety of phenylphosphate but not the phosphorylation of phenol. Phosphorylation of phenol requires protein 1, MgATP, and another protein, protein 2 (40 kDa), which resembles the N-terminal part of phosphoenol pyruvate synthase. Proteins 1 and 2 catalyze the following reaction: phenol + MgATP + H(2)O-->phenylphosphate + MgAMP + orthophosphate. The phosphoryl group in phenylphosphate is derived from the beta-phosphate group of ATP. The free energy of ATP hydrolysis obviously favors the trapping of phenol (K(m), 0.04 mM), even at a low ambient substrate concentration. The reaction is stimulated severalfold by another protein, protein 3 (24 kDa), which contains two cystathionine-beta-synthase domains of unknown function but does not show significant overall similarity to known proteins. The molecular and catalytic features of phenylphosphate synthase resemble those of phosphoenolpyruvate synthase, albeit with interesting modifications.

The adaptation of the electron transfer chain of Rhodopseudomonas capsulata to different light intensities

(1) Cells of Rhodopseudomonas capsulata (wild-type) were grown photoheterotrophically in a turbidostat under very high and very low light intensity. Membranes were isolated from cells adapted to the respective light conditions and fractionated by sucrose density centrifugation. The molar ratios of ubiquinone and cytochromes c2, c1, b-561 and b-566 per reaction center were 3-fold to 5-fold higher in high-light than in low-light membranes. (2) Most of the Cyt(c1 + c2) and Cyt b-561 detected in dark redox titrations undergoes light-induced redox changes, both in high- and in low-light membranes. (3) The fractions of the total photooxidizable reaction center and Cyt(c1 + c2) oxidized under continuous light in the absence of antimycin are higher in membranes from low-light- than from high-light-grown cells. (4) From these data and results of kinetic studies it is proposed that cyclic electron flow under saturating light intensities is faster in high-light-grown cells.

Spectroscopical studies on the light-harvesting pigment-protein complex II from dark-aerobic and light-anaerobic grown cells of Rhodobacter sulfidophilus

The photosynthetic bacterium Rhodobacter sulfidophilus can grow and synthesize photosynthetic pigments under dark-aerobic as well as light-anaerobic growth conditions. Under both growth conditions intracytoplasmic membrane vesicles (diameter about 35 nm) are formed. The light-harvesting (LH) pigment-protein complex II, isolated from dark-aerobic and light-anaerobic grown cells, consists of two small polypeptides (

Crystallization and spectroscopic investigation with polarized light of the reaction center-B875 light-harvesting complex of Rhodopseudomonas palustris

A pigment‐protein complex containing a reaction center and the B875 light‐harvesting complex from Rhodopseudomonas palustris was purified and crystallized in the presence of detergent. Thin, rectangular crystals were obtained and used for optical spectroscopy. The protein pattern as well as the spectrum of the solubilized and crystallized complex show that the purified complex was crystallized without a major change in its composition and conformation. The absorption band intensities in the plane of the crystal as well as the spectra of the tilted crystal indicate that the 590 nm Qx transitions are aligned along the long axis of the crystal, while the 875 nm Qy transitions are aligned along the short axis of the crystal plane. The results favor a model where the antenna bacteriochlorophylls associated with one reaction center have a common Qx direction and perpendicular to it a distribution of 875 nm Qy transitions exhibiting a maximum along one direction.

The major part of polar carotenoids of the aerobic bacteria Roseococcus thiosulfatophilus RB3 and Erythromicrobium ramosum E5 is not bound to the bacteriochlorophyll a-complexes of the photosynthetic apparatus

The obligate aerobic bacteria Roseococcus thiosulfatophilus RB3 and Erythromicrobium ramosum E5 contain numerous polar carotenoids. The major carotenoid of the strain RB3 was the C30 carotene-dioate (4,4′-diapocarotene-4,4′-dioate) and the respective diglycosyl ester which have never been isolated before from a bacteriochlorophyll containing bacterium. Strain E5 contains the very polar erythroxanthin sulphate. The major carotenoid bound to reaction center and light-harvesting complexes is bacteriorubixanthinal. Most of the carotenoids of both strains are not bound to the pigment-protein complexes of the photosynthetic apparatus but to the envelope fraction (cytoplasmic membrane and cell wall).

Bacteriochlorophyll-protein interaction in the light-harvesting complex B800-850 from Rhodobacter sulfidophilus: A Fourier-transform Raman spectroscopic investigation

Near-infrared excited Fourier-transform Raman spectra have been obtained from different spectral forms of Rhodobacter sulfidophilus light-harvesting II complexes. This complex, when isolated in lauryldimethylamine oxide, exists in a 805–828 nm form, which can be reversibly converted to the native 805–851 nm form upon addition of salt. The FT-Raman spectra predominantly show contributions of the carotenoid in the light-harvesting complex, with small but significant contributions of the bacteriochlorophylls excited in preresonance in the Q y transition. One strongly and one weakly interacting 2a acetyl C=O group as well as one moderately strong interacting and one non-interacting 9-keto C=O carbonyl modes of the bacteriochlorophylls can be discerned for the 805–828 nm form. Changes of relative band intensities caused by different resonance conditions for the different spectral forms lead to an assignment of the strongly interacting 2a acetyl C=O and the moderately strong interacting 9 keto C=O to bacteriochlorophylls organized in the 828 pigment moiety. Shifts of these bands to higher frequencies upon the salt-induced transition indicate a perturbation of the pigment-protein interaction, probably caused by a local protein conformational change.