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Membranes of aerobically grown Rhodobacter capsulatus contain only one type of NADH:ubiquinone oxidoreductase which is homologous to the proton-translocating complex I. The K(m) value of the enzyme for NADH was determined to be 8 microM. After solubilization of the membranes with an alkylglucoside detergent, two fragments of complex I with molecular masses of 110 kDa and 140 kDa were isolated by chromatographic steps in the presence of detergent. Both fragments contain at least two polypeptides with apparent molecular masses of 46 kDa and 42 kDa. FMN was identified as cofactor in the preparations. Degenerative oligonucleotide primers were used to amplify a part of the sequence coding for the NADH-binding subunit of complex I by PCR. With the PCR product as probe, a genomic fragment was cloned and sequenced containing the genes encoding the two purified polypeptides and additional reading frames. The two genes are named nuoE and nuoF and are homologous to nqo2 and nqo1 of Paracoccus denitrificans. However, NuoE contains a C-terminal extension of 149 amino acids compared with Nqo2. NuoE and NuoF have molecular masses of 41259 Da and 47133 Da and contain the NADH-, FMN- and FeS-cluster-binding motifs.

Mutants of Rhodobacter capsulatus, blocked at different steps of bacteriochlorophyll a (BChl) synthesis between protoporphyrin IX and 2-hydroxyethyl bacteriochlorophyllide a, were induced to synthesize the photosynthetic apparatus by lowering of oxygen tension in dark cultures. The cells were pulse-labeled with [35S]methionine and the radioactivity chased after dilution of [35S]. The specific radioactivity in the pigment-binding proteins of light-harvesting and reaction center proteins of the wild-type strain was not lowered during the chase period of three hours but in the BChl-free mutants the label disappeared within five to thirty minutes. The polypeptides were inserted into the membrane but did not remain stably incorporated. In the mutant strain NK9 the synthesis of the carotenoid spheroidenone/spheroidene was inhibited by insertion of Tn5 in the crtI gene (phytoene desaturase), which blocked completely the formation of the light-harvesting (LH) complex II (B800–850) but not of the LHI (B870) complex. In this mutant the polypeptides of the LH complexes were synthesized in a lower amount than in the wild-type cells and were inserted into the membrane. The LHIIα poly-peptide disappeared after 60 min of chase while the LHIIβ was more stable. It was concluded that the pigments are not only necessary for absorption of photons and efficient transfer of excitation energy but have a structural role by stabilizing the oligomeric LH complexes. This is in accordance with the crystal structure data of LHII complexes.

Heinrich Anton de Bary (1831–1888), Professor of Botany at the German universities in Freiburg i.Br., Halle a.S. and Strassbourg (now in France), discovered sexual and asexual propagation of fungi by microscopically observing the different stages of development. His experimental approach and concept, based on Darwin's theory of evolution, were the starting points for a phylogenetic taxonomy, which, however, could not realise before present time. He defined several species and subspecies and their delimitation experimentally. de Bary also exactly described the entire process of infection of plants by fungi. The terms parasitism, symbiosis, heterospory and heteroecy were coined or redefined based on his experimental studies. His numerous studies on algae, lichens, ferns and higher plants enriched the knowledge of the time. As a teacher, de Bary was very modern – not authoritarian and promoted self-reliance, observation skills, self-control and a critical evaluation of one's own results and conclusions. He was internationally acknowledged, and many scientists worldwide came to visit his modern-equipped laboratory. Key Concepts: The great achievements of biological research in the nineteenth century were the setting up of cell biology, disproof of spontaneous generation and Charles Darwin's proposal of the formation of species by natural selection. Anton de Bary founded the developmental biology of fungi, that is, he identified the sequence of sexual and asexual stages and their fructification organs. Different rust fungi are heteroecic, that is, the different developmental stages are dependent on different host plants. de Bary proposed that the classification of fungi should be based on their evolution instead of on pure morphological criteria. de Bary is the one who first defined the term symbiosis as a closed spatial coexistence of different species. de Bary mastered his life as Professor of Botany, Director of Botanical Gardens, successful researcher and teacher of students through a high degree of self-discipline and a sense of duty. Keywords: de Bary; evolutionary biology; fungal development

Chemotrophe Bakterien, die unter Verwendung von Sauerstoff organische Substrate abbauen und Bacteriochlorophyll a synthetisieren, sind an marinen und Suswasserstandorten weit verbreitet. Sie gehoren phylogenetisch verschiedenen Gruppen der Proteobakterien an und bilden einen Photosyntheseapparat aus, der in seiner Organisation dem der Purpurbakterien entspricht, physiologisch aber keine Bedeutung zu haben scheint. Er ist unter der obligat aeroben chemotrophen Lebensweise der Bakterien praktisch funktionslos. Diese Bakterien konnen nicht wie die roten und grunen photosynthetischen Bakterien anaerob photoheterotroph oder photoautotroph im Licht wachsen. Im folgenden wird die monophyletische Evolution des Photosyntheseapparates und seine spezielle Funktion und Entwicklung bei diesen obligat aeroben Bakterien diskutiert.

The gene encoding catalase-peroxidase was cloned from chromosomal DNA of Rhodobacter capsulatus B10. The nucleotide sequence of a 3.7-kb SacI-HindIII fragment, containing the catalase-peroxidase gene (cpeA) and its flanking regions were determined. A 1728-bp open reading frame, coding for 576 amino acid residues (molecular mass 61516 Da) of the enzyme, was observed. A Shine-Dalgarno sequence was found 5 bp upstream from the translational start site. The deduced amino acid sequence coincides with that of the amino terminus and of four peptides derived from trypsin digestion of the purified catalase-peroxidase of R. capsulatus B10. The amino acid sequence of R. capsulatus catalase-peroxidase shows interesting similarities to the amino acid sequences of the hydroperoxidases of Escherichia coli (42.7%) and Salmonella typhimurium (39.9%), the peroxidase of Bacillus stearothermophilus (32.1%) and the catalase-peroxidase of Mycobacterium intracellulare (42.2%). As shown by a cpeA::lacZ fusion in trans in R. capsulatus, the expression of the catalase-peroxidase gene is regulated by oxygen. The promoter of the cpeA gene was localized within 320 bp upstream of the ATG start codon.

Formation of the light harvesting complex B800-850 (LHII) of Rhodobacter capsulatus requires the expression of more than the three known genes specific for that complex (pucA, pucB and pucE) encoding the alpha, beta and gamma subunits of LHII, respectively. In this work evidence is presented that the product of the gene pucC, which is located downstream from pucA, is essential for high-level transcription of the pucBACDE operon and formation of LHII. Plasmids were constructed containing deletions in one or several puc genes and transferred to a pucC::Tn5 mutant in which the puc operon is not expressed. It was found that the LHII- phenotype of the mutant was due to the missing PucC protein and that all known puc genes are located in one operon. To dissect the pucC, pucD and pucE genes from pucB and pucA and independently regulate them, they were placed under control of the nifHDK promoter. Only under nitrogen-fixing growth conditions was the LHII- pucC::Tn5 mutant complemented by this construction. It is concluded that expression of pucC is essential for formation of the LHII complex in R.capsulatus. Analysis of the pucD and pucE genes led to the conclusion that the products of these genes stabilize the B800-850 complex.

Trp-8 and Pro-13 of the Rhodobacter capsulatus light-harvesting (LH) I alpha polypeptide are highly conserved among LHI and LHII alpha proteins of several species of the Rhodospirillaceae. Exchange of Trp-8 and Pro-13 to other amino acyl residues similar in structure and/or hydrophobicity indicates that Trp-8 is involved in the insertion of the LHI alpha polypeptide into the intracytoplasmic membrane (ICM). Pro-13, however, seems not to participate in the integration process of the LHI alpha protein but seems to be important for stable insertion of the LHI beta partner protein in the ICM.

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 Q x transitions are aligned along the long axis of the crystal, while the 875 nm Q y 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 Q x direction and perpendicular to it a distribution of 875 nm Q y transitions exhibiting a maximum along one direction.

The cytochrome oxidase (EC 1.9.3.1) of Rhodopseudomonas palustris was extracted with Triton X-100 plus KCl, from the membrane fraction of cells grown aerobically in the dark. The solubilized enzyme was purified by (NH4)2SO4 precipitation and chromatography on DEAE-cellulose. The purification resulted in a 108-fold enrichment of cytochrome oxidase on the basis of specific activity when compared to the membrane fraction. The purified enzyme was phosphate-sensitive (less than mM), oxidized reduced bovine, horse and yeast cytochrome c, and was inhibited 50% by 0.5 muM KCN or 7 muM NaN3. The native purified preparation migrated as one band in polyacrylamide gel electrophoresis. In the presence of dodecylsulfate four major polypeptides with apparent molecular weights of 30500, 25500, 12200 and 9500 were observed. The enzyme reacted with oxygen via cytochrome o. The purified preparation contained cytochrome c but was free of flavoproteins and NADH-linked and succinate-linked enzyme activities of the respiratory chain.

A new procedure for isolation, purification and identification of the three polypeptides of the membranebound light-harvesting complex I1 (BSOO-850) of Rhodopseudomonas capsulata has been developed. The polypeptides were extracted from crude intracytoplasmic membranes with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60. The peak fractions were transferred to solvents of different polarity and separated by gel filtration or ion-exchange chromatography. The three major polypeptides isolated by this two-step chromatography were found to be homogenous and identical with the three polypeptides of the light-harvesting complex 11, as judged by amino acid analysis and N-terminal sequence determination. Contaminating minor polypeptides, of which the functions are unknown, were different from the polypeptides of the B800-850 complex studied by the same criteria. The photosynthetic apparatus of Rhodopseudomonas capsuluta contains three bacteriochlorophyll-carotenoid-protein complexes, the photochemical reaction center and the two light-harvesting complexes I (B870) and I1 (BSOO-850). These complexes have been solubilized by detergent treatment from the membrane and purified [I, 21. The light-harvesting complex I1 contains three polypeptides. The smaller polypeptides are stoichiometrically associated with bacteriochlorophyll a and carotenoids forming the pigment moieties absorbing at 800 nm and 855 nm respectively [2,3]. The native B800-850 complex seems to consist of a tetramer of the basic unit, having an apparent M, of 180000 [4]. The three polypeptides have been isolated from the complex and characterized by amino acid composition and N-terminal sequence [2]. The isolation procedure was time-consuming and resulted in a low yield. In order to obtain highly purified polypeptides in larger amounts simple and efficient methods were developed. The polypeptides were extracted directly from freeze-dried crude membranes and purified to homogeneity in two chromatographic steps. The relevance of this procedure for isolation of integral membrane proteins and some properties of the isolated polypeptides are discussed.

The small bacteriochlorophyll-binding polypeptide of the light-harvesting complex B870 was extracted from the intracytoplasmic membrane of the strain A1a+ of Rhodopseudomonas capsulata with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60 using the same solvent. The polypeptide obtained from the peak fraction III was found to be homogeneous and identical with the small polypeptide isolated from the B870 complex as shown by dodecyl sulfate/polyacrylamide gel electrophoresis, amino acid composition and N-terminal sequence. The complete amino acid sequence is given. The relative molecular mass based on the amino acid sequence is 5341. The polarity of amino acids is 35.42%. The C-terminal part of the peptide chain from residue 29 to 48 is hydrophobic and includes one His residue.

A cytochrome c (cyt. c ) was solubilized with Triton-X-100 and co-purified with cytochrome c oxidase from membranes of chemotrophically grown cells of Rhodopseudomonas capsulata . Cyt. c and cytochrome oxidase were separated on Sephadex G-50 columns. Antibodies against cytochrome c 2 from the same bacterium did not cross react with the membrane-bound cyt. c . The IEP of the membrane-bound cyt. c was found to be pH 8.2 the midpoint potential was 234 ± 11 mV at pH 7.0. This cyt. c binds CO. The native cyt. c is a dimer with an apparent M r of 25 000 containing 2 mol heme per mol dimer, which is believed to function as an electron donor for the high-potential cytochrome c oxidase.

The small bacteriochlorophyll-binding polypeptide(β) of the light-harvesting complex B800-850 from the phototrophic bacterium Rhodopseudomonas capsulata was purified from the complex and the membrane, respectively. The complete amino-acid sequence has been determined. The M r of the polypeptide is 4597. The protein consists of 49 amino acids, 64% of which are hydrophobic. Homology with other bacteriochlorophyll-binding polypeptides is discussed. Bacteriochlorophyll-binding polypeptide Primary structure Rhodopseudomonas capsulata B800-850 antenna pigment complex

From the lipopolysaccharide of the gram-negative bacterium Rhodopseudomonas capsulata (Athiorhodaceae) a lipophilic sugar was isolated by chromatographic procedures and characterized by mass spectrometry as belonging to the group of 3-O-methyl-6-deoxy-hexoses. Demethylation showed that the parental sugar had the same properties in borate electrophoresis as rhamnose. Comparison with an authentic sample of 3-O-methyl-rhamnose in gas liquid chromatography, paper chromatography and borate electrophoresis showed that the sugar under examination is 3-O-methylrhamnose (acofriose). Investigation of its optical rotation indicates that the sugar has the l-configuration. l-Acofriose (3-O-methyl-l-rhamnose) has not hitherto been found in lipopolysaccharides of gram-negative bacteria.