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The beginning of modern microbiology can be traced back to the 1870s, and it was based on the development of new concepts that originated during the two preceding centuries on the role of microorganisms, new experimental methods, and discoveries in chemistry, physics, and evolutionary cell biology. The crucial progress was the isolation and growth on solid media of clone cultures arising from single cells and the demonstration that these pure cultures have specific, inheritable characteristics and metabolic capacities. The doctrine of the spontaneous generation of microorganisms, which stimulated research for a century, lost its role as an important concept. Microorganisms were discovered to be causative agents of infectious diseases and of specific metabolic processes. Microscopy techniques advanced studies on microorganisms. The discovery of sexuality and development in microorganisms and Darwin's theory of evolution contributed to the founding of microbiology as a science. Ferdinand Cohn (1828-1898), a pioneer in the developmental biology of lower plants, considerably promoted the taxonomy and physiology of bacteria, discovered the heat-resistant endospores of bacilli, and was active in applied microbiology.

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.

The expression of the Rhodobacter capsulatus catalase-peroxidase gene (cpeA) was studied by in-frame fusions of the upstream region of the cpeA gene to a promoter-less lacZ gene. The transcription of the cpeA gene is about 20-50-fold higher under aerobic-dark than under anaerobic-light conditions. The promoter was localized within a 69-bp upstream DNA region. The transcription start site, determined by primer extension, is 28 bases upstream from the initiation codon, confirming the postulated promoter localized by deletion analysis. Deletion of the part of the upstream region specifically responsible for oxygen regulation resulted in constitutive expression of the cpeA gene.

Synthesis and phosphorylation of the proteins α and β of the light-harvesting (LH) complex I (B870) were investigated in a cell-free translation system of Rhodobacter capsulatus. Both proteins were incorporated into the membrane fraction; LHIβ was inserted in the absence of LHIα was phosphorylated in the presence of [γ-32P]ATP only when membranes were present. Phosphorylated LHIβ was found only in the absence of membranes. The phosphate group bound to LHIβ was not transferred to LHIα during insertion. The results indicate that a membrane-bound and a soluble protein kinase are involved. Strong light reduced the amount of phosphorylated LHIα. The results are discussed with respect to the assembly and function of LHI.

The spectral analysis of membrane fractions and a fraction enriched in light-harvesting (LH) complex I + reaction center (RC) of Roseococcus thiosulfatophilus, RB3 and of the membrane fraction and of the isolated LH II and LH I + RC complexes of Erythromicrobium ramosum, E5 is reported. Quantum yields of singlet energy transfer between carotenoids and bacteriochlorophyll (light-harvesting function) were calculated from comparison of absorption and fluorescence excitation spectra of the respective preparations. The results indicate that the excess of carotenoids in the membrane does not contribute to the lightharvesting function of the LH complexes.

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.

Light-harvesting complex I (LHI) of Rhodobacter capsulatus contains bacteriochlorophyll and carotenoids which are noncovalently bound to two different apoproteins (alpha and beta polypeptides) carrying oppositely charged N-terminal ends. The contribution of these charged segments to the assembly of LHI was studied with mutants having oppositely charged amino acids in the alpha or beta polypeptide. The influence of these mutations on the insertion and assembly process of the LHI complex was investigated by means of spectroscopic analysis of isolated intracytoplasmic membranes and pulse-chase experiments. Exchange of four positively charged amino acids to negatively charged amino acids on the N-terminal domain of the alpha subunit inhibited completely the assembly of the LHI complex. Although this mutant has no antenna, the reaction center is active and the cells were able to grow anaerobically in the light. Conversely, mutation of the four negatively charged amino acids of the N-terminal segment of the beta polypeptide did not prevent the assembly of the LHI complex, although the stability of the complex and the size of the photosynthetic unit were affected. The presence of the mutated beta polypeptide was confirmed by protein sequencing.

Most of the bacterial photosynthetic reaction centres known to date contain a cytochrome subunit with four covalently bound haem groups. In the case of Blastochloris viridis, this reaction centre subunit is anchored in the membrane by a lipid molecule covalently attached to the cysteine which forms the N-terminus of the mature protein after processing by a signal peptidase. We show that posttranslational N-terminal cleavage of the cytochrome subunit does not occur in the aerobic photosynthetic bacterium Roseobacter denitrificans. From sequence analysis of the resulting elongated N-terminus it follows that a transmembrane helix is anchoring the reaction centre-bound cytochrome in the membrane. Comparative sequence analysis strongly suggests that all cytochrome subunits lacking the lipid coupling cysteine share this structural feature. Comparison of the N-terminal segment of the cytochrome subunit of Roseobacter denitrificans with the sequences of the PufX proteins from Rhodobacter sphaeroides and Rhodobacter capsulatus suggests a phylogenetic relation.

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.

A respiring culture of Rhodobacter sphaeroides, grown in the dark under defined aerobic conditions, produced cells capable of immediately commencing adaptation to photosynthetic growth on exposure to light and further reduction of oxygen tension. Adaptation was complete after 12 h and the bacteriochlorophyll a content increased 10–20-fold. This adaptation was performed in the presence of either H218O or 18O2. The extracted bacteriochlorophyll a was examined by mass spectrometry to determine the origin of both the 3-acetyl and 131-oxo oxygen atoms: both were derived from water. The derivation of the 131-oxo group from water in R. sphaeroides indicates that the formation of isocyclic ring E from the 13-propionic acid methylester side chain of Mg2+-protoporphyrin IX mono-methylester is an anaerobic process involving a hydratase. This is very different to the situation in higher plants and green algae where the formation of isocyclic ring E is an aerobic process in which the 131-oxo group is derived from molecular oxygen via an oxygenase. In contrast to adapting R. sphaeroides cells, the 131-oxo group of bacteriochlorophyll a in growing cells of the obligate aerobic chemotrophic bacterium Roseobacter denitrificans, was labelled by 18O2 and is, therefore, derived from molecular oxygen like in higher plants and green algae; however, the 3-acetyl group was not labelled by 18O2. Thus, while the 131-oxo group has different origins in R. sphaeroides and R. denitrificans, the 3-acetyl group arises in both bacteria by enzymic hydration of the vinyl group of a chlorophyll a derivative.

The photosynthetic bacterium Rhodobacter sulfidophilus is able to grow chemotrophically and phototrophically at a broad range of light intensities. In contrast to other facultative phototrophs, R. sulfidophilus synthesizes reaction center and light-harvesting (LH) complexes, B870 (LHI) and B800-850 (LHII) even under full aerobic conditions in the dark. The content of bacteriochlorophyll (BChl) varied from 3.8 micrograms Bchl per mg cell protein when grown at high light intensity (20,000 lux) to 60 micrograms Bchl per mg cell protein when grown at low light intensities (6 lux). After a shift from high light to low light conditions, the size of the photosynthetic unit increased by a factor of 4. Chromatographic analysis of the LHII complex, isolated and purified from cells grown phototrophically (at high and low light intensities) and chemotrophically, could resolve only one type of alpha and one type of beta polypeptide in the purified complex, of which the N-terminal sequences have been determined.

We analyzed the 16S ribosomal DNAs of three obligately aerobic, bacteriochlorophyll a-containing bacteria, "Roseococcus thiosulfatophilus," "Erythromicrobium ramosum," and new isolate T4T (T = type strain), which was obtained from a marine cyanobacterial mat. "Roseococcus thiosulfatophilus" is a member of the alpha-1 subclass of the Proteobacteria and is moderately related to Rhodopila globiformis, Thiobacillus acidophilus, and Acidiphilium cryptum (level of sequence similarity, 90%). "Erythromicrobium ramosum" and isolate T4T are closely related to Erythrobacter longus and Porphyrobacter neustonensis (level of sequence similarity, 95%). These organisms are members of the alpha-4 subclass of the Proteobacteria. Strain T4T is a motile, red or orange bacterium. The major carotenoids are bacteriorubixanthinal and erythroxanthin sulfate. In vivo measurements revealed bacteriochlorophyll absorption maxima at 377, 590, 800, and 868 nm. Strain T4T grows in the presence of 5 to 96/1000 salinity and uses glucose, fructose, acetate, pyruvate, glutamate, succinate, and lactate as substrates. On the basis of its distinct phylogenetic position and phenotypic characteristics which are different from those of Erythrobacter longus, we propose that strain T4T should be placed in a new species of the genus Erythrobacter, Erythrobacter litoralis. The descriptions of "Roseococcus thiosulfatophilus" and "Erythromicrobium ramosum" are emended.

The NH2 termini of light-harvesting complex I (LHI) polypeptides alpha and beta of Rhodobacter capsulatus are thought to be involved in the assembly of the LHI complex. For a more detailed study of the role of the NH2-terminal segment of the LHI alpha protein in insertion into the intracytoplasmic membrane (ICM) of R. capsulatus, amino acids 6 to 8, 9 to 11, 12 and 13, or 14 and 15 of the LHI alpha protein were deleted. Additionally, the hydrophobic stretch of the amino acids 7 to 11 was lengthened by insertion of hydrophobic or hydrophilic amino acids. All mutations abolished the ability of the mutant strains to form a functional LHI antenna complex. All changes introduced into the LHI alpha protein strongly reduced the stability of its LHI beta partner protein in the ICM. The effects on the mutated protein itself, however, were different. Deletion of amino acids 6 to 8, 9 to 11, or 14 and 15 drastically reduced the amount of the LHI alpha protein inserted into the membrane or prevented its insertion. Deletion of amino acids 12 and 13 and lengthening of the stretch of amino acids 7 to 11 reduced the half-life of the mutated LHI alpha protein in the ICM in comparison with the wild-type LHI alpha protein. Under the selective pressure of low light, revertants which regained a functional LHI antenna complex were identified only for the mutant strain deleted of amino acids 9 to 11 of the LHI alpha polypeptide [U43 (pTPR15)]. The restoration of the LHI+ phenotype was due to an in-frame duplication of 9 bp in the pufA gene directly upstream of the site of deletion present in strain U43(pTPR15). The duplicated nucleotides code for the amino acids Lys, Ile, and Trp. Membranes purified from the revertants were different from that of the reaction center-positive LHI+ LHII- control strain U43(pTX35) in doubling of the carotenoid content and increase of the size of the photosynthetic unit. By separating the reaction center and LHI complexes of the revertants by native preparative gel electrophoresis, we confirmed that the higher amount of carotenoids was associated with the LHI proteins.

Strain-specific low-molecular-weight polysaccharides of different chemical compositions were obtained from cells of nine different wild-type strains of the phototrophic bacterium Rhodopseudomonas gelatinosa. The polysaccharides are free of typical capsule components like hexuronic or aminohexuronic acids but contain (except that of strain 39/2) substantial amounts of phosphorus. A number of unusual o-methyl sugars (2-o-methyl-D-galactose, 2,3-di-o-methyl-D-galactose, 2-o-methyl-L-fucose) as well as 3,6-dideoxy-D-xylo-hexose (abequose) were identified in the R. gelatinosa polysaccharides. o-Methyl and dideoxy sugars however, are typical constituents of O-specific chains of the lipopolysaccharides of gram-negative bacteria (Rhodospirillaceae and Enterobacteriaceae, respectively). Considering both the R-type character of the R. gelatinosa lipopolysaccharides and the occurrence of these strain-specific ETEROPOLYSACCHARIDES, THE ASSUMPTION SEEMS TO BE JUSTIFIED THAT THE LOW-MOLECULAR-WEIGHT POLYSACCHARIDES ARE RELATED TO O-specific chains of lipopolysaccharides (haptens) rather than to capsular or slime antigens. In serological terms the polysaccharides of R. gelatinosa have to be classified as K-antigens. They are able to cover the O-specificity of the respective different strains and confer on them additional specificity which is demonstrable by bacterial agglutination.

SUMMARY: A high molecular weight polysaccharide sedimenting at 105 000 g was extracted into the water phase of phenol/water extracts of Chlorogloeopsis PCC 6912 cells. The main sugars of the heteropolysaccharide were mannose, 6-O-methyl-d-mannose, glucose, rhamnose, and glucuronic and galacturonic acids. 6-O-Methyl-d-mannose was present partly in terminal linkage and partly 1,3-chain-linked, as revealed by methylation analysis. 3-O-Methyl-mannose, however, was found in only trace amounts and exclusively occupied terminal positions. The heteropolysaccharide presumably represented a polysaccharide of the cell wall. There was no indication of a lipid moiety. Application of procedures commonly used for lipopolysaccharide extraction from whole cells resulted in very low yields or even indicated complete lack of a typical lipopolysaccharide.

The near infrared absorption spectra and their fourth derivative were measured in membrane preparations or in the isolated light harvesting pigment-protein complex B 800-850 from Rhodopseudomonas capsulata in order to know how the underlying molecular absorption spectra contribute to the observed absorption curve. In contrast to the observations of Cogdell and Crofts [Biochim. Biophys. Acta 502, 409 (1978)] no splitting of the 855 nm absorption bands was observed at 300 K, 100 K, and 4 K. However, a small but significant splitting of the 870 nm band in all derivates (2nd, 4th, 6th) was observed at 300 K and 4 K. The lack of splitting of the 855 nm absorption band will be discussed in the light of the molecular organization of the B 855 moiety of this light harvesting bacteriochlorophyll-protein complex.

Lipopolysaccharides (LPS, O-antigens) of 12 strains of the photosynthetic bacterium Rhodopseudomonas gelatinosa were obtained by the phenol/chloroform/petroleum ether method, recommended for extracting lipophilic glycolipids of enterobacterial R-mutants. All R. gelatinosa LPS have essentially the same chemical composition. Similar to LPS of Salmonella R-mutants of chemotypes Rd1 and Rd2, the sole neutral sugar constituent is an aldoheptose. The heptose of R. gelatinosa LPS has the D-glycero-D-manno- configuration, in contrast to the L-glycero-D-mannoheptose of enterobacterial LPS. 2-Keto-3-deoxyoctonate forms the acid-labile linkage between the lipid moiety (lipid A) and the oligosaccharide moiety of R. gelatinosa LPS. Like enterobacterial lipid A, lipid A of this species contains phosphate and D-glucosamine as the sole amino sugar. The fatty acid spectrum conprises beta-hydroxycapric, lauric, and myristic acids. Beta-Hydroxymyristic acid, the typical fatty acid of enterobacterial LPS, is lacking. The R. gelatinosa LPS show O-antigenic acitivity; passive hemagglutinations with untreated or heat-treated (not well alkali-treated) LPS and antisera prepared against heat-killed cells yield high titers. According to the serological cross-reactions observed, the LPS of the 12 strains could be arranged into two different serotypes: serotype I comprising strains 29/1, 29/2, 25/2, and serotype II comprising strains 44/K/6, 3/1, IS/10, 39/2, Dr2, 2150, P8P9, K32, P18f3.1. No serological cross-reactions were observed between LPS of these two different serotypes in passive hemagglutinations.

The light-harvesting antenna complex II of Rhodopseudomonas capsulata is characterized by two prominent IR-absorption maxima at 802 and 855 nm and three polypeptides of 14,000, 10,000 and 8,000 molecular weight (SDS-polyacrylamide gel electrophoresis). Bacteriochlorophyll (Bchl) is associated with the two lower molecular weight polypeptides. The membranes of the mutant Y 5, which lades reaction center and light harvesting complex I (B 875), were treated with trypsin. The 14,000 polypeptide was rapidly digested by trypsin, but the absorption spectrum of the membrane and the activity of the cytochrome c oxidase were not altered. Subsequently the 8,000 polypeptide was degraded. The digestion of the 8,000 polypeptide was concomitant with and proportional to the loss of absorbance at 802 nm. The absorption peak at 855 nm and the content of the 10,000 molecular weight polypeptide were, in contrast, stable for a longer time, but were also lost simultaneously. These results, in combination with the recent publications of Sauer and Austin (Biochemistry 17, 2011, 1978), and Cogdell and Crofts (Biochim. Biophys. Acta 502, 409, 1978) support the idea that the two molecules of Bchl associated with the 10,000 polypeptide are responsible for the 855 nm peak while the one or two mol of Bchl associated with 8,000 polypeptide result in the 802 nm absorption maximum.

The photosynthetically negative mutant strain Y142 of Rhodopseudomonas capsulata, which synthesizes bacteriochlorophyll (Bchl), carotenoids and the light-harvesting (LH) complex B800–850, but no reaction center and LH complex B870, is capable of synthesizing the Bchl-binding polypeptide (α, 12 kDa) of B870. In contrast to the high stability of the polypeptides of the B800–850 complex, the 12 kDa polypeptide was rapidly degraded after synthesis and insertion into the membrane.

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.

The photosynthetic chromatophore membranes of Rhodopseudomonas capsulata were fused with liposomes to investigate the effects of lipid dilution on energy transfer between the bacteriochlorophyll-protein complexes of this membrane. Phosphatidylcholine-containing liposomes were mixed with chromatophores at pH 6.0 to 6.2, and the mixture was fractionated on discontinuous sucrose gradients into four membrane fractions with lipid-to-protein ratios that varied 11-fold. Freeze-fracture electron microscopy revealed that the fractions contained closed vesicles formed by the fusion of liposomes to chromatophores. Particles with 9-nm diameters on the P fracture faces did not appear to change in size with increasing lipid content, but the number of particles per membrane area decreased proportionally with increases in the lipid-to-protein ratio. The bacteriochlorophyll-to-protein ratios, electrophoretic polypeptide profiles on sodium dodecyl sulfate-polyacrylamide gels, and light-induced absorbance changes at 595 nm caused by photosynthetic reaction centers were not altered by fusion. The relative fluorescence emission intensities due to the B875 light-harvesting complex increased significantly with increasing lipid content, but no increases in fluorescence due to the B800-B850 light-harvesting complex were observed. Electron transport rates, measured as succinate-cytochrome c reductase activities, decreased with increased lipid content. The results indicate an uncoupling of energy transfer between the B875 light-harvesting and reaction center complexes with lipid dilution of the chromatophore membrane.

Absorption, linear dichroism and circular dichroism spectra of Rhodopseudomonas capsulata (wild-type-St. Louis strain, mutant Y5 and mutant Ala+) are particularly sensitive to the nature of the light-harvesting bacteriochlorophyll-carotenoid-protein complexes. Evidence for exciton-type interactions is seen near 855 nm in the membranes from the wild-type and from mutant Y5, as well as in an isolated B-800 + 850 light-harvesting complex from mutant Y5. The strong circular dichroism that reflects these interactions is attenuated more than 10-fold in membranes from the Ala+ mutant, which lacks both B-800 + 850 and colored carotenoids and contains only the B-875 light-harvesting complex. These results lead to the conclusion that these two light-harvesting complexes have significantly different chromophore arrangements or local environments.

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 mutant strains A l a+ and Y 5 of Rhodopseudomonas capsulata are defective in formation of the light-harvesting complexes B800-850 and B870, respectively, but synthesize the large bacteriochlorophyll-binding polypeptides of the respectively complexes, shown by amino acid composition, N- or C-terminal sequence and immunoblotting. The stable assembly of the light-harvesting complexes in the membrane seems to be dependent from the coordinated synthesis of all pigment and protein components.

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.

Proteinase K and trypsin were used to determine the orientation of the light-harvesting B800-850 alpha and beta polypeptides within the chromatophores (inside-out membrane vesicles) of the mutant strain Y5 of Rhodopseudomonas capsulata. With proteinase K 7 amino acid residues of the B800-850 alpha polypeptide were cleaved off up to position Trp-7--Thr-8 of the N terminus, and 11 residues were cleaved off up to position Leu-11-Ser-12 of the beta chain N terminus. The C termini of the B800-850 alpha and beta polypeptides, including the hydrophobic transmembrane portions, remained intact. It is proposed that the N termini of the alpha and beta subunits, each containing one transmembrane alpha-helical span, are exposed on the cytoplasmic membrane surface and the C termini are exposed to or directed toward the periplasm.

A cell surface protein (Mr 68,000) of the moderately but obligately halophilic phototrophic bacterium Rhodospirillum salexigens was identified by two independent methods: first, by labeling the cell surface with radioactive iodine and lactoperoxidase, and second, by washing cells in 30% sucrose to remove proteins attached to the cell surface by ionic bonds. The identified protein very likely represents the outermost layer of the cell envelope of R. salexigens as observed by electron microscopy. The protein was isolated. Its isoelectric point was determined to be 4.4; the excess of acidic over basic amino acids was found to be 18.3 mol%; and its average hydrophobicity was 2.26 kJ per residue.

A cytochrome oxidase (EC 1.9.3.1) was solubilized from the membrane fraction of aerobically grown cells of Rhodopseudomonas capsulata by treatment with Triton X-100. The enzyme was purified 160 fold by chromatography on DEAE-Sepharose CL-6B and affinity chromatography on cytochrome c-thiol activated Sepharose 4B. The purified enzyme has a pH-optimum at 8.5 and a temperature optimum at 35 °C. The ap­ parent Km for reduced horse cytochrome c is 24 μм (at pH 8 and 30 °C). The purified cytochrome oxidase was 50% inhibited by 1.5 μм KCN and 10 μм NaN3. The purified enzyme contained one polypeptide of mr 65,000 and 6-type cytochrome.

Soluble NADH dehydrogenase was purified to homogeneity from chemotrophically grown cells of Rhodopseudomonas capsulata by ammonium sulfate fractionation, AH -Sepharose 4B chromatography and FMN-Sepharose 6B affinity chromatography. The enzyme contains a single polypeptide chain of an apparent M, of 37000, suggesting that the subunit structure is different from that of the membrane-bound enzyme. The purified soluble NADH dehydrogenase requires flavin compounds, e.g., FMN, FAD and riboflavin, for activity. Addition of FMN and FAD. but not riboflavin, to the enzyme solution stabilized the enzyme. The pH optimum for activity was at 7.5. The enzyme was specific for NADH as an electron donor while NADPH was inert. Menadione, ferricyanide, cytochrome c and DCIP served as an electron acceptor. The M ichaelis constants for NADH. DCIP, FM N. and cytochrome c were 45, 2.9. 7.9 and 15 μM, respectively. Many properties of soluble NADH dehydrogenase were substantially different from those of the membrane-bound enzyme, suggesting different functions.

Chemotrophically grown cells of Rhodopseudomonas capsulata contain at least three different pyridine nucleotide dehydrogenases, i) a soluble, found in the supernatant (144000 × g) of cell free extracts, NADH-dependent, ii) a mem brane-bound, NADH-dependent, and iii) a soluble, found in the supernatant N AD PH dependent. The membrane-bound NADH dehydrogenase (E.C. 1.6.99.3) has been solubilized by sodium deoxycholate treatm ent of m em branes and purified 75 fold by column chrom atography on Sephadex G-150 and DEAE cellulose in the presence of sodium cholate. The native enzyme has an apparent molecular mass (Mr) of 97 000, containing polypeptides of Mr of about 15 000. The pH optim um was at 7.5. The enzyme was specific for NADH. The Michaelis constant for NADH and DCIP were 4.0 and 63 μm, respectively. The enzyme was inactivated by FMN, riboflavin and NADH. In contrast, the soluble NADH-dehydrogenase (i) was activated by FMN.

The formation of the light-harvesting complex B800-850 (LH-II) of Rhodobacter capsulatus requires, in addition to the synthesis of the polypeptides alpha and beta (the gene products of pucA and pucB), the synthesis of bacteriochlorophyll and carotenoids and the expression of at least one gene localized downstream from the pucBA operon. This was concluded from the observation that a Tn5 insertion downstream from pucBA inhibited the formation of the LH-II complex and the formation of the pucBA mRNA. The Tn5 insertion point was mapped and found to be over 500 base pairs (bp) downstream from the end of the pucA gene, suggesting the presence of additional puc genes. A region of about 3,000 bp including the pucB and pucA genes and DNA downstream from pucA was sequenced and found to contain three open reading frames (ORFs C, D, and E). The polypeptide deduced from the first ORF (C) contains 403 amino acids with strongly hydrophobic stretches and one large and three small hydrophilic domains carrying many charged residues. The other two ORFs contain 113 (D) and 118 (E) codons. The amino acid sequences of the N terminus and two tryptic peptides of an alkaline-soluble Mr-14,000 subunit of the isolated LH-II complex were identical with the deduced amino acid sequence of ORF E.

SUMMARY: A sheath fraction was isolated from cells of the cyanobacterium Chlorogloeopsis PCC 6912. It had a fibrillar fine structure, a high density and contained 38% (w/w) carbohydrates and 22% (w/w) protein. Glucose, mannose, galactose, arabinose, xylose, an unknown sugar and glucuronic acid were the major carbohydrates. Lipids were almost entirely absent. The protein was not solubilized by treatment with hot phenol/water (68 °C; 20 min) or Triton X-100 (2%, w/v; 20 °C; 3 h). The carbohydrates of the sheath belong to two different polysaccharides. The major one was not separated from the protein moiety of the sheath by hot phenol/water. It contained glucose as the main constituent, arabinose and xylose but little galactose, in addition to the other carbohydrates. In contrast, the minor polysaccharide was soluble in the water phase of hot phenol/water extracts and contained galactose as a major constituent in addition to glucose and mannose, but very little arabinose and xylose. The polysaccharide enriched in galactose could also be obtained from the water phase by precipitation with Cetavlon when sheath-containing whole cells were extracted by phenol/water.

Intracytoplasmic membrane vesicles (chromatophores) isolated from Rhodopseudomonas capsulata cells were fused with liposomes by a pH transition procedure. Vesicles of lower density and higher lipid contents and larger diameter than chromatophores were obtained. Similar results were observed by Ca2+ induced fusion and by the freeze-thawing method. Respiratory and light-induced electron transport were measured in chromatophores and fused vesicles. Light-induced reaction center bleaching was observed in all types of vesicles, whereas repiratory electron transport was substantially diminished by lipid incorporation. Ubiquinone 10 restored to some extent respiratory electron transport and oxidative phosphorylation and it modified the photophosphorylation kinetics under continuous light. Electrochromic carotenoid band-shift and the 9-aminoacridine fluorescence quenching indicate that the capacity of the fused vesicles to maintain an electrochemical proton gradient has not been substantially diminished. From the kinetics of 9-aminoacridine quenching an increased K+-permeability seems to be apparent.

Lowering of oxygen partial pressure in chemotrophic cultures or reduction of light intensity in phototrophic cultures of Rhodopseudomonas capsulata induced formation of the photosynthetic apparatus. A maximum of mRNA coding for the reaction center (RC) and the light-harvesting 1 B870 antenna complex polypeptides occurred 30 min after induction. Maximal expression of mRNA for B800-B850 antenna proteins appeared with a lag time of about 25 min after RC/B870 mRNA. Pigment-binding polypeptides were inserted into the membrane immediately after mRNA synthesis. It is concluded that the delayed formation of the B800-B850 complex compared to the RC and the B870 complex is caused by sequential expression of the corresponding genes. Biological activity of pigment-protein complexes increased after the incorporation of their polypeptides parallel to the maximum of bacteriochlorophyll synthesis. Studies on mutant strains defective in the formation of pigment-protein complexes suggested that pigment synthesis is of importance for assembly of stable complexes.

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.

Aerobically in the dark grown cells were incubated semiaerobically (30 min) and afterwards 180 min anaerobically in the light During phototrophic induction the bacteriochlorophyll concentra­ tion increased from 0.26 to 2.10 nmol/mg cell protein. In samples taken at different times after lowering of oxygen partial pressure the following tetrapyrrol derivatives were identified by fluorescence emission and excitation spectroscopy at 1.7 K: Mg-protoporphyrin EX-monomethylester, Mg-2,4-divinylphaeoporphyrin a5-monomethylester, 2-de-vinyl-2 -hydroxyethyl-chlorophyllide, 2 -devinyl-2 -hydroxyethyl-pheophorbide, chlorophyllide a, pheophorbide, bacteriopheophorbide, bacteriopheophytin, bacteriochlorophyllide and bacte­ riochlorophyll a in different pigment complexes. The highest relative concentrations of bacte­ riochlorophyll precursors normalized to the total amount o f bacteriochlorophyll a were found in cells during the first hour of adaptation at 0.5 μg Bchl/mg cell protein or less.

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