LHII Complex Research Articles

Effect of light intensity and various organic acids on the growth of Rhodobacter sphaeroides LHII-deficient mutant in a turbidostat culture.

The composition of photosynthetic apparatus of Rhodobacter sphaeroides wild strain 2.4.1 and its LHII-deficient mutant DBCΩ was compared. The absence of LHII in the mutant was confirmed by comparison of chromatophores spectra and by the absence of electrophoretic band corresponding to LHII complex. Continuous turbidostat cultures of wild strain and its LHII-deficient mutant were compared in response to different light intensities. Cultures were grown using lactate, mixture of lactate and acetate or succinate as carbon source. For comparative analysis, an approximation of experimental data by Monod and Gompertz equations were used. Cultures of DBCΩ had lower growth rates than wild strain when grown on lactate as electron donor and carbon source. Cultures of both strains grown on lactate and acetate or on succinate had similar growth rates. The cultures showed maximum growth rates when grown with succinate. Bacteriochlorophyll a content increased in both strains with decrease of incident light intensity. However, the variation of Bchl a content in wild strain was much more significant. Under light-limiting conditions, bacteriochlorophyll a content in DBCΩ was 4-5 times lower than in the wild strain. Under light-saturating conditions, it was only 1.5-2.5 times lower. Growing with lactate or with lactate and acetate, the mutant switched from light limitation under low light intensities to limitation by organic acids under higher light, whereas the parental strain had similar switch of limiting factor only when growing with lactate and acetate mixture. DBCΩ mutant has higher minimal light intensity enabling growth on any organic acid as a substrate. When growing with lactate or with lactate and acetate, the mutant reached maximum growth rate at lower light intensities than the wild strain. This phenomenon was observed for the first time. Taking into account the concentration of BChl a under light-limiting conditions, the thickness of the suspension capable of effective light absorption could be increased by 4-5 times, which is favorable for intensive cultivation.

Polymorphobacter fuscus sp. nov., isolated from permafrost soil, and emended description of the genus Polymorphobacter.

Strain D40PT, representing a novel Gram-stain-negative, obligately aerobic, bacteriochlorophyll a-containing bacterium of the α-4 subgroup of the phylum Proteobacteria, was isolated from permafrost soil of Kunlun mountains gap, Qinghai-Tibet plateau. Cells were non-motile rod-cocci and formed brown-pigmented colonies. According to the absorption spectrum, carotenoids and two different photosynthetic light-harvesting complexes, an LHI complex and a B800-835-type peripheral LHII complex, were present in the cells. The strain was oxidase-negative and catalase-positive. The predominant fatty acids of strain D40PT were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C17 : 1ω6c and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The main polar lipids were phosphatidylethanolamine, phosphatidylglycerol, unidentified phospholipid, two glycolipids and sphingoglycolipid. The major respiratory quinone was ubiquinone-10, whereas ubiquinone-9 was present in smaller amounts. The 16S rRNA gene sequence similarity to the closest phylogenetic relative, Polymorphobacter multimanifer JCM 18140T, was 97.5 %. DNA-DNA relatedness (ΔTm) between strain D40PT and P. multimanifer was 12.4 °C. The G+C content of the genomic DNA of strain D40PT was 67.4 mol%. Accordingly, the strain represents a novel species, for which the name Polymorphobacter fuscus sp. nov. is proposed. The type strain is D40PT ( = CGMCC 1.12714T = JCM 19740T). An emended description of the genus Polymorphobacter is also proposed.

Reconstitution Approach to Tune Spectral Features of Light Harvesting Complexes for Improved Light Absorption and Energy Transfer

Light harvesting complexes developed by living organisms render themselves as an excellent system for understanding basic physical and chemical processes behind the conversion of sunlight energy. Although light harvesting complexes are pretty robust, biochemical reconstitution and genetic modifications have proven the flexibility to tailor their absorption spectra and energy transfer. Importantly, the refolding of the protein and the exchanging of the pigment in micellar media results in very similar pigment arrangement within the native complexes. Here, we show reconstitution approaches with different pigments that have been carried out in PCP, LHCII, and LHI complexes. Monitoring on the spectral changes and energy transfer has also been described.

Speciation of chitosan–phosphate and chitosan–nucleotide systems in NaCI aqueous solution

The speciation of chitosan (310 kDa) with organic (adenosine 5’-monophosphate, AMP, and adenosine 5’-triphosphate, ATP), and inorganic phosphorus containing ligands (phosphate and pyrophosphate) was investigated in NaCl aqueous solutions at I = 0.1mol L−1 and T = 25°C. For all the systems, the investigated results obtained gave evidence for the formation of (chitosan)LHi complex species (L = nucleotides, phosphate and pyrophosphate; i = 1 to 4, but for AMP, i = 1 to 3). The stability data of complex species were used to calculate the sequestering ability of chitosan towards phosphorus compounds considered here, expressed as pL50 i.e., – log(total chitosan concentration) necessary to bind 50% of ligand. The pL50 values, calculated at different pH values, show quite a good sequestering ability of chitosan towards phosphorus compounds, in particular for the ones having higher negative charge, such as pyrophosphate and ATP.

Heterologous synthesis and assembly of functional LHII antenna complexes from Rhodovulum sulfidophilum in Rhodobacter sphaeroides mutant

The light harvesting complexes, including LHII and LHI, are the important components of photosynthetic apparatus. Rhodovulum (Rdv.) sulfidophilum and Rhodobacter (R.) sphaeroides belong to two genera of photosynthetic bacteria, and they are very different in some physiological characteristics and light harvesting complexes structure. The LHII structural genes (pucBsAs) from Rdv. sulfidophilum and the LHI structural genes (pufBA) from R. sphaeroides were amplified, and cloned into an expression vector controlled by puc promoter from R. sphaeroides, which was then introduced into LHI and LHII-minus R. sphaeroides mutants; the transconjugant strains synthesized heterologous LHII and native LHI complexes, which played normal roles in R. sphaeroides. The Rdv. sulfidophilum LHII complex from pucBsAs had near-infrared absorption bands at ~801-853 nm in R. sphaeroides, and was able to transfer energy efficiently to the native LHI complex. The results show that the pucBsAs genes from Rdv. sulfidophilum could be expressed in R. sphaeroides, and the functional foreign LHII and native LHI were assembled into the membrane of R. sphaeroides.

Sandarakinorhabdus limnophila gen. nov., sp. nov., a novel bacteriochlorophyll a-containing, obligately aerobic bacterium isolated from freshwater lakes

Three strains (so36, so42T and wo26) representing a novel Gram-negative, obligately aerobic, bacteriochlorophyll a-containing species of the alpha-4 subgroup of the Proteobacteria were isolated from freshwater lakes using a high-throughput cultivation technique. The non-motile and slender rod-shaped cells formed orange-red-pigmented colonies. The main carotenoids were nostoxanthin and keto-nostoxanthin. According to the absorption spectrum, two different photosynthetic light-harvesting complexes, an LHI complex and a B800-830-type peripheral LHII complex, were present in the cells. The predominant fatty acids of strain so42T were hexadecenoic acid (16 : 1omega7c) and octadecenoic acid (18 : 1omega7c), whereas 17 : 1omega6c and 14 : 0 iso 2-OH were present in smaller amounts. The main polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol, glycolipid and sphingoglycolipids. The major respiratory lipoquinone was ubiquinone-10, whereas ubiquinone-9 was present in smaller amounts. The three strains were cytochrome oxidase-negative and catalase-positive and formed alkaline and acid phosphatases. The strains grew chemoorganoheterotrophically in mineral media supplemented with various organic acids, amino acids or complex substrates such as peptone and yeast extract. The G+C content of the genomic DNA of strain so42T was 64.3 mol%. The three novel isolates contained the same 16S rRNA gene sequence. The 16S rRNA gene sequence similarity to the closest phylogenetic relative Sandaracinobacter sibiricus was only 92.8 %. Accordingly, the three strains represent a new genus and species, for which the name Sandarakinorhabdus limnophila gen. nov., sp. nov., is proposed, with strain so42T (=DSM 17366T = CECT 7086T) as the designated type strain.

Effect of light and oxygen and adaptation to changing light conditions in a photosynthetic mutant in which the LHII complex of Rhv. sulfidophilum was heterologously expressed in a strain of Rb. capsulatus whose puc operon was deleted.

In this paper we show the effect of oxygen and light on the expression of the photosynthetic apparatus of a mutant heterologously expressing the puc operon. This mutant was obtained by introducing in trans an expression plasmid, bearing the puc A, B, and C genes of Rhv. sulfidophilum, as well as its own promoter, in an LHII(-) mutant of Rb. capsulatus. The results showed that oxygen and light repressed LHII expression. Even low-light intensities lowered the LHII content to undetectable levels by spectrophotometry or by SDS-PAGE. In high-light grown cells, where the relative ratios of LHI and LHII complexes were significantly diminished, we were able to detect LHII complexes. Under the latter condition, the absorption spectrum showed that some pigment accumulated in the membrane even in the absence of cell division. These pigments were used in a later step to assemble LHII complexes, when the high-light grown cells were transferred to semiaerobiosis in the dark. Transition of high-light grown cells to low-light conditions allowed us to study the adaptability of these heterologous mutant cells. We observed that adaptation never occurred, in part probably owing to energy limitation.

Excitons in a photosynthetic light-harvesting system: a combined molecular dynamics, quantum chemistry, and polaron model study.

The dynamics of pigment-pigment and pigment-protein interactions in light-harvesting complexes is studied with an approach that combines molecular dynamics simulations with quantum chemistry calculations and a polaron model analysis. The molecular dynamics simulation of light-harvesting (LH) complexes was performed on an 87 055 atom system comprised of a LH-II complex of Rhodospirillum molischianum embedded in a lipid bilayer and surrounded with appropriate water layers. For each of the 16 B850 bacteriochlorophylls (BChls), we performed 400 ab initio quantum chemistry calculations on geometries that emerged from the molecular dynamical simulations, determining the fluctuations of pigment excitation energies as a function of time. From the results of these calculations we construct a time-dependent Hamiltonian of the B850 exciton system from which we determine within linear response theory the absorption spectrum. Finally, a polaron model is introduced to describe both the excitonic and coupled phonon degrees of freedom by quantum mechanics. The exciton-phonon coupling that enters into the polaron model, and the corresponding phonon spectral function, are derived from the molecular dynamics and quantum chemistry simulations. The model predicts that excitons in the B850 BChl ring are delocalized over five pigments at room temperature. Also, the polaron model permits the calculation of the absorption and circular dichroism spectra of the B850 excitons from the sole knowledge of the autocorrelation function of the excitation energies of individual BChls, which is readily available from the combined molecular dynamics and quantum chemistry simulations. The obtained results are found to be in good agreement with the experimentally measured absorption and circular dichroism spectra.

Spectral Changes Induced by Alkaline pH and Specific Chemical Modification of Amino Acid Residues in the Light‐Harvesting II Antenna Complex from Ectothiorhodospira sp.

Abstract— The absorption spectrum of the membrane‐bound light‐harvesting (LH)II antenna complex from Ectothiorhodospira sp. has two characteristic near‐infrared bands at 797 (B800 band) and 857 (B850 band) nm. Alkaline pH induced a B850 band blue shift of 17–21 nm depending on experimental conditions. The blue shift was totally reversible when the original experimental conditions were re‐established. No significant effect was observed, however, on the B800 band under the same experimental conditions. The intensity and shape of the pigment circular dichroism signals were maintained with the exception of a blue shift of the signal from the B850 band concomitant with the blue shift of that absorption band. Specific chemical modification of the LHII complex with salicylaldehyde allowed correlation of the alkaline pH effect with the neutralization of a lysine positive charge. We propose that the observed blue shift of the B850 band is due to distortion of the bacteriochlorophyll domain as a consequence of electrostatic and probably hydrogen‐bonding changes but not due to modification of the pigment excitonic interactions within the pigment‐protein complex.

The role of pigments in the assembly of photosynthetic complexes in Rhodobacter capsulatus

AbstractMutants 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.

Two-dimensional crystallization of the light-harvesting I - reaction centre photounit from Rhodospirillum rubrum

A stoichiometric unit of the light-harvesting complex I and the reaction centre (LHI-RC complex) has been isolated from a carotenoid-less mutant of the purple non-sulphur bacterium Rhodospirillum rubrum by mild solubilization of photosynthetic membranes with the phospholipid detergent diheptylphosphatidylcholine. Dialysis of the isolated LHI-RC complexes in the presence of added dioleoyl- sn-phosphatidylcholine produced ordered two-dimensional crystals. Digital image processing revealed that the LHI-RC are packed together in a square lattice ( a = b = 16.3 nm). The dimensions of the LHI ring are essentially identical with those determined from two-dimensional (2D) crystals of purified carotenoid-containing light-harvesting I complexes after analysis by cryo-electron microscopic techniques or from negatively stained 2D crystals of purified LHI complexes from a carotenoid-less mutant. Each LHI ring of the LHI-RC complex contains a central diffuse stain-excluding region, which is assigned to the reaction centre. The analysis of the LHI-RC 2D crystals strongly suggests that the geometry and subunit stoichiometry of the LHI ring is unaffected by the presence of a reaction centre that can probably assume various orientations within the ring.

The effects of the detergent LDAO on the carotenoid metabolism and growth of Rhodovulum sulfidophilum

Carotenoids play an important role in the purple non-sulfur bacteria. In this work we show that addition of the zwitterionic detergent LDAO (lauryldimethylamine N-oxide) to the growth media of Rhodovulum sulfidophilum results in a drastic alteration of the carotenoid biosynthesis, leading to the accumulation of the precursor neurosporene instead of carotenoids of the spheroidene series. This is probably due to a specific inhibition of the hydroxylation of neurosporene either by direct inhibition of the hydroxylase or by disruption of a presumed carotenoid synthesis multienzyme complex coordinated by the crtK encoded protein. Neurosporene can be integrated into peripheral light harvesting complexes (LHII), and is capable of efficient singlet energy transfer, but the resulting LHII complexes show a reduced stability relative to those containing normal carotenoids.

Formation of the light-harvesting complex I (B870) of anoxygenic phototrophic purple bacteria.

The light-harvesting (LH) complex I (B870) of anoxygenic photosynthetic purple bacteria is the oligomeric form of its subunit B820 consisting of the low-molecular-weight polypeptides alpha, beta, bacteriochlorophyll (BChl), and carotenoids in the stoichiometric ratio [alpha1 beta1 (BChl2) Crt1-2]n. LHI surrounds the photochemical reaction center (RC). The major absorption band of the LHI complex is species-specific and is found at 870-890 nm; those of the subunit and the monomeric BChl a (dissolved in methanol) absorb at 820 and 770 nm, respectively. The isolated LHI complex can be reversibly dissociated to the B820 subunit or to the polypeptides and pigments by addition of detergents. Reconstitution of the B820 or the functional B870 complex is still possible after partial truncation of the N- or C-terminal regions of the alpha- or beta-polypeptide or of the beta-polypeptide only. The minimal structural requirements for reconstitution of a spectrally wild-type form after truncation of the polypeptides and/or modifications of the BChl molecule are described. The insertion of the LHIalpha- and LHIbeta-polypeptides into the membrane and the in vivo assembly of LHI, studied in a cell-free system and in whole cells of Rhodobacter capsulatus, depend on the primary structures of both polypeptides, BChl, the chaperones DnaK and GroEL, membrane-bound proteins, and energized membranes. Exchanges, deletions, or insertions of amino acyl residues, especially in the conserved region of the N-terminus of the LHIalpha-polypeptide, prevent or reduce the efficiency and stability of the LHI assembly. Therefore, reconstitution of LHI in a detergent micelle does not exactly reproduce the formation of the LHI complex in the photosynthetic membrane in vivo. The N-terminal domains play a crucial role in the formation of the oligomeric protein scaffold and of the pigment array. Facultatively phototrophic bacteria such as Rhodospirillum (Rsp.) rubrum or Rhodobacter (Rba.) capsulatus can adjust to changes in oxygen tension, light intensity, temperature, and substrates to grow under chemotrophic or phototrophic conditions. The photosynthetic apparatus (PSA), localized mainly on intracytoplasmic membranes (ICM), is usually synthesized only under low oxygen partial pressure. The cellular amount and composition of the PSA are modified upon changing light intensity in relation to cell growth (Drews and Golecki 1995). The morphogenesis of cellular structures like ICM is quite different from self-assembly. Self-assembly is a reversible process of aggregation of the constituents of a complex structure without protein synthesis and is driven by weak or strong forces in the interactions of the constituents. Morphogenesis results from the interplay of numerous gene products and the cellular organization and is always dependent upon pre-existent structures (Harold 1995). The morphogenesis of the photosynthetic membrane in purple bacteria has been studied in its different steps. The regulation at the transcriptional and post-transcriptional levels in purple bacteria, and the structure and morphogenesis of the ICM have been described recently (Armstrong 1995; Bauer 1995; Biel 1995; Drews and Golecki 1995; Klug 1995). In this mini-review, I will focus on the minimal requirements for the in vitro assembly of light-harvesting (LH) complex I (B870) from its constituents in detergent micelles and compare the results with observations on the complex process of targeting and import of LHI polypeptides into the membrane and assembly of B870.

Import and assembly of the ? and ?-polypeptides of the light-harvesting complex I (B870) in the membrane system of Rhodobacter capsulatus investigated in an in vitro translation system

Transcripts of the genes pufBA, pufB or pufA from Rhodobacter capsulatus were translated in a cell-free system of R. capsulatus. The incorporation of the nascent polypeptides LHIα and β in various types of membranes and the assembly of the light-harvesting (LH) complex I (B870) were investigated. The highest rate of stable incorporation of LHIα and β into the membrane was observed with membranes from the wild type strain grown under chemotrophic conditions. Addition of membranes from cells defective in biosynthesis of pigment-binding proteins resulted in a less efficient or less stable incorporation of LHIαβ. The single polypeptides LHIα or β were synthesized and inserted into the membrane but were extractable to a higher percentage by 6 M urea than the pairwise inserted LHI polypeptides.If the ribosomes and the S135 extract were depleted of DnaK the rate of synthesis of both polypeptides, LHIα and β, was strongly reduced. Removal of GroEL from the cell-free system did not impair the synthesis and membrane association of both proteins, but affected the stable insertion. A high percentage of the LHIαβ polypeptides became extractable by 6 M urea if the cell-free system was depleted of GroEL. Addition of GroEL to the cell-free system restored the capacity of stable insertion of both proteins into the membrane. GroEL interacted with LHIα and β before membrane targeting as shown by immunological means.A protein fraction, which can be removed from the membrane with a low-salt buffer, supported the effective and stable incorporation of LHIαβ into the membrane. It is concluded that the assembly of the LHI complex in the membrane system of R. capsulatus is a multistep process guided and supported by polypeptides located in the cytoplasm and in the membrane. In the cell-free in vitro system not only the correct insertion of the LHI polypeptides but also an assembly with bacteriochlorophyll was observed. BChl was synthesized from δ-amino levulinate in the cell free system.

The light-harvesting complex II (B800-850) of Rhodobacter sulfidophilus: Characterization and formation under different growth conditions

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 μg Bchl per mg cell protein when grown at high light intensity (20 000 lux) to 60 μg 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. Chromatographie 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 a and one type of β polypeptide in the purified complex, of which the N-terminal sequences have been determined.

Electrochromic responses of carotenoid absorbance bands in purified light-harvesting complexes from Rhodobacter capsulatus reconstituted into liposomes

Light-Harvesting Complexes I and II (LHI and LHII) were extracted from chromatophores of Rhodobacter capsulatus, purified in Triton X-100 and reconstituted into phospholipid vesicles. Application of membrane potentials (K + diffusion potentials) to LHII proteoliposomes led to absorbance changes in the carotenoid bands which were spectrally similar to those in chromatophores. These (electrochromic) absorbance changes were linear with the applied membrane potential between −107 mV and +105 mV. The data were consistent with the existence of two forms of carotenoid in LHII. One form, comprising 2 3 of the total and with a long wavelength absorbance maximum at 510 nm, was not significantly affected by membrane potential. The other component, comprising 1 3 of the total and with a long wavelength absorbance maximum at 516.5 nm, was shifted by approx. 1.6 nm to the red by a membrane potential of 105 mV. Reduction of the B800 bacteriochlorophyll in LHII with NaBH 4 before reconstitution did not affect the absorbance spectrum of the carotenoids and it did not affect their response to applied membrane potentials in proteoliposomes. Although the electrochromically-sensitive carotenoids might be associated with B800, interactions with the bacteriochlophyll are perhaps not the cause of the polarisation of the carotenoid that is responsible for the linearity of the response. The carotenoids in reconstituted LHI complexes were not detectably electrochromic. The electrochromic absorbance changes of carotenoids in LHII could be useful for membrane potential measurement in liposomes containing ion-translocating proteins.

Rhodobacter capsulatus puc operon: promoter location, transcript sizes and effects of deletions on photosynthetic growth.

Gene deletions of the puc operon of Rhodobacter capsulatus showed that the pucC and pucE genes, but not pucD, were required for formation of wild-type levels of the LHII complex. Deletion of pucC or pucE also impaired photosynthetic growth. The effects of pucC deletion were suppressed by secondary mutations that mapped outside the puc operon. Fusion of a lac'Z gene to pucE' showed that most of pucE transcription originated from upstream of pucB. RNA blot analysis revealed a 2.4 kb transcript that hybridized to probes specific for the pucBA, pucC and pucDE regions, indicating that some puc operon messages extend from just before the pucB gene to just after the pucE gene.

DNA sequencing and complementation/deletion analysis of the bchA‐puf operon region of Rhodobacter sphaeroides: in vivo mapping of the oxygen‐regulated puf promoter

Within the photosynthetic gene cluster of Rhodobacter sphaeroides the genes encoding light-harvesting LHI and reaction-centre complexes are transcriptionally linked in the order pufBALMX. The region stretching 1.6 kb upstream of pufB has been examined by DNA sequencing and by complementation/deletion analysis. These studies demonstrate that three open reading frames are located upstream of pufB. One open reading frame, designated bchA, terminates just inside pufQ, which is located proximal to pufB. BchA contains a 37 bp region that functions as the oxygen-regulated promoter for pufQ, and probably for the puf operon as a whole. We also demonstrate that the protein encoded by pufQ appears to play a role in bacteriochlorophyll biosynthesis.

Analysis of the Rhodobacter capsulatus puc operon: the pucC gene plays a central role in the regulation of LHII (B800-850 complex) expression.

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.

Characterization of reaction center/antenna complexes from bacteriochlorophyll a containing Ectothiorhodospiraceae

Pigment-protein complexes have been isolated from three bacteriochlorophyll- a -containing Ectothiorhodospira species: E. mobilis, E. shaposhnikovii and E. halophila , using detergent solubilisation of photosynthetic membranes and sucrose-density gradient centrifugation. This isolation procedure yields pure LHII complexes and reaction centers with either one or both antenna complexes attached (i.e., RC/LHI and RC/LHI/LHII complexes). The RC/LHI/LHII complexes consist of three reaction center polypeptides, H (35 kDa), M (34 kDa), and L (26.5 kDa), plus two LHI and two LHII antenna polypeptides. In photosynthetic membranes of the three Ectothiorhodospira species two major types of heme c are present, of low (25 mV) and high (297 mV) midpoint potential, respectively. These heme groups belong to separate cytochromes. During solubilisation and purification, no loss of the H subunit from the reaction centers is observed. However, the reaction centers do lose their c -type cytochromes. The high-potential cytochrome c is completely lost during solubilisation of RC complexes from photosynthetic membranes, while the low-potential cytochrome is largely lost during isolation of RC/LHI complexes. The residual amount of this low-potential cytochrome, associated with the RC/LHI complexes, varies with the salt concentration used during the isolation, but remains below 0.2 mol heme c per mol reaction center. This is less than 20% of the amount originally present in photosynthetic membranes.