Characterization of expressed pigmented core light harvesting complex (LH 1) in a reaction center deficient mutant of Blastochloris viridis

The utility of photosynthetically defective mutants in the purple photosynthetic bacterium Blastochloris viridis (formerly Rhodopseudomonas viridis)was demonstrated with construction of a reaction-center deficient mutant, LH 1-H. This LH 1-H mutant has a photosynthetic apparatus in which most of the puf operon genes were deleted, resulting in an organism containing only the genes for the light harvesting polypeptides and the H subunit of the reaction center. This B. viridisstrain containing a truncation of the puf operon was characterized by gel electrophoresis, lipid-to-protein ratio analysis, optical spectroscopy, electron paramagnetic resonance and transmission electron microscopy. Optical and electron paramagnetic resonance spectroscopies revealed no photoactivity in this LH 1-H mutant consistent with the absence of intact reaction centers. Electron paramagnetic resonance evidence for assembled LH 1 complexes suggested that the interactions between light harvesting polypeptide complexes in membranes were largely unchanged despite the absence of their companion reaction center cores. The observed increase in the lipid-to-protein ratio was consistent with modified interactions between LH 1s, a view supported by transmission electron microscopy analysis of membrane fragments. The results show that B. viridis can serve as a practical system for investigating structure-function relationships in membranes and photosynthesis through the construction of photosynthetically defective mutants. This revised version was published online in August 2006 with corrections to the Cover Date.     

The formation of bacteriochlorophyll · protein complexes of the photosynthetic apparatus of Rhodopseudomonas capsulata during early stages of development

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg (pO₂). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll.Cells which were incubated at 0.5 mmHg (pO₂) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of ¹⁴C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.     

A Rhodobacter sphaeroides puf L, M and X deletion mutant and its complementation in trans with a 5.3 kb puf operon shuttle fragment

A Rhodobacter sphaeroides puf L, M and X deletion mutant was constructed using interposon mutagenesis. The puf L, M and X genes were replaced with a kanamycin resistance cartridge isolated from the transposon Tn5. The deletion strain PUFLMX 21 did not grow photoheterotrophically and was resistant to kanamycin. Southern blot analysis of genomic DNA from the deletion strain confirmed that the kanamycin resistance was inserted specifically into the puf operon and that the L, M and X genes were deleted. A spontaneous carotenoid mutant of PUFLMX was selected and was found to accumulate primarily neurosporene. Spectroscopic analysis of chromatophores isolated from the deletion strain showed normal B875 and B800-850 expression providing further evidence that the photosynthetic minus phenotype was not the result of insertional inactivation of the promoter region of the puf operon, or the puf Q region. The deletion strain could be returned to the photosynthetic plus phenotype by complementation in trans with a 5.3 kb puf operon shuttle fragment, although the generation time of the complemented strain was 30% longer than wild type.     

Functional Organization of the Chlorophyll-Containing Complexes of Chlamydomonas reinhardi: A Study of Their Formation and Interconnection with Reaction Centers in the Greening Process of the y-1 Mutant

The stepwise synthesis and assembly of photosynthetic membrane components in the y-1 mutant of Chlamydomonas reinhardi have been previously demonstrated (Ohad 1975 In Membrane Biogenesis, Mitochondria, Chloroplasts and Bacteria, Plenum, pp 279-350). This experimental system was used here in order to investigate the process of formation and interconnection of the energy collecting chlorophylls with the reaction centers of both photosystems I and II. The following measurements were carried out: photosynthetic electron flow at various light intensities, including parts or the entire electron transfer chain; analysis of the kinetics of fluorescence emission at room temperature and fluorescence emission spectra at 77 K, and electrophoretic separation of membrane polypeptides and chlorophyll protein complexes. Based on the data obtained it is concluded that: (a) each photosystem (PSI and PSII) contains, in addition to the reaction center, an interconnecting antenna and a main or light harvesting antenna complex; (b) the formation of the light harvesting complex, interconnecting antenna, and reaction centers for each photosystem can occur independently. (c) the interconnecting antennae link the light harvesting complexes with the respective reaction centers. In their absence, energy transfer between the light harvesting chlorophylls and the reaction centers is inefficient. The formation of the interconnecting antennae and efficient assembly of photosystem components occur simultaneously with the de novo synthesis of chlorophyll and at least three polypeptides, one translated in the cytoplasm and two translated in the chloroplast. The synthesis of these polypeptides was found to be light dependent.     

A powerful hybrid <Emphasis Type="Italic">puc</Emphasis> operon promoter tightly regulated by both IPTG and low oxygen level

Rhodobacter sphaeroides has been intensively studied and provides an excellent model for studying both photo-synthesis and membrane development. The photosynthetic apparatus (LH2 and LH1-RC complexes) can be synthesized in large scale and integrated into the intracytoplasmic membrane system under specific conditions, which thus provides us insight to utilize the puc or(and) puf operon to heterologously express recombinant proteins in the intracytoplasmic membrane using Rb. sphaeroides as a novel expression system. However, basal level of expression of puc and puf promoter is uncontrolled. We report the construction of LH2 polypeptide expression vector that contains a reengineered lacI ^q-puc promoter-lac operator hybrid promoter, which allows the puc operon to be regulated by both IPTG and low oxygen level. Synthesis of LH2 complexes was completely repressed in the absence of isopropyl β-D-thiogalactoside (IPTG), and the degree of induction was controlled by varying the concentration of IPTG. The optimal concentration of IPTG was determined. SDS-PAGE and Western blot were employed for further analysis. Our results suggest that the reengineered hybrid promoter is efficient to tightly regulate the expression of the puc operon, and our strategy can open up a new approach in the study of the membrane protein expression system.     

Characterization of a mutant strain of Rhodovulum sulfidophilum lacking the pufA and pufB genes encoding the polypeptides for the light-harvesting complex 1 (B 870)

Contradictory results on the effectiveness of energy transfer from the light harvesting complex 2 (LH2) directly to the reaction center (RC) in mutant strains lacking the core light-harvesting complex 1 (LH1) have been obtained with cells of Rhodobacter capsulatus and Rhodobacter sphaeroides. A LH1⁻ mutant of Rhodovulum sulfidophilum, named rsLRI, was constructed by deletion of the pufBA genes, resulting in a kanamycin resistant photosynthetically positive clone. To restore the wild type phenotype, a complemented strain C2 was constructed by inserting in trans a DNA segment containing the pufBA genes. Light-induced FTIR difference spectra indicate that the RC in the rsLRI mutant and in the C2 complemented strains are functionally and structurally identical with those in the wild type strain, demonstrating that the assembly and the function of the RC is not impaired by the LH1 deletion. The photosynthetic growth rate of the rsLRI strain increased with decreasing light intensity. At 50 W m⁻² no photosynthetic growth was observed. These results indicate that the light energy harvested by the LH2 complex was not or inefficiently transferred to the RC; thus most of the energy necessary for photosynthetic growth is in the LH1⁻ strain directly absorbed by the RC. It is supposed that in the mutant strain, RC and LH2 cannot interact in an efficient way.     

Pleiotropic effects of localized Rhodobacter capsulatus puf operon deletions on production of light-absorbing pigment-protein complexes.

The polycistronic puf operon of Rhodobacter capsulatus encodes protein components for the photosynthetic reaction center and one of the two antenna complexes involved in the capture of light energy. We report here that deletions within specific puf genes alter the synthesis and/or assembly in the photosynthetic membranes of pigment-protein complexes not affected genetically by the deletion. The pufX gene is required for normal ratios of antenna complexes, and its deletion results in an increase of membrane-bound light-harvesting I (LHI) complex-specific proteins. Expression of pufQ in strains deleted for the genes encoding the LHI and the photosynthetic reaction center (RC) yields a novel A868 peak that has not been associated with any of the pigment-protein complexes described previously. While deletions in the RC-coding region resulted in decreased LHI absorbance, no quantitative alteration in membrane-bound LHI protein was observed, suggesting that an intact RC complex is required for correct assembly of LHI in the membrane.     

The reaction center is the sensitive target of the mercury(II) ion in intact cells of photosynthetic bacteria

The sensitivity of intact cells of purple photosynthetic bacterium Rhodobacter sphaeroides wild type to low level (<100 μM) of mercury (Hg2?) contamination was evaluated by absorption and fluorescence spectroscopies of the bacteriochlorophyll-protein complexes. All assays related to the function of the reaction center (RC) protein (induction of the bacteriochlorophyll fluorescence, delayed fluorescence and light-induced oxidation and reduction of the bacteriochlorophyll dimer and energization of the photosynthetic membrane) showed prompt and later effects of the mercury ions. The damage expressed by decrease of the magnitude and changes of rates of the electron transfer kinetics followed complex (spatial and temporal) pattern according to the different Hg2? sensitivities of the electron transport (donor/acceptor) sites including the reduced bound and free cytochrome c? and the primary reduced quinone. In contrast to the RC, the light harvesting system and the bc? complex demonstrated much higher resistance against the mercury pollution. The 850 and 875 nm components of the peripheral and core complexes were particularly insensitive to the mercury(II) ions. The concentration of the photoactive RCs and the connectivity of the photosynthetic units decreased upon mercury treatment. The degree of inhibition of the photosynthetic apparatus was always higher when the cells were kept in the light than in the dark indicating the importance of metabolism in active transport of the mercury ions from outside to the intracytoplasmic membrane. Any of the tests applied in this study can be used for detection of changes in photosynthetic bacteria at the early stages of the action of toxicants.     

Adaptation of the Photosynthetic Apparatus to Irradiance in Dunaliella tertiolecta: A Kinetic Study

The time course of adaptation from a high to a low photon flux density was studied in the marine chlorophyte Dunaliella tertiolecta. A one-step transition from 700 to 70 micromole quanta per square meter per second resulted in a reduction of doubling rate from 1.1 to 0.4 per day within 24 hours, followed by a slower accumulation of photosynthetic pigments, light harvesting antenna complexes, Photosystem II reaction centers and structural lipids that constitute the thylakoid membranes. Photoregulated changes in the biochemical composition of the thylakoid proteins and lipids were functionally accompanied by decreases in the minimal photosynthetic quantum requirement and photosynthetic capacity, and an increase in the minimal turnover time for in vivo electron transport from water to CO₂. Analysis of de novo synthesis of thylakoid membranes and proteins indicates that a high light to low light transition leads to a transient in carbon metabolism away from lipid biosynthesis toward the synthesis of the light harvesting antenna protein complexes, accompanied by a slower restoration rate of reaction centers and thylakoid membranes. This pattern of sequential synthesis of light harvesting complexes followed by reaction centers and membranes, appears to optimize light harvesting capabilities as cells adapt to low photon flux densities.     

PucC and LhaA direct efficient assembly of the light‐harvesting complexes in Rhodobacter sphaeroides

The mature architecture of the photosynthetic membrane of the purple phototroph Rhodobacter sphaeroides has been characterised to a level where an atomic‐level membrane model is available, but the roles of the putative assembly proteins LhaA and PucC in establishing this architecture are unknown. Here we investigate the assembly of light‐harvesting LH2 and reaction centre‐light‐harvesting1‐PufX (RC‐LH1‐PufX) photosystem complexes using spectroscopy, pull‐downs, native gel electrophoresis, quantitative mass spectrometry and fluorescence lifetime microscopy to characterise a series of lhaA and pucC mutants. LhaA and PucC are important for specific assembly of LH1 or LH2 complexes, respectively, but they are not essential; the few LH1 subunits found in ΔlhaA mutants assemble to form normal RC‐LH1‐PufX core complexes showing that, once initiated, LH1 assembly round the RC is cooperative and proceeds to completion. LhaA and PucC form oligomers at sites of initiation of membrane invagination; LhaA associates with RCs, bacteriochlorophyll synthase (BchG), the protein translocase subunit YajC and the YidC membrane protein insertase. These associations within membrane nanodomains likely maximise interactions between pigments newly arriving from BchG and nascent proteins within the SecYEG‐SecDF‐YajC‐YidC assembly machinery, thereby co‐ordinating pigment delivery, the co‐translational insertion of LH polypeptides and their folding and assembly to form photosynthetic complexes.     

Assembly of photosynthetic apparatus in Rhodobacter sphaeroides as revealed by functional assessments at different growth phases and in synchronized and greening cells

The development of photosynthetic membranes of intact cells of Rhodobacter sphaeroides was tracked by light-induced absorption spectroscopy and induction and relaxation of the bacteriochlorophyll fluorescence. Changes in membrane structure were induced by three methods: synchronization of cell growth, adjustment of different growth phases and transfer from aerobic to anaerobic conditions (greening) of the bacteria. While the production of the bacteriochlorophyll and carotenoid pigments and the activation of light harvesting and reaction center complexes showed cell-cycle independent and continuous increase with characteristic lag phases, the accumulation of phospholipids and membrane potential (electrochromism) exhibited stepwise increase controlled by cell division. Cells in the stationary phase of growth demonstrated closer packing and tighter energetic coupling of the photosynthetic units (PSU) than in their early logarithmic stage. The greening resulted in rapid (within 0–4 h) induction of BChl synthesis accompanied with a dominating role for the peripheral light harvesting system (up to LH2/LH1 ~2.5), significantly increased rate (~7·10⁴ s^?1) and yield (F _v/F _max ~0.7) of photochemistry and modest (~2.5-fold) decrease of the rate of electron transfer (~1.5·10⁴ s^?1). The results are discussed in frame of a model of sequential assembly of the PSU with emphasis on crowding the LH2 complexes resulting in an increase of the connectivity and yield of light capture on the one hand and increase of hindrance to diffusion of mobile redox agents on the other hand.     

Photosynthetic reaction center mutagenesis via chimeric rescue of a non-functional Rhodobacter capsulatus puf operon with sequences from Rhodobacter sphaeroides

Photosynthetically active chimeric reaction centers which utilize genetic information from both Rhodobacter capsulatus and Rb. sphaeroides puf operons were isolated using a novel method termed chimeric rescue. This method involves in vivo recombination repair of a Rb. capsulatus host operon harboring a deletion in pufM with a non-expressed Rb. sphaeroides donor puf operon. Following photosynthetic selection, three revertant classes were recovered: 1) those which used Rb. sphaeroides donor sequence to repair the Rb. capsulatus host operon without modification of Rb. sphaeroides puf operon sequences (conversions), 2) those which exchanged sequence between the two operons (inversions), and 3) those which modified plasmid or genomic sequences allowing expression of the Rb. sphaeroides donor operon. The distribution of recombination events across the Rb. capsulatus puf operon was decidedly non-random and could be the result of the intrinsic recombination systems or could be a reflection of some species-specific, functionally distinct characteristic(s). The minimum region required for chimeric rescue is the D-helix and half of the D/E-interhelix of M. When puf operon sequences 3 of nucleotide M882 are exchanged, significant impairment of excitation trapping is observed. This region includes both the 3 end of pufM and sequences past the end of pufM.     

Circular dichroism and absorption spectra of bacteriochlorophyll-protein and reaction center complexes from Chlorobium thiosulfatophilum

A water-soluble bacteriochlorophyll-protein and a complex which also contained photochemically-active reaction centers were isolated from Chlorobium thiosulfatophilum. The procedures were similar to those used previously on Chloropseudomonas ethylica (Fowler, C. F., Nugent, N. A. and Fuller, R. C. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2278–2282), and the corresponding complexes from the two organisms exhibit marked similarities. They are characterized in terms of their absorption spectra at 100 and 300 °K, circular dichroism spectra at 300 °K, and, in the case of the reaction center complexes, by the light- or oxidation-induced changes in the absorption and circular dichroism spectra. On the basis of exciton interactions observed in the circular dichroism and low-temperature absorption spectra, we conclude that the predominant pigment arrangement in the bacteriochlorophyll-reaction center complex is distinctly different from that in the bacteriochlorophyll-protein.     

Effects of light intensity on membrane differentiation in Rhodopseudomonas capsulata

Cells of Rhodopseudomonas capsulata, strain 37b4, leu^?, precultivated anaerobically under low light intensity, were exposed to high light intensity (2000 W · m^?2). The cells grew with a mass doubling time of 3 h. The synthesis of bacteriochlorophyll (BChl) began after two doublings of cell mass. Reaction center and light-harvesting BChl I (B-875) were the main constituents of the photosynthetic apparatus incorporated into the membrane. The size of the photosynthetic unit (total BChl/reaction center) decreased and light-harvesting BChl I became the dominating BChl species. Concomitant with the appearance of the different spectral forms of BChl the respective proteins were incorporated into the membrane, i.e. the three reaction center polypeptides, the polypeptide associated with light-harvesting BChl I, the two polypeptides associated with BChl II. A polypeptide of an apparent molecular weight of 45 000 was also incorporated. A lowering of the light intensity to 7 W · m^?2 resulted in a lag phase of growth for 6 h. Afterwards, the time for doubling of cell mass was 11 h. The concentration of all three BChl complexes (reaction center, light-harvesting BChl I and II complexes)/cell and per membrane protein increased immediately. Also the size of the photosynthetic unit and the amount of intracytoplasmic membranes/cell increased.The activities of photophosphorylation, succinate dehydrogenase, NADH dehydrogenase and NADH oxidation (respiratory chain)/membrane protein are higher in membrane preparations isolated from cells grown at high light intensities than in such preparations from cells grown at low light intensities.     

Variable fluorescence in green sulfur bacteria

Green sulfur bacteria possess a complex photosynthetic machinery. The dominant light harvesting systems are chlorosomes, which consist of bacteriochlorophyll c, d or e oligomers with small amounts of protein. The chlorosomes are energetically coupled to the membrane-embedded iron sulfur-type reaction center via a bacteriochlorophyll a-containing baseplate protein and the Fenna-Matthews-Olson (FMO) antenna protein. The fluorescence yield and spectral properties of these photosynthetic complexes were investigated in intact cells of several species of green sulfur bacteria under physiological, anaerobic conditions. Surprisingly, green sulfur bacteria show a complex modulation of fluorescence yield upon illumination that is very similar to that observed in oxygenic phototrophs. Within a few seconds of illumination, the fluorescence reaches a maximum, which decreases within a minute of illumination to a lower steady state. Fluorescence spectroscopy reveals that the fluorescence yield during both processes is primarily modulated on the FMO-protein level, while the emission from chlorosomes remains mostly unchanged. The two most likely candidates that modulate bacteriochlorophyll fluorescence are (1) direct excitation quenching at the FMO-protein level and (2) indirect modulation of FMO-protein fluorescence by the reduction state of electron carriers that are part of the reaction center.     

Construction of a physical map of the 45 kb photosynthetic gene cluster of Rhodobacter sphaeroides

1) A number of overlapping clones have been isolated from a Rhodobacter sphaeroides gene bank. Following conjugative gene transfer from Escherichia coli these clones restore a wild type phenotype to several mutants unable to synthesise bacteriochlorophyll. 2) The insert DNA was analysed by restriction mapping and together the clones form the basis of the first restriction map of the 45 kb photosynthetic gene cluster of Rb. sphaeroides. 3) This cluster is bounded on one side by puh A encoding the reaction centre H polypeptide and on the other by the puf operon encoding reaction centre L and M apoproteins and light harvesting LH1 and polypeptides. 4) DNA fragments from the 45 kb cluster were used to probe genomic DNA from other photosynthetic bacteria. Using heterologous hybridisation conditions, a significant degree of homology is shown between Rb. sphaeroides and these other bacteria, suggesting close evolutionary links with Rb. capsulatus in particular.     

Membrane adhesion in photosynthetic bacterial membranes. Light harvesting complex I (LHI) appears to be the main adhesion factor

We have reconstituted pigment-protein complexes isolated from Rhodopseudomonas palustris photosynthetic membranes into phospholipid liposomes. The various complexes were tested for their ability to promote adhesion of the liposome membrane in the presence and absence of Mg²⁺ ions. Samples containing a reaction center (RC)/light-harvesting I (LHI) complex appeared to stack in a manner resembling control thylakoids in 2 and 5 mM Mg²⁺. We also tested for the effects of Mg²⁺ on detergent extractablity of pigment-protein complexes from intact membranes. Mg²⁺ sharply reduced the amount of LHI solubilized from membranes, while having little effect on the extractability of the light harvesting II complex (LHII) and the RC. Based on these results we suggest that LHI is the principal adhesion factor of R. palustris thylakoids.Abbreviations LHC light harvesting complex - OG octyl glucoside - RC reaction center This paper is dedicated to Professor G. Drews on the occasion of his 60th birthday