Roles of bacteriochlorophyll and carotenoid synthesis in formation of intracytoplasmic membrane systems and pigment-protein complexes in an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114.

Synthesis of bacteriochlorophyll and carotenoids was inhibited in an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114, by alpha, alpha'-dipyridyl and diphenylamine. Formation of two pigment-protein complexes, reaction center-B870 (RC-B870) and B806, and development of the intracytoplasmic membranes of the cells were studied by spectral analysis and electron microscopy. Inhibition of bacteriochlorophyll synthesis by alpha, alpha'-dipyridyl, which was accompanied by a decrease in carotenoid synthesis, suppressed formation of intracytoplasmic membranes in the cells. Growth under illumination had a similar effect on formation of pigments and membranes. On the other hand, inhibition of carotenoid synthesis by diphenylamine did not suppress either development of the membrane system or bacteriochlorophyll synthesis. Formation of RC-B870 and B806 complexes, however, was differentially affected by blockage of carotenoid synthesis. In the presence of diphenylamine, the B806 complex was formed in a much smaller amount than the RC-B870 complex. These results suggest that, in Erythrobacter sp. strain OCh114, bacteriochlorophyll plays an essential role in intracytoplasmic membrane development, and carotenoids are important for assembly of pigment-protein complexes.     

PufQ regulates porphyrin flux at the haem/bacteriochlorophyll branchpoint of tetrapyrrole biosynthesis via interactions with ferrochelatase

Facultative phototrophs such as Rhodobacter sphaeroides can switch between heterotrophic and photosynthetic growth. This transition is governed by oxygen tension and involves the large‐scale production of bacteriochlorophyll, which shares a biosynthetic pathway with haem up to protoporphyrin IX. Here, the pathways diverge with the insertion of Fe²⁺ or Mg²⁺ into protoporphyrin by ferrochelatase or magnesium chelatase, respectively. Tight regulation of this branchpoint is essential, but the mechanisms for switching between respiratory and photosynthetic growth are poorly understood. We show that PufQ governs the haem/bacteriochlorophyll switch; pufQ is found within the oxygen‐regulated pufQBALMX operon encoding the reaction centre–light‐harvesting photosystem complex. A pufQ deletion strain synthesises low levels of bacteriochlorophyll and accumulates the biosynthetic precursor coproporphyrinogen III; a suppressor mutant of this strain harbours a mutation in the hemH gene encoding ferrochelatase, substantially reducing ferrochelatase activity and increasing cellular bacteriochlorophyll levels. FLAG‐immunoprecipitation experiments retrieve a ferrochelatase‐PufQ‐carotenoid complex, proposed to regulate the haem/bacteriochlorophyll branchpoint by directing porphyrin flux toward bacteriochlorophyll production under oxygen‐limiting conditions. The co‐location of pufQ and the photosystem genes in the same operon ensures that switching of tetrapyrrole metabolism toward bacteriochlorophyll is coordinated with the production of reaction centre and light‐harvesting polypeptides.     

Bacteriochlorophyll and Heme Synthesis in Rhodopseudomonas spheroides: Possible Role of Heme in Regulation of the Branched Biosynthetic Pathway

Synthesis of heme, measured by incorporation of iron-59, and of bacteriochlorophyll was studied with wild-type and mutant strains of Rhodopseudomonas spheroides. The wild type formed heme from glycine and succinate at one-fortieth the rate of bacteriochlorophyll under anaerobic-light conditions. Added delta-aminolevulinate stimulated heme synthesis 10-fold without increasing bacteriochlorophyll production. Heme synthesis from glycine and succinate was increased when the magnesium branch of the biosynthetic path was curtailed by mutation or by p-fluorophenylalanine or 8-azaguanine. Synthesis of bacteriochlorophyll by the wild type from glycine and succinate stopped immediately after addition of puromycin, but heme production continued for a period. Porphyrins and other precursors did not appear upon addition of puromycin alone, but simultaneous addition of o-phenanthroline resulted in the accumulation of coproporphyrin. Production of this porphyrin by a mutant strain with impaired ability to form heme was unaffected by puromycin. Heme synthesis from glycine and succinate or from delta-aminolevulinate was decreased by limitation of methionine; it is suggested that coproporphyrin accumulation from glycine and succinate under conditions of methionine deficiency results from relief of feedback inhibition of delta-aminolevulinate synthase by heme. The development of delta-aminolevulinate synthase activity in response to low aeration is prevented by addition of delta-aminolevulinate. This repressive action of the latter is abolished when its conversion to heme is impeded by mutation or by methionine deficiency. It is suggested that heme, the quantitatively minor end product of the branched biosynthetic pathway, may regulate the flow of common intermediates when utilization of protoporphyrin by the magnesium branch is diminished. This regulation may be exerted by feedback inhibition of delta-aminolevulinate synthase and also by repression of enzyme formation.     

Influence of blue light on the structure stability of antenna complexes from Allochromatium minutissimum with different content of carotenoids

Effects of photooxidation of bacteriochlorophyll (absorbtion at 850 nm) from the light-harvesting complex LH2 of Alc. minutissimum membranes on the LH2 complex structure have been studied. Photooxidation was induced by blue light that is absorbed by carotenoids. Four samples with different levels (from 100% to 3–5%) and composition of carotenoids were obtained by inhibiting the carotenoid biosynthesis in bacteria with diphenylamine. Electrophoresis in polyacrylamide gel showed that after illumination LH2 complex contained all the oxidized bacteriochlorophyll. The carotenoid composition did not change after the oxidation of the main part of bacteriochlorophyll in the LH2 complex. The results suggest that oxidation takes place in the bacteriochlorophyll part, which is essential for the molecule optical properties (the system of double conjugated bonds is changed), but does not influence the stability of the structure of the LH2 complex.     

Mutant Strains of Rhodopseudomonas spheroides Lacking δ-Aminolevulinate Synthase: Growth, Heme, and Bacteriochlorophyll Synthesis

Two mutant strains of Rhodopseudomonas spheroides were described which lacked delta-aminolevulinate synthase activity. They required delta-aminolevulinate for growth; they did not respond to protoporphyrin or magnesium photoporphyrin, and only poorly to hemin. Synthesis of cytochromes and heme by mutant H-4 was dependent upon delta-aminolevulinate; this strain did not form bacteriochlorophyll either with or without delta-aminolevulinate and, consequently, grew only under aerobic conditions. Mutant H-5 formed bacteriochlorophyll in response to delta-aminolevulinate and grew both anaerobically in the light and aerobically in the dark; the amount of delta-aminolevulinate needed for optimal anaerobic growth was higher than that required aerobically. Synthesis of bacteriochlorophyll and heme by suspensions of mutant H-5 incubated anaerobically in the light was dependent upon delta-aminolevulinate; bacteriochlorophyll production was completely inhibited by high aeration and by puromycin. The mutants differed in their ability to take up radioactive delta-aminolevulinate from the external environment; mutant H-5 was less active than mutant H-4 or the wild type. It was suggested that R. spheroides made only one form of delta-aminolevulinate synthase, which provided delta-aminolevulinate for bacteriochlorophyll and heme synthesis.     

Isolation and characterization of a glycerol auxotroph of Rhodopseudomonas capsulata: effect of lipid synthesis on the synthesis of photosynthetic pigments.

A glycerol auxotroph was isolated from Rhodopseudomonas capsulata for use as a system for studying membrane synthesis and function. When the mutant was deprived of glycerol, net phospholipid synthesis ceased immediately and a small amount of free fatty acids accumulated. A turnover of lipid occurred in both deprived and supplemented cultures. Deoxyribonucleic acid and protein synthesis continued for one doubling of cell massand then slowed down in deprived cells. Net ribonucleic acid synthesis slowed down more dramatically. Oxidative phosphorylation activity of membrane preparations from aerobically and semi-anaerobically grown cells appeared unaffected by glycerol deprivation, indicating that simultaneous lipid synthesis is not a requirement for new oxidative phosphorylating activity. In the absence of net phospholipid synthesis, bacteriochlorophyll and carotenoid syntheses were reduced to 30% of the activity of supplemented cultures. Delta-Aminolevulinic acid synthase, the first enzyme on the bacteriochlorophyll pathway that is subject to regulatory control, increased in activity in deprived cultures. Lascelles and Szilagyi (1965) showed an association between phospholipid synthesis and pigment production. They found an increased lipid content associated with pigmented cells. The present results indicate that not only is there an association between lipid and pigment synthesis, but also there is actually a dependence of bacteriochlorophyll synthesis on phospholipid synthesis.     

Magnesium protoporphyrin chelatase activity in Rhodopseudomonas spheroides. Studies with whole cells

1. Whole cells of Rhodopseudomonas spheroides grown under semi-anaerobic conditions in the light incorporated magnesium into exogenous protoporphyrin when incubated with EDTA or the related chelators EGTA, N-(2-hydroxyethyl)-ethylenediamine-NN'N'- triacetate and trans-1,2-diaminocyclohexanetetra-acetate. 2. The reaction was demonstrated under anaerobic conditions in the light or at low oxygen partial pressure in the dark. Partial pressures of oxygen greater than 15% inhibited the reaction. 3. Cells grown under pure oxygen were completely inactive, but on adaptation to growth under low oxygen partial pressure (O(2)+N(2), 5:95) the development of activity paralleled the synthesis of bacteriochlorophyll. 4. The reaction with normal cells did not require protein synthesis, but cells that had lost their activity by being illuminated in Mg(2+)-deficient medium did not recover it in the absence of protein synthesis. 5. The product of the reaction was magnesium protoporphyrin monomethyl ester. 6. Evidence is presented that insertion of magnesium is obligatorily coupled with methylation and it is concluded that the reaction is dependent on a multienzyme complex.     

Ferricyanide-induced absorbance change of carotenoid in chromatophores of Rhodopseudomonas spheroides correlated to the oxidation of bacteriochlorophyll 885

Addition of high concentrations (e.g., 1–100 mM) of ferricyanideto a chromatophorc suspension of Rhodopseudomonas spheroidescaused a change in the absorption spectrum of carotenoid (spheroidene),which was completely reversed by adding reducing reagents suchas ferrocyanide and ascorbate. The spectral change is representedby a shift in the absorption spectrum of carotenoid by 2 to2.5 nm towards the longer wavelength side. The presence of piericidinA, o-phenanthroline or Cl-CCP in the reaction mixture did notaffect the ferricyanide-induced absorbance change. Triton X-100markedly suppressed the magnitude of the change. The additionof ferricyanide also caused simultaneous absorbance changeswith maxima at 590 and 885 nm. These are ascribed to oxidationof the (bulk) bacteriochlorophyll, BChl 885. There was no absorptionchange at other peaks of bacteriochlorophyll in the infraredregion (i.e., 800 and 855 nm). Therefore, the ferricyanide-inducedabsorbance change of carotenoid did not represent an oxidation-reductionreaction of carotenoid but was intimately correlated with oxidationof BChl 885 in the chromatophores, as judged from similaritiesobserved with respect to the time course patterns, midpointpotential (545–555 mv) in the ferriferrocyanide reactionsystem, as well as behavior towards various reagents and inhibitorsadded. A similar change of carotenoid (i.e., 2–2.5 nmshift of absorption spectrum) was caused by addition of MgCl₂to the chromatophores, but this did not induce any change inthe absorption spectrum of bacteriochlorophyll. The nature ofthe spectral change of carotenoid in chromatophores is discussed. (Received April 16, 1970; )     

Untersuchungen über die lichtabhängige Carotinoidsynthese

Summary Under anaerobic conditions Fusarium aquaeductuum is able to synthesize carotenogenic enzymes but does not produce pigments. If illumination of the mycelia in the presence of oxygen is followed by an incubation in the dark under N₂ atmosphere, the strictly concurrent formation of the different carotenoids sets off as soon as aerobic conditions are restored. The paraboloidal increase of pigment production possibly indicates that synthesis of carotenogenic enzymes is also resumed. Blocking this enzyme synthesis by addition of cycloheximide leads to a simultaneous and linear increase of each carotenoid portion as soon as oxygen is replenished. This is interpreted to mean that light induces carotenogenic enzymes in a coupled group. On the other hand, our present and earlier results do not support any hypothesis on the existence of a carotenogenic multienzyme complex. The composition of the pigment after carotenoid production has ceased provides evidence for a selective inhibition of the synthesis of individual carotenogenic enzymes. Changes in pigment composition caused by an incubation of the mycelia for 12 h under anaerobic conditions are also reported.     

Semiaerobic induction of bacteriochlorophyll synthesis in the green bacterium Chloroflexus aurantiacus.

Comparison of Chloroflexus aurantiacus J-10-fl cells by freeze-fracture electron microscopy showed that cell shape and dimensions did not depend on oxygen tension or light intensity during growth. The major morphological difference between cells cultured anaerobically in the light and aerobically in the dark was the absence of chlorosomes in aerobically grown cells. C. aurantiacus cells cultured aerobically in the dark began bacteriochlorophyll synthesis immediately when shifted to either phototrophic or semiaerobic conditions. Cells adapting to phototrophic conditions grew to the same density and synthesized as much bacteriochlorophyll as nonadapting phototrophic cultures grown at the same light intensity. Cells adapting to reduced oxygen tension (semiaerobic conditions) in the dark entered an 8- to 12-h growth lag during which the bacteriochlorophyll content increased significantly. Despite variations in the initial bacteriochlorophyll content and in the length of the growth lag, the amounts of bacteriochlorophyll a and c were constant at the end of the semiaerobic growth lag. At later times during adaptation to semiaerobic conditions, after growth resumed, variations in the ratio of bacteriochlorophyll c/bacteriochlorophyll a were observed and suggested independent regulation of the two bacteriochlorophylls.     

Map of genes for carotenoid and bacteriochlorophyll biosynthesis in Rhodopseudomonas capsulata.

The recently discovered gene transfer system of Rhodopseudomonas capsulata was used to construct a genetic map of a region concerned with bacteriochlorophyll and carotenoid production. Mutants blocked in the biosynthesis of these compounds were isolated, and each was characterized on the basis of pigments accumulated during growth under low pO2. One-point, two-point, three-point, and ratio test crosses were performed between various mutant strains, and the results were amenable to conventional genetic analyses. A mapping function was found that related cotransfer frequency to map distance. Seven clusters of mutations, five affecting carotenoid and two affecting bacteriochlorophyll biosynthesis, were arranged in one linkage group. Each cluster of mutations is thought to represent a gene. The length of the mapped region is estimated to be less than 1% of the genome. Cotransfer is observed between markers separated by about 5 to 10 genes.     

Growth and metabolism of the purple sulfur bacterium Thiocapsa roseopersicina under combined light/dark and oxic/anoxic regimens

The dominant purple sulfur bacterium of laminated sediment ecosystems in temperate environments, Thiocapsa roseopersicina, was cultivated in sulfide-limited continuous cultures (D=0.03 h^-1) subjected to various combined diel regimen of aeration and illumination in order to simulate environmental conditions in microbial mats. For comparison, cultures were grown under similar illumination regimens but continuously anoxic conditions.Bacteriochlorophyll a (BChla) and carotenoid synthesis was restricted to anoxic-dark periods and did not occur during oxic-light periods. An increase in the length of the oxic-light periods resulted in decreased pigment contents. However, phototrophic growth remained possible even at 20 h oxic-light/4 h anoxic-dark regimens. When anoxic conditions were maintained throughtout, BChla synthesis occurred both during light and dark periods.Glycogen was synthesized in the light and degraded in the dark. Calculations showed that degradation of 1/4–1/5 of the glycogen is sufficient to account for the BChla and carotenoid synthesis in the dark.The data showed that T. roseopersicina is very well adapted to cope with the combined oxygen and light regimes as they occur in microbial mats, which may explain the dominance of this bacterium in the purple layer of these sediment ecosystems.Non-standard abbreviations BChl bacteriochlorophyll - specific growth rate - D dilution rate - S_R concentration of limiting substrate in reservoir bottle     

The Effects of ultraviolet irradiation on hybrid films of photosynthetic reaction centers and quantum dots in various organic matrices

The effects of ultraviolet radiation (up to 0.6 J/cm²) on the absorption spectra and electron transfer in dehydrated films of photosynthetic reaction centers from purple bacteria Rb. sphaeroides and hybrid structures that included reaction centers, quantum dots, and protein structure stabilizers (trehalose, polyvinyl alcohol, and methylcellulose) have been studied. Ultraviolet irradiation led to partial destruction of bacteriochlorophyll molecules (pheophytinization) and the reaction center carotenoid. In this case, ultraviolet irradiation did not exert a significant effect on electron transfer between the photoactive bacteriochlorophyll and quinone electron acceptors. The incorporation of reaction centers into organic matrices reduced pheophytinization. Trehalose was the most efficient in reducing the damage evoked by ultraviolet irradiation of the carotenoid molecule. Hybrid films that contained quantum dots were resistant to pheophytinization upon ultraviolet irradiation, but the presence of quantum dots did not affect the processes of carotenoid destruction upon exposure to ultraviolet radiation. Ultraviolet radiation had an insignificant effect on the characteristics of quantum dots (the fluorescence lifetime).     

Oxygen and light effects on the expression of the photosynthetic apparatus in Bradyrhizobium sp. C7T1 strain

Photosynthetic bradyrhizobia are nitrogen-fixing symbionts colonizing the stem and roots of some leguminous plants like Aeschynomene. The effect of oxygen and light on the formation of the photosynthetic apparatus of Bradyrhizobium sp. C7T1 strain is described here. Oxygen is required for growth, but at high concentration inhibits the synthesis of bacteriochlorophyll (BChl) and of the photosynthetic apparatus. However, we show that in vitro, aerobic photosynthetic electron transport occurred leading to ADP photophosphorylation. The expression of the photosynthetic apparatus was regulated by oxygen in a manner which did not agree with earlier results in other photosynthetic bradyrhizobia since BChl accumulation was the highest under microaerobic conditions. This strain produces photosynthetic pigments when grown under cyclic illumination or darkness. However, under continuous white light illumination, a Northern blot analysis of the puf operon showed that, the expression of the photosynthetic genes of the antenna was considerable. Under latter conditions BChl accumulation in the cells was dependent on the oxygen concentration. It was not detectable at high oxygen tensions but became accumulated under low oxygen (microaerobiosis). It is known that in photosynthetic bradyrhizobia bacteriophytochrome photoreceptor (BphP) partially controls the synthesis of the photosystem in response to light. In C7T1 strain far-red light illumination did not stimulate the synthesis of the photosynthetic apparatus suggesting the presence of a non-functional BphP-mediated light regulatory mechanism.     

Energy transfer between the carotenoid and the bacteriochlorophyll within the B-800-850 light-harvesting pigment-protein complex of Rhodopseudomonas sphaeroides

Energy transfer between carotenoid and bacteriochlorophyll has been studied in isolated B-800-850 antenna pigment-protein complexes from different strains of Rhodopseudomonas sphaeroides which contain different types of carotenoid. Singlet-singlet energy transfer from the carotenoid to the bacteriochlorophyll is efficient (75-100%) and is rather insensitive to carotenoid type, over the range of carotenoids tested. The yield of carotenoid triplets is low (2-15%) but this arises from a low yield of bacteriochlorophyll triplet formation rather than from an inefficient triplet-triplet exchange reaction. The rate of the triplet-triplet exchange reaction between the bacteriochlorophyll and the carotenoid is fast (Ktt greater than or equal to 1.4 . 10(8) S-1) and also relatively independent of the type of carotenoid present.     

Membranes of Rhodopseudomonas sphaeroides: effect of cerulenin on assembly of chromatophore membrane.

The effects of cerulenin were investigated in Rhodopseudomonas sphaeroides to elucidate further the mechanisms controlling the assembly of the chromatophore membrane. When this potent inhibitor of fatty acid biosynthesis was added to photosynthetically grown cultures, there was an immediate cessation of phospholipid, bacteriochlorophyll a, carotenoid, and ubiquinone formation. Concurrently, there was also a marked decrease in the rate of incorporation of protein into the chromatophore membrane. In contrast, only a small decrease in the rate of soluble and cell envelope protein synthesis was observed and, in chemotrophically grown cells, protein continued to be incorporated into both the cytoplasmic and outer membranes. The removal of delta-aminolaevulinate from mutant H-5 of R. sphaeroides, which requires this porphyrin precursor, was reexamined to determine whether cerulenin-induced cessation of chromatophore protein incorporation was due solely to blocked bacteriochlorophyll a synthesis. In the deprived H-5 cells, inhibition of [35S]methionine incorporation into chromatophores was confined mainly to apoproteins of bacteriochlorophyll a complexes. Other minor chromatophore proteins continued to be inserted to a greater extent than in cerulenin-treated wild type where phospholipid synthesis has also ceased. These results indicated that the assembly of the chromatophore membrane is under strict regulatory control involving concomitant phospholipid, pigment, and protein syntheses.     

Singlet and triplet excited carotenoid and antenna bacteriochlorophyll of the photosynthetic purple bacterium Rhodospirillum rubrum as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation pulse and kinetics of absorbance changes were measured in chromatophores of the photosynthetic purple bacterium Rhodospirillum rubrum after chemical oxidation of the primary electron donor P-875. Kinetics and spectra were measured of the excited singlet states of carotenoid and bacteriochlorophyll a and also of the triplet state of the carotenoid. The excited singlet state of carotenoid, produced by direct excitation at 532 nm, is characterized by a bleaching of the ground state absorption bands in the region 450–490 nm and by an absorbance increase with a maximum near 570 nm. Its lifetime was calculated to be 0.6 ± 0.1 ps in vitro and less than 1 ps in vivo. The triplet state of carotenoid in vivo is formed within 100 ps after direct carotenoid excitation via a pathway that does not involve excited states of bacteriochlorophyll. Singlet excitation of a bacteriochlorophyll a molecule causes the bleaching of its Q_x and Q_y absorption bands, and is probably associated with blue shifts of the Q_y absorption band of about six neighboring bacteriochlorophyll molecules. Upon increasing the excitation density, the average lifetime of the singlet excitations on bacteriochlorophyll decreased from about 350 ps to about 10 ps or less. The results are in quantitative agreement with the known effect of singlet-singlet annihilation upon the fluorescence yield, and furthermore show that no bacteriochlorophyll or carotenoid triplet formation is associated with this annihilation.     

Identification of the carotenoid present in the B-800–850 antenna complex from Rhodopseudomonas capsulata as that which responds electrochromically to transmembrane electric fields

Mild proteolysis of Rhodopseudomonas capsulata chromatophores results in a parallel loss of the 800 nm bacteriochlorophyll absorption band and a blue shift in the carotenoid absorption bands associated with the B-800–850 light-harvesting complex. Both the light-induced and the salt-induced electrochromic carotenoid band shift disappear in parallel to the loss of the 800 nm bacteriochlorophyll absorption upon pronase treatment of chromatophores. During the time required for the loss of the 800 nm bacteriochlorophyll absorption and the loss of the electrochromic carotenoid band shift photochemistry is not inhibited and the ionic conductance of the membrane remains very low. We conclude that the carotenoid associated with the B-800–850 light-harvesting complex is the one that responds electrochromically to the transmembrane electric field. Analysis of the pigment content of Rps. capsulata chromatophores indicates that all of the carotenoid may be accounted for in the well defined pigment-protein complexes.     

The type, amount, location, and energy transfer properties of the carotenoid in reaction centers from Rhodopseudomonas sphaeroides.

Analysis of photosynthetic reaction centers from Rhodopseudomonas sphaeroides strains 2.4.1 and Ga shows that each contains approx. 1 mol of a specific carotenoid per mol of reaction center. In strain 2.4.1. the carotenoid is spheroidene (1-methoxy-3,4-didehydro-1,2,7',8',-tetrahydro-psi,psi-carotene); in strain Ga, it is chloroxanthin (1-hydroxy-1, 2, 7', 8'-tetrahydro-psi,psi-carotene). The carotenoid is bound to the same pair of proteins as are the bacteriochlorophylls and bacteriopheophytins of the reaction center. This binding induces strong circular dichroism in the absorption bands of the carotenoid. The carotenoid is close enough to the other pigments of the reaction center so that light energy transfers efficiently from the carotenoid to the bacteriochlorophyll, sensitizing bacteriochlorophyll fluorescence. The fluorescence polarization spectrum of the reaction centers shows that the transition vectors for the visible absorption bands of the carotenoid lie approximately parallel to the 600 nm (Qx) transition of the bacteriochlorophyll complex.