Specific Bchl Research Articles

Phosphorylation of LHI beta during membrane synthesis in the photosynthetic bacterium Rhodovulum sulfidophilum.

Cells of Rhv. sulfidophilum were grown under different conditions in the presence of 32P-phosphate and the corresponding H and L membrane fractions obtained and fractionated by SDS-PAGE. Both membranes showed almost identical polypeptide composition. The bacteriochlorophyll (Bchl) specific content in H was always lower that in L. As described before, oxygen did not regulate gene expression. Under high light, an almost two- to threefold decrease of the cellular specific Bchl content was observed. Pulse and chase experiments showed that transitions from aerobiosis to light-anaerobiosis did not quantitatively affect the Bchl content of the membranes, although a turnover of the 32P-phosphate and 35S-methionine was observed. LHI beta was the only polypeptidic subunit of the Bchl-binding polypeptides that was phosphorylated in vivo, and phosphotyrosine was the only phosphorylated amino acid detectable. The phosphorylated LHI beta was determined to be insoluble in the organic solvent mixture of (vol/vol) 1:1 chloroform-methanol containing ammonium acetate (0.1 m final concentration). Treatment with a chaotropic agent such as Na2CO3 solubilized the phosphorylated LHI beta, indicating that part of this posttranslationally modified polypeptide was not inserted in a transmembrane position. These results were used to speculate about the regulatory properties of this posttranslational modification of LHI beta on membrane differentiation.

Control by light of whole-cell nitrogenase activity and of nitrogenase and bacteriochlorophyll a formation in Rhodobacter capsulatus strain B10S

Control of nitrogenase and bacteriochlorophyll a (BChl) by light was studied under steady-state conditions with continuous cultures of Rhodobacter capsulatus B10S supplied with malate and growth-limiting amounts of ammonium. Consumption of malate and, correspondingly, the C/N ratio at which malate and ammonium were consumed increased when illumination was increased from 3 to approximately 20 klx and became constant at higher illuminations of up to 40 klx. Essentially the same kinetics were observed with respect to nitrogenase activity of cells, contents of nitrogenase polypeptides, and nifH promoter activity. Substrate consumption was half-maximal at 8 klx and was independent of the presence of nitrogenase. Therefore, it is concluded that light controls the C/N ratio (a quantitative measure of the nitrogen status of cells), which in turn is involved in the control of nitrogenase at the level of nif promoter activity. Post-translational regulation of nitrogenase activity by ADP-ribosylation was not observed under steady-state conditions, but it took place when illumination was suddenly decreased to the range where malate consumption and, consequently, the C/N ratio decreased. Irrespective of the presence or absence of nitrogenase, specific BChl contents of the cultures were constant above 20 klx, and they increased at lower illuminations. These results do not confirm a recently proposed link between nitrogen fixation and photosynthesis as represented by BChl.

Influence of vitamin B12 and light on the formation of chlorosomes in green- and brown-colored Chlorobium species

The specific Bchl a and c content of the vitamin B12-dependent Chlorobium limicola strain 1230 decreased strongly under vitamin B12 limitation. In comparison to a regularly grown culture (20 μg vitamin B12/l) the specific Bchl c content of a B12-limited culture was reduced to 20% and the specific Bchl a content to 42%. By ultrathin sections it could be clearly demonstrated that B12-deficient cells contained no chlorosomes. After the addition of vitamin B12 to a deficient culture, chlorosomes were formed and the Bchl a and c content increased again to the level of regularly grown cells. The brown-colored Chlorobium phaeobacteroides strain 2430 (type strain) and the extremely low-light-adapted strain MN1 were compared with respect to the influence of light on the formation of chlorosomes and the Bchl e and carotenoid content. By ultrathin sections it could be demonstrated that strain MN1 produced two-fold larger chlorosomes. Chlorosome dimensions of strain MN1 decreased with increasing light intensities. The number of chlorosomes per cell in both strains did not change with different light intensities. Strain MN1 formed twice as much Bchl e as the type strain when grown at 30 or below 1 μmol · m-2 · s-1. Under comparable light conditions strain MN1 formed 14–57% more carotenoids than the type strain. Low light intensities aaused the carotenoid content to increase by 25% in strain 2430 in comparison to high light intensity.

Abundance and salt tolerance of obligately aerobic, phototrophic bacteria in a marine microbial mat

Data have been collected on the abundance of obligately aerobic, bacteriochlorophyll- a-containing bacteria in a marine microbial mat on the West Frisian Island of Texel, The Netherlands. Plate counts on media rich in organic matter revealed average numbers of 3 ∗10 5·cm −3 sediment in the top 10 mm of the mat; the number of purple non-sulphur bacteria was of the same magnitude. Due to the relatively small dimensions of obligately aerobic anoxygenic phototrophic bacteria and purple non-sulphur bacteria, compared to those of purple sulphur bacteria, the contributions of either of the two former groups to the biomass of Bchl- a-containing organisms was approximately 3%. The specific Bchl- a-content of the isolated obligately aerobic phototrophs was very low (0.8 to 1.0 μg·mg −1 protein) compared to that of purple non-sulphur bacteria (16 to 20 μg·mg −1 protein), and purple sulphur bacteria (27 to 30 μg·mg −1). As a consequence, the relative contribution to the total Bchl a concentration of the two former groups (0.1% and 2.1%, respectively) was negligible, compared to that of the purple sulphur bacteria (97.8%). Salinities <50 had little effect on growth rate and yield of isolates; at salinities between 50 and 100 the doubling time increased progressively with a concomitant decrease in yield; no growth occurred at salinities > 140.

Light and oxygen regulation of the synthesis of bacteriochlorophylls a and c in Chloroflexus aurantiacus

Control of the synthesis of bacteriochlorophylls (Bchls) a and c by light and oxygen was studied in Chloroflexus aurantiacus grown in batch or chemostat culture with serine as the growth-limiting substrate. For comparison, inhibition by gabaculine of the formation of selected tetrapyrroles was studied. The inhibitory effect of gabaculine decreased in the following order of tetrapyrrole formation: coproporphyrin greater than Bchl c greater than Bchl a. Not only did addition of 5-aminolevulinate (ALA) reverse the inhibition by gabaculine, it also caused an increase in Bchl c content when the cultures grew at high concentrations of ALA. Inhibition of Bchl a, Bchl c, and coproporphyrin formation by oxygen was similar to inhibition by gabaculine. Addition of ALA to aerated cultures led to significant accumulation of coproporphyrin. These results suggest that oxygen inhibits tetrapyrrole formation at a site before ALA formation. Control by light was studied with chemostat cultures transferred from 5 klx to 25 klx. This resulted in only a transient increase of the protein level of the culture, while specific contents of Bchls c and a and the ratio Bchl c/Bchl a decreased to lower steady states. However, the specific content of coproporphyrin increased. Addition of ALA to chemostat cultures adapted to 50 klx increased specific coproporphyrin and Bchl c contents by factors of about 20 and 4, respectively, while the specific Bchl a content was only slightly increased and protein levels were unaffected. Increasing the serine concentration caused an initial increase in the specific Bchl c content, which returned to the original value as soon as the protein content had attained its maximal level. These results suggest that light does not control ALA formation as strictly as oxygen and that competition of biomass formation and tetrapyrrole synthesis for common precursors may be influenced by light.

Differentiation of the photosynthetic apparatus of Chloroflexus aurantiacus depending on growth with different amino acids

The phototrophic green bacterium Chloroflexus aurantiacus was grown anaerobically in batch culture with different amino acids at 56°C and constant illumination of 25 klx. The composition of the photosynthetic apparatus was measured by quantitation of the bacteriochlorophylls (Bchl) a and c (representing the membrane-bound and the chlorosomal moieties, respectively). Ser added at concentrations up to 15 mM stimulated protein formation and Bchl a and c syntheses. A comparable stimulation was found with Glu and Ala. Coproporphyrin accumulation approached saturation at 5 mM of Ala, Asp, Orn, and Ser, while with Glu and Arg saturating concentrations were above 5 mM. Protein and tetrapyrrole syntheses became saturated at 2.5 to 5 mM of Asp, Ile, and Val. However, with Arg and Orn Bchl c synthesis was stimulated up to 2.5 mM, growth and Bchl a synthesis up to 5 mM. At higher Arg or Orn concentrations these activities were inhibited. Coproporphyrin accumulation was highest with Arg or Orn, at concentrations which inhibited growth and Bchl formation. Stimulation of Bchl synthesis took place preferentially at the level of Bchl c, while Bchl a was more sensitive toward inhibition. In both cases however, the ratio of Bchl c to Bchl a increased with higher amino acid concentrations. Nevertheless, each amino acid induced a typical effect. To understand different effects exerted by different amino acids, chemostat cultures were grown limited by either Ser or Glu. With Ser, steady state protein levels and specific Bchl a contents decreased slightly when increasing the dilution rate (D). Concomitantly Bchl c and coproporphyrin levels as well as the ratio of Bchl a/Bchl c increased. With Glu as the limiting substrate, all of the above mentioned parameters decreased. Since all of the Ser was consumed and increasing amounts of Glu remained unutilized in the spent medium, it is concluded that differences in the formation of the three pyrrole derivatives tested are due to differences in the affinities of uptake systems for Ser and Glu.

Growth of the phototrophic purple sulfur bacterium Thiocapsa roseopersicina under oxic/anoxic regimens in the light

The phototrophic purple sulfur bacterium Thiocapsa roseopersicina was grown in sulfide-limited continuous cultures exposed to oxic/anoxic regimens in continuous light. Synthesis of bacteriochlorophyll a (BChl a) did not occur during the oxic periods, but started immediately upon the creation of anoxic conditions. In contrast, protein synthesis continued during both oxic and anoxic periods. Consequently, the specific content of BChla fluctuated. Despite the presence of oxygen and the fluctuating BChl a content, growth occurred predominantly in a phototrophic mode and respiration was virtually zero. BChl a synthesis continued at high rates during anoxic periods, thus compensating for the lack of synthesis during oxic periods. When cultivated under regimens with oxic periods shorter than 12 h the highest specific BCh a content was 27 μg·mg − protein. In contrast, when cultivated under regimens with oxic periods longer than 12 h the specific BChl a content was always lower than 27μg·mg − length of the oxic periods. During the anoxic periods, BChl a synthesis occurred at the maximal velocity of 1.2 μg·mg −1 protein·h −, but the length of the anoxic periods was not sufficient to allow the BChl a content to reach the maximum level. Cultivation under continuously oxic conditions eventually resulted in pigmentless cells growing chemolithotrophically. The BChl a synthesizing ability was not lost during prolonged exposure to oxygen. It was concluded that T. roseopersicina is very well adapted to oxic/anoxic cycles.

Growth of the phototrophic purple sulfur bacteriumThiocapsaroseopersicinaunder oxic/anoxic regimens in the light

Abstract The phototrophic purple sulfur bacterium Thiocapsa roseopersicina was grown in sulfide-limited continuous cultures exposed to oxic/anoxic regimens in continuous light. Synthesis of bacteriochlorophyll a (BChl a ) did not occur during the oxic periods, but started immediately upon the creation of anoxic conditions. In contrast, protein synthesis continued during both oxic and anoxic periods. Consequently, the specific content of BChla fluctuated. Despite the presence of oxygen and the fluctuating BChl a content, growth occurred predominantly in a phototrophic mode and respiration was virtually zero. BChl a synthesis continued at high rates during anoxic periods, thus compensating for the lack of synthesis during oxic periods. When cultivated under regimens with oxic periods shorter than 12 h the highest specific BCh a content was 27 μg·mg − protein. In contrast, when cultivated under regimens with oxic periods longer than 12 h the specific BChl a content was always lower than 27μg·mg − length of the oxic periods. During the anoxic periods, BChl a synthesis occurred at the maximal velocity of 1.2 μg·mg −1 protein·h − , but the length of the anoxic periods was not sufficient to allow the BChl a content to reach the maximum level. Cultivation under continuously oxic conditions eventually resulted in pigmentless cells growing chemolithotrophically. The BChl a synthesizing ability was not lost during prolonged exposure to oxygen. It was concluded that T. roseopersicina is very well adapted to oxic/anoxic cycles.

Regulation of tetrapyrrole synthesis by light in chemostat cultures of Rhodobacter sphaeroides.

Control of bacteriochlorophyll (Bchl), magnesium protoporphyrin monomethyl ester (MgPME), cytochromes, and coproporphyrin by light was studied with chemostat cultures of Rhodobacter sphaeroides growing at a constant dilution rate. By increasing the growth-limiting light energy flux from 10 to 55 W/m2, specific Bchl contents decreased from 19.3 to 7.9 nmol/mg of protein. This was strictly proportional to a decrease in the ratio of B800-850 to B875 light-harvesting complexes. MgPME levels increased from 1.5 to 5.3 nmol/mg of protein, while cytochrome as well as coproporphyrin levels stayed constant at 0.46 and 1.95 nmol/mg of protein, respectively. Since in chemostat cultures steady-state levels of a product represent the rate of synthesis, these results infer only slight control of the rate-limiting step of total tetrapyrrol formation by light. In substrate-limited cultures MgPME was accumulated when growth and Bchl formation approached substrate saturation. This suggests that light controls a second step, i.e., MgPME conversion, whenever too much precursor is available, owing to the low sensitivity of the initial step of control. MgPME was preferentially localized in a subcellular fraction with high contents of B875 complexes. A second fraction exhibiting increased contents of B800-850 complexes lacked significant levels of MgPME. These results are discussed in terms of localization of Bchl synthesis in the membrane system of R. sphaeroides.

Growth rate and control of development of the photosynthetic apparatus in Chloroflexus aurantiacus

Chloroflexus aurantiacus was grown photoheterotrophically in a chemostat in order to study the influence of growth rate on the formation of bacteriochlorophyll a (Bchl a) which represents the membrane-bound photosynthetic pigment complexes, and of Bchl c which represents the light harvesting pigment-proteins of the chlorosome. Steady state cell protein levels as well as specific Bchl a contents increased linearly and specific Bchl c contents exponentially when the dilution rate, representing growth rate, was decreased. In spite of differences in the light intensities, continuous cultures growing at comparable growth rates and densities exhibited comparable specific contents of both Bchls and largely identical molar ratios of Bchl c/Bchl a. The growth rate of constantly illuminated batch cultures was varied by changing the concentration of growth-limiting nutrients. Cultures growing at higher growth rates showed higher cell densities but lower specific Bchl levels as well as lower molar ratios of Bchl c/Bchl a than cultures growing at low growth rate. Determination of the light energy flux required for half-maximal saturation of photosynthetic activity (light dependent proton extrusion) by chemostat cultures showed a dependency of that activity by the content of cellular Bchl c. In summary, the results suggest that, growth rate or a factor regulating growth rate, rather than light affected specific Bchl levels and because of the increasing molar ratio of Bchl c to Bchl a, the light harvesting capacity and photosynthetic efficiency of the photosynthetic apparatus.

Porphyrin biosynthesis in Rhodopseudomonas palustris—XII. δ-aminolevulinate synthetase switch—off/on regulation

The high levels of δ-aminolevulinate synthetase (ALA-S) in Rhodopseudomonas palustris cells grown anaerobically in the light (Ph) decrease to those found in cells grown aerobically in the dark (A), when the former cultures were vigorously oxygenated; simultaneously bacteriochlorophyll (Bchl) synthesis abruptly halted leading to diminished steady-state specific Bchl content. When flushing oxygen was interrupted, enzymic activity increased, whether chloramphenicol was present or not in the medium; if the protein synthesis inhibitor was added when oxygenation started, ALA-S declined in the same fashion as in its absence, but thereafter reactivation of the enzyme was lower than before. Succinyl-CoA-synthetase and ALA-dehydratase activities were also measured under the conditions described, and no changes at all have been observed. The existence of different forms of ALA-S in R. palustris depending on growth conditions is postulated along with the formation of low molecular weight factors which can modulate ALA-S activity by binding to the enzyme; a widespread mechanism in the adaptation of micro-organisms to changes in environment. It is also proposed that this particular regulatory phenomenon, could be referred to as a switch off/on mechanism controlling ALA-S activity in R. palustris.

Chemoautotrophic growth of Thiocystis violacea, Chromatium gracile and C. vinosum in the dark at various O2‐concentrations

AbstractThe influence of various O2‐concentrations on chemoautotrophic growth was determined in the range from microaerobic to aerobic conditions. Chromatium vinosum D, Thiocystis violacea 2311 and Chromatium gracile 25‐1 were grown chemoautotrophically using consecutively repeated batch cultures. Rapid growth of all three strains occurred only under microaerobic conditions. The fastest growth (μ = 0.058) was obtained with Chromatium gracile 25‐1 grown in the presence of 1 mg O2 · 1−1. For optimal chemoautotrophic growth sulfur‐containing cells had to be supplied continuously with extracellular electron donor. Multiplication of thiocystis violacea 2311 and Chromatium gracile 25‐1 was significantly inhibited after several cell generations, while Chromatium vinosum D could be grown unlimited. In case of the latter bacterium any given O2‐concentration determined a unique growth rate and a unique specific bchl α content. The analysis of absorption spectra of whole cells revealed that different pigment complexes were regulated separately by oxygen. Growth yields were not dependent on the O2‐concentration. A growth yield of 12 g dry cell mass per mol of thiosulfate oxidized was determined for Chromatium vinosum D growing in the micro‐ to semiaerobic O2‐concentration range. When compared to phototrophically grown cells, the catalase content of chemotrophically grown cells was 2 times lower while the maximal specific respiration rate of the cells was 7 times higher. The ecological significance of the kinetics of chemotrophic growth under various growth conditions is discussed.

Energetic aspects of photophosphorylation capacity and reaction center content of Rhodopseudomonas capsulata, grown in a turbidostat under different irradiances

Cells of Rhodopseudomonas capsulata were grown in a turbido-stat and adapted to high (1400 W/m 2) or low (40 W/m 2) light intensities. In high-light-grown cells the specific BChl content was about 10-times lower, the number of intracytoplasmatic membrane vesicles smaller by a factor of about 20, the photosynthetic unit smaller by a factor of 1.9 and the reaction center content about 5-times lower than in low-light-grown cells. However, the photophosphorylation rate per reaction center under saturating light was higher in high-light-grown cells by a factor of 7.7, apparently compensating the lower amount of reaction centers. Adaptation of the cells to different irradiances not only seems to comprise a variation of the size and composition of the antennae, but also a change in the affinity of the photosynthetic system to light, as concluded from saturation curves obtained from the two adaptation stages of cells.

A comparative study on the composition of chlorosomes (Chlorobium vesicles) and cytoplasmic membranes from Chloroflexus aurantiacus strain Ok-70-fl and Chlorobium limicola f. thiosulfatophilum strain 6230

Highly purified fractions of chlorosomes and cytoplasmic membranes were isolated from Chloroflexus aurantiacus Ok-70-fl and Chlorobium limicola 6230. These fractions were comparatively analyzed for their pigmentation, phospholipid, glycolipid, and cytochrome c content as well as for their specific activities of succinate dehydrogenase and NADH-oxidase. The data showed that there are some differences in pigmentation and phospholipid content between the isolated fractions of Chloroflexus and Chlorobium. Chlorosomes of Chloroflexus contained a specific BChl a-complex with a characteristic absorption maximum at about 790 nm. This BChl a-complex could not be detected in spectra of chlorosomes from Chlorobium. The near infrared region of the spectra of the isolated cytoplasmic membranes of both organisms revealed considerable differences: The BChl a-complexes of Chloroflexus membranes exhibited peaks at 806 and 868 nm whereas the membranes of Chlorobium had a single BChl a-peak at 710 nm. In contrast to the findings with Chlorobium the chlorosomes of Chloroflexus contained at least twice as much phospholipids as did the cytoplasmic membranes. In Chlorobium the phospholipid content of cytoplasmic membranes is three times that of their chlorosomes. The distribution of all other components (carotenoid composition, enzyme activities, cytochrome c content, and glycolipids) was about the same in both strains. From the data it was concluded that differences in the organization of the photosynthetic apparatus are mainly based on differences of the organization of the photosynthetic units in the cytoplasmic membrane and probably the kind of linkage of the light harvesting system in the chlorosomes with the reaction center in the cytoplasmic membranes.

Control of synthesis of reaction center bacteriochlorophyll in photosynthetic bacteria.

Abstract— The ratio of total bacteriochlorophyll (BChl) to reaction center BChl has been determined in the wild‐type and carotenoid‐less mutants of Rhodopseudomonas spheroides and Rhodospirillum rubrum with various specific BChl contents. In wild‐type R. spheroides the ratio increases as the specific BChl content increases; in the other strains the ratio is almost invariant.The ability of the wild‐type of R. spheroides to alter the size of the photosynthetic unit is apparently correlated with its multi‐component BChl spectrum. The amount of reaction center BChl relative to the amount of the longest wavelength component of the spectrum is constant. The variation in the size of the photosynthetic unit is due to variation in the amount of this component relative to total BChl.The size of the photosynthetic unit does not change as the specific BChl content decreases during growth under aerobic conditions of a culture grown previously under photosynthetic conditions.

Growth and ultrastructure of Rhodomicrobium vannielii as a function of light intensity.

Cells of Rhodomicrobium vannielii were grown in a controlled environment at several different light intensities. Differential rates of bacteriochlorophyll (BChl) synthesis and specific BChl contents were inversely related to the light intensity. On the other hand, the specific rate of growth-before reaching a maximal value-was directly related to the intensity of the light. Thin sections of cells grown at moderately low light showed the typical peripherally located, symmetrically distributed lamellate system, whereas an asymmetrical distribution of a less extensive lamellate system occurred in cells grown at high light intensities. It is proposed that a limited number of individual units of the lamellate system are originally derived from inward folds of the cytoplasmic membrane, and that subsequent lamellae arise by proliferation, including possible forking and definite folding back, of the few original lamellar membranes.