Bacteriochlorophyll Synthesis and the Ultrastructure of Wild Type and Mutant Strains of Rhodopseudomonas spheroides

The ultrastructure of sectioned cells of mutant and wild type Rhodopseudomonas spheroides has been examined by electron microscopy. The characteristic vesicles associated with the presence of bacteriochlorophyll were found in wild type cells grown with low aeration. These were also found in mutant TA-R which forms bacteriochlorophyll under high aeration. None of the mutants with blocks in bacteriochlorophyll synthesis contained intracytoplasmic membrane. These included mutant 8-17 which accumulates bacteriochlorophyllide but fails at the phytolation step. We conclude that the intact bacteriochlorophyll molecule, or some particular membrane protein associated with it, is needed for the development of the characteristic intracytoplasmic membrane system in R. spheroides.     

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.     

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.     

Bacteriophages of Rhodopseudomonas spheroides: Isolation and Characterization of a Rhodopseudomonas spheroides Bacteriophage

A DNA-containing bacteriophage, designated RS1, infecting Rhodopseudomonas spheroides 2.4.1, has been isolated from sewage. The buoyant density of RS1 in CsCl equilibrium centrifugation is 1.50 g/cm(3), and the buoyant density of RS1 DNA is 1.706. The phage possesses a polyhedral head, approximately 65 nm in diameter, and a tail 60 nm long. When grown on aerobic cells, RS1 has a latent period of 120 min and an average burst size of 20. When grown on anaerobic cells, RS1 has a latent period of 150 min, and a burst size similar to that observed during aerobic infection. The adsorption rate constant of RS1 to aerobic cells is 1.2 x 10(-9) ml/min, and 0.58 x 10(-9) ml/min to anaerobic cells. Adsorption of RS1 to R. spheroides requires the presence of divalent cations.     

Ferrochelatase of Rhodopseudomonas spheroides

Extracts of Rhodopseudomonas spheroides contain two ferrochelatases: one is soluble and forms metalloporphyrins from deuteroporphyrin and haematoporphyrin; the other is particulate and forms metalloporphyrins from protoporphyrin, mesoporphyrin, deuteroporphyrin and haematoporphyrin. Neither enzyme incorporates Mg²⁺ into porphyrins or Fe²⁺ into porphyrin cytochrome c. By using the particulate enzyme, plots of 1/v versus 1/s when one substrate was varied and the other kept constant showed that neither substrate affected the K_m of the other. The suggested sequential mechanism for the reaction is supported by derivative plots of slopes and intercepts. The K_m for deuteroporphyrin was 21.3μm and that for Co²⁺ was 6.13μm. The enzyme incorporated Co²⁺, Fe²⁺, Zn²⁺, Ni²⁺ and Mn²⁺; Cd²⁺ was not incorporated and was an inhibitor, competitive with respect to Co²⁺, non-competitive with respect to deuteroporphyrin. The K_i for Cd²⁺ was 0.73μm. Ferrochelatase was inhibited by protohaem, non-competitively with respect to Co²⁺ or with respect to deuteroporphyrin. Inhibition by magnesium protoporphyrin was non-competitive with respect to deuteroporphyrin, uncompetitive with respect to Co²⁺. The inhibitory concentrations of the metalloporphyrins are lower than those required for the inhibition of δ-aminolaevulate synthetase by protohaem. Fe²⁺ is not incorporated aerobically into porphyrins unless an electron donor, succinate or NADH, is supplied; the low aerobic rate of metalloporphyrin synthesis obtained is insensitive to rotenone and antimycin. The rate of Fe³⁺ incorporation increases as anaerobic conditions are achieved.     

Regulation of Bacteriochlorophyll Synthesis by Oxygen in Respiratory Mutants of Rhodopseudomonas capsulata

Respiratory mutants of the facultative photosynthetic bacterium Rhodopseudomonas capsulata were used to investigate the mechanism of (reversible) inhibition of bacteriochlorophyll (BChl) synthesis by molecular oxygen. Although mutant strain M5 lacks cytochrome oxidase activity, it closely resembles the parental wild-type strain in respect to the effect of O(2) on BChl formation. This observation does not support an earlier hypothesis that O(2) regulates BChl synthesis through an effect on the redox state of a component of the respiratory electron transport system. Mutant strain M2 shows normal cytochrome oxidase activity, but lacks both reduced nicotinamide adenine dinucleotide and succinate dehydrogenase activities; relative to the parental strain, BChl synthesis in M2 is more sensitive to O(2) inhibition. The foregoing and results of related experiments can be accounted for by a revised interpretation of the O(2) effect, which proposes that O(2) directly inactivates a "factor" necessary for BChl formation and that, at relatively low O(2) tension, the inactivation can be reversed by a flow of electrons (derived from reduced nicotinamide adenine dinucleotide and succinate) diverted from a portion of the electron transport system delimited by the mutational blocks in M2 and M5.     

Electron microscopy of chromatophores of Rhodopseudomonas spheroides

Gibson, K. D. (St. Mary's Hospital Medical School, London, England). Electron microscopy of Rhodopseudomonas spheroides. J. Bacteriol. 90:1059-1072. 1965.-Fixed and stained chromatophores and whole cells of anaerobically grown Rhodopseudomonas spheroides were examined in thin sections in the electron microscope. Both purified chromatophores and intracellular membrane-bound vesicles had exactly the same appearance, namely that of spheres or ellipsoids with a thin electron-dense shell surrounding an electron-lucent interior. The distribution of diameters in the two types of structure was also found to be the same, and was compatible with a normal distribution, with a mean of 570 A and a standard deviation 40 A. Negatively stained chromatophores appeared like discs or collapsed spheres. The presence of invaginations of the cytoplasmic membrane in this species was confirmed, and a new structure resembling a twin chromatophore was observed. The bearing of these results on theories of the origin of chromatophores is discussed, and it is concluded that they offer some support for each one of the three main theories about the origin of particulate organelles.