Preferential inhibition by ethidium bromide of photosynthetic apparatus formation in Rhodopseudomonas spheroides cells

Ethidium bromide (EB) inhibited growth of cells of the non-sulfurpurple photosynthetic bacterium Rhodopseudomonas spheroides.The inhibitory action of EB on the light-anaerobic culturedcells was stronger than on dark-aerobic cultured ones. EB alsosuppressed the induced synthesis of bacteriochlorophyll (Bchl)and carotenoids in the dark-aerobically grown cells incubatedwith gentle aeration under no-growth conditions, suggestingthat the target of the inhibitory action of EB on the photosyntheticgrowth of R. spheroides cells is chromatophore formation. EBdepressed the incorporation of ³H- or ¹⁴C-uracil into both RNAand DNA fractions from cells incubated with gentle aeration.In contrast, inhibition by EB of ³H-uracil incorporation intothe DNA fraction was not observed under vigorous aeration. Ourfindings seemed to favor the hypothesis described previously(10) that lowering the intracellular oxidation-reduction potentialmight bring about a unique synthesis or turnover of DNA responsiblefor chromatophore formation. (Received December 22, 1977; )     

Kinetics of photosynthetic membrane protein assembly in Rhodopseudomonas spheroides

Glycogen phosphorylase in cell-free extracts of Neurospora crassa is activated 10- to 15-fold by incubation with MgATP^2?. When the MgATP^2? is removed, the active form (a form) reverts to the inactive form (b form). The inactivation requires Mg²⁺ and is inhibited by NaF. The results confirm that Neurospora crassa glycogen phosphorylase exists in two interconvertible forms and strongly suggests that the interconversion is catalyzed by a kinase and phosphatase. The a form was partially purified. The enzyme has a molecular weight of 320,000. Uridine diphosphate glucose is a linear competitive inhibitor with respect to glucose-1-phosphate and a linear non-competitive inhibitor with respect to glycogen. Glucose-6-phosphate is a hyperbolic (partial) noncompetitive inhibitor with respect to all substrates in both directions. The b form of the enzyme in crude cell-free extracts is stimulated 2- to 3-fold by 5′-AMP. As the b form is purified, the 5′-AMP activation is diminished. The molecular weight of the partially purified “b” form was also 320,000.     

Isolation and fractionation of the photosynthetic membranous organelles from Rhodopseudomonas spheroides

Molecular sieve chromatography and sucrose gradient centrifugation were used to prepare large quantities of purified chromatophores from Rhodopseudomonas spheroides. Electron micrographs of these chromatophores revealed that the final preparations were very homogeneous and free of non-chromatophore particulate material. As an additional check on purity, (14)C-l-phenylalanine-labeled aerobic cells, devoid of chromatophores, were mixed with unlabeled photosynthetic cells. The resulting preparation contained less than 1% of the radioactivity, originally located in non-chromatophore protein. The purified chromatophores were solubilized in 2-chloroethanol and separated into two fractions. Fraction P(1) contained 3 to 5% of the total chromatophore protein and could be resolved into 10 electrophoretic components. The second fraction, P(II), contained five electrophoretic components. One of these components had associated with it all of the pigment and phospholipid present in P(II). Preliminary immunochemical studies on these fractions are also reported.     

Vitamin B(12) in photosynthetic bacteria and methionine synthesis by Rhodopseudomonas spheroides

1. Assay of some photosynthetic bacteria for vitamin B₁₂ showed them to be relatively rich in this factor. Rhodopseudomonas spheroides, grown photosynthetically in Co²⁺-supplemented medium, contained about 100μg./g. dry wt. 2. Extracts of wild-type Rps. spheroides methylated homocysteine by a mechanism similar to the cobalamin-dependent pathway present in Escherichia coli. However, no mechanism similar to the cobalamin-independent N⁵-methyltetrahydrofolate–homocysteine transmethylase of E. coli could be detected in Rps. spheroides. 3. N⁵N¹⁰-Methylenetetrahydrofolate-reductase activity was found in Rps. spheroides. 4. A methionine-requiring mutant strain of Rps. spheroides (strain 2/33), which does not respond to homocysteine, made the same amount of vitamin B₁₂ as the parent organism. Extracts did not form methionine from N⁵-methyltetrahydrofolate and homocysteine even in the presence of cofactors shown to be necessary with the parent strain, and it is concluded that the mutant is blocked in the formation of the apoenzyme of a homocysteine-methylating system similar to the vitamin B₁₂-dependent one in E. coli.     

Light-induced absorbance change of carotenoid in chromatophores of photosynthetic bacterium, Rhodopseudomonas spheroides

The nature of the light-induced absorbance change of carotenoid,spheroidene, was investigated with the chromatophores of Rhodopseudomonasspheroides. The experimental results indicate that the changedoes not represent an oxidation-reduction reaction of the carotenoid,but is caused by a change in the state of the chromatophoresclosely related to the high energy state of the photophosphorylation.Since the change almost vanishes at liquid nitrogen temperature,it probably does not represent a primary photochemical reactionin the chromatophores. The values of the quantum yield for thechange of carotenoid were above unity ; 2.5 on an avera (Received November 20, 1969; )     

Heterogeneous profile in thermal denaturation of DNA preparation from the non-sulfur purple photosynthetic bacterium Rhodopseudomonas spheroides

Thermal denaturation profiles of DNA preparations from fourstrains of Rhodopseudomonas spheroides were comparatively studied.All the melting curves in 0.1 ? SSC displayed Tm at 82 ?0.5?and a faint bimodal transition near 78?. The differential meltingrates as a function of temperature exhibited several peaks,suggesting that inter- or intra-molecular structural heterogeneitymay be present in R. spheroides DNA. ¹ Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Japan. (Received February 15, 1975; )