Attenuated effect of oxygen on photopigment synthesis in Rhodospirillum centenum.

Rhodospirillum centenum resembles typical nonsulfur photosynthetic bacteria in a number of respects, including its ability to grow either anaerobically as a phototroph or aerobically as a heterotroph. We demonstrate, however, that R. centenum is unusual in its ability to synthesize a functional photosynthetic apparatus regardless of the presence of molecular oxygen. Aerobically expressed photopigments were shown to be functionally active, as demonstrated by the ability of heterotrophically grown cells to grow photosynthetically, without a lag, when suddenly placed under anaerobic conditions. An R. centenum mutant that has acquired the ability to repress synthesis of photopigments in the presence of oxygen was also characterized. Both the wild type and the oxygen-repressed mutant of R. centenum were found to exhibit high light intensity repression of photopigment biosynthesis. The latter result suggests that R. centenum contains separate regulatory circuits for controlling synthesis of its photochemical apparatus by light intensity and oxygen.     

Photoresponses in Rhodobacter sphaeroides: role of photosynthetic electron transport.

Rhodobacter sphaeroides responds to a decrease in light intensity by a transient stop followed by adaptation. There is no measurable response to increases in light intensity. We confirmed that photosynthetic electron transport is essential for a photoresponse, as (i) inhibitors of photosynthetic electron transport inhibit photoresponses, (ii) electron transport to oxidases in the presence of oxygen reduces the photoresponse, and (iii) the magnitude of the response is dependent on the photopigment content of the cells. The photoresponses of cells grown in high light, which have lower concentrations of light-harvesting photopigment and reaction centers, saturated at much higher light intensities than the photoresponses of cells grown in low light, which have high concentrations of light-harvesting pigments and reaction centers. We examined whether the primary sensory signal from the photosynthetic electron transport chain was a change in the electrochemical proton gradient or a change in the rate of electron transport itself (probably reflecting redox sensing). R. sphaeroides showed no response to the addition of the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, which decreased the electrochemical proton gradient, although a behavioral response was seen to a reduction in light intensity that caused an equivalent reduction in proton gradient. These results strongly suggest that (i) the photosynthetic apparatus is the primary photoreceptor, (ii) the primary signal is generated by a change in the rate of electron transport, (iii) the change in the electrochemical proton gradient is not the primary photosensory signal, and (iv) stimuli affecting electron transport rates integrate via the electron transport chain.     

Role of proton motive force in phototactic and aerotactic responses of Rhodopseudomonas sphaeroides.

Rhodopseudomonas sphaeroides grown under nonrigorous anaerobic conditions in the light developed components of a branched respiratory electron transfer chain, and a photosynthetic electron transfer chain. Both respiratory pathways were sensitive to rotenone and high concentrations of cyanide, but oxygen uptake was only partially inhibited by the addition of low concentrations of cyanide or antimycin A. When incubated anaerobically in the dark, R. sphaeroides responded positively to an oxygen gradient in the absence of rotenone. In the presence of rotenone, aerotaxis only occurred when the antimycin A-sensitive branch of the pathway was functioning, although both branches still reduced oxygen. Although there was electron movement along the respiratory chain, aerotaxis only occurred in response to a change in proton motive force. When incubated anaerobically in the light, the movement of R. sphaeroides up a light gradient depended on photosynthetic electron transport. When incubated aerobically, high-intensity actinic illumination inhibited oxygen uptake and aerotaxis. In a low-intensity light gradient the phototactic response was inhibited by oxygen. These results are discussed in relation to the interaction of the electron transfer chains and their roles in controlling tactic responses in R. sphaeroides.     

The Phycomyces lens: measurement of the sporangiophore intensity profile using a fiber optic microprobe

A fiber optic microprobe, 5.5 m in diameter, was used as a detector to measure the light intensity profile at the distal cell surface of Phycomyces blakesleeanus (Burgeff) sporangiophores that were irradiated unilaterally by a collimated xenon source. The light intensity at a fixed location of the cell surface showed large random variations over time which were probably the result of optical effects of particles being carried past the probe by cytoplasmic streaming. The intensity profile, formed around the distal periphery of the cell by the lens action of the sporangiophore, was determined from intensity measurements made while the probe was held fixed and the incident beam direction was varied in angle of azimuth. The resulting profile consisted of two steeply rising sides enclosing a central plateau or shallow well which ranged in fluence rate from 1.6 to 2.2 times that of the incident beam. These experimental findings differ from theoretical modeling where much greater contrast between the sides and central portion of the lens profile was predicted. These results also indicate that the mechanism of phototropic sensory perception in Phycomyces may filter out cytoplasmic light flicker and may not require strong contrasting regions within the lens profile to detect light direction.     

Localization and environmental regulation of MCP-like proteins in Rhodobacter sphaeroides

Chemotaxis to many compounds by Rhodobacter sphaeroides requires transport and at least partial metabolism of the chemoeffector. Previous investigations using phototrophically grown cells have failed to find any homologues of the MCP chemoreceptors identified in Escherichia coli. However, using an antibody raised against the highly conserved domain of E. coli Tsr, MCP-like proteins were identified in R. sphaeroides WS8N. Analysis using Western blotting and immunogold electron microscopy showed that expression of these MCP-like proteins is environmentally regulated and that receptors are targeted to two different cellular locations: the poles of the cells and the cytoplasm. In aerobically grown cells, these proteins were shown by immunoelectron microscopy to localize predominantly to the cell poles and to an electron-dense body in the cytoplasm. Western blot analysis indicated a 17-fold reduction in protein concentration when cells were grown in the light. The number of immunogold particles was also dramatically reduced in anaerobically light-grown cells and their cellular distribution was altered. Fewer receptors localized to the cell poles and more particles randomly distributed within the cell, but the cytoplasmic cluster remained. These trends were more pronounced in cells grown anaerobically under dim light than in those grown anaerobically under bright light, suggesting that expression is controlled by redox state and either light intensity or the extent of photosynthetic membrane synthesis. Recent work on E. coli chemosensing suggests that oligomerization of receptors and chemosensory proteins is important for sensory signalling. The data presented here suggest that this oligomerization can occur with cytoplasmic receptors and also provides an explanation for the multiple copies of chemosensory proteins in R. sphaeroides.     

Chromosome transfer in Rhodobacter sphaeroides: Hfr formation and genetic evidence for two unique circular chromosomes.

A 600-bp oriT-containing DNA fragment from the Rhodobacter sphaeroides 2.4.1 S factor (oriTs) (A. Suwanto and S. Kaplan, J. Bacteriol. 174:1124-1134, 1992) was shown to promote polarized chromosomal transfer when provided in cis. A Kmr-oriTs-sacR-sacB (KTS) DNA cassette was constructed by inserting oriTs-sacR-sacB into a pUTmini-Tn5 Km1 derivative. With this delivery system, KTS appeared to be randomly inserted into the genome of R. sphaeroides, generating mutant strains which also gained the ability to act as Hfr donors. An AseI site in the Kmr cartridge (from Tn903) and DraI and SnaBI sites in sacR-sacB (the levansucrase gene from Bacillus subtilis) were employed to localize the KTS insertion definitively by pulsed-field gel electrophoresis. The orientation of oriTs at the site of insertion was determined by Southern hybridization analysis. Interrupted mating experiments performed with some of the Hfr strains exhibited a gradient of marker transfer and further provided genetic evidence for the circularity and presence of two chromosomal linkage groups in this bacterium. The genetic and environmental conditions for optimized mating between R. sphaeroides strains were also defined. The results presented here and our physical map of the R. sphaeroides 2.4.1 genome are discussed in light of the presence of two chromosomes.     

Electron transport-dependent taxis in Rhodobacter sphaeroides.

Rhodobacter sphaeroides showed chemotaxis to the terminal electron acceptors oxygen and dimethyl sulfoxide, and the responses to these effectors were shown to be influenced by the relative activities of the different electron transport pathways. R. sphaeroides cells tethered by their flagella showed a step-down response to a decrease in the oxygen or dimethyl sulfoxide concentration when using them as terminal acceptors. Bacteria using photosynthetic electron transport, however, showed a step-down response to oxygen addition. Addition of the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone did not cause a transient behavioral response, although it decreased the electrochemical proton gradient (delta p) and increased the rate of electron transport. However, removal of the ionophore, which caused an increase in delta p and a decrease in the electron transport rate, resulted in a step-down response. Together, these data suggest that behavioral responses of R. sphaeroides to electron transport effectors are caused by changes in the rate of electron transport rather than changes in delta p.     

Conservation of the photosynthesis gene cluster in Rhodospirillum centenum

Intraspecies and intergenus complementation analysis were utilized to demonstrate that photosynthesis genes are clustered in distantly related purple photosynthetic bacteria. Specifically, we show that the linkage order for genes involved in bacteriochlorophyll and carotenoid biosynthesis in Rhodospirillum centenum are arranged essentially as in Rhodobacter capsulatus and Rhodobacter sphaeroides. In addition, the location and relative distance observed between the puf and puh operons which encode for light harvesting and reaction-centre structural genes are also conserved between these species. Conservation of the photosynthesis gene cluster implies either that there are structural or regulatory constraints that limit rearrangement of the photosynthesis gene cluster or that there may have been lateral transfer of the photosynthesis gene cluster among different species of phototrophic bacteria.     

Reductive effect of H(2) uptake and poly-beta-hydroxybutyrate formation on nitrogenase-mediated H(2) accumulation of Rhodobacter sphaeroides according to light intensity

Nitrogenase-mediated H(2) accumulation of Rhodobacter sphaeroides under photoheterotrophic conditions is reduced directly by the hydrogenase activity catalyzing H(2) uptake and indirectly by energy-demanding metabolic processes such as poly-beta-hydroxybutyrate (PHB) formation. H(2) accumulation of R. sphaeroides was examined during cell growth under illumination of 15, 7, and 3 W/m(2). Mutations in either hupSL (H(2)-uptake hydrogenase) or phbC (PHB synthase) had no effect on nitrogenase activity. The nitrogenase activity of R. sphaeroides grown at 15 W/m(2), however, was 70% higher than that of cells grown at 3 W/m(2), while the H(2)-uptake hydrogenase activity was approximately 3-fold higher in the same comparison. Accordingly, H(2) uptake by hydrogenase, monitored by measuring the difference in H(2) accumulation between a hupSL-deletion mutant and the corresponding parental strain, appeared to reach a maximum level as illumination was increased to 15 W/m(2). On the other hand, the surplus energy due to lack of PHB formation led to a fixed increase in H(2) accumulation independent of light intensity, reflecting the fact that the cellular PHB content was not changed significantly depending on light intensity. Therefore, H(2) uptake by hydrogenase should be suppressed to achieve higher H(2) accumulation of R. sphaeroides, especially at 15 W/m(2).     

Differential carotenoid composition of the B875 and B800-850 photosynthetic antenna complexes in Rhodobacter sphaeroides 2.4.1: involvement of spheroidene and spheroidenone in adaptation to changes in light intensity and oxygen availability.

Rhodobacter sphaeroides 2.4.1 is a member of the nonsulfur purple facultative photosynthetic proteobacteria, capable of growth under a variety of cultivation conditions. In addition to the structural polypeptides and bacteriochlorophyll, the two major antenna complexes, B875 and B800-850, contain a variety of carotenoids which are an important structural and functional component of the membrane-bound photosynthetic complexes of this bacterium. Two major carotenoids, spheroidene and its keto derivative, spheroidenone, are differentially synthesized by R. sphaeroides, depending on the growth conditions. Spheroidene prevails during growth under anaerobic conditions and low light intensities, whereas spheroidenone is predominant in semiaerobically grown cells or during anaerobic growth at high light intensities. In this study, we demonstrate that in wild-type cells, spheroidene is predominantly associated with the B800-850 photosynthetic antenna complex and spheroidenone is more abundant in the B875 complex. Exploiting mutants defective in the biosynthesis of either the B875 or B800-850 light-harvesting complex, we demonstrate an association between the formation of either the B875 or B800-850 complex, on the one hand, and the accumulation of spheroidenone or spheroidene, on the other. The possible involvement of the conversion of spheroidene to spheroidenone as a significant control mechanism involved in the adaptation of R. sphaeroides to changes in light intensity and oxygen tension is discussed.     

Role of three basic light reactions in photomovement of desmids

Photoaccumulations in light trap experiments have been studied in the desmids, Cosmarium, Micrasterias and Euastrum. Dependence of accumulation density on exposure time follows saturation curves, while dose response curves show optima. Time-lapse microcinematography and population methods have revealed that all three basic light-induced motor responses known in microorganisms participate in producing photoaccumulations in desmids. During the initial phase the cells are phototactically attracted towards the trap by scattered light. In low light intensity traps photokinetic reactions may play only a minor role, since photokinesis could be evoked only by light intensities100 lx in Cosmarium cucumis. True photophobic reactions have been demonstrated for the first time in desmids. There are two types of phobic responses in desmids: either the cell reverses its movement or it swings sidewise into the new direction. Behaviour of partially shadowed cells suggests that perception of light direction is brough about by simultaneous intensity measurement at two or more sites within the cell.     

Mechanism of Photoaccumulation in Paramecium bursaria

Responses of Paramecium bursaria to light intensity changes were investigated. The resting paramecia show a direction changing response (photophobic response) to a sudden decrease of light intensity, whereas no response was shown to an increase in intensity. The critical intensity decrease dI_c necessary to show the response was measured at various values of initial light intensity, and the ratio dI_c/I was found to be equal to ～0.15. The swimming paramecia show different behavior depending on their swimming direction in the spatial gradient of light intensity. Paramecia show direction change more frequently when they are swimming down the gradient than in the opposite direction. This difference in the rate of direction changing is 13–17%. These results may offer an explanation for the mechanism of photoaccumulation.     

Light-induced division and genomic synchrony in phototrophically growing cultures of Rhodopseudomonas sphaeroides.

An experimental procedure for rapidly obtaining cell populations of phototrophically growing Rhodopseudomonas sphaeroides which display division and genomic synchrony has been developed. The basis of the procedure resides with the normal physiological response displayed by cells of R. sphaeroides that have been subjected to an immediate decrease in incident light intensity. After an abrupt high- to low-light transition of an asynchronously dividing cell population, an immediate cessation of increases in culture turbidity, total cell number, and net accumulations of culture deoxyribonucleic acid and phospholipid occurs. Total cell number remains constant for 2.5 h after the transition to low light, after which time, it undergoes a sharp increase. Reinitiation of high-light conditions of growth 1 h subsequent to this increase in total cell number results in a cell population possessing a high degree of division and genomic synchrony. A characterization of this procedure, together with a demonstration of its utility for studies on intracytoplasmic membrane assembly, is presented.     

Use of the plasmid R89S replicon for constructing integration vectors

It has been shown that the plasmid R89S derivatives can be used as integrative vectors for bacteria in which the plasmid is unable to replicate autonomously. The chromosomal and plasmid fragments of phototrophic bacterium Rhodobacter sphaeroides have been cloned in plasmid pVZ365, a SmRKmR-derivative of R89S. The obtained recombinant plasmids were mobilized into R. sphaeroides cells by the I pcP-group conjugative plasmid R751. The frequencies of the SmR-transconjugants formation are 3.7.10(-5) to 5.6.10(-3) per recipient cell. The formation of the SmR-transconjugants has not been revealed in case of the plasmid pVZ365 mobilization. The recombinant molecules containing R. sphaeroides plasmid fragments have been shown to integrate into endogenous plasmids and form cointegrates with them.     

Expression of the gltP gene of Escherichia coli in a glutamate transport-deficient mutant of Rhodobacter sphaeroides restores chemotaxis to glutamate

Rhodobacter sphaeroides is chemotactic to glutamate and most other amino acids. In Escherichia coli , chemotaxis involves a membrane-bound sensor that either binds the amino acid directly or interacts with the binding protein loaded with the amino acid. In R. sphaeroides , chemotaxis is thought to require both the uptake and the metabolism of the amino acid. Glutamate is accumulated by the cells via a binding protein-dependent system. To determine the role of the binding protein and transport in glutamate taxis, mutants were created by Tn 5 insertion mutagenesis and selected for growth in the presence of the toxic glutamine analogue γ-glutamyl-hydrazide. One of the mutants, R. sphaeroides MJ7, was defective in glutamate uptake but showed wild-type levels of binding protein. The mutant showed no chemotactic response to glutamate. Both glutamate uptake and chemotaxis were recovered when the gltP gene, coding for the H⁺-linked glutamate carrier of E. coli , was expressed in R. sphaeroides MJ7. It is concluded that the chemotactic response to glutamate strictly requires uptake of glutamate, supporting the view that intracellular metabolism is needed for chemotaxis in R. sphaeroides .     

强脉冲光治疗鲜红斑痣的血管能量分布模拟

根据鲜红斑痣的病理特点,建立分层模型中包含分立血管的皮肤模型,以血管为主要研究对象,用蒙特卡罗的模拟方法模拟在强脉冲光系统治疗鲜红斑痣中,血管中光能量密度的分布规律.结果显示与单波长激光相比,强脉冲光照射下血管中光能量的分布更加均匀,在光束传输方向上能量梯度更平坦;且血管中能量极大值更小.     

The resonance Raman spectrum of carotenoids as an intrinsic probe for membrane potential. Oscillatory changes in the spectrum of neurosporene in the chromatophores of Rhodopseudomonas sphaeroides.

The resonance Raman spectrum of the carotenoid neurosporene is shown to be a sensitive monitor of absorption shifts, and thus changes in membrane potential, in chromatophores of the GlC mutant of Rhodopseudomonas sphaeroides. For a Raman excitation wavelength at 472.7 nm, the intensities of the two most prominent resonance Raman features (v1 and v2) respond very differently to small shifts in the absorption maxima. Thus, the ratio intensity v1/intensity v2 is a sensitive probe for absorption shifts. Changes in this ratio of approximately 20% were observed during a valinomycin induced diffusion potential. At 5 degrees C changes in the average intensity ratio of +6, -4 and -14% were brought about by oligomycin, FCCP and sodium deoxycholate, respectively. The changes in intensity ratio were temperature dependent and, in addition, effects due to the laser beam acting as an actinic light could be detected. Oscillatory changes were observed in absolute Raman and Rayleigh scattering intensities for chromatophores at 5 degrees C and for intact cells under growing conditions.     

Color-sensitive motility and methanol release responses in Rhodobacter sphaeroides

Blue-light-induced repellent and demethylation responses, characteristic of behavioral adaptation, were observed in Rhodobacter sphaeroides. They were analyzed by computer-assisted motion analysis and through the release of volatile tritiated compounds from [methyl-(3)H]methionine-labeled cells, respectively. Increases in the stop frequency and the rate of methanol release were induced by exposure of cells to repellent light signals, such as an increase in blue- and a decrease in infrared-light intensity. At a lambda of >500 nm the amplitude of the methanol release response followed the absorbance spectrum of the photosynthetic pigments, suggesting that they function as photosensors for this response. In contrast to the previously reported motility response to a decrease in infrared light, the blue-light response reported here does not depend on the number of photosynthetic pigments per cell, suggesting that it is mediated by a separate sensor. Therefore, color discrimination in taxis responses in R. sphaeroides involves two photosensing systems: the photosynthetic pigments and an additional photosensor, responding to blue light. The signal generated by the former system could result in the migration of cells to a light climate beneficial for photosynthesis, while the blue-light system could allow cells to avoid too-high intensities of (harmful) blue light.