Quantum efficiency of native and mutant bacteriorhodopsin obtained from blue light induced relaxation experiments

Bacteriorhodopsin was continuously excited with green background light. In this way a steady state distribution of all intermediates of the photocycle was obtained. Then a perturbation of the system was induced by a blue laser flash and the resulting absorption changes were measured. The experiments were done with native bacteriorhodopsin and with the point mutant BR _Asp96Asn , in which aspartate 96 is changed to asparagine. Blue light induced relaxation experiments revealed a rate constant belonging to the excitation of bacteriorhodopsin by the green background. With this rate constant the quantum efficiency of native bacteriorhodopsin and of BR _Asp96Asn was determined to be 0.60 ± 0.10. Signals obtained with native bacteriorhodopsin could be explained with a simple model of the photocycle consisting of three consecutive intermediates BR ₅₆₈, L ₅₅₀ and M ₄₁₂. To describe the behavior of BR _Asp96Asn , a further photoactive intermediate after the M ₄₁₂ state had to be postulated. Properties of this intermediate are similar to those of the N ₅₅₀ state.     

Selective enrichment and characterization of Roseospirillum parvum, gen. nov. and sp. nov., a new purple nonsulfur bacterium with unusual light absorption properties

A new type of phototrophic purple bacterium, strain 930I, was isolated from a microbial mat covering intertidal sandy sediments of Great Sippewissett Salt Marsh (Woods Hole, Mass., USA). The bacterium could only be enriched at a wavelength of 932 (± 10) nm. Cells were vibrioid- to spirilloid-shaped and motile by means of bipolar monotrichous flagellation. The intracytoplasmic membranes were of the lamellar type. Photosynthetic pigments comprised bacteriochlorophyll a and the carotenoids spirilloxanthin and lycopenal. The isolated strain exhibited an unusual, long-wavelength absorption maximum at 911 nm. Sulfide or thiosulfate served as electron donor for anoxygenic phototrophic growth. During growth on sulfide, elemental sulfur globules formed outside the cells. Elemental sulfur could not be further oxidized to sulfate. In the presence of sulfide plus bicarbonate, fructose, acetate, propionate, butyrate, valerate, 2-oxoglutarate, pyruvate, lactate, malate, succinate, fumarate, malonate, casamino acids, yeast extract, L(+)-alanine, and L(+)-glutamate were assimilated. Sulfide, thiosulfate, or elemental sulfur served as a reduced sulfur source for photosynthetic growth. Maximum growth rates were obtained at pH 7.9, 30 °C, 50 μmol quanta m^–2 s^–1 of daylight fluorescent tubes, and a salinity of 1–2% NaCl. The strain grew microaerophilically in the dark at a partial pressure of 1 kPa O₂. The DNA base composition was 71.2 mol% G + C. Sequence comparison of 16S rRNA genes indicated that the isolate is a member of the α-Proteobacteria and is most closely related to Rhodobium orientis at a similarity level of 93.5%. Because of the large phylogenetic distance to known phototrophic species of the α-Proteobacteria and of its unique absorption spectrum, strain 930I is described as a new genus and species, Roseospirillum parvum gen. nov. and sp. nov. Received: 29 December 1998 / Accepted: 17 March 1999     

Influence of stray capacitance and sample resistance on the kinetics of fast photovoltages from oriented purple membranes

Flash-induced photovoltages were measured with metal electrodes in two experimental systems of purple membranes oriented by an electric field. One system consisted of a suspension of purple membranes cooled to 80 K. The photovoltage evoked by a xenon flash lamp displayed a single phase with a fast rise and a slow RC-decay. The signal shape is consistent with a fast charge separation occurring before the decay of the K-intermediate. The other system consisted of purple membranes embedded and stabilized in polyacrylamide gel. At room temperature, the photovoltage, evoked by a 10 ns laser flash, displayed a negative phase in the submicrosecond range and a slower positive one. The shape of the signals were altered in a complex manner by the stray capacitance and the ionic strength. The rise-time of the negative phase was approx. 14 and approx. 40 ns at ionic strengths of 10 and 1 mM, respectively. The initial peak amplitudes of the photovoltage from both experimental systems depended on the external capacitance in an inverse manner, indicating that both experimental systems were not impedance-matched. The evaluation of kinetic data of molecular reactions from measurements of the photovoltage is discussed.     

Sulfate activation in Rhodobacter sulfidophilus and other species of the Rhodospirillaceae

Rhodobacter sulfidophilus, R. capsulatus, R. sphaeroides, Rhodospirillum rubrum, Rhodopseudomonas palustris, R. viridis and Rhodocyclus gelatinosus were found to be able to synthesize adenylylsulfate and 3-phosphoadenylylsulfate from sulfate and ATP. The presence of ATP sulfurylase was proven for the soluble protein fractions of all these species. ADP sulfurylase was not found. ATP sulfurylase was purified from R. sulfidophilus. Its molecular weight was 290,000. The enzyme is stabilized by magnesium ions and elevated salinities. The optimal pH was 8.0, activity was found between pH 6.8 and 9.4. The enzyme is inactivated at temperatures above 40°C. Kinetic studies resulted in K _m(ATP)=0.26 mM, K _m(sulfate)=0.33 mM; K _i(AMP)-2.1 mM, K _i(ADP)=1.15 mM; K _i(APS)=0.8 M; K _i(sulfite)=0.4. mM; K _i(sulfide)=0.66mM.Uncommon abbreviations APS adenylylsulfate - PAPS 3-phosphoadenylylsulfate - PEP phosphoenolpyruvate Dedicated to Professor Gerhart Drews on the occasion of his 60th birthday     

Association of a new type of gliding,filamentous, purple phototrophic bacterium inside bundles of Microcoleus chthonoplastes in hypersaline cyanobacterial mats

An unidentified filamentous purple bacterium, probably belonging to a new genus or even a new family, is found in close association with the filamentous, mat-forming cyanobacterium Microcoleus chthonoplastes in a hypersaline pond at Guerrero Negro, Baja California Sur, Mexico, and in Solar Lake, Sinai, Egypt. This organism is a gliding, segmented trichome, 0.8–0.9 m wide. It contains intracytoplasmic stacked lamellae which are perpendicular and obliquely oriented to the cell wall, similar to those described for the purple sulfur bacteria Ectothiorhodospira. These bacteria are found inside the cyanobacterial bundle, enclosed by the cyanobacterial sheath. Detailed transmission electron microscopical analyses carried out in horizontal sections of the upper 1.5 mm of the cyanobacterial mat show this cyanobacterial-purple bacterial association at depths of 300–1200 m, corresponding to the zone below that of maximal oxygenic photosynthesis. Sharp gradients of oxygen and sulfide are established during the day at this microzone in the two cyanobacterial mats studied. The close association, the distribution pattern of this association and preliminary physiological experiments suggest a co-metabolism of sulfur by the two-membered community. This probable new genus of purple bacteria may also grow photoheterotrophically using organic carbon excreted by the cyanobacterium. Since the chemical gradients in the entire photic zone fluctuate widely in a diurnal cycle, both types of metabolism probably take place. During the morning and afternoon, sulfide migrates up to the photic zone allowing photoautotrophic metabolism with sulfide as the electron donor. During the day the photic zone is highly oxygenated and the purple bacteria may either use oxidized species of sulfur such as elemental sulfur and thiosulfate in the photoautotrophic mode or grow photoheterotrophically using organic carbon excreted by M. chthonoplastes. The new type of filamentous purple sulfur bacteria is not available yet in pure culture, and its taxonomical position cannot be fully established. This organism is suggested to be a new type of gliding, filamentous, purple phototroph.     

Evidence for chromophore-chromophore interactions in the Purple Membrane from reconstitution experiments of the chromophore-free membrane

We recently presented evidence showing that the visible CD spectrum of the purple membrane from Halobacterium halobium consists of two contributions: a broad positive band centered at the absorption maximum due to the interaction of the chromophore with the protein to which it is bound, and an exciton coupling band due to the interaction between chromophores of adjacent bacteriorhodopsin molecules in the hexagonal surface lattice (Heyn et al., 1975). This interpretation receives strong support from the present experiments in which the chromophore-free membrane is reconstituted by the addition of retinal. Since the coupling signal arises from the interaction between pairs of neighboring chromophores, its contribution to the spectrum would be expected to be very small in the initial stages of the titration experiment, but increasing quadratically with the percentage reconstitution. The broad positive band, on the other hand, is expected to increase linearly with the percentage reconstitution. On the basis of these considerations a satisfactory explanation of the CD reconstitution experiments could be given. Since it appears to be impossible to explain the titration experiments without the quadratic term, we conclude that chromophore-chromophore interactions play an important role. No significant changes in secondary structure upon reconstitution could be detected consistent with our binding model which neglects cooperativity.Abbreviations CD circular dichroism - UV ultraviolet     

Thymidine incorporation in Lake Cisó: Problems in estimating bacterial secondary production across oxic-anoxic interfaces

Abstract Heterotrophic bacterial activity was measured by means of the ³H-thymidine (³H-TdR) incorporation technique in Lake Cisó, a small holomictic lake with anoxic hypolimnion. We tested several methodological questions across the vertical profile: TdR concentration at which maximal incorporation is reached, linearity of incorporation and isotope dilution, during holomixis and stratification periods. The TdR concentration at which maximal incorporation is reached changed seasonally and vertically. During holomixis, maximal incorporation was not always reached at concentrations up to 40 nM. Uptake was always linear in short incubation times and decreased from epi- to hypolimnion. The isotope dilution technique indicated a degree of participation in DNA synthesis higher than 50%, although a linear relationship between the inverse of ³H-TdR incorporation and increasing ‘cold’ thymidine concentration was not always observed. Autoradiographic experiments showed a low percentage of bacteria taking up ³H-TdR in both aerobic and anaerobic samples. The percentage of total labeled bacteria seemed to be generally higher in the metalimnion (11% maximal value) than in the hypolimnion. Labeled Amoebobacter and Chromatium cells were detected in field samples. Amoebobacter cells photoassimilated TdR in culture. Therefore, our results show that ³H-TdR incorporation is not an appropriate technique to estimate bacterial secondary production in anaerobic systems and in oxic-anoxic interfaces.     

Susceptibility of various purple and green sulfur bacteria to different antimicrobial agents

Abstract Several purple and green sulfur bacteria (genera Chromatium, Thiocapsa and Chlorobium ) were tested for their sensitivity to different antimicrobial agents by a disc diffusion assay, using thioacetamide as a source of hydrogen sulfide for plate growth. Chlorobium limicola strains were more sensitive to amoxicillin, erythromycin and nalidixic acid, whereas gentamicin and netilmicin were more active against the purple bacteria tested. None of the organisms were sensitive to oxacillin and trimethoprim + sulfamethoxazole. The critical concentrations at the edge of the inhibition zone were also calculated for three organisms and the antimicrobials colistin, mitomycin C, penicillin G, rifampicin, and streptomycin. The results obtained suggest that colistin, mitomycin C, penicillin G would provide selective conditions against the growth of Chlorobium limicola strains, while streptomycin and other aminoglycoside antibiotics would select against purple bacteria.     

中性红再生紫膜的光化学性质

研究了中性红再生紫膜从先适应状态到暗适应状态的反应及再生紫膜中中性红的光吸收变化。实验结果说明紫膜上的金属离子结合位点可能深入膜内的质子通道,与构成质子通道的一些重要氨基酸残基相互作用。紫膜经去离子化处理变成蓝膜后,带有正电荷的质子化中性红也可以占据此金属离子结合位点,使蓝膜再生为紫膜。但金属离子与结合位点具有更强的亲和力,使蓝膜再生为紫膜的能力比质子化中性红强。