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Sample records for a-proteobacterium rhodobacter sphaeroides

  1. Bioremediation of lead contaminated soil with Rhodobacter sphaeroides.

    PubMed

    Li, Xiaomin; Peng, Weihua; Jia, Yingying; Lu, Lin; Fan, Wenhong

    2016-08-01

    Bioremediation with microorganisms is a promising technique for heavy metal contaminated soil. Rhodobacter sphaeroides was previously isolated from oil field injection water and used for bioremediation of lead (Pb) contaminated soil in the present study. Based on the investigation of the optimum culturing conditions and the tolerance to Pb, we employed the microorganism for the remediation of Pb contaminated soil simulated at different contamination levels. It was found that the optimum temperature, pH, and inoculum size for R. sphaeroides is 30-35 °C, 7, and 2 × 10(8) mL(-1), respectively. Rhodobacter sphaeroides did not remove the Pb from soil but did change its speciation. During the bioremediation process, more available fractions were transformed to less accessible and inert fractions; in particular, the exchangeable phase was dramatically decreased while the residual phase was substantially increased. A wheat seedling growing experiment showed that Pb phytoavailability was reduced in amended soils. Results inferred that the main mechanism by which R. sphaeroides treats Pb contaminated soil is the precipitation formation of inert compounds, including lead sulfate and lead sulfide. Although the Pb bioremediation efficiency on wheat was not very high (14.78% root and 24.01% in leaf), R. sphaeroides remains a promising alternative for Pb remediation in contaminated soil. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Structural Characterization of the Fla2 Flagellum of Rhodobacter sphaeroides

    PubMed Central

    de la Mora, Javier; Uchida, Kaoru; del Campo, Ana Martínez; Camarena, Laura; Aizawa, Shin-Ichi

    2015-01-01

    ABSTRACT Rhodobacter sphaeroides is a free-living alphaproteobacterium that contains two clusters of functional flagellar genes in its genome: one acquired by horizontal gene transfer (fla1) and one that is endogenous (fla2). We have shown that the Fla2 system is normally quiescent and under certain conditions produces polar flagella, while the Fla1 system is always active and produces a single flagellum at a nonpolar position. In this work we purified and characterized the structure and analyzed the composition of the Fla2 flagellum. The number of polar filaments per cell is 4.6 on average. By comparison with the Fla1 flagellum, the prominent features of the ultra structure of the Fla2 HBB are the absence of an H ring, thick and long hooks, and a smoother zone at the hook-filament junction. The Fla2 helical filaments have a pitch of 2.64 μm and a diameter of 1.4 μm, which are smaller than those of the Fla1 filaments. Fla2 filaments undergo polymorphic transitions in vitro and showed two polymorphs: curly (right-handed) and coiled. However, in vivo in free-swimming cells, we observed only a bundle of filaments, which should probably be left-handed. Together, our results indicate that Fla2 cell produces multiple right-handed polar flagella, which are not conventional but exceptional. IMPORTANCE R. sphaeroides possesses two functional sets of flagellar genes. The fla1 genes are normally expressed in the laboratory and were acquired by horizontal transfer. The fla2 genes are endogenous and are expressed in a Fla1− mutant grown phototrophically and in the absence of organic acids. The Fla1 system produces a single lateral or subpolar flagellum, and the Fla2 system produces multiple polar flagella. The two kinds of flagella are never expressed simultaneously, and both are used for swimming in liquid media. The two sets of genes are certainly ready for responding to specific environmental conditions. The characterization of the Fla2 system will help us to understand

  3. The two photocycles of photoactive yellow protein from Rhodobacter sphaeroides.

    PubMed

    Haker, Andrea; Hendriks, Johnny; van Stokkum, Ivo H M; Heberle, Joachim; Hellingwerf, Klaas J; Crielaard, Wim; Gensch, Thomas

    2003-03-07

    The absorption spectrum of the photoactive yellow protein from Rhodobacter sphaeroides (R-PYP) shows two maxima, absorbing at 360 nm (R-PYP(360)) and 446 nm (R-PYP(446)), respectively. Both forms are photoactive and part of a temperature- and pH-dependent equilibrium (Haker, A., Hendriks, J., Gensch, T., Hellingwerf, K. J., and Crielaard, W. (2000) FEBS Lett. 486, 52-56). At 20 degrees C, for PYP characteristic, the 446-nm absorbance band displays a photocycle, in which the depletion of the 446-nm ground state absorption occurs in at least three phases, with time constants of <30 ns, 0.5 micros, and 17 micros. Intermediates with both blue- and red-shifted absorption maxima are transiently formed, before a blue-shifted intermediate (pB(360), lambda(max) = 360 nm) is established. The photocycle is completed with a monophasic recovery of the ground state with a time constant of 2.5 ms. At 7 degrees C these photocycle transitions are slowed down 2- to 3-fold. Upon excitation of R-PYP(360) with a UV-flash (330 +/- 50 nm) a species with a difference absorption maximum at approximately 435 nm is observed that returns to R-PYP(360) on a minute time scale. Recovery can be accelerated by a blue light flash (450 nm). R-PYP(360) and R-PYP(446) differ in their overall protein conformation, as well as in the isomerization and protonation state of the chromophore, as determined with the fluorescent polarity probe Nile Red and Fourier Transform Infrared spectroscopy, respectively.

  4. Hydrogen Production by Co-cultures of Rhizopus oryzae and a Photosynthetic Bacterium, Rhodobacter sphaeroides RV

    NASA Astrophysics Data System (ADS)

    Asada, Yasuo; Ishimi, Katsuhiro; Nagata, Yoko; Wakayama, Tatsuki; Miyake, Jun; Kohno, Hideki

    Hydrogen production with glucose by using co-immobilized cultures of a fungus, Rhizopus oryzae NBRC5384, and a photosynthetic bacterium, Rhodobacter sphaeroides RV, in agar gels was studied. The co-immobilized cultures converted glucose to hydrogen via lactate in a high molar yield of about 8moles of hydrogen per glucose at a maximum under illuminated conditions.

  5. DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1.

    PubMed

    Choudhary, M; Kaplan, S

    2000-02-15

    This paper describes the DNA sequence of the photosynthesis region of Rhodobacter sphaeroides 2.4.1 (T). The photosynthesis gene cluster is located within a approximately 73 kb Ase I genomic DNA fragment containing the puf, puhA, cycA and puc operons. A total of 65 open reading frames (ORFs) have been identified, of which 61 showed significant similarity to genes/proteins of other organisms while only four did not reveal any significant sequence similarity to any gene/protein sequences in the database. The data were compared with the corresponding genes/ORFs from a different strain of R.sphaeroides and Rhodobacter capsulatus, a close relative of R. sphaeroides. A detailed analysis of the gene organization in the photosynthesis region revealed a similar gene order in both species with some notable differences located to the pucBAC = cycA region. In addition, photosynthesis gene regulatory protein (PpsR, FNR, IHF) binding motifs in upstream sequences of a number of photosynthesis genes have been identified and shown to differ between these two species. The difference in gene organization relative to pucBAC and cycA suggests that this region originated independently of the photosynthesis gene cluster of R.sphaeroides.

  6. Gene co-expression network analysis in Rhodobacter capsulatus and application to comparative expression analysis of Rhodobacter sphaeroides

    SciTech Connect

    Pena-Castillo, Lourdes; Mercer, Ryan; Gurinovich, Anastasia

    2014-08-28

    The genus Rhodobacter contains purple nonsulfur bacteria found mostly in freshwater environments. Representative strains of two Rhodobacter species, R. capsulatus and R. sphaeroides, have had their genomes fully sequenced and both have been the subject of transcriptional profiling studies. Gene co-expression networks can be used to identify modules of genes with similar expression profiles. Functional analysis of gene modules can then associate co-expressed genes with biological pathways, and network statistics can determine the degree of module preservation in related networks. In this paper, we constructed an R. capsulatus gene co-expression network, performed functional analysis of identified gene modules, and investigatedmore » preservation of these modules in R. capsulatus proteomics data and in R. sphaeroides transcriptomics data. Results: The analysis identified 40 gene co-expression modules in R. capsulatus. Investigation of the module gene contents and expression profiles revealed patterns that were validated based on previous studies supporting the biological relevance of these modules. We identified two R. capsulatus gene modules preserved in the protein abundance data. We also identified several gene modules preserved between both Rhodobacter species, which indicate that these cellular processes are conserved between the species and are candidates for functional information transfer between species. Many gene modules were non-preserved, providing insight into processes that differentiate the two species. In addition, using Local Network Similarity (LNS), a recently proposed metric for expression divergence, we assessed the expression conservation of between-species pairs of orthologs, and within-species gene-protein expression profiles. Conclusions: Our analyses provide new sources of information for functional annotation in R. capsulatus because uncharacterized genes in modules are now connected with groups of genes that constitute a joint functional

  7. Biosorption of heavy metal ions on Rhodobacter sphaeroides and Alcaligenes eutrophus H16

    SciTech Connect

    Seki, Hideshi; Suzuki, Akira; Mitsueda, Shinichiro

    1998-01-15

    A fundamental study of the application of bacteria to the recovery of toxic heavy metals from aqueous environments was carried out. The biosorption characteristics of cadmium and lead ions were determined with purple nonsulfur bacteria, Rhodobacter sphaeroides and hydrogen bacteria, Alcaligenes eutrophus H16 that were inactivated by steam sterilization. A simplified version of the metal binding model proposed by Plette et al. was used for the description of meal binding data. The results showed that the biosorption of bivalent metal ions to whole cell bodies of the bacteria was due to monodentate binding to two different types of acidic sites:more » carboxilic and phosphatic-type sites. The number of metal binding sites of A. eutrophus was 2.4-fold larger than that of R. sphaeroides.« less

  8. Phytoavailability and geospeciation of cadmium in contaminated soil remediated by Rhodobacter sphaeroides.

    PubMed

    Fan, Wenhong; Jia, Yingying; Li, Xiaomin; Jiang, Wei; Lu, Lin

    2012-07-01

    A microorganism was isolated from oil field injection water and identified as Rhodobacter sphaeroides. It was used for the remediation of simulated cadmium-contaminated soil. The phytoavailability of Cd was investigated through wheat seedling method to determine the efficiency of remediation. It was found that after remediation, the accumulation of Cd in wheat roots and leaves decreased by 67% and 53%, respectively. The Cd speciation in soil was determined with Tessier extraction procedure. It was found that the total Cd content in soil did not change during the experiments, but the geo-speciation of Cd changed remarkably. Among the five fractions, the concentration of exchangeable phases decreased by 27-46% and that of the phases bound to Fe-Mn oxides increased by 22-44%. The decrease of Cd accumulation in wheat showed significant positive correlation with the decrease of exchangeable phases. It could be concluded that the remediation of R. sphaeroides was carried out through the conversion of Cd to more stable forms. The decrease of sulfate concentration in supernatant indicated that the R. sphaeroides consumed sulfate. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Cloning and heterologous expression of chlorophyll a synthase in Rhodobacter sphaeroides.

    PubMed

    Ipekoğlu, Emre M; Göçmen, Koray; Öz, Mehmet T; Gürgan, Muazzez; Yücel, Meral

    2017-03-01

    Rhodobacter sphaeroides is a purple non-sulfur bacterium which photoheterotrophically produces hydrogen from organic acids under anaerobic conditions. A gene coding for putative chlorophyll a synthase (chlG) from cyanobacterium Prochlorococcus marinus was amplified by nested polymerase chain reaction and cloned into an inducible-expression plasmid which was subsequently transferred to R. sphaeroides for heterologous expression. Induced expression of chlG in R. sphaeroides led to changes in light absorption spectrum within 400-700 nm. The hydrogen production capacity of the mutant strain was evaluated on hydrogen production medium with 15 mM malate and 2 mM glutamate. Hydrogen yield and productivity were increased by 13.6 and 22.6%, respectively, compared to the wild type strain. The results demonstrated the feasibility of genetic engineering to combine chlorophyll and bacteriochlorophyll biosynthetic pathways which utilize common intermediates. Heterologous expression of key enzymes from biosynthetic pathways of various pigments is proposed here as a general strategy to improve absorption spectra and yield of photosynthesis and hydrogen gas production in bacteria. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Bioremediation of cadmium- and zinc-contaminated soil using Rhodobacter sphaeroides.

    PubMed

    Peng, Weihua; Li, Xiaomin; Song, Jingxiang; Jiang, Wei; Liu, Yingying; Fan, Wenhong

    2018-04-01

    Bioremediation using microorganisms is a promising technique to remediate soil contaminated with heavy metals. In this study, Rhodobacter sphaeroides was used to bioremediate soils contaminated with cadmium (Cd) and zinc (Zn). The study found that the treatment reduced the overall bioavailable fractions (e.g., exchangeable and carbonate bound phases) of Cd and Zn. More stable fractions (e.g., Fe-Mn oxide, organic bound, and residual phases (only for Zn)) increased after bioremediation. A wheat seedling experiment revealed that the phytoavailability of Cd was reduced after bioremediation using R. sphaeroides. After bioremediation, the exchangeable phases of Cd and Zn in soil were reduced by as much as 30.7% and 100.0%, respectively; the Cd levels in wheat leaf and root were reduced by as much as 62.3% and 47.2%, respectively. However, when the soils were contaminated with very high levels of Cd and Zn (Cd 54.97-65.33 mg kg -1 ; Zn 813.4-964.8 mg kg -1 ), bioremediation effects were not clear. The study also found that R. sphaeroides bioremediation in soil can enhance the Zn/Cd ratio in the harvested wheat leaf and root overall. This indicates potentially favorable application in agronomic practice and biofortification. Although remediation efficiency in highly contaminated soil was not significant, R. sphaeroides may be potentially and practically applied to the bioremediation of soils co-contaminated by Cd and Zn. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Timescales of Coherent Dynamics in the Light Harvesting Complex 2 (LH2) of Rhodobacter sphaeroides.

    PubMed

    Fidler, Andrew F; Singh, Ved P; Long, Phillip D; Dahlberg, Peter D; Engel, Gregory S

    2013-05-02

    The initial dynamics of energy transfer in the light harvesting complex 2 from Rhodobacter sphaeroides were investigated with polarization controlled two-dimensional spectroscopy. This method allows only the coherent electronic motions to be observed revealing the timescale of dephasing among the excited states. We observe persistent coherence among all states and assign ensemble dephasing rates for the various coherences. A simple model is utilized to connect the spectroscopic transitions to the molecular structure, allowing us to distinguish coherences between the two rings of chromophores and coherences within the rings. We also compare dephasing rates between excited states to dephasing rates between the ground and excited states, revealing that the coherences between excited states dephase on a slower timescale than coherences between the ground and excited states.

  12. Time-resolved tryptophan fluorescence in photosynthetic reaction centers from Rhodobacter sphaeroides

    NASA Technical Reports Server (NTRS)

    Godik, V. I.; Blankenship, R. E.; Causgrove, T. P.; Woodbury, N.

    1993-01-01

    Tryptophan fluorescence of reaction centers isolated from Rhodobacter sphaeroides, both stationary and time-resolved, was studied. Fluorescence kinetics were found to fit best a sum of four discrete exponential components. Half of the initial amplitude was due to a component with a lifetime of congruent to 60 ps, belonging to Trp residues, capable of efficient transfer of excitation energy to bacteriochlorophyll molecules of the reaction center. The three other components seem to be emitted by Trp ground-state conformers, unable to participate in such a transfer. Under the influence of intense actinic light, photooxidizing the reaction centers, the yield of stationary fluorescence diminished by congruent to 1.5 times, while the number of the kinetic components and their life times remained practically unchanged. Possible implications of the observed effects for the primary photosynthesis events are considered.

  13. Modelling and analysis of bacterial tracks suggest an active reorientation mechanism in Rhodobacter sphaeroides

    PubMed Central

    Rosser, Gabriel; Baker, Ruth E.; Armitage, Judith P.; Fletcher, Alexander G.

    2014-01-01

    Most free-swimming bacteria move in approximately straight lines, interspersed with random reorientation phases. A key open question concerns varying mechanisms by which reorientation occurs. We combine mathematical modelling with analysis of a large tracking dataset to study the poorly understood reorientation mechanism in the monoflagellate species Rhodobacter sphaeroides. The flagellum on this species rotates counterclockwise to propel the bacterium, periodically ceasing rotation to enable reorientation. When rotation restarts the cell body usually points in a new direction. It has been assumed that the new direction is simply the result of Brownian rotation. We consider three variants of a self-propelled particle model of bacterial motility. The first considers rotational diffusion only, corresponding to a non-chemotactic mutant strain. Two further models incorporate stochastic reorientations, describing ‘run-and-tumble’ motility. We derive expressions for key summary statistics and simulate each model using a stochastic computational algorithm. We also discuss the effect of cell geometry on rotational diffusion. Working with a previously published tracking dataset, we compare predictions of the models with data on individual stopping events in R. sphaeroides. This provides strong evidence that this species undergoes some form of active reorientation rather than simple reorientation by Brownian rotation. PMID:24872500

  14. Novel Methods for Analysing Bacterial Tracks Reveal Persistence in Rhodobacter sphaeroides

    PubMed Central

    Rosser, Gabriel; Fletcher, Alexander G.; Wilkinson, David A.; de Beyer, Jennifer A.; Yates, Christian A.; Armitage, Judith P.; Maini, Philip K.; Baker, Ruth E.

    2013-01-01

    Tracking bacteria using video microscopy is a powerful experimental approach to probe their motile behaviour. The trajectories obtained contain much information relating to the complex patterns of bacterial motility. However, methods for the quantitative analysis of such data are limited. Most swimming bacteria move in approximately straight lines, interspersed with random reorientation phases. It is therefore necessary to segment observed tracks into swimming and reorientation phases to extract useful statistics. We present novel robust analysis tools to discern these two phases in tracks. Our methods comprise a simple and effective protocol for removing spurious tracks from tracking datasets, followed by analysis based on a two-state hidden Markov model, taking advantage of the availability of mutant strains that exhibit swimming-only or reorientating-only motion to generate an empirical prior distribution. Using simulated tracks with varying levels of added noise, we validate our methods and compare them with an existing heuristic method. To our knowledge this is the first example of a systematic assessment of analysis methods in this field. The new methods are substantially more robust to noise and introduce less systematic bias than the heuristic method. We apply our methods to tracks obtained from the bacterial species Rhodobacter sphaeroides and Escherichia coli. Our results demonstrate that R. sphaeroides exhibits persistence over the course of a tumbling event, which is a novel result with important implications in the study of this and similar species. PMID:24204227

  15. Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides

    DOE PAGES

    Burger, Brian T.; Imam, Saheed; Scarborough, Matthew J.; ...

    2017-06-06

    Rhodobacter sphaeroides is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosyntheticmore » growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for R. sphaeroides. Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species.« less

  16. A cryptochrome-like protein is involved in the regulation of photosynthesis genes in Rhodobacter sphaeroides.

    PubMed

    Hendrischk, Anne-Kathrin; Frühwirth, Sebastian Walter; Moldt, Julia; Pokorny, Richard; Metz, Sebastian; Kaiser, Gebhard; Jäger, Andreas; Batschauer, Alfred; Klug, Gabriele

    2009-11-01

    Blue light receptors belonging to the cryptochrome/photolyase family are found in all kingdoms of life. The functions of photolyases in repair of UV-damaged DNA as well as of cryptochromes in the light-dependent regulation of photomorphogenetic processes and in the circadian clock in plants and animals are well analysed. In prokaryotes, the only role of members of this protein family that could be demonstrated is DNA repair. Recently, we identified a gene for a cryptochrome-like protein (CryB) in the alpha-proteobacterium Rhodobacter sphaeroides. The protein lacks the typical C-terminal extension of cryptochromes, and is not related to the Cry DASH family. Here we demonstrate that CryB binds flavin adenine dinucleotide that can be photoreduced by blue light. CryB binds single-stranded DNA with very high affinity (K(d) approximately 10(-8) M) but double-stranded DNA and single-stranded RNA with far lower affinity (K(d) approximately 10(-6) M). Despite of that, no in vitro repair activity for pyrimidine dimers in single-stranded DNA could be detected. However, we show that CryB clearly affects the expression of genes for pigment-binding proteins and consequently the amount of photosynthetic complexes in R. sphaeroides. Thus, for the first time a role of a bacterial cryptochrome in gene regulation together with a biological function is demonstrated.

  17. The organization of LH2 complexes in membranes from Rhodobacter sphaeroides.

    PubMed

    Olsen, John D; Tucker, Jaimey D; Timney, John A; Qian, Pu; Vassilev, Cvetelin; Hunter, C Neil

    2008-11-07

    The mapping of the photosynthetic membrane of Rhodobacter sphaeroides by atomic force microscopy (AFM) revealed a unique organization of arrays of dimeric reaction center-light harvesting I-PufX (RC-LH1-PufX) core complexes surrounded and interconnected by light-harvesting LH2 complexes (Bahatyrova, S., Frese, R. N., Siebert, C. A., Olsen, J. D., van der Werf, K. O., van Grondelle, R., Niederman, R. A., Bullough, P. A., Otto, C., and Hunter, C. N. (2004) Nature 430, 1058-1062). However, membrane regions consisting solely of LH2 complexes were under-represented in these images because these small, highly curved areas of membrane rendered them difficult to image even using gentle tapping mode AFM and impossible with contact mode AFM. We report AFM imaging of membranes prepared from a mutant of R. sphaeroides, DPF2G, that synthesizes only the LH2 complexes, which assembles spherical intracytoplasmic membrane vesicles of approximately 53 nm diameter in vivo. By opening these vesicles and adsorbing them onto mica to form small, < or =120 nm, largely flat sheets we have been able to visualize the organization of these LH2-only membranes for the first time. The transition from highly curved vesicle to the planar sheet is accompanied by a change in the packing of the LH2 complexes such that approximately half of the complexes are raised off the mica surface by approximately 1 nm relative to the rest. This vertical displacement produces a very regular corrugated appearance of the planar membrane sheets. Analysis of the topographs was used to measure the distances and angles between the complexes. These data are used to model the organization of LH2 complexes in the original, curved membrane. The implications of this architecture for the light harvesting function and diffusion of quinones in native membranes of R. sphaeroides are discussed.

  18. Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway

    SciTech Connect

    Chi, Shuang C.; Mothersole, David J.; Dilbeck, Preston

    Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon-carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N=10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoeamore » agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2'-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC-LH1-PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2'-diketo-spirilloxanthin (15 conjugated CC bonds; N=15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N=9; 94%), spheroidene (N=10; 96%) and spheroidenone (N=11; 95%), whereas intermediate values were measured for lycopene (N=11; 64%), rhodopin (N=11; 62%) and spirilloxanthin (N=13; 39%). In conclusion, the variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis.« less

  19. Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway

    DOE PAGES

    Chi, Shuang C.; Mothersole, David J.; Dilbeck, Preston; ...

    2014-10-27

    Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon-carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N=10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoeamore » agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2'-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC-LH1-PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2'-diketo-spirilloxanthin (15 conjugated CC bonds; N=15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N=9; 94%), spheroidene (N=10; 96%) and spheroidenone (N=11; 95%), whereas intermediate values were measured for lycopene (N=11; 64%), rhodopin (N=11; 62%) and spirilloxanthin (N=13; 39%). In conclusion, the variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis.« less

  20. Free Fe(3+)/Fe(2+) improved the biomass resource recovery and organic matter removal in Rhodobacter sphaeroides purification of sewage.

    PubMed

    Liu, Rijia; Wu, Pan; Lang, Lang; Xu, Changru; Wang, Yanling

    2016-01-01

    The enhancement in biomass production and organic matter removal of Rhodobacter sphaeroides (R. sphaeroides) through iron ions in soybean protein wastewater treatment was investigated. Different dosages of ferric ions were introduced in the reactors under light-anaerobic conditions. Free ferric and ferrous ions in wastewater were formed and their concentrations were the optimal for the growth of R. sphaeroides when the total Fe dosage was 20 mg/L. At the optimal dosage, biomass production (4000 mg/L) and protease activity improved by 50% and 48% when compared to the controls, respectively. The organic matter and protein removal reached above 90% and hydraulic retention time was shortened from 96 to 72 h. A mechanism analysis indicated that iron ions can effectively improve the adenosine triphosphate production, which may furthermore encourage the synthesis of protease and the cellular material.

  1. Connectivity of the intracytoplasmic membrane of Rhodobacter sphaeroides: a functional approach.

    PubMed

    Verméglio, André; Lavergne, Jérôme; Rappaport, Fabrice

    2016-01-01

    The photosynthetic apparatus in the bacterium Rhodobacter sphaeroides is mostly present in intracytoplasmic membrane invaginations. It has long been debated whether these invaginations remain in topological continuity with the cytoplasmic membrane, or form isolated chromatophore vesicles. This issue is revisited here by functional approaches. The ionophore gramicidin was used as a probe of the relative size of the electro-osmotic units in isolated chromatophores, spheroplasts, or intact cells. The decay of the membrane potential was monitored from the electrochromic shift of carotenoids. The half-time of the decay induced by a single channel in intact cells was about 6 ms, thus three orders of magnitude slower than in isolated chromatophores. In spheroplasts obtained by lysis of the cell wall, the single channel decay was still slower (~23 ms) and the sensitivity toward the gramicidin concentration was enhanced 1,000-fold with respect to isolated chromatophores. These results indicate that the area of the functional membrane in cells or spheroplasts is about three orders of magnitude larger than that of isolated chromatophores. Intracytoplasmic vesicles, if present, could contribute to at most 10% of the photosynthetic apparatus in intact cells of Rba. sphaeroides. Similar conclusions were obtained from the effect of a ∆pH-induced diffusion potential in intact cells. This caused a large electrochromic response of carotenoids, of similar amplitude as the light-induced change, indicating that most of the system is sensitive to a pH change of the external medium. A single internal membrane and periplasmic space may offer significant advantages concerning renewal of the photosynthetic apparatus and reallocation of the components shared with other bioenergetic pathways.

  2. Construction, expression, and localization of a CycA::PhoA fusion protein in Rhodobacter sphaeroides and Escherichia coli.

    PubMed Central

    Varga, A R; Kaplan, S

    1989-01-01

    We demonstrated the utility of Escherichia coli alkaline phosphatase, encoded by phoA, as a reporter molecule for genetic fusions in Rhodobacter sphaeroides. A portion of the R. sphaeroides cycA gene was fused to phoA, yielding a fusion protein comprising the putative signal sequence and first 10 amino acids of the cytochrome c2 apoprotein joined to the sixth amino acid of alkaline phosphatase. The fusion protein was efficiently transported to the periplasm of R. sphaeroides as determined by enzyme activity, Western immunoblot analysis, and immunogold electron microscopy. We also documented the ability of an R. sphaeroides mutant, RS104, with gross defects in photosynthetic membrane morphology to efficiently recognize and translocate the fusion protein to the periplasmic compartment. The inclusion of 500 base pairs of R. sphaeroides DNA in cis to the cycA structural gene resulted in a 2.5-fold increase in alkaline phosphatase activity in photosynthetically grown cells compared with the activity in aerobically grown cells, demonstrating that the fusion protein is regulated in a manner similar to that of cytochrome c2 regulation. We also constructed two pUC19-based plasmids suitable for the construction of translational fusions to phoA. In these plasmids, translational fusions of phoA to the gene under consideration can be made in all three reading frames, thus facilitating construction and expression of fusion protein systems utilizing phoA. Images PMID:2553661

  3. Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway

    PubMed Central

    Chi, Shuang C.; Mothersole, David J.; Dilbeck, Preston; Niedzwiedzki, Dariusz M.; Zhang, Hao; Qian, Pu; Vasilev, Cvetelin; Grayson, Katie J.; Jackson, Philip J.; Martin, Elizabeth C.; Li, Ying; Holten, Dewey; Neil Hunter, C.

    2015-01-01

    Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon–carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N = 10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2′-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC–LH1–PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2′-diketo-spirilloxanthin (15 conjugated C 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 C bonds; N = 15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N = 9; 94%), spheroidene (N = 10; 96%) and spheroidenone (N = 11; 95%), whereas intermediate values were measured for lycopene (N = 11; 64%), rhodopin (N = 11; 62%) and spirilloxanthin (N = 13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the

  4. Bioremediation of petroleum hydrocarbon contaminated soil by Rhodobacter sphaeroides biofertilizer and plants.

    PubMed

    Jiao, Haihua; Luo, Jinxue; Zhang, Yiming; Xu, Shengjun; Bai, Zhihui; Huang, Zhanbin

    2015-09-01

    Bio-augmentation is a promising technique for remediation of polluted soils. This study aimed to evaluate the bio-augmentation effect of Rhodobacter sphaeroides biofertilizer (RBF) on the bioremediation of total petroleum hydrocarbons (TPH) contaminated soil. A greenhouse pot experiment was conducted over a period of 120 days, three methods for enhancing bio-augmentation were tested on TPH contaminated soils, including single addition RBF, planting, and combining of RBF and three crop species, such as wheat (W), cabbage (C) and spinach (S), respectively. The results demonstrated that the best removal of TPH from contaminated soil in the RBF bio-augmentation rhizosphere soils was found to be 46.2%, 65.4%, 67.5% for W+RBF, C+RBF, S+RBF rhizosphere soils respectively. RBF supply impacted on the microbial community diversity (phospholipid fatty acids, PLFA) and the activity of soil enzymes, such as dehydrogenase (DH), alkaline phosphatase (AP) and urease (UR). There were significant difference among the soil only containing crude oil (CK), W, C and S rhizosphere soils and RBF bio-augmentation soils. Moreover, the changes were significantly distinct depended on crops species. It was concluded that the RBF is a valuable material for improving effect of remediation of TPH polluted soils.

  5. Kinetic and spectroscopic characterization of tungsten-substituted DMSO reductase from Rhodobacter sphaeroides.

    PubMed

    Pacheco, Josué; Niks, Dimitri; Hille, Russ

    2018-03-01

    We have examined the kinetic and spectroscopic properties of a tungsten-substituted form of DMSO reductase from Rhodobacter sphaeroides, an enzyme that normally possesses molybdenum. Partial reduction with sodium dithionite yields a well-resolved W(V) EPR signal of the so-called "high-g split" type that exhibits markedly greater g-anisotropy than the corresponding Mo(V) signal of the native form of the enzyme, with the g values shifted to higher magnetic field by as much as Δg ave  = 0.056. Deuteration of the enzyme confirms that the coupled proton is solvent-exchangeable, allowing us to accurately simulate the tungsten hyperfine coupling. Global curve-fitting analysis of UV/vis absorption spectra observed in the course of the reaction of the tungsten-substituted enzyme with sodium dithionite affords a well-defined absorption spectrum for the W(V) species. Surprisingly, the absorption spectrum for this species exhibits significantly larger molar extinction coefficients than either the reduced or the oxidized spectrum. This spectrum, in conjunction with those for fully oxidized W(VI) and fully reduced W(IV) enzyme, has been used to deconvolute the absorption spectra seen in the course of turnover, in the which enzyme is reacted with sodium dithionite and DMSO, demonstrating that the W(V) is an authentic catalytic intermediate that accumulates to approximately 50% of the total enzyme in the steady state.

  6. Early bacteriopheophytin reduction in charge separation in reaction centers of Rhodobacter sphaeroides.

    PubMed

    Zhu, Jingyi; van Stokkum, Ivo H M; Paparelli, Laura; Jones, Michael R; Groot, Marie Louise

    2013-06-04

    A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and electron transfers among a small number of tetrapyrrole pigments embedded in the interior of a protein. In the purple bacterial reaction center (RC), a highly efficient ultrafast charge separation takes place between a pair of bacteriochlorophylls: an accessory bacteriochlorophyll (B) and bacteriopheophytin (H). In this work, we applied ultrafast spectroscopy in the visible and near-infrared spectral region to Rhodobacter sphaeroides RCs to accurately track the timing of the electron on BA and HA via the appearance of the BA and HA anion bands. We observed an unexpectedly early rise of the HA⁻ band that challenges the accepted simple picture of stepwise electron transfer with 3 ps and 1 ps time constants. The implications for the mechanism of initial charge separation in bacterial RCs are discussed in terms of a possible adiabatic electron transfer step between BA and HA, and the effect of protein conformation on the electron transfer rate. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  7. Redox potential tuning through differential quinone binding in the photosynthetic reaction center of Rhodobacter sphaeroides

    DOE PAGES

    Vermaas, Josh V.; Taguchi, Alexander T.; Dikanov, Sergei A.; ...

    2015-03-03

    Ubiquinone forms an integral part of the electron transport chain in cellular respiration and photosynthesis across a vast number of organisms. Prior experimental results have shown that the photosynthetic reaction center (RC) from Rhodobacter sphaeroides is only fully functional with a limited set of methoxy-bearing quinones, suggesting that specific interactions with this substituent are required to drive electron transport and the formation of quinol. The nature of these interactions has yet to be determined. Through parameterization of a CHARMM-compatible quinone force field and subsequent molecular dynamics simulations of the quinone-bound RC, in this paper we have investigated and characterized themore » interactions of the protein with the quinones in the Q A and Q B sites using both equilibrium simulation and thermodynamic integration. In particular, we identify a specific interaction between the 2-methoxy group of ubiquinone in the Q B site and the amide nitrogen of GlyL225 that we implicate in locking the orientation of the 2-methoxy group, thereby tuning the redox potential difference between the quinones occupying the Q A and Q B sites. Finally, disruption of this interaction leads to weaker binding in a ubiquinone analogue that lacks a 2-methoxy group, a finding supported by reverse electron transfer electron paramagnetic resonance experiments of the Q A–Q B– biradical and competitive binding assays.« less

  8. Absorption and fluorescence spectroscopic characterization of BLUF domain of AppA from Rhodobacter sphaeroides

    NASA Astrophysics Data System (ADS)

    Zirak, P.; Penzkofer, A.; Schiereis, T.; Hegemann, P.; Jung, A.; Schlichting, I.

    2005-08-01

    The BLUF domain of the transcriptional anti-repressor protein AppA from the non-sulfur anoxyphototrophic purple bacterium Rhodobacter sphaeroides was characterized by absorption and emission spectroscopy. The BLUF domain constructs AppA 148 (consisting of amino-acid residues 1-148) and AppA 126 (amino-acid residues 1-126) are investigated. The cofactor of the investigated domains is found to consist of a mixture of the flavins riboflavin, FMN, and FAD. The dark-adapted domains exist in two different active receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF r,f and BLUF r,sl) and a small non-interacting conformation (BLUF nc). The active receptor conformations are transformed to putative signalling states (BLUF s,f and BLUF s,sl) of low fluorescence efficiency and picosecond fluorescence lifetime by blue-light excitation (light-adapted domains). In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 17 min. A quantum yield of signalling state formation of about 25% was determined by intensity dependent transmission measurements. A photo-cycle scheme is presented including photo-induced charge transfer complex formation, charge recombination, and protein binding pocket reorganisation.

  9. Photodynamics of the small BLUF protein BlrB from Rhodobacter sphaeroides.

    PubMed

    Zirak, P; Penzkofer, A; Schiereis, T; Hegemann, P; Jung, A; Schlichting, I

    2006-06-01

    The BLUF protein BlrB from the non-sulphur anoxyphototrophic purple bacterium Rhodobacter sphaeroides is characterized by absorption and emission spectroscopy. BlrB expressed from E. coli binding FAD, FMN, and riboflavin (called BrlB(I)) and recombinant BlrB containing only FAD (called BlrB(II)) are investigated. The dark-adapted proteins exist in two different receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF(r,f) and BLUF(r,sl)). Some of the flavin-cofactor (ca. 8%) is unbound in thermodynamic equilibrium with the bound cofactor. The two receptor conformations are transformed to putative signalling states (BLUF(s,f) and BLUF(s,sl)) of decreased fluorescence efficiency and shortened fluorescence lifetime by blue-light excitation. In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 2s. Quantum yields of signalling state formation of about 90% for BlrB(II) and about 40% for BlrB(I) were determined by intensity dependent transmission measurements. Extended blue-light excitation causes unbound flavin degradation (formation of lumichrome and lumiflavin-derivatives) and bound cofactor conversion to the semiquinone form. The flavin-semiquinone further reduces and the reduced flavin re-oxidizes back in the dark. A photo-dynamics scheme is presented and relevant quantum efficiencies and time constants are determined.

  10. Delipidation of Cytochrome c Oxidase from Rhodobacter sphaeroides Destabilizes its Quaternary Structure

    PubMed Central

    Musatov, Andrej; Varhač, Rastislav; Hosler, Jonathan P.; Sedlák, Erik

    2016-01-01

    Delipidation of detergent-solubilized cytochrome c oxidase isolated from Rhodobacter sphaeroides (Rbs-CcO) has no apparent structural and/or functional effect on the protein, however affects its resistance against thermal or chemical denaturation. Phospholipase A2 (PLA2) hydrolysis of phospholipids that are co-purified with the enzyme removes all but two tightly bound phosphatidylethanolamines. Replacement of the removed phospholipids with nonionic detergent decreases both thermal stability of the enzyme and its resilience against the effect of chemical denaturants such as urea. In contrast to nondelipidated Rbs-CcO, the enzymatic activity of PLA2-treated Rbs-CcO is substantially diminished after exposure to high (>4M) urea concentration at room temperature without an alteration of its secondary structure. Absorbance spectroscopy and sedimentation velocity experiments revealed a strong correlation between intact tertiary structure of heme regions and quaternary structure, respectively, and the enzymatic activity of the protein. We concluded that phospholipid environment of Rbs-CcO has the protective role for stability of its tertiary and quaternary structures. PMID:26923069

  11. Redox potential tuning through differential quinone binding in the photosynthetic reaction center of Rhodobacter sphaeroides.

    PubMed

    Vermaas, Josh V; Taguchi, Alexander T; Dikanov, Sergei A; Wraight, Colin A; Tajkhorshid, Emad

    2015-03-31

    Ubiquinone forms an integral part of the electron transport chain in cellular respiration and photosynthesis across a vast number of organisms. Prior experimental results have shown that the photosynthetic reaction center (RC) from Rhodobacter sphaeroides is only fully functional with a limited set of methoxy-bearing quinones, suggesting that specific interactions with this substituent are required to drive electron transport and the formation of quinol. The nature of these interactions has yet to be determined. Through parameterization of a CHARMM-compatible quinone force field and subsequent molecular dynamics simulations of the quinone-bound RC, we have investigated and characterized the interactions of the protein with the quinones in the Q(A) and Q(B) sites using both equilibrium simulation and thermodynamic integration. In particular, we identify a specific interaction between the 2-methoxy group of ubiquinone in the Q(B) site and the amide nitrogen of GlyL225 that we implicate in locking the orientation of the 2-methoxy group, thereby tuning the redox potential difference between the quinones occupying the Q(A) and Q(B) sites. Disruption of this interaction leads to weaker binding in a ubiquinone analogue that lacks a 2-methoxy group, a finding supported by reverse electron transfer electron paramagnetic resonance experiments of the Q(A)⁻Q(B)⁻ biradical and competitive binding assays.

  12. An Extract of Rhodobacter sphaeroides Reduces Cisplatin-Induced Nephrotoxicity in Mice

    PubMed Central

    Chang, Wen-Wei; Liu, Jau-Jin; Liu, Chi-Fan; Liu, Wen-Sheng; Lim, Yun-Ping; Cheng, Yu-Jung; Lee, Che-Hsin

    2013-01-01

    Cisplatin is used as a treatment for various types of solid tumors. Renal injury severely limits the use of cisplatin. Renal cell apoptosis, oxidative stress, and inflammation contribute to cisplatin-induced nephrotoxicity. Previously, we found that an extract of Rhodobacter sphaeroides (Lycogen™) inhibited proinflammatory cytokines and the production of nitric oxide in activated macrophages in a dextran sodium sulfate (DSS)-induced colitis model. Here, we evaluated the effect of Lycogen™, a potent anti-inflammatory agent, in mice with cisplatin-induced renal injury. We found that attenuated renal injury correlated with decreased apoptosis due to a reduction in caspase-3 expression in renal cells. Oral administration of Lycogen™ significantly reduced the expression of tumor necrosis factor-α and interleukin-1β in mice with renal injury. Lycogen™ reduces renal dysfunction in mice with cisplatin-induced renal injury. The protective effects of the treatment included blockage of the cisplatin-induced elevation in serum urea nitrogen and creatinine. Meanwhile, Lycogen™ attenuated body weight loss and significantly prolonged the survival of mice with renal injury. We propose that Lycogen™ exerts anti-inflammatory activities that represent a promising strategy for the treatment of cisplatin-induced renal injury. PMID:24335753

  13. Spectral and kinetic effects accompanying the assembly of core complexes of Rhodobacter sphaeroides.

    PubMed

    Freiberg, Arvi; Chenchiliyan, Manoop; Rätsep, Margus; Timpmann, Kõu

    2016-11-01

    In the present work, spectral and kinetic changes accompanying the assembly of the light-harvesting 1 (LH1) complex with the reaction center (RC) complex into monomeric RC-LH1 and dimeric RC-LH1-PufX core complexes of the photosynthetic purple bacterium Rhodobacter sphaeroides are systematically studied over the temperature range of 4.5-300K. The samples were interrogated with a combination of optical absorption, hole burning, fluorescence excitation, steady state and picosecond time resolved fluorescence spectroscopy. Fair additivity of the LH1 and RC absorption spectra suggests rather weak electronic coupling between them. A low-energy tail revealed at cryogenic temperatures in the absorption spectra of both monomeric and dimeric core complexes is proved to be due to the special pair of the RC. At selected excitation intensity and temperature, the fluorescence decay time of core complexes is shown to be a function of multiple factors, most importantly of the presence/absence of RCs, the supramolecular architecture (monomeric or dimeric) of the complexes, and whether the complexes were studied in a native membrane environment or in a detergent - purified state. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization.

    PubMed

    Wang, Hualei; Li, Guinan; Li, Mingyang; Wei, Dongzhi; Wang, Xuedong

    2014-01-01

    In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli. The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography. This nitrilase showed one single band on SDS-PAGE with a molecular weight of 40 kDa. This suggested that the native nitrilase consisted of 14 subunits with identical size. The optimal pH and temperature of the purified enzyme were 7.0 and 40 °C, respectively. The kinetic parameters V max and K m toward 3-cyanopyridine were 77.5 μmol min(-1) mg(-1) and 73.1 mmol/l, respectively. The enzyme can easily convert aliphatic nitrile and aromatic nitriles to their corresponding acids. Furthermore, this enzyme demonstrated regioselectivity in hydrolysis of aliphatic dinitriles. This specific characteristic makes this nitrilase have a great potential for commercial production of various cyanocarboxylic acids by hydrolyzing readily available dinitriles.

  15. A new nitrilase-producing strain named Rhodobacter sphaeroides LHS-305: biocatalytic characterization and substrate specificity.

    PubMed

    Yang, Chunsheng; Wang, Xuedong; Wei, Dongzhi

    2011-12-01

    The characteristics of the new nitrilase-producing strain Rhodobacter sphaeroides LHS-305 were investigated. By investigating several parameters influencing nitrilase production, the specific cell activity was ultimately increased from 24.5 to 75.0 μmol g(-1) min(-1), and hereinto, the choice of inducer proved the most important factor. The aromatic nitriles (such as 3-cyanopyridine and benzonitrile) were found to be the most favorable substrates of the nitrilase by analyzing the substrate spectrum. It was speculated that the unsaturated carbon atom attached to the cyano group was crucial for this type of nitrilase. The value of apparent K (m), substrate inhibition constant, and product inhibition constant of the nitrilase against 3-cyanopyridine were 4.5 × 10(-2), 29.2, and 8.6 × 10(-3) mol L(-1), respectively. When applied in nicotinic acid preparation, the nitrilase is able to hydrolyze 200 mmol L(-1) 3-cyanopyridine with 93% conversion rate in 13 h by 6.1 g L(-1) cells (dry cell weight).

  16. Native Mass Spectrometry Characterizes the Photosynthetic Reaction Center Complex from the Purple Bacterium Rhodobacter sphaeroides

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Harrington, Lucas B.; Lu, Yue; Prado, Mindy; Saer, Rafael; Rempel, Don; Blankenship, Robert E.; Gross, Michael L.

    2017-01-01

    Native mass spectrometry (MS) is an emerging approach to study protein complexes in their near-native states and to elucidate their stoichiometry and topology. Here, we report a native MS study of the membrane-embedded reaction center (RC) protein complex from the purple photosynthetic bacterium Rhodobacter sphaeroides. The membrane-embedded RC protein complex is stabilized by detergent micelles in aqueous solution, directly introduced into a mass spectrometer by nano-electrospray (nESI), and freed of detergents and dissociated in the gas phase by collisional activation. As the collision energy is increased, the chlorophyll pigments are gradually released from the RC complex, suggesting that native MS introduces a near-native structure that continues to bind pigments. Two bacteriochlorophyll a pigments remain tightly bound to the RC protein at the highest collision energy. The order of pigment release and their resistance to release by gas-phase activation indicates the strength of pigment interaction in the RC complex. This investigation sets the stage for future native MS studies of membrane-embedded photosynthetic pigment-protein and related complexes.

  17. Effect of high pressure on the photochemical reaction center from Rhodobacter sphaeroides R26.1.

    PubMed Central

    Gall, A; Ellervee, A; Bellissent-Funel, M C; Robert, B; Freiberg, A

    2001-01-01

    High-pressure studies on the photochemical reaction center from the photosynthetic bacterium Rhodobacter sphaeroides, strain R26.1, shows that, up to 0.6 GPa, this carotenoid-less membrane protein does not loose its three-dimensional structure at room temperature. However, as evidenced by Fourier-transform preresonance Raman and electronic absorption spectra, between the atmospheric pressure and 0.2 GPa, the structure of the bacterial reaction center experiences a number of local reorganizations in the binding site of the primary electron donor. Above that value, the apparent compressibility of this membrane protein is inhomogeneous, being most noticeable in proximity to the bacteriopheophytin molecules. In this elevated pressure range, no more structural reorganization of the primary electron donor binding site can be observed. However, its electronic structure becomes dramatically perturbed, and the oscillator strength of its Q(y) electronic transition drops by nearly one order of magnitude. This effect is likely due to very small, pressure-induced changes in its dimeric structure. PMID:11222309

  18. Structural and genetic analysis of a mutant of Rhodobacter sphaeroides WS8 deficient in hook length control.

    PubMed Central

    González-Pedrajo, B; Ballado, T; Campos, A; Sockett, R E; Camarena, L; Dreyfus, G

    1997-01-01

    Motility in the photosynthetic bacterium Rhodobacter sphaeroides is achieved by the unidirectional rotation of a single subpolar flagellum. In this study, transposon mutagenesis was used to obtain nonmotile flagellar mutants from this bacterium. We report here the isolation and characterization of a mutant that shows a polyhook phenotype. Morphological characterization of the mutant was done by electron microscopy. Polyhooks were obtained by shearing and were used to purify the hook protein monomer (FlgE). The apparent molecular mass of the hook protein was 50 kDa. N-terminal amino acid sequencing and comparisons with the hook proteins of other flagellated bacteria indicated that the Rhodobacter hook protein has consensus sequences common to axial flagellar components. A 25-kb fragment from an R. sphaeroides WS8 cosmid library restored wild-type flagellation and motility to the mutant. Using DNA adjacent to the inserted transposon as a probe, we identified a 4.6-kb SalI restriction fragment that contained the gene responsible for the polyhook phenotype. Nucleotide sequence analysis of this region revealed an open reading frame with a deduced amino acid sequence that was 23.4% identical to that of FliK of Salmonella typhimurium, the polypeptide responsible for hook length control in that enteric bacterium. The relevance of a gene homologous to fliK in the uniflagellated bacterium R. sphaeroides is discussed. PMID:9352903

  19. Multi-PAS domain-mediated protein oligomerization of PpsR from Rhodobacter sphaeroides

    SciTech Connect

    Heintz, Udo; Meinhart, Anton; Winkler, Andreas, E-mail: andreas.winkler@mpimf-heidelberg.mpg.de

    2014-03-01

    Crystal structures of two truncated variants of the transcription factor PpsR from R. sphaeroides are presented that enabled the phasing of a triple PAS domain construct. Together, these structures reveal the importance of α-helical PAS extensions for multi-PAS domain-mediated protein oligomerization and function. Per–ARNT–Sim (PAS) domains are essential modules of many multi-domain signalling proteins that mediate protein interaction and/or sense environmental stimuli. Frequently, multiple PAS domains are present within single polypeptide chains, where their interplay is required for protein function. Although many isolated PAS domain structures have been reported over the last decades, only a few structures of multi-PAS proteinsmore » are known. Therefore, the molecular mechanism of multi-PAS domain-mediated protein oligomerization and function is poorly understood. The transcription factor PpsR from Rhodobacter sphaeroides is such a multi-PAS domain protein that, in addition to its three PAS domains, contains a glutamine-rich linker and a C-terminal helix–turn–helix DNA-binding motif. Here, crystal structures of two N-terminally and C-terminally truncated PpsR variants that comprise a single (PpsR{sub Q-PAS1}) and two (PpsR{sub N-Q-PAS1}) PAS domains, respectively, are presented and the multi-step strategy required for the phasing of a triple PAS domain construct (PpsR{sub ΔHTH}) is illustrated. While parts of the biologically relevant dimerization interface can already be observed in the two shorter constructs, the PpsR{sub ΔHTH} structure reveals how three PAS domains enable the formation of multiple oligomeric states (dimer, tetramer and octamer), highlighting that not only the PAS cores but also their α-helical extensions are essential for protein oligomerization. The results demonstrate that the long helical glutamine-rich linker of PpsR results from a direct fusion of the N-cap of the PAS1 domain with the C-terminal extension of the N

  20. Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences.

    PubMed Central

    Sockett, R E; Donohue, T J; Varga, A R; Kaplan, S

    1989-01-01

    A reaction center H- strain (RCH-) of Rhodobacter sphaeroides, PUHA1, was made by in vitro deletion of an XhoI restriction endonuclease fragment from the puhA gene coupled with insertion of a kanamycin resistance gene cartridge. The resulting construct was delivered to R. sphaeroides wild-type 2.4.1, with the defective puhA gene replacing the wild-type copy by recombination, followed by selection for kanamycin resistance. When grown under conditions known to induce intracytoplasmic membrane development, PUHA1 synthesized a pigmented intracytoplasmic membrane. Spectral analysis of this membrane showed that it was deficient in B875 spectral complexes as well as functional reaction centers and that the level of B800-850 spectral complexes was greater than in the wild type. The RCH- strain was photosythetically incompetent, but photosynthetic growth was restored by complementation with a 1.45-kilobase (kb) BamHI restriction endonuclease fragment containing the puhA gene carried in trans on plasmid pRK404. B875 spectral complexes were not restored by complementation with the 1.45-kb BamHI restriction endonuclease fragment containing the puhA gene but were restored along with photosynthetic competence by complementation with DNA from a cosmid carrying the puhA gene, as well as a flanking DNA sequence. Interestingly, B875 spectral complexes, but not photosynthetic competence, were restored to PUHA1 by introduction in trans of a 13-kb BamHI restriction endonuclease fragment carrying genes encoding the puf operon region of the DNA. The effect of the puhA deletion was further investigated by an examination of the levels of specific mRNA species derived from the puf and puc operons, as well as by determinations of the relative abundances of polypeptides associated with various spectral complexes by immunological methods. The roles of puhA and other genetic components in photosynthetic gene expression and membrane assembly are discussed. Images PMID:2644200

  1. Ultrafast Electron Transfer Kinetics in the LM Dimer of Bacterial Photosynthetic Reaction Center from Rhodobacter sphaeroides.

    PubMed

    Sun, Chang; Carey, Anne-Marie; Gao, Bing-Rong; Wraight, Colin A; Woodbury, Neal W; Lin, Su

    2016-06-23

    It has become increasingly clear that dynamics plays a major role in the function of many protein systems. One system that has proven particularly facile for studying the effects of dynamics on protein-mediated chemistry is the bacterial photosynthetic reaction center from Rhodobacter sphaeroides. Previous experimental and computational analysis have suggested that the dynamics of the protein matrix surrounding the primary quinone acceptor, QA, may be particularly important in electron transfer involving this cofactor. One can substantially increase the flexibility of this region by removing one of the reaction center subunits, the H-subunit. Even with this large change in structure, photoinduced electron transfer to the quinone still takes place. To evaluate the effect of H-subunit removal on electron transfer to QA, we have compared the kinetics of electron transfer and associated spectral evolution for the LM dimer with that of the intact reaction center complex on picosecond to millisecond time scales. The transient absorption spectra associated with all measured electron transfer reactions are similar, with the exception of a broadening in the QX transition and a blue-shift in the QY transition bands of the special pair of bacteriochlorophylls (P) in the LM dimer. The kinetics of the electron transfer reactions not involving quinones are unaffected. There is, however, a 4-fold decrease in the electron transfer rate from the reduced bacteriopheophytin to QA in the LM dimer compared to the intact reaction center and a similar decrease in the recombination rate of the resulting charge-separated state (P(+)QA(-)). These results are consistent with the concept that the removal of the H-subunit results in increased flexibility in the region around the quinone and an associated shift in the reorganization energy associated with charge separation and recombination.

  2. Rhodobacter sphaeroides spd mutations allow cytochrome c2-independent photosynthetic growth.

    PubMed Central

    Rott, M A; Donohue, T J

    1990-01-01

    In Rhodobacter sphaeroides, cytochrome c2 (cyt c2) is a periplasmic redox protein required for photosynthetic electron transfer. cyt c2-deficient mutants created by replacing the gene encoding the apoprotein for cyt c2 (cycA) with a kanamycin resistance cartridge are photosynthetically incompetent. Spontaneous mutations that suppress this photosynthesis deficiency (spd mutants) arise at a frequency of 1 to 10 in 10(7). We analyzed the cytochrome content of several spd mutants spectroscopically and by heme peroxidase assays. These suppressors lacked detectable cyt c2, but they contained a new soluble cytochrome which was designated isocytochrome c2 (isocyt c2) that was not detectable in either cycA+ or cycA mutant cells. When spd mutants were grown photosynthetically, isocyt c2 was present at approximately 20 to 40% of the level of cyt c2 found in photosynthetically grown wild type cells, and it was found in the periplasm with cytochromes c' and c554. These spd mutants also had several other pleiotropic phenotypes. Although photosynthetic growth rates of the spd mutants were comparable to those of wild-type strains at all light intensities tested, they contained elevated levels of B800-850 pigment-protein complexes. Several spd mutants contained detectable amounts of isocyt c2 under aerobic conditions. Finally, heme peroxidase assays indicated that, under anaerobic conditions, the spd mutants may contain another new cytochrome in addition to isocyt c2. These pleiotropic phenotypes, the frequency at which the spd mutants arise, and the fact that a frameshift mutagen is very effective in generating the spd phenotype suggest that some spd mutants contain a mutation in loci which regulate cytochrome synthesis. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 PMID:2156806

  3. Photo dynamics of BLUF domain mutant H44R of AppA from Rhodobacter sphaeroides

    NASA Astrophysics Data System (ADS)

    Zirak, P.; Penzkofer, A.; Hegemann, P.; Mathes, T.

    2007-05-01

    The photo-cycle dynamics of the H44R mutant of the BLUF domain of the transcriptional anti-repressor protein AppA (AppA-H44R) from the non-sulfur anoxyphototropic purple bacterium Rhodobacter sphaeroides is studied in order to gain information on the involvement of His44 in the photo-cyclic mechanism of the AppA BLUF domain and to add information to the involved processes. The amino acid residue histidine at position 44 is replaced by arginine. A 12 nm red-shifted signalling state is formed upon blue-light excitation, while in wild-type AppA (AppA-wt) the red-shift is 16 nm. The recovery to the receptor dark state is approximately a factor of 2.5 faster ( τrec ≈ 6.5 min) than the recovery of the wild-type counterpart. Extended light exposure of the mutant causes photo-degradation of flavin (mainly free flavin conversion to lumichrome and re-equilibration between free and non-covalently bound flavin) and protein aggregation (showing up as light scattering). No photo-degradation was observed for AppA-wt. The quantum efficiency of signalling-state formation determined by intensity dependent absorption measurements is found to be ϕs ≈ 0.3 (for AppA-wt: ϕs ≈ 0.24). A two-component single-exponential fluorescence relaxation was observed, which is interpreted as fast fluorescence quenching to an equilibrium value by photo-induced electron transfer followed by slower fluorescence decay due to charge recombination. Based on the experimental findings, an extended photo-cycle model for BLUF domains is proposed.

  4. Supplementing Rhodobacter sphaeroides in the diet of lactating Holstein cows may naturally produce coenzyme Q10-enriched milk

    PubMed Central

    Bae, Gui-Seck; Choi, Ahreum; Yeo, Joon Mo; Kim, Jong Nam; Song, Jaeyong; Kim, Eun Joong; Chang, Moon Baek

    2018-01-01

    Objective To examine the effects of Rhodobacter sphaeroides (R. sphaeroides) supplementation as a direct-fed microbial (DFM) on rumen fermentation in dairy cows and on coenzyme Q10 (CoQ10) transition into milk, an in vitro rumen simulation batch culture and an in vivo dairy cow experiment were conducted. Methods The characteristics of in vitro ruminal fermentation were investigated using rumen fluids from six cannulated Holstein dairy cows at 2 h post-afternoon feeding. A control treatment was included in the experiments based on a typified total mixed ration (TMR) for lactating dairy cows, which was identical to the one used in the in vivo study, plus R. sphaeroides at 0.1%, 0.3%, and 0.5% TMR dry matter. The in vivo study employed six ruminally cannulated lactating Holstein cows randomly allotted to either the control TMR (C-TMR) treatment or to a diet supplemented with a 0.5% R. sphaeroides culture (S-TMR, dry matter basis) ad libitum. The presence of R. sphaeroides was verified using denaturing gradient gel electrophoresis (DGGE) applied to the bacterial samples obtained from the in vivo study. The concentration of CoQ10 in milk and in the supernatant from the in vitro study was determined using high performance liquid chromatography. Results The results of the in vitro batch culture and DGGE showed that the concentration of CoQ10 significantly increased after 2 h of R. sphaeroides supplementation above 0.1%. When supplemented to the diet of lactating cows at the level of 0.5%, R. sphaeroides did not present any adverse effect on dry matter intake and milk yield. However, the concentration of CoQ10 in milk dramatically increased, with treated cows producing 70.9% more CoQ10 than control cows. Conclusion The CoQ10 concentration in milk increased via the use of a novel DFM, and R. sphaeroides might be used for producing value-added milk and dairy products in the future. PMID:28427254

  5. Construction and Validation of the Rhodobacter sphaeroides 2.4.1 DNA Microarray: Transcriptome Flexibility at Diverse Growth Modes

    PubMed Central

    Pappas, Christopher T.; Sram, Jakub; Moskvin, Oleg V.; Ivanov, Pavel S.; Mackenzie, R. Christopher; Choudhary, Madhusudan; Land, Miriam L.; Larimer, Frank W.; Kaplan, Samuel; Gomelsky, Mark

    2004-01-01

    A high-density oligonucleotide DNA microarray, a genechip, representing the 4.6-Mb genome of the facultative phototrophic proteobacterium, Rhodobacter sphaeroides 2.4.1, was custom-designed and manufactured by Affymetrix, Santa Clara, Calif. The genechip contains probe sets for 4,292 open reading frames (ORFs), 47 rRNA and tRNA genes, and 394 intergenic regions. The probe set sequences were derived from the genome annotation generated by Oak Ridge National Laboratory after extensive revision, which was based primarily upon codon usage characteristic of this GC-rich bacterium. As a result of the revision, numerous missing ORFs were uncovered, nonexistent ORFs were deleted, and misidentified start codons were corrected. To evaluate R. sphaeroides transcriptome flexibility, expression profiles for three diverse growth modes—aerobic respiration, anaerobic respiration in the dark, and anaerobic photosynthesis—were generated. Expression levels of one-fifth to one-third of the R. sphaeroides ORFs were significantly different in cells under any two growth modes. Pathways involved in energy generation and redox balance maintenance under three growth modes were reconstructed. Expression patterns of genes involved in these pathways mirrored known functional changes, suggesting that massive changes in gene expression are the major means used by R. sphaeroides in adaptation to diverse conditions. Differential expression was observed for genes encoding putative new participants in these pathways (additional photosystem genes, duplicate NADH dehydrogenase, ATP synthases), whose functionality has yet to be investigated. The DNA microarray data correlated well with data derived from quantitative reverse transcription-PCR, as well as with data from the literature, thus validating the R. sphaeroides genechip as a powerful and reliable tool for studying unprecedented metabolic versatility of this bacterium. PMID:15231807

  6. Photochemical hole-burned spectra of protonated and deuterated reaction centers of Rhodobacter sphaeroides

    SciTech Connect

    Lyle, P.A.; Kolaczkowski, S.V.; Small, G.J.

    1993-07-01

    Photochemical hole-burned spectra with improved signal-to-noise ratio ([times]20) are reported for the protonated and deuterated reaction center of the purple bacterium Rhodobacter sphaeroides. Spectra obtained as a function of burn frequency ([omega][sub B]) establish that the lifetime of P870*, the primary electron-donor state, is invariant to location of [omega][sub B] within the inhomogeneous distribution of P870 zero-phonon line transition frequencies. For both the protonated and deuterated RC, which exhibit P870 absorption widths at 4.2 K of only 440 and 420 cm[sup [minus]1], the zero-phonon holes yield a lifetime of 0.93 [+-] 0.10 ps. This lifetime is independent of temperature betweenmore » 1.6 and 8.0 K (range over which the zero-phonon hole could be studied). The invariance of the P870* lifetime to [omega][sub B] and other data indicates that the nonexponential decay of P870* (Vos et al. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 8885) is due neither to a distribution of values from the electronic coupling matrix element associated with electron transfer, which one might expect from the normal glasslike structural heterogeneity of the RC, nor to gross heterogeneity. The higher quality of the hole spectra has allowed for more stringent testing of the theoretical model previously used to simulate the P870 hole profiles and absorption spectrum. Although the essential findings reported earlier (see, e.g., Reddy et al. Photosyn. Res. 1992, 31, 167) are not altered, it is concluded that the modeling of the distribution of low-frequency phonons (mean frequency approximately 30 cm[sup [minus]1]), which couples to P870*, in terms of a Debye distribution is inadequate. The anomalous low-frequency modes of glasses and polymers are suggested to be important also for proteins. 60 refs., 8 figs., 2 tabs.« less

  7. Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

    DOE PAGES

    Cartron, Michaël L.; Olsen, John D.; Sener, Melih; ...

    2014-02-13

    Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH 2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc 1 (cytbc 1) complexes that oxidise QH 2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc 1 complexes with gold nanobeads, each attached by a Histidine 10 (His 10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesiclesmore » showed that the majority of the cytbc 1 complexes occur as dimers in the membrane. The cytbc 1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc 1, whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc 1-RC-LH1- PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc 1, ATP synthase, cytaa 3 and cytcbb 3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc 1 dimers and 2 ATP synthases. In conclusion, simulation of the interconnected energy, electron and proton transfer processes showed a halfmaximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.« less

  8. Biochemical analysis and the preliminary crystallographic characterization of D-tagatose 3-epimerase from Rhodobacter sphaeroides.

    PubMed

    Qi, Zhengliang; Zhu, Zhangliang; Wang, Jian-Wen; Li, Songtao; Guo, Qianqian; Xu, Panpan; Lu, Fuping; Qin, Hui-Min

    2017-11-09

    D-Tagatose 3-epimerase epimerizes D-fructose to yield D-psicose, which is a rare sugar that exists in small quantities in nature and is difficult to synthesize chemically. We aim to explore potential industrial biocatalysts for commercial-scale manufacture of this rare sugar. A D-tagatose 3-epimerase from Rhodobacter sphaeroides (RsDTE) has recently been identified as a D-tagatose 3-epimerase that can epimerize D-fructose to yield D-psicose with a high conversion rate. The purified RsDTE by Ni-affinity chromatography, ionic exchange chromatography and gel filtration forms a tetramer in solution. The maximal activity was in Tris-HCl buffer pH 8.5, and the optimal temperature was at 35 °C. The product, D-psicose, was confirmed using HPLC and NMR. Crystals of RsDTE were obtained using crystal kits and further refined under crystallization conditions such as 10% PEG 8000,0.1 M HEPES pH 7.5, and 8% ethylene glycol at 20 °C using the sitting-drop vapor diffusion method. The RsDTE homology model showed that it possessed the characteristic TIM-barrel fold. Four residues, Glu156, Asp189, Gln215 and Glu250, forms a hydrogen bond network with the active Mn(II) for the hydride transfer reaction. These residues may constitute the catalytic tetrad of RsDTE. The residues around O1, O2 and O3 of the substrates were conserved. However, the binding-site residues are different at O4, O5 and O6. Arg118 formed the unique hydrogen bond with O4 of D-fructose which indicates RsDTE's preference of D-fructose more than any other family enzymes. RsDTE possesses a different metal-binding site. Arg118, forming unique hydrogen bond with O4 of D-fructose, regulates the substrate recognition. The research on D-tagatose 3-epimerase or D-psicose 3-epimerase enzymes attracts enormous commercial interest and would be widely used for rare sugar production in the future.

  9. Grain dust-induced lung inflammation is reduced by Rhodobacter sphaeroides diphosphoryl lipid A.

    PubMed

    Jagielo, P J; Quinn, T J; Qureshi, N; Schwartz, D A

    1998-01-01

    To further determine the importance of endotoxin in grain dust-induced inflammation of the lower respiratory tract, we evaluated the efficacy of pentaacylated diphosphoryl lipid A derived from the lipopolysaccharide of Rhodobacter sphaeroides (RsDPLA) as a partial agonist of grain dust-induced airway inflammation. RsDPLA is a relatively inactive compound compared with lipid A derived from Escherichia coli (LPS) and has been demonstrated to act as a partial agonist of LPS-induced inflammation. To assess the potential stimulatory effect of RsDPLA in relation to LPS, we incubated THP-1 cells with RsDPLA (0.001-100 micrograms/ml), LPS (0.02 microgram endotoxin activity/ml), or corn dust extract (CDE; 0.02 microgram endotoxin activity/ml). Incubation with RsDPLA revealed a tumor necrosis factor (TNF)-alpha stimulatory effect at 100 micrograms/ml. In contrast, incubation with LPS or CDE resulted in TNF-alpha release at 0.02 microgram/ml. Pretreatment of THP-1 cells with varying concentrations of RsDPLA before incubation with LPS or CDE (0.02 microgram endotoxin activity/ml) resulted in a dose-dependent reduction in the LPS- or CDE-induced release of TNF-alpha with concentrations of RsDPLA of up to 10 micrograms/ml but not at 100 micrograms/ml. To further understand the role of endotoxin in grain dust-induced airway inflammation, we utilized the unique LPS inhibitory property of RsDPLA to determine the inflammatory response to inhaled CDE in mice in the presence of RsDPLA. Ten micrograms of RsDPLA intratracheally did not cause a significant inflammatory response compared with intratracheal saline. However, pretreatment of mice with 10 micrograms of RsDPLA intratracheally before exposure to CDE (5.4 and 0.2 micrograms/m3) or LPS (7.2 and 0.28 micrograms/m3) resulted in significant reductions in the lung lavage concentrations of total cells, neutrophils, and specific proinflammatory cytokines compared with mice pretreated with sterile saline. These results confirm the LPS

  10. Biocontrol activity and patulin-removal effects of Bacillus subtilis, Rhodobacter sphaeroides and Agrobacterium tumefaciens against Penicillium expansum.

    PubMed

    Wang, Y; Yuan, Y; Liu, B; Zhang, Z; Yue, T

    2016-11-01

    This study was conducted to evaluate the biocontrol potential of Bacillus subtilis CICC 10034, Rhodobacter sphaeroides CGMCC 1.2182 and Agrobacterium tumefaciens CGMCC 1.2554 against patulin (PAT)-producer Penicillium expansum and their ability to remove PAT. Bacillus subtilis effectively inhibited P. expansum both on apples and in in vitro experiments, which reduced the rot diameter on apples by 38% compared with the control. The reduction was followed by those induced by A. tumefaciens (27·63%) and R. sphaeroides (23·67%). None of the cell-free supernatant (CFS) was able to prevent pathogen growth. Three antagonists could suppress PAT production by P. expansum on apples by 98·5, 93·7 and 94·99% after treatment with B. subtilis, R. sphaeroides and A. tumefaciens respectively. In addition, the three strains led to a 0·56-1·47 log CFU g -1 reduction in colony number of P. expansum on apples. Survival of antagonists on apple wounds revealed their tolerance to PAT. Furthermore, both live and autoclaved cells of three strains efficiently adsorbed artificially spiked PAT from medium. The selected antagonists could be applied before harvesting to control apple infection by PAT-producing fungi and also during processing to act as PAT detoxifiers. Since little information related to the capability of R. sphaeroides and A. tumefaciens to inhibit P. expansum is currently available, the results of this study provide some new perspectives to the biocontrol field. © 2016 The Society for Applied Microbiology.

  11. Transient absorption study of two-photon excitation mechanism in the LH2 complex from purple bacterium Rhodobacter sphaeroides.

    PubMed

    Stepanenko, Ilya; Kompanetz, Viktor; Makhneva, Zoya; Chekalin, Sergey; Moskalenko, Andrei; Razjivin, Andrei

    2012-03-08

    The mechanism of two-photon excitation of a peripheral light-harvesting complex LH2 (B800-850) from purple bacterium Rhodobacter sphaeroides was explained on the basis of femtosecond transient absorption data. Fast bleaching of the B850 absorption band was measured under two-photon excitation by 1350 nm femtosecond pulses, showing fast subpicosecond arrival of excitation energy to B850 circular aggregates. Any spectral changes connected with the B800 absorption band of B800-BChl molecules were absent. A similar picture was observed under one-photon excitation of the LH2 complex by 675 nm femtosecond pulses. We believe these effects may be attributed to direct excitation of high-energy excitonic states of a B850 circular aggregate or its vibrational manifold in accordance with the model of Abe [Chem. Phys. 2001, 264, 355-363].

  12. Femtosecond dynamics of energy transfer in B800-850 light-harvesting complexes of Rhodobacter sphaeroides.

    PubMed Central

    Trautman, J K; Shreve, A P; Violette, C A; Frank, H A; Owens, T G; Albrecht, A C

    1990-01-01

    We report femtosecond transient absorption studies of energy transfer dynamics in the B800-850 light-harvesting complex (LHC) of Rhodobacter sphaeroides 2.4.1. For complexes solubilized in lauryldimethylamine-N-oxide (LDAO), the carotenoid to bacteriochlorophyll (Bchl) B800 and carotenoid to Bchl B850 energy transfer times are 0.34 and 0.20 ps, respectively. The B800 to B850 energy transfer time is 2.5 ps. For complexes treated with lithium dodecyl sulfate (LDS), a carotenoid to B850 energy transfer time of less than or equal to 0.2 ps is seen, and a portion of the total carotenoid population is decoupled from Bchl. In both LDAO-solubilized and LDS-treated complexes an intensity-dependent picosecond decay component of the excited B850 population is ascribed to excitation annihilation within minimal units of the LHC. PMID:2404276

  13. Direct Visualization of Exciton Reequilibration in the LH1 and LH2 Complexes of Rhodobacter sphaeroides by Multipulse Spectroscopy

    PubMed Central

    Cohen Stuart, Thomas A.; Vengris, Mikas; Novoderezhkin, Vladimir I.; Cogdell, Richard J.; Hunter, C. Neil; van Grondelle, Rienk

    2011-01-01

    The dynamics of the excited states of the light-harvesting complexes LH1 and LH2 of Rhodobacter sphaeroides are governed, mainly, by the excitonic nature of these ring-systems. In a pump-dump-probe experiment, the first pulse promotes LH1 or LH2 to its excited state and the second pulse dumps a portion of the excited state. By selective dumping, we can disentangle the dynamics normally hidden in the excited-state manifold. We find that by using this multiple-excitation technique we can visualize a 400-fs reequilibration reflecting relaxation between the two lowest exciton states that cannot be directly explored by conventional pump-probe. An oscillatory feature is observed within the exciton reequilibration, which is attributed to a coherent motion of a vibrational wavepacket with a period of ∼150 fs. Our disordered exciton model allows a quantitative interpretation of the observed reequilibration processes occurring in these antennas. PMID:21539791

  14. Characterization of D-tagatose-3-epimerase from Rhodobacter sphaeroides that converts D-fructose into D-psicose.

    PubMed

    Zhang, Longtao; Mu, Wanmeng; Jiang, Bo; Zhang, Tao

    2009-06-01

    A non-characterized gene, previously proposed as the D-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with D-fructose and decreased for other substrates in the order: D-tagatose, D-psicose, D-ribulose, D-xylulose and D-sorbose. Its activity was maximal at pH 9 and 40 degrees C while being enhanced by Mn(2+). At pH 9 and 40 degrees C, 118 g D-psicose l(-1) was produced from 700 g D-fructose l(-1) after 3 h.

  15. Biohydrogen and polyhydroxyalkanoate co-production by Enterobacter aerogenes and Rhodobacter sphaeroides from Calophyllum inophyllum oil cake.

    PubMed

    Arumugam, A; Sandhya, M; Ponnusami, V

    2014-07-01

    The feasibility of coupled biohydrogen and polyhydroxyalkanoate production by Enterobacter aerogenes and Rhodobacter sphaeroides using Calophyllum inophyllum oil cake was studied under dark and photo fermentation conditions. The utilization of a non-edible acidic oil cake (C. inophyllum), and exploitation of a modified minimal salt media led to reduction in the cost of media. Cost of fermentation is reduced by implementation of alternate dark-photo fermentative periods and through the use of a co-culture consisting of a dark fermentative (E. aerogenes) and a photo fermentative (R. sphaeroides) bacterium. The biohydrogen and polyhydroxyalkanoate produced were 7.95 L H2/L media and 10.73 g/L media, respectively, under alternate dark and photo fermentation and were 3.23 L H2/L media and 5.6g/L media, respectively under complete dark fermentation. The characteristics of the oil cake and alternate dark (16 h) and photo (8h) fermentative conditions were found to be supportive in producing high biohydrogen and polyhydroxyalkanoate (PHA) yield. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Resonance Raman spectroscopy of 2H-labelled spheroidenes in petroleum ether and in the Rhodobacter sphaeroides reaction centre.

    PubMed

    Kok, P; Köhler, J; Groenen, E J; Gebhard, R; van der Hoef, I; Lugtenburg, J; Farhoosh, R; Frank, H A

    1997-03-01

    As a step towards the structural analysis of the carotenoid spheroidene in the Rhodobacter sphaeroides reaction centre, we present the resonance Raman spectra of 14-2H, 15-2H, 15'-2H, 14'-2H, 14,15'-2H2 and 15-15'-2H2 spheroidenes in petroleum ether and, except for 14,15'-2H2 spheroidene, in the Rb. sphaeroides R26 reaction center (RC). Analysis of the spectral changes upon isotopic substitution allows a qualitative assignment of most of the vibrational bands to be made. For the all-trans spheroidenes in solution the resonance enhancement of the Raman bands is determined by the participation of carbon carbon stretching modes in the centre of the conjugated chain, the C9 to C15' region. For the RC-bound 15,15'-cis spheroidenes, enhancement is determined by the participation of carbon-carbon stretching modes in the centre of the molecule, the C13 to C13' region. Comparison of the spectra in solution and in the RC reveals evidence for an out-of-plane distortion of the RC-bound spheroidene in the central C14 to C14' region of the carotenoid. The characteristic 1240 cm-1 band in the spectrum of the RC-bound spheroidene has been assigned to a normal mode that contains the coupled C12-C13 and C13'-C12' stretch vibrations.

  17. Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation.

    PubMed

    Benning, C; Huang, Z H; Gage, D A

    1995-02-20

    Cells of the photosynthetic bacterium Rhodobacter sphaeroides grown under phosphate-limiting conditions accumulated nonphosphorous glycolipids and lipids carrying head groups derived from amino acids. Concomitantly, the relative amount of phosphoglycerolipids decreased from 90 to 22 mol% of total polar lipids in the membranes. Two lipids, not detectable in cells grown under standard conditions, were synthesized during phosphate-limited growth. Fast atom bombardment mass spectroscopy, exact mass measurements, 1H NMR spectroscopy, sugar composition analysis, and methylation analysis of the predominant glycolipid led to the identification of the novel compound 1,2-di-O-acyl-3-O-[alpha-D-glucopyranosyl-(1-->4)-O-beta-D-galactopyr anosyl]glycerol. The second lipid was identified as the betaine lipid 1,2-di-O-acyl-[4'-(N,N,N-trimethyl)-homoserine]glycerol by cochromatography employing an authentic standard from Chlamydomonas reinhardtii, fast atom bombardment mass spectroscopy, exact mass measurements, and 1H NMR spectroscopy. Prior to this observation, the occurrence of this lipid was thought to be restricted to lower plants and algae. Apparently, these newly synthesized nonphosphorous lipids, in addition to the sulfo- and the ornithine lipid also found in R. sphaeroides grown under optimal conditions, take over the role of phosphoglycerolipids in phosphate-deprived cells.

  18. Biohydrogen production by purple non-sulfur bacteria Rhodobacter sphaeroides: Effect of low-intensity electromagnetic irradiation.

    PubMed

    Gabrielyan, Lilit; Sargsyan, Harutyun; Trchounian, Armen

    2016-09-01

    The present work was focused on the effects of low-intensity (the flux capacity was of 0.06mWcm(-2)) electromagnetic irradiation (EMI) of extremely high frequencies or millimeter waves on the growth and hydrogen (H2) photoproduction by purple non-sulfur bacteria Rhodobacter sphaeroides MDC6521 (from Armenian mineral springs). After exposure of R. sphaeroides, grown under anaerobic conditions upon illumination, to EMI (51.8GHz and 53.0GHz) for 15min an increase of specific growth rate by ~1.2-fold, in comparison with control (non-irradiated cells), was obtained. However, the effect of EMI depends on the duration of irradiation: the exposure elongation up to 60min caused the delay of the growth lag phase and the decrease specific growth rate by ~1.3-fold, indicating the bactericidal effect of EMI. H2 yield of the culture, irradiated by EMI for 15min, determined during 72h growth, was ~1.2-fold higher than H2 yield of control cells, whereas H2 production by cultures, irradiated by EMI for 60min was not observed during 72h growth. This difference in the effects of extremely high frequency EMI indicates a direct effect of radiation on the membrane transfer and the enzymes of these bacteria. Moreover, EMI increased DCCD-inhibited H(+) fluxes across the bacterial membrane and DCCD-sensitive ATPase activity of membrane vesicles, indicating that the proton FoF1-ATPase is presumably a basic target for extremely high frequency EMI related to H2 production by cultures. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Aspects of Rhodobacter sphaeroides ChrR required for stimuli to promote dissociation of σE/ChrR complexes

    PubMed Central

    Greenwell, Roger; Nam, Tae-Wook; Donohue, Timothy J.

    2011-01-01

    In the photosynthetic bacterium Rhodobacter sphaeroides, a transcriptional response to the reactive oxygen species singlet oxygen (1O2) is mediated by ChrR, a zinc metalloprotein that binds to and inhibits activity of the alternative sigma factor, σE. We provide evidence that 1O2 promotes dissociation of σE from ChrR to activate transcription in vivo. To identify what is required for 1O2 to promote dissociation of σE/ChrR complexes, we analyzed the in vivo properties of variant ChrR proteins with amino acid changes in conserved residues of the C-terminal cupin-like domain (ChrR-CLD). We found that 1O2 was unable to promote detectable dissociation of σE/ChrR complexes when the ChrR-CLD zinc ligands (His141, His143, Glu147, and His177) were substituted with alanine, even though individual substitutions caused a 2- to 10-fold decrease in zinc affinity for this domain relative to that of wild-type ChrR (Kd ∼4.6 × 10−10 M). We conclude that the side chains of these invariant residues play a crucial role in the response to 1O2. Additionally, we found that cells containing variant ChrR proteins with single amino acid substitutions at Cys187 or Cys189 exhibited σE activity similar to those containing wild-type ChrR when exposed to 1O2, suggesting that these thiol side chains are not required for 1O2 to induce σE activity in vivo. Finally, we found that the same aspects of R. sphaeroides ChrR needed for a response to 1O2 are required for dissociation of σE/ChrR in the presence of the organic hydroperoxide, tert-butyl hydroperoxide (t-BOOH). PMID:21295582

  20. Probing energy transfer events in the light harvesting complex 2 (LH2) of Rhodobacter sphaeroides with two-dimensional spectroscopy.

    PubMed

    Fidler, Andrew F; Singh, Ved P; Long, Phillip D; Dahlberg, Peter D; Engel, Gregory S

    2013-10-21

    Excitation energy transfer events in the photosynthetic light harvesting complex 2 (LH2) of Rhodobacter sphaeroides are investigated with polarization controlled two-dimensional electronic spectroscopy. A spectrally broadened pulse allows simultaneous measurement of the energy transfer within and between the two absorption bands at 800 nm and 850 nm. The phased all-parallel polarization two-dimensional spectra resolve the initial events of energy transfer by separating the intra-band and inter-band relaxation processes across the two-dimensional map. The internal dynamics of the 800 nm region of the spectra are resolved as a cross peak that grows in on an ultrafast time scale, reflecting energy transfer between higher lying excitations of the B850 chromophores into the B800 states. We utilize a polarization sequence designed to highlight the initial excited state dynamics which uncovers an ultrafast transfer component between the two bands that was not observed in the all-parallel polarization data. We attribute the ultrafast transfer component to energy transfer from higher energy exciton states to lower energy states of the strongly coupled B850 chromophores. Connecting the spectroscopic signature to the molecular structure, we reveal multiple relaxation pathways including a cyclic transfer of energy between the two rings of the complex.

  1. Insights into the species-specific TLR4 signaling mechanism in response to Rhodobacter sphaeroides lipid A detection

    NASA Astrophysics Data System (ADS)

    Anwar, Muhammad Ayaz; Panneerselvam, Suresh; Shah, Masaud; Choi, Sangdun

    2015-01-01

    TLR4 in complex with MD2 senses the presence of lipid A (LA) and initiates a signaling cascade that curb the infection. This complex is evolutionarily conserved and can initiate the immune system in response to a variety of LAs. In this study, molecular dynamics simulation (25 ns) was performed to elucidate the differential behavior of TLR4/MD2 complex in response to Rhodobacter sphaeroides lipid A (RsLA). Penta-acyl chain-containing RsLA is at the verge of agonist (6 acyl-chains) and antagonist (4 acyl-chains) structure, and activates the TLR4 pathway in horses and hamsters, while inhibiting in humans and murine. In the time-evolved coordinates, the promising factors that dictated the differential response included the local and global mobility pattern of complexes, solvent-accessible surface area of ligand, and surface charge distributions of TLR4 and MD2. We showed that the GlcN1-GlcN2 backbone acquires agonist (3FXI)-like configurations in horses and hamsters, while acquiring antagonist (2E59)-like configurations in humans and murine systems. Moreover, analysis of F126 behavior in the MD2 F126 loop (amino acids 123-129) and loop EF (81-89) suggested that certain sequence variations also contribute to species-specific response. This study underlines the TLR4 signaling mechanism and provides new therapeutic opportunities.

  2. Direct visualization of exciton reequilibration in the LH1 and LH2 complexes of Rhodobacter sphaeroides by multipulse spectroscopy.

    PubMed

    Cohen Stuart, Thomas A; Vengris, Mikas; Novoderezhkin, Vladimir I; Cogdell, Richard J; Hunter, C Neil; van Grondelle, Rienk

    2011-05-04

    The dynamics of the excited states of the light-harvesting complexes LH1 and LH2 of Rhodobacter sphaeroides are governed, mainly, by the excitonic nature of these ring-systems. In a pump-dump-probe experiment, the first pulse promotes LH1 or LH2 to its excited state and the second pulse dumps a portion of the excited state. By selective dumping, we can disentangle the dynamics normally hidden in the excited-state manifold. We find that by using this multiple-excitation technique we can visualize a 400-fs reequilibration reflecting relaxation between the two lowest exciton states that cannot be directly explored by conventional pump-probe. An oscillatory feature is observed within the exciton reequilibration, which is attributed to a coherent motion of a vibrational wavepacket with a period of ∼150 fs. Our disordered exciton model allows a quantitative interpretation of the observed reequilibration processes occurring in these antennas. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. Stereoselective oxidation of aliphatic diols and reduction of hydroxy-ketones with galactitol dehydrogenase from Rhodobacter sphaeroides D.

    PubMed

    Kohring, G W; Wiehr, P; Jeworski, M; Giffhorn, F

    2003-01-01

    From the Rhodobacter sphaeroides mutant D a galactitol dehydrogenase (GDH) was isolated and characterized in an earlier investigation (1). The enzyme expressed activity with a wide spread substrate spectrum, like sugars, sugar alcohols, secondary alcohols or the corresponding ketones and it can be used for the production of the rare sugar L-tagatose by regioselective oxidation of galactitol (2). This study focuses on the preparation of optically pure aliphatic diols by oxidation of one enantiomer or stereospecific reduction of keto-alcohols and diketones. The oxidation of 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol and 1,2-hexanediol occurred highly specific with the S-enantiomer leaving the R-enantiomer of the diols in the reaction vessel. Also (S)-1,2,6-hexanetriol was oxidized by GDH to 1,6-dihydroxy-2-hexanone. The Km values of these reactions decreased with increasing length of the carbon chain. Reduction of hydroxyacetone or 1-hydroxy-2-butanone resulted in an excess of 93% (S)-1,2-propanediol and more than 98% of (S)-1,2-butanediol, respectively. The diketone 2,3-hexanedione was only reduced to (2R,3S)-2,3-hexanediol, one of the possible four configurations. The wide substrate spectrum on one hand and the selectivity in the reaction on the other hand make GDH a very interesting enzyme for the production of optically pure building blocks in the chemical synthesis of bioactive compounds.

  4. Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre

    PubMed Central

    Grayson, Katie J.; Faries, Kaitlyn M.; Huang, Xia; Qian, Pu; Dilbeck, Preston; Martin, Elizabeth C.; Hitchcock, Andrew; Vasilev, Cvetelin; Yuen, Jonathan M.; Niedzwiedzki, Dariusz M.; Leggett, Graham J.; Holten, Dewey; Kirmaier, Christine; Neil Hunter, C.

    2017-01-01

    Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP–RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (QX) absorption bands of the RC allow energy transfer via a Förster mechanism, with an efficiency of 40±10%. This proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis. PMID:28054547

  5. Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre

    DOE PAGES

    Grayson, Katie J.; Faries, Kaitlyn M.; Huang, Xia; ...

    2017-01-05

    Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP–RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (Qmore » X) absorption bands of the RC allow energy transfer via a Fo¨rster mechanism, with an efficiency of 40±10%. Finally, this proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis.« less

  6. Characterization of the flgG operon of Rhodobacter sphaeroides WS8 and its role in flagellum biosynthesis.

    PubMed

    González-Pedrajo, Bertha; de la Mora, Javier; Ballado, Teresa; Camarena, Laura; Dreyfus, Georges

    2002-11-13

    In this work, we show evidence regarding the functionality of a large cluster of flagellar genes in Rhodobacter sphaeroides. The genes of this cluster, flgGHIJKL and orf-1, are mainly involved in the formation of the basal body, and flgK and flgL encode the hook-associated proteins HAP1 and HAP3. In general, these genes showed a good similarity as compared with those reported for Salmonella enterica. However, flgJ and flgK showed particular features that make them unique among the flagellar sequences already reported. flgJ is only a third of the size reported for flgJ from Salmonella; whereas flgK is about three times larger than any other flgK sequence previously known. Our results indicate that both genes are functional, and their products are essential for flagellar assembly. In contrast, the interruption of orf-1, did not affect motility suggesting that this sequence, if functional, is not indispensable for flagellar assembly. Finally, we present genetic evidence suggesting that the flgGHIJKL genes are expressed as a single transcriptional unit depending on the sigma-54 factor.

  7. Optimization of Biomass and 5-Aminolevulinic Acid Production by Rhodobacter sphaeroides ATCC17023 via Response Surface Methodology.

    PubMed

    Liu, Shuli; Zhang, Guangming; Li, Jianzheng; Li, Xiangkun; Zhang, Jie

    2016-06-01

    Microbial 5-aminolevulinic acid (ALA) produced from wastewater is considered as potential renewable energy. However, many hurdles are needed to be overcome such as the regulation of key influencing factors on ALA yield. Biomass and ALA production by Rhodobacter sphaeroides was optimized using response surface methodology. The culturing medium was artificial volatile fatty acids wastewater. Three additives were optimized, namely succinate and glycine that are precursors of ALA biosynthesis, and D-glucose that is an inhibitor of ALA dehydratase. The optimal conditions were achieved by analyzing the response surface plots. Statistical analysis showed that succinate at 8.56 mmol/L, glycine at 5.06 mmol/L, and D-glucose at 7.82 mmol/L were the best conditions. Under these optimal conditions, the highest biomass production and ALA yield of 3.55 g/L and 5.49 mg/g-biomass were achieved. Subsequent verification experiments at optimal values had the maximum biomass production of 3.41 ± 0.002 g/L and ALA yield of 5.78 ± 0.08 mg/g-biomass.

  8. Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre

    NASA Astrophysics Data System (ADS)

    Grayson, Katie J.; Faries, Kaitlyn M.; Huang, Xia; Qian, Pu; Dilbeck, Preston; Martin, Elizabeth C.; Hitchcock, Andrew; Vasilev, Cvetelin; Yuen, Jonathan M.; Niedzwiedzki, Dariusz M.; Leggett, Graham J.; Holten, Dewey; Kirmaier, Christine; Neil Hunter, C.

    2017-01-01

    Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP-RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (QX) absorption bands of the RC allow energy transfer via a Förster mechanism, with an efficiency of 40+/-10%. This proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis.

  9. Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides.

    PubMed Central

    Falcone, D L; Tabita, F R

    1991-01-01

    A Rhodobacter sphaeroides ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strain was constructed that was complemented by plasmids containing either the form I or form II CO2 fixation gene cluster. This strain was also complemented by genes encoding foreign RubisCO enzymes expressed from a Rhodospirillum rubrum RubisCO promoter. In R. sphaeroides, the R. rubrum promoter was regulated, resulting in variable levels of disparate RubisCO molecules under different growth conditions. Photosynthetic growth of the R. sphaeroides deletion strain complemented with cyanobacterial RubisCO revealed physiological properties reflective of the unique cellular environment of the cyanobacterial enzyme. The R. sphaeroides RubisCO deletion strain and R. rubrum promoter system may be used to assess the properties of mutagenized proteins in vivo, as well as provide a potential means to select for altered RubisCO molecules after random mutagenesis of entire genes or gene regions encoding RubisCO enzymes. Images PMID:1900508

  10. Trypsin treatment of reaction centers from Rhodobacter sphaeroides in the dark and under illumination: protein structural changes follow charge separation.

    PubMed

    Brzezinski, P; Andréasson, L E

    1995-06-06

    Reaction centers from Rhodobacter sphaeroides R-26 were treated with trypsin in the dark and during illumination (in the charge-separated state). Trypsination resulted in a time-dependent modification of the reaction centers, reflected in changes in the charge recombination rate, in the inhibition of QA- to QB electron transfer, and eventually to inhibition of charge separation. Comparisons of centers with ubiquinone or anthraquinone in the QA site, in which the charge recombination pathways are different, indicate that trypsination affects charges close to the QA(-)-binding site. Studies of light-induced voltage changes from moving charges in reaction centers incorporated in lipid layers on a Teflon film, a technique which allows the discrimination of effects on donor and acceptor sides, indicate that the acceptor side is preferentially degraded by trypsin in the dark. Tryptic digestion during illumination generally resulted in a marked strengthening and acceleration of the effects seen already during dark treatment, but new effects were also detected in gel electrophoretic peptide patterns, in optical spectra, and in the kinetic measurements. Optical kinetic measurements revealed that the donor side of the reaction centers became susceptible to modification by trypsin during illumination as seen in the value of the binding constant for soluble cytochrome c2 which increased by a factor of 2, whereas it was much less affected after trypsination of reaction centers in the dark. The influence of illumination on the rate and mode by which trypsin acts on reaction centers indicates that changes in the protein conformation follow charge separation.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. An extended model for the repression of photosynthesis genes by the AppA/PpsR system in Rhodobacter sphaeroides.

    PubMed

    Pandey, Rakesh; Flockerzi, Dietrich; Hauser, Marcus J B; Straube, Ronny

    2012-09-01

    Purple bacteria derive energy from aerobic respiration or photosynthesis depending on the availability of oxygen and light. Under aerobic conditions, photosynthesis genes are specifically repressed by the PpsR protein. In Rhodobacter sphaeroides, the repressive action of PpsR is antagonized by the blue-light and redox-sensitive flavoprotein AppA, which sequesters PpsR under anaerobic conditions into transcriptionally inactive complexes. However, under semi-aerobic conditions, blue-light excitation of AppA causes the AppA-PpsR complexes to dissociate, again leading to a repression of photosynthesis genes. We have recently developed a simple mathematical model suggesting that this phenotype arises from the formation of a maximum in the response curve of reduced PpsR at intermediate oxygen concentrations. However, this model focused mainly on the oxygen-dependent interactions whereas light regulation was only implemented in a simplified manner. In the present study, we incorporate a more detailed mechanism for the light-dependent interaction between AppA and PpsR, which now allows for a direct comparison with experiments. Specifically, we take into account that, upon blue-light excitation, AppA undergoes a conformational change, creating a long-lived signalling state causing the dissociation of the AppA-PpsR complexes. The predictions of the extended model are found to be in good agreement with experimental results on the light-dependent repression of photosynthesis genes under semi-aerobic conditions. We also identify the potential kinetic and stoichiometric constraints that the interplay between light and redox regulation imposes on the functionality of the AppA/PpsR system, especially with respect to a possible bistable response. © 2012 The Authors Journal compilation © 2012 FEBS.

  12. Transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration in Rhodobacter sphaeroides 2.4.1.

    PubMed

    Arai, Hiroyuki; Roh, Jung Hyeob; Kaplan, Samuel

    2008-01-01

    Rhodobacter sphaeroides 2.4.1 is a facultative photosynthetic anaerobe that grows by anoxygenic photosynthesis under anaerobic-light conditions. Changes in energy generation pathways under photosynthetic and aerobic respiratory conditions are primarily controlled by oxygen tensions. In this study, we performed time series microarray analyses to investigate transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration. Major changes in gene expression profiles occurred in the initial 15 min after the shift from anaerobic-light to aerobic-dark conditions, with changes continuing to occur up to 4 hours postshift. Those genes whose expression levels changed significantly during the time series were grouped into three major classes by clustering analysis. Class I contained genes, such as that for the aa3 cytochrome oxidase, whose expression levels increased after the shift. Class II contained genes, such as those for the photosynthetic apparatus and Calvin cycle enzymes, whose expression levels decreased after the shift. Class III contained genes whose expression levels temporarily increased during the time series. Many genes for metabolism and transport of carbohydrates or lipids were significantly induced early during the transition, suggesting that those endogenous compounds were initially utilized as carbon sources. Oxidation of those compounds might also be required for maintenance of redox homeostasis after exposure to oxygen. Genes for the repair of protein and sulfur groups and uptake of ferric iron were temporarily upregulated soon after the shift, suggesting they were involved in a response to oxidative stress. The flagellar-biosynthesis genes were expressed in a hierarchical manner at 15 to 60 min after the shift. Numerous transporters were induced at various time points, suggesting that the cellular composition went through significant changes during the transition from anaerobic photosynthesis to aerobic respiration

  13. Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway

    PubMed Central

    2016-01-01

    Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC=C + NC=O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids. PMID:27285777

  14. Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway.

    PubMed

    Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Hunter, C Neil; Bocian, David F; Holten, Dewey; Niedzwiedzki, Dariusz M

    2016-06-23

    Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC═C + NC═O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids.

  15. Use of transcriptomic data for extending a model of the AppA/PpsR system in Rhodobacter sphaeroides.

    PubMed

    Pandey, Rakesh; Armitage, Judith P; Wadhams, George H

    2017-12-28

    Photosynthetic (PS) gene expression in Rhodobacter sphaeroides is regulated in response to changes in light and redox conditions mainly by PrrB/A, FnrL and AppA/PpsR systems. The PrrB/A and FnrL systems activate the expression of them under anaerobic conditions while the AppA/PpsR system represses them under aerobic conditions. Recently, two mathematical models have been developed for the AppA/PpsR system and demonstrated how the interaction between AppA and PpsR could lead to a phenotype in which PS genes are repressed under semi-aerobic conditions. These models have also predicted that the transition from aerobic to anaerobic growth mode could occur via a bistable regime. However, they lack experimentally quantifiable inputs and outputs. Here, we extend one of them to include such quantities and combine all relevant micro-array data publically available for a PS gene of this bacterium and use that to parameterise the model. In addition, we hypothesise that the AppA/PpsR system alone might account for the observed trend of PS gene expression under semi-aerobic conditions. Our extended model of the AppA/PpsR system includes the biological input of atmospheric oxygen concentration and an output of photosynthetic gene expression. Following our hypothesis that the AppA/PpsR system alone is sufficient to describe the overall trend of PS gene expression we parameterise the model and suggest that the rate of AppA reduction in vivo should be faster than its oxidation. Also, we show that despite both the reduced and oxidised forms of PpsR binding to the PS gene promoters in vitro, binding of the oxidised form as a repressor alone is sufficient to reproduce the observed PS gene expression pattern. Finally, the combination of model parameters which fit the biological data well are broadly consistent with those which were previously determined to be required for the system to show (i) the repression of PS genes under semi-aerobic conditions, and (ii) bistability. We found

  16. Enhanced photo-fermentative H2 production using Rhodobacter sphaeroides by ethanol addition and analysis of soluble microbial products

    PubMed Central

    2014-01-01

    Background Biological fermentation routes can provide an environmentally friendly way of producing H2 since they use renewable biomass as feedstock and proceed under ambient temperature and pressure. In particular, photo-fermentation has superior properties in terms of achieving high H2 yield through complete degradation of substrates. However, long-term H2 production data with stable performance is limited, and this data is essential for practical applications. In the present work, continuous photo-fermentative H2 production from lactate was attempted using the purple non-sulfur bacterium, Rhodobacter sphaeroides KD131. As a gradual drop in H2 production was observed, we attempted to add ethanol (0.2% v/v) to the medium. Results As continuous operation went on, H2 production was not sustained and showed a negligible H2 yield (< 0.5 mol H2/mol lactateadded) within two weeks. Electron balance analysis showed that the reason for the gradual drop in H2 production was ascribed to the increase in production of soluble microbial products (SMPs). To see the possible effect of ethanol addition, a batch test was first conducted. The presence of ethanol significantly increased the H2 yield from 1.15 to 2.20 mol H2/mol lactateadded, by suppressing the production of SMPs. The analysis of SMPs by size exclusion chromatography showed that, in the later period of fermentation, more than half of the low molecular weight SMPs (< 1 kDa) were consumed and used for H2 production when ethanol had been added, while the concentration of SMPs continuously increased in the absence of ethanol. It was found that the addition of ethanol facilitated the utilization of reducing power, resulting in an increase in the cellular levels of NAD+ and NADP+. In continuous operation, ethanol addition was effective, such that stable H2 production was attained with an H2 yield of 2.5 mol H2/mol lactateadded. Less than 15% of substrate electrons were used for SMP production, whereas 35% were used in

  17. The observation of ultrafast excited-state dynamical evolution in B800- partially or completely released LH2 of Rhodobacter sphaeroides 601 at room temperature.

    PubMed

    Liu, Weimin; Liu, Yuan; Yan, Yongli; Liu, Kangjun; Guo, Lijun; Xu, Chunhe; Qian, Shixiong

    2006-04-01

    Photodynamics of two kinds of peripheral antenna complexes (LH2 of Rhodobacter sphaeroides, native LH2 (RS601) and B800-released LH2 where B800-BChls were partially or completely removed with different pH treatments), were studied using femtosecond pump-probe technique at different laser wavelengths. The obtained results for these samples with different B800/B850 ratios demonstrated that under the excitation around B800 nm, the photoabsorption and photobleaching dynamics were caused by the direct excitation of upper excitonic levels of B850 and excited state of B800 pigments, respectively. Furthermore, the removal of B800 pigments had little effect on the energy transfer processes of B850 interband/intraband transfer.

  18. Structural and preliminary molecular dynamics studies of the Rhodobacter sphaeroides reaction center and its mutant form L(M196)H + H(M202)L

    NASA Astrophysics Data System (ADS)

    Klyashtorny, V. G.; Fufina, T. Yu.; Vasilieva, L. G.; Shuvalov, V. A.; Gabdulkhakov, A. G.

    2014-07-01

    Pigment-protein interactions are responsible for the high efficiency of the light-energy transfer and conversion in photosynthesis. The reaction center (RC) from the purple bacterium Rhodobacter sphaeroides is the most convenient model for studying the mechanisms of primary processes of photosynthesis. Site-directed mutagenesis can be used to study the effect of the protein environment of electron-transfer cofactors on the optical properties, stability, pigment composition, and functional activity of RC. The preliminary analysis of RC was performed by computer simulation of the amino acid substitutions L(M196)H + H(M202)L at the pigment-protein interface and by estimating the stability of the threedimensional structure of the mutant RC by the molecular dynamics method. The doubly mutated reaction center was overexpressed, purified, and crystallized. The three-dimensional structure of this mutant was determined by X-ray crystallography and compared with the molecular dynamics model.

  19. Effect of pH on optimization of photofermentative hydrogen production by co-culture of Rhodobacter sphaeroides-NMBL-02 and Bacillus firmus-NMBL-03.

    PubMed

    Pandey, A; Dolly, S; Semwal, D; Pandey, A

    2017-07-31

    Rhodobacter sphaeroides NMBL-02, photosynthetic purple non sulfur (PNS) bacteria and associated Bacillus firmus NMBL-03 were isolated from water sample collected from 15-20 inches beneath the surface of ponds from Northern region of India in modified Sistrom's media (120 ml) containing 3 g/L malate and 1.2 g/L ammonium sulfate. The isolation was done in air tight serum bottles (120 ml) under tungsten bulb (1.8 kLux light intensity) at 30 oC ± 2 oC. The PNS and heterotrophic bacteria associated with the culture was purified by clonal selection method and characterized by 16S rDNA sequencing. The PNS isolate was identified as Rhodobacter sphaeroides NMBL-02 (ID: 1467407, Accession BANKIT: JN256030) and associated heterotroph as Bacillus firmus NMBL-03 (Gene Bank Accession no.: JN 256029). The effect of initial medium pH on optimization of hydrogen production was investigated in batch process. The maximum hydrogen potential and hydrogen production rate was 2310 ± 55 ml/L and 4.75 ml/L culture/h respectively using glutamate (1.7 mmol/L) as nitrogen source and malate (22.38 mmol/L) as carbon source with 76.39% malate conversion efficiency at initial medium pH 5.0. This co-culture has the ability to produce significant amount of hydrogen in the pH range of 5.0 to 10.0 with 76.39% to 35.71% malate conversion respectively.

  20. The nature of the lower excited state of the special pair of bacterial photosynthetic reaction center of Rhodobacter Sphaeroides and the dynamics of primary charge separation

    NASA Astrophysics Data System (ADS)

    Ivashin, N. V.; Shchupak, E. E.

    2016-08-01

    Quantum-chemical calculations of the structure in the ground and lower singlet excited states and the vibrations (in the ground state) of special pair P of photosynthetic reaction center of purple bacteria (RCPb) Rhodobacter Sphaeroides, consisting of two bacteriochlorophyll molecules PA and PB, have been carried out. It is shown that excitation of the special pair is followed by fast relaxation dynamics, accompanied by the transformation of the initial P* state into the P A δ+ P B δ- state (δ ~ 0.5) with charge separation. This behavior is due to the presence of several nonplanar vibrations with participation of the acetyl group of macrocycle PB in the nuclear wave packet on the potential surface of the P* state; these vibrations facilitate destabilization of the lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) of the macrocycle PA and formation of the P A δ+ P B δ- state. The structural transformations in the P* state are due to its linking character in the contact region of the acetyl group-containing pyrrole rings of PA and PB. The transition from the P* state to specifically the P A δ+ P B δ- state is related to the fact that the acetyl group PA is involved in the intermolecular hydrogen bond with amino acid residue HisL168; for this reason, this group and the pyrrole ring linked with it can hardly participate in structural transformations. The electronic matrix element H12 of the electron transfer from the special pair in the P A δ+ P B δ- state to a molecule of accessory bacteriochlorophyll BA greatly exceeds that for the transfer to BB. This circumstance and the fact that the P A δ+ P B δ- state is energetically more favorable than the P* state facilitate the preferred directionality of the electron transfer in RCPb Rhodobacter Sphaeroides with participation of the cofactors located in its subunit L.

  1. Profiling of ornithine lipids in bacterial extracts of Rhodobacter sphaeroides by reversed-phase liquid chromatography with electrospray ionization and multistage mass spectrometry (RPLC-ESI-MS(n)).

    PubMed

    Granafei, Sara; Losito, Ilario; Trotta, Massimo; Italiano, Francesca; de Leo, Vincenzo; Agostiano, Angela; Palmisano, Francesco; Cataldi, Tommaso R I

    2016-01-15

    Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the α-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MS(n), n = 2-4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M-H](-) at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M-H](-) ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic

  2. Physical Mapping of bchG, orf427, and orf177 in the Photosynthesis Gene Cluster of Rhodobacter sphaeroides: Functional Assignment of the Bacteriochlorophyll Synthetase Gene

    PubMed Central

    Addlesee, Hugh A.; Fiedor, Leszek; Hunter, C. Neil

    2000-01-01

    The purple photosynthetic bacterium Rhodobacter sphaeroides has within its genome a cluster of photosynthesis-related genes approximately 41 kb in length. In an attempt to identify genes involved in the terminal esterification stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5-kb region of this cluster was sequenced. Four open reading frames (ORFs) were identified, and each was analyzed by transposon mutagenesis. The product of one of these ORFs, bchG, shows close homologies with (bacterio)chlorophyll synthetases, and mutants in this gene were found to accumulate bacteriopheophorbide, the metal-free derivative of the bacteriochlorophyll precursor bacteriochlorophyllide, suggesting that bchG is responsible for the esterification of bacteriochlorophyllide with an alcohol moiety. This assignment of function to bchG was verified by the performance of assays demonstrating the ability of BchG protein, heterologously synthesized in Escherichia coli, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate in vitro, thereby generating bacteriochlorophyll. This step is pivotal to the assembly of a functional photosystem in R. sphaeroides, a model organism for the study of structure-function relationships in photosynthesis. A second gene, orf177, is a member of a large family of isopentenyl diphosphate isomerases, while sequence homologies suggest that a third gene, orf427, may encode an assembly factor for photosynthetic complexes. The function of the remaining ORF, bchP, is the subject of a separate paper (H. Addlesee and C. N. Hunter, J. Bacteriol. 181:7248–7255, 1999). An operonal arrangement of the genes is proposed. PMID:10809697

  3. Quenching capabilities of long-chain carotenoids in light-harvesting-2 complexes from Rhodobacter sphaeroides with an engineered carotenoid synthesis pathway

    SciTech Connect

    Dilbeck, Preston L.; Tang, Qun; Mothersole, David J.

    Here, six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = N C=C + N C=O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid firstmore » singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S 1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a lightharvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S 1 excited state B850*. This quenching is manifested as a substantial (~2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids« less

  4. Quenching capabilities of long-chain carotenoids in light-harvesting-2 complexes from Rhodobacter sphaeroides with an engineered carotenoid synthesis pathway

    DOE PAGES

    Dilbeck, Preston L.; Tang, Qun; Mothersole, David J.; ...

    2016-06-10

    Here, six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = N C=C + N C=O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid firstmore » singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S 1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a lightharvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S 1 excited state B850*. This quenching is manifested as a substantial (~2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids« less

  5. Functional characteristics of spirilloxanthin and keto-bearing Analogues in light-harvesting LH2 complexes from Rhodobacter sphaeroides with a genetically modified carotenoid synthesis pathway.

    PubMed

    Niedzwiedzki, Dariusz M; Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Bocian, David F; Holten, Dewey; Hunter, C Neil

    2015-01-01

    Light-harvesting 2 (LH2) complexes from a genetically modified strain of the purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. Carotenoid synthesis in the Rba. sphaeroides strain was engineered to redirect carotenoid production away from spheroidene into the spirilloxanthin synthesis pathway. The strain assembles LH2 antennas with substantial amounts of spirilloxanthin (total double-bond conjugation length N=13) if grown anaerobically and of keto-bearing long-chain analogs [2-ketoanhydrorhodovibrin (N=13), 2-ketospirilloxanthin (N=14) and 2,2'-diketospirilloxanthin (N=15)] if grown semi-aerobically (with ratios that depend on growth conditions). We present the photophysical, electronic, and vibrational properties of these carotenoids, both isolated in organic media and assembled within LH2 complexes. Measurements of excited-state energy transfer to the array of excitonically coupled bacteriochlorophyll a molecules (B850) show that the mean lifetime of the first singlet excited state (S1) of the long-chain (N≥13) carotenoids does not change appreciably between organic media and the protein environment. In each case, the S1 state appears to lie lower in energy than that of B850. The energy-transfer yield is ~0.4 in LH2 (from the strain grown aerobically or semi-aerobically), which is less than half that achieved for LH2 that contains short-chain (N≤11) analogues. Collectively, the results suggest that the S1 excited state of the long-chain (N≥13) carotenoids participates little if at all in carotenoid-to-BChl a energy transfer, which occurs predominantly via the carotenoid S2 excited state in these antennas. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Differences in the binding of the primary quinone receptor in Photosystem I and reaction centres of Rhodobacter sphaeroides-R26 studied with transient EPR spectroscopy

    NASA Astrophysics Data System (ADS)

    van der Est, A.; Sieckmann, I.; Lubitz, W.; Stehlik, D.

    1995-05-01

    The binding of the primary quinone acceptor, Q, in Photosystem I (PS I) and reaction centres (RC's) of Rhodobacter Sphaeroide-R26 in which, the non-heme iron has been replaced by zinc (Zn-bRC's) is studied using transient EPR spectroscopy. In PS I, Q is phylloquinone (vitamin K 1, VK 1) and is referred to as A 1. In Zn-bRC's, it is ubiquinone-10 (UQ 10) and called Q A. Native samples of the two RC's as well as those in which A 1 and Q A have been replaced by perdeuterated napthoquinone (NQ- d6) and duroquinone (DQ- d12) are compared. The spin polarized K-band (24 GHz) spectra of the charge separated state P +.Q -. (P = primary chlorophyll donor) in Zn-bRC's show that substitution of Q A, with NQ- d6 and DQ- d12 does not have a measurable effect on the quinone orientation in the Q A site. In contrast, large differences in the orientation of VK 1, NQ- d6 and DQ- d12 in the A 1 site in PS I are found. In addition, all three quinones in PS I are oriented differently than Q A in Zn-bRC's. Further, the x and y principal values of the g-tensors of VK 1-., NQ -. and DQ -. in PS I are shown to be significantly larger than in frozen alcohol and Zn-bRC's. It is suggested that the differences in the orientation and a g-values of the quinones in the two RC's arise from a weaker binding to the protein in PS I.

  7. Hierarchical regulation of photosynthesis gene expression by the oxygen-responsive PrrBA and AppA-PpsR systems of Rhodobacter sphaeroides.

    PubMed

    Gomelsky, Larissa; Moskvin, Oleg V; Stenzel, Rachel A; Jones, Denise F; Donohue, Timothy J; Gomelsky, Mark

    2008-12-01

    In the facultatively phototrophic proteobacterium Rhodobacter sphaeroides, formation of the photosynthetic apparatus is oxygen dependent. When oxygen tension decreases, the response regulator PrrA of the global two-component PrrBA system is believed to directly activate transcription of the puf, puh, and puc operons, encoding structural proteins of the photosynthetic complexes, and to indirectly upregulate the photopigment biosynthesis genes bch and crt. Decreased oxygen also results in inactivation of the photosynthesis-specific repressor PpsR, bringing about derepression of the puc, bch, and crt operons. We uncovered a hierarchical relationship between these two regulatory systems, earlier thought to function independently. We also more accurately assessed the spectrum of gene targets of the PrrBA system. First, expression of the appA gene, encoding the PpsR antirepressor, is PrrA dependent, which establishes one level of hierarchical dominance of the PrrBA system over AppA-PpsR. Second, restoration of the appA transcript to the wild-type level is insufficient for rescuing phototrophic growth impairment of the prrA mutant, whereas inactivation of ppsR is sufficient. This suggests that in addition to controlling appA transcription, PrrA affects the activity of the AppA-PpsR system via an as yet unidentified mechanism(s). Third, PrrA directly activates several bch and crt genes, traditionally considered to be the PpsR targets. Therefore, in R. sphaeroides, the global PrrBA system regulates photosynthesis gene expression (i) by rigorous control over the photosynthesis-specific AppA-PpsR regulatory system and (ii) by extensive direct transcription activation of genes encoding structural proteins of photosynthetic complexes as well as genes encoding photopigment biosynthesis enzymes.

  8. Exploitation of dark fermented effluent of cheese whey by co-culture of Rhodobacter sphaeroides and Bacillus firmus for photo-hydrogen production.

    PubMed

    Pandey, A; Pandey, A

    2017-07-31

    In this study photo-hydrogen production from cheese whey dark fermentation (DF) effluent by the co-culture of Rhodobacter sphaeroides -NMBL-01 and Bacillus firmus - NMBL-03 has been reported. The effect of pH, initial chemical oxygen demand (COD) and the concentration effect of FeSO4.7H2O on photo-hydrogen production have been investigated. The end products of dark fermentation effluent of cheese whey were mainly comprised of soluble organic acids, i.e. butyric acid and lactic acid. The batch process was carried out under light intensity of 2.5 kLux at 32 ± 2oC without any addition of extra carbon and nitrogen source. The single parameter optimization studies revealed optimum pH 6.5, initial COD 4.71 g/L and supplementation of Fe2+ concentration 100 mg/L. The maximum cumulative hydrogen production and yield were found to be 469 ± 45.8 ml H2/L and 146.56 ± 14.31 ml H2/g COD reduced (67.9% reduction in COD) respectively. The mutual interactions among the process parameters were also investigated by three factorial Box-Behnken design of response surface methodology. The optimized experimental values were found concurrent with the calculated values obtained from the theoretical model.

  9. Expression of glnB and a glnB-Like Gene (glnK) in a Ribulose Bisphosphate Carboxylase/Oxygenase-Deficient Mutant of Rhodobacter sphaeroides

    PubMed Central

    Qian, Yilei; Tabita, F. Robert

    1998-01-01

    In a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant of Rhodobacter sphaeroides, strain 16PHC, nitrogenase activity was derepressed in the presence of ammonia under photoheterotrophic growth conditions. Previous studies also showed that reintroduction of a functional RubisCO and Calvin-Benson-Bassham (CBB) pathway suppressed the deregulation of nitrogenase synthesis in this strain. In this study, the derepression of nitrogenase synthesis in the presence of ammonia in strain 16PHC was further explored by using a glnB::lacZ fusion, since the product of the glnB gene is known to have a negative effect on ammonia-regulated nif control. It was found that glnB expression was repressed in strain 16PHC under photoheterotrophic growth conditions with either ammonia or glutamate as the nitrogen source; glutamine synthetase (GS) levels were also affected in this strain. However, when cells regained a functional CBB pathway by trans complementation of the deleted genes, wild-type levels of GS and glnB expression were restored. Furthermore, a glnB-like gene, glnK, was isolated from this organism, and its expression was found to be under tight nitrogen control in the wild type. Surprisingly, glnK expression was found to be derepressed in strain 16PHC under photoheterotrophic conditions in the presence of ammonia. PMID:9721307

  10. Resonance Raman and surface-enhanced resonance Raman spectra of LH2 antenna complex from Rhodobacter sphaeroides and Ectothiorhodospira sp. excited in the Qx and Qy transitions.

    PubMed

    Chumanov, G; Picorel, R; Ortiz de Zarate, I; Cotton, T M; Seibert, M

    2000-05-01

    Well-resolved vibrational spectra of LH2 complex isolated from two photosynthetic bacteria, Rhodobacter sphaeroides and Ectothiorhodospira sp., were obtained using surface-enhanced resonance Raman scattering (SERRS) exciting into the Qx and the Qy transitions of bacteriochlorophyll a. High-quality SERRS spectra in the Qy region were accessible because the strong fluorescence background was quenched near the roughened Ag surface. A comparison of the spectra obtained with 590 nm and 752 nm excitation in the mid- and low-frequency regions revealed spectral differences between the two LH2 complexes as well as between the LH2 complexes and isolated bacteriochlorophyll a. Because peripheral modes of pigments contribute mainly to the low-frequency spectral region, frequencies and intensities of many vibrational bands in this region are affected by interactions with the protein. The results demonstrate that the microenvironment surrounding the pigments within the two LH2 complexes is somewhat different, despite the fact that the complexes exhibit similar electronic absorption spectra. These differences are most probably due to specific pigment-pigment and pigment-protein interactions within the LH2 complexes, and the approach might be useful for addressing subtle static and dynamic structural variances between pigment-protein complexes from different sources or in complexes altered chemically or genetically.

  11. Chronic Exposure to Rhodobacter Sphaeroides Extract Lycogen™ Prevents UVA-Induced Malondialdehyde Accumulation and Procollagen I Down-Regulation in Human Dermal Fibroblasts

    PubMed Central

    Yang, Tsai-Hsiu; Lai, Ying-Hsiu; Lin, Tsuey-Pin; Liu, Wen-Sheng; Kuan, Li-Chun; Liu, Chia-Chyuan

    2014-01-01

    UVA contributes to the pathogenesis of skin aging by downregulation of procollagen I content and induction of matrix metalloproteinase (MMP)-associated responses. Application of antioxidants such as lycopene has been demonstrated as a convenient way to achieve protection against skin aging. Lycogen™, derived from the extracts of Rhodobacter sphaeroides, exerts several biological effects similar to that of lycopene whereas most of its anti-aging efficacy remains uncertain. In this study, we attempted to examine whether Lycogen™ could suppress malondialdehyde (MDA) accumulation and restore downregulated procollagen I expression induced by UVA exposure. In human dermal fibroblasts Hs68 cells, UVA repressed cell viability and decreased procollagen I protein content accompanied with the induction of MMP-1 and MDA accumulation. Remarkably, incubation with 50 μM Lycogen™ for 24 h ameliorated UVA-induced cell death and restored UVA-induced downregulation of procollagen in a dose-related manner. Lycogen™ treatment also prevented the UVA-induced MMP-1 upregulation and intracellular MDA generation in Hs68 cells. Activation of NFκB levels, one of the downstream events induced by UVA irradiation and MMP-1 induction, were also prevented by Lycogen™ administration. Taken together, our findings demonstrate that Lycogen™ may be an alternative agent that prevents UVA-induced skin aging and could be used in cosmetic and pharmaceutical applications. PMID:24463291

  12. In vivo effects on photosynthesis gene expression of base pair exchanges in the gene encoding the light-responsive BLUF domain of AppA in Rhodobacter sphaeroides.

    PubMed

    Metz, Sebastian; Hendriks, Johnny; Jäger, Andreas; Hellingwerf, Klaas; Klug, Gabriele

    2010-01-01

    The Rhodobacter sphaeroides protein AppA has the unique quality of sensing and transmitting light and redox signals. By acting as antirepressor to the PpsR protein, it acts as a major regulator in photosynthesis gene expression. In this study, we show that by introducing amino acid exchanges into the AppA protein, the in vivo activity as an antirepressor can be greatly altered. The tryptophan 104 to phenylalanine (W104F) base exchange greatly diminished blue-light sensitivity of the BLUF domain. From the obtained in vivo data, the difference in thermal recovery rate of the signaling state of the BLUF domain between the wild type and mutated protein was calculated, predicting an about 10-fold faster recovery in the mutant, which is consistent with in vitro data. Introduction of a tyrosine 21 to phenylalanine (Y21F) or to cysteine (Y21C) mutation led to a complete loss of AppA antirepressor activity, while additionally leading to an increase of photosynthesis gene expression after illumination with high blue-light quantities. Interestingly, this effect is not visible in a W104F/Y21F double mutant that again shows a wild-type-like behavior of the BLUF domain after blue-light illumination, thus restoring the activity of AppA.

  13. Dynamics of energy transfer from lycopene to bacteriochlorophyll in genetically-modified LH2 complexes of Rhodobacter sphaeroides.

    PubMed

    Hörvin Billsten, H; Herek, J L; Garcia-Asua, G; Hashøj, L; Polívka, T; Hunter, C N; Sundström, V

    2002-03-26

    LH2 complexes from Rb. sphaeroides were modified genetically so that lycopene, with 11 saturated double bonds, replaced the native carotenoids which contain 10 saturated double bonds. Tuning the S1 level of the carotenoid in LH2 in this way affected the dynamics of energy transfer within LH2, which were investigated using both steady-state and time-resolved techniques. The S1 energy of lycopene in n-hexane was determined to be approximately 12 500 +/- 150 cm(-1), by direct measurement of the S1-S2 transient absorption spectrum using a femtosecond IR-probing technique, thus placing an upper limit on the S1 energy of lycopene in the LH2 complex. Fluorescence emission and excitation spectra demonstrated that energy can be transferred from lycopene to the bacteriochlorophyll molecules within this LH2 complex. The energy-transfer dynamics within the mutant complex were compared to wild-type LH2 from Rb. sphaeroides containing the carotenoid spheroidene and from Rs. molischianum, in which lycopene is the native carotenoid. The results show that the overall efficiency for Crt --> B850 energy transfer is approximately 80% in lyco-LH2 and approximately 95% in WT-LH2 of Rb. sphaeroides. The difference in overall Crt --> BChl transfer efficiency of lyco-LH2 and WT-LH2 mainly relates to the low efficiency of the Crt S(1) --> BChl pathway for complexes containing lycopene, which was 20% in lyco-LH2. These results show that in an LH2 complex where the Crt S1 energy is sufficiently high to provide efficient spectral overlap with both B800 and B850 Q(y) states, energy transfer via the Crt S1 state occurs to both pigments. However, the introduction of lycopene into the Rb. sphaeroides LH2 complex lowers the S1 level of the carotenoid sufficiently to prevent efficient transfer of energy to the B800 Q(y) state, leaving only the Crt S1 --> B850 channel, strongly suggesting that Crt S1 --> BChl energy transfer is controlled by the relative Crt S1 and BChl Q(y) energies.

  14. Engineering of a calcium-ion binding site into the RC-LH1-PufX complex of Rhodobacter sphaeroides to enable ion-dependent spectral red-shifting.

    PubMed

    Swainsbury, David J K; Martin, Elizabeth C; Vasilev, Cvetelin; Parkes-Loach, Pamela S; Loach, Paul A; Neil Hunter, C

    2017-11-01

    The reaction centre-light harvesting 1 (RC-LH1) complex of Thermochromatium (Tch.) tepidum has a unique calcium-ion binding site that enhances thermal stability and red-shifts the absorption of LH1 from 880nm to 915nm in the presence of calcium-ions. The LH1 antenna of mesophilic species of phototrophic bacteria such as Rhodobacter (Rba.) sphaeroides does not possess such properties. We have engineered calcium-ion binding into the LH1 antenna of Rba. sphaeroides by progressively modifying the native LH1 polypeptides with sequences from Tch. tepidum. We show that acquisition of the C-terminal domains from LH1 α and β of Tch. tepidum is sufficient to activate calcium-ion binding and the extent of red-shifting increases with the proportion of Tch. tepidum sequence incorporated. However, full exchange of the LH1 polypeptides with those of Tch. tepidum results in misassembled core complexes. Isolated α and β polypeptides from our most successful mutant were reconstituted in vitro with BChl a to form an LH1-type complex, which was stabilised 3-fold by calcium-ions. Additionally, carotenoid specificity was changed from spheroidene found in Rba. sphaeroides to spirilloxanthin found in Tch. tepidum, with the latter enhancing in vitro formation of LH1. These data show that the C-terminal LH1 α/β domains of Tch. tepidum behave autonomously, and are able to transmit calcium-ion induced conformational changes to BChls bound to the rest of a foreign antenna complex. Thus, elements of foreign antenna complexes, such as calcium-ion binding and blue/red switching of absorption, can be ported into Rhodobacter sphaeroides using careful design processes. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  15. Monomeric RC-LH1 core complexes retard LH2 assembly and intracytoplasmic membrane formation in PufX-minus mutants of Rhodobacter sphaeroides

    SciTech Connect

    Adams, Peter G.; Mothersole, David J.; Ng, Irene W.

    2011-01-01

    In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre–light-harvesting 1–PufX (RC–LH1–PufX) ‘core’ complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX -). Lower rates of LH2more » assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX - mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC–LH1–PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX - membranes, resulting in locally ordered clusters of monomeric RC–LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation.« less

  16. Monomeric RC-LH1 core complexes retard LH2 assembly and intracytoplasmic membrane formation in PufX-minus mutants of Rhodobacter sphaeroides.

    PubMed

    Adams, Peter G; Mothersole, David J; Ng, Irene W; Olsen, John D; Hunter, C Neil

    2011-09-01

    In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre-light-harvesting 1-PufX (RC-LH1-PufX) 'core' complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX(-)). Lower rates of LH2 assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX(-) mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC-LH1-PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX(-) membranes, resulting in locally ordered clusters of monomeric RC-LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation. 2011 Elsevier B.V. All rights reserved.

  17. Role of B800 in carotenoid-bacteriochlorophyll energy and electron transfer in LH2 complexes from the purple bacterium Rhodobacter sphaeroides.

    PubMed

    Polívka, Tomas; Niedzwiedzki, Dariusz; Fuciman, Marcel; Sundström, Villy; Frank, Harry A

    2007-06-28

    The role of the B800 in energy and electron transfer in LH2 complexes has been studied using femtosecond time-resolved transient absorption spectroscopy. The B800 site was perturbed by application of lithium dodecyl sulfate (LDS), and comparison of treated and untreated LH2 complexes from Rhodobacter sphaeroides incorporating carotenoids neurosporene, spheroidene, and spheroidenone was used to explore the role of B800 in carotenoid to bacteriochlorophyll-a (BChla) energy transfer and carotenoid radical formation. Efficiencies of the S1-mediated energy transfer in the LDS-treated complexes were 86, 61, and 57% in the LH2 complexes containing neurosporene, spheroidene, and spheroidenone, respectively. Analysis of the carotenoid S1 lifetimes in solution, LDS-treated, and untreated LH2 complexes allowed determination of B800/B850 branching ratio in the S1-mediated energy transfer. It is shown that B800 is a major acceptor, as approximately 60% of the energy from the carotenoid S1 state is accepted by B800. This value is nearly independent of conjugation length of the carotenoid. In addition to its role in energy transfer, the B800 BChla is the only electron acceptor in the event of charge separation between carotenoid and BChla in LH2 complexes, which is demonstrated by prevention of carotenoid radical formation in the LDS-treated LH2 complexes. In the untreated complexes containing neurosporene and spheroidene, the carotenoid radical is formed with a time constant of 300-400 fs. Application of different excitation wavelengths and intensity dependence of the carotenoid radical formation showed that the carotenoid radical can be formed only after excitation of the S2 state of carotenoid, although the S2 state itself is not a precursor of the charge-separated state. Instead, either a hot S1 state or a charge-transfer state lying between S2 and S1 states of the carotenoid are discussed as potential precursors of the charge-separated state.

  18. Binding and Energetics of Electron Transfer between an Artificial Four-Helix Mn-Protein and Reaction Centers from Rhodobacter sphaeroides.

    PubMed

    Espiritu, Eduardo; Olson, Tien L; Williams, JoAnn C; Allen, James P

    2017-12-12

    The ability of an artificial four-helix bundle Mn-protein, P1, to bind and transfer an electron to photosynthetic reaction centers from the purple bacterium Rhodobacter sphaeroides was characterized using optical spectroscopy. Upon illumination of reaction centers, an electron is transferred from P, the bacteriochlorophyll dimer, to Q A , the primary electron acceptor. The P1 Mn-protein can bind to the reaction center and reduce the oxidized bacteriochlorophyll dimer, P + , with a dissociation constant of 1.2 μM at pH 9.4, comparable to the binding constant of c-type cytochromes. Amino acid substitutions of surface residues on the Mn-protein resulted in increases in the dissociation constant to 8.3 μM. The extent of reduction of P + by the P1 Mn-protein was dependent on the P/P + midpoint potential and the pH. Analysis of the free energy difference yielded a midpoint potential of approximately 635 mV at pH 9.4 for the Mn cofactor of the P1 Mn-protein, a value similar to those found for other Mn cofactors in proteins. The linear dependence of -56 mV/pH is consistent with one proton being released upon Mn oxidation, allowing the complex to maintain overall charge neutrality. These outcomes demonstrate the feasibility of designing four-helix bundles and other artificial metalloproteins to bind and transfer electrons to bacterial reaction centers and establish the usefulness of this system as a platform for designing sites to bind novel metal cofactors capable of performing complex oxidation-reduction reactions.

  19. Mutation-Induced Changes in the Protein Environment and Site Energies in the (M)L214G Mutant of the Rhodobacter sphaeroides Bacterial Reaction Center.

    PubMed

    Jankowiak, Ryszard; Rancova, Olga; Chen, Jinhai; Kell, Adam; Saer, Rafael G; Beatty, J Thomas; Abramavicius, Darius

    2016-08-18

    This work focuses on the low-temperature (5 K) photochemical (transient) hole-burned (HB) spectra within the P870 absorption band, and their theoretical analysis, for the (M)L214G mutant of the photosynthetic Rhodobacter sphaeroides bacterial reaction center (bRC). To provide insight into system-bath interactions of the bacteriochlorophyll a (BChl a) special pair, i.e., P870, in the mutated bRC, the optical line shape function for the P870 band is calculated numerically. On the basis of the modeling studies, we demonstrate that (M)L214G mutation leads to a heterogeneous population of bRCs with modified (increased) total electron-phonon coupling strength of the special pair BChl a and larger inhomogeneous broadening. Specifically, we show that after mutation in the (M)L214G bRC a large fraction (∼50%) of the bacteriopheophytin (HA) chromophores shifts red and the 800 nm absorption band broadens, while the remaining fraction of HA cofactors retains nearly the same site energy as HA in the wild-type bRC. Modeling using these two subpopulations allowed for fits of the absorption and nonresonant (transient) HB spectra of the mutant bRC in the charge neutral, oxidized, and charge-separated states using the Frenkel exciton Hamiltonian, providing new insight into the mutant's complex electronic structure. Although the average (M)L214G mutant quantum efficiency of P(+)QA(-) state formation seems to be altered in comparison with the wild-type bRC, the average electron transfer time (measured via resonant transient HB spectra within the P870 band) was not affected. Thus, mutation in the vicinity of the electron acceptor (HA) does not tune the charge separation dynamics. Finally, quenching of the (M)L214G mutant excited states by P(+) is addressed by persistent HB spectra burned within the B band in chemically oxidized samples.

  20. Role of Rhodobacter sphaeroides photosynthetic reaction center residue M214 in the composition, absorbance properties, and conformations of H(A) and B(A) cofactors.

    PubMed

    Saer, Rafael G; Hardjasa, Amelia; Rosell, Federico I; Mauk, A Grant; Murphy, Michael E P; Beatty, J Thomas

    2013-04-02

    In the native reaction center (RC) of Rhodobacter sphaeroides, the side chain of (M)L214 projects orthogonally toward the plane and into the center of the A branch bacteriopheophytin (BPhe) macrocycle. The possibility that this side chain is responsible for the dechelation of the central Mg(2+) of bacteriochlorophyll (BChl) was investigated by replacement of (M)214 with residues possessing small, nonpolar side chains that can neither coordinate nor block access to the central metal ion. The (M)L214 side chain was also replaced with Cys, Gln, and Asn to evaluate further the requirements for assembly of the RC with BChl in the HA pocket. Photoheterotrophic growth studies showed no difference in growth rates of the (M)214 nonpolar mutants at a low light intensity, but the growth of the amide-containing mutants was impaired. The absorbance spectra of purified RCs indicated that although absorbance changes are associated with the nonpolar mutations, the nonpolar mutant RC pigment compositions are the same as in the wild-type protein. Crystal structures of the (M)L214G, (M)L214A, and (M)L214N mutants were determined (determined to 2.2-2.85 Å resolution), confirming the presence of BPhe in the HA pocket and revealing alternative conformations of the phytyl tail of the accessory BChl in the BA site of these nonpolar mutants. Our results demonstrate that (i) BChl is converted to BPhe in a manner independent of the aliphatic side chain length of nonpolar residues replacing (M)214, (ii) BChl replaces BPhe if residue (M)214 has an amide-bearing side chain, (iii) (M)214 side chains containing sulfur are not sufficient to bind BChl in the HA pocket, and (iv) the (M)214 side chain influences the conformation of the phytyl tail of the BA BChl.

  1. Electron-Transfer Secondary Reaction Matrices for MALDI MS Analysis of Bacteriochlorophyll a in Rhodobacter sphaeroides and Its Zinc and Copper Analogue Pigments

    NASA Astrophysics Data System (ADS)

    Calvano, Cosima Damiana; Ventura, Giovanni; Trotta, Massimo; Bianco, Giuliana; Cataldi, Tommaso R. I.; Palmisano, Francesco

    2017-01-01

    Bacteriochlorophyll a ( BChl a), a photosynthetic pigment performing the same functions of chlorophylls in plants, features a bacteriochlorin macrocycle ring (18 π electrons) with two reduced pyrrole rings along with a hydrophobic terpenoid side chain (i.e., the phytol residue). Chlorophylls analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is not so straightforward since pheophytinization (i.e., release of the central metal ion) and cleavage of the phytol-ester linkage are invariably observed by employing protonating matrices such as 2,5-dihydroxybenzoic acid, sinapinic acid, and α-cyano-4-hydroxycinnamic acid. Using BChl a from Rhodobacter sphaeroides R26 strain as a model system, different electron-transfer (ET) secondary reaction matrices, leading to the formation of almost stable radical ions in both positive ([M]+•) and negative ([M]-•) ionization modes at m/z 910.55, were evaluated. Compared with ET matrices such as trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), 2,2':5',2''-terthiophene (TER), anthracene (ANT), and 9,10-diphenylanthracene (DP-ANT), 1,5-diaminonaphthalene (DAN) was found to provide the highest ionization yield with a negligible fragmentation. DAN also displayed excellent ionization properties for two metal ion-substituted bacteriochlorophylls, (i.e., Zn- and Cu-BChl a at m/z 950.49 and 949.49), respectively. MALDI MS/MS of both radical charged molecular species provide complementary information, thus making analyte identification more straightforward.

  2. PucC and LhaA direct efficient assembly of the light‐harvesting complexes in Rhodobacter sphaeroides

    PubMed Central

    Mothersole, David J.; Jackson, Philip J.; Vasilev, Cvetelin; Tucker, Jaimey D.; Brindley, Amanda A.; Dickman, Mark J.

    2015-01-01

    Summary The mature architecture of the photosynthetic membrane of the purple phototroph R hodobacter sphaeroides has been characterised to a level where an atomic‐level membrane model is available, but the roles of the putative assembly proteins LhaA and PucC in establishing this architecture are unknown. Here we investigate the assembly of light‐harvesting LH2 and reaction centre‐light‐harvesting1‐PufX (RC‐LH1‐PufX) photosystem complexes using spectroscopy, pull‐downs, native gel electrophoresis, quantitative mass spectrometry and fluorescence lifetime microscopy to characterise a series of lha A and puc C mutants. LhaA and PucC are important for specific assembly of LH1 or LH2 complexes, respectively, but they are not essential; the few LH1 subunits found in Δlha A mutants assemble to form normal RC‐LH1‐PufX core complexes showing that, once initiated, LH1 assembly round the RC is cooperative and proceeds to completion. LhaA and PucC form oligomers at sites of initiation of membrane invagination; LhaA associates with RCs, bacteriochlorophyll synthase (BchG), the protein translocase subunit YajC and the YidC membrane protein insertase. These associations within membrane nanodomains likely maximise interactions between pigments newly arriving from BchG and nascent proteins within the SecYEG‐SecDF‐YajC‐YidC assembly machinery, thereby co‐ordinating pigment delivery, the co‐translational insertion of LH polypeptides and their folding and assembly to form photosynthetic complexes. PMID:26419219

  3. Bioaugmentation of Lactobacillus delbrueckii ssp. bulgaricus TISTR 895 to enhance bio-hydrogen production of Rhodobacter sphaeroides KKU-PS5.

    PubMed

    Laocharoen, Sucheera; Reungsang, Alissara; Plangklang, Pensri

    2015-01-01

    Bioaugmentation or an addition of the desired microorganisms or specialized microbial strains into the anaerobic digesters can enhance the performance of microbial community in the hydrogen production process. Most of the studies focused on a bioaugmentation of native microorganisms capable of producing hydrogen with the dark-fermentative hydrogen producers while information on bioaugmentation of purple non-sulfur photosynthetic bacteria (PNSB) with lactic acid-producing bacteria (LAB) is still limited. In our study, bioaugmentation of Rhodobacter sphaeroides KKU-PS5 with Lactobacillus delbrueckii ssp. bulgaricus TISTR 895 was conducted as a method to produce hydrogen. Unfortunately, even though well-characterized microorganisms were used in the fermentation system, a cultivation of two different organisms in the same bioreactor was still difficult because of the differences in their metabolic types, optimal conditions, and nutritional requirements. Therefore, evaluation of the physical and chemical factors affecting hydrogen production of PNSB augmented with LAB was conducted using a full factorial design followed by response surface methodology (RSM) with central composite design (CCD). A suitable LAB/PNSB ratio and initial cell concentration were found to be 1/12 (w/w) and 0.15 g/L, respectively. The optimal initial pH, light intensity, and Mo concentration obtained from RSM with CCD were 7.92, 8.37 klux and 0.44 mg/L, respectively. Under these optimal conditions, a cumulative hydrogen production of 3396 ± 66 mL H2/L, a hydrogen production rate (HPR) of 9.1 ± 0.2 mL H2/L h, and a hydrogen yield (HY) of 9.65 ± 0.23 mol H2/mol glucose were obtained. KKU-PS5 augmented with TISTR 895 produced hydrogen from glucose at a relatively high HY, 9.65 ± 0.23 mol H2/mol glucose, i.e., 80 % of the theoretical yield. The ratio of the strains TISTR 895/KKU-PS5 and their initial cell concentrations affected the rate of lactic acid production and its

  4. Structure and function of cytochrome c2 in electron transfer complexes with the photosynthetic reaction center of Rhodobacter sphaeroides: optical linear dichroism and EPR.

    PubMed

    Drepper, F; Mathis, P

    1997-02-11

    The photosynthetic reaction center (RC) and its secondary electron donor the water-soluble cytochrome (cyt) c2 from the purple bacterium Rhodobacter sphaeroides have been used in cross-linked and non-cross-linked complexes, oriented in compressed gels or partially dried multilayers, to study the respective orientation of the primary donor P (BChl dimer) and of cyt c2. Three methods were used: (i) Polarized optical absorption spectra at 295 and 10 K were measured and the linear dichroism of the two individual transitions (Qx, Qy), which are nearly degenerate within the alpha-band of reduced cyt c2, was determined. Attribution of the polarization directions to the molecular axes within the heme plane yielded the average cyt orientation in the complexes. (ii) Time-resolved flash absorption measurements using polarized light allowed determination of the orientation of cyt c2 in complexes which differ in their kinetics of electron transfer. (iii) EPR spectroscopy of ferricyt c2 in cross-linked RC-cyt c2 complexes was used to determine the angle between the heme and the membrane plane. The results suggest the following structural properties for the docking of cyt c2 to the RC: (i) In cross-linked complexes, the two cytochromes displaying half-lives of 0.7 and 60 micros for electron transfer to P+ are similarly oriented (difference < 10 degrees). (ii) For cross-linked cyt c2 the heme plane is parallel to the symmetry axis of the RC (0 degrees +/- 10 degrees). Moreover, the Qy transition, which is assumed to be polarized within the ring III-ring I direction of the heme plane, makes an angle of 56 degrees +/- 1 degree with the symmetry axis. (iii) The dichroism spectrum for the fast phase (0.7 micros) for the non-cross-linked cyt c2-RC complex suggests an orientation similar to that of cross-linked cyt c2, but the heme plane is tilted about 20 degrees closer to the membrane. An alternative model is that two or more bound states of cyt c2 with heme plane tilt angles between

  5. Elimination of polarity in the carotenoid terminus promotes the exposure of B850-binding sites (Tyr 44, 45) and ANS-mediated energy transfer in LH2 complexes of Rhodobacter sphaeroides.

    PubMed

    Liu, Yuan; Wu, Yongqiang; Xu, Chunhe

    2004-12-10

    Carotenoids in the peripheral light-harvesting complexes (LH2) of the green mutant (GM309) of Rhodobacter sphaeroides were identified as containing neurosporenes, which lack the polar CH(3)O group, compared to spheroidenes in native-LH2 of R. sphaeroides 601. After LH2 complexes were treated with 1-anilino-8-naphthalene sulfonate (ANS), new energy transfer pathways from ANS or tryptophan to carotenoids were discovered in both native- and GM309-LH2. The carotenoid fluorescence intensity of GM309-LH2 was greater than that of native-LH2 when bound with ANS, suggesting that the elimination of polarity in the neurosporene increases the energy transfer from ANS to carotenoid. The fact that two alpha-tyrosines (alpha-Tyr 44, 45, B850-binding sites) in each alpha-apoprotein of GM309-LH2 were more easily modified than those of native-LH2 by N-acetylimidazole (NAI) indicates that the elimination of polarity in the neurosporene terminus increases the exposure of these sites to solution.

  6. Regulation of a polyamine transporter by the conserved 3′ UTR-derived sRNA SorX confers resistance to singlet oxygen and organic hydroperoxides in Rhodobacter sphaeroides

    PubMed Central

    Peng, Tao; Berghoff, Bork A.; Oh, Jeong-Il; Weber, Lennart; Schirmer, Jasmin; Schwarz, Johannes; Glaeser, Jens; Klug, Gabriele

    2016-01-01

    ABSTRACT Singlet oxygen is generated by bacteriochlorophylls when light and oxygen are simultaneously present in Rhodobacter sphaeroides. Singlet oxygen triggers a specific response that is partly regulated by the alternative sigma factor RpoHI/HII. The sRNA RSs2461 has previously been identified as an RpoHI/HII-dependent sRNA and is derived from the 3′ UTR of the mRNA for an OmpR-type transcriptional regulator. Similar to the RpoHI/HII-dependent CcsR and SorY sRNAs, RSs2461 affects the resistance of R. sphaeroides against singlet oxygen and was therefore renamed here SorX. Furthermore, SorX has a strong impact on resistance against organic hydroperoxides that usually occur as secondary damages downstream of singlet oxygen. The 75-nt SorX 3′ fragment, which is generated by RNase E cleavage and highly conserved among related species, represents the functional entity. A target search identified potA mRNA, which encodes a subunit of a polyamine transporter, as a direct SorX target and stress resistance via SorX could be linked to potA. The PotABCD transporter is an uptake system for spermidine in E. coli. While spermidine is generally described as beneficial during oxidative stress, we observed significantly increased sensitivity of R. sphaeroides to organic hydroperoxides in the presence of spermidine. We therefore propose that the diminished import of spermidine, due to down-regulation of potA by SorX, counteracts oxidative stress. Together with results from other studies this underlines the importance of regulated transport to bacterial stress defense. PMID:27420112

  7. Regulation of a polyamine transporter by the conserved 3' UTR-derived sRNA SorX confers resistance to singlet oxygen and organic hydroperoxides in Rhodobacter sphaeroides.

    PubMed

    Peng, Tao; Berghoff, Bork A; Oh, Jeong-Il; Weber, Lennart; Schirmer, Jasmin; Schwarz, Johannes; Glaeser, Jens; Klug, Gabriele

    2016-10-02

    Singlet oxygen is generated by bacteriochlorophylls when light and oxygen are simultaneously present in Rhodobacter sphaeroides. Singlet oxygen triggers a specific response that is partly regulated by the alternative sigma factor RpoHI/HII. The sRNA RSs2461 has previously been identified as an RpoHI/HII-dependent sRNA and is derived from the 3' UTR of the mRNA for an OmpR-type transcriptional regulator. Similar to the RpoHI/HII-dependent CcsR and SorY sRNAs, RSs2461 affects the resistance of R. sphaeroides against singlet oxygen and was therefore renamed here SorX. Furthermore, SorX has a strong impact on resistance against organic hydroperoxides that usually occur as secondary damages downstream of singlet oxygen. The 75-nt SorX 3' fragment, which is generated by RNase E cleavage and highly conserved among related species, represents the functional entity. A target search identified potA mRNA, which encodes a subunit of a polyamine transporter, as a direct SorX target and stress resistance via SorX could be linked to potA. The PotABCD transporter is an uptake system for spermidine in E. coli. While spermidine is generally described as beneficial during oxidative stress, we observed significantly increased sensitivity of R. sphaeroides to organic hydroperoxides in the presence of spermidine. We therefore propose that the diminished import of spermidine, due to down-regulation of potA by SorX, counteracts oxidative stress. Together with results from other studies this underlines the importance of regulated transport to bacterial stress defense.

  8. Evolutionary implications of phylogenetic analyses of the gene transfer agent (GTA) of Rhodobacter capsulatus.

    PubMed

    Lang, Andrew S; Taylor, Terumi A; Beatty, J Thomas

    2002-11-01

    The gene transfer agent (GTA) of the a-proteobacterium Rhodobacter capsulatus is a cell-controlled genetic exchange vector. Genes that encode the GTA structure are clustered in a 15-kb region of the R. capsulatus chromosome, and some of these genes show sequence similarity to known bacteriophage head and tail genes. However, the production of GTA is controlled at the level of transcription by a cellular two-component signal transduction system. This paper describes homologues of both the GTA structural gene cluster and the GTA regulatory genes in the a-proteobacteria Rhodopseudomonas palustris, Rhodobacter sphaeroides, Caulobacter crescentus, Agrobacterium tumefaciens and Brucella melitensis. These sequences were used in a phylogenetic tree approach to examine the evolutionary relationships of selected GTA proteins to these homologues and (pro)phage proteins, which was compared to a 16S rRNA tree. The data indicate that a GTA-like element was present in a single progenitor of the extant species that contain both GTA structural cluster and regulatory gene homologues. The evolutionary relationships of GTA structural proteins to (pro)phage proteins indicated by the phylogenetic tree patterns suggest a predominantly vertical descent of GTA-like sequences in the a-proteobacteria and little past gene exchange with (pro)phages.

  9. The crystal structures of the tri-functional Chloroflexus aurantiacus and bi-functional Rhodobacter sphaeroides malyl-CoA lyases and comparison with CitE-like superfamily enzymes and malate synthases.

    PubMed

    Zarzycki, Jan; Kerfeld, Cheryl A

    2013-11-09

    Malyl-CoA lyase (MCL) is a promiscuous carbon-carbon bond lyase that catalyzes the reversible cleavage of structurally related Coenzyme A (CoA) thioesters. This enzyme plays a crucial, multifunctional role in the 3-hydroxypropionate bi-cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus. A second, phylogenetically distinct MCL from Rhodobacter sphaeroides is involved in the ethylmalonyl-CoA pathway for acetate assimilation. Both MCLs belong to the large superfamily of CitE-like enzymes, which includes the name-giving β-subunit of citrate lyase (CitE), malyl-CoA thioesterases and other enzymes of unknown physiological function. The CitE-like enzyme superfamily also bears sequence and structural resemblance to the malate synthases. All of these different enzymes share highly conserved catalytic residues, although they catalyze distinctly different reactions: C-C bond formation and cleavage, thioester hydrolysis, or both (the malate synthases). Here we report the first crystal structures of MCLs from two different phylogenetic subgroups in apo- and substrate-bound forms. Both the C. aurantiacus and the R. sphaeroides MCL contain elaborations on the canonical β8/α8 TIM barrel fold and form hexameric assemblies. Upon ligand binding, changes in the C-terminal domains of the MCLs result in closing of the active site, with the C-terminal domain of one monomer forming a lid over and contributing side chains to the active site of the adjacent monomer. The distinctive features of the two MCL subgroups were compared to known structures of other CitE-like superfamily enzymes and to malate synthases, providing insight into the structural subtleties that underlie the functional versatility of these enzymes. Although the C. aurantiacus and the R. sphaeroides MCLs have divergent primary structures (~37% identical), their tertiary and quaternary structures are very similar. It can be assumed that the C-C bond formation catalyzed by the MCLs occurs as proposed for

  10. The unusually strong hydrogen bond between the carbonyl of Q(A) and His M219 in the Rhodobacter sphaeroides reaction center is not essential for efficient electron transfer from Q(A)(-) to Q(B).

    PubMed

    Breton, Jacques; Lavergne, Jérôme; Wakeham, Marion C; Nabedryk, Eliane; Jones, Michael R

    2007-06-05

    In native reaction centers (RCs) from photosynthetic purple bacteria the primary quinone (QA) and the secondary quinone (QB) are interconnected via a specific His-Fe-His bridge. In Rhodobacter sphaeroides RCs the C4=O carbonyl of QA forms a very strong hydrogen bond with the protonated Npi of His M219, and the Ntau of this residue is in turn coordinated to the non-heme iron atom. The second carbonyl of QA is engaged in a much weaker hydrogen bond with the backbone N-H of Ala M260. In previous work, a Trp side chain was introduced by site-directed mutagenesis at the M260 position in the RC of Rb. sphaeroides, resulting in a complex that is completely devoid of QA and therefore nonfunctional. A photochemically competent derivative of the AM260W mutant was isolated that contains a Cys side chain at the M260 position (denoted AM260(W-->C)). In the present work, the interactions between the carbonyl groups of QA and the protein in the AM260(W-->C) suppressor mutant have been characterized by light-induced FTIR difference spectroscopy of the photoreduction of QA. The QA-/QA difference spectrum demonstrates that the strong interaction between the C4=O carbonyl of QA and His M219 is lost in the mutant, and the coupled CO and CC modes of the QA- semiquinone are also strongly perturbed. In parallel, a band assigned to the perturbation of the C5-Ntau mode of His M219 upon QA- formation in the native RC is lacking in the spectrum of the mutant. Furthermore, a positive band between 2900 and 2400 cm-1 that is related to protons fluctuating within a network of highly polarizable hydrogen bonds in the native RC is reduced in amplitude in the mutant. On the other hand, the QB-/QB FTIR difference spectrum is essentially the same as for the native RC. The kinetics of electron transfer from QA- to QB were measured by the flash-induced absorption changes at 780 nm. Compared to native RCs the absorption transients are slowed by a factor of about 2 for both the slow phase (in the

  11. Role of PufX protein in photosynthetic growth of Rhodobacter sphaeroides. 1. PufX is required for efficient light-driven electron transfer and photophosphorylation under anaerobic conditions.

    PubMed

    Barz, W P; Francia, F; Venturoli, G; Melandri, B A; Verméglio, A; Oesterhelt, D

    1995-11-21

    The pufX gene is essential for photoheterotrophic growth of the purple bacterium Rhodobacter sphaeroides. In order to analyze the molecular function of the PufX membrane protein, we constructed a chromosomal pufX deletion mutant and phenotypically compared it to a pufX+ control strain and to two suppressor mutants which are able to grow photosynthetically in the absence of pufX. Using this genetic background, we confirmed that PufX is required for photoheterotrophic growth under anaerobic conditions, although all components of the photosynthetic apparatus were present in similar amounts in all strains investigated. We show that the deletion of PufX is not lethal for illuminated pufX- cells, suggesting that PufX is required for photosynthetic cell division. Since chromatophores isolated from the pufX- mutant were found to be unsealed vesicles, the role of PufX in photosynthetic energy transduction was studied in vivo. We show that PufX is essential for light-induced ATP synthesis (photophosphorylation) in anaerobically incubated cells. Measurements of absorption changes induced by a single turnover flash demonstrated that PufX is not required for electron flow through the reaction center and the cytochrome bc1 complex under anaerobic conditions. During prolonged illumination, however, PufX is essential for the generation of a sufficiently large membrane potential to allow photosynthetic growth. These in vivo results demonstrate that under anaerobic conditions PufX plays an essential role in facilitating effective interaction of the components of the photosynthetic apparatus.

  12. Mechanism of energy transfer from carotenoids to bacteriochlorophyll : light-harvesting by carotenoids having different extents of {pi}-electron conjugation incorporated into the B850 antenna complex from the carotenoidless bacterium Rhodobacter sphaeroides R-26.1.

    SciTech Connect

    Desamero, R. Z. B.; Chynwat, V.; van der Hoef, I.

    1998-10-15

    Spheroidene and a series of spheroidene analogues with extents of p-electron conjugation ranging from 7 to 13 carbon-carbon double bonds were incorporated into the B850 light-harvesting complex of Rhodobacter sphaeroides R-26.1. The structures and spectroscopic properties of the carotenoids and the dynamics of energy transfer from the carotenoid to bacteriochlorophyll (BChl) in the B850 complex were studied by using steady-state absorption, fluorescence, fluorescence excitation, resonance Raman, and time-resolved absorption spectroscopy. The spheroidene analogues used in this study were 5',6'-dihydro-7',8'-didehydrospheroidene, 7',8'-didehydrospheroidene, and 1',2'-dihydro-3',4',7',8'-tetradehydrospheroidene. These data, taken together with results from 3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene, and spheroidene already published (Frank, H. A.; Farhoosh,more » R.; Aldema, M. L.; DeCoster, B.; Christensen, R. L.; Gebhard, R.; Lugtenburg, J. Photochem. Photobiol. 1993, 57, 49. Farhoosh, R.; Chynwat, V.; Gebhard, R.; Lugtenburg, J.; Frank, H. A. Photosynth. Res. 1994, 42, 157), provide a systematic series of molecules for understanding the molecular features that determine the mechanism of energy transfer from carotenoids to BChl in photosynthetic bacterial light-harvesting complexes. The data support the hypothesis that only carotenoids having 10 or less carbon-carbon double bonds transfer energy via their 21Ag (S1) states to BChl to any significant degree. Energy transfer via the 11Bu (S2) state of the carotenoid becomes more important than the S1 route as the number of conjugated carbon-carbon double bonds increases. The results also suggest that the S2 state associated with the Qx transition of the B850 BChl is the most likely acceptor state for energy transfer originating from both the 2{sup 1}A{sub g} (S{sub 1}) and 1{sup 1}B{sub u} (S{sub 2}) states of all carotenoids.« less

  13. Role of the PufX protein in photosynthetic growth of Rhodobacter sphaeroides. 2. PufX is required for efficient ubiquinone/ubiquinol exchange between the reaction center QB site and the cytochrome bc1 complex.

    PubMed

    Barz, W P; Verméglio, A; Francia, F; Venturoli, G; Melandri, B A; Oesterhelt, D

    1995-11-21

    The PufX membrane protein is essential for photosynthetic growth of Rhodobacter sphaeroides because it is required for multiple-turnover electron transfer under anaerobic conditions [see accompanying article; Barz, W. P., Francia, F., Venturoli, G., Melandri, B. A., Verméglio, A., & Oesterhelt, D. (1995) Biochemistry 34, 15235-15247]. In order to understand the molecular role of PufX, light-induced absorption spectroscopy was performed using a pufX- mutant, a pufX+ strain, and two suppressor mutants. We show that the reaction center (RC) requires PufX for its functionality under different redox conditions than the cytochrome bc1 complex: When the kinetics of flash-induced reduction of cytochrome b561 were monitored in chromatophores, we observed a requirement of PufX for turnover of the cytochrome bc1 complex only at high redox potential (Eh > 140 mV), suggesting a function of PufX in lateral ubiquinol transfer from the RC. In contrast, PufX is required for multiple turnover of the RC only under reducing conditions: When the Q pool was partially oxidized in vivo using oxygen or electron acceptors like dimethyl sulfoxide or trimethylamine N-oxide, the deletion of PufX had no effect on light-driven electron flow through the RC. Flash train experiments under anaerobic in vivo conditions revealed that RC photochemistry does not depend on PufX for the first two flash excitations. Following the third and subsequent flashes, however, efficient charge separation requires PufX, indicating an important role of PufX for fast Q/QH2 exchange at the QB site of the RC. We show that the Q/QH2 exchange rate is reduced approximately 500-fold by the deletion of PufX when the Q pool is nearly completely reduced, demonstrating an essential role of PufX for the access of ubiquinone to the QB site. The fast ubiquinone/ubiquinol exchange is partially restored by suppressor mutations altering the macromolecular antenna structure. These results suggest an indirect role of PufX in

  14. Analysis of the kinetics of P+ HA- recombination in membrane-embedded wild-type and mutant Rhodobacter sphaeroides reaction centers between 298 and 77 K indicates that the adjacent negatively charged QA ubiquinone modulates the free energy of P+ HA- and may influence the rate of the protein dielectric response.

    PubMed

    Gibasiewicz, Krzysztof; Pajzderska, Maria; Dobek, Andrzej; Brettel, Klaus; Jones, Michael R

    2013-09-26

    Time-resolved spectroscopic studies of recombination of the P(+)HA(-) radical pair in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides give an opportunity to study protein dynamics triggered by light and occurring over the lifetime of P(+)HA(-). The state P(+)HA(-) is formed after the ultrafast light-induced electron transfer from the primary donor pair of bacteriochlorophylls (P) to the acceptor bacteriopheophytin (HA). In order to increase the lifetime of this state, and thus increase the temporal window for the examination of protein dynamics, it is possible to block forward electron transfer from HA(-) to the secondary electron acceptor QA. In this contribution, the dynamics of P(+)HA(-) recombination were compared at a range of temperatures from 77 K to room temperature, electron transfer from HA(-) to QA being blocked either by prereduction of QA or by genetic removal of QA. The observed P(+)HA(-) charge recombination was significantly slower in the QA-deficient RCs, and in both types of complexes, lowering the temperature from RT to 77 K led to a slowing of charge recombination. The effects are explained in the frame of a model in which charge recombination occurs via competing pathways, one of which is thermally activated and includes transient formation of a higher-energy state, P(+)BA(-). An internal electrostatic field supplied by the negative charge on QA increases the free energy levels of the state P(+)HA(-), thus decreasing its energetic distance to the state P(+)BA(-). In addition, the dielectric response of the protein environment to the appearance of the state P(+)HA(-) is accelerated from ∼50-100 ns in the QA-deficient mutant RCs to ∼1-16 ns in WT RCs with a negatively charged QA(-). In both cases, the temperature dependence of the protein dynamics is weak.

  15. Comparison of the fluorescence kinetics of detergent-solubilized and membrane-reconstituted LH2 complexes from Rps. acidophila and Rb. sphaeroides.

    PubMed

    Pflock, Tobias; Dezi, Manuela; Venturoli, Giovanni; Cogdell, Richard J; Köhler, Jürgen; Oellerich, Silke

    2008-01-01

    Picosecond time-resolved fluorescence spectroscopy has been used in order to compare the fluorescence kinetics of detergent-solubilized and membrane-reconstituted light-harvesting 2 (LH2) complexes from the purple bacteria Rhodopseudomonas (Rps.) acidophila and Rhodobacter (Rb.) sphaeroides. LH2 complexes were reconstituted in phospholipid model membranes at different lipid:protein-ratios and all samples were studied exciting with a wide range of excitation densities. While the detergent-solubilized LH2 complexes from Rps. acidophila showed monoexponential decay kinetics (tau(f )= 980 ps) for excitation densities of up to 3.10(13) photons/(pulse.cm(2)), the membrane-reconstituted LH2 complexes showed multiexponential kinetics even at low excitation densities and high lipid:protein-ratios. The latter finding indicates an efficient clustering of LH2 complexes in the phospholipid membranes. Similar results were obtained for the LH2 complexes from Rb. sphaeroides.

  16. Structure of the dimeric PufX-containing core complex of Rhodobacter blasticus by in situ atomic force microscopy.

    PubMed

    Scheuring, Simon; Busselez, Johan; Lévy, Daniel

    2005-01-14

    We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 alpha/beta-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of approximately 25 A. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a approximately 25-A wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.

  17. Alternative initial proton acceptors for the D pathway of Rhodobacter sphaeroides cytochrome c oxidase

    PubMed Central

    Varanasi, Lakshman; Hosler, Jonathan

    2011-01-01

    In order to characterize protein structures that control proton uptake, forms of cytochrome c oxidase (CcO) containing a carboxyl or a thiol group in line with the initial, internal waters of the D pathway for proton transfer have been assayed in the presence and absence of subunit III. Subunit III provides approximately half of the protein surrounding the entry region of the D pathway. The mutant N139D-D132N contains a carboxyl group 6Å within the D pathway and lacks the normal, surface-exposed proton acceptor, Asp-132. With subunit III, the steady-state activity of this mutant is slow but once subunit III is removed its activity is the same as wild-type CcO lacking subunit III (∼1800 H+ s-1). Thus, a carboxyl group ∼25% within the pathway enhances proton uptake even though the carboxyl has no direct contact with bulk solvent. Protons from solvent apparently move to internal Asp-139 through a short file of waters, normally blocked by subunit III. Cysteine-139 also supports rapid steady-state proton uptake, demonstrating that an anion other than a carboxyl can attract and transfer protons into the D pathway. When both Asp-132 and Asp/Cys-139 are present, the removal of subunit III increases CcO activity to rates greater than that of normal CcO due to simultaneous proton uptake by two initial acceptors. The results show how the environment of the initial proton acceptor for the D pathway in these CcO forms dictates the pH range of CcO activity, with implications for the function of Asp-132, the normal proton acceptor. PMID:21344856

  18. The Flagellar Protein FliL Is Essential for Swimming in Rhodobacter sphaeroides▿ †

    PubMed Central

    Suaste-Olmos, Fernando; Domenzain, Clelia; Mireles-Rodríguez, José Cruz; Poggio, Sebastian; Osorio, Aurora; Dreyfus, Georges; Camarena, Laura

    2010-01-01

    In this work we characterize the function of the flagellar protein FliL in Rhodobacter sphaeroides. Our results show that FliL is essential for motility in this bacterium and that in its absence flagellar rotation is highly impaired. A green fluorescent protein (GFP)-FliL fusion forms polar and lateral fluorescent foci that show different spatial dynamics. The presence of these foci is dependent on the expression of the flagellar genes controlled by the master regulator FleQ, suggesting that additional components of the flagellar regulon are required for the proper localization of GFP-FliL. Eight independent pseudorevertants were isolated from the fliL mutant strain. In each of these strains a single nucleotide change in motB was identified. The eight mutations affected only three residues located on the periplasmic side of MotB. Swimming of the suppressor mutants was not affected by the presence of the wild-type fliL allele. Pulldown and yeast two-hybrid assays showed that that the periplasmic domain of FliL is able to interact with itself but not with the periplasmic domain of MotB. From these results we propose that FliL could participate in the coupling of MotB with the flagellar rotor in an indirect fashion. PMID:20889747

  19. Treatment of anaerobically digested swine wastewater by Rhodobacter blasticus and Rhodobacter capsulatus.

    PubMed

    Wen, Shan; Liu, Hongyu; He, Huijun; Luo, Le; Li, Xiang; Zeng, Guangming; Zhou, Zili; Lou, Wei; Yang, Chunping

    2016-12-01

    Two strains of photosynthetic bacteria, Rhodobacter blasticus and Rhodobacter capsulatus, were used in this work to investigate the feasibility of using photosynthetic bacteria for the treatment of anaerobically digested swine wastewater. The effects of crucial factors which influence the pollutants removal efficiency were also examined. Results showed that anaerobically digested swine wastewater could be treated effectively by photosynthetic bacteria. The treatment efficiency was significantly higher by the mixed photosynthetic bacteria than that by any unitary bacterium. The optimal treatment condition by mixed bacteria was inoculation of 10.0%(v/v) of the two bacteria by 1:1, initial pH of 7.0 and initial chemical oxygen demand of 4800mgL -1 . Under these conditions, the removal rate of chemical oxygen demand was 83.3%, which was 19.3% higher than when using Rhodobacter blasticus or 10.6% higher than when using Rhodobacter capsulatus separately. This mixed photosynthetic bacteria achieved high chemical oxygen demand removal and cell yields. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Electrostatic dominoes: long distance propagation of mutational effects in photosynthetic reaction centers of Rhodobacter capsulatus.

    PubMed

    Sebban, P; Maróti, P; Schiffer, M; Hanson, D K

    1995-07-04

    Two point mutants from the purple bacterium Rhodobacter capsulatus, both modified in the M protein of the photosynthetic reaction center, have been studied by flash-induced absorbance spectroscopy. These strains carry either the M231Arg --> Leu or M43ASN --> Asp mutations, which are located 9 and 15 A, respectively, from the terminal electron acceptor QB. In the wild-type Rb. sphaeroides structure, M231Arg is involved in a conserved salt bridge with H125Glu and H232Glu and M43Asn is located among several polar residues that form or surround the QB binding site. These substitutions were originally uncovered in phenotypic revertants isolated from the photosynthetically incompetent L212Glu-L213Asp --> Ala-Ala site-specific double mutant. As second-site suppressor mutations, they have been shown to restore the proton transfer function that is interrupted in the L212Ala-L213Ala double mutant. The electrostatic effects that are induced in reaction centers by the M231Arg --> Leu and M43Asn --> Asp substitutions are roughly the same in either the double-mutant or wild-type backgrounds. In a reaction center that is otherwise wild type in sequence, they decrease the free energy gap between the QA- and QB- states by 24 +/- 5 and 45 +/- 5 meV, respectively. The pH dependences of K2, the QA-QB <--> QAQB- equilibrium constant, are altered in reaction centers that carry either of these substitutions, revealing differences in the pKas of titratable groups compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Soluble Variants of Rhodobacter capsulatus Membrane-anchored Cytochrome cy Are Efficient Photosynthetic Electron Carriers*

    PubMed Central

    Öztürk, Yavuz; Lee, Dong-Woo; Mandaci, Sevnur; Osyczka, Artur; Prince, Roger C.; Daldal, Fevzi

    2008-01-01

    Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c2 and the membrane-anchored cyt cy. In species like R. capsulatus, cyt cy functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt cy (S-cy), by fusing genetically the cyt c2 signal sequence to the cyt c domain of cyt cy. This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-cy regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-cy variants at amounts comparable with that of cyt c2, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-cy was about 340 mV, similar to that of cyts c2 or cy. Remarkably, electron transfer kinetics from the cyt bc1 complex to the photochemical reaction center (RC) mediated by cyt S-cy was distinct from those seen with the cyt c2 or cyt cy. The kinetics exhibited a pronounced slow phase, suggesting that cyt S-cy interacted with the RC less tightly than cyt c2. Comparison of structural models of cyts c2 and S-cy revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c2 to the RC are not conserved in cyt cy, whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers. PMID:18343817

  2. Triazine herbicide resistance in the photosynthetic bacterium Rhodopseudomonas sphaeroides

    PubMed Central

    Brown, Alfred E.; Gilbert, Carl W.; Guy, Rachel; Arntzen, Charles J.

    1984-01-01

    The photoaffinity herbicide azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine) selectively labels the L subunit of the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides. Herbicide-resistant mutants retain the L subunit and have altered binding properties for methylthio- and chloro-substituted triazines as well as altered equilibrium constants for electron transfer between primary and secondary electron acceptors. We suggest that a subtle alteration in the L subunit is responsible for herbicide resistance and that the L subunit is the functional analog of the 32-kDa QB protein of chloroplast membranes. Images PMID:16593520

  3. Electron spin relaxation enhancement measurements of interspin distances in human, porcine, and Rhodobacter electron transfer flavoprotein ubiquinone oxidoreductase (ETF QO)

    NASA Astrophysics Data System (ADS)

    Fielding, Alistair J.; Usselman, Robert J.; Watmough, Nicholas; Simkovic, Martin; Frerman, Frank E.; Eaton, Gareth R.; Eaton, Sandra S.

    2008-02-01

    Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a membrane-bound electron transfer protein that links primary flavoprotein dehydrogenases with the main respiratory chain. Human, porcine, and Rhodobacter sphaeroides ETF-QO each contain a single [4Fe-4S] 2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated enzyme and become paramagnetic on reduction with the enzymatic electron donor or with dithionite. The anionic flavin semiquinone can be reduced further to diamagnetic hydroquinone. The redox potentials for the three redox couples are so similar that it is not possible to poise the proteins in a state where both the [4Fe-4S] + cluster and the flavoquinone are fully in the paramagnetic form. Inversion recovery was used to measure the electron spin-lattice relaxation rates for the [4Fe-4S] + between 8 and 18 K and for semiquinone between 25 and 65 K. At higher temperatures the spin-lattice relaxation rates for the [4Fe-4S] + were calculated from the temperature-dependent contributions to the continuous wave linewidths. Although mixtures of the redox states are present, it was possible to analyze the enhancement of the electron spin relaxation of the FAD semiquinone signal due to dipolar interaction with the more rapidly relaxing [4Fe-4S] + and obtain point-dipole interspin distances of 18.6 ± 1 Å for the three proteins. The point-dipole distances are within experimental uncertainty of the value calculated based on the crystal structure of porcine ETF-QO when spin delocalization is taken into account. The results demonstrate that electron spin relaxation enhancement can be used to measure distances in redox poised proteins even when several redox states are present.

  4. A glimpse into the proteome of phototrophic bacterium Rhodobacter capsulatus.

    PubMed

    Onder, Ozlem; Aygun-Sunar, Semra; Selamoglu, Nur; Daldal, Fevzi

    2010-01-01

    A first glimpse into the proteome of Rhodobacter capsulatus revealed more than 450 (with over 210 cytoplasmic and 185 extracytoplasmic known as well as 55 unknown) proteins that are identified with high degree of confidence using nLC-MS/MS analyses. The accumulated data provide a solid platform for ongoing efforts to establish the proteome of this species and the cellular locations of its constituents. They also indicate that at least 40 of the identified proteins, which were annotated in genome databases as unknown hypothetical proteins, correspond to predicted translation products that are indeed present in cells under the growth conditions used in this work. In addition, matching the identification labels of the proteins reported between the two available R. capsulatus genome databases (ERGO-light with RRCxxxxx and NT05 with NT05RCxxxx numbers) indicated that 11 such proteins are listed only in the latter database.

  5. Electron Spin Relaxation Enhancement Measurements of Interspin Distances in Human, Porcine, and Rhodobacter Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETF-QO)

    PubMed Central

    Fielding, Alistair J.; Usselman, Robert J.; Watmough, Nicholas; Simkovic, Martin; Frerman, Frank E.; Eaton, Gareth R.; Eaton, Sandra S.

    2008-01-01

    Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a membrane-bound electron transfer protein that links primary flavoprotein dehydrogenases with the main respiratory chain. Human, porcine, and Rhodobacter sphaeroides ETF-QO each contain a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated enzyme and become paramagnetic on reduction with the enzymatic electron donor or with dithionite. The anionic flavin semiquinone can be reduced further to diamagnetic hydroquinone. The redox potentials for the three redox couples are so similar that it is not possible to poise the proteins in a state where both the [4Fe-4S]+ cluster and the flavoquinone are fully in the paramagnetic form. Inversion recovery was used to measure the electron spin-lattice relaxation rates for the [4Fe-4S]+ between 8 and 18 K and for semiquinone between 25 and 65 K. At higher temperatures the spin-lattice relaxation rates for the [4Fe-4S]+ were calculated from the temperature-dependent contributions to the continuous wave linewidths. Although mixtures of the redox states are present, it was possible to analyze the enhancement of the electron spin relaxation of the FAD semiquinone signal due to dipolar interaction with the more rapidly relaxing [4Fe-4S]+ and obtain point dipole interspin distances of 18.6 ± 1 Å for the three proteins. The point-dipole distances are within experimental uncertainty of the value calculated based on the crystal structure of porcine ETF-QO when spin delocalization is taken into account. The results demonstrate that electron spin relaxation enhancement can be used to measure distances in redox poised proteins even when several redox states are present. PMID:18037314

  6. Electron spin relaxation enhancement measurements of interspin distances in human, porcine, and Rhodobacter electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO).

    PubMed

    Fielding, Alistair J; Usselman, Robert J; Watmough, Nicholas; Simkovic, Martin; Frerman, Frank E; Eaton, Gareth R; Eaton, Sandra S

    2008-02-01

    Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a membrane-bound electron transfer protein that links primary flavoprotein dehydrogenases with the main respiratory chain. Human, porcine, and Rhodobacter sphaeroides ETF-QO each contain a single [4Fe-4S](2+,1+) cluster and one equivalent of FAD, which are diamagnetic in the isolated enzyme and become paramagnetic on reduction with the enzymatic electron donor or with dithionite. The anionic flavin semiquinone can be reduced further to diamagnetic hydroquinone. The redox potentials for the three redox couples are so similar that it is not possible to poise the proteins in a state where both the [4Fe-4S](+) cluster and the flavoquinone are fully in the paramagnetic form. Inversion recovery was used to measure the electron spin-lattice relaxation rates for the [4Fe-4S](+) between 8 and 18K and for semiquinone between 25 and 65K. At higher temperatures the spin-lattice relaxation rates for the [4Fe-4S](+) were calculated from the temperature-dependent contributions to the continuous wave linewidths. Although mixtures of the redox states are present, it was possible to analyze the enhancement of the electron spin relaxation of the FAD semiquinone signal due to dipolar interaction with the more rapidly relaxing [4Fe-4S](+) and obtain point-dipole interspin distances of 18.6+/-1A for the three proteins. The point-dipole distances are within experimental uncertainty of the value calculated based on the crystal structure of porcine ETF-QO when spin delocalization is taken into account. The results demonstrate that electron spin relaxation enhancement can be used to measure distances in redox poised proteins even when several redox states are present.

  7. Culturable Rhodobacter and Shewanella species are abundant in estuarine turbidity maxima of the Columbia River

    PubMed Central

    Bräuer, S. L.; Adams, C.; Kranzler, K.; Murphy, D.; Xu, M.; Zuber, P.; Simon, H. M.; Baptista, A. M.; Tebo, B. M.

    2017-01-01

    Summary Measurements of dissolved, ascorbate-reducible and total Mn by ICP-OES revealed significantly higher concentrations during estuarine turbidity maxima (ETM) events, compared with non-events in the Columbia River. Most probable number (MPN) counts of Mn-oxidizing or Mn-reducing heterotrophs were not statistically different from that of other heterotrophs (103–104 cells ml−1) when grown in defined media, but counts of Mn oxidizers were significantly lower in nutrient-rich medium (13 cells ml−1). MPN counts of Mn oxidizers were also significantly lower on Mn(III)-pyrophosphate and glycerol (21 cells ml−1). Large numbers of Rhodobacter spp. were cultured from dilutions of 10−2 to 10−5, and many of these were capable of Mn(III) oxidation. Up to c. 30% of the colonies tested LBB positive, and all 77 of the successfully sequenced LBB positive colonies (of varying morphology) yielded sequences related to Rhodobacter spp. qPCR indicated that a cluster of Rhodobacter isolates and closely related strains (95–99% identity) represented approximately 1–3% of the total Bacteria, consistent with clone library results. Copy numbers of SSU rRNA genes for either Rhodobacter spp. or Bacteria were four to eightfold greater during ETM events compared with non-events. Strains of a Shewanella sp. were retrieved from the highest dilutions (10−5) of Mn reducers, and were also capable of Mn oxidation. The SSU rRNA gene sequences from these strains shared a high identity score (98%) with sequences obtained in clone libraries. Our results support previous findings that ETMs are zones with high microbial activity. Results indicated that Shewanella and Rhodobacter species were present in environmentally relevant concentrations, and further demonstrated that a large proportion of culturable bacteria, including Shewanella and Rhodobacter spp., were capable of Mn cycling in vitro. PMID:20977571

  8. The Single Superoxide Dismutase of Rhodobacter capsulatus Is a Cambialistic, Manganese-Containing Enzyme

    PubMed Central

    Tabares, Leandro C.; Bittel, Cristian; Carrillo, Néstor; Bortolotti, Ana; Cortez, Néstor

    2003-01-01

    The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SODRc) homologous to iron-containing superoxide dismutase enzymes. Recombinant SODRc, however, displayed higher activity after refolding with Mn2+, especially when the pH of the assay mixture was raised. SODRc isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SODRc behaves as a Mn-containing dismutase with cambialistic properties. PMID:12730184

  9. Nostoc sphaeroides Kützing, an excellent candidate producer for CELSS

    NASA Astrophysics Data System (ADS)

    Hao, Zongjie; Li, Dunhai; Li, Yanhui; Wang, Zhicong; Xiao, Yuan; Wang, Gaohong; Liu, Yongding; Hu, Chunxiang; Liu, Qifang

    2011-11-01

    Some phytoplankton can be regarded as possible candidates in the establishment of Controlled Ecological Life Support System (CELSS) for some intrinsic characteristics, the first characteristic is that they should grow rapidly, secondly, they should be able to endure some stress factors and develop some corresponding adaptive strategies; also it is very important that they could provide food rich in nutritious protein and vitamins for the crew; the last but not the least is they can also fulfill the other main functions of CELSS, including supplying oxygen, removing carbon dioxide and recycling the metabolic waste. According to these characteristics, Nostoc sphaeroides, a potential healthy food in China, was selected as the potential producer in CELSS. It was found that the oxygen average evolution rate of this algae is about 150 μmol O 2 mg -1 h -1, and the size of them are ranged from 2 to 20 mm. Also it can be cultured with high population density, which indicated that the potential productivity of Nostoc sphaeroides is higher than other algae in limited volume. We measured the nutrient contents of the cyanobacterium and concluded it was a good food for the crew. Based on above advantages, Nostoc sphaeroides was assumed to a suitable phytoplankton for the establishment of Controlled Ecological Life Support System. We plan to develop suitable bioreactor with the cyanobacterium for supplying oxygen and food in future space missions.

  10. Effects of light intensity and quality on phycobiliprotein accumulation in the cyanobacterium Nostoc sphaeroides Kützing.

    PubMed

    Ma, Rui; Lu, Fan; Bi, Yonghong; Hu, Zhengyu

    2015-08-01

    To assess the effects of light intensity and quality on the growth and phycobiliproteins (PBP) accumulation in Nostoc sphaeroides Kützing (N. sphaeroides). Dry weights, dry matter, protein, chlorophyll and PBP contents were higher under 90 μmol m(-2) s(-1) than under other intensities (both higher and lower). Phycocyanin and allophycocyanin increased with light intensity while phycoerythrin decreased. Fresh weights, protein and PBP contents increased at the highest rates under blue light. Red light resulted in higher values of dry matter, phycocyanin and chlorophyll a. White light at 90 μmol m(-2) s(-1) or blue light 30 μmol m(-2) s(-1) were optimal for the growth and phycobiliprotein accumulation in N. sphaeroides.

  11. Nostoc sphaeroides Kütz, a candidate producer par excellence for CELSS

    NASA Astrophysics Data System (ADS)

    Wang, Gaohong; Hao, Zongjie; Liu, Yongding

    A lot of aquatic organisms could be regarded as suitable candidates par excellence in the establishment of CELSS, since they are relatively easy and fast to grow and resistant to changes in environmental condition as well as providing nutritious, protein-and vitamin-rich foods for the crew, which can fulfill the main functions of CELSS, including supplying oxygen, water and food, removing carbon dioxide and making daily life waste reusable. Our labotory has developed mass culture of Nostoc sphaeroides Kütz, which is one of traditional healthy food in China and. The oxygen evolution rate of the cyanobacterium is about 150 molO2.mg-1.h-1, and it usually grows into colony with size between 2-20mm, which is easy to be harvested. It also can be cultured with high density, which show that the productivity of the cyanobacterium in limited volume is higher than other microalgae. We had measured the nutrient content of the cyanobacterium and developed some Chinese Dishes and Soups with Nostoc sphaeroides Kütz, which showed that it was a good food for crew. Using remote sensing technique, we also investigated its growth in Closed System under microgravity by SHENZHOU-2 spacecraft in January 2001. We plan to develop suitable bioreactor with the cyanobacterium for supplying oxygen and food to crew in future.

  12. Effects of radio frequency and high pressure steam sterilisation on the colour and flavour of prepared Nostoc sphaeroides.

    PubMed

    Xu, Jicheng; Zhang, Min; An, Yanjun; Roknul, Azam Sm; Adhikari, Benu

    2018-03-01

    Nostoc sphaeroides has been used as a highly effective herbal medicine and dietary supplement for thousands of years. The desired dark green colour of fresh N. sphaeroides is converted into an undesirable dark brown during conventional high pressure (HP) steam sterilisation. Radio frequency (RF) sterilisation technology was used in this study to determine its effectiveness in sterilising N. sphaeroides and to achieve better preservation of natural colour and desirable flavour. Sterilisation was carried out using a 6 kW, 27 MHz RF instrument for 10, 20 and 30 min. The degree of microbial kill and the effects of RF sterilisation on colour and flavour were determined and compared with those obtained from HP steam (121 °C, 30 min) sterilisation. The effects of RF sterilisation on colour and flavour (measured using electronic nose) parameters were significantly lower than that in HP steam sterilisation. The RF sterilisation carried out for 20 min achieved logarithmic reduction of bacterial population and met China's national standard while preserving the colour and flavour better. Results of the present study indicated that application of RF sterilisation would improve the quality of sterilised N. sphaeroides and broaden its application in the food and health food industries. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  13. The mechanisms of protection of antioxidants on Nostoc sphaeroides against UV-B radiation

    NASA Astrophysics Data System (ADS)

    Wang, G. H.

    UV radiation is one of space harmful factor for earth organisms in space exploration In the present work we studied on the role of antioxidant system in Nostoc sphaeroides K u tz Cyanobacteria and the effects of exogenous antioxidant molecules on its photosynthetic rate under UV-B radiation It was found that UV-B radiation decreased the photosynthetic activity of cyanobacterium but promoted the activity of antioxidant system to protect photosystem II PSII and exogenous antioxidant sodium nitroprusside SNP N-acetylcysteine NAC had an obvious protection on PSII activity under UV-B radiation The activity of SOD Superoxide Dismutase EC 1 15 1 1 CAT Catalase EC 1 11 1 6 POD Peroxidase EC 1 11 1 7 and content of MDA and ASC were improved by 0 5mM and 1mM SNP but 0 1mM SNP decreased the activity of antioxide system Exogenous NAC addition decreased the activity of SOD POD CAT and the content MDA and ASC but exogenous NAC addition increased the content of GSH The results suggested that exogenous SNP and NAC may protect algae by different mechanisms in which SNP maybe play double roles as sources of reactive free radicals or ROS scavengers in formation of algae s protection of PSII under UV-B radiation while NAC does function as antioxidant reagent or precursor of glutathione which could protect PSII directly from UV-B radiation Keyword antioxidant system exogenous or endogenous antioxidant Nostoc sphaeroides photosynthesis UV-B radiation

  14. Expression in Escherichia coli and characterization of a recombinant 7Fe ferredoxin of Rhodobacter capsulatus.

    PubMed Central

    Jouanneau, Y; Duport, C; Meyer, C; Gaillard, J

    1992-01-01

    The 7Fe ferredoxin of Rhodobacter capsulatus (FdII) could be expressed in Escherichia coli by cloning the fdxA gene coding for FdII downstream from the lac promoter. The expressed recombinant ferredoxin appeared as a brown protein which was specifically recognized in E. coli cell-free extracts by anti-FdII serum. The purified recombinant ferredoxin was indistinguishable from R. capsulatus FdII on the basis of its molecular, redox and spectroscopic properties. These results indicate that the [3Fe-4S] and [4Fe-4S] clusters were correctly inserted into the recombinant ferredoxin. Images Fig. 2. PMID:1325780

  15. Gene transfer agent (GTA) genes reveal diverse and dynamic Roseobacter and Rhodobacter populations in the Chesapeake Bay.

    PubMed

    Zhao, Yanlin; Wang, Kui; Budinoff, Charles; Buchan, Alison; Lang, Andrew; Jiao, Nianzhi; Chen, Feng

    2009-03-01

    Within the bacterial class Alphaproteobacteria, the order Rhodobacterales contains the Roseobacter and Rhodobacter clades. Roseobacters are abundant and play important biogeochemical roles in marine environments. Roseobacter and Rhodobacter genomes contain a conserved gene transfer agent (GTA) gene cluster, and GTA-mediated gene transfer has been observed in these groups of bacteria. In this study, we investigated the genetic diversity of these two groups in Chesapeake Bay surface waters using a specific PCR primer set targeting the conserved Rhodobacterales GTA major capsid protein gene (g5). The g5 gene was successfully amplified from 26 Rhodobacterales isolates and the bay microbial communities using this primer set. Four g5 clone libraries were constructed from microbial assemblages representing different regions and seasons of the bay and yielded diverse sequences. In total, 12 distinct g5 clusters could be identified among 158 Chesapeake Bay clones, 11 fall within the Roseobacter clade, and one falls in the Rhodobacter clade. The vast majority of the clusters (10 out of 12) lack cultivated representatives. The composition of g5 sequences varied dramatically along the bay during the wintertime, and a distinct Roseobacter population composition between winter and summer was observed. The congruence between g5 and 16S rRNA gene phylogenies indicates that g5 may serve as a useful genetic marker to investigate diversity and abundance of Roseobacter and Rhodobacter in natural environments. The presence of the g5 gene in the natural populations of Roseobacter and Rhodobacter implies that genetic exchange through GTA transduction could be an important mechanism for maintaining the metabolic flexibility of these groups of bacteria.

  16. Investigation on characteristics of 3D printing using Nostoc sphaeroides biomass.

    PubMed

    An, Yan-Jun; Guo, Chao-Fan; Zhang, Min; Zhong, Ze-Ping

    2018-06-27

    Gel-like constructs can be produced using an extrusion-based 3D food printing (3D-FP) technique. Nostoc sphaeroides biomass is a natural gel material. Considering its good nutrition and rheological properties, these algae were chosen in this study as supply material (ink) for 3D-FP. With this gel material, the extrusion-based 3D printing system was set as a model, and the printing behavior was investigated. Furthermore, the 3D-FP mechanisms were explained through low-field nuclear magnetic resonance and rheological measurements. Results indicated that although fresh biomass gel was printable, nonuniformity and instability occurred during printing. Blanched inks showed nonsmooth printing behavior, which was associated with a decrease in elasticity and viscosity. The printability was improved by increasing the rehydration time to 24 h when rehydrated powder was used. Increasing the rehydration time increased the water-binding degree. Pre-gelatinized potato starch was added to the mixture at ratios ranging from 1 to 100 g kg -1 . The best printing outcome was observed at 40 g kg -1 potato starch. We emphasize that elasticity and viscosity balance is an essential parameter to achieve printability. The strategies adopted in this work provide new insights into the development of personalized food regarding texture and nutritional additive content. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  17. Inactivation of suppressor T cell activity by the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides.

    PubMed Central

    Baker, P J; Taylor, C E; Stashak, P W; Fauntleroy, M B; Hasløv, K; Qureshi, N; Takayama, K

    1990-01-01

    Antibody responses of mice immunized with type III pneumococcal polysaccharide were examined with and without treatment with nontoxic lipopolysaccharide from Rhodopseudomonas sphaeroides (Rs-LPS). The results obtained were similar to those described previously for mice treated with monophosphoryl lipid A (MPL) except that lower amounts of Rs-LPS were needed. Both were without effect when given at the time of immunization with type III pneumococcal polysaccharide but elicited significant enhancement when given 2 to 3 days later. Such enhancement was T cell dependent and not due to polyclonal activation of immunoglobulin M synthesis by B cells. Treatment with either Rs-LPS or MPL abolished the expression but not induction of low-dose paralysis, a form of immunological unresponsiveness known to be mediated by suppressor T cells (Ts). The in vitro treatment of cell suspensions containing Ts with extremely small amounts of Rs-LPS or MPI completely eliminated the capacity of such cells to transfer suppression to other mice. These findings indicate that the immunomodulatory effects of both MPL and Rs-LPS are mainly the result of eliminating the inhibitors effects of Ts; this permits the positive effects of amplifier T cells to be more fully expressed, thereby resulting in an increased antibody response. The significance of these and other findings to the use of Rs-LPS as a pharmacotherapeutic agent for gram-negative bacterial sepsis is discussed. PMID:2143752

  18. Structural and phylogenetic analysis of Rhodobacter capsulatus NifF: uncovering general features of nitrogen-fixation (nif)-flavodoxins.

    PubMed

    Pérez-Dorado, Inmaculada; Bortolotti, Ana; Cortez, Néstor; Hermoso, Juan A

    2013-01-09

    Analysis of the crystal structure of NifF from Rhodobacter capsulatus and its homologues reported so far reflects the existence of unique structural features in nif flavodoxins: a leucine at the re face of the isoalloxazine, an eight-residue insertion at the C-terminus of the 50's loop and a remarkable difference in the electrostatic potential surface with respect to non-nif flavodoxins. A phylogenetic study on 64 sequences from 52 bacterial species revealed four clusters, including different functional prototypes, correlating the previously defined as "short-chain" with the firmicutes flavodoxins and the "long-chain" with gram-negative species. The comparison of Rhodobacter NifF structure with other bacterial flavodoxin prototypes discloses the concurrence of specific features of these functional electron donors to nitrogenase.

  19. Long-term biological hydrogen production by agar immobilized Rhodobacter capsulatus in a sequential batch photobioreactor.

    PubMed

    Elkahlout, Kamal; Alipour, Siamak; Eroglu, Inci; Gunduz, Ufuk; Yucel, Meral

    2017-04-01

    In this study, agar immobilization technique was employed for biological hydrogen production using Rhodobacter capsulatus DSM 1710 (wild type) and YO3 (hup-mutant) strains in sequential batch process. Different agar and glutamate concentrations were tested with defined nutrient medium. Agar concentration 4% (w/v) and 4 mM glutamate were selected for bacterial immobilization in terms of rate and longevity of hydrogen production. Acetate concentration was increased from 40 to 60-100 and 60 mM gave best results with both bacterial strains immobilized in 4% (w/v) agar. Cell concentration was increased from 2.5 to 5 mg dcw mL -1 agar and it was found that increasing cell concentration of wild-type strain caused decrease in yield and productivity while these parameters improved by increasing cell concentration of mutant strain. Also, the hydrogen production time has extended from 17 days up to 60 days according to the process conditions and parameters. Hydrogen production by immobilized photosynthetic bacteria is a convenient technology for hydrogen production as it enables to produce hydrogen with high organic acid concentrations comparing to suspended cultures. Besides, immobilization increases the stability of the system and allowed sequential batch operation for long-term application.

  20. Field evidence for the potential of Rhodobacter capsulatus as Biofertilizer for flooded rice.

    PubMed

    Gamal-Eldin, Hosny; Elbanna, Khaled

    2011-02-01

    In a previous study, we evaluated the effects of inoculating rice plants with the phototrophic purple nonsulfur bacterium Rhodobacter capsulatus (Rc) on growth and yield of rice in pots and lysimeter experiments and the results obtained have been highly encouraging. In this study, we carried out two field experiments: one in the experimental farm of the Faculty of Agriculture, Fayoum University, and the second in a farmer's field in Kafr El-sheikh, to assess the effects of Rc on growth and yield of rice in comparison and in combination with chemical nitrogen fertilizer (CNF) and farmyard manure. The results indicated that both biological and grain yields in all the Rc inoculated treatments were significantly higher than those in the uninoculated corresponding treatments in both fields. With regard to grain yield, the major factor for determining the effectiveness of any agricultural treatment, inoculation with Rc in combination with 50% of the recommended CNF rate gave a grain yield that was statistically equivalent to that obtained with 100% of the recommended CNF rate. These results provide a clear evidence for the potential of Rc as biofertilizer for flooded rice under field conditions.

  1. Genetic analysis of a bacterial genetic exchange element: The gene transfer agent of Rhodobacter capsulatus

    PubMed Central

    Lang, Andrew S.; Beatty, J. T.

    2000-01-01

    An unusual system of genetic exchange exists in the purple nonsulfur bacterium Rhodobacter capsulatus. DNA transmission is mediated by a small bacteriophage-like particle called the gene transfer agent (GTA) that transfers random 4.5-kb segments of the producing cell's genome to recipient cells, where allelic replacement occurs. This paper presents the results of gene cloning, analysis, and mutagenesis experiments that show that GTA resembles a defective prophage related to bacteriophages from diverse genera of bacteria, which has been adopted by R. capsulatus for genetic exchange. A pair of cellular proteins, CckA and CtrA, appear to constitute part of a sensor kinase/response regulator signaling pathway that is required for expression of GTA structural genes. This signaling pathway controls growth-phase-dependent regulation of GTA gene messages, yielding maximal gene expression in the stationary phase. We suggest that GTA is an ancient prophage remnant that has evolved in concert with the bacterial genome, resulting in a genetic exchange process controlled by the bacterial cell. PMID:10639170

  2. In vitro assembly of a prohead-like structure of the Rhodobacter capsulatus gene transfer agent

    SciTech Connect

    Spano, Anthony J.; Chen, Frank S.; Goodman, Benjamin E.

    2007-07-20

    The gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli, can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27-35 nm. The analysis of scanning TEM images revealed particles of mass {approx} 4.3 MDa, representing 101 {+-} 11 copies ofmore » the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring.« less

  3. Effects of karaya saponin and Rhodobacter capsulatus on yolk cholesterol in laying hens.

    PubMed

    Afrose, S; Hossain, M S; Maki, T; Tsujii, H

    2010-06-01

    1. It has been reported that karaya saponin and Rhodobacter capsulatus individually have hypocholesterolaemic activity in laying hens. This study focuses on the effect of adding karaya saponin with R. capsulatus to hen's diet with regard to serum and egg yolk cholesterol and triglycerides. 2. A total of 56 Boris Brown laying hens were divided into 7 groups at 20 weeks of age. Combinations of 25, 50, 75 mg kg(-1) karaya saponin and R. capsulatus 200 and 400 mg kg(-1) were used as treatment groups. 3. After 8 weeks of supplementation, the effects of all the combinations of karaya saponin and R. capsulatus on serum and egg yolk cholesterol, triglycerides, and high-density lipoprotein (HDL)-cholesterol were greater than either karaya saponin or R. capsulatus alone. The combination of karaya saponin 50 mg kg(-1)+ R. capsulatus 400 mg kg(-1) exhibited the greatest reduction of serum (325%) and yolk (225%) cholesterol and the greatest increase of faecal, liver bile acids and yolk fatty acid (oleic, linoleic and linolenic) concentrations. In addition, egg production and yolk colour were significantly improved by the combined use of karaya saponin and R. capsulatus supplementation. 4. Therefore, the dietary supplementation of karaya saponin and R. capsulatus may lead to the production of a low-cholesterol egg, with production performance maintained at a standard level.

  4. Functional Characterization of the FoxE Iron Oxidoreductase from the Photoferrotroph Rhodobacter ferrooxidans SW2*

    PubMed Central

    Saraiva, Ivo H.; Newman, Dianne K.; Louro, Ricardo O.

    2012-01-01

    Photoferrotrophy is presumed to be an ancient type of photosynthetic metabolism in which bacteria use the reducing power of ferrous iron to drive carbon fixation. In this work the putative iron oxidoreductase of the photoferrotroph Rhodobacter ferrooxidans SW2 was cloned, purified, and characterized for the first time. This protein, FoxE, was characterized using spectroscopic, thermodynamic, and kinetic techniques. It is a c-type cytochrome that forms a trimer or tetramer in solution; the two hemes of each monomer are hexacoordinated by histidine and methionine. The hemes have positive reduction potentials that allow downhill electron transfer from many geochemically relevant ferrous iron forms to the photosynthetic reaction center. The reduction potentials of the hemes are different and are cross-assigned to fast and slow kinetic phases of ferrous iron oxidation in vitro. Lower reactivity was observed at high pH and may contribute to prevent ferric iron precipitation inside or at the surface of the cell. These results help fill in the molecular details of a metabolic process that likely contributed to the deposition of precambrian banded iron formations, globally important sedimentary rocks that are found on every continent today. PMID:22661703

  5. Replacement of sugars to hydrogen production by Rhodobacter capsulatus using dark fermentation effluent as substrate.

    PubMed

    Silva, Felipe Thales Moreira; Moreira, Luiza Rojas; de Souza Ferreira, Juliana; Batista, Fabiana Regina Xavier; Cardoso, Vicelma Luiz

    2016-01-01

    Hydrogen is a promising alternative for the increased global energy demand since it has high energy density and is a clean fuel. The aim of this work was to evaluate the photo-fermentation by Rhodobacter capsulatus, using the dark fermentation effluent as substrate. Different systems were tested by changing the type of sugar in the dark fermentation, investigating the influence of supplementing DFE with sugar and adding alternate and periodically lactose and glucose throughout the process. The supplementation of the DFE with sugar resulted in higher H2 productivity and the replacement of the sugars repeatedly during the photo-fermentation process was important to maintain the cell culture active. By controlling the residual amount of sugar, bacteria inhibition was avoided; lactic acid, that was toxic to the biomass, was consumed and the metabolic route of butyric acid production was predominant. Under optimum conditions, the H2 productivity reached 208.40mmolH2/Ld in 52h. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Extracellular production of tellurium nanoparticles by the photosynthetic bacterium Rhodobacter capsulatus.

    PubMed

    Borghese, Roberto; Brucale, Marco; Fortunato, Gianuario; Lanzi, Massimiliano; Mezzi, Alessio; Valle, Francesco; Cavallini, Massimiliano; Zannoni, Davide

    2016-05-15

    The toxic oxyanion tellurite (TeO3(2-)) is acquired by cells of Rhodobacter capsulatus grown anaerobically in the light, via acetate permease ActP2 and then reduced to Te(0) in the cytoplasm as needle-like black precipitates. Interestingly, photosynthetic cultures of R. capsulatus can also generate Te(0) nanoprecipitates (TeNPs) outside the cells upon addition of the redox mediator lawsone (2-hydroxy-1,4-naphtoquinone). TeNPs generation kinetics were monitored to define the optimal conditions to produce TeNPs as a function of various carbon sources and lawsone concentration. We report that growing cultures over a 10 days period with daily additions of 1mM tellurite led to the accumulation in the growth medium of TeNPs with dimensions from 200 up to 600-700 nm in length as determined by atomic force microscopy (AFM). This result suggests that nucleation of TeNPs takes place over the entire cell growth period although the addition of new tellurium Te(0) to pre-formed TeNPs is the main strategy used by R. capsulatus to generate TeNPs outside the cells. Finally, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR) analysis of TeNPs indicate they are coated with an organic material which keeps the particles in solution in aqueous solvents. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Role of Rhodobacter sp. Strain PS9, a Purple Non-Sulfur Photosynthetic Bacterium Isolated from an Anaerobic Swine Waste Lagoon, in Odor Remediation

    PubMed Central

    Do, Young S.; Schmidt, Thomas M.; Zahn, James A.; Boyd, Eric S.; de la Mora, Arlene; DiSpirito, Alan A.

    2003-01-01

    Temporal pigmentation changes resulting from the development of a purple color in anaerobic swine waste lagoons were investigated during a 4-year period. The major purple photosynthetic bacterium responsible for these color changes and the corresponding reductions in odor was isolated from nine photosynthetic lagoons. By using morphological, physiological, and phylogenetic characterization methods we identified the predominant photosynthetic bacterium as a new strain of Rhodobacter, designated Rhodobacter sp. strain PS9. Rhodobacter sp. strain PS9 is capable of photoorganotrophic growth on a variety of organic compounds, including all of the characteristic volatile organic compounds (VOC) responsible for the odor associated with swine production facilities (J. A. Zahn, A. A. DiSpirito, Y. S. Do, B. E. Brooks, E. E. Copper, and J. L. Hatfield, J. Environ. Qual. 30:624-634, 2001). The seasonal variations in airborne VOC emitted from waste lagoons showed that there was a 80 to 93% decrease in the concentration of VOC during a photosynthetic bloom. During the height of a bloom, the Rhodobacter sp. strain PS9 population accounted for 10% of the total community and up to 27% of the eubacterial community based on 16S ribosomal DNA signals. Additional observations based on seasonal variations in meteorological, biological, and chemical parameters suggested that the photosynthetic blooms of Rhodobacter sp. strain PS9 were correlated with lagoon water temperature and with the concentrations of sulfate and phosphate. In addition, the photosynthetic blooms of Rhodobacter sp. strain PS9 were inversely correlated with the concentrations of protein and fluoride. PMID:12620863

  8. Rhodobacter capsulatus gains a competitive advantage from respiratory nitrate reduction during light-dark transitions.

    PubMed

    Ellington, M J K; Richardson, D J; Ferguson, S J

    2003-04-01

    Rhodobacter capsulatus N22DNAR(+) possesses a periplasmic nitrate reductase and is capable of reducing nitrate to nitrite under anaerobic conditions. In the absence of light this ability cannot support chemoheterotrophic growth in batch cultures. This study investigated the effect of nitrate reduction on the growth of R. capsulatus N22DNAR(+) during multiple light-dark cycles of anaerobic photoheterotrophic/dark chemoheterotrophic growth conditions in carbon-limited continuous cultures. The reduction of nitrate did not affect the photoheterotrophic growth yield of R. capsulatus N22DNAR(+). After a transition from photoheterotrophic to dark chemoheterotrophic growth conditions, the reduction of nitrate slowed the initial washout of a R. capsulatus N22DNAR(+) culture. Towards the end of a period of darkness nitrate-reducing cultures maintained higher viable cell counts than non-nitrate-reducing cultures. During light-dark cycling of a mixed culture, the strain able to reduce nitrate (N22DNAR(+)) outcompeted the strain which was unable to reduce nitrate (N22). The evidence indicates that the periplasmic nitrate reductase activity supports slow growth that retards the washout of a culture during anaerobic chemoheterotrophic conditions, and provides a protonmotive force for cell maintenance during the dark period before reillumination. This translates into a selective advantage during repeated light-dark cycles, such that in mixed culture N22DNAR(+) outcompetes N22. Exposure to light-dark cycles will be a common feature for R. capsulatus in its natural habitats, and this study shows that nitrate respiration may provide a selective advantage under such conditions.

  9. Zinc biosorption by the purple non-sulfur bacterium Rhodobacter capsulatus.

    PubMed

    Magnin, Jean-Pierre; Gondrexon, Nicolas; Willison, John C

    2014-12-01

    This paper presents the first report providing information on the zinc (Zn) biosorption potentialities of the purple non-sulfur bacterium Rhodobacter capsulatus. The effects of various biological, physical, and chemical parameters on Zn biosorption were studied in both the wild-type strain B10 and a strain, RC220, lacking the endogenous plasmid. At an initial Zn concentration of 10 mg·L(-1), the Zn biosorption capacity at pH 7 for bacterial biomass grown in synthetic medium containing lactate as carbon source was 17 and 16 mg Zn·(g dry mass)(-1) for strains B10 and RC220, respectively. Equilibrium was achieved in a contact time of 30-120 min, depending on the initial Zn concentration. Zn sorption by live biomass was modelled, at equilibrium, according to the Redlich-Peterson and Langmuir isotherms, in the range of 1-600 mg Zn·L(-1). The wild-type strain showed a maximal Zn uptake capacity (Qm) of 164 ± 8 mg·(g dry mass)(-1) and an equilibrium constant (Kads) of 0.017 ± 0.00085 L·(mg Zn)(-1), compared with values of 73.9 mg·(g dry mass)(-1) and 0.361 L·mg(-1) for the strain lacking the endogenous plasmid. The Qm value observed for R. capsulatus B10 is one of the highest reported in the literature, suggesting that this strain may be useful for Zn bioremediation. The lower Qm value and higher equilibrium constant observed for strain RC220 suggest that the endogenous plasmid confers an enhanced biosorption capacity in this bacterium, although no genetic determinants for Zn resistance appear to be located on the plasmid, and possible explanations for this are discussed.

  10. Effect of dietary Rhodobacter capsulatus on cholesterol concentration and fatty acid composition in broiler meat.

    PubMed

    Salma, U; Miah, A G; Maki, T; Nishimura, M; Tsujii, H

    2007-09-01

    The study was designed to investigate the effects of dietary Rhodobacter capsulatus on cholesterol concentration and fatty acid composition in broiler meat. A total of 45 two-week-old male broiler chicks were randomly assigned into 3 treatment groups and fed ad libitum diets supplemented with 0 (control), 0.02, and 0.04% R. capsulatus for a 6-wk feeding period. The results of this study revealed that the supplementation of 0.04% R. capsulatus in diet reduced (P < 0.05) cholesterol and triglyceride concentrations in broiler meat. The concentrations (expressed as a percentage of total fatty acids) of oleic acid (18:1), linoleic acid (18:2), and linolenic (18:3) acid in thigh muscle and breast muscle were higher (P < 0.05) in the broilers fed the 0.04% R. capsulatus supplemented diet than in the broilers fed the control diet. The ratio of unsaturated fatty acids to saturated fatty acids was greater (P < 0.05) in both muscles of broilers fed the 0.04% R. capsulatus supplemented diet than the control diet. In addition, the concentrations of serum cholesterol and triglyceride, and hepatic cholesterol and triglyceride were also reduced (P < 0.05) by dietary R. capsulatus. Compared with the control diet, the 0.04% R. capsulatus supplemented diet reduced (P < 0.05) the ratio of low-density lipoprotein-cholesterol to high-density lipoprotein-cholesterol. Moreover, the supplementation of R. capsulatus in broiler diets did not show any adverse effect on production performance. Therefore, these results conclude that the application of R. capsulatus into diet may be feasible to reduce cholesterol concentration and improve the ratio of unsaturated fatty acids to saturated fatty acids in broiler meat.

  11. Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus

    SciTech Connect

    Faries, Kaitlyn M.; Kressel, Lucas L.; Dylla, Nicholas P.

    Using high-throughput methods for mutagenesis, protein isolation and charge-separation functionality, we have assayed 40 Rhodobacter capsulatus reaction center (RC) mutants for their P+ QB- yield (P is a dimer of bacteriochlorophylls and Q is a ubiquinone) as produced using the normally inactive B-side cofactors BB and HB (where B is a bacteriochlorophyll and H is a bacteriopheophytin). Two sets of mutants explore all possible residues at M131 (M polypeptide, native residue Val near HB) in tandem with either a fixed His or a fixed Asn at L181 (L polypeptide, native residue Phe near BB). A third set of mutants exploresmore » all possible residues at L181 with a fixed Glu at M131 that can form a hydrogen bond to HB. For each set of mutants, the results of a rapid millisecond screening assay that probes the yield of P+ QB- are compared among that set and to the other mutants reported here or previously. For a subset of eight mutants, the rate constants and yields of the individual B-side electron transfer processes are determined via transient absorption measurements spanning 100 fs to 50 μs. The resulting ranking of mutants for their yield of P+ QB- from ultrafast experiments is in good agreement with that obtained from the millisecond screening assay, further validating the efficient, high-throughput screen for B-side transmembrane charge separation. Results from mutants that individually show progress toward optimization of P+ HB- → P+ QB- electron transfer or initial P* → P+ HB- conversion highlight unmet challenges of optimizing both processes simultaneously.« less

  12. Bacterial Survival under Extreme UV Radiation: A Comparative Proteomics Study of Rhodobacter sp., Isolated from High Altitude Wetlands in Chile

    PubMed Central

    Pérez, Vilma; Hengst, Martha; Kurte, Lenka; Dorador, Cristina; Jeffrey, Wade H.; Wattiez, Ruddy; Molina, Veronica; Matallana-Surget, Sabine

    2017-01-01

    Salar de Huasco, defined as a polyextreme environment, is a high altitude saline wetland in the Chilean Altiplano (3800 m.a.s.l.), permanently exposed to the highest solar radiation doses registered in the world. We present here the first comparative proteomics study of a photoheterotrophic bacterium, Rhodobacter sp., isolated from this remote and hostile habitat. We developed an innovative experimental approach using different sources of radiation (in situ sunlight and UVB lamps), cut-off filters (Mylar, Lee filters) and a high-throughput, label-free quantitative proteomics method to comprehensively analyze the effect of seven spectral bands on protein regulation. A hierarchical cluster analysis of 40 common proteins revealed that all conditions containing the most damaging UVB radiation induced similar pattern of protein regulation compared with UVA and visible light spectral bands. Moreover, it appeared that the cellular adaptation of Rhodobacter sp. to osmotic stress encountered in the hypersaline environment from which it was originally isolated, might further a higher resistance to damaging UV radiation. Indeed, proteins involved in the synthesis and transport of key osmoprotectants, such as glycine betaine and inositol, were found in very high abundance under UV radiation compared to the dark control, suggesting the function of osmolytes as efficient reactive oxygen scavengers. Our study also revealed a RecA-independent response and a tightly regulated network of protein quality control involving proteases and chaperones to selectively degrade misfolded and/or damaged proteins. PMID:28694800

  13. Characterization of a tungsten-substituted nitrogenase isolated from Rhodobacter capsulatus.

    PubMed

    Siemann, Stefan; Schneider, Klaus; Oley, Mareke; Müller, Achim

    2003-04-08

    In the phototrophic non-sulfur bacterium Rhodobacter capsulatus, the biosynthesis of the conventional Mo-nitrogenase is strictly Mo-regulated. Significant amounts of both dinitrogenase and dinitrogenase reductase were only formed when the growth medium was supplemented with molybdate (1 microM). During cell growth under Mo-deficient conditions, tungstate, at high concentrations (1 mM), was capable of partially (approximately 25%) substituting for molybdate in the induction of nitrogenase synthesis. On the basis of such conditions, a tungsten-substituted nitrogenase was isolated from R. capsulatus with the aid of anfA (Fe-only nitrogenase defective) mutant cells and partially purified by Q-sepharose chromatography. Metal analyses revealed the protein to contain an average of 1 W-, 16 Fe-, and less than 0.01 Mo atoms per alpha(2)beta(2)-tetramer. The tungsten-substituted (WFe) protein was inactive in reducing N(2) and marginally active in acetylene reduction, but it was found to show considerable activity with respect to the generation of H(2) from protons. The EPR spectrum of the WFe protein, recorded at 4 K, exhibited three distinct signals: (i) an S = 3/2 signal, which dominates the low-field region of the spectrum (g = 4.19, 3.93) and is indicative of a tungsten-substituted cofactor (termed FeWco), (ii) a marginal S = 3/2 signal (g = 4.29, 3.67) that can be attributed to residual amounts of FeMoco present in the protein, and (iii) a broad S = 1/2 signal (g = 2.09, 1.95, 1.86) arising from at least two paramagnetic species. Redox titrational analysis of the WFe protein revealed the midpoint potential of the FeWco (E(m) < -200 mV) to be shifted to distinctly lower potentials as compared to that of the FeMoco (E(m) approximately -50 mV) present in the native enzyme. The P clusters of both the WFe and the MoFe protein appear indistinguishable with respect to their midpoint potentials. EPR spectra recorded with the WFe protein under turnover conditions exhibited a 20

  14. Photo-biohydrogen production potential of Rhodobacter capsulatus-PK from wheat straw

    PubMed Central

    2013-01-01

    Background Biotechnological exploitation of lignocellulosic biomass is promising for sustainable and environmentally sound energy provision strategy because of the abundant availability of the renewable resources. Wheat straw (WS) comprising of 75-80% cellulose and hemicellulose is one of widely available, inexpensive and renewable lignocellulosic biomass types. The cellulosic and hemicellulose substrate can be hydrolyzed into monomeric sugars by chemical and/or biological methods. Results This study examined comparative potential of dilute acid and pre-ammonia pretreated and enzymatically hydrolyzed wheat straw (WS) for hydrogen production by purple non sulfur bacterium Rhodobacter capsulatus-PK. Gas production became noticeable after 14 h of inoculation in WS pretreated with 4% H2SO4. The detoxified liquid hydrolyzate (DLH) after overliming attained a production level of 372 mL-H2/L after 16 h under illumination of 120-150 W/m2 at 30 ± 2.0°C. Whereas the non-detoxified acid pretreated hydrolyzate (NDLH) of WS could produce only upto 254 mL-H2/L after 21 h post inoculation. Evolution of H2 became observable just after 10 ± 2.0 h of inoculation by employing 48 h age inoculum on the WS pretreated with 30% ammonia, hydrolyzed with cellulase 80 FPU/g and β-glucosidase 220 CbU/ml at 50°C. Upto 712 ml/L of culture was measured with continuous shaking for 24 h. The 47.5% and 64.2% higher hydrogen volume than the DLH and NDLH substrates, respectively appeared as a function of significantly higher monomeric sugar contents of the enzymatically hydrolyzed substrate and lesser/zero amounts of toxic derivatives including pH reducing agents. Conclusion Photofermentative hydrogen production from lignocellulosic waste is a feasible approach for eco-friendly sustainable supply of bioenergy in a cost-effective way. Results of this study provide new insight for addressing biotechnological exploitation of abundantly available and low-cost cellulosic substrates

  15. Characterization of LHI- and LHI+ Rhodobacter capsulatus pufA mutants.

    PubMed Central

    Richter, P; Brand, M; Drews, G

    1992-01-01

    The NH2 termini of light-harvesting complex I (LHI) polypeptides alpha and beta of Rhodobacter capsulatus are thought to be involved in the assembly of the LHI complex. For a more detailed study of the role of the NH2-terminal segment of the LHI alpha protein in insertion into the intracytoplasmic membrane (ICM) of R. capsulatus, amino acids 6 to 8, 9 to 11, 12 and 13, or 14 and 15 of the LHI alpha protein were deleted. Additionally, the hydrophobic stretch of the amino acids 7 to 11 was lengthened by insertion of hydrophobic or hydrophilic amino acids. All mutations abolished the ability of the mutant strains to form a functional LHI antenna complex. All changes introduced into the LHI alpha protein strongly reduced the stability of its LHI beta partner protein in the ICM. The effects on the mutated protein itself, however, were different. Deletion of amino acids 6 to 8, 9 to 11, or 14 and 15 drastically reduced the amount of the LHI alpha protein inserted into the membrane or prevented its insertion. Deletion of amino acids 12 and 13 and lengthening of the stretch of amino acids 7 to 11 reduced the half-life of the mutated LHI alpha protein in the ICM in comparison with the wild-type LHI alpha protein. Under the selective pressure of low light, revertants which regained a functional LHI antenna complex were identified only for the mutant strain deleted of amino acids 9 to 11 of the LHI alpha polypeptide [U43 (pTPR15)]. The restoration of the LHI+ phenotype was due to an in-frame duplication of 9 bp in the pufA gene directly upstream of the site of deletion present in strain U43(pTPR15). The duplicated nucleotides code for the amino acids Lys, Ile, and Trp. Membranes purified from the revertants were different from that of the reaction center-positive LHI+ LHII- control strain U43(pTX35) in doubling of the carotenoid content and increase of the size of the photosynthetic unit. By separating the reaction center and LHI complexes of the revertants by native

  16. Rhodobacter sp. Rb3, an aerobic anoxygenic phototroph which thrives in the polyextreme ecosystem of the Salar de Huasco, in the Chilean Altiplano.

    PubMed

    Pérez, Vilma; Dorador, Cristina; Molina, Verónica; Yáñez, Carolina; Hengst, Martha

    2018-03-22

    The Salar de Huasco is an evaporitic basin located in the Chilean Altiplano, which presents extreme environmental conditions for life, i.e. high altitude (3800 m.a.s.l.), negative water balance, a wide salinity range, high daily temperature changes and the occurrence of the highest registered solar radiation on the planet (> 1200 W m -2 ). This ecosystem is considered as a natural laboratory to understand different adaptations of microorganisms to extreme conditions. Rhodobacter, an anoxygenic aerobic phototrophic bacterial genus, represents one of the most abundant groups reported based on taxonomic diversity surveys in this ecosystem. The bacterial mat isolate Rhodobacter sp. strain Rb3 was used to study adaptation mechanisms to stress-inducing factors potentially explaining its success in a polyextreme ecosystem. We found that the Rhodobacter sp. Rb3 genome was characterized by a high abundance of genes involved in stress tolerance and adaptation strategies, among which DNA repair and oxidative stress were the most conspicuous. Moreover, many other molecular mechanisms associated with oxidative stress, photooxidation and antioxidants; DNA repair and protection; motility, chemotaxis and biofilm synthesis; osmotic stress, metal, metalloid and toxic anions resistance; antimicrobial resistance and multidrug pumps; sporulation; cold shock and heat shock stress; mobile genetic elements and toxin-antitoxin system were detected and identified as potential survival mechanism features in Rhodobacter sp. Rb3. In total, these results reveal a wide set of strategies used by the isolate to adapt and thrive under environmental stress conditions as a model of polyextreme environmental resistome.

  17. The response of antioxidant systems in Nostoc sphaeroides against UV-B radiation and the protective effects of exogenous antioxidants

    NASA Astrophysics Data System (ADS)

    Wang, Gaohong; Hu, Chunxiang; Li, Dunhai; Zhang, Delu; Li, Xiaoyan; Chen, Kun; Liu, Yongding

    UV radiation is one of many harmful factors found in space that are detrimental to organisms on earth in space exploration. In the present work, we examined the role of antioxidant system in Nostoc sphaeroides Kütz (Cyanobacterium) and the effects of exogenously applied antioxidant molecules on its photosynthetic rate under UV-B radiation. It was found that UV-B radiation promoted the activity of antioxidant system to protect photosystem II (PSII) and exogenously applied antioxidant: sodium nitroprusside (SNP) and N-acetylcysteine (NAC) had an obvious protection on PSII activity under UV-B radiation. The activity of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and content of MDA (malondialdehyde) and ASC (ascorbate) were improved by 0.5 mM and 1 mM SNP, but 0.1 mM SNP decreased the activity of antioxidant system. Addition of exogenous NAC decreased the activity of SOD, POD, CAT and the content MDA and ASC. In contrast, exogenously applied NAC increased GSH content. The results suggest that exogenous SNP and NAC may protect algae by different mechanisms: SNP may play double roles as both sources of reactive free radicals as well as ROS scavengers in mediating the protective role of PSII on algae under UV-B radiation. On the other hand, NAC functions as an antioxidant or precursor of glutathione, which could protect PSII directly from UV-B radiation.

  18. Proteomic Analysis and Identification of the Structural and Regulatory Proteins of the Rhodobacter capsulatus Gene Transfer Agent

    PubMed Central

    Chen, Frank; Spano, Anthony; Goodman, Benjamin E.; Blasier, Kiev R.; Sabat, Agnes; Jeffery, Erin; Norris, Andrew; Shabanowitz, Jeffrey; Hunt, Donald F.; Lebedev, Nikolai

    2010-01-01

    The gene transfer agent of Rhodobacter capsulatus (GTA) is a unique phage-like particle that exchanges genetic information between members of this same species of bacterium. Besides being an excellent tool for genetic mapping, the GTA has a number of advantages for biotechnological and nanoengineering purposes. To facilitate the GTA purification and identify the proteins involved in GTA expression, assembly and regulation, in the present work we construct and transform into R. capsulatus Y262 a gene coding for a C-terminally His-tagged capsid protein. The constructed protein was expressed in the cells, assembled into chimeric GTA particles inside the cells and excreted from the cells into surrounding medium. Transmission electron micrographs of phosphotungstate-stained, NiNTA-purified chimeric GTA confirm that its structure is similar to normal GTA particles, with many particles composed both of a head and a tail. The mass spectrometric proteomic analysis of polypeptides present in the GTA recovered outside the cells shows that GTA is composed of at least 9 proteins represented in the GTA gene cluster including proteins coded for by Orf’s 3, 5, 6–9, 11, 13, and 15. PMID:19105630

  19. Proteomic analysis and identification of the structural and regulatory proteins of the Rhodobacter capsulatus gene transfer agent.

    PubMed

    Chen, Frank; Spano, Anthony; Goodman, Benjamin E; Blasier, Kiev R; Sabat, Agnes; Jeffery, Erin; Norris, Andrew; Shabanowitz, Jeffrey; Hunt, Donald F; Lebedev, Nikolai

    2009-02-01

    The gene transfer agent of Rhodobacter capsulatus (GTA) is a unique phage-like particle that exchanges genetic information between members of this same species of bacterium. Besides being an excellent tool for genetic mapping, the GTA has a number of advantages for biotechnological and nanoengineering purposes. To facilitate the GTA purification and identify the proteins involved in GTA expression, assembly and regulation, in the present work we construct and transform into R. capsulatus Y262 a gene coding for a C-terminally His-tagged capsid protein. The constructed protein was expressed in the cells, assembled into chimeric GTA particles inside the cells and excreted from the cells into surrounding medium. Transmission electron micrographs of phosphotungstate-stained, NiNTA-purified chimeric GTA confirm that its structure is similar to normal GTA particles, with many particles composed both of a head and a tail. The mass spectrometric proteomic analysis of polypeptides present in the GTA recovered outside the cells shows that GTA is composed of at least 9 proteins represented in the GTA gene cluster including proteins coded for by Orf's 3, 5, 6-9, 11, 13, and 15.

  20. The Presence of ADP-Ribosylated Fe Protein of Nitrogenase in Rhodobacter capsulatus Is Correlated with Cellular Nitrogen Status

    PubMed Central

    Yakunin, Alexander F.; Laurinavichene, Tatyana V.; Tsygankov, Anatoly A.; Hallenbeck, Patrick C.

    1999-01-01

    The photosynthetic bacterium Rhodobacter capsulatus has been shown to regulate its nitrogenase by covalent modification via the reversible ADP-ribosylation of Fe protein in response to darkness or the addition of external NH4+. Here we demonstrate the presence of ADP-ribosylated Fe protein under a variety of steady-state growth conditions. We examined the modification of Fe protein and nitrogenase activity under three different growth conditions that establish different levels of cellular nitrogen: batch growth with limiting NH4+, where the nitrogen status is externally controlled; batch growth on relatively poor nitrogen sources, where the nitrogen status is internally controlled by assimilatory processes; and continuous culture. When cultures were grown to stationary phase with different limiting concentrations of NH4+, the ADP-ribosylation state of Fe protein was found to correlate with cellular nitrogen status. Additionally, actively growing cultures (grown with N2 or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in the modified state. The correlation between cellular nitrogen status and ADP-ribosylation state was corroborated with continuous cultures grown under various degrees of nitrogen limitation. These results show that in R. capsulatus the modification system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions. PMID:10094674

  1. The reductive half-reaction of xanthine dehydrogenase from Rhodobacter capsulatus: the role of Glu232 in catalysis.

    PubMed

    Hall, James; Reschke, Stefan; Cao, Hongnan; Leimkühler, Silke; Hille, Russ

    2014-11-14

    The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Rhodobacter capsulatus contains a novel cb-type cytochrome c oxidase without a CuA center.

    PubMed

    Gray, K A; Grooms, M; Myllykallio, H; Moomaw, C; Slaughter, C; Daldal, F

    1994-03-15

    The facultative phototrophic bacterium Rhodobacter capsulatus is capable of growth in a wide range of environmental conditions using a highly branched electron-transfer chain. During respiratory growth of this organism reducing equivalents are conveyed to oxygen via two terminal oxidases, previously called "cyt b410" (cytochrome c oxidase) and "cyt b260" (quinol oxidase). The cytochrome c oxidase was purified to homogeneity from a semiaerobically grown R. capsulatus strain. The purified enzyme consumes oxygen at a rate of 600 s-1, oxidizes reduced equine cyt c and R. capsulatus cyt c2, and has high sensitivity to cyanide. The complex is composed of three major polypeptides of apparent molecular masses 45, 32, and 28 kDa on SDS-PAGE. The 32- and 28-kDa proteins also stain with tetramethylbenzidine, indicating that they are c-type cytochromes. Partial amino acid sequences obtained from each of the subunits reveal significant homology to the fixN, fixO, and fixP gene products of Bradyrhizobium japonicum and Rhizobium meliloti. The reduced enzyme has an optical absorption spectrum with distinct features near 550 and 560 nm and an asymmetric Soret band centered at 418 nm, indicating the presence of both c- and b-type cytochromes. Two electrochemically distinct cyt c are apparent, with redox midpoint potentials (Em7) of 265 and 320 mV, while the low-spin cyt b has an Em7 value of 385 mV. The enzyme binds carbon monoxide, and the CO difference spectrum indicates that CO binds to a high-spin cyt b. Pyridine hemochrome and HPLC analyses suggest that the complex contains 1 mol of heme C to 1 mol of protoheme and that neither heme O nor heme A is present.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Manipulating the Energetics and Rates of Electron Transfer in Rhodobacter capsulatus Reaction Centers with Asymmetric Pigment Content

    SciTech Connect

    Faries, Kaitlyn M.; Dylla, Nicholas P.; Hanson, Deborah K.

    2017-07-17

    Seemingly redundant parallel pathways for electron transfer (ET), composed of identical sets of cofactors, are a cornerstone feature of photosynthetic reaction centers (RCs) involved in light-energy conversion. In native bacterial RCs, both A and B branches house one bacteriochlorophyll (BChl) and one bacteriopheophytin (BPh), but the A branch is used exclusively. Described herein are the results-obtained for two Rhodobacter capsulatus RCs with an unnaturally high degree of cofactor asymmetry, two BPh on the RC's B side and two BChl on the A side. These pigment changes derive, respectively, from the His(M180)Leu mutation [a BPh ((Phi(B)) replaces the B-side BChl (BB)],more » and the Leu(M212)His mutation [a BChl (beta(A))) replaces the A-side BPh (H-A)]. Additionally, Tyr(M208)Phe was employed to disfavor ET to the A branch; in one mutant, Val(M131)Glu creates a hydrogen bond to H-B to enhance ET to H-B. In both Phi(B) mutants, the decay kinetics of the excited primary ET donor (P*) resolve three populations with lifetimes of similar to 9 ps (50-60%), similar to 40 ps (10-20%), and similar to 200 ps (20-30%), with P+Phi(-)(B) formed predominantly from the 9 ps fraction. The 50-60% yield of P+Phi(B)- is the highest yet observed for a Phi(B)-containing RC. The results provide insight into factors needed for efficient multistep ET.« less

  4. Assignment of the sup 1 H and sup 15 N NMR spectra of Rhodobacter capsulatus ferrocytochrome c sub 2

    SciTech Connect

    Gooley, P.R.; Caffrey, M.S.; Cusanovich, M.A.

    1990-03-06

    The peptide resonances of the {sup 1}H and {sup 15}N nuclear magnetic resonance spectra of ferrocytochrome c{sub 2} from Rhodobacter capsulatus are sequentially assigned by a combination of 2D {sup 1}H-{sup 1}H and {sup 1}H-{sup 15}N spectroscopy, the latter performed on {sup 15}N-enriched protein. Short-range nuclear Overhauser effect (NOE) data show {alpha}-helices from residues 3-17, 55-65, 69-88, and 103-115. Within the latter two {alpha}-helices, there are three single 3{sub 10} turns, 70-72, 76-78, and 107-109. In addition {alpha}H-NH{sub i+1} and {alpha}H-NH{sub i+2} NOEs indicate that the N-terminal helix (3-17) is distorted. Compared to horse or tuna cytochrome c and cytochromemore » c{sub 2} of Rhodospirillium rubrum, there is a 6-residue insertion at residues 23-29 in R. capsulatus cytochrome c{sub 2}. The NOE data show that this insertion forms a loop, probably an {Omega} loop. {sup 1}H-{sup 15}N heteronuclear multiple quantum correlation experiments are used to follow NH exchange over a period of 40 h. As the 2D spectra are acquired in short time periods (30 min), rates for intermediate exchanging protons can be measured. Comparison of the NH exchange data for the N-terminal helix of cytochrome c{sub 2} of R. capsulatus with the highly homologous horse heart cytochrome c shows that this helix is less stable in cytochrome c{sub 2}.« less

  5. The photosynthetic bacteria Rhodobacter capsulatus and Synechocystis sp. PCC 6803 as new hosts for cyclic plant triterpene biosynthesis

    PubMed Central

    Hage-Hülsmann, Jennifer; Dietsch, Maximilian; Kranz-Finger, Sarah; Hüren, Vanessa; Metzger, Sabine; Urlacher, Vlada B.; Gigolashvili, Tamara; Kopriva, Stanislav; Axmann, Ilka M.; Jaeger, Karl-Erich

    2017-01-01

    Cyclic triterpenes constitute one of the most diverse groups of plant natural products. Besides the intriguing biochemistry of their biosynthetic pathways, plant triterpenes exhibit versatile bioactivities, including antimicrobial effects against plant and human pathogens. While prokaryotes have been extensively used for the heterologous production of other classes of terpenes, the synthesis of cyclic triterpenes, which inherently includes the two-step catalytic formation of the universal linear precursor 2,3-oxidosqualene, is still a major challenge. We thus explored the suitability of the metabolically versatile photosynthetic α-proteobacterium Rhodobacter capsulatus SB1003 and cyanobacterium Synechocystis sp. PCC 6803 as alternative hosts for biosynthesis of cyclic plant triterpenes. Therefore, 2,3-oxidosqualene production was implemented and subsequently combined with different cyclization reactions catalyzed by the representative oxidosqualene cyclases CAS1 (cycloartenol synthase), LUP1 (lupeol synthase), THAS1 (thalianol synthase) and MRN1 (marneral synthase) derived from model plant Arabidopsis thaliana. While successful accumulation of 2,3-oxidosqualene could be detected by LC-MS analysis in both hosts, cyclase expression resulted in differential production profiles. CAS1 catalyzed conversion to only cycloartenol, but expression of LUP1 yielded lupeol and a triterpenoid matching an oxidation product of lupeol, in both hosts. In contrast, THAS1 expression did not lead to cyclic product formation in either host, whereas MRN1-dependent production of marnerol and hydroxymarnerol was observed in Synechocystis but not in R. capsulatus. Our findings thus indicate that 2,3-oxidosqualene cyclization in heterologous phototrophic bacteria is basically feasible but efficient conversion depends on both the respective cyclase enzyme and individual host properties. Therefore, photosynthetic α-proteo- and cyanobacteria are promising alternative candidates for providing

  6. Spectral diffusion and electron-phonon coupling of the B800 BChl a molecules in LH2 complexes from three different species of purple bacteria.

    PubMed

    Baier, J; Gabrielsen, M; Oellerich, S; Michel, H; van Heel, M; Cogdell, R J; Köhler, J

    2009-11-04

    We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlorophyll a molecules in the peripheral light-harvesting complex LH2 for three different species of purple bacteria, Rhodobacter sphaeroides, Rhodospirillum molischianum, and Rhodopseudomonas acidophila. We come to the conclusion that B800 binding pockets for Rhodobacter sphaeroides and Rhodopseudomonas acidophila are rather similar with respect to the polarity of the protein environment but that the packaging of the alphabeta-polypeptides seems to be less tight in Rb. sphaeroides with respect to the other two species.

  7. Identification of "Haematobacter," a new genus of aerobic Gram-negative rods isolated from clinical specimens, and reclassification of Rhodobacter massiliensis as "Haematobacter massiliensis comb. nov.".

    PubMed

    Helsel, Leta O; Hollis, Dannie; Steigerwalt, Arnold G; Morey, Roger E; Jordan, Jean; Aye, Tin; Radosevic, Jon; Jannat-Khah, Deanna; Thiry, Dorothy; Lonsway, David R; Patel, Jean B; Daneshvar, Maryam I; Levett, Paul N

    2007-04-01

    Twelve strains of gram-negative, nonfermenting rods recovered mainly from septicemic patients were studied using conventional and molecular methods. The phenotypic profiles of these strains most closely resembled Psychrobacter phenylpyruvicus. They produced catalase, oxidase, urease, and H(2)S (lead acetate paper) but did not produce indole, reduce nitrate or nitrite, or hydrolyze gelatin or esculin. No acid production was observed in a King's oxidation-fermentation base containing d-glucose, d-xylose, d-mannitol, sucrose, lactose, or maltose. All strains were nonmotile and nonpigmented. Most strains produced green discoloration on blood agar. All strains grew at 25 degrees C and 35 degrees C and most grew on MacConkey agar. They shared a common cellular fatty acid (CFA) profile characterized by large amounts (56% to 90%) of 18:1omega7c and the presence of 3-OH-10:0, 16:1omega7c, 16:0, and 19:0cycomega8c that overall was most similar to that of Rhodobacter species but was quite distinct from that of P. phenylpyruvicus. The MICs for most beta-lactams, fluoroquinolones, aminoglycosides, and carbapenems were low. MICs for aztreonam and piperacillin were higher, with MICs for some strains of > 64 mg/liter and > 128 mg/liter, respectively. Polyphasic analysis of these strains, including morphological, biochemical, CFA composition, DNA-DNA hybridization, 16S rRNA gene sequencing, and percent guanine-plus-cytosine (G+C) content analysis, demonstrated that these strains and Rhodobacter massiliensis represent a new genus, "Haematobacter" (proposed name), with the species H. missouriensis (type strain H1892(T) = CCUG 52307(T) = CIP 109176(T)) and H. massiliensis comb. nov. (type strain Framboise(T) = CCUG 47968(T) = CIP 107725(T)) and an unnamed genomospecies.

  8. The SOS Response Master Regulator LexA Regulates the Gene Transfer Agent of Rhodobacter capsulatus and Represses Transcription of the Signal Transduction Protein CckA.

    PubMed

    Kuchinski, Kevin S; Brimacombe, Cedric A; Westbye, Alexander B; Ding, Hao; Beatty, J Thomas

    2016-02-01

    The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a genetic exchange element that combines central aspects of bacteriophage-mediated transduction and natural transformation. RcGTA particles resemble a small double-stranded DNA bacteriophage, package random ∼4-kb fragments of the producing cell genome, and are released from a subpopulation (<1%) of cells in a stationary-phase culture. RcGTA particles deliver this DNA to surrounding R. capsulatus cells, and the DNA is integrated into the recipient genome though a process that requires homologs of natural transformation genes and RecA-mediated homologous recombination. Here, we report the identification of the LexA repressor, the master regulator of the SOS response in many bacteria, as a regulator of RcGTA activity. Deletion of the lexA gene resulted in the abolition of detectable RcGTA production and an ∼10-fold reduction in recipient capability. A search for SOS box sequences in the R. capsulatus genome sequence identified a number of putative binding sites located 5' of typical SOS response coding sequences and also 5' of the RcGTA regulatory gene cckA, which encodes a hybrid histidine kinase homolog. Expression of cckA was increased >5-fold in the lexA mutant, and a lexA cckA double mutant was found to have the same phenotype as a ΔcckA single mutant in terms of RcGTA production. The data indicate that LexA is required for RcGTA production and maximal recipient capability and that the RcGTA-deficient phenotype of the lexA mutant is largely due to the overexpression of cckA. This work describes an unusual phenotype of a lexA mutant of the alphaproteobacterium Rhodobacter capsulatus in respect to the phage transduction-like genetic exchange carried out by the R. capsulatus gene transfer agent (RcGTA). Instead of the expected SOS response characteristic of prophage induction, this lexA mutation not only abolishes the production of RcGTA particles but also impairs the ability of cells to receive Rc

  9. Effect of light-dark cycles on hydrogen and poly-β-hydroxybutyrate production by a photoheterotrophic culture and Rhodobacter capsulatus using a dark fermentation effluent as substrate.

    PubMed

    Montiel Corona, Virginia; Le Borgne, Sylvie; Revah, Sergio; Morales, Marcia

    2017-02-01

    A Rhodobacter capsulatus strain and a photoheterotrophic culture (IZT) were cultivated to produce hydrogen under different light-dark cycles. A dark fermentation effluent (DFE) was used as substrate. It was found that IZT culture had an average cumulative hydrogen production (Paccum H 2 ) of 1300±43mLH 2 L -1 under continuous illumination and light-dark cycles of 30 or 60min. In contrast, R. capsulatus reduced its Paccum H 2 by 20% under 30:30min light-dark cycles, but tripled its poly-β-hydroxybutyrate (PHB) content (308±2mgPHB gdw -1 ) compared to continuous illumination. The highest PHB content by IZT culture was 178±10mgPHB gdw -1 under 15:15min light-dark cycles. PCR-DGGE analysis revealed that the IZT culture was mainly composed of Rhodopseudomonas palustris identified with high nucleotide similarity (99%). The evaluated cultures might be used for hydrogen and PHB production. They might provide energy savings by using light-dark cycles and DFE valorization. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Cooperation between two periplasmic copper chaperones is required for full activity of the cbb 3-type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus

    SciTech Connect

    Trasnea, Petru -Iulian; Utz, Marcel; Khalfaoui-Hassani, Bahia

    Copper (Cu) is an essential micronutrient that functions as a cofactor in several important enzymes, like respiratory heme-copper oxygen reductases. Yet, Cu is also toxic and therefore cells engage a highly coordinated Cu uptake and delivery system to prevent the accumulation of toxic Cu concentrations. In the current work we analyzed Cu delivery to the cbb 3-type cytochrome c oxidase ( cbb 3-Cox) of Rhodobacter capsulatus. We identified the PCu AC-like periplasmic chaperone PccA and analyzed its contribution to cbb 3-Cox assembly. Our data demonstrate that PccA is a Cu-binding protein with a preference for Cu(I), which is required formore » efficient cbb 3-Cox assembly, in particular at low Cu concentrations. By using in vivo and in vitro crosslinking we show that PccA forms a complex with the Sco1-homologue SenC. This complex is stabilized in the absence of the cbb 3-Cox specific assembly factors CcoGHIS. In cells lacking SenC, the cytoplasmic Cu content is significantly increased, but the simultaneous absence of PccA prevents this Cu accumulation. Lastly, these data demonstrate that the interplay between PccA and SenC is not only required for Cu delivery during cbb 3-Cox assembly, but that it also regulates Cu homeostasis in R. capsulatus.« less

  11. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H 2 production

    SciTech Connect

    Wecker, Matt S. A.; Beaton, Stephen E.; Chado, Robert A.

    The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H 2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H 2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system. Here we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H 2. The resulting strain photoproduces H 2 and self-reports its own H 2 production through fluorescence. Furthermore, this model system represents amore » unique method of developing hydrogenase-based H 2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H 2.« less

  12. Dietary Karaya Saponin and Rhodobacter capsulatus Exert Hypocholesterolemic Effects by Suppression of Hepatic Cholesterol Synthesis and Promotion of Bile Acid Synthesis in Laying Hens.

    PubMed

    Afrose, Sadia; Hossain, Md Sharoare; Salma, Ummay; Miah, Abdul Gaffar; Tsujii, Hirotada

    2010-01-01

    This study was conducted to elucidate the mechanism underlying the hypolipidemic action of karaya saponin or Rhodobacter (R.) capsulatus. A total of 40 laying hens (20-week-old) were assigned into four dietary treatment groups and fed a basal diet (as a control) or basal diets supplemented with either karaya saponin, R. capsulatus, or both for 60 days. The level of serum low-density-lipoprotein cholesterol and the levels of cholesterol and triglycerides in the serum, liver, and egg yolk were reduced by all the supplementations (P < .05). Liver bile acid concentration and fecal concentrations of cholesterol, triacylglycerol, and bile acid were simultaneously increased by the supplementation of karaya saponin, R. capsulatus, and the combination of karaya saponin and R. capsulatus (P < .05). The supplementation of karaya saponin, R. capsulatus, and the combination of karaya saponin and R. capsulatus suppressed the incorporation of (14)C from 1-(14)C-palmitic acid into the fractions of total lipids, phospholipids, triacylglycerol, and cholesterol in the liver in vitro (P < .05). These findings suggest that the hypocholesterolemic effects of karaya saponin and R. capsulatus are caused by the suppression of the cholesterol synthesis and the promotion of cholesterol catabolism in the liver.

  13. Dietary Karaya Saponin and Rhodobacter capsulatus Exert Hypocholesterolemic Effects by Suppression of Hepatic Cholesterol Synthesis and Promotion of Bile Acid Synthesis in Laying Hens

    PubMed Central

    Afrose, Sadia; Hossain, Md. Sharoare; Salma, Ummay; Miah, Abdul Gaffar; Tsujii, Hirotada

    2010-01-01

    This study was conducted to elucidate the mechanism underlying the hypolipidemic action of karaya saponin or Rhodobacter (R.) capsulatus. A total of 40 laying hens (20-week-old) were assigned into four dietary treatment groups and fed a basal diet (as a control) or basal diets supplemented with either karaya saponin, R. capsulatus, or both for 60 days. The level of serum low-density-lipoprotein cholesterol and the levels of cholesterol and triglycerides in the serum, liver, and egg yolk were reduced by all the supplementations (P < .05). Liver bile acid concentration and fecal concentrations of cholesterol, triacylglycerol, and bile acid were simultaneously increased by the supplementation of karaya saponin, R. capsulatus, and the combination of karaya saponin and R. capsulatus (P < .05). The supplementation of karaya saponin, R. capsulatus, and the combination of karaya saponin and R. capsulatus suppressed the incorporation of 14C from 1-14C-palmitic acid into the fractions of total lipids, phospholipids, triacylglycerol, and cholesterol in the liver in vitro (P < .05). These findings suggest that the hypocholesterolemic effects of karaya saponin and R. capsulatus are caused by the suppression of the cholesterol synthesis and the promotion of cholesterol catabolism in the liver. PMID:21490913

  14. Cooperation between two periplasmic copper chaperones is required for full activity of the cbb 3-type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus

    DOE PAGES

    Trasnea, Petru -Iulian; Utz, Marcel; Khalfaoui-Hassani, Bahia; ...

    2016-02-28

    Copper (Cu) is an essential micronutrient that functions as a cofactor in several important enzymes, like respiratory heme-copper oxygen reductases. Yet, Cu is also toxic and therefore cells engage a highly coordinated Cu uptake and delivery system to prevent the accumulation of toxic Cu concentrations. In the current work we analyzed Cu delivery to the cbb 3-type cytochrome c oxidase ( cbb 3-Cox) of Rhodobacter capsulatus. We identified the PCu AC-like periplasmic chaperone PccA and analyzed its contribution to cbb 3-Cox assembly. Our data demonstrate that PccA is a Cu-binding protein with a preference for Cu(I), which is required formore » efficient cbb 3-Cox assembly, in particular at low Cu concentrations. By using in vivo and in vitro crosslinking we show that PccA forms a complex with the Sco1-homologue SenC. This complex is stabilized in the absence of the cbb 3-Cox specific assembly factors CcoGHIS. In cells lacking SenC, the cytoplasmic Cu content is significantly increased, but the simultaneous absence of PccA prevents this Cu accumulation. Lastly, these data demonstrate that the interplay between PccA and SenC is not only required for Cu delivery during cbb 3-Cox assembly, but that it also regulates Cu homeostasis in R. capsulatus.« less

  15. Nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus; the H+/H- ratio and the activation state of the enzyme during reduction of acetyl pyridine adenine dinucleotide.

    PubMed

    Palmer, T; Jackson, J B

    1992-02-21

    Chromatophores from Rhodobacter capsulatus were incubated in the dark with NADPH and acetylpyridineadenine dinucleotide (AcPdAD+) in the presence of different concentrations of myxothiazol. The transhydrogenase activity was monitored until an appropriate mass action ratio, [AcPdAD+][NADPH]/[AcPdADH][NADP+], was reached. The sample was then illuminated and the initial rate of either AcPdAD+ reduction by NADPH or AcPdADH oxidation by NADP+ was recorded. The ratio of H+ translocated per H- equivalent transferred by transhydrogenase was calculated from the value of the membrane potential (delta pH = 0) at which illumination caused no net reaction in either direction. The mean value for the H+/H- ratio was 0.55. At greater values of [AcPdAD+][NADPH]/[AcPdADH][NADP+] than were employed in the above experiments and over a wider range of concentrations of myxothiazol, it was found that incremental increases in the membrane potential always gave rise to a decrease, never an increase in the rate of AcPdAD+ reduction. In contrast to the H(+)-ATP synthase, there is no evidence of any activation/deactivation of H(+)-transhydrogenase by the protonmotive force.

  16. Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H 2 production

    DOE PAGES

    Wecker, Matt S. A.; Beaton, Stephen E.; Chado, Robert A.; ...

    2016-08-17

    The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H 2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H 2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system. Here we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H 2. The resulting strain photoproduces H 2 and self-reports its own H 2 production through fluorescence. Furthermore, this model system represents amore » unique method of developing hydrogenase-based H 2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H 2.« less

  17. Synergistic dark and photo-fermentation continuous system for hydrogen production from molasses by Clostridium acetobutylicum ATCC 824 and Rhodobacter capsulatus DSM 1710.

    PubMed

    Morsy, Fatthy Mohamed

    2017-04-01

    This study investigated synergistic dark and photo-fermentation using continuous fermentation system (CFS). The system relies on connecting several fermenters from bottom of one to top culture level of the next in a manner that allows for delaying movement of the substrate and thus for its full consumption. While H 2 was collected, CFS allowed for moving liquid byproducts toward the outlet and hence continuous productivity. CFS could be efficiently used for: (1) Continuous dark and photo-fermentation H 2 production by Clostridium acetobutylicum and Rhodobacter capsulatus producing 5.65moleH 2 mole -1 hexose; (2) Continuous dark-fermentation synergistic H 2 , acetone, butanol and ethanol (ABE) production by C. acetobutylicum which produced per mole hexose, 2.43mol H 2 along with 73.08g ABE (3) Continuous H 2 and methane production by C. acetobutylicum and bacterial sludge producing, per mole hexose, 1.64mol pure H 2 and 2.56mol CH 4 mixed with 0.37mol H 2 ·The hydraulic retention time (HRT) for whole system was short where organic acids produced in dark-fermentation in first fermenter were synergistically utilized for H 2 production by R. capsulatus in subsequent fermenters. CFS is suitable for fast-digestible sugars but not lignocelluloses or other hard-digestible organics, requiring prolonged HRT, unless such polymeric organics were hydrolyzed prior to fermentation. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Functional assignment of gene AAC16202.1 from Rhodobacter capsulatus SB1003: new insights into the bacterial SDR sorbitol dehydrogenases family.

    PubMed

    Sola-Carvajal, Agustín; García-García, María Inmaculada; Sánchez-Carrón, Guiomar; García-Carmona, Francisco; Sánchez-Ferrer, Alvaro

    2012-11-01

    Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with over 60,000 non-redundant sequences in the database, many of which need a correct functional assignment. Among them, the gene AAC16202.1 (NCBI) from Rhodobacter capsulatus SB1003 has been assigned in Uniprot both as a sorbitol dehydrogenase (#D5AUY1) and, as an N-acetyl-d-mannosamine dehydrogenase (#O66112), both enzymes being of biotechnological interest. When the gene was overexpressed in Escherichia coli Rosetta (DE3)pLys, the purified enzyme was not active toward N-acetyl-d-mannosamine, whereas it was active toward d-sorbitol and d-fructose. However, the relative activities toward xylitol and l-iditol (0.45 and 6.9%, respectively) were low compared with that toward d-sorbitol. Thus, the enzyme could be considered sorbitol dehydrogenase (SDH) with very low activity toward xylitol, which could increase its biotechnological interest for determining sorbitol without the unspecific cross-determination of added xylitol in food and pharma compositions. The tetrameric enzyme (120 kDa) showed similar catalytic efficiency (2.2 × 10(3) M(-1) s(-1)) to other sorbitol dehydrogenases for d-sorbitol, with an optimum pH of 9.0 and an optimum temperature of 37 °C. The enzyme was also more thermostable than other reported SDH, ammonium sulfate being the best stabilizer in this respect, increasing the melting temperature (T(m)) up to 52.9 °C. The enzyme can also be considered as a new member of the Zn(2+) independent SDH family since no effect on activity was detected in the presence of divalent cations or chelating agents. Finally, its in silico analysis enabled the specific conserved sequence blocks that are the fingerprints of bacterial sorbitol dehydrogenases and mainly located at C-terminal of the protein, to be determined for the first time. This knowledge will facilitate future data curation of present databases and a better functional assignment of newly described

  19. Transformable Rhodobacter strains, method for producing transformable Rhodobacter strains

    DOEpatents

    Laible, Philip D.; Hanson, Deborah K.

    2018-05-08

    The invention provides an organism for expressing foreign DNA, the organism engineered to accept standard DNA carriers. The genome of the organism codes for intracytoplasmic membranes and features an interruption in at least one of the genes coding for restriction enzymes. Further provided is a system for producing biological materials comprising: selecting a vehicle to carry DNA which codes for the biological materials; determining sites on the vehicle's DNA sequence susceptible to restriction enzyme cleavage; choosing an organism to accept the vehicle based on that organism not acting upon at least one of said vehicle's sites; engineering said vehicle to contain said DNA; thereby creating a synthetic vector; and causing the synthetic vector to enter the organism so as cause expression of said DNA.

  20. The PpaA/AerR regulators of photosynthesis gene expression from anoxygenic phototrophic proteobacteria contain heme-binding SCHIC domains.

    PubMed

    Moskvin, Oleg V; Gilles-Gonzalez, Marie-Alda; Gomelsky, Mark

    2010-10-01

    The SCHIC domain of the B12-binding domain family present in the Rhodobacter sphaeroides AppA protein binds heme and senses oxygen. Here we show that the predicted SCHIC domain PpaA/AerR regulators also bind heme and respond to oxygen in vitro, despite their low sequence identity with AppA.

  1. Open reading frame 5 (ORF5), encoding a ferredoxinlike protein, and nifQ are cotranscribed with nifE, nifN, nifX, and ORF4 in Rhodobacter capsulatus.

    PubMed Central

    Moreno-Vivian, C; Hennecke, S; Pühler, A; Klipp, W

    1989-01-01

    DNA sequence analysis of a 1,600-base-pair fragment located downstream of nifENX in nif region A of Rhodobacter capsulatus revealed two additional open reading frames (ORFs): ORF5, encoding a ferredoxinlike protein, and nifQ. The ferredoxinlike gene product contained two cysteine motifs, typical of ferredoxins coordinating two 4Fe-4S clusters, but the distance between these two motifs was unusual for low-molecular-weight ferredoxins. The R. capsulatus nifQ gene product shared a high degree of homology with Klebsiella pneumoniae and Azotobacter vinelandii NifQ, including a typical cysteine motif located in the C-terminal part. nifQ insertion mutants and also an ORF5-nifQ double deletion mutant showed normal diazotrophic growth only in the presence of high concentrations of molybdate. This demonstrated that the gene encoding the ferredoxinlike protein is not essential for nitrogen fixation. No NifA-activated consensus promoter could be found in the intergenic region between nifENX-ORF4 and ORF5-nifQ. Analyses of a nifQ-lacZYA fusion revealed that transcription of nifQ was initiated at a promoter in front of nifE. In contrast to other nitrogen-fixing organisms, R. capsulatus nifE, nifN, nifX, ORF4, ORF5, and nifQ were organized in one transcriptional unit. PMID:2708314

  2. SciTech Connect

    Tabita, Fred Robert

    The overall objective of this project is to determine the mechanism by which a transcriptional activator protein affects CO 2 fixation (cbb) gene expression in nonsulfur purple photosynthetic bacteria, with special emphasis to Rhodobacter sphaeroides and with comparison to Rhodopseudomonas palustris. These studies culminated in several publications which indicated that additional regulators interact with the master regulator CbbR in both R. sphaeroides and R. palustris. In addition, the interactive control of the carbon and nitrogen assimilatory pathways was studied and unique regulatory signals were discovered.

  3. Harnessing Solar Power: Novel Strategies for Rational Design of Photocatalysts and Photovoltaic Materials

    DTIC Science & Technology

    2015-09-01

    Complex 2 ( LH2 ) of Rhodobacter sphaeroides with Two- Dimensional Spectroscopy” J. Chem. Phys. 139, 155101 2013. A.F. Fidler, V.P. Singh, P.D. Long...P.D. Dahlberg, and G.S. Engel, “Time Scales of Coherent Dynamics in the Light-Harvesting Complex 2 ( LH2 ) of Rhodobacter sphaeroides” J. Phys. Chem...Spectroscopy of the Light-harvesting Complex LH2 ” Opt. Lett. 36:9 1665-1667 2011 E. Harel, A.F. Fidler, and G.S. Engel, “Single-Shot GRadient

  4. Biosynthesis of Chlorophyll a in a Purple Bacterial Phototroph and Assembly into a Plant Chlorophyll-Protein Complex.

    PubMed

    Hitchcock, Andrew; Jackson, Philip J; Chidgey, Jack W; Dickman, Mark J; Hunter, C Neil; Canniffe, Daniel P

    2016-09-16

    Improvements to photosynthetic efficiency could be achieved by manipulating pigment biosynthetic pathways of photosynthetic organisms in order to increase the spectral coverage for light absorption. The development of organisms that can produce both bacteriochlorophylls and chlorophylls is one way to achieve this aim, and accordingly we have engineered the bacteriochlorophyll-utilizing anoxygenic phototroph Rhodobacter sphaeroides to make chlorophyll a. Bacteriochlorophyll and chlorophyll share a common biosynthetic pathway up to the precursor chlorophyllide. Deletion of genes responsible for the bacteriochlorophyll-specific modifications of chlorophyllide and replacement of the native bacteriochlorophyll synthase with a cyanobacterial chlorophyll synthase resulted in the production of chlorophyll a. This pigment could be assembled in vivo into the plant water-soluble chlorophyll protein, heterologously produced in Rhodobacter sphaeroides, which represents a proof-of-principle for the engineering of novel antenna complexes that enhance the spectral range of photosynthesis.

  5. SciTech Connect

    Norris, J.R.; Budil, D.E.; Gast, P.

    The orientation of the principal axes of the primary electron donor triplet state measured in single crystals of photosynthetic reaction centers is compared to the x-ray structures of the bacteria Rhodobacter (Rb.) sphaeroides R-26 and Rhodopseudomonas (Rps.) viridis. The primary donor of Rps. viridis is significantly different from that of Rb. sphaeroides. The measured directions of the axes indicate that triplet excitation is almost completely localized on the L-subunit half of the dimer in Rps. viridis but is more symmetrically distributed on the dimeric donor in Rb. sphaeroides R-26. The large reduction of the zero field splitting parameters relative tomore » monomeric bacteriochlorophyll triplet in vitro suggests significant participation of asymmetrical charge transfer electronic configurations in the special pair triplet state of both organisms.« less

  6. SciTech Connect

    Elkabbani, Ossama; Chang, Chonghwan; Tiede, D.

    Photosynthetic reaction centers (RCs) from the photosynthetic bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis are protein complexes closely related in both structure and function. The structure of the Rps. viridis RC was used to determine the structure of the RC from Rb. sphaeroides. Small but meaningful differences between the positions of the helices and the cofactors in the two complexes were identified. The distances between helices A{sub L} adn A{sub M}, between B{sub L} and B{sub M}, and between bacteriopheophytins BP{sub L} and BP{sub M} are significantly shorter in Rps. viridis than they are in Rb. sphaeroides RCs. There are amore » number of differences in the amino acid residues that surround the cofactors; some of these residues form hydrogen bonds with the cofactors. Differences in chemical properties of the two RCs.« less

  7. My daily constitutional in martinsried.

    PubMed

    Allen, James P

    2004-01-01

    The three-dimensional structures of bacterial reaction centers have served as the framework for much of our understanding of anoxygenic photosynthesis. A key step in the determination of the structure of the reaction center from Rhodobacter sphaeroides was the use the molecular replacement technique. For this technique, we made use of two sets of data. First, X-ray diffraction data had been measured from crystals of the reaction center from R. sphaeroides by our research group in California, led by George Feher and Douglas Rees. The second data set consisted of the coordinates of the three-dimensional structure of the reaction center from Rhodopseudomonas (now Blastochloris) viridis, which had been solved in the pioneering efforts of a group in Martinsried, led by Johann Deisenhofer, Robert Huber and Hartmut Michel. The collaborative efforts of these two groups to determine the structure of the reaction center from R. sphaeroides is described.

  8. The energies and kinetics of triplet carotenoids in the LH2 antenna complexes as determined by phosphorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Rondonuwu, Ferdy S.; Taguchi, Tokio; Fujii, Ritsuko; Yokoyama, Kyosuke; Koyama, Yasushi; Watanabe, Yasutaka

    2004-01-01

    The triplet (T 1) states of carotenoids (Cars) and bacteriochlorophyll a (BChl) in the LH2 antenna complexes from Rhodobacter sphaeroides G1C, Rba. sphaeroides 2.4.1 and Rhodospirillum molischianum, containing neurosporene, spheroidene and lycopene, respectively, were examined by stationary-state and time-resolved phosphorescence spectroscopy. The T 1 energies of Cars were determined, irrespective of the Car or BChl excitation, to be 7030 cm -1 (neurosporene), 6920 cm -1 (spheroidene) and 6870 cm -1 (lycopene), respectively, whereas that of BChl to be 7590 cm -1. In the Rba. sphaeroides G1C, the Car and BChl triplet states decayed in similar time constant as the BChl Q y state, a fact which indicates that the pair of triplet states decays through the triplet-triplet annihilation mechanism.

  9. Phylogenetic origins of the plant mitochondrion based on a comparative analysis of 5S ribosomal RNA sequences

    NASA Technical Reports Server (NTRS)

    Villanueva, E.; Delihas, N.; Luehrsen, K. R.; Fox, G. E.; Gibson, J.

    1985-01-01

    The complete nucleotide sequences of 5S ribosomal RNAs from Rhodocyclus gelatinosa, Rhodobacter sphaeroides, and Pseudomonas cepacia were determined. Comparisons of these 5S RNA sequences show that rather than being phylogenetically related to one another, the two photosynthetic bacterial 5S RNAs share more sequence and signature homology with the RNAs of two nonphotosynthetic strains. Rhodobacter sphaeroides is specifically related to Paracoccus denitrificans and Rc. gelatinosa is related to Ps. cepacia. These results support earlier 16S ribosomal RNA studies and add two important groups to the 5S RNA data base. Unique 5S RNA structural features previously found in P. denitrificans are present also in the 5S RNA of Rb. sphaeroides; these provide the basis for subdivisional signatures. The immediate consequence of obtaining these new sequences is that it is possible to clarify the phylogenetic origins of the plant mitochondrion. In particular, a close phylogenetic relationship is found between the plant mitochondria and members of the alpha subdivision of the purple photosynthetic bacteria, namely, Rb. sphaeroides, P. denitrificans, and Rhodospirillum rubrum.

  10. Studies on Hydrogen Production by Photosynthetic Bacteria after Anaerobic Fermentation of Starch by a Hyperthermophile, Pyrococcus furiosus

    NASA Astrophysics Data System (ADS)

    Sugitate, Toshihiro; Fukatsu, Makoto; Ishimi, Katsuhiro; Kohno, Hideki; Wakayama, Tatsuki; Nakamura, Yoshihiro; Miyake, Jun; Asada, Yasuo

    In order to establish the sequential hydrogen production from waste starch using a hyperthermophile, Pyrococcus furiosus, and a photosynthetic bacterium, basic studies were done. P. furiosus produced hydrogen and acetate by anaerobic fermentation at 90°C. A photosynthetic bacterium, Rhodobacter sphaeroides RV, was able to produce hydrogen from acetate under anaerobic and light conditions at 30°C. However, Rb. sphaeroides RV was not able to produce hydrogen from acetate in the presence of sodium chloride that was essential for the growth and hydrogen production of P. furiosus although it produced hydrogen from lactate at a reduced rate with 1% sodium chloride. A newly isolated strain, CST-8, from natural environment was, however, able to produce hydrogen from acetate, especially with 3 mM L-alanine and in the presence of 1% sodium chloride. The sequential hydrogen production with P. furiosus and salt-tolerant photosynthetic bacteria could be probable at least in the laboratory experiment scale.

  11. Rhodobase, a meta-analytical tool for reconstructing gene regulatory networks in a model photosynthetic bacterium.

    PubMed

    Moskvin, Oleg V; Bolotin, Dmitry; Wang, Andrew; Ivanov, Pavel S; Gomelsky, Mark

    2011-02-01

    We present Rhodobase, a web-based meta-analytical tool for analysis of transcriptional regulation in a model anoxygenic photosynthetic bacterium, Rhodobacter sphaeroides. The gene association meta-analysis is based on the pooled data from 100 of R. sphaeroides whole-genome DNA microarrays. Gene-centric regulatory networks were visualized using the StarNet approach (Jupiter, D.C., VanBuren, V., 2008. A visual data mining tool that facilitates reconstruction of transcription regulatory networks. PLoS ONE 3, e1717) with several modifications. We developed a means to identify and visualize operons and superoperons. We designed a framework for the cross-genome search for transcription factor binding sites that takes into account high GC-content and oligonucleotide usage profile characteristic of the R. sphaeroides genome. To facilitate reconstruction of directional relationships between co-regulated genes, we screened upstream sequences (-400 to +20bp from start codons) of all genes for putative binding sites of bacterial transcription factors using a self-optimizing search method developed here. To test performance of the meta-analysis tools and transcription factor site predictions, we reconstructed selected nodes of the R. sphaeroides transcription factor-centric regulatory matrix. The test revealed regulatory relationships that correlate well with the experimentally derived data. The database of transcriptional profile correlations, the network visualization engine and the optimized search engine for transcription factor binding sites analysis are available at http://rhodobase.org. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  12. Species differences in unlocking B-side electron transfer in bacterial reaction centers

    DOE PAGES

    Dylla, Nicholas P.; Faries, Kaitlyn M.; Wyllie, Ryan M.; ...

    2016-06-21

    The structure of the bacterial photosynthetic reaction center (RC) reveals symmetry-related electron transfer (ET) pathways, but only one path is used in native RCs. Analogous mutations have been made in two Rhodobacter (R.) species. A glutamic acid at position 133 in the M subunit increases transmembrane charge separation via the naturally inactive (B-side) path through impacts on primary ET in mutant R. sphaeroidesRCs. Prior work showed that the analogous substitution in the R. capsulatusRC also increases B-side activity, but mainly affects secondary ET. Finally, the overall yields of transmembrane ET are similar, but enabled in fundamentally different ways.

  13. SciTech Connect

    Thompson, M.A.; Zerner, M.C.

    Photosynthetic electron transfer is arguably the most important series of chemical transformations for life on this planet. In recent years the structure of the reaction centers (RC) from the photosynthetic bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides have been presented. On the basis of these structures, several mechanisms have been proposed to explain the primary electron-transfer event with as yet no consensus. The authors report here INDO/S calculations of the excited states of a model of the RC of Rps. viridis in both the absence and presence of a polarizable medium.

  14. Species differences in unlocking B-side electron transfer in bacterial reaction centers

    SciTech Connect

    Dylla, Nicholas P.; Faries, Kaitlyn M.; Wyllie, Ryan M.

    The structure of the bacterial photosynthetic reaction center (RC) reveals symmetry-related electron transfer (ET) pathways, but only one path is used in native RCs. Analogous mutations have been made in two Rhodobacter (R.) species. A glutamic acid at position 133 in the M subunit increases transmembrane charge separation via the naturally inactive (B-side) path through impacts on primary ET in mutant R. sphaeroidesRCs. Prior work showed that the analogous substitution in the R. capsulatusRC also increases B-side activity, but mainly affects secondary ET. Finally, the overall yields of transmembrane ET are similar, but enabled in fundamentally different ways.

  15. A Conserved Steroid Binding Site in Cytochrome c Oxidase

    SciTech Connect

    Qin, Ling; Mills, Denise A.; Buhrow, Leann

    2010-09-02

    Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

  16. Arbitrary-detuning asynchronous optical sampling pump-probe spectroscopy of bacterial reaction centers.

    PubMed

    Antonucci, Laura; Bonvalet, Adeline; Solinas, Xavier; Jones, Michael R; Vos, Marten H; Joffre, Manuel

    2013-09-01

    A recently reported variant of asynchronous optical sampling compatible with arbitrary unstabilized laser repetition rates is applied to pump-probe spectroscopy. This makes possible the use of a 5.1 MHz chirped pulse oscillator as the pump laser, thus extending the available time window to almost 200 ns with a time resolution as good as about 320 fs. The method is illustrated with the measurement in a single experiment of the complete charge transfer dynamics of the reaction center from Rhodobacter sphaeroides.

  17. Effect of integral membrane proteins on the lateral mobility of plastoquinone in phosphatidylcholine proteoliposomes.

    PubMed

    Blackwell, M F; Whitmarsh, J

    1990-11-01

    PYRENE FLUORESCENCE QUENCHING BY PLASTOQUINONE WAS USED TO ESTIMATE THE RATE OF PLASTOQUINONE LATERAL DIFFUSION IN SOYBEAN PHOSPHATIDYLCHOLINE PROTEOLIPOSOMES CONTAINING THE FOLLOWING INTEGRAL MEMBRANE PROTEINS: gramicidin D, spinach cytochrome bf complex, spinach cytochrome f, reaction centers from Rhodobacter sphaeroides, beef heart mitochondrial cytochrome bc(1), and beef heart mitochondrial cytochrome oxidase. The measured plastoquinone lateral diffusion coefficient varied between 1 and 3 . 10(-7) cm(2) s(-1) in control liposomes that lacked protein. When proteins were added, these values decreased: a 10-fold decrease was observed when 16-26% of the membrane surface area was occupied by protein for all the proteins but gramicidin. The larger protein complexes (cytochrome bf, Rhodobacter sphaeroides reaction centers, cytochrome bc(1), and cytochrome oxidase), whose hydrophobic volumes were 15-20 times as large as that of cytochrome f and the gramicidin transmembrane dimer, were 15-20 times as effective in decreasing the lateral-diffusion coefficient over the range of concentrations studied. These proteins had a much stronger effect than that observed for bacteriorhodopsin in fluorescence photobleaching recovery measurements. The effect of high-protein concentrations in gramicidin proteoliposomes was in close agreement with fluorescence photobleaching measurements. The results are compared with the predictions of several theoretical models of lateral mobility as a function of integral membrane concentration.

  18. Effect of integral membrane proteins on the lateral mobility of plastoquinone in phosphatidylcholine proteoliposomes

    PubMed Central

    Blackwell, Mary F.; Whitmarsh, John

    1990-01-01

    Pyrene fluorescence quenching by plastoquinone was used to estimate the rate of plastoquinone lateral diffusion in soybean phosphatidylcholine proteoliposomes containing the following integral membrane proteins: gramicidin D, spinach cytochrome bf complex, spinach cytochrome f, reaction centers from Rhodobacter sphaeroides, beef heart mitochondrial cytochrome bc1, and beef heart mitochondrial cytochrome oxidase. The measured plastoquinone lateral diffusion coefficient varied between 1 and 3 · 10-7 cm2 s-1 in control liposomes that lacked protein. When proteins were added, these values decreased: a 10-fold decrease was observed when 16-26% of the membrane surface area was occupied by protein for all the proteins but gramicidin. The larger protein complexes (cytochrome bf, Rhodobacter sphaeroides reaction centers, cytochrome bc1, and cytochrome oxidase), whose hydrophobic volumes were 15-20 times as large as that of cytochrome f and the gramicidin transmembrane dimer, were 15-20 times as effective in decreasing the lateral-diffusion coefficient over the range of concentrations studied. These proteins had a much stronger effect than that observed for bacteriorhodopsin in fluorescence photobleaching recovery measurements. The effect of high-protein concentrations in gramicidin proteoliposomes was in close agreement with fluorescence photobleaching measurements. The results are compared with the predictions of several theoretical models of lateral mobility as a function of integral membrane concentration. PMID:19431774

  19. Engineered photoproteins that give rise to photosynthetically-incompetent bacteria are effective as photovoltaic materials for biohybrid photoelectrochemical cells.

    PubMed

    Liu, Juntai; Friebe, Vincent M; Swainsbury, David J K; Crouch, Lucy I; Szabo, David A; Frese, Raoul N; Jones, Michael R

    2018-04-17

    Reaction centre/light harvesting proteins such as the RCLH1X complex from Rhodobacter sphaeroides carry out highly quantum-efficient conversion of solar energy through ultrafast energy transfer and charge separation, and these pigment-proteins have been incorporated into biohybrid photoelectrochemical cells for a variety of applications. In this work we demonstrate that, despite not being able to support normal photosynthetic growth of Rhodobacter sphaeroides, an engineered variant of this RCLH1X complex lacking the PufX protein and with an enlarged light harvesting antenna is unimpaired in its capacity for photocurrent generation in two types of bio-photoelectrochemical cells. Removal of PufX also did not impair the ability of the RCLH1 complex to act as an acceptor of energy from synthetic light harvesting quantum dots. Unexpectedly, the removal of PufX led to a marked improvement in the overall stability of the RCLH1 complex under heat stress. We conclude that PufX-deficient RCLH1 complexes are fully functional in solar energy conversion in a device setting and that their enhanced structural stability could make them a preferred choice over their native PufX-containing counterpart. Our findings on the competence of RCLH1 complexes for light energy conversion in vitro are discussed with reference to the reason why these PufX-deficient proteins are not capable of light energy conversion in vivo.

  20. Engineered photoproteins that give rise to photosynthetically-incompetent bacteria are effective as photovoltaic materials for biohybrid photoelectrochemical cells

    PubMed Central

    Liu, Juntai; Friebe, Vincent M.; Swainsbury, David J. K.; Crouch, Lucy I.; Szabo, David A.; Frese, Raoul N.

    2018-01-01

    Reaction centre/light harvesting proteins such as the RCLH1X complex from Rhodobacter sphaeroides carry out highly quantum-efficient conversion of solar energy through ultrafast energy transfer and charge separation, and these pigment-proteins have been incorporated into biohybrid photoelectrochemical cells for a variety of applications. In this work we demonstrate that, despite not being able to support normal photosynthetic growth of Rhodobacter sphaeroides, an engineered variant of this RCLH1X complex lacking the PufX protein and with an enlarged light harvesting antenna is unimpaired in its capacity for photocurrent generation in two types of bio-photoelectrochemical cells. Removal of PufX also did not impair the ability of the RCLH1 complex to act as an acceptor of energy from synthetic light harvesting quantum dots. Unexpectedly, the removal of PufX led to a marked improvement in the overall stability of the RCLH1 complex under heat stress. We conclude that PufX-deficient RCLH1 complexes are fully functional in solar energy conversion in a device setting and that their enhanced structural stability could make them a preferred choice over their native PufX-containing counterpart. Our findings on the competence of RCLH1 complexes for light energy conversion in vitro are discussed with reference to the reason why these PufX-deficient proteins are not capable of light energy conversion in vivo. PMID:29364305

  1. SciTech Connect

    Utschig, L. M.; Poluektov, O.; Schlesselman, S. L.

    The interaction of metal ions with isolated photosynthetic reaction centers (RCs) from the purple bacteria Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas viridis has been investigated with transient optical and magnetic resonance techniques. In RCs from all species, the electrochromic response of the bacteriopheophytin cofactors associated with Q{sub A}{sup -}Q{sub B} {yields} Q{sub A}Q{sub B}{sup -} electron transfer is slowed in the presence of Cu{sup 2+}. This slowing is similar to the metal ion effect observed for RCs from Rb. sphaeroides where Zn{sup 2+} was bound to a specific site on the surface of the RC [Utschig et al. (1998) Biochemistrymore » 37, 8278]. The coordination environments of the Cu{sup 2+} sites were probed with electron paramagnetic resonance (EPR) spectroscopy, providing the first direct spectroscopic evidence for the existence of a second metal site in RCs from Rb. capsulatus and Rps. viridis. In the dark, RCs with Cu{sup 2+} bound to the surface exhibit axially symmetric EPR spectra. Electron spin echo envelope modulation (ESEEM) spectral results indicate multiple weakly hyperfine coupled {sup 14}N nuclei in close proximity to Cu{sup 2+}. These ESEEM spectra resemble those observed for Cu{sup 2+} RCs from Rb. sphaeroides [Utschig et al. (2000) Biochemistry 39, 2961] and indicate that two or more histidines ligate the Cu{sup 2+} at the surface site in each RC. Thus, RCs from Rb. sphaeroides, Rb. capsulatus, and Rps. viridis each have a structurally analogous Cu{sup 2+} binding site that is involved in modulating the Q{sub A}{sup -}Q{sub B} {yields} Q{sub A}Q{sub B}{sup -} electron-transfer process. Inspection of the Rps. viridis crystal structure reveals four potential histidine ligands from three different subunits (M16, H178, H72, and L211) located beneath the Q{sub B} binding pocket. The location of these histidines is surprisingly similar to the grouping of four histidine residues (H68, H126, H128, and L211

  2. Different Functions of Phylogenetically Distinct Bacterial Complex I Isozymes

    SciTech Connect

    Spero, Melanie A.; Brickner, Joshua R.; Mollet, Jordan T.

    NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethylmore » sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroides complex I enzymes (complex I A and complex I E) in maintaining a cellular redox state during photoheterotrophic growth. We propose that the function of each isozyme during photoheterotrophic growth is either NADH synthesis (complex I A) or NADH oxidation (complex I E). The canonical alphaproteobacterial complex I isozyme (complex I A) was also shown to be important for routing electrons to nitrogenase-mediated H 2 production, while the horizontally acquired enzyme (complex I E) was dispensable in this process. Unlike the singular role of complex I in mitochondria, we predict that the phylogenetically distinct complex I enzymes found across bacterial species have evolved to enhance the functions of their respective electron transport chains. Cells use a proton motive force (PMF), NADH, and ATP to support numerous processes. In mitochondria, complex I uses NADH oxidation to generate a PMF, which can drive ATP synthesis. This study analyzed the function of complex I in bacteria, which contain more

  3. Different Functions of Phylogenetically Distinct Bacterial Complex I Isozymes

    DOE PAGES

    Spero, Melanie A.; Brickner, Joshua R.; Mollet, Jordan T.; ...

    2016-02-01

    NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethylmore » sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroides complex I enzymes (complex I A and complex I E) in maintaining a cellular redox state during photoheterotrophic growth. We propose that the function of each isozyme during photoheterotrophic growth is either NADH synthesis (complex I A) or NADH oxidation (complex I E). The canonical alphaproteobacterial complex I isozyme (complex I A) was also shown to be important for routing electrons to nitrogenase-mediated H 2 production, while the horizontally acquired enzyme (complex I E) was dispensable in this process. Unlike the singular role of complex I in mitochondria, we predict that the phylogenetically distinct complex I enzymes found across bacterial species have evolved to enhance the functions of their respective electron transport chains. Cells use a proton motive force (PMF), NADH, and ATP to support numerous processes. In mitochondria, complex I uses NADH oxidation to generate a PMF, which can drive ATP synthesis. This study analyzed the function of complex I in bacteria, which contain more

  4. Communication: Coherences observed in vivo in photosynthetic bacteria using two-dimensional electronic spectroscopy

    NASA Astrophysics Data System (ADS)

    Dahlberg, Peter D.; Norris, Graham J.; Wang, Cheng; Viswanathan, Subha; Singh, Ved P.; Engel, Gregory S.

    2015-09-01

    Energy transfer through large disordered antenna networks in photosynthetic organisms can occur with a quantum efficiency of nearly 100%. This energy transfer is facilitated by the electronic structure of the photosynthetic antennae as well as interactions between electronic states and the surrounding environment. Coherences in time-domain spectroscopy provide a fine probe of how a system interacts with its surroundings. In two-dimensional electronic spectroscopy, coherences can appear on both the ground and excited state surfaces revealing detailed information regarding electronic structure, system-bath coupling, energy transfer, and energetic coupling in complex chemical systems. Numerous studies have revealed coherences in isolated photosynthetic pigment-protein complexes, but these coherences have not been observed in vivo due to the small amplitude of these signals and the intense scatter from whole cells. Here, we present data acquired using ultrafast video-acquisition gradient-assisted photon echo spectroscopy to observe quantum beating signals from coherences in vivo. Experiments were conducted on isolated light harvesting complex II (LH2) from Rhodobacter sphaeroides, whole cells of R. sphaeroides, and whole cells of R. sphaeroides grown in 30% deuterated media. A vibronic coherence was observed following laser excitation at ambient temperature between the B850 and the B850∗ states of LH2 in each of the 3 samples with a lifetime of ˜40-60 fs.

  5. Oxygen-­dependent regulation of bacterial lipid production

    DOE PAGES

    Lemmer, Kimberly C.; Dohnalkova, Alice C.; Noguera, Daniel R.; ...

    2015-05-02

    Understanding the mechanisms of lipid accumulation in microorganisms is important for several reasons. In addition to providing insight into assembly of biological membranes, lipid accumulation has important applications in the production of renewable fuels and chemicals. The photosynthetic bacterium Rhodobacter sphaeroides is an attractive organism to study lipid accumulation, as it has the somewhat unique ability to increase membrane production at low O₂ tensions. Under these conditions, R. sphaeroides develops invaginations of the cytoplasmic membrane to increase its membrane surface area for housing of the membrane-bound components of its photosynthetic apparatus. Here we use fatty acid levels as a reportermore » of membrane lipid content. We show that, under low-O₂ and anaerobic conditions, the total fatty acid content per cell increases 3-fold. We also find that the increases in the amount of fatty acid and photosynthetic pigment per cell are correlated as O₂ tensions or light intensity are changed. To ask if lipid and pigment accumulation were genetically separable, we analyzed strains with mutations in known photosynthetic regulatory pathways. While a strain lacking AppA failed to induce photosynthetic pigment-protein complex accumulation, it increased fatty acid content under low O2 conditions. We also found that an intact PrrBA pathway is required for low O2-induced fatty acid accumulation. In conclusion, our findings suggest a previously unknown role of R. sphaeroides transcriptional regulators in increasing fatty acid and phospholipid accumulation in response to decreased O₂ tension.« less

  6. Mechanisms for hydrogen production by different bacteria during mixed-acid and photo-fermentation and perspectives of hydrogen production biotechnology.

    PubMed

    Trchounian, Armen

    2015-03-01

    H2 has a great potential as an ecologically-clean, renewable and capable fuel. It can be mainly produced via hydrogenases (Hyd) by different bacteria, especially Escherichia coli and Rhodobacter sphaeroides. The operation direction and activity of multiple Hyd enzymes in E. coli during mixed-acid fermentation might determine H2 production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating the activity of different Hyd enzymes is an effective way to enhance H2 production by E. coli in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H2 production. Mixed carbon (sugar and glycerol) utilization studies enlarge the kind of organic wastes used in biotechnology. During photo-fermentation under limited nitrogen conditions, H2 production by Rh. sphaeroides is observed when carbon and nitrogen sources are supplemented. The relationship of H2 production with H(+) transport across the membrane and membrane-associated ATPase activity is shown. On the other hand, combination of carbon sources (succinate, malate) with different nitrogen sources (yeast extract, glutamate, glycine) as well as different metal (Fe, Ni, Mg) ions might regulate H2 production. All these can enhance H2 production yield by Rh. sphaeroides in biotechnology Finally, two of these bacteria might be combined to develop and consequently to optimize two stages of H2 production biotechnology with high efficiency transformation of different organic sources.

  7. An integrated approach to reconstructing genome-scale transcriptional regulatory networks

    DOE PAGES

    Imam, Saheed; Noguera, Daniel R.; Donohue, Timothy J.; ...

    2015-02-27

    Transcriptional regulatory networks (TRNs) program cells to dynamically alter their gene expression in response to changing internal or environmental conditions. In this study, we develop a novel workflow for generating large-scale TRN models that integrates comparative genomics data, global gene expression analyses, and intrinsic properties of transcription factors (TFs). An assessment of this workflow using benchmark datasets for the well-studied γ-proteobacterium Escherichia coli showed that it outperforms expression-based inference approaches, having a significantly larger area under the precision-recall curve. Further analysis indicated that this integrated workflow captures different aspects of the E. coli TRN than expression-based approaches, potentially making themmore » highly complementary. We leveraged this new workflow and observations to build a large-scale TRN model for the α-Proteobacterium Rhodobacter sphaeroides that comprises 120 gene clusters, 1211 genes (including 93 TFs), 1858 predicted protein-DNA interactions and 76 DNA binding motifs. We found that ~67% of the predicted gene clusters in this TRN are enriched for functions ranging from photosynthesis or central carbon metabolism to environmental stress responses. We also found that members of many of the predicted gene clusters were consistent with prior knowledge in R. sphaeroides and/or other bacteria. Experimental validation of predictions from this R. sphaeroides TRN model showed that high precision and recall was also obtained for TFs involved in photosynthesis (PpsR), carbon metabolism (RSP_0489) and iron homeostasis (RSP_3341). In addition, this integrative approach enabled generation of TRNs with increased information content relative to R. sphaeroides TRN models built via other approaches. We also show how this approach can be used to simultaneously produce TRN models for each related organism used in the comparative genomics analysis. Our results highlight the advantages of

  8. Albirhodobacter marinus gen. nov., sp. nov., a member of the family Rhodobacteraceae isolated from sea shore water of Visakhapatnam, India.

    PubMed

    Nupur; Vaidya, Bhumika; Tanuku, Naga Radha Srinivas; Pinnaka, Anil Kumar

    2013-02-01

    A novel marine, Gram-negative, rod-shaped bacterium, designated strain N9(T), was isolated from a water sample of the sea shore at Visakhapatnam, Andhra Pradesh (India). Strain N9(T) was found to be positive for oxidase and catalase activities. The fatty acids were found to be dominated by C(16:0), C(18:1) ω7c and summed in feature 3 (C(16:1) ω7c and/or C(16:1) ω6c). Strain N9(T) was determined to contain Q-10 as the major respiratory quinone and phosphatidylethanolamine, phosphatidylglycerol, two aminophospholipids, two phospholipids and four unidentified lipids as polar lipids. The DNA G+C content of the strain N9(T) was found to be 63 mol%. 16S rRNA gene sequence analysis indicated that Rhodobacter sphaeroides, Rhodobacter johrii, Pseudorhodobacter ferrugineus, Rhodobacter azotoformans, Rhodobacter ovatus and Pseudorhodobacter aquimaris were the nearest phylogenetic neighbours, with pair-wise sequence similarities of 95.43, 95.36, 94.24, 95.31, 95.60 and 94.74 %, respectively. Phylogenetic analysis showed that strain N9(T) formed a distinct branch within the family Rhodobacteraceae and clustered with the clade comprising species of the genus Pseudorhodobacter, together with species of the genera Roseicitreum, Roseinatronobacter, Roseibaca and Rhodobaca. Species of the genus Pseudorhodobacter are phylogenetically close with a 16S rRNA gene sequence dissimilarity of 5.9-7.3 % (92.7-94.1 % similarity). Based on the above-mentioned phenotypic characteristics and on phylogenetic inference, strain N9(T) is proposed as a representative of a new genus and a novel species of the family Rhodobacteraceae as Albirhodobacter marinus gen. nov., sp. nov. The type strain of Albirhodobacter marinus is N9 (= MTCC 11277(T) = JCM 17680(T)).

  9. Interface for Light-Driven Electron Transfer by Photosynthetic Complexes Across Block Copolymer Membranes.

    PubMed

    Kuang, Liangju; Olson, Tien L; Lin, Su; Flores, Marco; Jiang, Yunjiang; Zheng, Wan; Williams, JoAnn C; Allen, James P; Liang, Hongjun

    2014-03-06

    Incorporation of membrane proteins into nanodevices to mediate recognition and transport in a collective and scalable fashion remains a challenging problem. We demonstrate how nanoscale photovoltaics could be designed using robust synthetic nanomembranes with incorporated photosynthetic reaction centers (RCs). Specifically, RCs from Rhodobacter sphaeroides are reconstituted spontaneously into rationally designed polybutadiene membranes to form hierarchically organized proteopolymer membrane arrays via a charge-interaction-directed reconstitution mechanism. Once incorporated, the RCs are fully active for prolonged periods based upon a variety of spectroscopic measurements, underscoring preservation of their 3D pigment configuration critical for light-driven charge transfer. This result provides a strategy to construct solar conversion devices using structurally versatile proteopolymer membranes with integrated RC functions to harvest broad regions of the solar spectrum.

  10. Directed formation of micro- and nanoscale patterns of functional light-harvesting LH2 complexes.

    PubMed

    Reynolds, Nicholas P; Janusz, Stefan; Escalante-Marun, Maryana; Timney, John; Ducker, Robert E; Olsen, John D; Otto, Cees; Subramaniam, Vinod; Leggett, Graham J; Hunter, C Neil

    2007-11-28

    The precision placement of the desired protein components on a suitable substrate is an essential prelude to any hybrid "biochip" device, but a second and equally important condition must also be met: the retention of full biological activity. Here we demonstrate the selective binding of an optically active membrane protein, the light-harvesting LH2 complex from Rhodobacter sphaeroides, to patterned self-assembled monolayers at the micron scale and the fabrication of nanometer-scale patterns of these molecules using near-field photolithographic methods. In contrast to plasma proteins, which are reversibly adsorbed on many surfaces, the LH2 complex is readily patterned simply by spatial control of surface polarity. Near-field photolithography has yielded rows of light-harvesting complexes only 98 nm wide. Retention of the native optical properties of patterned LH2 molecules was demonstrated using in situ fluorescence emission spectroscopy.

  11. Two-photon excitation spectroscopy of carotenoid-containing and carotenoid-depleted LH2 complexes from purple bacteria.

    PubMed

    Stepanenko, Ilya; Kompanetz, Viktor; Makhneva, Zoya; Chekalin, Sergey; Moskalenko, Andrei; Razjivin, Andrei

    2009-08-27

    We applied two-photon fluorescence excitation spectroscopy to LH2 complex from purple bacteria Allochromatium minutissimum and Rhodobacter sphaeroides . Bacteriochlorophyll fluorescence was measured under two-photon excitation of the samples within the 1200-1500 nm region. Spectra were obtained for both carotenoid-containing and -depleted complexes of each bacterium to allow their direct comparison. The depletion of carotenoids did not alter the two-photon excitation spectra of either bacteria. The spectra featured a wide excitation band around 1350 nm (2x675 nm, 14,800 cm(-1)) which strongly resembled two-photon fluorescence excitation spectra of similar complexes published by other authors. We consider obtained experimental data to be evidence of direct two-photon excitation of bacteriochlorophyll excitonic states in this spectral region.

  12. Prediction and Biochemical Demonstration of a Catabolic Pathway for the Osmoprotectant Proline Betaine

    PubMed Central

    Kumar, Ritesh; Zhao, Suwen; Vetting, Matthew W.; Wood, B. McKay; Sakai, Ayano; Cho, Kyuil; Solbiati, José; Almo, Steven C.; Sweedler, Jonathan V.; Jacobson, Matthew P.; Gerlt, John A.; Cronan, John E.

    2014-01-01

    ABSTRACT Through the use of genetic, enzymatic, metabolomic, and structural analyses, we have discovered the catabolic pathway for proline betaine, an osmoprotectant, in Paracoccus denitrificans and Rhodobacter sphaeroides. Genetic and enzymatic analyses showed that several of the key enzymes of the hydroxyproline betaine degradation pathway also function in proline betaine degradation. Metabolomic analyses detected each of the metabolic intermediates of the pathway. The proline betaine catabolic pathway was repressed by osmotic stress and cold stress, and a regulatory transcription factor was identified. We also report crystal structure complexes of the P. denitrificans HpbD hydroxyproline betaine epimerase/proline betaine racemase with l-proline betaine and cis-hydroxyproline betaine. PMID:24520058

  13. A Conserved Structural Module Regulates Transcriptional Responses to Diverse Stress Signals in Bacteria

    PubMed Central

    Campbell, Elizabeth A.; Greenwell, Roger; Anthony, Jennifer R.; Wang, Sheng; Lim, Lionel; Das, Kalyan; Sofia, Heidi J.; Donohue, Timothy J.; Darst, Seth A.

    2008-01-01

    SUMMARY A transcriptional response to singlet oxygen in Rhodobacter sphaeroides is controlled by the group IV σ factor σE and its cognate anti-σ ChrR. Crystal structures of the σE/ChrR complex reveal a modular, two-domain architecture for ChrR. The ChrR N-terminal anti-σ domain (ASD) binds a Zn2+ ion, contacts σE, and is sufficient to inhibit σE-dependent transcription. The ChrR C-terminal domain adopts a cupin fold, can coordinate an additional Zn2+, and is required for the transcriptional response to singlet oxygen. Structure-based sequence analyses predict that the ASD defines a common structural fold among predicted group IV antiσs. These ASDs are fused to diverse C-terminal domains that are likely involved in responding to specific environmental signals that control the activity of their cognate σ factor. PMID:17803943

  14. Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI.

    PubMed

    Greene, P J; Ballard, B T; Stephenson, F; Kohr, W J; Rodriguez, H; Rosenberg, J M; Boyer, H W

    1988-08-15

    Rhodobacter sphaeroides strain 630 produces restriction enzyme RsrI which is an isoschizomer of EcoRI. We have purified this enzyme and initiated a comparison with the EcoRI endonuclease. The properties of RsrI are consistent with a reaction mechanism similar to that of EcoRI: the position of cleavage within the -GAATTC-site is identical, the MgCl2 optimum for the cleavage is identical, and the pH profile is similar. Methylation of the substrate sequence by the EcoRI methylase protects the site from cleavage by the RsrI endonuclease. RsrI cross-reacts strongly with anti-EcoRI serum indicating three-dimensional structural similarities. We have determined the sequence of 34 N terminal amino acids for RsrI and this sequence possesses significant similarity to the EcoRI N terminus.

  15. Cellulose synthases: new insights from crystallography and modeling.

    PubMed

    Slabaugh, Erin; Davis, Jonathan K; Haigler, Candace H; Yingling, Yaroslava G; Zimmer, Jochen

    2014-02-01

    Detailed information about the structure and biochemical mechanisms of cellulose synthase (CelS) proteins remained elusive until a complex containing the catalytic subunit (BcsA) of CelS from Rhodobacter sphaeroides was crystalized. Additionally, a 3D structure of most of the cytosolic domain of a plant CelS (GhCESA1 from cotton, Gossypium hirsutum) was produced by computational modeling. This predicted structure contributes to our understanding of how plant CelS proteins may be similar and different as compared with BcsA. In this review, we highlight how these structures impact our understanding of the synthesis of cellulose and other extracellular polysaccharides. We show how the structures can be used to generate hypotheses for experiments testing mechanisms of glucan synthesis and translocation in plant CelS. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Tuning cofactor redox potentials: the 2-methoxy dihedral angle generates a redox potential difference of >160 mV between the primary (Q(A)) and secondary (Q(B)) quinones of the bacterial photosynthetic reaction center.

    PubMed

    Taguchi, Alexander T; Mattis, Aidas J; O'Malley, Patrick J; Dikanov, Sergei A; Wraight, Colin A

    2013-10-15

    Only quinones with a 2-methoxy group can act simultaneously as the primary (QA) and secondary (QB) electron acceptors in photosynthetic reaction centers from Rhodobacter sphaeroides. (13)C hyperfine sublevel correlation measurements of the 2-methoxy in the semiquinone states, SQA and SQB, were compared with quantum mechanics calculations of the (13)C couplings as a function of the dihedral angle. X-ray structures support dihedral angle assignments corresponding to a redox potential gap (ΔEm) between QA and QB of ~180 mV. This is consistent with the failure of a ubiquinone analogue lacking the 2-methoxy to function as QB in mutant reaction centers with a ΔEm of ≈160-195 mV.

  17. Bacterial cell identification in differential interference contrast microscopy images.

    PubMed

    Obara, Boguslaw; Roberts, Mark A J; Armitage, Judith P; Grau, Vicente

    2013-04-23

    Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins.

  18. Strong Coupling of Localized Surface Plasmons to Excitons in Light-Harvesting Complexes

    SciTech Connect

    Tsargorodska, Anna; Cartron, Michaël L.; Vasilev, Cvetelin

    Gold nanostructure arrays exhibit surface plasmon resonances that split after attaching light harvesting complexes 1 and 2 (LH1 and LH2) from purple bacteria. The splitting is attributed to strong coupling between the localized surface plasmon resonances and excitons in the light-harvesting complexes. Wild-type and mutant LH1 and LH2 from Rhodobacter sphaeroides containing different carotenoids yield different splitting energies, demonstrating that the coupling mechanism is sensitive to the electronic states in the light harvesting complexes. Plasmon–exciton coupling models reveal different coupling strengths depending on the molecular organization and the protein coverage, consistent with strong coupling. Strong coupling was also observed formore » self-assembling polypeptide maquettes that contain only chlorins. However, it is not observed for monolayers of bacteriochlorophyll, indicating that strong plasmon–exciton coupling is sensitive to the specific presentation of the pigment molecules.« less

  19. Demonstration of asymmetric electron conduction in pseudosymmetrical photosynthetic reaction centre proteins in an electrical circuit

    PubMed Central

    Kamran, Muhammad; Friebe, Vincent M.; Delgado, Juan D.; Aartsma, Thijs J.; Frese, Raoul N.; Jones, Michael R.

    2015-01-01

    Photosynthetic reaction centres show promise for biomolecular electronics as nanoscale solar-powered batteries and molecular diodes that are amenable to atomic-level re-engineering. In this work the mechanism of electron conduction across the highly tractable Rhodobacter sphaeroides reaction centre is characterized by conductive atomic force microscopy. We find, using engineered proteins of known structure, that only one of the two cofactor wires connecting the positive and negative termini of this reaction centre is capable of conducting unidirectional current under a suitably oriented bias, irrespective of the magnitude of the bias or the applied force at the tunnelling junction. This behaviour, strong functional asymmetry in a largely symmetrical protein–cofactor matrix, recapitulates the strong functional asymmetry characteristic of natural photochemical charge separation, but it is surprising given that the stimulus for electron flow is simply an externally applied bias. Reasons for the electrical resistance displayed by the so-called B-wire of cofactors are explored. PMID:25751412

  20. Strong Coupling of Localized Surface Plasmons to Excitons in Light-Harvesting Complexes

    DOE PAGES

    Tsargorodska, Anna; Cartron, Michaël L.; Vasilev, Cvetelin; ...

    2016-09-30

    Gold nanostructure arrays exhibit surface plasmon resonances that split after attaching light harvesting complexes 1 and 2 (LH1 and LH2) from purple bacteria. The splitting is attributed to strong coupling between the localized surface plasmon resonances and excitons in the light-harvesting complexes. Wild-type and mutant LH1 and LH2 from Rhodobacter sphaeroides containing different carotenoids yield different splitting energies, demonstrating that the coupling mechanism is sensitive to the electronic states in the light harvesting complexes. Plasmon–exciton coupling models reveal different coupling strengths depending on the molecular organization and the protein coverage, consistent with strong coupling. Strong coupling was also observed formore » self-assembling polypeptide maquettes that contain only chlorins. However, it is not observed for monolayers of bacteriochlorophyll, indicating that strong plasmon–exciton coupling is sensitive to the specific presentation of the pigment molecules.« less

  1. Demonstration of asymmetric electron conduction in pseudosymmetrical photosynthetic reaction centre proteins in an electrical circuit.

    PubMed

    Kamran, Muhammad; Friebe, Vincent M; Delgado, Juan D; Aartsma, Thijs J; Frese, Raoul N; Jones, Michael R

    2015-03-09

    Photosynthetic reaction centres show promise for biomolecular electronics as nanoscale solar-powered batteries and molecular diodes that are amenable to atomic-level re-engineering. In this work the mechanism of electron conduction across the highly tractable Rhodobacter sphaeroides reaction centre is characterized by conductive atomic force microscopy. We find, using engineered proteins of known structure, that only one of the two cofactor wires connecting the positive and negative termini of this reaction centre is capable of conducting unidirectional current under a suitably oriented bias, irrespective of the magnitude of the bias or the applied force at the tunnelling junction. This behaviour, strong functional asymmetry in a largely symmetrical protein-cofactor matrix, recapitulates the strong functional asymmetry characteristic of natural photochemical charge separation, but it is surprising given that the stimulus for electron flow is simply an externally applied bias. Reasons for the electrical resistance displayed by the so-called B-wire of cofactors are explored.

  2. Optimal chemotactic responses in stochastic environments

    PubMed Central

    Godány, Martin

    2017-01-01

    Although the “adaptive” strategy used by Escherichia coli has dominated our understanding of bacterial chemotaxis, the environmental conditions under which this strategy emerged is still poorly understood. In this work, we study the performance of various chemotactic strategies under a range of stochastic time- and space-varying attractant distributions in silico. We describe a novel “speculator” response in which the bacterium compare the current attractant concentration to the long-term average; if it is higher then they tumble persistently, while if it is lower than the average, bacteria swim away in search of more favorable conditions. We demonstrate how this response explains the experimental behavior of aerobically-grown Rhodobacter sphaeroides and that under spatially complex but slowly-changing nutrient conditions the speculator response is as effective as the adaptive strategy of E. coli. PMID:28644830

  3. A ternary AppA-PpsR-DNA complex mediates light regulation of photosynthesis-related gene expression.

    PubMed

    Winkler, Andreas; Heintz, Udo; Lindner, Robert; Reinstein, Jochen; Shoeman, Robert L; Schlichting, Ilme

    2013-07-01

    The anoxygenic phototrophic bacterium Rhodobacter sphaeroides uses different energy sources, depending on environmental conditions including aerobic respiration or, in the absence of oxygen, photosynthesis. Photosynthetic genes are repressed at high oxygen tension, but at intermediate levels their partial expression prepares the bacterium for using light energy. Illumination, however, enhances repression under semiaerobic conditions. Here, we describe molecular details of two proteins mediating oxygen and light control of photosynthesis-gene expression: the light-sensing antirepressor AppA and the transcriptional repressor PpsR. Our crystal structures of both proteins and their complex and hydrogen/deuterium-exchange data show that light activation of AppA-PpsR2 affects the PpsR effector region within the complex. DNA binding studies demonstrate the formation of a light-sensitive ternary AppA-PpsR-DNA complex. We discuss implications of these results for regulation by light and oxygen, highlighting new insights into blue light-mediated signal transduction.

  4. A mixed incoherent feed-forward loop contributes to the regulation of bacterial photosynthesis genes.

    PubMed

    Mank, Nils N; Berghoff, Bork A; Klug, Gabriele

    2013-03-01

    Living cells use a variety of regulatory network motifs for accurate gene expression in response to changes in their environment or during differentiation processes. In Rhodobacter sphaeroides, a complex regulatory network controls expression of photosynthesis genes to guarantee optimal energy supply on one hand and to avoid photooxidative stress on the other hand. Recently, we identified a mixed incoherent feed-forward loop comprising the transcription factor PrrA, the sRNA PcrZ and photosynthesis target genes as part of this regulatory network. This point-of-view provides a comparison to other described feed-forward loops and discusses the physiological relevance of PcrZ in more detail.

  5. A mixed incoherent feed-forward loop contributes to the regulation of bacterial photosynthesis genes

    PubMed Central

    Mank, Nils N.; Berghoff, Bork A.; Klug, Gabriele

    2013-01-01

    Living cells use a variety of regulatory network motifs for accurate gene expression in response to changes in their environment or during differentiation processes. In Rhodobacter sphaeroides, a complex regulatory network controls expression of photosynthesis genes to guarantee optimal energy supply on one hand and to avoid photooxidative stress on the other hand. Recently, we identified a mixed incoherent feed-forward loop comprising the transcription factor PrrA, the sRNA PcrZ and photosynthesis target genes as part of this regulatory network. This point-of-view provides a comparison to other described feed-forward loops and discusses the physiological relevance of PcrZ in more detail. PMID:23392242

  6. Investigation of Stability of Photosynthetic Reaction Center and Quantum Dot Hybrid Films.

    PubMed

    Lukashev, E P; Knox, P P; Oleinikov, I P; Seifullina, N Kh; Grishanova, N P

    2016-01-01

    The efficiency of interaction (efficiency of energy transfer) between various quantum dots (QDs) and photosynthetic reaction centers (RCs) from the purple bacterium Rhodobacter sphaeroides and conditions of long-term stability of functioning of such hybrid complexes in film preparations were investigated. It was found that dry films containing RCs and QDs and maintained at atmospheric humidity are capable to keep their functional activity for at least some months as judging by results of measurement of their spectral characteristics, efficiency of energy transfer from QDs to RCs, and RC electron-transport activity. Addition of trehalose to the films giving them still greater stability is especially expressed for films maintained at low humidity. These stable hybrid film structures are promising for further biotechnological studies for developing new phototransformation devices.

  7. Crystallographic snapshot of cellulose synthesis and membrane translocation.

    PubMed

    Morgan, Jacob L W; Strumillo, Joanna; Zimmer, Jochen

    2013-01-10

    Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.

  8. Energies and excited-state dynamics of 1Bu+, 1Bu- and 3Ag- states of carotenoids bound to LH2 antenna complexes from purple photosynthetic bacteria

    NASA Astrophysics Data System (ADS)

    Christiana, Rebecca; Miki, Takeshi; Kakitani, Yoshinori; Aoyagi, Shiho; Koyama, Yasushi; Limantara, Leenawaty

    2009-10-01

    Time-resolved pump-probe stimulated-emission and transient-absorption spectra were recorded after excitation with ˜30 fs pulses to the 1Bu+(0) and optically-forbidden diabatic levels of carotenoids, neurosporene, spheroidene and lycopene having n = 9-11 double bonds, bound to LH2 antenna complexes from Rhodobacter sphaeroides G1C, 2.4.1 and Rhodospirillum molischianum. The low-energy shift of stimulated emission from the covalent 1Bu-(0) and 3Ag-(0) levels slightly larger than that from the ionic 1Bu+(0) state suggests the polarization, whereas more efficient triplet generation suggests the twisting of the conjugated chain in Cars bound to the LH2 complexes, when compared to Cars free in solution.

  9. Incorporation of spheroidene and spheroidenone into light-harvesting complexes from purple sulfur bacteria.

    PubMed

    Ashikhmin, Aleksandr; Makhneva, Zoya; Bolshakov, Maksim; Moskalenko, Andrey

    2017-05-01

    Spheroidene and spheroidenone from the non-sulfur bacterium Rhodobacter (Rba.) sphaeroides were incorporated into diphenylamine (DPA) LH1-RC and LH2 complexes from sulfur bacteria Allochromatium (Alc.) minutissimum and Ectothiorhodospira (Ect.) haloalkaliphila in which carotenoid (Car) biosynthesis was inhibited by ~95%. A series of biochemical characteristics of the modified LH2 complexes was studied (electrophoretic mobility, absorption and CD spectra, Car composition, Car-to-BChl energy transfer and thermal stability). It was found that the electrophoretic mobility of the complexes with incorporated Cars did not change compared to that of the control and DPA-complexes, indicating the absence of any significant change in the structure of LH complexes upon DPA-treatment and subsequent incorporation of Cars. The analysis of fluorescence excitation spectra of the spheroidene-incorporated LH2 complex (LH2:sph) and the spheroidenone-incorporated LH2 complex (LH2:sph-ne) showed that spheroidene and spheroidenone exhibited relatively low efficiencies of energy transfer to BChl, when incorporated into the LH2 DPA-complexes from Alc. minutissimum and Ect. haloalkaliphila, although, they showed high efficiencies, being in their natural state in the LH2 complexes from Rba. sphaeroides. A significant increase in thermostability observed for the LH2:sph and LH2:sph-ne complexes with respect to the LH2 DPA-complexes indicated that the two incorporated Cars stabilized the structure of the LH2 complexes. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Response kinetics of tethered bacteria to stepwise changes in nutrient concentration.

    PubMed

    Chernova, Anna A; Armitage, Judith P; Packer, Helen L; Maini, Philip K

    2003-09-01

    We examined the changes in swimming behaviour of the bacterium Rhodobacter sphaeroides in response to stepwise changes in a nutrient (propionate), following the pre-stimulus motion, the initial response and the adaptation to the sustained concentration of the chemical. This was carried out by tethering motile cells by their flagella to glass slides and following the rotational behaviour of their cell bodies in response to the nutrient change. Computerised motion analysis was used to analyse the behaviour. Distributions of run and stop times were obtained from rotation data for tethered cells. Exponential and Weibull fits for these distributions, and variability in individual responses are discussed. In terms of parameters derived from the run and stop time distributions, we compare the responses to stepwise changes in the nutrient concentration and the long-term behaviour of 84 cells under 12 propionate concentration levels from 1 nM to 25 mM. We discuss traditional assumptions for the random walk approximation to bacterial swimming and compare them with the observed R. sphaeroides motile behaviour.

  11. Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases.

    PubMed

    Stephenson, F H; Ballard, B T; Boyer, H W; Rosenberg, J M; Greene, P J

    1989-12-21

    The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase. A clone containing an 11-kb BamHI fragment was isolated from an R. sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI. Extracts of E. coli containing a subclone of the 11-kb fragment display RsrI activity. Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa. A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI. Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI. The type-II ENases have many common properties, and a common origin might have been expected. Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms.

  12. Light-field-characterization in a continuous hydrogen-producing photobioreactor by optical simulation and computational fluid dynamics.

    PubMed

    Krujatz, Felix; Illing, Rico; Krautwer, Tobias; Liao, Jing; Helbig, Karsten; Goy, Katharina; Opitz, Jörg; Cuniberti, Gianaurelio; Bley, Thomas; Weber, Jost

    2015-12-01

    Externally illuminated photobioreactors (PBRs) are widely used in studies on the use of phototrophic microorganisms as sources of bioenergy and other photobiotechnology research. In this work, straightforward simulation techniques were used to describe effects of varying fluid flow conditions in a continuous hydrogen-producing PBR on the rate of photofermentative hydrogen production (rH2 ) by Rhodobacter sphaeroides DSM 158. A ZEMAX optical ray tracing simulation was performed to quantify the illumination intensity reaching the interior of the cylindrical PBR vessel. 24.2% of the emitted energy was lost through optical effects, or did not reach the PBR surface. In a dense culture of continuously producing bacteria during chemostatic cultivation, the illumination intensity became completely attenuated within the first centimeter of the PBR radius as described by an empirical three-parametric model implemented in Mathcad. The bacterial movement in chemostatic steady-state conditions was influenced by varying the fluid Reynolds number. The "Computational Fluid Dynamics" and "Particle Tracing" tools of COMSOL Multiphysics were used to visualize the fluid flow pattern and cellular trajectories through well-illuminated zones near the PBR periphery and dark zones in the center of the PBR. A moderate turbulence (Reynolds number = 12,600) and fluctuating illumination of 1.5 Hz were found to yield the highest continuous rH2 by R. sphaeroides DSM 158 (170.5 mL L(-1) h(-1) ) in this study. © 2015 Wiley Periodicals, Inc.

  13. The electron transfer flavoprotein: ubiquinone oxidoreductases.

    PubMed

    Watmough, Nicholas J; Frerman, Frank E

    2010-12-01

    Electron transfer flavoprotein: ubiqionone oxidoreductase (ETF-QO) is a component of the mitochondrial respiratory chain that together with electron transfer flavoprotein (ETF) forms a short pathway that transfers electrons from 11 different mitochondrial flavoprotein dehydrogenases to the ubiquinone pool. The X-ray structure of the pig liver enzyme has been solved in the presence and absence of a bound ubiquinone. This structure reveals ETF-QO to be a monotopic membrane protein with the cofactors, FAD and a [4Fe-4S](+1+2) cluster, organised to suggests that it is the flavin that serves as the immediate reductant of ubiquinone. ETF-QO is very highly conserved in evolution and the recombinant enzyme from the bacterium Rhodobacter sphaeroides has allowed the mutational analysis of a number of residues that the structure suggested are involved in modulating the reduction potential of the cofactors. These experiments, together with the spectroscopic measurement of the distances between the cofactors in solution have confirmed the intramolecular pathway of electron transfer from ETF to ubiquinone. This approach can be extended as the R. sphaeroides ETF-QO provides a template for investigating the mechanistic consequences of single amino acid substitutions of conserved residues that are associated with a mild and late onset variant of the metabolic disease multiple acyl-CoA dehydrogenase deficiency (MADD). Copyright © 2010 Elsevier B.V. All rights reserved.

  14. SciTech Connect

    Burger, Brian T.; Imam, Saheed; Scarborough, Matthew J.

    Rhodobacter sphaeroides is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosyntheticmore » growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for R. sphaeroides. Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species.« less

  15. Evaluation of Lighting Systems, Carbon Sources, and Bacteria Cultures on Photofermentative Hydrogen Production.

    PubMed

    Hu, Chengcheng; Choy, Sing-Ying; Giannis, Apostolos

    2018-05-01

    Fluorescent and incandescent lighting systems were applied for batch photofermentative hydrogen production by four purple non-sulfur photosynthetic bacteria (PNSB). The hydrogen production efficiency of Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodospirillum rubrum was evaluated using different carbon sources (acetate, butyrate, lactate, and malate). Incandescent light was found to be more effective for bacteria cell growth and hydrogen production. It was observed that PNSB followed substrate selection criteria for hydrogen production. Only R. palustris was able to produce hydrogen using most carbon sources. Cell density was almost constant, but cell growth rate and hydrogen production were significantly varied under the different lighting systems. The kinetics study suggested that initial substrate concentration had a positive correlation with lag phase duration. Among the PNSB, R. palustris grew faster and had higher hydrogen yields of 1.58, 4.92, and 2.57 mol H 2 /mol using acetate, butyrate, and lactate, respectively. In the integrative approach with dark fermentation effluents rich in organic acids, R. palustris should be enriched in the phototrophic microbial consortium of the continuous hydrogen production system.

  16. Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ

    PubMed Central

    Mank, Nils N.; Berghoff, Bork A.; Hermanns, Yannick N.; Klug, Gabriele

    2012-01-01

    The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA. PMID:22988125

  17. Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ.

    PubMed

    Mank, Nils N; Berghoff, Bork A; Hermanns, Yannick N; Klug, Gabriele

    2012-10-02

    The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.

  18. Proton conduction within the reaction centers of Rhodobacter capsulatus: the electrostatic role of the protein.

    PubMed

    Maróti, P; Hanson, D K; Baciou, L; Schiffer, M; Sebban, P

    1994-06-07

    Light-induced charge separation in the photosynthetic reaction center results in delivery of two electrons and two protons to the terminal quinone acceptor QB. In this paper, we have used flash-induced absorbance spectroscopy to study three strains that share identical amino acid sequences in the QB binding site, all of which lack the protonatable amino acids Glu-L212 and Asp-L213. These strains are the photosynthetically incompetent site-specific mutant Glu-L212/Asp-L213-->Ala-L212/Ala-L213 and two different photocompetent derivatives that carry both alanine substitutions and an intergenic suppressor mutation located far from QB (class 3 strain, Ala-Ala + Arg-M231-->Leu; class 4 strain, Ala-Ala + Asn-M43-->Asp). At pH 8 in the double mutant, we observe a concomitant decrease of nearly 4 orders of magnitude in the rate constants of second electron and proton transfer to QB compared to the wild type. Surprisingly, these rates are increased to about the same extent in both types of suppressor strains but remain > 2 orders of magnitude smaller than those of the wild type. In the double mutant, at pH 8, the loss of Asp-L213 and Glu-L212 leads to a substantial stabilization (> or = 60 meV) of the semiquinone energy level. Both types of compensatory mutations partially restore, to nearly the same level, the original free energy difference for electron transfer from primary quinone QA to QB. The pH dependence of the electron and proton transfer processes in the double-mutant and the suppressor strains suggests that when reaction centers of the double mutant are shifted to lower pH (1.5-2 units), they function like those of the suppressor strains at physiological pH. Our data suggest that the main effect of the compensatory mutations is to partially restore the negative electrostatic environment of QB and to increase an apparent "functional" pK of the system for efficient proton transfer to the active site. This emphasizes the role of the protein in tuning the electrostatic environment of its cofactors and highlights the possible long-range electrostatic effects.

  19. Stoichiometry of ATP hydrolysis and chlorophyllide formation of dark-operative protochlorophyllide oxidoreductase from Rhodobacter capsulatus

    SciTech Connect

    Nomata, Jiro; Terauchi, Kazuki; Fujita, Yuichi, E-mail: fujita@agr.nagoya-u.ac.jp

    Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing a reduction of the C17 = C18 double bond of Pchlide to form chlorophyllide a (Chlide) in bacteriochlorophyll biosynthesis. DPOR consists of an ATP-dependent reductase component, L-protein (a BchL dimer), and a catalytic component, NB-protein (a BchN–BchB heterotetramer). The L-protein transfers electrons to the NB-protein to reduce Pchlide, which is coupled with ATP hydrolysis. Here we determined the stoichiometry of ATP hydrolysis and the Chlide formation of DPOR. The minimal ratio of ATP to Chlide (ATP/2e{sup –}) was 4, which coincides with that of nitrogenase. The ratio increases with increasing molar ratiomore » of L-protein to NB-protein. This profile differs from that of nitrogenase. These results suggest that DPOR has a specific intrinsic property, while retaining the common features shared with nitrogenase. - Highlights: • The stoichiometry of nitrogenase-like protochlorophyllide reductase was determined. • The minimal ATP/2e{sup –} ratio was 4, which coincides with that of nitrogenase. • The ATP/2e{sup –} ratio increases with increasing L-protein/NB-protein molar ratio. • DPOR has an intrinsic property, but retains features shared with nitrogenase.« less

  20. High yield of secondary B-side electron transfer in mutant Rhodobacter capsulatus reaction centers

    SciTech Connect

    Kressel, Lucas; Faries, Kaitlyn M.; Wander, Marc J.

    2014-08-01

    From the crystal structures of reaction centers (RCs) from purple photosynthetic bacteria, two pathways for electron transfer (ET) are apparent but only one pathway (the A side) operates in the native protein-cofactor complex. Partial activation of the B-side pathway has unveiled the true inefficiencies of ET processes on that side in comparison to analogous reactions on the A side. Of significance are the relative rate constants for forward ET and the competing charge recombination reactions. On the B side, these rate constants are nearly equal for the secondary charge-separation step (ET from bacteriopheophytin to quinone), relegating the yield of thismore » process to < 50%. Herein we report efforts to optimize this step. In surveying all possible residues at position 131 in the M subunit, we discovered that when glutamic acid replaces the native valine the efficiency of the secondary ET is nearly two-fold higher than in the wild-type RC. The positive effect of M131 Glu is likely due to formation of a hydrogen bond with the ring V keto group of the B-side bacteriopheophytin leading to stabilization of the charge-separated state involving this cofactor. In conclusion, this change slows charge recombination by roughly a factor of two and affords the improved yield of the desired forward ET to the B-side quinone terminal acceptor.« less

  1. The effects of protein crowding in bacterial photosynthetic membranes on the flow of quinone redox species between the photochemical reaction center and the ubiquinol-cytochrome c2 oxidoreductase.

    PubMed

    Woronowicz, Kamil; Sha, Daniel; Frese, Raoul N; Sturgis, James N; Nanda, Vikas; Niederman, Robert A

    2011-08-01

    Atomic force microscopy (AFM) of the native architecture of the intracytoplasmic membrane (ICM) of a variety of species of purple photosynthetic bacteria, obtained at submolecular resolution, shows a tightly packed arrangement of light harvesting (LH) and reaction center (RC) complexes. Since there are no unattributed structures or gaps with space sufficient for the cytochrome bc(1) or ATPase complexes, they are localized in membrane domains distinct from the flat regions imaged by AFM. This has generated a renewed interest in possible long-range pathways for lateral diffusion of UQ redox species that functionally link the RC and the bc(1) complexes. Recent proposals to account for UQ flow in the membrane bilayer are reviewed, along with new experimental evidence provided from an analysis of intrinsic near-IR fluorescence emission that has served to test these hypotheses. The results suggest that different mechanism of UQ flow exist between species such as Rhodobacter sphaeroides, with a highly organized arrangement of LH and RC complexes and fast RC electron transfer turnover, and Phaeospirillum molischianum with a more random organization and slower RC turnover. It is concluded that packing density of the peripheral LH2 antenna in the Rba. sphaeroides ICM imposes constraints that significantly slow the diffusion of UQ redox species between the RC and cytochrome bc(1) complex, while in Phs. molischianum, the crowding of the ICM with LH3 has little effect upon UQ diffusion. This supports the proposal that in this type of ICM, a network of RC-LH1 core complexes observed in AFM provides a pathway for long-range quinone diffusion that is unaffected by differences in LH complex composition or organization.

  2. Automated ensemble assembly and validation of microbial genomes.

    PubMed

    Koren, Sergey; Treangen, Todd J; Hill, Christopher M; Pop, Mihai; Phillippy, Adam M

    2014-05-03

    The continued democratization of DNA sequencing has sparked a new wave of development of genome assembly and assembly validation methods. As individual research labs, rather than centralized centers, begin to sequence the majority of new genomes, it is important to establish best practices for genome assembly. However, recent evaluations such as GAGE and the Assemblathon have concluded that there is no single best approach to genome assembly. Instead, it is preferable to generate multiple assemblies and validate them to determine which is most useful for the desired analysis; this is a labor-intensive process that is often impossible or unfeasible. To encourage best practices supported by the community, we present iMetAMOS, an automated ensemble assembly pipeline; iMetAMOS encapsulates the process of running, validating, and selecting a single assembly from multiple assemblies. iMetAMOS packages several leading open-source tools into a single binary that automates parameter selection and execution of multiple assemblers, scores the resulting assemblies based on multiple validation metrics, and annotates the assemblies for genes and contaminants. We demonstrate the utility of the ensemble process on 225 previously unassembled Mycobacterium tuberculosis genomes as well as a Rhodobacter sphaeroides benchmark dataset. On these real data, iMetAMOS reliably produces validated assemblies and identifies potential contamination without user intervention. In addition, intelligent parameter selection produces assemblies of R. sphaeroides comparable to or exceeding the quality of those from the GAGE-B evaluation, affecting the relative ranking of some assemblers. Ensemble assembly with iMetAMOS provides users with multiple, validated assemblies for each genome. Although computationally limited to small or mid-sized genomes, this approach is the most effective and reproducible means for generating high-quality assemblies and enables users to select an assembly best tailored to

  3. Convergence of the transcriptional responses to heat shock and singlet oxygen stresses.

    PubMed

    Dufour, Yann S; Imam, Saheed; Koo, Byoung-Mo; Green, Heather A; Donohue, Timothy J

    2012-09-01

    Cells often mount transcriptional responses and activate specific sets of genes in response to stress-inducing signals such as heat or reactive oxygen species. Transcription factors in the RpoH family of bacterial alternative σ factors usually control gene expression during a heat shock response. Interestingly, several α-proteobacteria possess two or more paralogs of RpoH, suggesting some functional distinction. We investigated the target promoters of Rhodobacter sphaeroides RpoH(I) and RpoH(II) using genome-scale data derived from gene expression profiling and the direct interactions of each protein with DNA in vivo. We found that the RpoH(I) and RpoH(II) regulons have both distinct and overlapping gene sets. We predicted DNA sequence elements that dictate promoter recognition specificity by each RpoH paralog. We found that several bases in the highly conserved TTG in the -35 element are important for activity with both RpoH homologs; that the T-9 position, which is over-represented in the RpoH(I) promoter sequence logo, is critical for RpoH(I)-dependent transcription; and that several bases in the predicted -10 element were important for activity with either RpoH(II) or both RpoH homologs. Genes that are transcribed by both RpoH(I) and RpoH(II) are predicted to encode for functions involved in general cell maintenance. The functions specific to the RpoH(I) regulon are associated with a classic heat shock response, while those specific to RpoH(II) are associated with the response to the reactive oxygen species, singlet oxygen. We propose that a gene duplication event followed by changes in promoter recognition by RpoH(I) and RpoH(II) allowed convergence of the transcriptional responses to heat and singlet oxygen stress in R. sphaeroides and possibly other bacteria.

  4. The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein.

    PubMed

    Swanson, Michael A; Usselman, Robert J; Frerman, Frank E; Eaton, Gareth R; Eaton, Sandra S

    2008-08-26

    Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone pool. It contains one [4Fe-4S] (2+,1+) and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S] (2+,1+) to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S] (+) at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 and -30 mV for wild type to -11 and -19 mV, respectively. The N338A mutation decreased the potentials to -37 and -49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF 1e (-) catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO. Therefore, the iron-sulfur cluster is the immediate acceptor from ETF.

  5. The Iron-Sulfur Cluster of Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase Is the Electron Acceptor for Electron Transfer Flavoprotein†

    PubMed Central

    Swanson, Michael A.; Usselman, Robert J.; Frerman, Frank E.; Eaton, Gareth R.; Eaton, Sandra S.

    2009-01-01

    Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone pool. It contains one [4Fe-4S]2+,1+ and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S]2+,1+ to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S]+ at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 and -30 mV for wild type to -11 and -19 mV, respectively. The N338A mutation decreased the potentials to -37 and -49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF1e- catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO. Therefore, the iron-sulfur cluster is the immediate acceptor from ETF. PMID:9585549

  6. Proton environment of reduced Rieske iron-sulfur cluster probed by two-dimensional ESEEM spectroscopy

    PubMed Central

    Kolling, Derrick R. J.; Samoilova, Rimma I.; Shubin, Alexander A.; Crofts, Antony R.; Dikanov, Sergei A.

    2008-01-01

    The proton environment of the reduced [2Fe-2S] cluster in the water-soluble head domain of the Rieske iron—sulfur protein (ISF) from the cytochrome bc1 complex of Rhodobacter sphaeroides has been studied by orientation-selected X-band 2D ESEEM. The 2D spectra show multiple cross-peaks from protons, with considerable overlap. Samples in which 1H2O water was replaced by 2H2O were used to determine which of the observed peaks belong to exchangeable protons, likely involved in hydrogen bonds in the neighborhood of the cluster. By correlating the cross-peaks from 2D spectra recorded at different parts of the EPR spectrum, lines from nine distinct proton signals were identified. Assignment of the proton signals was based on a point-dipole model for interaction with electrons of Fe(III) and Fe(II) ions, using the high-resolution structure of ISF from Rb. sphaeroides. Analysis of experimental and calculated tensors has led us to conclude that even 2D spectra do not completely resolve all contributions from nearby protons. Particularly, the seven resolved signals from non-exchangeable protons could be produced by at least thirteen protons. The contributions from exchangeable protons were resolved by difference spectra (1H2O minus 2H2O), and assigned to two groups of protons with distinct anisotropic hyperfine values. The largest measured coupling exceeded any calculated value. This discrepancy could result from limitations of the point dipole approximation in dealing with the distribution of spin density over the sulfur atoms of the cluster and the cysteine ligands, or from differences between the structure in solution and the crystallographic structure. The approach demonstrated here provides a paradigm for a wide range of studies in which hydrogen-bonding interactions with metallic centers has a crucial role in understanding of function. PMID:19099453

  7. Absorption spectral change of peripheral-light harvesting complexes 2 induced by magnesium protoporphyrin IX monomethyl ester association

    NASA Astrophysics Data System (ADS)

    Yue, Huiying; Zhao, Chungui; Li, Kai; Yang, Suping

    2015-02-01

    Several spectrally different types of peripheral light harvesting complexes (LH) have been reported in anoxygenic phototrophic bacteria in response to environmental changes. In this study, two spectral forms of LH2 (T-LH2 and U-LH2) were isolated from Rhodobacter azotoformans. The absorption of T-LH2 was extremely similar to the LH2 isolated from Rhodobacter sphaeroides. U-LH2 showed an extra peak at ∼423 nm in the carotenoid region. To explore the spectral origin of this absorption peak, the difference in pigment compositions of two LH2 was analyzed. Spheroidene and bacteriochlorophyll aP were both contained in the two LH2. And magnesium protoporphyrin IX monomethyl ester (MPE) was only contained in U-LH2. It is known that spheroidene and bacteriochlorophyll aP do not produce ∼423 nm absorption peak either in vivo or in vitro. Whether MPE accumulation was mainly responsible for the formation of the ∼423 nm peak? The interactions between MPE and different proteins were further studied. The results showed that the maximum absorption of MPE was red-shifted from ∼415 nm to ∼423 nm when it was mixed with T-LH2 and its apoproteins, nevertheless, the Qy transitions of the bound bacteriochlorophylls in LH2 were almost unaffected, which indicated that the formation of the ∼423 nm peak was related to MPE-LH2 protein interaction. MPE did not bind to sites involved in the spectral tuning of BChls, but the conformation of integral LH2 was affected by MPE association, the alkaline stability of U-LH2 was lower than T-LH2, and the fluorescence intensity at 860 nm was decreased after MPE combination.

  8. Absorption spectral change of peripheral-light harvesting complexes 2 induced by magnesium protoporphyrin IX monomethyl ester association.

    PubMed

    Yue, Huiying; Zhao, Chungui; Li, Kai; Yang, Suping

    2015-02-25

    Several spectrally different types of peripheral light harvesting complexes (LH) have been reported in anoxygenic phototrophic bacteria in response to environmental changes. In this study, two spectral forms of LH2 (T-LH2 and U-LH2) were isolated from Rhodobacter azotoformans. The absorption of T-LH2 was extremely similar to the LH2 isolated from Rhodobacter sphaeroides. U-LH2 showed an extra peak at ∼423 nm in the carotenoid region. To explore the spectral origin of this absorption peak, the difference in pigment compositions of two LH2 was analyzed. Spheroidene and bacteriochlorophyll aP were both contained in the two LH2. And magnesium protoporphyrin IX monomethyl ester (MPE) was only contained in U-LH2. It is known that spheroidene and bacteriochlorophyll aP do not produce ∼423 nm absorption peak either in vivo or in vitro. Whether MPE accumulation was mainly responsible for the formation of the ∼423 nm peak? The interactions between MPE and different proteins were further studied. The results showed that the maximum absorption of MPE was red-shifted from ∼415 nm to ∼423 nm when it was mixed with T-LH2 and its apoproteins, nevertheless, the Qy transitions of the bound bacteriochlorophylls in LH2 were almost unaffected, which indicated that the formation of the ∼423 nm peak was related to MPE-LH2 protein interaction. MPE did not bind to sites involved in the spectral tuning of BChls, but the conformation of integral LH2 was affected by MPE association, the alkaline stability of U-LH2 was lower than T-LH2, and the fluorescence intensity at 860 nm was decreased after MPE combination. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Redox thermodynamics of the native and alkaline forms of eukaryotic and bacterial class I cytochromes c.

    PubMed

    Battistuzzi, G; Borsari, M; Sola, M; Francia, F

    1997-12-23

    The reduction potentials of beef heart cytochrome c and cytochromes c2 from Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Rhodobacter capsulatus were measured through direct electrochemistry at a surface-modified gold electrode as a function of temperature in nonisothermal experiments carried out at neutral and alkaline pH values. The thermodynamic parameters for protein reduction (DeltaS degrees rc and DeltaH degrees rc) were determined for the native and alkaline conformers. Enthalpy and entropy terms underlying species-dependent differences in E degrees and pH- and temperature-induced E degrees changes for a given cytochrome were analyzed. The difference of about +0.1 V in E degrees between cytochromes c2 and the eukaryotic species can be separated into an enthalpic term (-DeltaDeltaH degrees rc/F) of +0.130 V and an entropic term (TDeltaDeltaS degrees rc/F) of -0.040 V. Hence, the higher potential of the bacterial species appears to be determined entirely by a greater enthalpic stabilization of the reduced state. Analogously, the much lower potential of the alkaline conformer(s) as compared to the native species is by far enthalpic in origin for both protein families, and is largely determined by the substitution of Met for Lys in axial heme ligation. Instead, the biphasic E degrees /temperature profile for the native cytochromes is due to a difference in reduction entropy between the conformers at low and high temperatures. Temperature-dependent 1H NMR experiments suggest that the temperature-induced transition also involves a change in orientation of the axial methionine ligand with respect to the heme plane.

  10. Production of bioplastics and hydrogen gas by photosynthetic microorganisms

    NASA Astrophysics Data System (ADS)

    Yasuo, Asada; Masato, Miyake; Jun, Miyake

    1998-03-01

    Our efforts have been aimed at the technological basis of photosynthetic-microbial production of materials and an energy carrier. We report here accumulation of poly-(3-hydroxybutyrate) (PHB), a raw material of biodegradable plastics and for production of hydrogen gas, and a renewable energy carrier by photosynthetic microorganisms (tentatively defined as cyanobacteria plus photosynthetic bateria, in this report). A thermophilic cyanobacterium, Synechococcus sp. MA19 that accumulates PHB at more than 20% of cell dry wt under nitrogen-starved conditions was isolated and microbiologically identified. The mechanism of PHB accumulation was studied. A mesophilic Synechococcus PCC7942 was transformed with the genes encoding PHB-synthesizing enzymes from Alcaligenes eutrophus. The transformant accumulated PHB under nitrogen-starved conditions. The optimal conditions for PHB accumulation by a photosynthetic bacterium grown on acetate were studied. Hydrogen production by photosynthetic microorganisms was studied. Cyanobacteria can produce hydrogen gas by nitrogenase or hydrogenase. Hydrogen production mediated by native hydrogenase in cyanobacteria was revealed to be in the dark anaerobic degradation of intracellular glycogen. A new system for light-dependent hydrogen production was targeted. In vitro and in vivo coupling of cyanobacterial ferredoxin with a heterologous hydrogenase was shown to produce hydrogen under light conditions. A trial for genetic trasformation of Synechococcus PCC7942 with the hydrogenase gene from Clostridium pasteurianum is going on. The strong hydrogen producers among photosynthetic bacteria were isolated and characterized. Co-culture of Rhodobacter and Clostriumdium was applied to produce hydrogen from glucose. Conversely in the case of cyanobacteria, genetic regulation of photosynthetic proteins was intended to improve conversion efficiency in hydrogen production by the photosynthetic bacterium, Rhodobacter sphaeroides RV. A mutant acquired by

  11. Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus.

    PubMed

    Shimizu, Takayuki; Cheng, Zhuo; Matsuura, Katsumi; Masuda, Shinji; Bauer, Carl E

    2015-01-01

    PpsR is a major regulator of photosynthesis gene expression among all characterized purple photosynthetic bacteria. This transcription regulator has been extensively characterized in Rhodobacter (Rba.) capsulatus and Rba. sphaeroides which are members of the α-proteobacteria lineage. In this study, we have investigated the biochemical properties and mutational effects of a ppsR deletion strain in the β-proteobacterium Rubrivivax (Rvi.) gelatinosus in order to reveal phylogenetically conserved mechanisms and species-specific characteristics. A deletion of the ppsR gene resulted in de-repression of photosystem synthesis showing that PpsR functions as a repressor of photosynthesis genes in this species. We also constructed a Rvi. gelatinosus PpsR mutant in which a conserved cysteine at position 436 was changed to an alanine to examine whether or not this residue is important for sensing redox, as reported in Rhodobacter species. Surprisingly, the Cys436 Ala mutant retained the ability to repress photosynthesis gene expression under aerobic conditions, suggesting that PpsR from Rvi. gelatinosus has different redox-responding characteristics. Furthermore, biochemical analyses demonstrated that Rvi. gelatinosus PpsR only shows redox-dependent binding to promoters with 9-bp spacing, but not 8-bp spacing, between two PpsR-recognition sequences. These results indicate that redox-dependent binding of PpsR requires appropriate cis configuration of PpsR target sequences in Rvi. gelatinosus. These results also indicate that PpsR homologs from different species regulate photosynthesis genes with altered biochemical properties.

  12. Primary charge separation between P* and B A: Electron-transfer pathways in native and mutant GM203L bacterial reaction centers

    NASA Astrophysics Data System (ADS)

    Yakovlev, Andrey G.; Jones, Michael R.; Potter, Jane A.; Fyfe, Paul K.; Vasilieva, Lyudmila G.; Shkuropatov, Anatoli Ya.; Shuvalov, Vladimir A.

    2005-12-01

    Coherent components in the dynamics of decay of stimulated emission from the primary electron donor excited state P*, and of population of the product charge-separated states P+BA- and P+HA-, were studied in GM203L mutant reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides by measuring oscillations in the kinetics of absorbance changes at 940 nm (P* stimulated emission region), 1020 nm ( BA- absorption region) and 760 nm (H A bleaching region). Absorbance changes were induced by excitation of P (870 nm) with 18 fs pulses at 90 K. In the GM203L mutant, replacement of Gly M203 by Leu results in exclusion of the crystallographically defined water molecule (HOH55) located close to the oxygen of the 13 1-keto carbonyl group of B A and to His M202, which provides the axial ligand to the Mg of the P B bacteriochlorophyll. The results of femtosecond measurements were compared with those obtained with Rb. sphaeroides R-26 RCs containing an intact water HOH55. The main consequences of the GM203L mutation were found to be as follows: (i) a low-frequency oscillation at 32 cm -1, which is characteristic of the HOH55-containing RCs, disappears from the kinetics of absorbance changes at 1020 and 760 nm in the mutant RC; (ii) electron transfer from P* to B A in the wild type RC was characterized by two time constants of 1.1 ps (80%) and 4.3 ps (20%), but in the GM203L mutant was characterized by a single time constant of 4.3 ps, demonstrating a slowing of primary charge separation. The previously postulated rotation of water HOH55 with a fundamental frequency of 32 cm -1, triggered by electron transfer from P* to B A, was confirmed by observation of an isotopic shift of the 32 cm -1 oscillation in the kinetics of P+BA- population in deuterated, pheophytin-modified RCs of Rb. sphaeroides R-26, by a factor of 1.6. These data are discussed in terms of the influence of water HOH55 on the energetics of the P∗→P+BA- reaction, and protein dynamic events that occur on the time

  13. X-ray Transparent Microfluidic Chip for Mesophase-Based Crystallization of Membrane Proteins and On-Chip Structure Determination

    SciTech Connect

    Khvostichenko, Daria S.; Schieferstein, Jeremy M.; Pawate, Ashtamurthy S.

    2014-10-01

    Crystallization from lipidic mesophase matrices is a promising route to diffraction-quality crystals and structures of membrane proteins. The microfluidic approach reported here eliminates two bottlenecks of the standard mesophase-based crystallization protocols: (i) manual preparation of viscous mesophases and (ii) manual harvesting of often small and fragile protein crystals. In the approach reported here, protein-loaded mesophases are formulated in an X-ray transparent microfluidic chip using only 60 nL of the protein solution per crystallization trial. The X-ray transparency of the chip enables diffraction data collection from multiple crystals residing in microfluidic wells, eliminating the normally required manual harvesting and mounting ofmore » individual crystals. We validated our approach by on-chip crystallization of photosynthetic reaction center, a membrane protein from Rhodobacter sphaeroides, followed by solving its structure to a resolution of 2.5 Å using X-ray diffraction data collected on-chip under ambient conditions. A moderate conformational change in hydrophilic chains of the protein was observed when comparing the on-chip, room temperature structure with known structures for which data were acquired under cryogenic conditions.« less

  14. The Structure of L-Tyrosine 2,3-Aminomutase frmo the C-1027 Enediyne Antitumor Antibiotic Biosynthetic Pathway

    SciTech Connect

    Christianson,C.; Montavon, T.; Van Lanen, S.

    2007-01-01

    The SgcC4 L-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-{beta}-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate L-tyrosine to generate (S)-{beta}-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Heremore » we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the L-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations.« less

  15. A comparative spectroscopic and kinetic study of photoexcitations in detergent-isolated and membrane-embedded LH2 light-harvesting complexes.

    PubMed

    Freiberg, Arvi; Rätsep, Margus; Timpmann, Kõu

    2012-08-01

    Integral membrane proteins constitute more than third of the total number of proteins present in organisms. Solubilization with mild detergents is a common technique to study the structure, dynamics, and catalytic activity of these proteins in purified form. However beneficial the use of detergents may be for protein extraction, the membrane proteins are often denatured by detergent solubilization as a result of native lipid membrane interactions having been modified. Versatile investigations of the properties of membrane-embedded and detergent-isolated proteins are, therefore, required to evaluate the consequences of the solubilization procedure. Herein, the spectroscopic and kinetic fingerprints have been established that distinguish excitons in individual detergent-solubilized LH2 light-harvesting pigment-protein complexes from them in the membrane-embedded complexes of purple photosynthetic bacteria Rhodobacter sphaeroides. A wide arsenal of spectroscopic techniques in visible optical range that include conventional broadband absorption-fluorescence, fluorescence anisotropy excitation, spectrally selective hole burning and fluorescence line-narrowing, and transient absorption-fluorescence have been applied over broad temperature range between physiological and liquid He temperatures. Significant changes in energetics and dynamics of the antenna excitons upon self-assembly of the proteins into intracytoplasmic membranes are observed, analyzed, and discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. Copyright © 2011. Published by Elsevier B.V.

  16. Carotenoid radical cation formation in LH2 of purple bacteria: a quantum chemical study.

    PubMed

    Wormit, Michael; Dreuw, Andreas

    2006-11-30

    In LH2 complexes of Rhodobacter sphaeroides the formation of a carotenoid radical cation has recently been observed upon photoexcitation of the carotenoid S2 state. To shed more light onto the yet unknown molecular mechanism leading to carotenoid radical formation in LH2, the interactions between carotenoid and bacteriochlorophyll in LH2 are investigated by means of quantum chemical calculations for three different carotenoids--neurosporene, spheroidene, and spheroidenone--using time-dependent density functional theory. Crossings of the calculated potential energy curve of the electron transfer state with the bacteriochlorophyll Qx state and the carotenoid S1 and S2 states occur along an intermolecular distance coordinate for neurosporene and spheroidene, but for spheroidenone no crossing of the electron transfer state with the carotenoid S1 state could be found. By comparison with recent experiments where no formation of a spheroidenone radical cation has been observed, a molecular mechanism for carotenoid radical cation formation is proposed in which it is formed via a vibrationally excited carotenoid S1 or S*state. Arguments are given why the formation of the carotenoid radical cation does not proceed via the Qx, S2, or higher excited electron transfer states.

  17. Triplet Excitation Transfer between Carotenoids in the LH2 Complex from Photosynthetic Bacterium Rhodopseudomonas palustris.

    PubMed

    Feng, Juan; Wang, Qian; Wu, Yi-Shi; Ai, Xi-Cheng; Zhang, Xu-Jia; Huang, You-Guo; Zhang, Xing-Kang; Zhang, Jian-Ping

    2004-01-01

    We have studied, by means of sub-microsecond time-resolved absorption spectroscopy, the triplet-excited state dynamics of carotenoids (Cars) in the intermediate-light adapted LH2 complex (ML-LH2) from Rhodopseudomonas palustris containing Cars with different numbers of conjugated double bonds. Following pulsed photo-excitation at 590 nm at room temperature, rapid spectral equilibration was observed either as a red shift of the isosbestic wavelength on a time scale of 0.6-1.0 mus, or as a fast decay in the shorter-wavelength side of the T(n)<--T(1) absorption of Cars with a time constant of 0.5-0.8 mus. Two major spectral components assignable to Cars with 11 and 12 conjugated double bonds were identified. The equilibration was not observed in the ML-LH2 at 77 K, or in the LH2 complex from Rhodobacter sphaeroides G1C containing a single type of Car. The unique spectral equilibration was ascribed to temperature-dependent triplet excitation transfer among different Car compositions. The results suggest that Cars of 11 and 12 conjugated bonds, both in close proximity of BChls, may coexist in an alpha,beta-subunit of the ML-LH2 complex.

  18. Communication: Broad manifold of excitonic states in light-harvesting complex 1 promotes efficient unidirectional energy transfer in vivo

    NASA Astrophysics Data System (ADS)

    Sohail, Sara H.; Dahlberg, Peter D.; Allodi, Marco A.; Massey, Sara C.; Ting, Po-Chieh; Martin, Elizabeth C.; Hunter, C. Neil; Engel, Gregory S.

    2017-10-01

    In photosynthetic organisms, the pigment-protein complexes that comprise the light-harvesting antenna exhibit complex electronic structures and ultrafast dynamics due to the coupling among the chromophores. Here, we present absorptive two-dimensional (2D) electronic spectra from living cultures of the purple bacterium, Rhodobacter sphaeroides, acquired using gradient assisted photon echo spectroscopy. Diagonal slices through the 2D lineshape of the LH1 stimulated emission/ground state bleach feature reveal a resolvable higher energy population within the B875 manifold. The waiting time evolution of diagonal, horizontal, and vertical slices through the 2D lineshape shows a sub-100 fs intra-complex relaxation as this higher energy population red shifts. The absorption (855 nm) of this higher lying sub-population of B875 before it has red shifted optimizes spectral overlap between the LH1 B875 band and the B850 band of LH2. Access to an energetically broad distribution of excitonic states within B875 offers a mechanism for efficient energy transfer from LH2 to LH1 during photosynthesis while limiting back transfer. Two-dimensional lineshapes reveal a rapid decay in the ground-state bleach/stimulated emission of B875. This signal, identified as a decrease in the dipole strength of a strong transition in LH1 on the red side of the B875 band, is assigned to the rapid localization of an initially delocalized exciton state, a dephasing process that frustrates back transfer from LH1 to LH2.

  19. Solvation effect of bacteriochlorophyll excitons in light-harvesting complex LH2.

    PubMed

    Urboniene, V; Vrublevskaja, O; Trinkunas, G; Gall, A; Robert, B; Valkunas, L

    2007-09-15

    We have characterized the influence of the protein environment on the spectral properties of the bacteriochlorophyll (Bchl) molecules of the peripheral light-harvesting (or LH2) complex from Rhodobacter sphaeroides. The spectral density functions of the pigments responsible for the 800 and 850 nm electronic transitions were determined from the temperature dependence of the Bchl absorption spectra in different environments (detergent micelles and native membranes). The spectral density function is virtually independent of the hydrophobic support that the protein experiences. The reorganization energy for the B850 Bchls is 220 cm(-1), which is almost twice that of the B800 Bchls, and its Huang-Rhys factor reaches 8.4. Around the transition point temperature, and at higher temperatures, both the static spectral inhomogeneity and the resonance interactions become temperature-dependent. The inhomogeneous distribution function of the transitions exhibits less temperature dependence when LH2 is embedded in membranes, suggesting that the lipid phase protects the protein. However, the temperature dependence of the fluorescence spectra of LH2 cannot be fitted using the same parameters determined from the analysis of the absorption spectra. Correct fitting requires the lowest exciton states to be additionally shifted to the red, suggesting the reorganization of the exciton spectrum.

  20. Synchronous Measurement of Ultrafast Anisotropy Decay of the B850 in Bacterial LH2 Complex

    NASA Astrophysics Data System (ADS)

    Wang, Yun-Peng; Du, Lu-Chao; Zhu, Gang-Bei; Wang, Zhuan; Weng, Yu-Xiang

    2015-02-01

    Ultrafast anisotropic decay is a prominent parameter revealing ultrafast energy and electron transfer; however, it is difficult to be determined reliably owing to the requirement of a simultaneous availability of the parallel and perpendicular polarized decay kinetics. Nowadays, any measurement of anisotropic decay is a kind of approach to the exact simultaneity. Here we report a novel method for a synchronous ultrafast anisotropy decay measurement, which can well determine the anisotropy, even at a very early time, as the rising phase of the excitation laser pulse. The anisotropic decay of the B850 in bacterial light harvesting antenna complex LH2 of Rhodobacter sphaeroides in solution at room temperature with coherent excitation is detected by this method, which shows a polarization response time of 30 fs, and the energy transfer from the initial excitation to the bacteriochlorophylls in B850 ring takes about 70 fs. The anisotropic decay that is probed at the red side of the absorption spectrum, such as 880 nm, has an initial value of 0.4, corresponding to simulated emission, while the blue side with an anisotropy of 0.1 contributes to the ground-state bleaching. Our results show that the coherent excitation covering the whole ring might not be realized owing to the symmetry breaking of LH2: from C9 symmetry in membrane to C2 symmetry in solution.

  1. Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase

    PubMed Central

    Qin, Ling; Hiser, Carrie; Mulichak, Anne; Garavito, R. Michael; Ferguson-Miller, Shelagh

    2006-01-01

    Well ordered reproducible crystals of cytochrome c oxidase (CcO) from Rhodobacter sphaeroides yield a previously unreported structure at 2.0 Å resolution that contains the two catalytic subunits and a number of alkyl chains of lipids and detergents. Comparison with crystal structures of other bacterial and mammalian CcOs reveals that the positions occupied by native membrane lipids and detergent substitutes are highly conserved, along with amino acid residues in their vicinity, suggesting a more prevalent and specific role of lipid in membrane protein structure than often envisioned. Well defined detergent head groups (maltose) are found associated with aromatic residues in a manner similar to phospholipid head groups, likely contributing to the success of alkyl glycoside detergents in supporting membrane protein activity and crystallizability. Other significant features of this structure include the following: finding of a previously unreported crystal contact mediated by cadmium and an engineered histidine tag; documentation of the unique His–Tyr covalent linkage close to the active site; remarkable conservation of a chain of waters in one proton pathway (D-path); and discovery of an inhibitory cadmium-binding site at the entrance to another proton path (K-path). These observations provide important insight into CcO structure and mechanism, as well as the significance of bound lipid in membrane proteins. PMID:17050688

  2. Overall energy conversion efficiency of a photosynthetic vesicle

    PubMed Central

    Sener, Melih; Strumpfer, Johan; Singharoy, Abhishek; Hunter, C Neil; Schulten, Klaus

    2016-01-01

    The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytb⁢c1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in a quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12–0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination. DOI: http://dx.doi.org/10.7554/eLife.09541.001 PMID:27564854

  3. Construction and validation of an atomic model for bacterial TSPO from electron microscopy density, evolutionary constraints, and biochemical and biophysical data.

    PubMed

    Hinsen, Konrad; Vaitinadapoule, Aurore; Ostuni, Mariano A; Etchebest, Catherine; Lacapere, Jean-Jacques

    2015-02-01

    The 18 kDa protein TSPO is a highly conserved transmembrane protein found in bacteria, yeast, animals and plants. TSPO is involved in a wide range of physiological functions, among which the transport of several molecules. The atomic structure of monomeric ligand-bound mouse TSPO in detergent has been published recently. A previously published low-resolution structure of Rhodobacter sphaeroides TSPO, obtained from tubular crystals with lipids and observed in cryo-electron microscopy, revealed an oligomeric structure without any ligand. We analyze this electron microscopy density in view of available biochemical and biophysical data, building a matching atomic model for the monomer and then the entire crystal. We compare its intra- and inter-molecular contacts with those predicted by amino acid covariation in TSPO proteins from evolutionary sequence analysis. The arrangement of the five transmembrane helices in a monomer of our model is different from that observed for the mouse TSPO. We analyze possible ligand binding sites for protoporphyrin, for the high-affinity ligand PK 11195, and for cholesterol in TSPO monomers and/or oligomers, and we discuss possible functional implications. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Structure of Methylobacterium extorquens malyl-CoA lyase: CoA-substrate binding correlates with domain shift

    SciTech Connect

    Gonzalez, Javier M.; Marti-Arbona, Ricardo; Chen, Julian C. -H.

    Malyl-CoA lyase (MCL) is an Mg 2+-dependent enzyme that catalyzes the reversible cleavage of (2 S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg 2+is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl-CoA lyase complexed with Mg 2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding.more » Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. Furthermore, this domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.« less

  5. The swimming of a perfect deforming helix

    NASA Astrophysics Data System (ADS)

    Koens, Lyndon; Zhang, Hang; Mourran, Ahmed; Lauga, Eric

    2017-11-01

    Many bacteria rotate helical flagellar filaments in order to swim. When at rest or rotated counter-clockwise these flagella are left handed helices but they undergo polymorphic transformations to right-handed helices when the motor is reversed. These helical deformations themselves can generate motion, with for example Rhodobacter sphaeroides using the polymorphic transformation of the flagellum to generate rotation, or Spiroplasma propagating a change of helix handedness across its body's length to generate forward motion. Recent experiments reported on an artificial helical microswimmer generating motion without a propagating change in handedness. Made of a temperature sensitive gel, these swimmers moved by changing the dimensions of the helix in a non-reciprocal way. Inspired by these results and helix's ubiquitous presence in the bacterial world, we investigate how a deforming helix moves within a viscous fluid. Maintaining a single handedness along its entire length, we discuss how a perfect deforming helix can create a non-reciprocal swimming stroke, identify its principle directions of motion, and calculate the swimming kinematics asymptotically.

  6. Isolation and characterization of heterotrophic bacteria able to grow aerobically with quaternary ammonium alcohols as sole source of carbon and nitrogen.

    PubMed

    Kaech, Andres; Vallotton, Nathalie; Egli, Thomas

    2005-04-01

    The quaternary ammonium alcohols (QAAs) 2,3-dihydroxypropyl-trimethyl-ammonium (TM), dimethyl-diethanol-ammonium (DM) and methyl-triethanol-ammonium (MM) are hydrolysis products of their parent esterquat surfactants, which are widely used as softeners in fabric care. We isolated several bacteria growing with QAAs as the sole source of carbon and nitrogen. The strains were compared with a previously isolated TM-degrading bacterium, which was identified as a representative of the species Pseudomonas putida (Syst. Appl. Microbiol. 24 (2001) 252). Two bacteria were isolated with DM, referred to as strains DM 1 and DM 2, respectively. Based on 16S-rDNA analysis, they provided 97% (DM 1) and 98% (DM 2) identities to the closest related strain Zoogloea ramigera Itzigsohn 1868AL. Both strains were long, slim, motile rods but only DM 1 showed the floc forming activity, which is typical for representatives of the genus Zoogloea. Using MM we isolated a Gram-negative, non-motile rod referred to as strain MM 1. The 16S-rDNA sequence of the isolated bacterium revealed 94% identities (best match) to Rhodobacter sphaeroides only. The strains MM 1 and DM 1 exclusively grew with the QAA which was used for their isolation. DM 2 was also utilizing TM as sole source of carbon and nitrogen. However, all of the isolated bacteria were growing with the natural and structurally related compound choline.

  7. Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars.

    PubMed

    Jose, Joachim; von Schwichow, Steffen

    2004-04-02

    Whole cell biocatalysts are attractive technological tools for the regio- and enantioselective synthesis of products, especially from substrates with several identical reactive groups. In the present study, a whole cell biocatalyst for the synthesis of rare sugars from polyalcohols was constructed. For this purpose, sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase/reductase (SDR) family, was expressed on the surface of Escherichia coli using Autodisplay. Autodisplay is an efficient surface display system for Gram-negative bacteria and is based on the autotransporter secretion pathway. Transport of SDH to the outer membrane was monitored by SDS-PAGE and Western blotting of different cell fractions. The surface exposure of the enzyme could be verified by immunofluorescence microscopy and fluorescence activated cell sorting (FACS). The activity of whole cells displaying SDH at the surface was determined in an optical test. Specific activities were found to be 12 mU per 3.3 x 10(8) cells for the conversion of D-glucitol (sorbitol) to D-fructose, 7 mU for the conversion D-galactitol to D-tagatose, and 17 mU for the conversion of L-arabitol to L-ribulose. The whole cell biocatalyst obtained by surface display of SDH could also produce D-glucitol from D-fructose (29 mU per 3.3 x 10(8) cells).

  8. Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute

    PubMed Central

    Miyoshi, Tomohiro; Ito, Kosuke; Murakami, Ryo; Uchiumi, Toshio

    2016-01-01

    Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson–Crick base-pairing even in the 3′-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5′ base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region. PMID:27325485

  9. A comprehensive evaluation of assembly scaffolding tools

    PubMed Central

    2014-01-01

    Background Genome assembly is typically a two-stage process: contig assembly followed by the use of paired sequencing reads to join contigs into scaffolds. Scaffolds are usually the focus of reported assembly statistics; longer scaffolds greatly facilitate the use of genome sequences in downstream analyses, and it is appealing to present larger numbers as metrics of assembly performance. However, scaffolds are highly prone to errors, especially when generated using short reads, which can directly result in inflated assembly statistics. Results Here we provide the first independent evaluation of scaffolding tools for second-generation sequencing data. We find large variations in the quality of results depending on the tool and dataset used. Even extremely simple test cases of perfect input, constructed to elucidate the behaviour of each algorithm, produced some surprising results. We further dissect the performance of the scaffolders using real and simulated sequencing data derived from the genomes of Staphylococcus aureus, Rhodobacter sphaeroides, Plasmodium falciparum and Homo sapiens. The results from simulated data are of high quality, with several of the tools producing perfect output. However, at least 10% of joins remains unidentified when using real data. Conclusions The scaffolders vary in their usability, speed and number of correct and missed joins made between contigs. Results from real data highlight opportunities for further improvements of the tools. Overall, SGA, SOPRA and SSPACE generally outperform the other tools on our datasets. However, the quality of the results is highly dependent on the read mapper and genome complexity. PMID:24581555

  10. Field-cycling NMR with high-resolution detection under magic-angle spinning: determination of field-window for nuclear hyperpolarization in a photosynthetic reaction center.

    PubMed

    Gräsing, Daniel; Bielytskyi, Pavlo; Céspedes-Camacho, Isaac F; Alia, A; Marquardsen, Thorsten; Engelke, Frank; Matysik, Jörg

    2017-09-21

    Several parameters in NMR depend on the magnetic field strength. Field-cycling NMR is an elegant way to explore the field dependence of these properties. The technique is well developed for solution state and in relaxometry. Here, a shuttle system with magic-angle spinning (MAS) detection is presented to allow for field-dependent studies on solids. The function of this system is demonstrated by exploring the magnetic field dependence of the solid-state photochemically induced nuclear polarization (photo-CIDNP) effect. The effect allows for strong nuclear spin-hyperpolarization in light-induced spin-correlated radical pairs (SCRPs) under solid-state conditions. To this end, 13 C MAS NMR is applied to a photosynthetic reaction center (RC) of the purple bacterium Rhodobacter (R.) sphaeroides wildtype (WT). For induction of the effect in the stray field of the magnet and its subsequent observation at 9.4 T under MAS NMR conditions, the sample is shuttled by the use of an aerodynamically driven sample transfer technique. In the RC, we observe the effect down to 0.25 T allowing to determine the window for the occurrence of the effect to be between about 0.2 and 20 T.

  11. Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

    PubMed Central

    Inui, Masayuki; Roh, Jung Hyeob; Zahn, Kenneth; Yukawa, Hideaki

    2000-01-01

    A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of α-proteobacteria. PMID:10618203

  12. Hydrogen bonds in the vicinity of the special pair of the bacterial reaction center probed by hydrostatic high-pressure absorption spectroscopy.

    PubMed

    Kangur, Liina; Jones, Michael R; Freiberg, Arvi

    2017-12-01

    Using the native bacteriochlorophyll a pigment cofactors as local probes, we investigated the response to external hydrostatic high pressure of reaction center membrane protein complexes from the photosynthetic bacterium Rhodobacter sphaeroides. Wild-type and engineered complexes were used with a varied number (0, 1 or 2) of hydrogen bonds that bind the reaction center primary donor bacteriochlorophyll cofactors to the surrounding protein scaffold. A pressure-induced breakage of hydrogen bonds was established for both detergent-purified and membrane-embedded reaction centers, but at rather different pressures: between 0.2 and 0.3GPa and at about 0.55GPa, respectively. The free energy change associated with the rupture of the single hydrogen bond present in wild-type reaction centers was estimated to be equal to 13-14kJ/mol. In the mutant with two symmetrical hydrogen bonds (FM197H) a single cooperative rupture of the two bonds was observed corresponding to an about twice stronger bond, rather than a sequential rupture of two individual bonds. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Exciton exciton annihilation dynamics in chromophore complexes. II. Intensity dependent transient absorption of the LH2 antenna system.

    PubMed

    Bruggemann, B; May, V

    2004-02-01

    Using the multiexciton density matrix theory of excitation energy transfer in chromophore complexes developed in a foregoing paper [J. Chem. Phys. 118, 746 (2003)], the computation of ultrafast transient absorption spectra is presented. Beside static disorder and standard mechanisms of excitation energy dissipation the theory incorporates exciton exciton annihilation (EEA) processes. To elucidate signatures of EEA in intensity dependent transient absorption data the approach is applied to the B850 ring of the LH2 found in rhodobacter sphaeroides. As main indications for two-exciton population and resulting EEA we found (i) a weakening of the dominant single-exciton bleaching structure in the transient absorption, and (ii) an intermediate suppression of long-wavelength and short-wavelength shoulders around the bleaching structure. The suppression is caused by stimulated emission from the two-exciton to the one-exciton state and the return of the shoulders follows from a depletion of two-exciton population according to EEA. The EEA-signature survives as a short-wavelength shoulder in the transient absorption if orientational and energetic disorder are taken into account. Therefore, the observation of the EEA-signatures should be possible when doing frequency resolved transient absorption experiments with a sufficiently strongly varying pump-pulse intensity. Copyright 2004 American Institute of Physics

  14. Overall energy conversion efficiency of a photosynthetic vesicle

    SciTech Connect

    Sener, Melih; Strumpfer, Johan; Singharoy, Abhishek

    The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytbc1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in amore » quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.« less

  15. SciTech Connect

    Grayson, Katie J.; Faries, Kaitlyn M.; Huang, Xia

    Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP–RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (Qmore » X) absorption bands of the RC allow energy transfer via a Fo¨rster mechanism, with an efficiency of 40±10%. Finally, this proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis.« less

  16. Structure of Methylobacterium extorquens malyl-CoA lyase: CoA-substrate binding correlates with domain shift

    DOE PAGES

    Gonzalez, Javier M.; Marti-Arbona, Ricardo; Chen, Julian C. -H.; ...

    2017-01-27

    Malyl-CoA lyase (MCL) is an Mg 2+-dependent enzyme that catalyzes the reversible cleavage of (2 S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg 2+is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl-CoA lyase complexed with Mg 2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding.more » Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. Furthermore, this domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.« less

  17. RpoH2 sigma factor controls the photooxidative stress response in a non-photosynthetic rhizobacterium, Azospirillum brasilense Sp7.

    PubMed

    Kumar, Santosh; Rai, Ashutosh Kumar; Mishra, Mukti Nath; Shukla, Mansi; Singh, Pradhyumna Kumar; Tripathi, Anil Kumar

    2012-12-01

    Bacteria belonging to the Alphaproteobacteria normally harbour multiple copies of the heat shock sigma factor (known as σ(32), σ(H) or RpoH). Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours five copies of rpoH genes, one of which is an rpoH2 homologue. The genes around the rpoH2 locus in A. brasilense show synteny with that found in rhizobia. The rpoH2 of A. brasilense was able to complement the temperature-sensitive phenotype of the Escherichia coli rpoH mutant. Inactivation of rpoH2 in A. brasilense results in increased sensitivity to methylene blue and to triphenyl tetrazolium chloride (TTC). Exposure of A. brasilense to TTC and the singlet oxygen-generating agent methylene blue induced several-fold higher expression of rpoH2. Comparison of the proteome of A. brasilense with its rpoH2 deletion mutant and with an A. brasilense strain overexpressing rpoH2 revealed chaperone GroEL, elongation factors (Ef-Tu and EF-G), peptidyl prolyl isomerase, and peptide methionine sulfoxide reductase as the major proteins whose expression was controlled by RpoH2. Here, we show that the RpoH2 sigma factor-controlled photooxidative stress response in A. brasilense is similar to that in the photosynthetic bacterium Rhodobacter sphaeroides, but that RpoH2 is not involved in the detoxification of methylglyoxal in A. brasilense.

  18. Photocurrent generation by direct electron transfer using photosynthetic reaction centres

    NASA Astrophysics Data System (ADS)

    Mahmoudzadeh, A.; Saer, R.; Jun, D.; Mirvakili, S. M.; Takshi, A.; Iranpour, B.; Ouellet, E.; Lagally, E. T.; Madden, J. D. W.; Beatty, J. T.

    2011-09-01

    Photosynthetic reaction centres (RCs) convert light into separated charges with nearly perfect quantum efficiency, and have been used to generate photocurrent. Previous work has shown that electron tunnelling rates between redox centres in proteins depend exponentially on the tunnelling distance. In this work the RC from Rhodobacter sphaeroides was genetically modified with the aim of achieving the shortest tunnelling distances yet demonstrated between the RC's electron-accepting P site and underlying graphite and gold electrodes, and between the electron donor Q site and graphite electrodes. Opposite charges are carried to counter electrodes using mobile mediators, as in dye-sensitised solar cells. Native RCs are bound to graphite surfaces through N-(1-pyrene)iodoacetamide. Although the linker's length is only 4 Å, the electron transfer pathway between the Q electron donor site on the RC and the electrode surface is still too large for current to be significant. A mutant version with the electron acceptor P side close to the graphite surface produced currents of 15 nA cm-2 upon illumination. Direct binding of RCs to a gold surface is shown, resulting in currents of 5 nA cm-2. In both cases the current was unaffected by mediator concentration but increased with illumination, suggesting that direct electron transfer was achieved. The engineering of an RC to achieve direct electron transfer will help with long term efforts to demonstrate RC-based photovoltaic devices.

  19. Overall energy conversion efficiency of a photosynthetic vesicle

    DOE PAGES

    Sener, Melih; Strumpfer, Johan; Singharoy, Abhishek; ...

    2016-08-26

    The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytbc1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in amore » quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.« less

  20. Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

    PubMed

    Fredslund, Folmer; Otten, Harm; Gemperlein, Sabrina; Poulsen, Jens Christian N; Carius, Yvonne; Kohring, Gert Wieland; Lo Leggio, Leila

    2016-11-01

    Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the T m for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

  1. The role of pH control on biohydrogen production by single stage hybrid dark- and photo-fermentation.

    PubMed

    Zagrodnik, R; Laniecki, M

    2015-10-01

    The role of pH control on biohydrogen production by co-culture of dark-fermentative Clostridium acetobutylicum and photofermentative Rhodobacter sphaeroides was studied. Single stage dark fermentation, photofermentation and hybrid co-culture systems were studied at different values of controlled and uncontrolled pH. Increasing pH during dark fermentation resulted in lower hydrogen production rate (HPR) and longer lag time for both controlled and uncontrolled conditions. However, it only slightly affected cumulative H2 volume. Results have shown that pH control at pH 7.5 increased photofermentative hydrogen production from 0.966 to 2.502 L H2/L(medium) when compared to uncontrolled process. Fixed pH value has proven to be an important control strategy also for the hybrid process and resulted in obtaining balanced co-culture of dark and photofermentative bacteria. Control of pH at 7.0 was found optimum for bacteria cooperation in the co-culture what resulted in obtaining 2.533 L H2/L(medium) and H2 yield of 6.22 mol H2/mol glucose. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. A new inhibitor of the CoQ-dependent redox reactions in mitochondria and chromatophores.

    PubMed

    Kolesova, G M; Belyakova, M M; Mamedov, M D; Yaguzhinsky, L S

    2000-05-01

    The effects of 3,4-dimethoxyphenyl-1-amylketone (DPK) on the CoQ-dependent stages of the electron transport systems in mitochondria and Rhodobacter sphaeroides chromatophores were studied. The two systems contain the complete Q-cycle. The sensitivities of the Q-cycles of two electron transport systems to antimycin, myxothiazole, and other inhibitors are virtually indistinguishable from one another, but these systems have different CoQ reduction processes. The dependence of the inhibition extent of the mitochondrial succinate oxidase on the DPK concentration was studied. The effective concentration of DPK is 0.5-2.5 mM. The presence of the point of inflection in the titration curve indicates that there are two mechanisms of inhibition. The effects of DPK on the extent of reduction of cytochromes b and c1 + c in mitochondria as well as on the electrogenic stages of the Q-cycle in chromatophores were examined. The experiments showed that DPK prevents three CoQ-dependent reactions related to the Q-cycle: electron transport between succinate dehydrogenase and the Q-cycle in mitochondria and functioning of the Z (o) and C (i) sites of the Q-cycle in chromatophores. DPK does not affect the electrogenic reaction associated with protonation of the secondary quinone acceptor QB in the reaction center of chromatophores. The mitochondrial NADH-dehydrogenase is inhibited by DPK at lower but comparable concentrations (C50 = 0.2 mM).

  3. Electrostatics of the photosynthetic bacterial reaction center. Protonation of Glu L 212 and Asp L 213 - A new method of calculation.

    PubMed

    Ptushenko, Vasily V; Cherepanov, Dmitry A; Krishtalik, Lev I

    2015-12-01

    Continuum electrostatic calculation of the transfer energies of anions from water into aprotic solvents gives the figures erroneous by order of magnitude. This is due to the hydrogen bond disruption that suggests the necessity to reconsider the traditional approach of the purely electrostatic calculation of the transfer energy from water into protein. In this paper, the method combining the experimental estimates of the transfer energies from water into aprotic solvent and the electrostatic calculation of the transfer energies from aprotic solvent into protein is proposed. Hydrogen bonds between aprotic solvent and solute are taken into account by introducing an imaginary aprotic medium incapable to form hydrogen bonds with the solute. Besides, a new treatment of the heterogeneous intraprotein dielectric permittivity based on the microscopic protein structure and electrometric measurements is elaborated. The method accounts semi-quantitatively for the electrostatic effect of diverse charged amino acid substitutions in the donor and acceptor parts of the photosynthetic bacterial reaction center from Rhodobacter sphaeroides. Analysis of the volatile secondary acceptor site QB revealed that in the conformation with a minimal distance between quinone QB and Glu L 212 the proton uptake upon the reduction of QB is prompted by Glu L 212 in alkaline and by Asp L 213 in slightly acidic regions. This agrees with the pH dependences of protonation degrees and the proton uptake. The method of pK calculation was applied successfully also for dissociation of Asp 26 in bacterial thioredoxin. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. X-ray transparent microfluidic chip for mesophase-based crystallization of membrane proteins and on-chip structure determination

    DOE PAGES

    Khvostichenko, Daria S.; Schieferstein, Jeremy M.; Pawate, Ashtamurthy S.; ...

    2014-08-21

    Crystallization from lipidic mesophase matrices is a promising route to diffraction-quality crystals and structures of membrane proteins. The microfluidic approach reported here eliminates two bottlenecks of the standard mesophase-based crystallization protocols: (i) manual preparation of viscous mesophases and (ii) manual harvesting of often small and fragile protein crystals. In the approach reported here, protein-loaded mesophases are formulated in an X-ray transparent microfluidic chip using only 60 nL of the protein solution per crystallization trial. The X-ray transparency of the chip enables diffraction data collection from multiple crystals residing in microfluidic wells, eliminating the normally required manual harvesting and mounting ofmore » individual crystals. In addition, we validated our approach by on-chip crystallization of photosynthetic reaction center, a membrane protein from Rhodobacter sphaeroides, followed by solving its structure to a resolution of 2.5 Å using X-ray diffraction data collected on-chip under ambient conditions. A moderate conformational change in hydrophilic chains of the protein was observed when comparing the on-chip, room temperature structure with known structures for which data were acquired under cryogenic conditions.« less

  5. Purple non-sulfur photosynthetic bacteria monitor environmental stresses.

    PubMed

    Kis, Mariann; Sipka, Gábor; Asztalos, Emese; Rázga, Zsolt; Maróti, Péter

    2015-10-01

    Heavy metal ion pollution and oxygen deficiency are major environmental risks for microorganisms in aqueous habitat. The potential of purple non-sulfur photosynthetic bacteria for biomonitoring and bioremediation was assessed by investigating the photosynthetic capacity in heavy metal contaminated environments. Cultures of bacterial strains Rhodobacter sphaeroides, Rhodospirillum rubrum and Rubrivivax gelatinosus were treated with heavy metal ions in micromolar (Hg(2+)), submillimolar (Cr(6+)) and millimolar (Pb(2+)) concentration ranges. Functional assays (flash-induced absorption changes and bacteriochlorophyll fluorescence induction) and electron micrographs were taken to specify the harmful effects of pollution and to correlate to morphological changes of the membrane. The bacterial strains and functional tests showed differentiated responses to environmental stresses, revealing that diverse mechanisms of tolerance and/or resistance are involved. The microorganisms were vulnerable to the prompt effect of Pb(2+), showed weak tolerance to Hg(2+) and proved to be tolerant to Cr(6+). The reaction center controlled electron transfer in Rvx. gelatinosus demonstrated the highest degree of resistance against heavy metal exposure. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute.

    PubMed

    Miyoshi, Tomohiro; Ito, Kosuke; Murakami, Ryo; Uchiumi, Toshio

    2016-06-21

    Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson-Crick base-pairing even in the 3'-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5' base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region.

  7. SciTech Connect

    Utschig, L. M.; Dalosto, S. D.; Thurnauer, M. C.

    Metal ion binding to a surface site on photosynthetic reaction centers (RCs) modulates light-induced electron and proton transfer events in the RC. Whereas many studies have elucidated aspects of metal ion modulation events in Rhodobacter sphaeroides RCs, much less is understood about the surface site in Blastochloris viridis (Blc. viridis) RCs. Interestingly, electron paramagnetic resonance studies revealed two spectroscopically distinct Cu{sup 2+} surface site environments in Blc. viridis RCs. Herein, Cu{sup 2+} has been used to spectroscopically probe the structure of these Cu{sup 2+} site(s) in response to freezing conditions, temperature, and charge separation. One Cu{sup 2+} environment in Blc.more » viridis RCs, termed CuA, exhibits temperature-dependent conformational flexibility. Different conformation states of the CuA{sup 2+} site are trapped when the RC is frozen in the dark either by fast-freeze or slow-freeze procedure. The second Cu{sup 2+} environment, termed CuB, is structurally invariant to different freezing conditions and shows resolved hyperfine coupling to three nitrogen atoms. Cu{sup 2+} is most likely binding at the same location on the RC, but in different coordination environments which may reflect two distinct conformational states of the isolated Blc. viridis RC protein.« less

  8. Extracytoplasmic function σ factors of the widely distributed group ECF41 contain a fused regulatory domain

    PubMed Central

    Wecke, Tina; Halang, Petra; Staroń, Anna; Dufour, Yann S; Donohue, Timothy J; Mascher, Thorsten

    2012-01-01

    Bacteria need signal transducing systems to respond to environmental changes. Next to one- and two-component systems, alternative σ factors of the extra-cytoplasmic function (ECF) protein family represent the third fundamental mechanism of bacterial signal transduction. A comprehensive classification of these proteins identified more than 40 phylogenetically distinct groups, most of which are not experimentally investigated. Here, we present the characterization of such a group with unique features, termed ECF41. Among analyzed bacterial genomes, ECF41 σ factors are widely distributed with about 400 proteins from 10 different phyla. They lack obvious anti-σ factors that typically control activity of other ECF σ factors, but their structural genes are often predicted to be cotranscribed with carboxymuconolactone decarboxylases, oxidoreductases, or epimerases based on genomic context conservation. We demonstrate for Bacillus licheniformis and Rhodobacter sphaeroides that the corresponding genes are preceded by a highly conserved promoter motif and are the only detectable targets of ECF41-dependent gene regulation. In contrast to other ECF σ factors, proteins of group ECF41 contain a large C-terminal extension, which is crucial for σ factor activity. Our data demonstrate that ECF41 σ factors are regulated by a novel mechanism based on the presence of a fused regulatory domain. PMID:22950025

  9. Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes

    PubMed Central

    Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

    2012-01-01

    The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression. PMID:22434878

  10. Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes.

    PubMed

    Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

    2012-07-01

    The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression.

  11. The three-dimensional structures of bacterial reaction centers.

    PubMed

    Olson, T L; Williams, J C; Allen, J P

    2014-05-01

    This review presents a broad overview of the research that enabled the structure determination of the bacterial reaction centers from Blastochloris viridis and Rhodobacter sphaeroides, with a focus on the contributions from Duysens, Clayton, and Feher. Early experiments performed in the laboratory of Duysens and others demonstrated the utility of spectroscopic techniques and the presence of photosynthetic complexes in both oxygenic and anoxygenic photosynthesis. The laboratories of Clayton and Feher led efforts to isolate and characterize the bacterial reaction centers. The availability of well-characterized preparations of pure and stable reaction centers allowed the crystallization and subsequent determination of the structures using X-ray diffraction. The three-dimensional structures of reaction centers revealed an overall arrangement of two symmetrical branches of cofactors surrounded by transmembrane helices from the L and M subunits, which also are related by the same twofold symmetry axis. The structure has served as a framework to address several issues concerning bacterial photosynthesis, including the directionality of electron transfer, the properties of the reaction center-cytochrome c 2 complex, and the coupling of proton and electron transfer. Together, these research efforts laid the foundation for ongoing efforts to address an outstanding question in oxygenic photosynthesis, namely the molecular mechanism of water oxidation.

  12. Global analysis of photosynthesis transcriptional regulatory networks.

    PubMed

    Imam, Saheed; Noguera, Daniel R; Donohue, Timothy J

    2014-12-01

    Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

  13. Energetics of bacterial photosynthesis.

    PubMed

    Lebard, David N; Matyushov, Dmitry V

    2009-09-10

    We report the results of extensive numerical simulations and theoretical calculations of electronic transitions in the reaction center of Rhodobacter sphaeroides photosynthetic bacterium. The energetics and kinetics of five electronic transitions related to the kinetic scheme of primary charge separation have been analyzed and compared to experimental observations. Nonergodic formulation of the reaction kinetics is required for the calculation of the rates due to a severe breakdown of the system ergodicity on the time scale of primary charge separation, with the consequent inapplicability of the standard canonical prescription to calculate the activation barrier. Common to all reactions studied is a significant excess of the charge-transfer reorganization energy from the width of the energy gap fluctuations over that from the Stokes shift of the transition. This property of the hydrated proteins, breaking the linear response of the thermal bath, allows the reaction center to significantly reduce the reaction free energy of near-activationless electron hops and thus raise the overall energetic efficiency of the biological charge-transfer chain. The increase of the rate of primary charge separation with cooling is explained in terms of the temperature variation of induction solvation, which dominates the average donor-acceptor energy gap for all electronic transitions in the reaction center. It is also suggested that the experimentally observed break in the Arrhenius slope of the primary recombination rate, occurring near the temperature of the dynamical transition in proteins, can be traced back to a significant drop of the solvent reorganization energy close to that temperature.

  14. Analysis of the Electronic Structure of the Special Pair of a Bacterial Photosynthetic Reaction Center by 13 C Photochemically Induced Dynamic Nuclear Polarization Magic-Angle Spinning NMR Using a Double-Quantum Axis.

    PubMed

    Najdanova, Marija; Gräsing, Daniel; Alia, A; Matysik, Jörg

    2018-01-01

    The origin of the functional symmetry break in bacterial photosynthesis challenges since several decades. Although structurally very similar, the two branches of cofactors in the reaction center (RC) protein complex act very differently. Upon photochemical excitation, an electron is transported along one branch, while the other remains inactive. Photochemically induced dynamic nuclear polarization (photo-CIDNP) magic-angle spinning (MAS) 13 C NMR revealed that the two bacteriochlorophyll cofactors forming the "Special Pair" donor dimer are already well distinguished in the electronic ground state. These previous studies are relying solely on 13 C- 13 C correlation experiments as radio-frequency-driven recoupling (RFDR) and dipolar-assisted rotational resonance (DARR). Obviously, the chemical-shift assignment is difficult in a dimer of tetrapyrrole macrocycles, having eight pyrrole rings of similar chemical shifts. To overcome this problem, an INADEQUATE type of experiment using a POST C7 symmetry-based approach is applied to selectively isotope-labeled bacterial RC of Rhodobacter (R.) sphaeroides wild type (WT). We, therefore, were able to distinguish unresolved sites of the macromolecular dimer. The obtained chemical-shift pattern is in-line with a concentric assembly of negative charge within the common center of the Special Pair supermolecule in the electronic ground state. © 2017 The American Society of Photobiology.

  15. Detection of Singlet Oxygen Formation inside Photoactive Biohybrid Composite Material.

    PubMed

    Hajdu, Kata; Ur Rehman, Ateeq; Vass, Imre; Nagy, László

    2017-12-26

    Photosynthetic reaction center proteins (RCs) are the most efficient light energy converter systems in nature. The first steps of the primary charge separation in photosynthesis take place in these proteins. Due to their unique properties, combining RCs with nano-structures promising applications can be predicted in optoelectronic systems. In the present work RCs purified from Rhodobacter sphaeroides purple bacteria were immobilized on multiwalled carbon nanotubes (CNTs). Carboxyl-and amine-functionalised CNTs were used, so different binding procedures, physical sorption and chemical sorption as well, could be applied as immobilization techniques. Light-induced singlet oxygen production was measured in the prepared photoactive biocomposites in water-based suspension by histidine mediated chemical trapping. Carbon nanotubes were applied under different conditions in order to understand their role in the equilibration of singlet oxygen concentration in the suspension. CNTs acted as effective quenchers of ¹O₂ either by physical (resonance) energy transfer or by chemical (oxidation) reaction and their efficiency showed dependence on the diffusion distance of ¹O₂.

  16. Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production

    SciTech Connect

    Lemmer, Kimberly C.; Zhang, Weiping; Langer, Samantha J.

    ABSTRACT Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation inRhodobacter sphaeroides. By screening anR. sphaeroidesTn5mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their totalmore » lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals. IMPORTANCEThis paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase microbial lipid production. We also find that the utility of some of these

  17. Ultrafast time-resolved carotenoid to-bacteriochlorophyll energy transfer in LH2 complexes from photosynthetic bacteria.

    PubMed

    Cong, Hong; Niedzwiedzki, Dariusz M; Gibson, George N; LaFountain, Amy M; Kelsh, Rhiannon M; Gardiner, Alastair T; Cogdell, Richard J; Frank, Harry A

    2008-08-28

    Steady-state and ultrafast time-resolved optical spectroscopic investigations have been carried out at 293 and 10 K on LH2 pigment-protein complexes isolated from three different strains of photosynthetic bacteria: Rhodobacter (Rb.) sphaeroides G1C, Rb. sphaeroides 2.4.1 (anaerobically and aerobically grown), and Rps. acidophila 10050. The LH2 complexes obtained from these strains contain the carotenoids, neurosporene, spheroidene, spheroidenone, and rhodopin glucoside, respectively. These molecules have a systematically increasing number of pi-electron conjugated carbon-carbon double bonds. Steady-state absorption and fluorescence excitation experiments have revealed that the total efficiency of energy transfer from the carotenoids to bacteriochlorophyll is independent of temperature and nearly constant at approximately 90% for the LH2 complexes containing neurosporene, spheroidene, spheroidenone, but drops to approximately 53% for the complex containing rhodopin glucoside. Ultrafast transient absorption spectra in the near-infrared (NIR) region of the purified carotenoids in solution have revealed the energies of the S1 (2(1)Ag-)-->S2 (1(1)Bu+) excited-state transitions which, when subtracted from the energies of the S0 (1(1)Ag-)-->S2 (1(1)Bu+) transitions determined by steady-state absorption measurements, give precise values for the positions of the S1 (2(1)Ag-) states of the carotenoids. Global fitting of the ultrafast spectral and temporal data sets have revealed the dynamics of the pathways of de-excitation of the carotenoid excited states. The pathways include energy transfer to bacteriochlorophyll, population of the so-called S* state of the carotenoids, and formation of carotenoid radical cations (Car*+). The investigation has found that excitation energy transfer to bacteriochlorophyll is partitioned through the S1 (1(1)Ag-), S2 (1(1)Bu+), and S* states of the different carotenoids to varying degrees. This is understood through a consideration of the

  18. Atomic force microscopy studies of native photosynthetic membranes.

    PubMed

    Sturgis, James N; Tucker, Jaimey D; Olsen, John D; Hunter, C Neil; Niederman, Robert A

    2009-05-05

    In addition to providing the earliest surface images of a native photosynthetic membrane at submolecular resolution, examination of the intracytoplasmic membrane (ICM) of purple bacteria by atomic force microscopy (AFM) has revealed a wide diversity of species-dependent arrangements of closely packed light-harvesting (LH) antennae, capable of fulfilling the basic requirements for efficient collection, transmission, and trapping of radiant energy. A highly organized architecture was observed with fused preparations of the pseudocrystalline ICM of Blastochloris viridis, consiting of hexagonally packed monomeric reaction center light-harvesting 1 (RC-LH1) core complexes. Among strains which also form a peripheral LH2 antenna, images of ICM patches from Rhodobacter sphaeroides exhibited well-ordered, interconnected networks of dimeric RC-LH1 core complexes intercalated by rows of LH2, coexisting with LH2-only domains. Other peripheral antenna-containing species, notably Rhodospirillum photometricum and Rhodopseudomonas palustris, showed a less regular organization, with mixed regions of LH2 and RC-LH1 cores, intermingled with large, paracrystalline domains. The ATP synthase and cytochrome bc(1) complex were not observed in any of these topographs and are thought to be localized in the adjacent cytoplasmic membrane or in inaccessible ICM regions separated from the flat regions imaged by AFM. The AFM images have served as a basis for atomic-resolution modeling of the ICM vesicle surface, as well as forces driving segregation of photosynthetic complexes into distinct domains. Docking of atomic-resolution molecular structures into AFM topographs of Rsp. photometricum membranes generated precise in situ structural models of the core complex surrounded by LH2 rings and a region of tightly packed LH2 complexes. A similar approach has generated a model of the highly curved LH2-only membranes of Rba. sphaeroides which predicts that sufficient space exists between LH2 complexes

  19. Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production

    DOE PAGES

    Lemmer, Kimberly C.; Zhang, Weiping; Langer, Samantha J.; ...

    2017-05-23

    ABSTRACT Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation inRhodobacter sphaeroides. By screening anR. sphaeroidesTn5mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their totalmore » lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals. IMPORTANCEThis paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase microbial lipid production. We also find that the utility of some of these

  20. Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production.

    PubMed

    Lemmer, Kimberly C; Zhang, Weiping; Langer, Samantha J; Dohnalkova, Alice C; Hu, Dehong; Lemke, Rachelle A; Piotrowski, Jeff S; Orr, Galya; Noguera, Daniel R; Donohue, Timothy J

    2017-05-23

    Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation in Rhodobacter sphaeroides By screening an R. sphaeroides Tn 5 mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals. IMPORTANCE This paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase microbial lipid production. We also find that the utility of some of these alterations can be

  1. The Iron-Sulfur Cluster of Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETF-QO) is the Electron Acceptor for Electron Transfer Flavoprotein†

    PubMed Central

    Swanson, Michael A.; Usselman, Robert J.; Frerman, Frank E.; Eaton, Gareth R.; Eaton, Sandra S.

    2011-01-01

    Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone-pool. It contains one [4Fe-4S]2+,1+ and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S]2+,1+ to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S]+ at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 mV and −30 mV for wild type to −11 mV and −19 mV, respectively. The N338A mutation decreased the potentials to −37 mV and −49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF1e− catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone, but not in electron transfer from ETF to ETF-QO. Therefore the iron-sulfur cluster is the immediate acceptor from ETF. PMID:18672901

  2. Widespread Distribution and Functional Specificity of the Copper Importer CcoA: Distinct Cu Uptake Routes for Bacterial Cytochrome c Oxidases

    SciTech Connect

    Khalfaoui-Hassani, Bahia; Wu, Hongjiang; Blaby-Haas, Crysten E.

    ABSTRACT Cytochromecoxidases are members of the heme-copper oxidase superfamily. These enzymes have different subunits, cofactors, and primary electron acceptors, yet they all contain identical heme-copper (Cu B) binuclear centers within their catalytic subunits. The uptake and delivery pathways of the Cu Batom incorporated into this active site, where oxygen is reduced to water, are not well understood. Our previous work with the facultative phototrophic bacteriumRhodobacter capsulatusindicated that the copper atom needed for the Cu Bsite ofcbb 3-type cytochromecoxidase (cbb 3-Cox) is imported to the cytoplasm by a major facilitator superfamily-type transporter, CcoA. In this study, a comparative genomic analysis ofmore » CcoA orthologs in alphaproteobacterial genomes showed that CcoA is widespread among organisms and frequently co-occurs with cytochromecoxidases. To define the specificity of CcoA activity, we investigated its function inRhodobacter sphaeroides, a close relative ofR. capsulatusthat contains bothcbb 3- andaa 3-Cox. Phenotypic, genetic, and biochemical characterization of mutants lacking CcoA showed that in its absence, or even in the presence of its bypass suppressors, only the production ofcbb 3-Cox and not that ofaa 3-Cox was affected. We therefore concluded that CcoA is dedicated solely tocbb 3-Cox biogenesis, establishing that distinct copper uptake systems provide the Cu Batoms to the catalytic sites of these two similar cytochromecoxidases. These findings illustrate the large variety of strategies that organisms employ to ensure homeostasis and fine control of copper trafficking and delivery to the target cuproproteins under different physiological conditions. IMPORTANCEThecbb 3- andaa 3-type cytochromecoxidases belong to the widespread heme-copper oxidase superfamily. They are membrane-integral cuproproteins that catalyze oxygen reduction to water under hypoxic and normoxic growth conditions. These enzymes diverge in terms of subunit and

  3. Widespread Distribution and Functional Specificity of the Copper Importer CcoA: Distinct Cu Uptake Routes for Bacterial Cytochrome c Oxidases

    DOE PAGES

    Khalfaoui-Hassani, Bahia; Wu, Hongjiang; Blaby-Haas, Crysten E.; ...

    2018-02-27

    ABSTRACT Cytochromecoxidases are members of the heme-copper oxidase superfamily. These enzymes have different subunits, cofactors, and primary electron acceptors, yet they all contain identical heme-copper (Cu B) binuclear centers within their catalytic subunits. The uptake and delivery pathways of the Cu Batom incorporated into this active site, where oxygen is reduced to water, are not well understood. Our previous work with the facultative phototrophic bacteriumRhodobacter capsulatusindicated that the copper atom needed for the Cu Bsite ofcbb 3-type cytochromecoxidase (cbb 3-Cox) is imported to the cytoplasm by a major facilitator superfamily-type transporter, CcoA. In this study, a comparative genomic analysis ofmore » CcoA orthologs in alphaproteobacterial genomes showed that CcoA is widespread among organisms and frequently co-occurs with cytochromecoxidases. To define the specificity of CcoA activity, we investigated its function inRhodobacter sphaeroides, a close relative ofR. capsulatusthat contains bothcbb 3- andaa 3-Cox. Phenotypic, genetic, and biochemical characterization of mutants lacking CcoA showed that in its absence, or even in the presence of its bypass suppressors, only the production ofcbb 3-Cox and not that ofaa 3-Cox was affected. We therefore concluded that CcoA is dedicated solely tocbb 3-Cox biogenesis, establishing that distinct copper uptake systems provide the Cu Batoms to the catalytic sites of these two similar cytochromecoxidases. These findings illustrate the large variety of strategies that organisms employ to ensure homeostasis and fine control of copper trafficking and delivery to the target cuproproteins under different physiological conditions. IMPORTANCEThecbb 3- andaa 3-type cytochromecoxidases belong to the widespread heme-copper oxidase superfamily. They are membrane-integral cuproproteins that catalyze oxygen reduction to water under hypoxic and normoxic growth conditions. These enzymes diverge in terms of subunit and

  4. Lipopolysaccharide and Its Analog Antagonists Display Differential Serum Factor Dependencies for Induction of Cytokine Genes in Murine Macrophages

    PubMed Central

    Perera, Pin-Yu; Qureshi, Nilofer; Christ, William J.; Stütz, Peter; Vogel, Stefanie N.

    1998-01-01

    Monocytes/macrophages play a central role in mediating the effects of lipopolysaccharide (LPS) derived from gram-negative bacteria by the production of proinflammatory mediators. Recently, it was shown that the expression of cytokine genes for tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and interferon-inducible protein-10 (IP-10) by murine macrophages in response to low concentrations of LPS is entirely CD14 dependent. In this report, we show that murine macrophages respond to low concentrations of LPS (≤2 ng/ml) in the complete absence of serum, leading to the induction of TNF-α and IL-1β genes. In contrast to the TNF-α and IL-1β genes, the IP-10 gene is poorly induced in the absence of serum. The addition of recombinant human soluble CD14 (rsCD14) had very little effect on the levels of serum-free, LPS-induced TNF-α, IL-1β, and IP-10 genes. In contrast, the addition of recombinant human LPS-binding protein (rLBP) had opposing effects on the LPS-induced TNF-α or IL-1β and IP-10 genes. rLBP inhibited LPS-induced TNF-α and IL-1β genes, while it reconstituted IP-10 gene expression to levels induced in the presence of serum. These results provide further evidence that the induction of TNF-α or IL-1β genes occurs via a pathway that is distinct from one that leads to the induction of the IP-10 gene and that the pathways diverge at the level of the initial interaction between LPS and cellular CD14. Additionally, the results presented here indicate that LPS structural analog antagonists Rhodobacter sphaeroides diphosphoryl lipid A and SDZ 880.431 are able to inhibit LPS-induced TNF-α and IL-1β in the absence of serum, while a synthetic analog of Rhodobacter capsulatus lipid A (B 975) requires both rsCD14 and rLBP to function as an inhibitor. PMID:9596717

  5. The evolution of glutathione metabolism in phototrophic microorganisms

    NASA Technical Reports Server (NTRS)

    Fahey, R. C.; Buschbacher, R. M.; Newton, G. L.

    1987-01-01

    Of the many roles ascribed to glutathione (GSH) the one most clearly established is its role in the protection of higher eucaryotes against oxygen toxicity through destruction of thiol-reactive oxygen byproducts. If this is the primary function of GSH then GSH metabolism should have evolved during or after the evolution of oxygenic photosynthesis. That many bacteria do not produce GSH is consistent with this view. In the present study we have examined the low-molecular-weight thiol composition of a variety of phototrophic microorganisms to ascertain how evolution of GSH production is related to evolution of oxygenic photosynthesis. Cells were extracted in the presence of monobromobimane (mBBr) to convert thiols to fluorescent derivatives, which were analyzed by high-pressure liquid chromatography. Significant levels of GSH were not found in the green bacteria (Chlorobium thiosulfatophilum and Chloroflexus aurantiacus). Substantial levels of GSH were present in the purple bacteria (Chromatium vinosum, Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodocyclus gelatinosa), the cyanobacteria [Anacystis nidulans, Microcoleus chthonoplastes S.G., Nostoc muscorum, Oscillatoria amphigranulata, Oscillatoria limnetica, Oscillatoria sp. (Stinky Spring, Utah), Oscillatoria terebriformis, Plectonema boryanum, and Synechococcus lividus], and eucaryotic algae (Chlorella pyrenoidsa, Chlorella vulgaris, Euglena gracilis, Scenedesmus obliquus, and Chlamydomonas reinhardtii). Other thiols measured included cysteine, gamma-glutamylcysteine, thiosulfate, coenzyme A, and sulfide; several unidentified thiols were also detected. Many of the organisms examined also exhibited a marked ability to reduce mBBr to syn-(methyl,methyl)bimane, an ability that was quenched by treatment with 2-pyridyl disulfide or 5,5'-bisdithio-(2-nitrobenzoic acid) prior to reaction with mBBr. These observations indicate the presence of a reducing system capable of electron transfer to mBBr and reduction of

  6. Purple-bacterial photosynthetic reaction centers and quantum-dot hybrid-assemblies in lecithin liposomes and thin films.

    PubMed

    Lukashev, Eugeny P; Knox, Petr P; Gorokhov, Vladimir V; Grishanova, Nadezda P; Seifullina, Nuranija Kh; Krikunova, Maria; Lokstein, Heiko; Paschenko, Vladimir Z

    2016-11-01

    Quantum dots (QDs) absorb ultraviolet and long-wavelength visible light energy much more efficiently than natural bacterial light-harvesting proteins and can transfer the excitation energy to photosynthetic reaction centers (RCs). Inclusion of RCs combined with QDs as antennae into liposomes opens new opportunities for using such hybrid systems as a basis for artificial energy-transforming devices that potentially can operate with greater efficiency and stability than devices based only on biological components or inorganic components alone. RCs from Rhodobacter sphaeroides and QDs (CdSe/ZnS with hydrophilic covering) were embedded in lecithin liposomes by extrusion of a solution of multilayer lipid vesicles through a polycarbonate membrane or by dialysis of lipids and proteins dispersed with excess detergent. The efficiency of RC and QD interaction within the liposomes was estimated using fluorescence excitation spectra of the photoactive bacteriochlorophyll of the RCs and by measuring the fluorescence decay kinetics of the QDs. The functional activity of the RCs in hybrid complexes was fully maintained, and their stability was even increased. The efficiency of energy transfer between QDs and RCs and conditions of long-term stability of function of such hybrid complexes in film preparations were investigated as well. It was found that dry films containing RCs and QDs, maintained at atmospheric humidity, are capable of maintaining their functional activity for at least some months as judged by measurements of their spectral characteristics, efficiency of energy transfer from QDs to RCs and RC electron transport activity. Addition of trehalose to the films increases the stability further, especially for films maintained at low humidity. These stable hybrid film structures are promising for further studies towards developing new phototransformation devices for biotechnological applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Fine tuning of the spectral properties of LH2 by single amino acid residues.

    PubMed

    Silber, Martina V; Gabriel, Günther; Strohmann, Brigitte; Garcia-Martin, Adela; Robert, Bruno; Braun, Paula

    2008-05-01

    The peripheral light-harvesting complex, LH2, of Rhodobacter sphaeroides consists of an assembly of membrane-spanning alpha and beta polypeptides which assemble the photoactive bacteriochlorophyll and carotenoid molecules. In this study we systematically investigated bacteriochlorophyll-protein interactions and their effect on functional bacteriochlorophyll assembly by site-directed mutations of the LH2 alpha-subunit. The amino acid residues, isoleucine at position -1 and serine at position -4 were replaced by 12 and 13 other residues, respectively. All residues replacing isoleucine at position -1 supported the functional assembly of LH2. The replacement of isoleucine by glycine, glutamine or asparagine, however, produced LH2 complex with significantly altered spectral properties in comparison to LH2 WT. As indicated by resonance Raman spectroscopy extensive rearrangement of the bacteriochlorophyll-B850 macrocycle(s) took place in LH2 in which isoleucine -1 was replaced by glycine. The replacement results in disruption of the H-bond between the C3 acetyl groups and the aromatic residues +13/+14 without affecting the H-bond involving the C13(1) keto group. In contrast, nearly all amino acid replacements of serine at position -4 resulted in shifting of the bacteriochlorophyll-B850 red most absorption maximum. Interestingly, the extent of shifting closely correlated with the volume of the residue at position -4. These results illustrate that fine tuning of the spectral properties of the bacteriochlorophyll-B850 molecules depend on their packing with single amino acid residues at distinct positions.

  8. Photoprotection in a purple phototrophic bacterium mediated by oxygen-dependent alteration of carotenoid excited-state properties

    PubMed Central

    Šlouf, Václav; Chábera, Pavel; Olsen, John D.; Martin, Elizabeth C.; Qian, Pu; Hunter, C. Neil; Polívka, Tomáš

    2012-01-01

    Carotenoids are known to offer protection against the potentially damaging combination of light and oxygen encountered by purple phototrophic bacteria, but the efficiency of such protection depends on the type of carotenoid. Rhodobacter sphaeroides synthesizes spheroidene as the main carotenoid under anaerobic conditions whereas, in the presence of oxygen, the enzyme spheroidene monooxygenase catalyses the incorporation of a keto group forming spheroidenone. We performed ultrafast transient absorption spectroscopy on membranes containing reaction center-light-harvesting 1-PufX (RC-LH1-PufX) complexes and showed that when oxygen is present the incorporation of the keto group into spheroidene, forming spheroidenone, reconfigures the energy transfer pathway in the LH1, but not the LH2, antenna. The spheroidene/spheroidenone transition acts as a molecular switch that is suggested to twist spheroidenone into an s-trans configuration increasing its conjugation length and lowering the energy of the lowest triplet state so it can act as an effective quencher of singlet oxygen. The other consequence of converting carotenoids in RC-LH1-PufX complexes is that S2/S1/triplet pathways for spheroidene is replaced with a new pathway for spheroidenone involving an activated intramolecular charge-transfer (ICT) state. This strategy for RC-LH1-PufX-spheroidenone complexes maintains the light-harvesting cross-section of the antenna by opening an active, ultrafast S1/ICT channel for energy transfer to LH1 Bchls while optimizing the triplet energy for singlet oxygen quenching. We propose that spheroidene/spheroidenone switching represents a simple and effective photoprotective mechanism of likely importance for phototrophic bacteria that encounter light and oxygen. PMID:22586075

  9. Static and dynamic protein impact on electronic properties of light-harvesting complex LH2.

    PubMed

    Zerlauskiene, O; Trinkunas, G; Gall, A; Robert, B; Urboniene, V; Valkunas, L

    2008-12-11

    A comparative analysis of the temperature dependence of the absorption spectra of the LH2 complexes from different species of photosynthetic bacteria, i.e., Rhodobacter sphaeroides, Rhodoblastus acidophilus, and Phaeospirillum molischianum, was performed in the temperature range from 4 to 300 K. Qualitatively, the temperature dependence is similar for all of the species studied. The spectral bandwidths of both B800 and B850 bands increases with temperature while the band positions shift in opposite directions: the B800 band shifts slightly to the red while the B850 band to the blue. These results were analyzed using the modified Redfield theory based on the exciton model. The main conclusion drawn from the analysis was that the spectral density function (SDF) is the main factor underlying the strength of the temperature dependence of the bandwidths for the B800 and B850 electronic transitions, while the bandwidths themselves are defined by the corresponding inhomogeneous distribution function (IDF). Slight variation of the slope of the temperature dependence of the bandwidths between species can be attributed to the changes of the values of the reorganization energies and characteristic frequencies determining the SDF. To explain the shift of the B850 band position with temperature, which is unusual for the conventional exciton model, a temperature dependence of the IDF must be postulated. This dependence can be achieved within the framework of the modified (dichotomous) exciton model. The slope of the temperature dependence of the B850 bandwidth is then defined by the value of the reorganization energy and by the difference between the transition energies of the dichotomous states of the pigment molecules. The equilibration factor between these dichotomous states mainly determines the temperature dependence of the peak shift.

  10. Stability of integral membrane proteins under high hydrostatic pressure: the LH2 and LH3 antenna pigment-protein complexes from photosynthetic bacteria.

    PubMed

    Kangur, Liina; Timpmann, Kõu; Freiberg, Arvi

    2008-07-03

    The bacteriochlorophyll a-containing LH2 and LH3 antenna complexes are the integral membrane proteins that catalyze the photosynthetic process in purple photosynthetic bacteria. The LH2 complex from Rhodobacter sphaeroides shows characteristic strong absorbance at 800 and 850 nm due to the pigment molecules confined in two separate areas of the protein. In the LH3 complex from Rhodopesudomonas acidophila the corresponding bands peak at 800 and 820 nm. Using the bacteriochlorophyll a cofactors as intrinsic probes to monitor local changes in the protein structure, we investigate spectral responses of the antenna complexes to very high hydrostatic pressures up to 2.5 GPa when embedded into natural membrane environment or extracted with detergent. We first demonstrate that high pressure does induce significant alterations to the tertiary structure of the proteins not only in proximity of the 800 nm-absorbing bacteriochlorophyll a molecules known previously (Gall, A.; et al. Biochemistry 2003, 42, 13019) but also of the 850 nm- and 820 nm-absorbing molecules, including breakage of the hydrogen bond they are involved in. The membrane-protected complexes appear more resilient to damaging effects of the compression compared with the complexes extracted into mixed detergent-buffer environment. Increased resistance of the isolated complexes is observed at high protein concentration resulting aggregation as well as when cosolvent (glycerol) is added into the solution. These stability variations correlate with ability of penetration of the surrounding polar solvent (water) into the hydrophobic protein interiors, being thus the principal reason of the pressure-induced denaturation of the proteins. Considerable variability of elastic properties of the isolated complexes was also observed, tentatively assigned to heterogeneous protein packing in detergent micelles. While a number of the isolated complexes release most of their bacteriochlorophyll a content under high pressure

  11. Identification of protein W, the elusive sixth subunit of the Rhodopseudomonas palustris reaction center-light harvesting 1 core complex.

    PubMed

    Jackson, Philip J; Hitchcock, Andrew; Swainsbury, David J K; Qian, Pu; Martin, Elizabeth C; Farmer, David A; Dickman, Mark J; Canniffe, Daniel P; Hunter, C Neil

    2018-02-01

    The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc 1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  12. BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis

    PubMed Central

    Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Mélida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen

    2013-01-01

    Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200–300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP. PMID:24127606

  13. BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis.

    PubMed

    Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Mélida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen

    2013-10-29

    Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200-300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP.

  14. Atomic force microscopy reveals multiple patterns of antenna organization in purple bacteria: implications for energy transduction mechanisms and membrane modeling.

    PubMed

    Sturgis, James N; Niederman, Robert A

    2008-01-01

    Recent topographs of the intracytoplasmic membrane (ICM) of purple bacteria obtained by atomic force microscopy (AFM) have provided the first surface views of the native architecture of a multicomponent biological membrane at submolecular resolution, representing an important landmark in structural biology. A variety of species-dependent, closely packed arrangements of light-harvesting (LH) complexes was revealed: the most highly organized was found in Rhodobacter sphaeroides in which the peripheral LH2 antenna was seen either in large clusters or in fixed rows interspersed among ordered arrays of dimeric LH1-reaction center (RC) core complexes. A more random organization was observed in other species containing both the LH1 and LH2 complexes, as typified by Rhododspirillum photometricum with randomly packed monomeric LH1-RC core complexes intermingled with large, paracrystalline domains of LH2 antenna. Surprisingly, no structures that could be identified as the ATP synthase or cytochrome bc (1) complexes were observed, which may reflect their localization at ICM vesicle poles or in curved membrane areas, out of view from the flat regions imaged by AFM. This possible arrangement of energy transducing complexes has required a reassessment of energy tranduction mechanisms which place the cytochrome bc (1) complex in close association with the RC. Instead, more plausible proposals must account for the movement of quinone redox species over considerable membrane distances on appropriate time scales. AFM, together with atomic resolution structures are also providing the basis for molecular modeling of the ICM that is leading to an improved picture of the supramolecular organization of photosynthetic complexes, as well as the forces that drive their segregation into distinct domains.

  15. Exploring O2 Diffusion in A-Type Cytochrome c Oxidases: Molecular Dynamics Simulations Uncover Two Alternative Channels towards the Binuclear Site

    PubMed Central

    Oliveira, A. Sofia F.; Damas, João M.; Baptista, António M.; Soares, Cláudio M.

    2014-01-01

    Cytochrome c oxidases (Ccoxs) are the terminal enzymes of the respiratory chain in mitochondria and most bacteria. These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient. Despite decades of research and the availability of a large amount of structural and biochemical data available for the A-type Ccox family, little is known about the channel(s) used by O2 to travel from the solvent/membrane to the heme a3-CuB binuclear center (BNC). Moreover, the identification of all possible O2 channels as well as the atomic details of O2 diffusion is essential for the understanding of the working mechanisms of the A-type Ccox. In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC. For that, we use an integrated strategy combining atomistic molecular dynamics (MD) simulations (with and without explicit O2 molecules) and implicit ligand sampling (ILS) calculations. Based on the 3D free energy map for O2 inside Ccox, three channels were identified, all starting in the membrane hydrophobic region and connecting the surface of the protein to the BNC. One of these channels corresponds to the pathway inferred from the X-ray data available, whereas the other two are alternative routes for O2 to reach the BNC. Both alternative O2 channels start in the membrane spanning region and terminate close to Y288I. These channels are a combination of multiple transiently interconnected hydrophobic cavities, whose opening and closure is regulated by the thermal fluctuations of the lining residues. Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway. PMID:25474152

  16. The binding of quinone to the photosynthetic reaction centers: kinetics and thermodynamics of reactions occurring at the QB-site in zwitterionic and anionic liposomes.

    PubMed

    Mavelli, Fabio; Trotta, Massimo; Ciriaco, Fulvio; Agostiano, Angela; Giotta, Livia; Italiano, Francesca; Milano, Francesco

    2014-07-01

    Liposomes represent a versatile biomimetic environment for studying the interaction between integral membrane proteins and hydrophobic ligands. In this paper, the quinone binding to the QB-site of the photosynthetic reaction centers (RC) from Rhodobacter sphaeroides has been investigated in liposomes prepared with either the zwitterionic phosphatidylcholine (PC) or the negatively charged phosphatidylglycerol (PG) to highlight the role of the different phospholipid polar heads. Quinone binding (K Q) and interquinone electron transfer (L AB) equilibrium constants in the two type of liposomes were obtained by charge recombination reaction of QB-depleted RC in the presence of increasing amounts of ubiquinone-10 over the temperature interval 6-35 °C. The kinetic of the charge recombination reactions has been fitted by numerically solving the ordinary differential equations set associated with a detailed kinetic scheme involving electron transfer reactions coupled with quinone release and uptake. The entire set of traces at each temperature was accurately fitted using the sole quinone release constants (both in a neutral and a charge separated state) as adjustable parameters. The temperature dependence of the quinone exchange rate at the QB-site was, hence, obtained. It was found that the quinone exchange regime was always fast for PC while it switched from slow to fast in PG as the temperature rose above 20 °C. A new method was introduced in this paper for the evaluation of constant K Q using the area underneath the charge recombination traces as the indicator of the amount of quinone bound to the QB-site.

  17. Hydrogen bond network around the semiquinone of the secondary quinone acceptor Q(B) in bacterial photosynthetic reaction centers.

    PubMed

    Taguchi, Alexander T; O'Malley, Patrick J; Wraight, Colin A; Dikanov, Sergei A

    2015-05-07

    By utilizing a combined pulsed EPR and DFT approach, the high-resolution structure of the QB site semiquinone (SQB) was determined. The development of such a technique is crucial toward an understanding of protein-bound semiquinones on the structural level, as (i) membrane protein crystallography typically results in low resolution structures, and (ii) obtaining protein crystals in the semiquinone form is rarely feasible. The SQB hydrogen bond network was investigated with Q- (∼34 GHz) and X-band (∼9.7 GHz) pulsed EPR spectroscopy on fully deuterated reactions centers from Rhodobacter sphaeroides. Simulations in the SQB g-tensor reference frame provided the principal values and directions of the H-bond proton hyperfine tensors. Three protons were detected, one with an anisotropic tensor component, T = 4.6 MHz, assigned to the histidine NδH of His-L190, and two others with similar anisotropic constants T = 3.2 and 3.0 MHz assigned to the peptide NpH of Gly-L225 and Ile-L224, respectively. Despite the strong similarity in the peptide couplings, all hyperfine tensors were resolved in the Q-band ENDOR spectra. The Euler angles describing the series of rotations that bring the hyperfine tensors into the SQB g-tensor reference frame were obtained by least-squares fitting of the spectral simulations to the ENDOR data. These Euler angles show the locations of the hydrogen bonded protons with respect to the semiquinone. Our geometry optimized model of SQB used in previous DFT work is in strong agreement with the angular constraints from the spectral simulations, providing the foundation for future joint pulsed EPR and DFT semiquinone structural determinations in other proteins.

  18. [Improving Agricultural Safety of Soils Contaminated with Polycyclic Aromatic Hydrocarbons by In Situ Bioremediation].

    PubMed

    Jiao, Hai-huan; Pan, Jian-gang; Xu, Shena-jun; Bai, Zhi-hui; Wang, Dong; Huang, Zhan-bin

    2015-08-01

    In order to reduce the risk of enrichment of polycyclic aromatic hydrocarbons (PAHs) in crops, reduce the potential hazards of food-sourced PAHs to human and increase the agricultural safety of PAHs contaminated soils, the bio-augmented removal of polycyclic aromatic hydrocarbons (PAHs) was investigated through in situ remediation by introducing Rhodobacter sphaeroides (RS) into the agricultural soil contaminated by PAHs. The 50-times diluted RS was sprayed on leaf surface (in area B) or irrigated to roots (in area D). The treatment of spraying water of the equal amount was taken as the control (A) and the wheat field without any treatment as the blank (CK). Treatments were conducted since wheat seeding. Soil and wheat samples were collected in the mature period to analyze the changes of community structure of the soil microorganisms and the concentration of PAHs in soils and investigate the strengthening and restoration effects of RS on PAHs contaminated soils. Compared to the CK Area, the areas B and D revealed that the variation ratio of phospholipid fatty acids (PLFAs) that were the biomarker of soil microorganisms was 29.6%, and the ratio of total PAHs removed was increased 1.59 times and 1.68 times, respectively. The dry weight of wheat grain of 50 spikes was increased by 8.95% and 12.5%, respectively, and the enrichment factor of total PAHs was decreased by 58.9% and 62.2% respectively in the wheat grains. All the results suggested that RS reduced enrichment of PAHs in wheat grains and increased wheat yield, which had great exploitation and utilization potentiality in repairing and improving the agricultural safety of the soils contaminated with PHAs.

  19. Role of Small Subunit in Mediating Assembly of Red-type Form I Rubisco

    PubMed Central

    Joshi, Jidnyasa; Mueller-Cajar, Oliver; Tsai, Yi-Chin C.; Hartl, F. Ulrich; Hayer-Hartl, Manajit

    2015-01-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco. PMID:25371207

  20. Role of small subunit in mediating assembly of red-type form I Rubisco.

    PubMed

    Joshi, Jidnyasa; Mueller-Cajar, Oliver; Tsai, Yi-Chin C; Hartl, F Ulrich; Hayer-Hartl, Manajit

    2015-01-09

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Global Analysis of Photosynthesis Transcriptional Regulatory Networks

    PubMed Central

    Imam, Saheed; Noguera, Daniel R.; Donohue, Timothy J.

    2014-01-01

    Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis. PMID:25503406

  2. High-pressure modulation of the structure of the bacterial photochemical reaction center at physiological and cryogenic temperatures

    NASA Astrophysics Data System (ADS)

    Timpmann, Kõu; Kangur, Liina; Lõhmus, Ants; Freiberg, Arvi

    2017-07-01

    The optical absorption and fluorescence response to external high pressure of the reaction center membrane chromoprotein complex from the wild-type non-sulfur photosynthetic bacterium Rhodobacter sphaeroides was investigated using the native pigment cofactors as local molecular probes of the reaction center structure at physiological (ambient) and cryogenic (79 K) temperatures. In detergent-purified complexes at ambient temperature, abrupt blue shift and accompanied broadening of the special pair band was observed at about 265 MPa. These reversible in pressure features were assigned to a pressure-induced rupture of a lone hydrogen bond that binds the photo-chemically active L-branch primary electron donor bacteriochlorophyll cofactor to the surrounding protein scaffold. In native membrane-protected complexes the hydrogen bond rupture appeared significantly restricted and occurred close to about 500 MPa. The free energy change associated with the rupture of the special pair hydrogen bond in isolate complexes was estimated to be equal to about 12 kJ mol-1. In frozen samples at cryogenic temperatures the hydrogen bond remained apparently intact up to the maximum utilized pressure of 600 MPa. In this case, however, heterogeneous spectral response of the cofactors from the L-and M-branches was observed due to anisotropic build-up of the protein structure. While in solid phase, the special pair fluorescence as a function of pressure exactly followed the respective absorption spectrum at a constant Stokes shift, at ambient temperature, the two paths began to deviate strongly from one other at the hydrogen bond rupture pressure. This effect was tentatively interpreted by different emission properties of hydrogen-bound and hydrogen-unbound special pair exciton states.

  3. Langmuir-Blodgett and X-ray diffraction studies of isolated photosystem II reaction centers in monolayers and multilayers: physical dimensions of the complex.

    PubMed

    Uphaus, R A; Fang, J Y; Picorel, R; Chumanov, G; Wang, J Y; Cotton, T M; Seibert, M

    1997-04-01

    The photosystem II (PSII) reaction center (RC) is a hydrophobic intrinsic protein complex that drives the water-oxidation process of photosynthesis. Unlike the bacterial RC complex, an X-ray crystal structure of the PSII RC is not available. In order to determine the physical dimensions of the isolated PSII RC complex, we applied Langmuir techniques to determine the cross-sectional area of an isolated RC in a condensed monolayer film. Low-angle X-ray diffraction results obtained by examining Langmuir-Blodgett multilayer films of alternating PSII RC/Cd stearate monolayers were used to determine the length (or height; z-direction, perpendicular to the plane of the original membrane) of the complex. The values obtained for a PSII RC monomer were 26 nm2 and 4.8 nm, respectively, and the structural integrity of the RC in the multilayer film was confirmed by several approaches. Assuming a cylindrical-type RC structure, the above dimensions lead to a predicted volume of about 125 nm3. This value is very close to the expected volume of 118 nm3, calculated from the known molecular weight and partial specific volume of the PSII RC proteins. This same type of comparison was also made with the Rhodobacter sphaeroides RC based on published data, and we conclude that the PSII RC is much shorter in length and has a more regular solid geometric structure than the bacterial RC. Furthermore, the above dimensions of the PSII RC and those of PSII core (RC plus proximal antenna) proteins protruding outside the plane of the PSII membrane into the lumenal space as imaged by scanning tunneling microscopy (Seibert, Aust. J. Pl. Physiol. 22, 161-166, 1995) fit easily into the known dimensions of the PSII core complex visualized by others as electron-density projection maps. From this we conclude that the in situ PSII core complex is a dimeric structure containing two copies of the PSII RC.

  4. A unified description of the electrochemical, charge distribution, and spectroscopic properties of the special-pair radical cation in bacterial photosynthesis.

    PubMed

    Reimers, Jeffrey R; Hush, Noel S

    2004-04-07

    We apply our four-state 70-vibration vibronic-coupling model for the properties of the photosynthetic special-pair radical cation to: (1) interpret the observed correlations between the midpoint potential and the distribution of spin density between the two bacteriochlorophylls for 30 mutants of Rhodobacter sphaeroides, (2) interpret the observed average intervalence hole-transfer absorption energies as a function of spin density for six mutants, and (3) simulate the recently obtained intervalence electroabsorption Stark spectrum of the wild-type reaction center. While three new parameters describing the location of the sites of mutation with respect to the special pair are required to describe the midpoint-potential data, a priori predictions are made for the transition energies and the Stark spectrum. In general, excellent predictions are made of the observed quantities, with deviations being typically of the order of twice the experimental uncertainties. A unified description of many chemical and spectroscopic properties of the bacterial reaction center is thus provided. Central to the analysis is the assumption that the perturbations made to the reaction center, either via mutations of protein residues or by application of an external electric field, act only to independently modify the oxidation potentials of the two halves of the special pair and hence the redox asymmetry E0. While this appears to be a good approximation, clear evidence is presented that effects of mutation can be more extensive than what is allowed for. A thorough set of analytical equations describing the observed properties is obtained using the Born-Oppenheimer adiabatic approximation. These equations are generally appropriate for intervalence charge-transfer problems and include, for the first time, full treatment of both symmetric and antisymmetric vibrational motions. The limits of validity of the adiabatic approach to the full nonadiabatic problem are obtained.

  5. Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.

    PubMed

    Kong, Min Kyung; Kang, Hyun-Jun; Kim, Jin Ho; Oh, Soon Hwan; Lee, Pyung Cheon

    2015-11-20

    The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Monoolein Lipid Phases as Incorporation and Enrichment Materials for Membrane Protein Crystallization

    PubMed Central

    Wallace, Ellen; Dranow, David; Laible, Philip D.; Christensen, Jeff; Nollert, Peter

    2011-01-01

    The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization

  7. Location and magnetic relaxation properties of the stable tyrosine radical in photosystem II.

    PubMed

    Innes, J B; Brudvig, G W

    1989-02-07

    Dipolar interactions with neighboring metal ions can cause enhanced spin-lattice relaxation of free radicals. We have applied the theory of dipolar relaxation enhancement and shown that the dependence of the enhanced relaxation on the protein structure surrounding the free radical can be used to obtain distances from the free radical to the protein surface. To test the theoretical predictions, we have examined the effect of added Dy3+ complexes on the microwave power saturation of free radicals in two protein complexes of known structure: myoglobin nitroxide and the reaction center from Rhodobacter sphaeroides. Three cases have been considered: (1) metal ions bound to a specific site, (2) metal ions bound randomly over the protein surface, and (3) metal ions distributed randomly in solution. Only case 3, which assumes no specific binding, gave good agreement between the distances obtained by using the two model systems. The effect of added Dy3+ complexes on the microwave power saturation of signal IIslow from photosystem II (PSII) was used to determine the location of the stable tyrosine radical giving rise to signal IIslow. Assuming that the surface of a membrane-bound protein can be approximated as planar, we have obtained distances from the tyrosine radical to the membrane surface in thylakoids, in PSII membranes, and in Tris-washed PSII membranes. The distances we have determined are in good agreement with those predicted on the basis of a structural homology between the D1 and D2 subunits of PSII and the structurally characterized L and M subunits of the reaction center from purple non-sulfur bacteria. We have also examined the temperature dependence of the microwave power at half-saturation (P1/2) of signal IIslow from 4 to 200 K in dark-adapted PSII membranes. Above 70 K, the P1/2 increases as T2.5, which is consistent with a Raman relaxation mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. The reaction center is the sensitive target of the mercury(II) ion in intact cells of photosynthetic bacteria.

    PubMed

    Asztalos, Emese; Sipka, Gábor; Kis, Mariann; Trotta, Massimo; Maróti, Péter

    2012-06-01

    The sensitivity of intact cells of purple photosynthetic bacterium Rhodobacter sphaeroides wild type to low level (<100 μM) of mercury (Hg²⁺) contamination was evaluated by absorption and fluorescence spectroscopies of the bacteriochlorophyll-protein complexes. All assays related to the function of the reaction center (RC) protein (induction of the bacteriochlorophyll fluorescence, delayed fluorescence and light-induced oxidation and reduction of the bacteriochlorophyll dimer and energization of the photosynthetic membrane) showed prompt and later effects of the mercury ions. The damage expressed by decrease of the magnitude and changes of rates of the electron transfer kinetics followed complex (spatial and temporal) pattern according to the different Hg²⁺ sensitivities of the electron transport (donor/acceptor) sites including the reduced bound and free cytochrome c₂ and the primary reduced quinone. In contrast to the RC, the light harvesting system and the bc₁ complex demonstrated much higher resistance against the mercury pollution. The 850 and 875 nm components of the peripheral and core complexes were particularly insensitive to the mercury(II) ions. The concentration of the photoactive RCs and the connectivity of the photosynthetic units decreased upon mercury treatment. The degree of inhibition of the photosynthetic apparatus was always higher when the cells were kept in the light than in the dark indicating the importance of metabolism in active transport of the mercury ions from outside to the intracytoplasmic membrane. Any of the tests applied in this study can be used for detection of changes in photosynthetic bacteria at the early stages of the action of toxicants.

  9. The measured and calculated affinity of methyl and methoxy substituted benzoquinones for the QA site of bacterial reaction centers

    PubMed Central

    Zheng, Zhong; Dutton, P. Leslie; Gunner, M. R.

    2010-01-01

    Quinones play important roles in mitochondrial and photosynthetic energy conversion acting as intramembrane, mobile electron and proton carriers between catalytic sites in various electron transfer proteins. They display different affinity, selectivity, functionality and exchange dynamics in different binding sites. The computational analysis of quinone binding sheds light on the requirements for quinone affinity and specificity. The affinities of ten oxidized, neutral benzoquinones (BQs) were measured for the high affinity QA site in the detergent solubilized Rhodobacter sphaeroides bacterial photosynthetic reaction center. Multi-Conformation Continuum Electrostatics (MCCE) was then used to calculate their relative binding free energies by Grand Canonical Monte Carlo sampling with a rigid protein backbone, flexible ligand and side chain positions and protonation states. Van der Waals and torsion energies, Poisson-Boltzmann continuum electrostatics and accessible surface area dependent ligand-solvent interactions are considered. An initial, single cycle of GROMACS backbone optimization improves the match with experiment as do coupled ligand and side chain motions. The calculations match experiment with an RMSD of 2.29 and a slope of 1.28. The affinities are dominated by favorable protein-ligand van der Waals rather than electrostatic interactions. Each quinone appears in a closely clustered set of positions. Methyl and methoxy groups move into the same positions as found for the native quinone. Difficulties putting methyls into methoxy sites are observed. Calculations using an SAS dependent implicit van der Waals interaction smoothed out small clashes, providing a better match to experiment with a RMSD of 0.77 and a slope of 0.97. PMID:20607696

  10. Toll-like receptor 4 contributes to chronic itch, alloknesis and spinal astrocyte activation in male mice

    PubMed Central

    Liu, Tong; Han, Qingjian; Chen, Gang; Huang, Ya; Zhao, Lin-Xia; Berta, Temugin; Gao, Yong-Jing; Ji, Ru-Rong

    2016-01-01

    Increasing evidence suggests that Toll-like receptor 4 (TLR4) contributes importantly to spinal cord glial activation and chronic pain sensitization; however, its unique role in acute and chronic itch is unclear. In this study, we investigated the involvement of TLR4 in acute and chronic itch models in male mice using both transgenic and pharmacological approaches. Tlr4−/− mice exhibited normal acute itch induced by compound 48/80 and chloroquine, but these mice showed substantial reductions in scratching in chronic itch models of dry skin, induced by acetone and diethyether followed by water (AEW), contact dermatitis, and allergic contact dermatitis on the neck. Intrathecal (spinal) inhibition of TLR4 with lipopolysaccharide Rhodobacter sphaeroides (LPS-RS) did not affect acute itch but suppressed AEW-induced chronic itch. Compound 48/80 and AEW also produced robust alloknesis, a touch-elicited itch in wild-type mice, which was suppressed by intrathecal LPS-RS and Tlr4−/− deletion. AEW induced persistent upregulation of Tlr4 mRNA and increased TLR4 expression in GFAP-expressing astrocytes in spinal cord dorsal horn. AEW also induced TLR4-dependent astrogliosis (GFAP upregulation) in spinal cord. Intrathecal injection of astroglial inhibitor L-α-aminoadipate reduced AEW-induced chronic itch and alloknesis without affecting acute itch. Spinal TLR4 was also necessary for AEW-induced chronic itch in the cheek model. Interestingly, scratching plays an essential role in spinal astrogliosis, since AEW-induced astrogliosis was abrogated by putting Elizabethan Collars on the neck to prevent scratching the itchy skin. Our findings suggest that spinal TLR4 signaling is important for spinal astrocyte activation and astrogliosis that may underlie alloknesis and chronic itch. PMID:26645545

  11. Interaction and Synergism of Microbial Fuel Cell Bacteria within Methanogenesis

    NASA Technical Reports Server (NTRS)

    Klaus, David

    2004-01-01

    Biological hydrogen production from waste biomass has both terrestrial and Martian advanced life support applications. On earth, biological hydrogen production is being explored as a greenhouse neutral form of clean and efficient energy. In a permanently enclosed space habitat, carbon loop closure is required to reduce mission costs. Plants are grown to revitalize oxygen supply and are consumed by habitat inhabitants. Unharvested portions must then be recycled for reuse in the habitat. Several biological degradation techniques exist, but one process, biophotolysis, can be used to produce hydrogen from inedible plant biomass. This process is two-stage, with one stage using dark fermentation to convert plant wastes into organic acids. The second stage, photofermentation, uses photoheterotrophic purple non-sulfur bacteria with the addition of light to turn the organic acids into hydrogen and carbon dioxide. Such a system can prove useful as a co-generation scheme, providing some of the energy needed to power a larger primary carbon recovery system, such as composting. Since butyrate is expected as one of the major inputs into photofermentation, a characterization study was conducted with the bacterium Rhodobacter sphaeroides SCJ, a novel photoheterotrophic non-sulfur purple bacteria, to examine hydrogen production performance at 10 mM-100 mM butyrate concentrations. As butyrate levels increased, hydrogen production increased up to 25 mM, and then decreased and ceased by 100 mM. Additionally, lag phase increased with butyrate concentration, possibly indicating some product inhibition. Maximal substrate conversion efficiency was 8.0%; maximal light efficiency was 0.89%; and maximal hydrogen production rate was 7.7 Umol/mg/cdw/hr (173 ul/mg cdw/hr). These values were either consistent or lower than expected from literature.

  12. Impact of Mutations on the Midpoint Potential of the [4Fe-4S]+1,+2 Cluster and on Catalytic Activity in Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETF-QO)†

    PubMed Central

    Usselman, Robert J.; Fielding, Alistair J.; Frerman, Frank E.; Watmough, Nicholas J.; Eaton, Gareth R.; Eaton, Sandra S.

    2011-01-01

    Electron transfer flavoprotein - ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen bonding distance of the Sγ of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the mid-point potentials of the iron-sulfur cluster from +37 mV for wild type to −60 mV for Y501F and T525A and to −128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials. PMID:18069858

  13. Impact of mutations on the midpoint potential of the [4Fe-4S]+1,+2 cluster and on catalytic activity in electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO).

    PubMed

    Usselman, Robert J; Fielding, Alistair J; Frerman, Frank E; Watmough, Nicholas J; Eaton, Gareth R; Eaton, Sandra S

    2008-01-08

    Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen-bonding distance of the Sgamma of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the midpoint potentials of the iron-sulfur cluster from +37 mV for wild type to -60 mV for Y501F and T525A and to -128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials.

  14. Secondary coenzyme Q10 deficiency and oxidative stress in cultured fibroblasts from patients with riboflavin responsive multiple Acyl-CoA dehydrogenation deficiency.

    PubMed

    Cornelius, Nanna; Byron, Colleen; Hargreaves, Iain; Guerra, Paula Fernandez; Furdek, Andrea K; Land, John; Radford, Weston W; Frerman, Frank; Corydon, Thomas J; Gregersen, Niels; Olsen, Rikke K J

    2013-10-01

    Coenzyme Q10 (CoQ10) is essential for the energy production of the cells and as an electron transporter in the mitochondrial respiratory chain. CoQ10 links the mitochondrial fatty acid β-oxidation to the respiratory chain by accepting electrons from electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). Recently, it was shown that a group of patients with the riboflavin responsive form of multiple acyl-CoA dehydrogenation deficiency (RR-MADD) carrying inherited amino acid variations in ETF-QO also had secondary CoQ10 deficiency with beneficial effects of CoQ10 treatment, thus adding RR-MADD to an increasing number of diseases involving secondary CoQ10 deficiency. In this study, we show that moderately decreased CoQ10 levels in fibroblasts from six unrelated RR-MADD patients were associated with increased levels of mitochondrial reactive oxygen species (ROS). Treatment with CoQ10, but not with riboflavin, could normalize the CoQ10 level and decrease the level of ROS in the patient cells. Additionally, riboflavin-depleted control fibroblasts showed moderate CoQ10 deficiency, but not increased mitochondrial ROS, indicating that variant ETF-QO proteins and not CoQ10 deficiency are the causes of mitochondrial ROS production in the patient cells. Accordingly, the corresponding variant Rhodobacter sphaeroides ETF-QO proteins, when overexpressed in vitro, bind a CoQ10 pseudosubstrate, Q10Br, less tightly than the wild-type ETF-QO protein, suggesting that molecular oxygen can get access to the electrons in the misfolded ETF-QO protein, thereby generating superoxide and oxidative stress, which can be reversed by CoQ10 treatment.

  15. Energetics and kinetics of primary charge separation in bacterial photosynthesis.

    PubMed

    LeBard, David N; Kapko, Vitaliy; Matyushov, Dmitry V

    2008-08-21

    We report the results of molecular dynamics (MD) simulations and formal modeling of the free-energy surfaces and reaction rates of primary charge separation in the reaction center of Rhodobacter sphaeroides. Two simulation protocols were used to produce MD trajectories. Standard force-field potentials were employed in the first protocol. In the second protocol, the special pair was made polarizable to reproduce a high polarizability of its photoexcited state observed by Stark spectroscopy. The charge distribution between covalent and charge-transfer states of the special pair was dynamically adjusted during the simulation run. We found from both protocols that the breadth of electrostatic fluctuations of the protein/water environment far exceeds previous estimates, resulting in about 1.6 eV reorganization energy of electron transfer in the first protocol and 2.5 eV in the second protocol. Most of these electrostatic fluctuations become dynamically frozen on the time scale of primary charge separation, resulting in much smaller solvation contributions to the activation barrier. While water dominates solvation thermodynamics on long observation times, protein emerges as the major thermal bath coupled to electron transfer on the picosecond time of the reaction. Marcus parabolas were obtained for the free-energy surfaces of electron transfer by using the first protocol, while a highly asymmetric surface was obtained in the second protocol. A nonergodic formulation of the diffusion-reaction electron-transfer kinetics has allowed us to reproduce the experimental results for both the temperature dependence of the rate and the nonexponential decay of the population of the photoexcited special pair.

  16. Characterizing the proton loading site in cytochrome c oxidase.

    PubMed

    Lu, Jianxun; Gunner, M R

    2014-08-26

    Cytochrome c oxidase (CcO) uses the energy released by reduction of O2 to H2O to drive eight charges from the high pH to low pH side of the membrane, increasing the electrochemical gradient. Four electrons and protons are used for chemistry, while four more protons are pumped. Proton pumping requires that residues on a pathway change proton affinity through the reaction cycle to load and then release protons. The protonation states of all residues in CcO are determined in MultiConformational Continuum Electrostatics simulations with the protonation and redox states of heme a, a3, Cu(B), Y288, and E286 used to define the catalytic cycle. One proton is found to be loaded and released from residues identified as the proton loading site (PLS) on the P-side of the protein in each of the four CcO redox states. Thus, the same proton pumping mechanism can be used each time CcO is reduced. Calculations with structures of Rhodobacter sphaeroides, Paracoccus denitrificans, and bovine CcO derived by crystallography and molecular dynamics show the PLS functions similarly in different CcO species. The PLS is a cluster rather than a single residue, as different structures show 1-4 residues load and release protons. However, the proton affinity of the heme a3 propionic acids primarily determines the number of protons loaded into the PLS; if their proton affinity is too low, less than one proton is loaded.

  17. Characterizing the proton loading site in cytochrome c oxidase

    PubMed Central

    Lu, Jianxun; Gunner, M. R.

    2014-01-01

    Cytochrome c oxidase (CcO) uses the energy released by reduction of O2 to H2O to drive eight charges from the high pH to low pH side of the membrane, increasing the electrochemical gradient. Four electrons and protons are used for chemistry, while four more protons are pumped. Proton pumping requires that residues on a pathway change proton affinity through the reaction cycle to load and then release protons. The protonation states of all residues in CcO are determined in MultiConformational Continuum Electrostatics simulations with the protonation and redox states of heme a, a3, CuB, Y288, and E286 used to define the catalytic cycle. One proton is found to be loaded and released from residues identified as the proton loading site (PLS) on the P-side of the protein in each of the four CcO redox states. Thus, the same proton pumping mechanism can be used each time CcO is reduced. Calculations with structures of Rhodobacter sphaeroides, Paracoccus denitrificans, and bovine CcO derived by crystallography and molecular dynamics show the PLS functions similarly in different CcO species. The PLS is a cluster rather than a single residue, as different structures show 1–4 residues load and release protons. However, the proton affinity of the heme a3 propionic acids primarily determines the number of protons loaded into the PLS; if their proton affinity is too low, less than one proton is loaded. PMID:25114210

  18. Crystallographic Location and Mutational Analysis of Zn and Cd Inhibitory Sites and Role of Lipidic Carboxylates in Rescuing Proton Path Mutants in Cytochrome c Oxidase†

    PubMed Central

    Qin, Ling; Mills, Denise A.; Hiser, Carrie; Murphree, Anna; Garavito, R. Michael; Ferguson-Miller, Shelagh; Hosler, Jonathan

    2008-01-01

    Cytochrome c oxidase (CcO) transfers protons from the inner surface of the enzyme to the buried O2 reduction site through two different pathways, termed K and D, and from the outer surface via an undefined route. These proton paths can be inhibited by metals such as zinc or cadmium, but the sites of inhibition have not been established. Anomalous difference Fourier analyses of Rhodobacter sphaeroides CcO crystals, with cadmium added, reveal metal binding sites that include the proposed initial proton donor/acceptor of the K pathway, Glu-101 of subunit II. Mutant forms of CcO that lack Glu-101II (E101A and E101A/H96A) exhibit low activity and eliminate metal binding at this site. Significant activity is restored to E101A and E101A/H96A by adding the lipophilic carboxylic compounds, arachidonic acid and cholic acid, but not by their non-carboxylic analogues. These amphipathic acids likely provide their carboxylic groups as substitute proton donors/acceptors in the absence of Glu-101II, as previously observed for arachidonic acid in mutants that alter Asp-132I of the D pathway. The activity of E101A/H96A is still inhibited by zinc, but this remaining inhibition is nearly eliminated by removal of subunit III, which is known to alter the D pathway. The results identify the Glu-101/His-96 site of subunit II as the site of metal binding that inhibits the uptake of protons into the K pathway and indicate that subunit III contributes to zinc binding and/or inhibition of the D pathway. By removing subunit III from E101A/H96A, thereby eliminating zinc inhibition of the uptake of protons from the inner surface of CcO, we confirm that an external zinc binding site is involved in inhibiting the backflow of protons to the active site. PMID:17477548

  19. The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes.

    PubMed

    Swainsbury, David J K; Scheidelaar, Stefan; Foster, Nicholas; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R

    2017-10-01

    Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (<30kDa weight average molecular weight). The effectiveness of 10kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Transient binding of CO to Cu(B) in cytochrome c oxidase is dynamically linked to structural changes around a carboxyl group: a time-resolved step-scan Fourier transform infrared investigation.

    PubMed Central

    Heitbrink, Dirk; Sigurdson, Håkan; Bolwien, Carsten; Brzezinski, Peter; Heberle, Joachim

    2002-01-01

    The redox-driven proton pump cytochrome c oxidase is that enzymatic machinery of the respiratory chain that transfers electrons from cytochrome c to molecular oxygen and thereby splits molecular oxygen to form water. To investigate the reaction mechanism of cytochrome c oxidase on the single vibrational level, we used time-resolved step-scan Fourier transform infrared spectroscopy and studied the dynamics of the reduced enzyme after photodissociation of bound carbon monoxide across the mid-infrared range (2300-950 cm(-1)). Difference spectra of the bovine complex were obtained at -20 degrees C with 5 micros time resolution. The data demonstrate a dynamic link between the transient binding of CO to Cu(B) and changes in hydrogen bonding at the functionally important residue E(I-286). Variation of the pH revealed that the pK(a) of E(I-286) is >9.3 in the fully reduced CO-bound oxidase. Difference spectra of cytochrome c oxidase from beef heart are compared with those of the oxidase isolated from Rhodobacter sphaeroides. The bacterial enzyme does not show the environmental change in the vicinity of E(I-286) upon CO dissociation. The characteristic band shape appears, however, in redox-induced difference spectra of the bacterial enzyme but is absent in redox-induced difference spectra of mammalian enzyme. In conclusion, it is demonstrated that the dynamics of a large protein complex such as cytochrome c oxidase can be resolved on the single vibrational level with microsecond Fourier transform infrared spectroscopy. The applied methodology provides the basis for future investigations of the physiological reaction steps of this important enzyme. PMID:11751290

  1. Carotenoid biosynthesis in bacteria: In vitro studies of a crt/bch transcription factor from Rhodobacter capsulatus and carotenoid enzymes from Erwinia herbicola

    SciTech Connect

    O'Brien, D.A.

    1992-11-01

    A putative transcription factor in Rhodobactor capsulatus which binds upstream of the crt and bch pigment biosynthesis operons and appears to play a role in the adaptation of the organism from the aerobic to the anaerobic-photosynthetic growth mode was characterized. Chapter 2 describes the identification of this factor through an in vitro mobility shift assay, as well as the determination of its binding properties and sequence specificity. Chapter 3 focuses on the isolation of this factor. Biochemistry of later carotenoid biosynthesis enzymes derived from the non-photosynthetic bacterium, Erwinia herbicola. Chapter 4 describes the separate overexpression and in vitro analysis ofmore » two enzymes involved in the main sequence of the carotenoid biosynthesis pathway, lycopene cyclase and 5-carotene hydroxylase. Chapter 5 examines the overexpression and enzymology of functionally active zeaxanthin glucosyltransferase, an enzyme which carries out a more unusual transformation, converting a carotenoid into its more hydrophilic mono- and diglucoside derivatives. In addition, amino acid homology with other glucosyltransferases suggests a putative binding site for the UDP-activated glucose substrate.« less

  2. [Influence of LDAO on the conformation and release of bacteriochlorophyll of peripheral light-harvesting complex (LH2) from Rhodobacter azotoformans].

    PubMed

    Zhao, Gen-gui; Dong, Yan-min; Yang, Su-ping; Jiao, Nian-zhi; Qu, Yin-bo

    2010-10-01

    The aim of this study is to reveal the interaction relationships between lauryl dimethylamine N-oxide (LDAO) and peripheral light-harvesting complex (LH2) as well as the influence of LDAO on structure and function of LH2. In the present work, the effects of LDAO on the conformation and release processes of bacteriochlorophyll (BChl) of LH2 when incubated under different temperature and pH in the presence and absence of LDAO were investigated by spectroscopy. The results indicated that (1) the presence of LDAO resulted in alterations in the conformation, alpha-helix content, and spectra of Tyr and B850 band of LH2 at room temperature and pH 8.0. Moreover, energy transfer efficiency of LH2 was enhanced markedly in the presence of LDAO. (2) At 60 degrees C, both the B800 and B850 band of LH2 were released and transited into free BChl at pH 8.0. However, the release rates of bacteriochlorophylls of B800 and B850 band from LH2 were slowed down and the release processes were changed when incubated in the presence of LDAO. Hence, the stability of LH2 was improved in the presence of LDAO. (3) The accelerated release processes of bacteriochlorophylls of B800 and B850 band of LH2 were induced to transform into bacteriopheophytin (BPhe) and free BChl by LDAO in strong acid and strong alkalic solution at room temperature. However, the kinetic patterns of the B800 and B850 band were remarkably different. The release and self-assemble processes of B850 in LH2 were observed in strong acid solution without LDAO. Therefore, the different release behaviors of B800 and B850 of LH2 are induced by LDAO under different extreme environmental conditions.

  3. The protease ClpXP and the PAS-domain protein DivL regulate CtrA and gene transfer agent production in Rhodobacter capsulatus.

    PubMed

    Westbye, A B; Kater, L; Wiesmann, C; Ding, H; Yip, C K; Beatty, J T

    2018-04-06

    Several members of the Rhodobacterales ( Alphaproteobacteria ) produce a conserved horizontal gene transfer vector, called gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system to study GTA biology is the R. capsulatus GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the Alphaproteobacteria and is an essential regulator of RcGTA production: it controls production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that a dysregulation of the CckA-ChpT-CtrA phosphorelay - either by loss of the PAS-domain protein DivL or by substituting the autophosphorylation residue of the hybrid histidine kinase CckA - decreased CtrA phosphorylation and greatly increased RcGTA protein production in R. capsulatus We show that a loss of the ClpXP protease or the three C-terminal residues of CtrA resulted in increased CtrA levels in R. capsulatus, and identify ClpX(P) as essential for maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the Rhodobacterales Importance Members of the Rhodobacterales are abundant in ocean and fresh water environments. The conserved GTA produced by many Rhodobacterales may have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay, however several questions about GTA regulation remains. Our identification that a 'short' DivL homologue and ClpXP regulate CtrA in R. capsulatus extends the model of CtrA regulation from Caulobacter crescentus to a member of the Rhodobacterales. We found that the magnitude of RcGTA production greatly depended on DivL and CckA kinase activity, adding yet a layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles. Copyright © 2018 American Society for Microbiology.

  4. Identifying involvement of Lys251/Asp252 pair in electron transfer and associated proton transfer at the quinone reduction site of Rhodobacter capsulatus cytochrome bc1.

    PubMed

    Kuleta, Patryk; Sarewicz, Marcin; Postila, Pekka; Róg, Tomasz; Osyczka, Artur

    2016-10-01

    Describing dynamics of proton transfers in proteins is challenging, but crucial for understanding processes which use them for biological functions. In cytochrome bc1, one of the key enzymes of respiration or photosynthesis, proton transfers engage in oxidation of quinol (QH2) and reduction of quinone (Q) taking place at two distinct catalytic sites. Here we evaluated by site-directed mutagenesis the contribution of Lys251/Asp252 pair (bacterial numbering) in electron transfers and associated with it proton uptake to the quinone reduction site (Qi site). We showed that the absence of protonable group at position 251 or 252 significantly changes the equilibrium levels of electronic reactions including the Qi-site mediated oxidation of heme bH, reverse reduction of heme bH by quinol and heme bH/Qi semiquinone equilibrium. This implicates the role of H-bonding network in binding of quinone/semiquinone and defining thermodynamic properties of Q/SQ/QH2 triad. The Lys251/Asp252 proton path is disabled only when both protonable groups are removed. With just one protonable residue from this pair, the entrance of protons to the catalytic site is sustained, albeit at lower rates, indicating that protons can travel through parallel routes, possibly involving water molecules. This shows that proton paths display engineering tolerance for change as long as all the elements available for functional cooperation secure efficient proton delivery to the catalytic site. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Influence of hydrophobic micelle structure on crystallization of the photosynthetic RC-LH1-PufX complex from Rhodobacter blasticus.

    PubMed

    Barret, Laurie-Anne; Barrot-Ivolot, Cherone; Raynal, Simon; Jungas, Colette; Polidori, Ange; Bonneté, Françoise

    2013-07-25

    Small angle X-ray scattering (SAXS) experiments are performed on two non-ionic surfactants, the dodecyl β-maltoside (DDβM) and the propyl(bi)cyclohexyl α-maltoside (PCCαM), a maltoside derivative containing a rigid bicyclohexyl group as hydrophobic chain, in order to compare the influence of both hydrophobic moiety structure and anomeric form on micelle form factors and intermicellar interactions relevant for membrane protein crystallization. Density and refractive index measurements were performed in order to determine volumetric and optical properties of surfactants, essential for determination of micelle molar masses by both SAXS and SEC-MALLS. SAXS form factors were analyzed by Guinier approximation and inverse Fourier transformation, to obtain the radius of gyration (RG) and the pair distribution function (P(r)) of each surfactant. Form factor model fitting was also performed to describe the shape and the assembly of both surfactant micelles. Finally, second virial coefficients were measured at different percentages of polyethylene glycol 3350, in order to correlate surfactant intermicellar interactions and RC-LH1-PufX phase diagram. It is thus found that while size, shape, and dimensions of micelles are slightly similar for both surfactants, their molar mass and aggregation number differ significantly. PCCαM are more densely packed than DDβM, which reflects (1) an increase in van der Waals contacts between PCCαM hydrophobic chains in the micelle bulk and (2) a supplementary intermicellar attraction compared to DDβM. Finally addition of PEG, which induces a depletion attraction, decreases the solubility of the RC-LH1-PufX complex in PCCαM.

  6. Impact of the lipid bilayer on energy transfer kinetics in the photosynthetic protein LH2.

    PubMed

    Ogren, John I; Tong, Ashley L; Gordon, Samuel C; Chenu, Aurélia; Lu, Yue; Blankenship, Robert E; Cao, Jianshu; Schlau-Cohen, Gabriela S

    2018-03-28

    Photosynthetic purple bacteria convert solar energy to chemical energy with near unity quantum efficiency. The light-harvesting process begins with absorption of solar energy by an antenna protein called Light-Harvesting Complex 2 (LH2). Energy is subsequently transferred within LH2 and then through a network of additional light-harvesting proteins to a central location, termed the reaction center, where charge separation occurs. The energy transfer dynamics of LH2 are highly sensitive to intermolecular distances and relative organizations. As a result, minor structural perturbations can cause significant changes in these dynamics. Previous experiments have primarily been performed in two ways. One uses non-native samples where LH2 is solubilized in detergent, which can alter protein structure. The other uses complex membranes that contain multiple proteins within a large lipid area, which make it difficult to identify and distinguish perturbations caused by protein-protein interactions and lipid-protein interactions. Here, we introduce the use of the biochemical platform of model membrane discs to study the energy transfer dynamics of photosynthetic light-harvesting complexes in a near-native environment. We incorporate a single LH2 from Rhodobacter sphaeroides into membrane discs that provide a spectroscopically amenable sample in an environment more physiological than detergent but less complex than traditional membranes. This provides a simplified system to understand an individual protein and how the lipid-protein interaction affects energy transfer dynamics. We compare the energy transfer rates of detergent-solubilized LH2 with those of LH2 in membrane discs using transient absorption spectroscopy and transient absorption anisotropy. For one key energy transfer step in LH2, we observe a 30% enhancement of the rate for LH2 in membrane discs compared to that in detergent. Based on experimental results and theoretical modeling, we attribute this difference to

  7. Impact of the lipid bilayer on energy transfer kinetics in the photosynthetic protein LH2† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc04814a

    PubMed Central

    Ogren, John I.; Tong, Ashley L.; Gordon, Samuel C.; Chenu, Aurélia; Lu, Yue; Blankenship, Robert E.; Cao, Jianshu

    2018-01-01

    Photosynthetic purple bacteria convert solar energy to chemical energy with near unity quantum efficiency. The light-harvesting process begins with absorption of solar energy by an antenna protein called Light-Harvesting Complex 2 (LH2). Energy is subsequently transferred within LH2 and then through a network of additional light-harvesting proteins to a central location, termed the reaction center, where charge separation occurs. The energy transfer dynamics of LH2 are highly sensitive to intermolecular distances and relative organizations. As a result, minor structural perturbations can cause significant changes in these dynamics. Previous experiments have primarily been performed in two ways. One uses non-native samples where LH2 is solubilized in detergent, which can alter protein structure. The other uses complex membranes that contain multiple proteins within a large lipid area, which make it difficult to identify and distinguish perturbations caused by protein–protein interactions and lipid–protein interactions. Here, we introduce the use of the biochemical platform of model membrane discs to study the energy transfer dynamics of photosynthetic light-harvesting complexes in a near-native environment. We incorporate a single LH2 from Rhodobacter sphaeroides into membrane discs that provide a spectroscopically amenable sample in an environment more physiological than detergent but less complex than traditional membranes. This provides a simplified system to understand an individual protein and how the lipid–protein interaction affects energy transfer dynamics. We compare the energy transfer rates of detergent-solubilized LH2 with those of LH2 in membrane discs using transient absorption spectroscopy and transient absorption anisotropy. For one key energy transfer step in LH2, we observe a 30% enhancement of the rate for LH2 in membrane discs compared to that in detergent. Based on experimental results and theoretical modeling, we attribute this difference

  8. Structure of the Cellulose Synthase Complex of Gluconacetobacter hansenii at 23.4 Å Resolution

    PubMed Central

    Du, Juan; Vepachedu, Venkata; Cho, Sung Hyun; Kumar, Manish; Nixon, B. Tracy

    2016-01-01

    Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 Å for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into β-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. The results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the

  9. The influence of quorum sensing in compartment II of the MELiSSA loop

    NASA Astrophysics Data System (ADS)

    Condori, Sandra; Mastroleo, Felice; Wattiez, Ruddy; Leys, Natalie

    MELiSSA (Micro-Ecological Life Support System Alternative) has been conceived as a 5 compartments microorganisms and higher plants recycling system for long haul space flights. Rhodospirillum rubrum S1H colonizes compartment II. Previous work reported that continuous culture of the bacterium in a photobioreactor could lead to thick biofilm formation, leading to bioreactor arrest. Our aim is to investigate the unknown quorum sensing (QS) system of R. rubrum S1H, specifically under MELiSSA relevant culture conditions meaning light anaerobic (LAN) and using acetate as carbon source. In that purpose an autoinducer synthase gene (Rru_A3396) knockout mutant was constructed by allelic exchange generating strain M68. In addition phenotypic comparison between wild type (WT) and M68 was performed. Results of thin layer chromatography assay where Agrobacterium tumefaciens NT1 have been used as reporter strain showed that WT produces acyl-homoserine lactones (AHLs) from C4 to C12 acyl carbon chain length; however, in M68 no AHLs were detected confirming that gene Rru_A3396 (named rruI) encodes an autoinducer synthase. Interestingly under a low shear or static environment M68 showed cell aggregation similar as reported in a closely related bacterium Rhodobacter sphaeroides (cerI mutant). In contrast to WT, M68 did not form biofilm and exhibited a decreased motility and pigment content. M68 vs wild type transcriptomics results showed that 326 genes were statistically significant differentially expressed. Downregulation of genes related to photosynthesis e.g., reaction center subunits, light harvesting complex and photosynthetic assembly proteins was observed. Similar results were obtained for preliminary proteomic analysis. Results obtained showed that in R. rubrum S1H the AHL-based QS system regulates almost 8% of the genome which is linked to biofilm formation among other biological processes described above. Since strain M68 could not be used in compartment II due to its less

  10. SciTech Connect

    Breton, J.; Berger, G.; Nabedryk, E.

    The photoreduction of the secondary quinone acceptor Q{sub B} in reaction centers (RCs) of the photosynthetic bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis has been investigated by light-induced FTIR difference spectroscopy of RCs reconstituted with several isotopically labeled ubiquinones. The labels used were {sup 18}O on both carbonyls and {sup 13}C either uniformly or selectively at the 1- or the 4-position, i.e., on either one of the two carbonyls. The Q{sub B}{sup {minus}}/Q{sub B} spectra of RCs reconstituted with the isotopically labeled and unlabeled quinones as well as the double differences calculated form these spectra exhibit distinct isotopic shifts for amore » numer of bands attributed to vibrations of Q{sub B} and Q{sub B}{sup {minus}}. The vibrational modes of the quinone in the Q{sub B} site are compared to those of ubiquinone in vitro, leading to band assignments for the C{double_bond}O and C{double_bond}C vibrations of the neutral Q{sub B} and for the C---O and C---C of the semiquinone. The C{double_bond}O frequency of each of the carbonyls of the unlabeled quinone is revealed at 1641 cm{sup {minus}1} for both species. This demonstrates symmetrical and weak hydrogen bonding of the two C{double_bond}O groups to the protein at the Q{sub B} site. In contrast, the C{double_bond}C vibrations are not equivalent for selective labeling at C{sub 1} or at C{sub 4}, although they both contribute to the {approximately}1611-cm{sup {minus}1} band in the Q{sub B}{sup {minus}}/Q{sub B} spectra of the two species. Compared to the vibrations of isolated ubiquinone, the C{double_bond}C mode of Q{sub B} does not involve displacement of the C{sub 4} carbon atom, while the motion of C{sub 1} is not hindered. Further analysis of the spectra suggests that the protein at the binding site imposes a specific constraint on the methoxy and/or the methyl group proximal to the C{sub 4} carbonyl. 49 refs., 5 figs.« less

  11. Structure of the cellulose synthase complex of Gluconacetobacter hansenii at 23.4 Å resolution

    DOE PAGES

    Du, Juan; Vepachedu, Venkata; Cho, Sung Hyun; ...

    2016-05-23

    Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsDmore » in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 angstrom for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into β-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. Furthermore, the results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation

  12. Photobiological hydrogen production and carbon dioxide sequestration

    NASA Astrophysics Data System (ADS)

    Berberoglu, Halil

    Photobiological hydrogen production is an alternative to thermochemical and electrolytic technologies with the advantage of carbon dioxide sequestration. However, it suffers from low solar to hydrogen energy conversion efficiency due to limited light transfer, mass transfer, and nutrient medium composition. The present study aims at addressing these limitations and can be divided in three parts: (1) experimental measurements of the radiation characteristics of hydrogen producing and carbon dioxide consuming microorganisms, (2) solar radiation transfer modeling and simulation in photobioreactors, and (3) parametric experiments of photobiological hydrogen production and carbon dioxide sequestration. First, solar radiation transfer in photobioreactors containing microorganisms and bubbles was modeled using the radiative transport equation (RTE) and solved using the modified method of characteristics. The study concluded that Beer-Lambert's law gives inaccurate results and anisotropic scattering must be accounted for to predict the local irradiance inside a photobioreactor. The need for accurate measurement of the complete set of radiation characteristics of microorganisms was established. Then, experimental setup and analysis methods for measuring the complete set of radiation characteristics of microorganisms have been developed and successfully validated experimentally. A database of the radiation characteristics of representative microorganisms have been created including the cyanobacteria Anabaena variabilis, the purple non-sulfur bacteria Rhodobacter sphaeroides and the green algae Chlamydomonas reinhardtii along with its three genetically engineered strains. This enabled, for the first time, quantitative assessment of the effect of genetic engineering on the radiation characteristics of microorganisms. In addition, a parametric experimental study has been performed to model the growth, CO2 consumption, and H 2 production of Anabaena variabilis as functions of

  13. Stigmatellin Probes the Electrostatic Potential in the QB Site of the Photosynthetic Reaction Center

    SciTech Connect

    Gerencsér, László; Boros, Bogáta; Derrien, Valerie

    2015-01-01

    The electrostatic potential in the secondary quinone (QB) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to QB. It is controlled by the ionization states of residues in a strongly interacting cluster around the QB site. Reduction of the QB induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the QB binding pocket. It binds to themore » QB site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the QB pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the QB site was modulated by a single mutation, L213Asp/Ala, or double mutations, L213Asp-L212Glu/Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (DpK > 3.5) decreased dramatically to (DpK > 0.75) in the RC of the compensatory mutant (AAþM44Asn/AAþM44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the QB site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay.« less

  14. Modulation of the Lytic Activity of the Dedicated Autolysin for Flagellum Formation SltF by Flagellar Rod Proteins FlgB and FlgF

    PubMed Central

    Herlihey, Francesca A.; Osorio-Valeriano, Manuel; Dreyfus, Georges

    2016-01-01

    ABSTRACT SltF was identified previously as an autolysin required for the assembly of flagella in the alphaproteobacteria, but the nature of its peptidoglycan lytic activity remained unknown. Sequence alignment analyses suggest that it could function as either a muramidase, lytic transglycosylase, or β-N-acetylglucosaminidase. Recombinant SltF from Rhodobacter sphaeroides was purified to apparent homogeneity, and it was demonstrated to function as a lytic transglycosylase based on enzymatic assays involving mass spectrometric analyses. Circular dichroism (CD) analysis determined that it is composed of 83.4% α-structure and 1.48% β-structure and thus is similar to family 1A lytic transglycosylases. However, alignment of apparent SltF homologs identified in the genome database defined a new subfamily of the family 1 lytic transglycosylases. SltF was demonstrated to be endo-acting, cleaving within chains of peptidoglycan, with optimal activity at pH 7.0. Its activity is modulated by two flagellar rod proteins, FlgB and FlgF: FlgB both stabilizes and stimulates SltF activity, while FlgF inhibits it. Invariant Glu57 was confirmed as the sole catalytic acid/base residue of SltF. IMPORTANCE The bacterial flagellum is comprised of a basal body, hook, and helical filament, which are connected by a rod structure. With a diameter of approximately 4 nm, the rod is larger than the estimated pore size within the peptidoglycan sacculus, and hence its insertion requires the localized and controlled lysis of this essential cell wall component. In many beta- and gammaproteobacteria, this lysis is catalyzed by the β-N-acetylglucosaminidase domain of FlgJ. However, FlgJ of the alphaproteobacteria lacks this activity and instead it recruits a separate enzyme, SltF, for this purpose. In this study, we demonstrate that SltF functions as a newly identified class of lytic transglycosylases and that its autolytic activity is uniquely modulated by two rod proteins, FlgB and FlgF. PMID

  15. Structure of the cellulose synthase complex of Gluconacetobacter hansenii at 23.4 Å resolution

    SciTech Connect

    Du, Juan; Vepachedu, Venkata; Cho, Sung Hyun

    Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsDmore » in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 angstrom for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into β-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. Furthermore, the results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation

  16. Metabolic engineering of a tyrosine-overproducing yeast platform using targeted metabolomics.

    PubMed

    Gold, Nicholas D; Gowen, Christopher M; Lussier, Francois-Xavier; Cautha, Sarat C; Mahadevan, Radhakrishnan; Martin, Vincent J J

    2015-05-28

    L-tyrosine is a common precursor for a wide range of valuable secondary metabolites, including benzylisoquinoline alkaloids (BIAs) and many polyketides. An industrially tractable yeast strain optimized for production of L-tyrosine could serve as a platform for the development of BIA and polyketide cell factories. This study applied a targeted metabolomics approach to evaluate metabolic engineering strategies to increase the availability of intracellular L-tyrosine in the yeast Saccharomyces cerevisiae CEN.PK. Our engineering strategies combined localized pathway engineering with global engineering of central metabolism, facilitated by genome-scale steady-state modelling. Addition of a tyrosine feedback resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase Aro4 from S. cerevisiae was combined with overexpression of either a tyrosine feedback resistant yeast chorismate mutase Aro7, the native pentafunctional arom protein Aro1, native prephenate dehydrogenase Tyr1 or cyclohexadienyl dehydrogenase TyrC from Zymomonas mobilis. Loss of aromatic carbon was limited by eliminating phenylpyruvate decarboxylase Aro10. The TAL gene from Rhodobacter sphaeroides was used to produce coumarate as a simple test case of a heterologous by-product of tyrosine. Additionally, multiple strategies for engineering global metabolism to promote tyrosine production were evaluated using metabolic modelling. The T21E mutant of pyruvate kinase Cdc19 was hypothesized to slow the conversion of phosphoenolpyruvate to pyruvate and accumulate the former as precursor to the shikimate pathway. The ZWF1 gene coding for glucose-6-phosphate dehydrogenase was deleted to create an NADPH deficiency designed to force the cell to couple its growth to tyrosine production via overexpressed NADP(+)-dependent prephenate dehydrogenase Tyr1. Our engineered Zwf1(-) strain expressing TYRC ARO4(FBR) and grown in the presence of methionine achieved an intracellular L-tyrosine accumulation up to 520

  17. Structural Characterization and Function Determination of a Nonspecific Carboxylate Esterase from the Amidohydrolase Superfamily with a Promiscuous Ability To Hydrolyze Methylphosphonate Esters

    PubMed Central

    2015-01-01

    The uncharacterized protein Rsp3690 from Rhodobacter sphaeroides is a member of the amidohydrolase superfamily of enzymes. In this investigation the gene for Rsp3690 was expressed in Escherichia coli and purified to homogeneity, and the three-dimensional structure was determined to a resolution of 1.8 Å. The protein folds as a distorted (β/α)8-barrel, and the subunits associate as a homotetramer. The active site is localized to the C-terminal end of the β-barrel and is highlighted by the formation of a binuclear metal center with two manganese ions that are bridged by Glu-175 and hydroxide. The remaining ligands to the metal center include His-32, His-34, His-207, His-236, and Asp-302. Rsp3690 was shown to catalyze the hydrolysis of a wide variety of carboxylate esters, in addition to organophosphate and organophosphonate esters. The best carboxylate ester substrates identified for Rsp3690 included 2-naphthyl acetate (kcat/Km = 1.0 × 105 M–1 s–1), 2-naphthyl propionate (kcat/Km = 1.5 × 105 M–1 s–1), 1-naphthyl acetate (kcat/Km = 7.5 × 103 M–1 s–1), 4-methylumbelliferyl acetate (kcat/Km = 2.7 × 103 M–1 s–1), 4-nitrophenyl acetate (kcat/Km = 2.3 × 105 M–1 s–1), and 4-nitrophenyl butyrate (kcat/Km = 8.8 × 105 M–1 s–1). The best organophosphonate ester substrates included ethyl 4-nitrophenyl methylphosphonate (kcat/Km = 3.8 × 105 M–1 s–1) and isobutyl 4-nitrophenyl methylphosphonate (kcat/Km = 1.1 × 104 M–1 s–1). The (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate was hydrolyzed 10 times faster than the less toxic (RP)-enantiomer. The high inherent catalytic activity of Rsp3690 for the hydrolysis of the toxic enantiomer of methylphosphonate esters make this enzyme an attractive target for directed evolution investigations. PMID:24832101

  18. Cell biology and molecular basis of denitrification.

    PubMed Central

    Zumft, W G

    1997-01-01

    Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary

  19. Hydrogen bonding and spin density distribution in the QB semiquinone of bacterial reaction centers and comparison with the QA site

    PubMed Central

    Martin, Erik; Samoilova, Rimma I.; Narasimhulu, Kupala V.; Lin, Tzu-Jen; O’Malley, Patrick J.; Wraight, Colin A.; Dikanov, Sergei A.

    2011-01-01

    In the photosynthetic reaction center from Rhodobacter sphaeroides, the primary (QA) and secondary (QB) electron acceptors are both ubiquinone-10, but with very different properties and functions. To investigate the protein environment that imparts these functional differences, we have applied X-band HYSCORE, a 2D pulsed EPR technique, to characterize the exchangeable protons around the semiquinone (SQ) in the QA and QB sites, using samples of 15N-labeled reaction centers, with the native high spin Fe2+ exchanged for diamagnetic Zn2+, prepared in 1H2O and 2H2O solvent. The powder HYSCORE method is first validated against the orientation-selected Q-band ENDOR study of the QA SQ by Flores et al. (Biophys. J. 2007, 92, 671–682), with good agreement for two exchangeable protons with anisotropic hyperfine tensor components, T, both in the range 4.6–5.4 MHz. HYSCORE was then applied to the QB SQ where we found proton lines corresponding to T~5.2, 3.7 MHz and T~1.9 MHz. Density functional-based quantum mechanics/molecular mechanics (QM/MM) calculations, employing a model of the QB site, were used to assign the observed couplings to specific hydrogen bonding interactions with the QB SQ. These calculations allow us to assign the T=5.2 MHz proton to the His-L190 NδH…O4 (carbonyl) hydrogen bonding interaction. The T =3.7 MHz spectral feature most likely results from hydrogen bonding interactions of O1 (carbonyl) with both Gly-L225 peptide NH and Ser-L223 hydroxyl OH, which possess calculated couplings very close to this value. The smaller 1.9 MHz coupling is assigned to a weakly bound peptide NH proton of Ile-L224. The calculations performed with this structural model of the QB site show less asymmetric distribution of unpaired spin density over the SQ than seen for the QA site, consistent with available experimental data for 13C and 17O carbonyl hyperfine couplings. The implications of these interactions for QB function and comparisons with the QA site are discussed

  20. Whole genome sequence analyses revealed that strains of “Candidatus Liberibacter asiaticus” recently found in two California locations were different

    USDA-ARS?s Scientific Manuscript database

    ‘Candidatus Liberibacter asiaticus’ (CLas), an a-proteobacterium, is associated with citrus Huanglongbing (HLB; yellow shoot disease). In California, CLas was first detected in residential neighborhoods of Hacienda Heights (HH) in 2012 and San Gabriel (SG) in 2015. Although all infected trees were d...

  1. Two “Candidatus Liberibacter asiaticus” strains recently found in California harbor different prophages

    USDA-ARS?s Scientific Manuscript database

    “Candidatus Liberibacter asiaticus” (CLas), an a-proteobacterium, is associated with citrus Huanglongbing (HLB; yellow shoot disease). In California, CLas was first detected in the residential neighborhoods in Los Angeles County of Hacienda Heights (HH) in 2012 and in San Gabriel (SG) in 2015. Altho...

  2. A single prophage carrying a CRISPR/cas system in ‘Candidatus Liberibacter asiaticus’ strain A4 from Guangdong, China

    USDA-ARS?s Scientific Manuscript database

    “Candidatus Liberibacter asiaticus” (CLas) is an unculturable a-proteobacterium associated with citrus huanglongbing (HLB, yellow shoot disease), a highly destructive disease that affects citrus production worldwide. HLB was observed in Guangdong Province of China over a hundred years ago and remain...

  3. A New (Type 3) Prophage of “Candidatus Liberibacter asiaticus” in China

    USDA-ARS?s Scientific Manuscript database

    Prophages are important genetic entities of “Candidatus Liberibacter asiaticus” (CLas), a non-culturable a-proteobacterium associated with citrus Huanglongbing (HLB). Two CLas prophages have been described, SC1 (NC_019549.1, Type 1) and SC2 (NC_019550.1, Type 2). To explore the prophage repertoire, ...

  4. Membrane development in purple photosynthetic bacteria in response to alterations in light intensity and oxygen tension.

    PubMed

    Niederman, Robert A

    2013-10-01

    Studies on membrane development in purple bacteria during adaptation to alterations in light intensity and oxygen tension are reviewed. Anoxygenic phototrophic such as the purple α-proteobacterium Rhodobacter sphaeroides have served as simple, dynamic, and experimentally accessible model organisms for studies of the photosynthetic apparatus. A major landmark in photosynthesis research, which dramatically illustrates this point, was provided by the determination of the X-ray structure of the reaction center (RC) in Blastochloris viridis (Deisenhofer and Michel, EMBO J 8:2149-2170, 1989), once it was realized that this represented the general structure for the photosystem II RC present in all oxygenic phototrophs. This seminal advance, together with a considerable body of subsequent research on the light-harvesting (LH) and electron transfer components of the photosynthetic apparatus has provided a firm basis for the current understanding of how phototrophs acclimate to alterations in light intensity and quality. Oxygenic phototrophs adapt to these changes by extensive thylakoid membrane remodeling, which results in a dramatic supramolecular reordering to assure that an appropriate flow of quinone redox species occurs within the membrane bilayer for efficient and rapid electron transfer. Despite the high level of photosynthetic unit organization in Rba. sphaeroides as observed by atomic force microscopy (AFM), fluorescence induction/relaxation measurements have demonstrated that the addition of the peripheral LH2 antenna complex in cells adapting to low-intensity illumination results in a slowing of the rate of electron transfer turnover by the RC of up to an order of magnitude. This is ascribed to constraints in quinone redox species diffusion between the RC and cytochrome bc1 complexes arising from the increased packing density as the intracytoplasmic membrane (ICM) bilayer becomes crowded with LH2 rings. In addition to downshifts in light intensity as a paradigm

  5. SciTech Connect

    James C. Liao

    CBB reductive pentose phosphate pathway, whose key enzyme is ribulose 1,5-biphosphate carboxylase/oxygenase (RubisCO). In addition to providing virtually all cellular carbon during autotrophic metabolism, RubisCO-mediated CO{sub 2} assimilation is also very important for nonsulfur purple photosynthetic bacteria under photoheterotrophic growth conditions since CO{sub 2} becomes the major electron sink under these conditions. In this work, Ensemble Modeling (EM) was developed to examine the behavior of CBB-compromised RubisCO knockout mutant strains of the nonsulfur purple photosynthetic bacterium Rhodobacter sphaeroides. Mathematical models of metabolism can be a great aid in studying the effects of large perturbations to the system, such as the inactivation of RubisCO. Due to the complex and highly-interconnected nature of these networks, it is not a trivial process to understand what the effect of perturbations to the metabolic network will be, or vice versa, what enzymatic perturbations are necessary to yield a desired effect. Flux distribution is controlled by multiple enzymes in the network, often indirectly linked to the pathways of interest. Further, depending on the state of the cell and the environmental conditions, the effect of a perturbation may center around how it effects the carbon flow in the network, the balancing of cofactors, or both. Thus, it is desirable to develop mathematical models to describe, understand, and predict network behavior. Through the development of such models, one may gain the ability to generate a set of testable hypotheses for system behavior.« less

  6. Bio-Photoelectrochemical Solar Cells Incorporating Reaction Center and Reaction Center Plus Light Harvesting Complexes

    NASA Astrophysics Data System (ADS)

    Yaghoubi, Houman

    onto Au electrodes via surface exposed cysteine residues. This resulted in photocurrent densities as large as ~600 nA cm-2 while still the incident photon to generated electron quantum efficiency was as low as %3 x 10-4. 2- The second approach is to immobilize wild type RCs of Rhodobacter sphaeroides on the surface of a Au underlying electrode using self-assembled monolayers of carboxylic acid terminated oligomers and cytochrome c charge mediating layers, with a preferential orientation from the primary electron donor site. This approach resulted in EQE of up to 0.06%, which showed 200 times efficiency improvement comparing to the first approach. In the third approach, instead of isolated protein complexes, RCs plus light harvesting (LH) complexes were employed for a better photon absorption. Direct attachment of RC-LH1 complexes on Au working electrodes, resulted in 0.21% EQE which showed 3.5 times efficiency improvement over the second approach (700 times higher than the first approach). The main impact of this work is the harnessing of biological RCs for efficient energy harvesting in man-made structures. Specifically, the results in this work will advance the application of RCs in devices for energy harvesting and will enable a better understanding of bio and nanomaterial interfaces, thereby advancing the application of biological materials in electronic devices. At the end, this work offers general guidelines that can serve to improve the performance of bio-hybrid solar cells.

  7. Terminal steps of bacteriochlorophyll a phytol formation in purple photosynthetic bacteria

    SciTech Connect

    Shioi, Y.; Sasa, T.

    1984-04-01

    Four chemically different bacteriochlorophylls (Bchls) a esterified with geranylgeraniol, dihydrogeranyl-geraniol, tetrahydrogeraniol, and phytol have been detected by high-pressure liquid chromatography in cell extracts from Rhodopseudomonas sphaeroides and Chromatium vinosum. Bchl a containing phytol is the principal component, and the other three Bchls a comprise about 4% of the total Bchls a in stationary-phase cells of R. sphaeroides and C. vinosum. The high levels of the minor pigments occur in the beginning of Bchl a phytol formation, indicating that they are not degradation products, but intermediates of Bchl a phytol formation.

  8. A Study of the Interaction of Millimeter Wave Fields with Biological Systems.

    DTIC Science & Technology

    1984-07-01

    structurally complex proteins . The third issue is the relevance of the parameters used in previous modeling efforts. The strength of the exciton-phonon...modes of proteins in the millimeter and submillimeter regions of the electromagnetic spectrum. Specifically: o " Four separate groups of frequencies...Rhodopseudomonas Sphaeroides (4). In industrial or military environments a significant number of personnel are exposed to electromagnetic fields

  9. Antiphospholipid antibody-induced miR-146a-3p drives trophoblast interleukin-8 secretion through activation of Toll-like receptor 8.

    PubMed

    Gysler, Stefan M; Mulla, Melissa J; Guerra, Marta; Brosens, Jan J; Salmon, Jane E; Chamley, Lawrence W; Abrahams, Vikki M

    2016-07-01

    What is the role of microRNAs (miRs) in antiphospholipid antibody (aPL)-induced trophoblast inflammation? aPL-induced up-regulation of trophoblast miR-146a-3p is mediated by Toll-like receptor 4 (TLR4), and miR-146a-3p in turn drives the cells to secrete interleukin (IL)-8 by activating the RNA sensor, TLR8. Obstetric antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating aPL and an increased risk of pregnancy complications. We previously showed that aPL recognizing beta2 glycoprotein I (β2GPI) elicit human first trimester trophoblast secretion of IL-8 by activating TLR4. Since some miRs control TLR responses, their regulation in trophoblast cells by aPL and functional role in the aPL-mediated inflammatory response was investigated. miRs can be released from cells via exosomes, and therefore, miR exosome expression was also examined. A panel of miRs was selected based on their involvement with TLR signaling: miR-9; miR-146a-5p and its isomiR, miR-146a-3p; miR-155, miR-210; and Let-7c. Since certain miRs can activate the RNA sensor, TLR8, this was also investigated. For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR8 was studied. HTR8 cells transfected to express a TLR8 dominant negative (DN) were also used. Plasma was evaluated from pregnant women who have aPL, either with or without systemic lupus erythematous (SLE) (n = 39); SLE patients without aPL (n = 30); and healthy pregnant controls (n = 20). Trophoblast HTR8 wildtype and TLR8-DN cells were incubated with or without aPL (mouse anti-human β2GPI mAb) for 48-72 h. HTR8 cells were also treated with or without aPL in the presence and the absence of a TLR4 antagonist (lipopolysaccharide from Rhodobacter sphaeroides; LPS-RS), specific miR inhibitors or specific miR mimics. miR expression levels in trophoblast cells, trophoblast-derived exosomes and exosomes isolated from patient plasma were measured by qPCR. Trophoblast IL-8 secretion was

  10. Hemifluorinated maltose-neopentyl glycol (HF-MNG) amphiphiles for membrane protein stabilisation.

    PubMed

    Cho, Kyung Ho; Byrne, Bernadette; Chae, Pil Seok

    2013-03-04

    SOAP OPERA: Fluorinated amphiphile F4-MNG confers greater stability on Rhodobacter capsulatus superassembly relative to conventional detergents and nonfluorinated MNGs. Such amphiphiles are attractive as tools for membrane science because of their ease of preparation and structure variation. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Urinary tract infection caused by Chromobacterium violaceum.

    PubMed

    Pant, Narayan Dutt; Sharma, Manisha

    2015-01-01

    Chromobacterium violaceum, a proteobacterium, is a facultative anaerobe, which is generally present as the normal flora of water and soil in tropical and subtropical regions. The infection due to Chromobacterium violaceum is rare but mostly fatal. It is responsible for causing fatal cases of septicemia, visceral abscesses, skin and soft tissue infections, meningitis, diarrhea, and rarely urinary tract infection. The bacteria has high propensity to spread causing sepsis. Delayed proper treatment due to limited awareness related to the C. violaceum infection is responsible for the high mortality rate. Here, we describe a rare case of urinary tract infection by C. violaceum in a chronic kidney disease patient, which was managed with timely proper antimicrobial therapy as per the culture sensitivity report.

  12. The ring of life provides evidence for a genome fusion origin of eukaryotes.

    PubMed

    Rivera, Maria C; Lake, James A

    2004-09-09

    Genomes hold within them the record of the evolution of life on Earth. But genome fusions and horizontal gene transfer seem to have obscured sufficiently the gene sequence record such that it is difficult to reconstruct the phylogenetic tree of life. Here we determine the general outline of the tree using complete genome data from representative prokaryotes and eukaryotes and a new genome analysis method that makes it possible to reconstruct ancient genome fusions and phylogenetic trees. Our analyses indicate that the eukaryotic genome resulted from a fusion of two diverse prokaryotic genomes, and therefore at the deepest levels linking prokaryotes and eukaryotes, the tree of life is actually a ring of life. One fusion partner branches from deep within an ancient photosynthetic clade, and the other is related to the archaeal prokaryotes. The eubacterial organism is either a proteobacterium, or a member of a larger photosynthetic clade that includes the Cyanobacteria and the Proteobacteria.

  13. Visualizing tributyltin (TBT) in bacterial aggregates by specific rhodamine-based fluorescent probes.

    PubMed

    Jin, Xilang; Hao, Likai; She, Mengyao; Obst, Martin; Kappler, Andreas; Yin, Bing; Liu, Ping; Li, Jianli; Wang, Lanying; Shi, Zhen

    2015-01-01

    Here we present the first examples of fluorescent and colorimetric probes for microscopic TBT imaging. The fluorescent probes are highly selective and sensitive to TBT and have successfully been applied for imaging of TBT in bacterial Rhodobacter ferrooxidans sp. strain SW2 cell-EPS-mineral aggregates and in cell suspensions of the marine cyanobacterium Synechococcus PCC 7002 by using confocal laser scanning microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Photosynthetic Energy Transduction Publications | Bioenergy | NREL

    Science.gov Websites

    , Microbial Biotechnol. Image of two green spheres: one labeled Growth and the other labeled Catalysis. From Rhodobacter capsulatus, Int. J. Hydrogen Energy Image of two charts showing H2 sensitivity of the R NC74A, Planta Image of a diagrammatic view of current algal phylogeny illustrating common (e.g., Algae

  15. Surface-attached and suspended bacterial community structure as affected by C/N ratios: relationship between bacteria and fish production.

    PubMed

    Yu, Ermeng; Xie, Jun; Wang, Jinlin; Ako, Harry; Wang, Guangjun; Chen, Zhanghe; Liu, Yongfeng

    2016-07-01

    Bacteria play crucial roles in the combined system of substrate addition and C/N control, which has been demonstrated to improve aquaculture production. However, the complexity of surface-attached bacteria on substrates and suspended bacteria in the water column hamper further application of this system. This study firstly applied this combined system into the culture of grass carp, and then explored the relationship between microbial complexes from surface-attached and suspended bacteria in this system and the production of grass carp. In addition, this study investigated bacterial community structures as affected by four C/N ratios using Illumina sequencing technology. The results demonstrated that the weight gain rate and specific growth rate of grass carp in the CN20 group (C/N ratio 20:1) were the highest (P < 0.05), and dietary supplementation of the microbial complex had positive effects on the growth of grass carp (P < 0.05). Sequencing data revealed that, (1) the proportions of Verrucomicrobiae and Rhodobacter (surface-attached), sediminibacterium (suspended), and emticicia (surface-attached and suspended) were much higher in the CN20 group compared with those in the other groups (P < 0.05); (2) Rhodobacter, Flavobacterium, Acinetobacter, Pseudomonas, Planctomyces, and Cloacibacterium might be important for the microbial colonization on substrates; (3) as the C/N ratio increased, proportions of Hydrogenophaga (surface-attached and suspended), Zoogloea, and Flectobacillus (suspended) increased, but proportions of Bacillus, Clavibacter, and Cellvibro (surface-attached and suspended) decreased. In summary, a combined system of substrate addition and C/N control increased the production of grass carp, and Verrucomicrobiae and Rhodobacter in the surface-attached bacterial community were potential probiotic bacteria that contributed to the enhanced growth of grass carp.

  16. Characterization of a Major Cluster of nif, fix, and Associated Genes in a Sugarcane Endophyte, Acetobacter diazotrophicus

    PubMed Central

    Lee, Sunhee; Reth, Alexander; Meletzus, Dietmar; Sevilla, Myrna; Kennedy, Christina

    2000-01-01

    A major 30.5-kb cluster of nif and associated genes of Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus), a nitrogen-fixing endophyte of sugarcane, was sequenced and analyzed. This cluster represents the largest assembly of contiguous nif-fix and associated genes so far characterized in any diazotrophic bacterial species. Northern blots and promoter sequence analysis indicated that the genes are organized into eight transcriptional units. The overall arrangement of genes is most like that of the nif-fix cluster in Azospirillum brasilense, while the individual gene products are more similar to those in species of Rhizobiaceae or in Rhodobacter capsulatus. PMID:11092875

  17. Sequence of the bchG gene from Chloroflexus aurantiacus: relationship between chlorophyll synthase and other polyprenyltransferases

    NASA Technical Reports Server (NTRS)

    Lopez, J. C.; Ryan, S.; Blankenship, R. E.

    1996-01-01

    The sequence of the Chloroflexus aurantiacus open reading frame thought to be the C. aurantiacus homolog of the Rhodobacter capsulatus bchG gene is reported. The BchG gene product catalyzes esterification of bacteriochlorophyllide a by geranylgeraniol-PPi during bacteriochlorophyll a biosynthesis. Homologs from Arabidopsis thaliana, Synechocystis sp. strain PCC6803, and C. aurantiacus were identified in database searches. Profile analysis identified three related polyprenyltransferase enzymes which attach an aliphatic alcohol PPi to an aromatic substrate. This suggests a broader relationship between chlorophyll synthases and other polyprenyltransferases.

  18. The genome of obligately intracellular Ehrlichia canis revealsthemes of complex membrane structure and immune evasion strategies

    SciTech Connect

    Mavromatis, K.; Kuyler Doyle, C.; Lykidis, A.

    2005-09-01

    Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, a-proteobacterium is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, and 17 putative pseudogenes, and a substantial proportion of non-coding sequence (27 percent). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences, and a unique serine-threonine bias associated with the potential for O-glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein familiesmore » associated with immune evasion were identified, one of which contains poly G:C tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Proteins associated with pathogen-host interactions were identified including a small group of proteins (12) with tandem repeats and another with eukaryotic-like ankyrin domains (7).« less

  19. Microbial diversity in Los Azufres geothermal field (Michoacán, Mexico) and isolation of representative sulfate and sulfur reducers.

    PubMed

    Brito, Elcia M S; Villegas-Negrete, Norberto; Sotelo-González, Irene A; Caretta, César A; Goñi-Urriza, Marisol; Gassie, Claire; Hakil, Florence; Colin, Yannick; Duran, Robert; Gutiérrez-Corona, Felix; Piñón-Castillo, Hilda A; Cuevas-Rodríguez, Germán; Malm, Olaf; Torres, João P M; Fahy, Anne; Reyna-López, Georgina E; Guyoneaud, Rémy

    2014-03-01

    Los Azufres spa consists of a hydrothermal spring system in the Mexican Volcanic Axis. Five samples (two microbial mats, two mud pools and one cenote water), characterized by high acidity (pH between 1 and 3) and temperatures varying from 27 to 87 °C, were investigated for their microbial diversity by Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and 16S rRNA gene library analyses. These data are the first to describe microbial diversity from Los Azufres geothermal belt. The data obtained from both approaches suggested a low bacterial diversity in all five samples. Despite their proximity, the sampling points differed by their physico-chemical conditions (mainly temperature and matrix type) and thus exhibited different dominant bacterial populations: anoxygenic phototrophs related to the genus Rhodobacter in the biomats, colorless sulfur oxidizers Acidithiobacillus sp. in the warm mud and water samples, and Lyzobacter sp.-related populations in the hot mud sample (87 °C). Molecular data also allowed the detection of sulfate and sulfur reducers related to Thermodesulfobium and Desulfurella genera. Several strains affiliated to both genera were enriched or isolated from the mesophilic mud sample. A feature common to all samples was the dominance of bacteria involved in sulfur and iron biogeochemical cycles (Rhodobacter, Acidithiobacillus, Thiomonas, Desulfurella and Thermodesulfobium genera).

  20. Differing growth responses of major phylogenetic groups of marine bacteria to natural phytoplankton blooms in the western North Pacific Ocean.

    PubMed

    Tada, Yuya; Taniguchi, Akito; Nagao, Ippei; Miki, Takeshi; Uematsu, Mitsuo; Tsuda, Atsushi; Hamasaki, Koji

    2011-06-01

    Growth and productivity of phytoplankton substantially change organic matter characteristics, which affect bacterial abundance, productivity, and community structure in aquatic ecosystems. We analyzed bacterial community structures and measured activities inside and outside phytoplankton blooms in the western North Pacific Ocean by using bromodeoxyuridine immunocytochemistry and fluorescence in situ hybridization (BIC-FISH). Roseobacter/Rhodobacter, SAR11, Betaproteobacteria, Alteromonas, SAR86, and Bacteroidetes responded differently to changes in organic matter supply. Roseobacter/Rhodobacter bacteria remained widespread, active, and proliferating despite large fluctuations in organic matter and chlorophyll a (Chl-a) concentrations. The relative contribution of Bacteroidetes to total bacterial production was consistently high. Furthermore, we documented the unexpectedly large contribution of Alteromonas to total bacterial production in the bloom. Bacterial abundance, productivity, and growth potential (the proportion of growing cells in a population) were significantly correlated with Chl-a and particulate organic carbon concentrations. Canonical correspondence analysis showed that organic matter supply was critical for determining bacterial community structures. The growth potential of each bacterial group as a function of Chl-a concentration showed a bell-shaped distribution, indicating an optimal organic matter concentration to promote growth. The growth of Alteromonas and Betaproteobacteria was especially strongly correlated with organic matter supply. These data elucidate the distinctive ecological role of major bacterial taxa in organic matter cycling during open ocean phytoplankton blooms.

  1. Differing Growth Responses of Major Phylogenetic Groups of Marine Bacteria to Natural Phytoplankton Blooms in the Western North Pacific Ocean ▿ †

    PubMed Central

    Tada, Yuya; Taniguchi, Akito; Nagao, Ippei; Miki, Takeshi; Uematsu, Mitsuo; Tsuda, Atsushi; Hamasaki, Koji

    2011-01-01

    Growth and productivity of phytoplankton substantially change organic matter characteristics, which affect bacterial abundance, productivity, and community structure in aquatic ecosystems. We analyzed bacterial community structures and measured activities inside and outside phytoplankton blooms in the western North Pacific Ocean by using bromodeoxyuridine immunocytochemistry and fluorescence in situ hybridization (BIC-FISH). Roseobacter/Rhodobacter, SAR11, Betaproteobacteria, Alteromonas, SAR86, and Bacteroidetes responded differently to changes in organic matter supply. Roseobacter/Rhodobacter bacteria remained widespread, active, and proliferating despite large fluctuations in organic matter and chlorophyll a (Chl-a) concentrations. The relative contribution of Bacteroidetes to total bacterial production was consistently high. Furthermore, we documented the unexpectedly large contribution of Alteromonas to total bacterial production in the bloom. Bacterial abundance, productivity, and growth potential (the proportion of growing cells in a population) were significantly correlated with Chl-a and particulate organic carbon concentrations. Canonical correspondence analysis showed that organic matter supply was critical for determining bacterial community structures. The growth potential of each bacterial group as a function of Chl-a concentration showed a bell-shaped distribution, indicating an optimal organic matter concentration to promote growth. The growth of Alteromonas and Betaproteobacteria was especially strongly correlated with organic matter supply. These data elucidate the distinctive ecological role of major bacterial taxa in organic matter cycling during open ocean phytoplankton blooms. PMID:21515719

  2. Final technical report for award NO. DE-FG02-95ER20206

    SciTech Connect

    James P. Shapleigh

    2010-02-23

    ABSTRACT Initial work focused on the regulation of nitrite reductase, the defining reaction of denitrification as well as nitric oxide (NO) reductase. Expression of the genes encoding both proteins was controlled by NnrR. This regulator was shown to be responsive to NO. More recent work has shown NnrR function is also likely inhibited by oxygen. Therefore, it is this protein that sets the oxygen level at which nitrate respiration takes over from aerobic respiration. The gene encoding NO reductase appears to only require NnrR for expression. Expression of the gene encoding nitrite reductase is more complex. In addition to NnrR,more » a two component sensor regulator complex termed PrrA and PrrB is also required for expression. These proteins are global regulators and serve to link denitrification with other bioenergetic processes in the cell. They also provide an additional layer of oxygen dependent regulation. The sequencing of the R. sphaeroides 2.4.3 genome allowed us to identify several other genes regulated by NnrR. Surprisingly, most of the genes were not essential for denitrification. Their high level of conservation in related denitrifiers suggests they do provide a selectable benefit to the bacterium, however. We also examined the role of nitrate reductase in contributing to denitrification in R. sphaeroides. Strain 2.4.3 is unusual in having two distinct, but related clusters of genes encoding nitrate reductase. One of these genes clusters is expressed under high oxygen conditions but is repressed, likely by PrrB-PrrA, under low oxygen conditions. The other cluster is expressed only under low oxygen conditions. This cluster expresses the nitrate reductase used during denitrification. The high oxygen expressed cluster encodes a protein used for redox homeostasis. Surprisingly, both clusters are fully expressed even in the absence of nitrate. During the course of this work we found that the type strain of R. sphaeroides, 2.4.1, is a partial denitrifier

  3. Seeded Growth Route to Noble Calcium Carbonate Nanocrystal.

    PubMed

    Islam, Aminul; Teo, Siow Hwa; Rahman, M Aminur; Taufiq-Yap, Yun Hin

    2015-01-01

    A solution-phase route has been considered as the most promising route to synthesize noble nanostructures. A majority of their synthesis approaches of calcium carbonate (CaCO3) are based on either using fungi or the CO2 bubbling methods. Here, we approached the preparation of nano-precipitated calcium carbonate single crystal from salmacis sphaeroides in the presence of zwitterionic or cationic biosurfactants without external source of CO2. The calcium carbonate crystals were rhombohedron structure and regularly shaped with side dimension ranging from 33-41 nm. The high degree of morphological control of CaCO3 nanocrystals suggested that surfactants are capable of strongly interacting with the CaCO3 surface and control the nucleation and growth direction of calcium carbonate nanocrystals. Finally, the mechanism of formation of nanocrystals in light of proposed routes was also discussed.

  4. Seeded Growth Route to Noble Calcium Carbonate Nanocrystal

    PubMed Central

    Islam, Aminul; Teo, Siow Hwa; Rahman, M. Aminur; Taufiq-Yap, Yun Hin

    2015-01-01

    A solution-phase route has been considered as the most promising route to synthesize noble nanostructures. A majority of their synthesis approaches of calcium carbonate (CaCO3) are based on either using fungi or the CO2 bubbling methods. Here, we approached the preparation of nano-precipitated calcium carbonate single crystal from salmacis sphaeroides in the presence of zwitterionic or cationic biosurfactants without external source of CO2. The calcium carbonate crystals were rhombohedron structure and regularly shaped with side dimension ranging from 33–41 nm. The high degree of morphological control of CaCO3 nanocrystals suggested that surfactants are capable of strongly interacting with the CaCO3 surface and control the nucleation and growth direction of calcium carbonate nanocrystals. Finally, the mechanism of formation of nanocrystals in light of proposed routes was also discussed. PMID:26700479

  5. SciTech Connect

    Reppert, Mike; Kell, Adam; Pruitt, Thomas

    The vibrational spectral density is an important physical parameter needed to describe both linear and non-linear spectra of multi-chromophore systems such as photosynthetic complexes. Low-temperature techniques such as hole burning (HB) and fluorescence line narrowing are commonly used to extract the spectral density for a given electronic transition from experimental data. We report here that the lineshape function formula reported by Hayes et al. [J. Phys. Chem. 98, 7337 (1994)] in the mean-phonon approximation and frequently applied to analyzing HB data contains inconsistencies in notation, leading to essentially incorrect expressions in cases of moderate and strong electron-phonon (el-ph) coupling strengths.more » A corrected lineshape function L(ω) is given that retains the computational and intuitive advantages of the expression of Hayes et al. [J. Phys. Chem. 98, 7337 (1994)]. Although the corrected lineshape function could be used in modeling studies of various optical spectra, we suggest that it is better to calculate the lineshape function numerically, without introducing the mean-phonon approximation. New theoretical fits of the P870 and P960 absorption bands and frequency-dependent resonant HB spectra of Rb. sphaeroides and Rps. viridis reaction centers are provided as examples to demonstrate the importance of correct lineshape expressions. Comparison with the previously determined el-ph coupling parameters [Johnson et al., J. Phys. Chem. 94, 5849 (1990); Lyle et al., ibid. 97, 6924 (1993); Reddy et al., ibid. 97, 6934 (1993)] is also provided. The new fits lead to modified el-ph coupling strengths and different frequencies of the special pair marker mode, ω{sub sp}, for Rb. sphaeroides that could be used in the future for more advanced calculations of absorption and HB spectra obtained for various bacterial reaction centers.« less

  6. In Vitro Self-Assembly of the Light Harvesting Pigment-Protein LH2 Revealed by Ultrafast Spectroscopy and Electron Microscopy

    PubMed Central

    Schubert, Axel; Stenstam, Anna; Beenken, Wichard J. D.; Herek, Jennifer L.; Cogdell, Richard; Pullerits, Tõnu; Sundström, Villy

    2004-01-01

    Controlled ensemble formation of protein-surfactant systems provides a fundamental concept for the realization of nanoscale devices with self-organizing capability. In this context, spectroscopic monitoring of pigment-containing proteins yields detailed structural information. Here we have studied the association behavior of the bacterial light-harvesting protein LH2 from Rhodobacter spheroides in an n,n-dimethyldodecylamine-n-oxide/water environment. Time-resolved studies of the excitation annihilation yielded information about aggregate sizes and packing of the protein complexes therein. The results are compared to transmission electron microscopy images of instantaneously frozen samples. Our data indicate the manifestation of different phases, which are discussed with respect to the thermodynamic equilibrium in ternary protein-surfactant-water systems. Accordingly, by varying the concentration the formation of different types of aggregates can be controlled. Conditions for the appearance of isolated LH2 complexes are defined. PMID:15041674

  7. Guaranteeing a captive audience: coordinated regulation of gene transfer agent (GTA) production and recipient capability by cellular regulators.

    PubMed

    Westbye, Alexander B; Beatty, J Thomas; Lang, Andrew S

    2017-08-01

    Gene transfer agents (GTAs) are bacteriophage-like particles produced by many prokaryotes. Several members of the Alphaproteobacteria produce a class of genetically-related GTAs that is best studied in Rhodobacter capsulatus. DNA transfer by the R. capsulatus GTA (RcGTA) combines aspects of both transduction and natural transformation, as recipient cells require a natural transformation-like system to incorporate donated DNA. The genes involved in RcGTA production and recipient capability are located at multiple loci in the bacterial genome; however, a conserved phosphorelay containing the response regulator CtrA and a quorum sensing system regulate both RcGTA production and recipient capability. This review highlights recent discoveries in RcGTA biology, and focuses on the co-regulation of genes involved in RcGTA production and recipient capability. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Second-generation photosensitizers based on natural chlorines and bacteriochlorines

    NASA Astrophysics Data System (ADS)

    Mironov, Andrei F.

    1996-01-01

    New sensitizers for photodynamic therapy were synthesized on the base of biologically generated chlorins and bacteriochlorins. Derivatives of chlorophyll a and bacteriochlorophyll were prepared from the biomass of blue-green algae Spirulina Platensis and purple bacteria Rhodobacter Capsulatus, generated using specially designed photobioreactor. The strategy for chemical transformation of natural chlorophylls and bacteriochlorophyll has been discussed. Purpurin 18 and its dihydroanalogue bacteriopurpurin were chosen as the key intermediates. Modifications of peripheral substituents, such as introducing the new functional groups, hydrogenation of the B-pyrrolic ring, and insertion of amino acid residues gave the series of novel sensitizers, including water soluble chlorin p6 analogues, and derivatives with graded amphiphility for the studies of tumor accumulation in the malignant tissues.

  9. Synthesis and properties of new chlorin and bacteriochlorin photosensitizers

    NASA Astrophysics Data System (ADS)

    Mironov, Andrei F.

    1996-01-01

    A series of novel sensitizers, which absorb in the range of 660 - 820 nm, derived from natural occurring chlorophyll and bacteriochlorophyll was synthesized. Biomass of blue-green algae Spirulina platensis was used to prepare chlorophyll a derivatives, and biomass of purple bacteria Rhodobacter capsulatus was applied for preparation of bacteriochlorophyll a. The influence of different substituents on spectral characteristics and the amphipility of the sensitizer was investigated. The route for the synthesis of porphyrin macrocycle with the spacer that bears the isothiocyanate group capable for binding with proteins was proposed. Photophysical properties of chlorin p6, purpurin 18 and their esters in different solvents are investigated. Accumulation of two chlorins in the model Erlich tumor was studied.

  10. Tracking bio-hydrogen-mediated production of commodity chemicals from carbon dioxide and renewable electricity.

    PubMed

    Puig, Sebastià; Ganigué, Ramon; Batlle-Vilanova, Pau; Balaguer, M Dolors; Bañeras, Lluís; Colprim, Jesús

    2017-03-01

    This study reveals that reduction of carbon dioxide (CO 2 ) to commodity chemicals can be functionally compartmentalized in bioelectrochemical systems. In the present example, a syntrophic consortium composed by H 2 -producers (Rhodobacter sp.) in the biofilm is combined with carboxidotrophic Clostridium species, mainly found in the bulk liquid. The performance of these H 2 -mediated electricity-driven systems could be tracked by the activity of a biological H 2 sensory protein identified at cathode potentials between -0.2V and -0.3V vs SHE. This seems to point out that such signal is not strain specific, but could be detected in any organism containing hydrogenases. Thus, the findings of this work open the door to the development of a biosensor application or soft sensors for monitoring such systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants

    NASA Astrophysics Data System (ADS)

    Schulz, G. E.; Dreyer, M.; Klein, C.; Kreusch, A.; Mittl, P.; Mu¨ller, C. W.; Mu¨ller-Dieckmann, J.; Muller, Y. A.; Proba, K.; Schlauderer, G.; Spu¨rgin, P.; Stehle, T.; Weiss, M. S.

    1992-08-01

    Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8A˚resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0A˚resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.

  12. Different inhibitory potency of febuxostat towards mammalian and bacterial xanthine oxidoreductases: insight from molecular dynamics

    PubMed Central

    Kikuchi, Hiroto; Fujisaki, Hiroshi; Furuta, Tadaomi; Okamoto, Ken; Leimkühler, Silke; Nishino, Takeshi

    2012-01-01

    Febuxostat, a drug recently approved in the US, European Union and Japan for treatment of gout, inhibits xanthine oxidoreductase (XOR)-mediated generation of uric acid during purine catabolism. It inhibits bovine milk XOR with a Ki in the picomolar-order, but we found that it is a much weaker inhibitor of Rhodobacter capsulatus XOR, even though the substrate-binding pockets of mammalian and bacterial XOR are well-conserved as regards to catalytically important residues and three-dimensional structure, and both permit the inhibitor to be accommodated in the active site, as indicated by computational docking studies. To clarify the reason for the difference of inhibitory potency towards the two XORs, we performed molecular dynamics simulations. The results indicate that differences in mobility of hydrophobic residues that do not directly interact with the substrate account for the difference in inhibitory potency. PMID:22448318

  13. A novel directly coupled gradostat

    NASA Technical Reports Server (NTRS)

    Wimpenny, J. W.; Earnshaw, R. G.; Gest, H.; Hayes, J. M.; Favinger, J. L.

    1992-01-01

    The original bidirectional compound chemostat (gradostat) described by Lovitt and Wimpenny has been simplified by making a more compact apparatus in which chemical gradients are established by diffusion between adjacent culture chambers. The experimental model (diffusion coupled (DC) gradostat) consisted of five chambers whose contents could be agitated by turbines rotating in the horizontal plane on a common shaft. Two biological experiments were designed to reveal the value of the DC gradostat. A methylotroph (Methylophilus methylotrophus) grown in a methanol gradient showed expected changes in cell viability as a function of position in the five vessel array. Cells of two species of photosynthetic bacteria (Rhodobacter capsulata and Rhodopseudomonas marina/agilis) with different salt sensitivities could be mixed and subsequently separated by the DC gradostat operating with a NaCl gradient of 0-3% w/v.

  14. Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B

    PubMed Central

    Chénard, Caroline; Wirth, Jennifer F.

    2016-01-01

    ABSTRACT   Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages. PMID:27302758

  15. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

    PubMed

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F

    2015-08-18

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation.

  16. A Phylogenetic Analysis of the Globins in Fungi

    PubMed Central

    Hoogewijs, David; Dewilde, Sylvia; Vierstraete, Andy; Moens, Luc; Vinogradov, Serge N.

    2012-01-01

    Background All globins belong to one of three families: the F (flavohemoglobin) and S (sensor) families that exhibit the canonical 3/3 α-helical fold, and the T (truncated 3/3 fold) globins characterized by a shortened 2/2 α-helical fold. All eukaryote 3/3 hemoglobins are related to the bacterial single domain F globins. It is known that Fungi contain flavohemoglobins and single domain S globins. Our aims are to provide a census of fungal globins and to examine their relationships to bacterial globins. Results Examination of 165 genomes revealed that globins are present in >90% of Ascomycota and ∼60% of Basidiomycota genomes. The S globins occur in Blastocladiomycota and Chytridiomycota in addition to the phyla that have FHbs. Unexpectedly, group 1 T globins were found in one Blastocladiomycota and one Chytridiomycota genome. Phylogenetic analyses were carried out on the fungal globins, alone and aligned with representative bacterial globins. The Saccharomycetes and Sordariomycetes with two FHbs form two widely divergent clusters separated by the remaining fungal sequences. One of the Saccharomycete groups represents a new subfamily of FHbs, comprising a previously unknown N-terminal and a FHb missing the C-terminal moiety of its reductase domain. The two Saccharomycete groups also form two clusters in the presence of bacterial FHbs; the surrounding bacterial sequences are dominated by Proteobacteria and Bacilli (Firmicutes). The remaining fungal FHbs cluster with Proteobacteria and Actinobacteria. The Sgbs cluster separately from their bacterial counterparts, except for the intercalation of two Planctomycetes and a Proteobacterium between the Fungi incertae sedis and the Blastocladiomycota and Chytridiomycota. Conclusion Our results are compatible with a model of globin evolution put forward earlier, which proposed that eukaryote F, S and T globins originated via horizontal gene transfer of their bacterial counterparts to the eukaryote ancestor, resulting from

  17. Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B.

    PubMed

    Chénard, Caroline; Wirth, Jennifer F; Suttle, Curtis A

    2016-06-14

    Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages. Filamentous cyanobacteria belonging to the genus Nostoc are widespread and ecologically important in freshwater, yet little is known about the genomic content of their viruses. Here we report the first genomic analysis of cyanophages infecting

  18. Real-time studies on microalgae under microgravity

    NASA Astrophysics Data System (ADS)

    Wang, G. H.; Li, G. B.; Li, D. H.; Liu, Y. D.; Song, L. R.; Tong, G. H.; Liu, X. M.; Cheng, E. T.

    2004-07-01

    Using remote sensing technique, we investigated real-time Nostoc sphaeroides Kütz (Cyanobacterium) in Closed System under microgravity by SHENZHOU-2 spacecraft in January 2001. The experiments had 1 g centrifuges in space for control and ground control group experiments were also carried out in the same equipments and under the same controlled condition. The data about the population growth of Nostoc sp. of experiments and temperature changes of system were got from spacecraft every minute. From the data, we can find that population growth of Nostoc sp. in microgravity group was higher than that of other groups in space or on ground, even though both the control 1 g group in space and 1 g group on ground indicated same increasing characteristics in experiments. The growth rate of 1.4 g group (centrifuged group on ground) was also promoted during experiment. The temperature changes of systems are also affected by gravity and light. Some aspects about those differences were discussed. From the discussion of these results during experiment, it can be found that gravity is the major factor to lead to these changes.

  19. Carbon Isotope Fractionation during Catabolism and Anabolism in Acetogenic Bacteria Growing on Different Substrates

    PubMed Central

    Freude, Christoph

    2016-01-01

    Homoacetogenic bacteria are versatile microbes that use the acetyl coenzyme A (acetyl-CoA) pathway to synthesize acetate from CO2 and hydrogen. Likewise, the acetyl-CoA pathway may be used to incorporate other 1-carbon substrates (e.g., methanol or formate) into acetate or to homoferment monosaccharides completely to acetate. In this study, we analyzed the fractionation of pure acetogenic cultures grown on different carbon substrates. While the fractionation of Sporomusa sphaeroides grown on C1 compounds was strong (εC1, −49‰ to −64‰), the fractionation of Moorella thermoacetica and Thermoanaerobacter kivui using glucose (εGlu = −14.1‰) was roughly one-third as strong, suggesting a contribution of less-depleted acetate from fermentative processes. For M. thermoacetica, this could indeed be validated by the addition of nitrate, which inhibited the acetyl-CoA pathway, resulting in fractionation during fermentation (εferm = −0.4‰). In addition, we determined the fractionation into microbial biomass of T. kivui grown on H2/CO2 (εanabol. = −28.6‰) as well as on glucose (εanabol. = +2.9‰). PMID:26921422

  20. Evaluating the Nature of So-Called S*-State Feature in Transient Absorption of Carotenoids in Light-Harvesting Complex 2 (LH2) from Purple Photosynthetic Bacteria.

    PubMed

    Niedzwiedzki, Dariusz M; Hunter, C Neil; Blankenship, Robert E

    2016-11-03

    Carotenoids are a class of natural pigments present in all phototrophic organisms, mainly in their light-harvesting proteins in which they play roles of accessory light absorbers and photoprotectors. Extensive time-resolved spectroscopic studies of these pigments have revealed unexpectedly complex photophysical properties, particularly for carotenoids in light-harvesting LH2 complexes from purple bacteria. An ambiguous, optically forbidden electronic excited state designated as S* has been postulated to be involved in carotenoid excitation relaxation and in an alternative carotenoid-to-bacteriochlorophyll energy transfer pathway, as well as being a precursor of the carotenoid triplet state. However, no definitive and satisfactory origin of the carotenoid S* state in these complexes has been established, despite a wide-ranging series of studies. Here, we resolve the ambiguous origin of the carotenoid S* state in LH2 complex from Rba. sphaeroides by showing that the S* feature can be seen as a combination of ground state absorption bleaching of the carotenoid pool converted to cations and the Stark spectrum of neighbor neutral carotenoids, induced by temporal electric field brought by the carotenoid cation-bacteriochlorophyll anion pair. These findings remove the need to assign an S* state, and thereby significantly simplify the photochemistry of carotenoids in these photosynthetic antenna complexes.

  1. Evaluating the Nature of So-Called S*-State Feature in Transient Absorption of Carotenoids in Light-Harvesting Complex 2 (LH2) from Purple Photosynthetic Bacteria

    PubMed Central

    2016-01-01

    Carotenoids are a class of natural pigments present in all phototrophic organisms, mainly in their light-harvesting proteins in which they play roles of accessory light absorbers and photoprotectors. Extensive time-resolved spectroscopic studies of these pigments have revealed unexpectedly complex photophysical properties, particularly for carotenoids in light-harvesting LH2 complexes from purple bacteria. An ambiguous, optically forbidden electronic excited state designated as S* has been postulated to be involved in carotenoid excitation relaxation and in an alternative carotenoid-to-bacteriochlorophyll energy transfer pathway, as well as being a precursor of the carotenoid triplet state. However, no definitive and satisfactory origin of the carotenoid S* state in these complexes has been established, despite a wide-ranging series of studies. Here, we resolve the ambiguous origin of the carotenoid S* state in LH2 complex from Rba. sphaeroides by showing that the S* feature can be seen as a combination of ground state absorption bleaching of the carotenoid pool converted to cations and the Stark spectrum of neighbor neutral carotenoids, induced by temporal electric field brought by the carotenoid cation–bacteriochlorophyll anion pair. These findings remove the need to assign an S* state, and thereby significantly simplify the photochemistry of carotenoids in these photosynthetic antenna complexes. PMID:27726397

  2. Carbon Isotope Fractionation during Catabolism and Anabolism in Acetogenic Bacteria Growing on Different Substrates.

    PubMed

    Freude, Christoph; Blaser, Martin

    2016-05-01

    Homoacetogenic bacteria are versatile microbes that use the acetyl coenzyme A (acetyl-CoA) pathway to synthesize acetate from CO2 and hydrogen. Likewise, the acetyl-CoA pathway may be used to incorporate other 1-carbon substrates (e.g., methanol or formate) into acetate or to homoferment monosaccharides completely to acetate. In this study, we analyzed the fractionation of pure acetogenic cultures grown on different carbon substrates. While the fractionation of Sporomusa sphaeroides grown on C1 compounds was strong (εC1, -49‰ to -64‰), the fractionation of Moorella thermoacetica and Thermoanaerobacter kivui using glucose (εGlu= -14.1‰) was roughly one-third as strong, suggesting a contribution of less-depleted acetate from fermentative processes. ForM. thermoacetica, this could indeed be validated by the addition of nitrate, which inhibited the acetyl-CoA pathway, resulting in fractionation during fermentation (εferm= -0.4‰). In addition, we determined the fractionation into microbial biomass of T. kivui grown on H2/CO2(εanabol.= -28.6‰) as well as on glucose (εanabol.= +2.9‰). Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. The 2-Methoxy Group Orientation Regulates the Redox Potential Difference between the Primary (QA) and Secondary (QB) Quinones of Type II Bacterial Photosynthetic Reaction Centers.

    PubMed

    de Almeida, Wagner B; Taguchi, Alexander T; Dikanov, Sergei A; Wraight, Colin A; O'Malley, Patrick J

    2014-08-07

    Recent studies have shown that only quinones with a 2-methoxy group can act simultaneously as the primary (Q A ) and secondary (Q B ) electron acceptors in photosynthetic reaction centers from purple bacteria such as Rb. sphaeroides . 13 C HYSCORE measurements of the 2-methoxy group in the semiquinone states, SQ A and SQ B , were compared with DFT calculations of the 13 C hyperfine couplings as a function of the 2-methoxy dihedral angle. X-ray structure comparisons support 2-methoxy dihedral angle assignments corresponding to a redox potential gap (Δ E m ) between Q A and Q B of 175-193 mV. A model having a methyl group substituted for the 2-methoxy group exhibits no electron affinity difference. This is consistent with the failure of a 2-methyl ubiquinone analogue to function as Q B in mutant reaction centers with a Δ E m of ∼160-195 mV. The conclusion reached is that the 2-methoxy group is the principal determinant of electron transfer from Q A to Q B in type II photosynthetic reaction centers with ubiquinone serving as both acceptor quinones.

  4. Evaluating the nature of so-called S*-State feature in transient absorption of carotenoids in light-harvesting complex 2 (LH2) from purple photosynthetic bacteria

    DOE PAGES

    Niedzwiedzki, Dariusz M.; Hunter, C. Neil; Blankenship, Robert E.

    2016-10-11

    Carotenoids are a class of natural pigments present in all phototrophic organisms, mainly in their light-harvesting proteins in which they play roles of accessory light absorbers and photoprotectors. Extensive time-resolved spectroscopic studies of these pigments have revealed unexpectedly complex photophysical properties, particularly for carotenoids in light-harvesting LH2 complexes from purple bacteria. An ambiguous, optically forbidden electronic excited state designated as S* has been postulated to be involved in carotenoid excitation relaxation and in an alternative carotenoid-to-bacteriochlorophyll energy transfer pathway, as well as being a precursor of the carotenoid triplet state. However, no definitive and satisfactory origin of the carotenoid S*more » state in these complexes has been established, despite a wide-ranging series of studies. Here, we resolve the ambiguous origin of the carotenoid S* state in LH2 complex from Rba. sphaeroides by showing that the S* feature can be seen as a combination of ground state absorption bleaching of the carotenoid pool converted to cations and the Stark spectrum of neighbor neutral carotenoids, induced by temporal electric field brought by the carotenoid cation- bacteriochlorophyll anion pair. Lastly, these findings remove the need to assign an S* state, and thereby significantly simplify the photochemistry of carotenoids in these photosynthetic antenna complexes.« less

  5. Synthesis of covalently linked dimeric derivatives of chlorophyll a, pyrochlorophyll a, chlorophyll b, and bacteriochlorophyll a

    SciTech Connect

    Wasielewski, M.R.; Svec, W.A.

    1980-05-09

    Bis(chlorophyllide) ethylene glycol diesters were prepared for each of the title compounds. Pheophytins a and b isolated from alfalfa and bacteriochlorophyll a isolated from R. sphaeroides were treated with 80% aqueous trifluoroacetic acid to yield the corresponding pheophorbides. Pyropheophorbide was prepared by a literature procedure. Carbonic anhydride and benzotriazole-1-methanesulfonate activation methods were used in the esterification of the pheophorbides with ethylene glycol at ambient temperature. Each method yielded 75%+ of the pheophorbide ethylene glycol monoester. These monoesters were treated with equimolar amounts of the corresponding pheophorbide by using benzotriazol-1-methanesulfonate/4-(dimethylamino)pyridine in CH/sub 2/Cl/sub 2/ or dicyclohexylcarbodiimide/4-(dimethylamino)pyridine in CH/sub 2/Cl/sub 2/ atmore » ambient temperature. Yields of bis(phenophorbide) ethylene glycol diesters averaged about 50% for the former method and 70% for the latter method. Insertion of the magnesium atoms into the a series macrocycles was accomplished with iodomagnesium 2,6-di-tert-butyl-4-methylphenolate, IMgBHT, in CH/sub 2/Cl/sub 2/, while the metalation of the b and bacterial series macrocycles was carried out with a mixture of IMgBHT and lithium 2,2,6,6-tetramethylpiperidide in thiophen, all at ambient temperature. Both mono- and dimetalated derivatives were isolated and characterized in each case.« less

  6. Evaluating the nature of so-called S*-State feature in transient absorption of carotenoids in light-harvesting complex 2 (LH2) from purple photosynthetic bacteria

    SciTech Connect

    Niedzwiedzki, Dariusz M.; Hunter, C. Neil; Blankenship, Robert E.

    Carotenoids are a class of natural pigments present in all phototrophic organisms, mainly in their light-harvesting proteins in which they play roles of accessory light absorbers and photoprotectors. Extensive time-resolved spectroscopic studies of these pigments have revealed unexpectedly complex photophysical properties, particularly for carotenoids in light-harvesting LH2 complexes from purple bacteria. An ambiguous, optically forbidden electronic excited state designated as S* has been postulated to be involved in carotenoid excitation relaxation and in an alternative carotenoid-to-bacteriochlorophyll energy transfer pathway, as well as being a precursor of the carotenoid triplet state. However, no definitive and satisfactory origin of the carotenoid S*more » state in these complexes has been established, despite a wide-ranging series of studies. Here, we resolve the ambiguous origin of the carotenoid S* state in LH2 complex from Rba. sphaeroides by showing that the S* feature can be seen as a combination of ground state absorption bleaching of the carotenoid pool converted to cations and the Stark spectrum of neighbor neutral carotenoids, induced by temporal electric field brought by the carotenoid cation- bacteriochlorophyll anion pair. Lastly, these findings remove the need to assign an S* state, and thereby significantly simplify the photochemistry of carotenoids in these photosynthetic antenna complexes.« less

  7. Subunit III-depleted cytochrome c oxidase provides insight into the process of proton uptake by proteins

    PubMed Central

    Varanasi, Lakshman; Hosler, Jonathan P.

    2011-01-01

    We review studies of subunit III-depleted cytochrome c oxidase (CcO III (−)) that elucidate the structural basis of steady-state proton uptake from solvent into an internal proton transfer pathway. The removal of subunit III from R. sphaeroides CcO makes proton uptake into the D pathway a rate-determining step, such that measurements of the pH dependence of steady-state O2 consumption can be used to compare the rate and functional pKa of proton uptake by D pathways containing different initial proton acceptors. The removal of subunit III also promotes spontaneous suicide inactivation by CcO, greatly shortening its catalytic lifespan. Because the probability of suicide inactivation is controlled by the rate at which the D pathway delivers protons to the active site, measurements of catalytic lifespan provide a second method to compare the relative efficacy of proton uptake by engineered CcO III (−) forms. These simple experimental systems have been used to explore general questions of proton uptake by proteins, such as the functional value of an initial proton acceptor, whether an initial acceptor must be surface-exposed, which side chains will function as initial proton acceptors and whether multiple acceptors can speed proton uptake. PMID:22023935

  8. An x-ray absorption study of the iron site in bacterial photosynthetic reaction centers.

    PubMed Central

    Bunker, G; Stern, E A; Blankenship, R E; Parson, W W

    1982-01-01

    Measurements were made of the extended x-ray absorption fine structure (EXAFS) of the iron site in photosynthetic reaction centers from the bacterium Rhodopseudomonas sphaeroides. Forms with two quinones, two quinones with added o-phenanthroline, and one quinone were studied. Only the two forms containing two quinones maintained their integrity and were analyzed. The spectra show directly that the added o-phenanthroline does not chelate the iron atom. Further analysis indicates that the iron is octahedrally coordinated by nitrogen and/or oxygen atoms located at various distances, with the average value of about 2.14 A. The analysis suggests that most of the ligands are nitrogens and that three of the nitrogen ligands belong to histidine rings. This interpretation accounts for several unusual features of the EXAFS spectrum. We speculate that the quinones are bound to the histidine rings in some manner. Qualitative features of the absorption edge spectra also are discussed and are related to the Fe-ligand distance. PMID:6977382

  9. ASM Conference on Prokaryotic Development

    SciTech Connect

    Kaplan, H. B.

    2005-07-13

    Support was provided by DOE for the 2nd ASM Conference on Prokaryotic Development. The final conference program and abstracts book is attached. The conference presentations are organized around topics that are central to the current research areas in prokaryotic development. The program starts with topics that involve relatively simple models systems and ends with systems that are more complex. The topics are: i) the cell cycle, ii) the cytoskeleton, iii) morphogenesis, iv) developmental transcription, v) signaling, vi) multicellularity, and vii) developmental diversity and symbiosis. The best-studied prokaryotic development model systems will be highlighted at the conference through research presentations bymore » leaders in the field. Many of these systems are also model systems of relevance to the DOE mission including carbon sequestration (Bradyrizobium, Synechococcus), energy production (Anabaena, Rhodobacter) and bioremediation (Caulobacter, Mesorhizobium). In addition, many of the highlighted organisms have important practical applications; the actinomycetes and myxobacteria produce antimicrobials that are of commercial interest. It is certain that the cutting-edge science presented at the conference will be applicable to the large group of bacteria relevant to the DOE mission.« less

  10. Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotosynthetic and photosynthetic prokaryotes

    SciTech Connect

    Armstrong, G.A.; Hearst, J.E.; Alberti, M.

    1990-12-01

    Carotenoids comprise one of the most widespread classes of pigments found in nature. The first reactions of C{sub 40} carotenoid biosynthesis proceed through common intermediates in all organisms, suggesting the evolutionary conservation of early enzymes from this pathway. The authors report here the nucleotide sequence of three genes from the carotenoid biosynthesis gene cluster of Erwinia herbicola, a nonphotosynthetic epiphytic bacterium, which encode homologs of the CrtB, CrtE, and CrtI proteins of Rhodobacter capsulatus, a purple nonsulfur photosynthetic bacterium. CrtB (prephytoene pyrophosphate synthase), CrtE (phytoene synthase), and CrtI (phytoene dehydrogenase) are required for the first three reactions specific to themore » carotenoid branch of general isoprenoid metabolism. All three dehydrogenases possess a hydrophobic N-terminal domain containing a putative ADP-binding {beta}{alpha}{beta} fold characteristic of enzymes known to bind FAD or NAD(P) cofactors. These data indicate the structural conservation of early carotenoid biosynthesis enzymes in evolutionary diverse organisms.« less

  11. Determination of the topological shape of integral membrane protein light-harvesting complex LH2 from photosynthetic bacteria in the detergent solution by small-angle X-ray scattering.

    PubMed

    Hong, Xinguo; Weng, Yu-Xiang; Li, Ming

    2004-02-01

    The topological shape of the integral membrane protein light-harvesting complex LH2 from photosynthetic bacteria Rhodobacter spheroides 2.4.1 in detergent solution has been determined from synchrotron small-angle X-ray scattering data using direct curve-fitting by the ellipsoid, ab initio shape determination methods of simulated annealing algorithm and multipole expansion, respectively. The results indicate that the LH2 protein in aqueous solution is encapsulated by a monolayered detergent shell. The detergent-stabilized structure has the shape of an oblate plate, with a thickness of 40 A, a long axis of 110 A, and a short axis of 85 A. After correction for the detergent shell, the shape of the LH2 core is also an oblate plate with a height of 40 A, a long axis of 80 A, and a short axis of 55 A. In contrast to the cylindrical crystal structure with a height of 40 A and a diameter of 68 A, the molecular shape of the LH2 complex in detergent solution clearly deviates from the ringlike crystal structure, with an eccentricity found to be 0.59-consistent with the result of single molecular spectroscopy study of the isolated single LH2 molecules.

  12. Determination of the Topological Shape of Integral Membrane Protein Light-Harvesting Complex LH2 from Photosynthetic Bacteria in the Detergent Solution by Small-Angle X-Ray Scattering

    PubMed Central

    Hong, Xinguo; Weng, Yu-Xiang; Li, Ming

    2004-01-01

    The topological shape of the integral membrane protein light-harvesting complex LH2 from photosynthetic bacteria Rhodobacter spheroides 2.4.1 in detergent solution has been determined from synchrotron small-angle X-ray scattering data using direct curve-fitting by the ellipsoid, ab initio shape determination methods of simulated annealing algorithm and multipole expansion, respectively. The results indicate that the LH2 protein in aqueous solution is encapsulated by a monolayered detergent shell. The detergent-stabilized structure has the shape of an oblate plate, with a thickness of 40 Å, a long axis of 110 Å, and a short axis of 85 Å . After correction for the detergent shell, the shape of the LH2 core is also an oblate plate with a height of 40 Å, a long axis of 80 Å, and a short axis of 55 Å. In contrast to the cylindrical crystal structure with a height of 40 Å and a diameter of 68 Å, the molecular shape of the LH2 complex in detergent solution clearly deviates from the ringlike crystal structure, with an eccentricity found to be 0.59—consistent with the result of single molecular spectroscopy study of the isolated single LH2 molecules. PMID:14747343

  13. Diverse bacteria isolated from microtherm oil-production water.

    PubMed

    Sun, Ji-Quan; Xu, Lian; Zhang, Zhao; Li, Yan; Tang, Yue-Qin; Wu, Xiao-Lei

    2014-02-01

    In total, 435 pure bacterial strains were isolated from microtherm oil-production water from the Karamay Oilfield, Xinjiang, China, by using four media: oil-production water medium (Cai medium), oil-production water supplemented with mineral salt medium (CW medium), oil-production water supplemented with yeast extract medium (CY medium), and blood agar medium (X medium). The bacterial isolates were affiliated with 61 phylogenetic groups that belong to 32 genera in the phyla Actinobacteria, Firmicutes, and Proteobacteria. Except for the Rhizobium, Dietzia, and Pseudomonas strains that were isolated using all the four media, using different media led to the isolation of bacteria with different functions. Similarly, nonheme diiron alkane monooxygenase genes (alkB/alkM) also clustered according to the isolation medium. Among the bacterial strains, more than 24 % of the isolates could use n-hexadecane as the sole carbon source for growth. For the first time, the alkane-degrading ability and alkB/alkM were detected in Rhizobium, Rhodobacter, Trichococcus, Micrococcus, Enterococcus, and Bavariicoccus strains, and the alkM gene was detected in Firmicutes strains.

  14. Combined actions of multiple hairpin loop structures and sites of rate-limiting endonucleolytic cleavage determine differential degradation rates of individual segments within polycistronic puf operon mRNA.

    PubMed Central

    Klug, G; Cohen, S N

    1990-01-01

    Differential expression of the genes within the puf operon of Rhodobacter capsulatus is accomplished in part by differences in the rate of degradation of different segments of the puf transcript. We report here that decay of puf mRNA sequences specifying the light-harvesting I (LHI) and reaction center (RC) photosynthetic membrane peptides is initiated endoribonucleolytically within a discrete 1.4-kilobase segment of the RC-coding region. Deletion of this segment increased the half-life of the RC-coding region from 8 to 20 min while not affecting decay of LHI-coding sequences upstream from an intercistronic hairpin loop structure shown previously to impede 3'-to-5' degradation. Prolongation of RC segment half-life was dependent on the presence of other hairpin structures 3' to the RC region. Inserting the endonuclease-sensitive sites into the LHI-coding segment markedly accelerated its degradation. Our results suggest that differential degradation of the RC- and LHI-coding segments of puf mRNA is accomplished at least in part by the combined actions of RC region-specific endonuclease(s), one or more exonucleases, and several strategically located exonuclease-impeding hairpins. Images PMID:2394682

  15. Altered Microbiome Leads to Significant Phenotypic and Transcriptomic Differences in a Lipid Accumulating Chlorophyte.

    PubMed

    Richter, Lubna V; Mansfeldt, Cresten B; Kuan, Michael M; Cesare, Alexandra E; Menefee, Stephen T; Richardson, Ruth E; Ahner, Beth A

    2018-06-19

    Given the challenges facing the economically favorable production of products from microalgae, understanding factors that might impact productivity rates including growth rates and accumulation of desired products, for example, triacylglycerols (TAG) for biodiesel feedstock, remains critical. Although operational parameters such as media composition and reactor design can clearly effect growth rates, the role of microbe-microbe interactions is just beginning to be elucidated. In this study an oleaginous marine algae Chlorella spp. C596 culture is shown to be better described as a microbial community. Perturbations to this microbial community showed a significant impact on phenotypes including sustained differences in growth rate and TAG accumulation of 2.4 and 2.5 fold, respectively. Characterization of the associated community using Illumina 16S rRNA amplicon and random shotgun transcriptomic analyses showed that the fast growth rate correlated with two specific bacterial species ( Ruegeria and Rhodobacter spp). The transcriptomic response of the Chlorella species revealed that the slower growing algal consortium C596-S1 upregulated genes associated with photosynthesis and resource scavenging and decreased the expression of genes associated with transcription and translation relative to the initial C596-R1. Our studies advance the appreciation of the effects microbiomes can have on algal growth in bioreactors and suggest that symbiotic interactions are involved in a range of critical processes including nitrogen, carbon cycling, and oxidative stress.

  16. The gene transfer agent-like particle of the marine phototrophic bacterium Rhodovulum sulfidophilum.

    PubMed

    Nagao, Nobuyoshi; Yamamoto, Junya; Komatsu, Hiroyuki; Suzuki, Hiromichi; Hirose, Yuu; Umekage, So; Ohyama, Takashi; Kikuchi, Yo

    2015-12-01

    Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum , we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5 kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40 nm and a tail length of about 60 nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum . However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.

  17. Nonpigmented and Bacteriochlorophyll-Containing Bradyrhizobia Isolated from Aeschynomene indica

    PubMed Central

    van Berkum, P.; Tully, R. E.; Keister, D. L.

    1995-01-01

    The legume genus Aeschynomene is unusual, since many species develop stem nodules and the bradyrhizobia isolated from these nodules produce bacteriochlorophyll (Bchl). Evidence is presented that the bradyrhizobia of Aeschynomene indica have wide distribution throughout the world, since A. indica was nodulated when grown in 58 soils collected in 14 different countries. Only 38 of 79 isolates tested synthesized Bchl and carotenoids during heterotrophic growth. Nine isolates produced Bchl constitutively, and cultures were pigmented after growth in the dark. The other isolates required light for Bchl production. The DNA from seven pigmented and three nonpigmented bradyrhizobia hybridized with a DNA probe containing the genes for the photosynthetic apparatus of Rhodobacter capsulatus, but DNA from two other nonpigmented isolates did not hybridize with this probe. A relationship between pigmentation in culture and symbiotic phenotype was not evident, since bradyrhizobia of both Bchl phenotypes nodulated stems of A. indica and formed nitrogen-fixing symbioses. Several isolates, which were ineffective on A. indica, probably do belong to the proposed cross-inoculation group 3 (D. Alazard, Appl. Environ. Microbiol. 50:732-734, 1985), since they did not nodulate Aeschynomene americana or Macroptilium atropurpureum. Since it has been suggested that extant rhizobia arose from photosynthetic ancestors (J. I. Sprent, p. 45-54, in P. M. Gresshoff, L. E. Roth, G. Stacey, and W. E. Newton, ed., Nitrogen Fixation: Achievements and Objectives, 1990), we propose that the nonpigmented isolates may represent an extant lineage of an intermediate evolutionary stage. PMID:16534933

  18. High-Throughput Biosensor Discriminates Between Different Algal H 2-Photoproducing Strains

    SciTech Connect

    Wecker, Matt S. A.; Maria L. Ghirardi

    2014-02-27

    A number of species of microalgae and cyanobacteria photosynthetically produce H 2 gas by coupling water oxidation with the reduction of protons to molecular hydrogen, generating renewable energy from sunlight and water. Photosynthetic H 2 production, however, is transitory, and there is considerable interest in increasing and extending it for commercial applications. Here we report a Petri-plate version of our previous, microplate-based assay that detects photosynthetic H 2 production by algae. The assay consists of an agar overlay of H 2-sensing Rhodobacter capsulatus bacteria carrying a green fluorescent protein that responds to H 2 produced by single algal colonies inmore » the bottom agar layer. The assay distinguishes between algal strains that photoproduce H 2 at different levels under high light intensities, and it does so in a simple, inexpensive, and high-throughput manner. The assay will be useful for screening both natural populations and mutant libraries for strains having increased H 2 production, and useful for identifying various genetic factors that physiologically or genetically alter algal hydrogen production.« less

  19. Engineering electron metabolism to increase ethanol production in Clostridium thermocellum

    DOE PAGES

    Lo, Jonathan; Olson, Daniel G.; Murphy, Sean Jean-Loup; ...

    2016-10-28

    Here, the NfnAB (NADH-dependent reduced ferredoxin:NADP + oxidoreductase) and Rnf ( Rhodobacter nitrogen fixation) complexes are thought to catalyze electron transfer between reduced ferredoxin and NAD(P) +. Efficient electron flux is critical for engineering fuel production pathways, but little is known about the relative importance of these enzymes in vivo. In this study we investigate the importance of the NfnAB and Rnf complexes in Clostridium thermocellum for growth on cellobiose and Avicel using gene deletion, enzyme assays, and fermentation product analysis. The NfnAB complex does not seem to play a major role in metabolism, since deletion of nfnAB genes hadmore » little effect on the distribution of fermentation products. By contrast, the Rnf complex appears to play an important role in ethanol formation. Deletion of rnf genes resulted in a decrease in ethanol formation. Overexpression of rnf genes resulted in an increase in ethanol production of about 30%, but only in strains where the hydG hydrogenase maturation gene was also deleted.« less

  20. A process economic assessment of hydrocarbon biofuels production using chemoautotrophic organisms

    SciTech Connect

    Khan, NE; Myers, JA; Tuerk, AL

    Economic analysis of an ARPA-e Electrofuels (http://arpa-e.energy.gov/?q=arpa-e-programs/electrofuels) process is presented, utilizing metabolically engineered Rhodobacter capsulatus or Ralstonia eutropha to produce the C30+ hydrocarbon fuel, botryococcene, from hydrogen, carbon dioxide, and oxygen. The analysis is based on an Aspen plus (R) bioreactor model taking into account experimentally determined Rba. capsulatus and Rls. eutropha growth and maintenance requirements, reactor residence time, correlations for gas-liquid mass-transfer coefficient, gas composition, and specific cellular fuel productivity. Based on reactor simulation results encompassing technically relevant parameter ranges, the capital and operating costs of the process were estimated for 5000 bbl-fuel/day plant and used to predict fuelmore » cost. Under the assumptions used in this analysis and crude oil prices, the Levelized Cost of Electricity (LCOE) required for economic feasibility must be less than 2(sic)/kWh. While not feasible under current market prices and costs, this work identifies key variables impacting process cost and discusses potential alternative paths toward economic feasibility. (C) 2014 Elsevier Ltd. All rights reserved.« less

  1. Metabolic engineering in chemolithoautotrophic hosts for the production of fuels and chemicals.

    PubMed

    Nybo, S Eric; Khan, Nymul E; Woolston, Benjamin M; Curtis, Wayne R

    2015-07-01

    The ability of autotrophic organisms to fix CO2 presents an opportunity to utilize this 'greenhouse gas' as an inexpensive substrate for biochemical production. Unlike conventional heterotrophic microorganisms that consume carbohydrates and amino acids, prokaryotic chemolithoautotrophs have evolved the capacity to utilize reduced chemical compounds to fix CO2 and drive metabolic processes. The use of chemolithoautotrophic hosts as production platforms has been renewed by the prospect of metabolically engineered commodity chemicals and fuels. Efforts such as the ARPA-E electrofuels program highlight both the potential and obstacles that chemolithoautotrophic biosynthetic platforms provide. This review surveys the numerous advances that have been made in chemolithoautotrophic metabolic engineering with a focus on hydrogen oxidizing bacteria such as the model chemolithoautotrophic organism (Ralstonia), the purple photosynthetic bacteria (Rhodobacter), and anaerobic acetogens. Two alternative strategies of microbial chassis development are considered: (1) introducing or enhancing autotrophic capabilities (carbon fixation, hydrogen utilization) in model heterotrophic organisms, or (2) improving tools for pathway engineering (transformation methods, promoters, vectors etc.) in native autotrophic organisms. Unique characteristics of autotrophic growth as they relate to bioreactor design and process development are also discussed in the context of challenges and opportunities for genetic manipulation of organisms as production platforms. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  2. Metagenomics approach to the study of the gut microbiome structure and function in zebrafish Danio rerio fed with gluten formulated diet.

    PubMed

    Koo, Hyunmin; Hakim, Joseph A; Powell, Mickie L; Kumar, Ranjit; Eipers, Peter G; Morrow, Casey D; Crowley, Michael; Lefkowitz, Elliot J; Watts, Stephen A; Bej, Asim K

    2017-04-01

    In this study, we report the gut microbial composition and predictive functional profiles of zebrafish, Danio rerio, fed with a control formulated diet (CFD), and a gluten formulated diet (GFD) using a metagenomics approach and bioinformatics tools. The microbial communities of the GFD-fed D. rerio displayed heightened abundances of Legionellales, Rhizobiaceae, and Rhodobacter, as compared to the CFD-fed counterparts. Predicted metagenomics of microbial communities (PICRUSt) in GFD-fed D. rerio showed KEGG functional categories corresponding to bile secretion, secondary bile acid biosynthesis, and the metabolism of glycine, serine, and threonine. The CFD-fed D. rerio exhibited KEGG functional categories of bacteria-mediated cobalamin biosynthesis, which was supported by the presence of cobalamin synthesizers such as Bacteroides and Lactobacillus. Though these bacteria were absent in GFD-fed D. rerio, a comparable level of the cobalamin biosynthesis KEGG functional category was observed, which could be contributed by the compensatory enrichment of Cetobacterium. Based on these results, we conclude D. rerio to be a suitable alternative animal model for the use of a targeted metagenomics approach along with bioinformatics tools to further investigate the relationship between the gluten diet and microbiome profile in the gut ecosystem leading to gastrointestinal diseases and other undesired adverse health effects. Copyright © 2017. Published by Elsevier B.V.

  3. Dynamic modeling of temperature change in outdoor operated tubular photobioreactors.

    PubMed

    Androga, Dominic Deo; Uyar, Basar; Koku, Harun; Eroglu, Inci

    2017-07-01

    In this study, a one-dimensional transient model was developed to analyze the temperature variation of tubular photobioreactors operated outdoors and the validity of the model was tested by comparing the predictions of the model with the experimental data. The model included the effects of convection and radiative heat exchange on the reactor temperature throughout the day. The temperatures in the reactors increased with increasing solar radiation and air temperatures, and the predicted reactor temperatures corresponded well to the measured experimental values. The heat transferred to the reactor was mainly through radiation: the radiative heat absorbed by the reactor medium, ground radiation, air radiation, and solar (direct and diffuse) radiation, while heat loss was mainly through the heat transfer to the cooling water and forced convection. The amount of heat transferred by reflected radiation and metabolic activities of the bacteria and pump work was negligible. Counter-current cooling was more effective in controlling reactor temperature than co-current cooling. The model developed identifies major heat transfer mechanisms in outdoor operated tubular photobioreactors, and accurately predicts temperature changes in these systems. This is useful in determining cooling duty under transient conditions and scaling up photobioreactors. The photobioreactor design and the thermal modeling were carried out and experimental results obtained for the case study of photofermentative hydrogen production by Rhodobacter capsulatus, but the approach is applicable to photobiological systems that are to be operated under outdoor conditions with significant cooling demands.

  4. Structure of the dimeric RC–LH1–PufX complex from Rhodobaca bogoriensis investigated by electron microscopy

    PubMed Central

    Semchonok, Dmitry A.; Chauvin, Jean-Paul; Frese, Raoul N.; Jungas, Colette; Boekema, Egbert J.

    2012-01-01

    Electron microscopy and single-particle averaging were performed on isolated reaction centre (RC)—antenna complexes (RC–LH1–PufX complexes) of Rhodobaca bogoriensis strain LBB1, with the aim of establishing the LH1 antenna conformation, and, in particular, the structural role of the PufX protein. Projection maps of dimeric complexes were obtained at 13 Å resolution and show the positions of the 2 × 14 LH1 α- and β-subunits. This new dimeric complex displays two open, C-shaped LH1 aggregates of 13 αβ polypeptides partially surrounding the RCs plus two LH1 units forming the dimer interface in the centre. Between the interface and the two half rings are two openings on each side. Next to the openings, there are four additional densities present per dimer, considered to be occupied by four copies of PufX. The position of the RC in our model was verified by comparison with RC–LH1–PufX complexes in membranes. Our model differs from previously proposed configurations for Rhodobacter species in which the LH1 ribbon is continuous in the shape of an S, and the stoichiometry is of one PufX per RC. PMID:23148268

  5. RNA degradation in Archaea and Gram-negative bacteria different from Escherichia coli.

    PubMed

    Evguenieva-Hackenberg, Elena; Klug, Gabriele

    2009-01-01

    Exoribonucleolytic and endoribonucleolytic activities are important for controlled degradation of RNA and contribute to the regulation of gene expression at the posttranscriptional level by influencing the half-lives of specific messenger RNAs. The RNA half-lives are determined by the characteristics of the RNA substrates and by the availability and the properties of the involved proteins-ribonucleases and assisting polypeptides. Much is known about RNA degradation in Eukarya and Bacteria, but there is limited information about RNA-degrading enzymes and RNA destabilizing or stabilizing elements in the domain of the Archaea. The recent progress in the understanding of the structure and function of the archaeal exosome, a protein complex with RNA-degrading and RNA-tailing capabilities, has given some first insights into the mechanisms of RNA degradation in the third domain of life and into the evolution of RNA-degrading enzymes. Moreover, other archaeal RNases with degrading potential have been described and a new mechanism for protection of the 5'-end of RNA in Archaea was discovered. Here, we summarize the current knowledge on RNA degradation in the Archaea. Additionally, RNA degradation mechanisms in Rhodobacter capsulatus and Pseudomonas syringae are compared to those in the major model organism for Gram-negatives, Escherichia coli, which dominates our view on RNA degradation in Bacteria.

  6. Studies of DNA supercoiling in vivo and in vitro

    SciTech Connect

    Cook, David Nelson

    This thesis describes a number of diverse experiments whose common theme is to elaborate some aspect of DNA supercoiling. The torsion elastic constant of DNA is measure as a function of superhelix density using the technique of picosecond Time Resolved Fluorescence Polarization Anisotropy (FPA) of intercalated ethidium bromide. The results agree with theories which predict that the anisotropy decay should vary with the square root of the relative viscosity. This experiment furthermore demonstrates a sensitivity of FPA to a change in torsion elastic constant of less than 10%. A number of covalently closed DNA samples, ranging in superhelix density frommore » = -0.123 to +0.042, are then examined. A novel method for measuring changes in local supercoiling on a large PNA molecule which is sensitive to changes in supercoiling of regions of chromosomal DNA as short as 1 kilobase in length is presented. Study of chromosomal supercoiling regulating anaerobic gene expression in the facultative photosynthetic bacterium, Rhodobacter capsulatus showed that no stable change in chromosomal supercoiling upon a shift from aerobic respiratory growth to anaerobic photosynthetic conditions. Studies to detect transient changes in DNA supercoiling indicate that DNA downstream from heavily transcribed genes for the photosynthetic reaction center are relaxed or perhaps overwound upon the induction of photosynthetic metabolism. These results are interpreted in terms of the twin domain model of transcriptional supercoiling.« less

  7. Studies of DNA supercoiling in vivo and in vitro

    SciTech Connect

    Cook, D.N.

    This thesis describes a number of diverse experiments whose common theme is to elaborate some aspect of DNA supercoiling. The torsion elastic constant of DNA is measure as a function of superhelix density using the technique of picosecond Time Resolved Fluorescence Polarization Anisotropy (FPA) of intercalated ethidium bromide. The results agree with theories which predict that the anisotropy decay should vary with the square root of the relative viscosity. This experiment furthermore demonstrates a sensitivity of FPA to a change in torsion elastic constant of less than 10%. A number of covalently closed DNA samples, ranging in superhelix density frommore » = [minus]0.123 to [plus]0.042, are then examined. A novel method for measuring changes in local supercoiling on a large PNA molecule which is sensitive to changes in supercoiling of regions of chromosomal DNA as short as 1 kilobase in length is presented. Study of chromosomal supercoiling regulating anaerobic gene expression in the facultative photosynthetic bacterium, Rhodobacter capsulatus showed that no stable change in chromosomal supercoiling upon a shift from aerobic respiratory growth to anaerobic photosynthetic conditions. Studies to detect transient changes in DNA supercoiling indicate that DNA downstream from heavily transcribed genes for the photosynthetic reaction center are relaxed or perhaps overwound upon the induction of photosynthetic metabolism. These results are interpreted in terms of the twin domain model of transcriptional supercoiling.« less

  8. Ready to use bioinformatics analysis as a tool to predict immobilisation strategies for protein direct electron transfer (DET).

    PubMed

    Cazelles, R; Lalaoui, N; Hartmann, T; Leimkühler, S; Wollenberger, U; Antonietti, M; Cosnier, S

    2016-11-15

    Direct electron transfer (DET) to proteins is of considerable interest for the development of biosensors and bioelectrocatalysts. While protein structure is mainly used as a method of attaching the protein to the electrode surface, we employed bioinformatics analysis to predict the suitable orientation of the enzymes to promote DET. Structure similarity and secondary structure prediction were combined underlying localized amino-acids able to direct one of the enzyme's electron relays toward the electrode surface by creating a suitable bioelectrocatalytic nanostructure. The electro-polymerization of pyrene pyrrole onto a fluorine-doped tin oxide (FTO) electrode allowed the targeted orientation of the formate dehydrogenase enzyme from Rhodobacter capsulatus (RcFDH) by means of hydrophobic interactions. Its electron relays were directed to the FTO surface, thus promoting DET. The reduction of nicotinamide adenine dinucleotide (NAD(+)) generating a maximum current density of 1μAcm(-2) with 10mM NAD(+) leads to a turnover number of 0.09electron/s/molRcFDH. This work represents a practical approach to evaluate electrode surface modification strategies in order to create valuable bioelectrocatalysts. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Time-resolved spectroscopic studies of photosynthetic reaction centers and tetrapyrrole chromophores for biomedical and solar-energy applications

    NASA Astrophysics Data System (ADS)

    Kee, Hooi Ling

    2008-10-01

    The photophysical properties of diverse tetrapyrrole chromophores as well as energy and electron transfer processes in tetrapyrrole dyads are investigated using static and time-resolved (femtoseconds to seconds) absorption and fluorescence spectroscopy. The goal of these studies is to elucidate the molecular design principals necessary to construct chromophores with the specific and tunable properties that will enhance applications in optical molecular imaging, photodynamic therapy, and solar-energy conversion. The kinetic properties of the transient intermediate P+H B- involving the bacteriopheophytin molecule HB on the normally inactive (B) cofactor branch of the bacterial photosynthetic reaction center are examined in Rhodobacter capsulatus mutants. Using nanosecond flash photolysis and F(L181)Y/Y(M208)F/L(M212)H mutant, the decay pathways and yields of P+HB- were measured, giving an overall yield of 13% for B-side charge separation P* → P+HB- → P+ QB- in this mutant. The goal of these studies is to understand the fundamental differences in the rates, yields, and mechanisms of charge separation and charge recombination along the two parallel electron-transport chains in the bacterial reaction center.

  10. Understanding the degradation of Congo red and bacterial diversity in an air-cathode microbial fuel cell being evaluated for simultaneous azo dye removal from wastewater and bioelectricity generation.

    PubMed

    Sun, Jian; Li, Youming; Hu, Yongyou; Hou, Bin; Zhang, Yaping; Li, Sizhe

    2013-04-01

    We investigated the mechanism of Congo red degradation and bacterial diversity in a single-chambered microbial fuel cell (MFC) incorporating a microfiltration membrane and air-cathode. The MFC was operated continuously for more than 4 months using a mixture of Congo red and glucose as fuel. We demonstrated that the Congo red azo bonds were reduced at the anode to form aromatic amines. This is consistent with the known mechanism of anaerobic biodegradation of azo dyes. The MFC developed a less dense biofilm at the anode in the presence of Congo red compared to its absence indicating that Congo red degradation negatively affected biofilm formation. Denaturing gradient gel electrophoresis and direct 16S ribosomal DNA gene nucleotide sequencing revealed that the microbial communities differed depending on whether Congo red was present in the MFC. Geobacter-like species known to generate electricity were detected in the presence or absence of Congo red. In contrast, Azospirillum, Methylobacterium, Rhodobacter, Desulfovibrio, Trichococcus, and Bacteroides species were only detected in its presence. These species were most likely responsible for degrading Congo red.

  11. Effects of aeration and internal recycle flow on nitrous oxide emissions from a modified Ludzak-Ettinger process fed with glycerol.

    PubMed

    Song, Kang; Suenaga, Toshikazu; Harper, Willie F; Hori, Tomoyuki; Riya, Shohei; Hosomi, Masaaki; Terada, Akihiko

    2015-12-01

    Nitrous oxide (N2O) is emitted from a modified Ludzak-Ettinger (MLE) process, as a primary activated sludge system, which requires mitigation. The effects of aeration rates and internal recycle flow (IRF) ratios on N2O emission were investigated in an MLE process fed with glycerol. Reducing the aeration rate from 1.5 to 0.5 L/min increased gaseous the N2O concentration from the aerobic tank and the dissolved N2O concentration in the anoxic tank by 54.4 and 53.4 %, respectively. During the period of higher aeration, the N2O-N conversion ratio was 0.9 % and the potential N2O reducers were predominantly Rhodobacter, which accounted for 21.8 % of the total population. Increasing the IRF ratio from 3.6 to 7.2 decreased the N2O emission rate from the aerobic tank and the dissolved N2O concentration in the anoxic tank by 56 and 48 %, respectively. This study suggests effective N2O mitigation strategies for MLE systems.

  12. Comparison of two bioinformatics tools used to characterize the microbial diversity and predictive functional attributes of microbial mats from Lake Obersee, Antarctica.

    PubMed

    Koo, Hyunmin; Hakim, Joseph A; Morrow, Casey D; Eipers, Peter G; Davila, Alfonso; Andersen, Dale T; Bej, Asim K

    2017-09-01

    In this study, using NextGen sequencing of the collective 16S rRNA genes obtained from two sets of samples collected from Lake Obersee, Antarctica, we compared and contrasted two bioinformatics tools, PICRUSt and Tax4Fun. We then developed an R script to assess the taxonomic and predictive functional profiles of the microbial communities within the samples. Taxa such as Pseudoxanthomonas, Planctomycetaceae, Cyanobacteria Subsection III, Nitrosomonadaceae, Leptothrix, and Rhodobacter were exclusively identified by Tax4Fun that uses SILVA database; whereas PICRUSt that uses Greengenes database uniquely identified Pirellulaceae, Gemmatimonadetes A1-B1, Pseudanabaena, Salinibacterium and Sinobacteraceae. Predictive functional profiling of the microbial communities using Tax4Fun and PICRUSt separately revealed common metabolic capabilities, while also showing specific functional IDs not shared between the two approaches. Combining these functional predictions using a customized R script revealed a more inclusive metabolic profile, such as hydrolases, oxidoreductases, transferases; enzymes involved in carbohydrate and amino acid metabolisms; and membrane transport proteins known for nutrient uptake from the surrounding environment. Our results present the first molecular-phylogenetic characterization and predictive functional profiles of the microbial mat communities in Lake Obersee, while demonstrating the efficacy of combining both the taxonomic assignment information and functional IDs using the R script created in this study for a more streamlined evaluation of predictive functional profiles of microbial communities. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Identification of Microbial Communities in Open and Closed Circuit Bioelectrochemical MBRs by High-Throughput 454 Pyrosequencing

    PubMed Central

    Huang, Jian; Wang, Zhiwei; Zhu, Chaowei; Ma, Jinxing; Zhang, Xingran; Wu, Zhichao

    2014-01-01

    Two bioelectrochemical membrane bioreactors (MBRs) developed by integrating microbial fuel cell and MBR technology were operated under closed-circuit and open-circuit modes, and high-throughput 454 pyrosequencing was used to investigate the effects of the power generation on the microbial community of bio-anode and bio-cathode. Microbes on the anode under open-circuit operation (AO) were enriched and highly diverse when compared to those on the anode under closed-circuit operation (AC). However, among the cathodes the closed-circuit mode (CC) had richer and more diverse microbial community compared to the cathode under open-circuit mode (CO). On the anodes AO and AC, Proteobacteria and Bacteroidetes were the dominant phyla, while Firmicutes was enriched only on AC. Deltaproteobacteria affiliated to Proteobacteria were also more abundant on AC than AO. Furthermore, the relative abundance of Desulfuromonas, which are well-known electrogenic bacteria, were much higher on AC (10.2%) when compared to AO (0.11%), indicating that closed-circuit operation was more conducive for the growth of electrogenic bacteria on the anodes. On the cathodes, Protebacteria was robust on CC while Bacteroidetes was more abundant on CO. Rhodobacter and Hydrogenophaga were also enriched on CC than CO, suggesting that these genera play a role in electron transfer from the cathode surface to the terminal electron acceptors in the bioelectrochemical MBR under closed-circuit operation. PMID:24705450

  14. Chloroplast chlB gene is required for light-independent chlorophyll accumulation in Chlamydomonas reinhardtii.

    PubMed

    Liu, X Q; Xu, H; Huang, C

    1993-10-01

    Light-independent chlorophyll synthesis occurs in some algae, lower plants, and gymnosperms, but not in angiosperms. We have identified a new chloroplast gene, chlB, that is required for the light-independent accumulation of chlorophyll in the green alga Chlamydomonas reinhardtii. The chlB gene was cloned, sequenced, and then disrupted by performing particle gun-mediated chloroplast transformation. The resulting homoplasmic mutant was unable to accumulate chlorophyll in the dark and thus exhibited a 'yellow-in-the-dark' phenotype. The chlB gene encodes a polypeptide of 688 amino acid residues, and is distinct from two previously characterized chloroplast genes (chlN and chlL) also required for light-independent chlorophyll accumulation in C. reinhardtii. Three unidentified open reading frames in chloroplast genomes of liverwort, black pine, and Chlamydomonas moewusii were also identified as chlB genes, based on their striking sequence similarities to the C. reinhardtii chlB gene. A chlB-like gene is absent in chloroplast genomes of tobacco and rice, consistent with the lack of light-independent chlorophyll synthesis in these plants. Polypeptides encoded by the chloroplast chlB genes also show significant sequence similarities with the bchB gene product of Rhodobacter capsulatus. Comparisons among the chloroplast chlB and the bacterial bchB gene products revealed five highly conserved sequence areas that are interspersed by four stretches of highly variable and probably insertional sequences.

  15. Atomistic determinants of co-enzyme Q reduction at the Qi-site of the cytochrome bc1 complex

    NASA Astrophysics Data System (ADS)

    Postila, Pekka A.; Kaszuba, Karol; Kuleta, Patryk; Vattulainen, Ilpo; Sarewicz, Marcin; Osyczka, Artur; Róg, Tomasz

    2016-09-01

    The cytochrome (cyt) bc1 complex is an integral component of the respiratory electron transfer chain sustaining the energy needs of organisms ranging from humans to bacteria. Due to its ubiquitous role in the energy metabolism, both the oxidation and reduction of the enzyme’s substrate co-enzyme Q has been studied vigorously. Here, this vast amount of data is reassessed after probing the substrate reduction steps at the Qi-site of the cyt bc1 complex of Rhodobacter capsulatus using atomistic molecular dynamics simulations. The simulations suggest that the Lys251 side chain could rotate into the Qi-site to facilitate binding of half-protonated semiquinone - a reaction intermediate that is potentially formed during substrate reduction. At this bent pose, the Lys251 forms a salt bridge with the Asp252, thus making direct proton transfer possible. In the neutral state, the lysine side chain stays close to the conserved binding location of cardiolipin (CL). This back-and-forth motion between the CL and Asp252 indicates that Lys251 functions as a proton shuttle controlled by pH-dependent negative feedback. The CL/K/D switching, which represents a refinement to the previously described CL/K pathway, fine-tunes the proton transfer process. Lastly, the simulation data was used to formulate a mechanism for reducing the substrate at the Qi-site.

  16. Engineering electron metabolism to increase ethanol production in Clostridium thermocellum

    SciTech Connect

    Lo, Jonathan; Olson, Daniel G.; Murphy, Sean Jean-Loup

    Here, the NfnAB (NADH-dependent reduced ferredoxin:NADP + oxidoreductase) and Rnf ( Rhodobacter nitrogen fixation) complexes are thought to catalyze electron transfer between reduced ferredoxin and NAD(P) +. Efficient electron flux is critical for engineering fuel production pathways, but little is known about the relative importance of these enzymes in vivo. In this study we investigate the importance of the NfnAB and Rnf complexes in Clostridium thermocellum for growth on cellobiose and Avicel using gene deletion, enzyme assays, and fermentation product analysis. The NfnAB complex does not seem to play a major role in metabolism, since deletion of nfnAB genes hadmore » little effect on the distribution of fermentation products. By contrast, the Rnf complex appears to play an important role in ethanol formation. Deletion of rnf genes resulted in a decrease in ethanol formation. Overexpression of rnf genes resulted in an increase in ethanol production of about 30%, but only in strains where the hydG hydrogenase maturation gene was also deleted.« less

  17. Altered gut microbiota associated with intestinal disease in grass carp (Ctenopharyngodon idellus).

    PubMed

    Tran, Ngoc Tuan; Zhang, Jing; Xiong, Fan; Wang, Gui-Tang; Li, Wen-Xiang; Wu, Shan-Gong

    2018-05-18

    Gut microbiota plays a crucial importance in their host. Disturbance of the microbial structure and function is known to be associated with inflammatory intestinal disorders. Enteritis is a significant cause of high mortality in fish species, including grass carp (Ctenopharyngodon idellus). Study regarding the association between microbial alternations and enteritis in grass carp is still absent. In this study, changes in the gut microbiota of grass carp suffering from enteritis were investigated using NGS-based 16S rRNA sequencing. Six healthy and ten abnormal fish (showing reddening anus, red odiferous fluid accumulating in the abdominal capacity, and flatulence and haemorrhage in the intestine) were collected from a fish farm in Huanggang Fisheries Institute (Hubei, China). Our results revealed that the diversity, structure, and function of gut microbiota were significantly different between diseased and healthy fish (P < 0.05). Particularly, members of the genera Dechloromonas, Methylocaldum, Planctomyces, Rhodobacter, Caulobacter, Flavobacterium, and Pseudomonas were significantly increased in diseased fish compared with that in healthy fish (P < 0.05). Predicted function indicated that microbiota significantly changed the specific metabolic pathways (related to amino acid metabolism, xenobiotics biodegradation and metabolism, and carbohydrate metabolism) in diseased fish (P < 0.05). Taken together, our findings point out the association between changes of the gut microbiota and enteritis in grass carp, which provide basic information useful for diagnoses, prevention, and treatment of intestinal diseases occurring in cultured fish.

  18. Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

    PubMed

    Hirashima, Takashi; Toyoshima, Masakazu; Moriyama, Takashi; Sato, Naoki

    2018-01-01

    Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

  19. Isolation of Acetogenic Bacteria That Induce Biocorrosion by Utilizing Metallic Iron as the Sole Electron Donor

    PubMed Central

    Yumoto, Isao; Kamagata, Yoichi

    2014-01-01

    Corrosion of iron occurring under anoxic conditions, which is termed microbiologically influenced corrosion (MIC) or biocorrosion, is mostly caused by microbial activities. Microbial activity that enhances corrosion via uptake of electrons from metallic iron [Fe(0)] has been regarded as one of the major causative factors. In addition to sulfate-reducing bacteria and methanogenic archaea in marine environments, acetogenic bacteria in freshwater environments have recently been suggested to cause MIC under anoxic conditions. However, no microorganisms that perform acetogenesis-dependent MIC have been isolated or had their MIC-inducing mechanisms characterized. Here, we enriched and isolated acetogenic bacteria that induce iron corrosion by utilizing Fe(0) as the sole electron donor under freshwater, sulfate-free, and anoxic conditions. The enriched communities produced significantly larger amounts of Fe(II) than the abiotic controls and produced acetate coupled with Fe(0) oxidation prior to CH4 production. Microbial community analysis revealed that Sporomusa sp. and Desulfovibrio sp. dominated in the enrichments. Strain GT1, which is closely related to the acetogen Sporomusa sphaeroides, was eventually isolated from the enrichment. Strain GT1 grew acetogenetically with Fe(0) as the sole electron donor and enhanced iron corrosion, which is the first demonstration of MIC mediated by a pure culture of an acetogen. Other well-known acetogenic bacteria, including Sporomusa ovata and Acetobacterium spp., did not grow well on Fe(0). These results indicate that very few species of acetogens have specific mechanisms to efficiently utilize cathodic electrons derived from Fe(0) oxidation and induce iron corrosion. PMID:25304512

  20. Isolation of acetogenic bacteria that induce biocorrosion by utilizing metallic iron as the sole electron donor.

    PubMed

    Kato, Souichiro; Yumoto, Isao; Kamagata, Yoichi

    2015-01-01

    Corrosion of iron occurring under anoxic conditions, which is termed microbiologically influenced corrosion (MIC) or biocorrosion, is mostly caused by microbial activities. Microbial activity that enhances corrosion via uptake of electrons from metallic iron [Fe(0)] has been regarded as one of the major causative factors. In addition to sulfate-reducing bacteria and methanogenic archaea in marine environments, acetogenic bacteria in freshwater environments have recently been suggested to cause MIC under anoxic conditions. However, no microorganisms that perform acetogenesis-dependent MIC have been isolated or had their MIC-inducing mechanisms characterized. Here, we enriched and isolated acetogenic bacteria that induce iron corrosion by utilizing Fe(0) as the sole electron donor under freshwater, sulfate-free, and anoxic conditions. The enriched communities produced significantly larger amounts of Fe(II) than the abiotic controls and produced acetate coupled with Fe(0) oxidation prior to CH4 production. Microbial community analysis revealed that Sporomusa sp. and Desulfovibrio sp. dominated in the enrichments. Strain GT1, which is closely related to the acetogen Sporomusa sphaeroides, was eventually isolated from the enrichment. Strain GT1 grew acetogenetically with Fe(0) as the sole electron donor and enhanced iron corrosion, which is the first demonstration of MIC mediated by a pure culture of an acetogen. Other well-known acetogenic bacteria, including Sporomusa ovata and Acetobacterium spp., did not grow well on Fe(0). These results indicate that very few species of acetogens have specific mechanisms to efficiently utilize cathodic electrons derived from Fe(0) oxidation and induce iron corrosion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Edible blue-green algae reduce the production of pro-inflammatory cytokines by inhibiting NF-κB pathway in macrophages and splenocytes

    PubMed Central

    Ku, Chai Siah; Pham, Tho X.; Park, Youngki; Kim, Bohkyung; Shin, Min; Kang, Insoo; Lee, Jiyoung

    2013-01-01

    Background Chronic inflammation contributes to the development of pathological disorders including insulin resistance and atherosclerosis. Identification of anti-inflammatory natural products can prevent the inflammatory diseases. Methods Anti-inflammatory effects of blue-green algae (BGA), i.e., Nostoc commune var. Sphaeroides Kützing (NO) and Spirulina Platensis (SP), were compared in RAW 264.7 and mouse bone marrow-derived macrophages (BMM) as well as splenocytes from apolipoprotein E knockout (apoE−/−) mice fed BGA. Results When macrophages pretreated with 100 μg/ml NO lipid extract (NOE) or SP lipid extract (SPE) were activated by lipopolysaccharide (LPS), expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), and IL-6, were significantly repressed. NOE and SPE also significantly repressed the expression of TNFα and IL-1β in BMM. LPS-induced secretion of IL-6 was lower in splenocytes from apoE−/− fed an atherogenic diet containing 5% NO or SP for 12 weeks. In RAW 264.7 macrophages, NOE and SPE markedly decreased nuclear translocation of NF-κB. The degree of repression of pro-inflammatory gene expression by algal extracts was much stronger than that of SN50, an inhibitor of NF-κB nuclear translocation. Trichostatin A, a pan histone deacetylase inhibitor, increased basal expression of IL-1β and attenuated the repression of the gene expression by SPE. SPE significantly down-regulated mRNA abundance of 11 HDAC isoforms, consequently increasing acetylated histone 3 levels. Conclusion NOE and SPE repress pro-inflammatory cytokine expression and secretion in macrophages and splenocytes via inhibition of NF-κB pathway. Histone acetylation state is likely involved in the inhibition. General significance This study underscores natural products can exert anti-inflammatory effects by epigenetic modifications such as histone acetylation. PMID:23357040

  2. Volume changes and electrostriction in the primary photoreactions of various photosynthetic systems: estimation of dielectric coefficient in bacterial reaction centers and of the observed volume changes with the Drude-Nernst equation.

    PubMed

    Mauzerall, David; Hou, Jian-Min; Boichenko, Vladimir A

    2002-01-01

    Photoacoustics (PA) allows the determination of enthalpy and volume changes of photoreactions in photosynthetic reaction centers on the 0.1-10 mus time scale. These include the bacterial centers from Rb. sphaeroides, PS I and PS II centers from Synechocystis and in whole cells. In vitro and in vivo PA data on PS I and PS II revealed that both the volume change (-26 A(3)) and reaction enthalpy (-0.4 eV) in PS I are the same as those in the bacterial centers. However the volume change in PS II is small and the enthalpy far larger, -1 eV. Assigning the volume changes to electrostriction allows a coherent explanation of these observations. One can explain the large volume decrease in the bacterial centers with an effective dielectric coefficient of approximately 4. This is a unique approach to this parameter so important in estimation of protein energetics. The value of the volume contraction for PS I can only be explained if the acceptor is the super- cluster (Fe(4)S(4))(Cys(4)) with charge change from -1 to -2. The small volume change in PS II is explained by sub-mus electron transfer from Y(Z) anion to P(680) cation, in which charge is only moved from the Y(Z) anion to the Q(A) with no charge separation or with rapid proton transfer from oxidized Y(Z) to a polar region and thus very little change in electrostriction. At more acid pH equally rapid proton transfer from a neighboring histidine to a polar region may be caused by the electric field of the P(680) cation.

  3. Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

    PubMed Central

    Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

  4. [Construction and Characterization of B850-Only LH2 Energy Transfer System in Purple Bacteria].

    PubMed

    Li, Kai; Zhao, Chun-gui; Yue, Hui-ying; Yang, Su-ping; Qu, Yin-bo; Jiao, Nian-zhi

    2015-04-01

    To seek microscopic molecular mechanism of energy transfer and complex reconstitution in the photosynthesis, the conditions for construction of B850-only peripheral light-harvesting complex (LH2) and their properties were investigated using absorption, fluorescence spectroscopy, molecular sieve chromatography, ultrafiltration and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) from the purple bacteria. The results indicated that bacteriochlorophylls (BChl) of B800 incubated in 10 mmo · L(-1) Tris-HCl (pH 8.0) buffer are selectively released from their binding sites of LH2 of Rhodobacter azotoformans (A-LH2) by 0.08% (W/V) SDS. B850-only A-LH2 was constructed after removing free BChl mixing with 10% methyl alcohol by ultrafiltration. B850 BChl was released after A-LH2 was incubated for 240 min in dark at room temperature (RT). While BChl of B800 incubated in pH 1.9 buffer were selectively released from their binding sites of LH2 of Rhodopseudomonas palustris (P-LH2). The authors acquired two components using molecular sieve chromatography. Free BChl of one component was not removed and self-assembled to P-LH2. The other removed free BChl and B850-only P-LH2 was constructed. B850 unchanged after P-LH2 was incubated. P-LH2 α and β subunits have different molecular weights, but those of A-LH2 are in the contrary. It is concluded that B850-only P-LH2 is more stable than A-LH2. The enigmatic split of the B800 absorption band was not observed in these LH2, but we acquired two kinds of B800-released LH2 from Rhodopseudomonas palustris. The authors' results may provide a new light to separate homogeneous Apoprotein LH2.

  5. Insights into origin and evolution of α-proteobacterial gene transfer agents

    PubMed Central

    Shakya, Migun; Soucy, Shannon M

    2017-01-01

    Abstract Several bacterial and archaeal lineages produce nanostructures that morphologically resemble small tailed viruses, but, unlike most viruses, contain apparently random pieces of the host genome. Since these elements can deliver the packaged DNA to other cells, they were dubbed gene transfer agents (GTAs). Because many genes involved in GTA production have viral homologs, it has been hypothesized that the GTA ancestor was a virus. Whether GTAs represent an atypical virus, a defective virus, or a virus co-opted by the prokaryotes for some function, remains to be elucidated. To evaluate these possibilities, we examined the distribution and evolutionary histories of genes that encode a GTA in the α-proteobacterium Rhodobacter capsulatus (RcGTA). We report that although homologs of many individual RcGTA genes are abundant across bacteria and their viruses, RcGTA-like genomes are mainly found in one subclade of α-proteobacteria. When compared with the viral homologs, genes of the RcGTA-like genomes evolve significantly slower, and do not have higher %A+T nucleotides than their host chromosomes. Moreover, they appear to reside in stable regions of the bacterial chromosomes that are generally conserved across taxonomic orders. These findings argue against RcGTA being an atypical or a defective virus. Our phylogenetic analyses suggest that RcGTA ancestor likely originated in the lineage that gave rise to contemporary α-proteobacterial orders Rhizobiales, Rhodobacterales, Caulobacterales, Parvularculales, and Sphingomonadales, and since that time the RcGTA-like element has co-evolved with its host chromosomes. Such evolutionary history is compatible with maintenance of these elements by bacteria due to some selective advantage. As for many other prokaryotic traits, horizontal gene transfer played a substantial role in the evolution of RcGTA-like elements, not only in shaping its genome components within the orders, but also in occasional dissemination of Rc

  6. Environmental factors influencing gene transfer agent (GTA) mediated transduction in the subtropical ocean.

    PubMed

    McDaniel, Lauren D; Young, Elizabeth C; Ritchie, Kimberly B; Paul, John H

    2012-01-01

    Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs) of marine α-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain) were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's) in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10-30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI) and ambient bacterial abundance. These results indicate that GTA-mediated HGT in the

  7. Insights into origin and evolution of α-proteobacterial gene transfer agents.

    PubMed

    Shakya, Migun; Soucy, Shannon M; Zhaxybayeva, Olga

    2017-07-01

    Several bacterial and archaeal lineages produce nanostructures that morphologically resemble small tailed viruses, but, unlike most viruses, contain apparently random pieces of the host genome. Since these elements can deliver the packaged DNA to other cells, they were dubbed gene transfer agents (GTAs). Because many genes involved in GTA production have viral homologs, it has been hypothesized that the GTA ancestor was a virus. Whether GTAs represent an atypical virus, a defective virus, or a virus co-opted by the prokaryotes for some function, remains to be elucidated. To evaluate these possibilities, we examined the distribution and evolutionary histories of genes that encode a GTA in the α-proteobacterium Rhodobacter capsulatus (RcGTA). We report that although homologs of many individual RcGTA genes are abundant across bacteria and their viruses, RcGTA-like genomes are mainly found in one subclade of α-proteobacteria. When compared with the viral homologs, genes of the RcGTA-like genomes evolve significantly slower, and do not have higher %A+T nucleotides than their host chromosomes. Moreover, they appear to reside in stable regions of the bacterial chromosomes that are generally conserved across taxonomic orders. These findings argue against RcGTA being an atypical or a defective virus. Our phylogenetic analyses suggest that RcGTA ancestor likely originated in the lineage that gave rise to contemporary α-proteobacterial orders Rhizobiales , Rhodobacterales , Caulobacterales , Parvularculales , and Sphingomonadales , and since that time the RcGTA-like element has co-evolved with its host chromosomes. Such evolutionary history is compatible with maintenance of these elements by bacteria due to some selective advantage. As for many other prokaryotic traits, horizontal gene transfer played a substantial role in the evolution of RcGTA-like elements, not only in shaping its genome components within the orders, but also in occasional dissemination of Rc

  8. Functional and Evolutionary Characterization of a Gene Transfer Agent’s Multilocus “Genome”

    PubMed Central

    Hynes, Alexander P.; Shakya, Migun; Mercer, Ryan G.; Grüll, Marc P.; Bown, Luke; Davidson, Fraser; Steffen, Ekaterina; Matchem, Heidi; Peach, Mandy E.; Berger, Tim; Grebe, Katherine; Zhaxybayeva, Olga; Lang, Andrew S.

    2016-01-01

    Gene transfer agents (GTAs) are phage-like particles that can package and transfer a random piece of the producing cell’s genome, but are unable to transfer all the genes required for their own production. As such, GTAs represent an evolutionary conundrum: are they selfish genetic elements propagating through an unknown mechanism, defective viruses, or viral structures “repurposed” by cells for gene exchange, as their name implies? In Rhodobacter capsulatus, production of the R. capsulatus GTA (RcGTA) particles is associated with a cluster of genes resembling a small prophage. Utilizing transcriptomic, genetic and biochemical approaches, we report that the RcGTA “genome” consists of at least 24 genes distributed across five distinct loci. We demonstrate that, of these additional loci, two are involved in cell recognition and binding and one in the production and maturation of RcGTA particles. The five RcGTA “genome” loci are widespread within Rhodobacterales, but not all loci have the same evolutionary histories. Specifically, two of the loci have been subject to frequent, probably virus-mediated, gene transfer events. We argue that it is unlikely that RcGTA is a selfish genetic element. Instead, our findings are compatible with the scenario that RcGTA is a virus-derived element maintained by the producing organism due to a selective advantage of within-population gene exchange. The modularity of the RcGTA “genome” is presumably a result of selection on the host organism to retain GTA functionality. PMID:27343288

  9. Environmental Factors Influencing Gene Transfer Agent (GTA) Mediated Transduction in the Subtropical Ocean

    PubMed Central

    McDaniel, Lauren D.; Young, Elizabeth C.; Ritchie, Kimberly B.; Paul, John H.

    2012-01-01

    Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs) of marine α-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain) were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's) in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10–30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI) and ambient bacterial abundance. These results indicate that GTA-mediated HGT in the

  10. Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris*

    PubMed Central

    Satagopan, Sriram; Chan, Sum; Perry, L. Jeanne; Tabita, F. Robert

    2014-01-01

    The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a “closed” conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile165 and Met331) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala47) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme's kcat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature. PMID:24942737

  11. Structure-function studies with the unique hexameric form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodopseudomonas palustris.

    PubMed

    Satagopan, Sriram; Chan, Sum; Perry, L Jeanne; Tabita, F Robert

    2014-08-01

    The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a "closed" conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile(165) and Met(331)) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala(47)) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme's kcat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. A unique assemblage of cosmopolitan freshwater bacteria and higher community diversity differentiate an urbanized estuary from oligotrophic Lake Michigan

    PubMed Central

    Newton, Ryan J.; McLellan, Sandra L.

    2015-01-01

    Water quality is impacted significantly by urbanization. The delivery of increased nutrient loads to waterways is a primary characteristic of this land use change. Despite the recognized effects of nutrient loading on aquatic systems, the influence of urbanization on the bacterial community composition of these systems is not understood. We used massively-parallel sequencing of bacterial 16S rRNA genes to examine the bacterial assemblages in transect samples spanning the heavily urbanized estuary of Milwaukee, WI to the relatively un-impacted waters of Lake Michigan. With this approach, we found that genera and lineages common to freshwater lake epilimnia were common and abundant in both the high nutrient, urban-impacted waterways, and the low nutrient Lake Michigan. Although the two environments harbored many taxa in common, we identified a significant change in the community assemblage across the urban-influence gradient, and three distinct community features drove this change. First, we found the urban-influenced waterways harbored significantly greater bacterial richness and diversity than Lake Michigan (i.e., taxa augmentation). Second, we identified a shift in the relative abundance among common freshwater lineages, where acI, acTH1, Algoriphagus and LD12, had decreased representation and Limnohabitans, Polynucleobacter, and Rhodobacter had increased representation in the urban estuary. Third, by oligotyping 18 common freshwater genera/lineages, we found that oligotypes (highly resolved sequence clusters) within many of these genera/lineages had opposite preferences for the two environments. With these data, we suggest many of the defined cosmopolitan freshwater genera/lineages contain both oligotroph and more copiotroph species or populations, promoting the idea that within-genus lifestyle specialization, in addition to shifts in the dominance among core taxa and taxa augmentation, drive bacterial community change in urbanized waters. PMID:26483766

  13. Biodegradation Of Thiocyanate Using Microbial Consortia Cultured From Gold Mine Tailings

    NASA Astrophysics Data System (ADS)

    Moreau, J. W.; Watts, M. P.; Spurr, L. P.; Vu, H. P.

    2015-12-01

    Some bacteria possess the capability to degrade SCN-; therefore, harnessing this metabolic trait offers a biotechnological remediation strategy for SCN- produced in gold ore processing. A tailings storage facility (TSF) at a gold mine in Victoria, Australia holds large quantities of thiocyanate (SCN-) contaminated mine waste. The surface water in the TSF typically contains SCN- concentrations of >800 mg L-1, and seepage from the facility has contaminated the groundwater at the site. This study aimed to culture SCN-degrading microbes from the TSF, characterize the microbial consortia and test its operational parameters for use in a thiocyanate-degrading bioreactor. Surface samples were obtained from several locations around the TSF facility and used to inoculate medium reflective of the moderately saline and alkaline tailings water at the TSF, in the absence of organic carbon but subject to additions of phosphate and trace metals. Four microbial consortia capable of rapid SCN- degradation were successfully cultured. Sequencing of 16S rRNA genes found that the consortia were dominated by Thiobacillus species, a genus of known SCN- degraders. Lower abundances of other SCN- degraders; Sphingopyxis and Rhodobacter, were also identified. The impact of a number of geochemical conditions, including pH, temperature and SCN- concentration, upon the growth and SCN- degrading capacity of these consortia was determined. These results informed the optimization of a lab-scale thiocyanate degrading bioreactor. In summary, the cultured bacterial consortia proved effective towards SCN- degradation at the prevailing geochemical conditions of the TSF, requiring minimal nutrient additions. These consortia were dominated by genera of known autotrophic SCN- degraders. The comprehensive characterisation of these SCN- degrading consortia will provide the fundamental operational parameters required for deployme