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Effects of hydrogen bonding and the axial ligand interaction on the B800 band in two LH2 complexes Rhodopseudomonas (Rps.) acidophila and Rhodospirillum (Rs.) molischianum have been theo retically investigated by using density functional theory. The local electrostatic environment of the B800 bacteriochlorophyll is simulated as an atomic charge field consisting of the pigments in the protomer unit. Despite the fact that the B800 binding sites in two structures are very different, their absorption spectra are almost identical. Our calculations indicate that the charged axial ligand in Rs. molischianum and the hydrogen bonding in Rps. acidophila lead to similar red shifts, possibly explaining the above controversy. We also found (i) additional B800 bacteriochlorophyll transitions located between the Q and Soret regions for both LH2 complexes and (ii) the ligand to the B800 charge-transfer excited states in the long-wavelength region for the B800-alphaAsp(6) complex in,the Rs. molischianum LH2 system.

Plants use a set of light sensors to control their growth and development in response to changes in ambient light. In particular, phytochromes exert their regulatory activity by switching between a biologically inactive red-light-absorbing form (Pr) and an active far-red-light absorbing form (Pfr)1,2. Recently, biochemical and genetic studies have demonstrated the occurrence of phytochrome-like proteins in photosynthetic and non-photosynthetic bacteria3,4,5,6,7—but little is known about their functions. Here we report the discovery of a bacteriophytochrome located downstream from the photosynthesis gene cluster in a Bradyrhizobium strain symbiont of Aeschynomene. The synthesis of the complete photosynthetic apparatus is totally under the control of this bacteriophytochrome. A similar behaviour is observed for the closely related species Rhodopseudomonas palustris, but not for the more distant anoxygenic photosynthetic bacteria of the genus Rhodobacter, Rubrivivax or Rhodospirillum. Unlike other (bacterio)phytochromes, the carboxy-terminal domain of this bacteriophytochrome contains no histidine kinase features. This suggests a light signalling pathway involving direct protein–protein interaction with no phosphorelay cascade. This specific mechanism of regulation may represent an important ecological adaptation to optimize the plant–bacteria interaction.

1. Introduction 22. Structure of the bacterial PSU 52.1 Organization of the bacterial PSU 52.2 The crystal structure of the RC 92.3 The crystal structures of LH-II 112.4 Bacteriochlorophyll pairs in LH-II and the RC 132.5 Models of LH-I and the LH-I-RC complex 152.6 Model for the PSU 173. Excitation transfer in the PSU 183.1 Electronic excitations of BChls 22 3.1.1 Individual BChls 22 3.1.2 Rings of BChls 22 3.1.2.1 Exciton states 22 3.1.3 Effective Hamiltonian 24 3.1.4 Optical properties 25 3.1.5 The effect of disorder 263.2 Theory of excitation transfer 29 3.2.1 General theory 29 3.2.2 Mechanisms of excitation transfer 32 3.2.3 Approximation for long-range transfer 34 3.2.4 Transfer to exciton states 353.3 Rates for transfer processes in the PSU 37 3.3.1 Car→BChl transfer 37 3.3.1.1 Mechanism of Car→BChl transfer 39 3.3.1.2 Pathways of Car→BChl transfer 40 3.3.2 Efficiency of Car→BChl transfer 40 3.3.3 B800-B850 transfer 44 3.3.4 LH-II→LH-II transfer 44 3.3.5 LH-II→LH-I transfer 45 3.3.6 LH-I→RC transfer 45 3.3.7 Excitation migration in the PSU 46 3.3.8 Genetic basis of PSU assembly 494. Concluding remarks 535. Acknowledgments 556. References 55Life as we know it today exists largely because of photosynthesis, the process through which light energy is converted into chemical energy by plants, algae, and photosynthetic bacteria (Priestley, 1772; Barnes, 1893; Wurmser, 1925; Van Niel, 1941; Clayton & Sistrom, 1978; Blankenship et al. 1995; Ort & Yocum, 1996). Historically, photosynthetic organisms are grouped into two classes. When photosynthesis is carried out in the presence of air it is called oxygenic photosynthesis (Ort & Yocum, 1996). Otherwise, it is anoxygenic (Blankenship et al. 1995). Higher plants, algae and cyanobacteria perform oxygenic photosynthesis, which involves reduction of carbon dioxide to carbohydrate and oxidation of water to produce molecular oxygen. Some photosynthetic bacteria, such as purple bacteria, carry out anoxygenic photosynthesis that involves oxidation of molecules other than water. In spite of these differences, the general principles of energy transduction are the same in anoxygenic and oxygenic photosynthesis (Van Niel, 1931, 1941; Stanier, 1961; Wraight, 1982; Gest, 1993). The primary processes of photosynthesis involve absorption of photons by light-harvesting complexes (LHs), transfer of excitation energy from LHs to the photosynthetic reaction centers (RCs), and the primary charge separation across the photosynthetic membrane (Sauer, 1975; Knox, 1977; Fleming & van Grondelle, 1994; van Grondelle et al. 1994). In this article, we will focus on the anoxygenic photosynthetic process in purple bacteria, since its photosynthetic system is the most studied and best characterized during the past 50 years.

Cultured cells and environmental samples were used directly in PCRs without the isolation of DNA. Serial dilution was used to eliminate the inhibitory effect of materials in natural samples. Primers specific for pmoA, which encodes a subunit of the particulate methane monooxygenase, were used to detect and quantify methanotrophic bacteria by direct most probable number PCR. Phototrophic bacteria were detected in environmental samples by direct PCR with primers specific for pufM, and members of the bacterial domain were detected with primers for 16S rDNA. Direct PCR provides a rapid, simple, and sensitive method for detecting and quantifying bacteria in environmental samples. Detection of methanotrophic bacteria can be applied to monitoring bioremediation.

Changes in the carotenoid (Car) 1Bu+ absorption upon excitation (using a photon density of 1015photons cm-2) of bacteriochlorophyll (BChl) in the LH2 complex from Rhodospirillum molischianumwere traced with subpicosecond time resolution. The absorption changes could be empirically fit by two components showing a blue shift and a red shift in a ratio of 1:3.6. The shifts of Car absorption were explained in terms of interaction between change in dipole moment upon the 1Bu+ ← ground transition of Car and that upon the Qy ← ground transition of BChl. Calculations of the shifts for three different types of excitation in the B850 ring of the LH2 complex, i.e., (i) localized excitation on B850a or B850b, (ii) partially delocalized excitation over four B850s, and (iii) completely delocalized excitation over the B850 ring predicted the Car shifts with direction (relative magnitude), (i) a blue shift (1) or a red shift (3.3), (ii) a red shift (0.8), and (iii) a small red shift (0.2), respectively. Thus, the blue shift and the red shift components were attributed to localized excitations on B850a and B850b, respectively. The B850a excitation decayed rapidly (∼ 0.1 ps), whereas the B850b excitation decayed more slowly (∼ 0.4 ps) and then stayed; the former decay was ascribed to singlet−singlet annihilation between B850a and B850b, whereas the latter decay to singlet−singlet annihilation between B850b's. The unique feature of excitation with high photon density, which preferentially generates the localized excitations through multiple excitation, and enhances subsequent singlet−singlet annihilation reactions, is discussed.

Photosynthetic bacteria respond to alterations in light conditions by migrating to locations that allows optimal use of light as an energy source. Studies have indicated that photosynthesis-driven electron transport functions as an attractant signal for motility among purple photosynthetic bacteria. However, it is unclear just how the motility-based signal transduction system monitors electron flow through photosynthesis-driven electron transport. Recently, we have demonstrated that the purple photosynthetic bacterium Rhodospirillum centenum is capable of rapidly moving swarm cell colonies toward infrared light as well as away from visible light. Light-driven colony motility of R. centenum has allowed us to perform genetic dissection of the signaling pathway that affects photosynthesis-driven motility. In this study, we have undertaken sequence and mutational analyses of one of the components of a signal transduction pathway, Ptr, which appears responsible for transmitting a signal from the photosynthesis-driven electron transport chain to the chemotaxis signal transduction cascade. Mutational analysis demonstrates that cells disrupted for ptr are defective in altering motility in response to light, as well as defective in light-dependent release of methanol. We present a model which proposes that Ptr senses the redox state of a component in the photosynthetic cyclic electron transport chain and that Ptr is responsible for transmitting a signal to the chemotaxis machinery to induce a photosynthesis-dependent motility response.

International audience; The lateral organization of biological membranes is of great importance in many biological processes, both for the formation of specific structures such as super-complexes and for function as observed in signal transduction systems. Over the last years, AFM studies, particularly of bacterial photosynthetic membranes, have revealed that certain proteins are able to segregate into functional domains with a specific organization. Furthermore, the extended non-random nature of the organization has been suggested to be important for the energy and redox transport properties of these specialized membranes. In the work reported here, using a coarse-grained Monte Carlo approach, we have investigated the nature of interaction potentials able to drive the formation and segregation of specialized membrane domains from the rest of the membrane and furthermore how the internal organization of the segregated domains can be modulated by the interaction potentials. These simulations show that long-range interactions are necessary to allow formation of membrane domains of realistic structure. We suggest that such possibly non-specific interactions may be of great importance in the lateral organization of biological membranes in general and in photosynthetic systems in particular. Finally, we consider the possible molecular origins of such interactions and suggest a fundamental role for lipid-mediated interactions in driving the formation of specialized photosynthetic membrane domains. We call these lipid-mediated interactions a 'lipophobic effect.'

The carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum was examined at several potentials. The electron paramagnetic resonance (EPR) spectrum of CODH poised at approximately −295 mV exhibits a species (referred to as Cred1) that was previously attributed to [Fe4S4]C1+ (S = 1/2) weakly exchange-coupling with Ni2+ (S = 1) to yield apparent g-values of (gz,y,x = 2.03, 1.88, 1.71). UV−visible absorption spectroscopy showed only one [Fe4S4] cluster to be reduced at −295 mV. Based upon our assignment of S = 1/2 resonances in indigo carmine-poised C531A CODH (see Part 1: Staples, C. R.; Heo, J.; Spangler, N. J.; Kerby, R. L.; Roberts, G. P.; Ludden, P. W. J. Am. Chem. Soc. In press) to a [(COL)Fe3+-Ni2+-H-]4+ cluster, a careful search for similar resonances in the EPR spectrum of the enzyme state of wild-type CODH producing Cred1 was undertaken. Coupled putative [(COL)Fe3+-Ni2+-H-]4+ signals were observed in low intensity, which, in conjunction with the other assignments, prompted a reinterpretatio...

Nickel enzymes are a relatively new class of metalloenzymes. The seven known nickel enzymes are urease, hydrogenase, CO-dehydrogenase, methyl-coenzyme M reductase, Ni-superoxide dismutase, glyoxalase I and cis-trans isomerase. The requirement for nickel implies the presence of a nickel-processing system, since free transition metals are harmful to the cell. A nickel-processing system involves the recognition and transport of nickel into the cell and the handling of the nickel once it enters the cell until it is inserted into the nickel enzyme. Several mechanisms for nickel transport have been identified and will be reviewed here. Accessory proteins required for the biosynthesis of the nickel active site have been identified. Accessory proteins bind the nickel when it enters the cell and are proposed to assist with the insertion of nickel into the enzyme. The function of the characterized nickel-processing proteins is described, and models for nickel insertion into the nickel enzymes are presented.

The amino acid sequences of two very different high-potential iron-sulfur protein (HiPIP) isozymes have been determined from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salinarum. Iso-1 HiPIP, which is monomeric and contains 57 amino acid residues, is most similar to the Thiobacillus ferrooxidans iron-oxidizing enzyme (45% identity and a 6-residue deletion). On the other hand, iso-2 HiPIP, which is isolated as an oligomer, contains a peptide chain with 54 amino acid residues. It is the smallest reported to date and is only 31% identical to iso-1 HiPIP. A massive deletion of 17 residues is found at the N-terminus, such that only 2 residues remain prior to the first cysteine. Iso-2 HiPIP also has a 12-residue insertion and a 5-residue deletion. Prior to this study, there were only 2 absolutely conserved residues (Tyr 19 and Gly 75, Chromatium numbering) in addition to the 4 iron-sulfur cluster binding cysteine residues among the 13 HiPIPs sequenced to date. We found that Tyr 19 is absent in iso-2 HiPIP along with the entire N-terminal loop. Moreover, Gly 75 is substituted in both R. salinarum HiPIPs. These characteristics make the R. salinarum HiPIPs, and especially iso-2, the most divergent yet characterized.

Fourier transform near-infrared resonance Raman spectroscopy can be used to obtain information on the bacteriochlorophyll a (BChl a) molecules responsible for the redmost absorption band in photosynthetic complexes from purple bacteria. This technique is able to distinguish distortions of the bacteriochlorin macrocycle as small as 0.02 A, and a systematic analysis of those vibrational modes sensitive to BChl a macrocycle conformational changes was recently published [Näveke et al. (1997) J. Raman Spectrosc. 28, 599-604]. The conformation of the two BChl a molecules constituting the primary electron donor in bacterial reaction centers, and of the 850 and 880 nm-absorbing BChl a molecules in the light-harvesting LH2 and LH1 proteins, has been investigated using this technique. From this study it can be concluded that both BChl a molecules of the primary electron donor in the photochemical reaction center are in a conformation close to the relaxed conformation observed for pentacoordinate BChl a in diethyl ether. In contrast, the BChl a molecules responsible for the long-wavelength absorption transition in both LH1 and LH2 antenna complexes are considerably distorted, and furthermore there are noticeable differences between the conformations of the BChl molecules bound to the alpha- and beta-apoproteins. The molecular conformations of the pigments are very similar in all the antenna complexes investigated.

A phytochrome-like protein called Ppr was discovered in the purple photosynthetic bacterium Rhodospirillum centenum . Ppr has a photoactive yellow protein (PYP) amino-terminal domain, a central domain with similarity to phytochrome, and a carboxyl-terminal histidine kinase domain. Reconstitution experiments demonstrate that Ppr covalently attaches the blue light–absorbing chromophore p -hydroxycinnamic acid and that it has a photocycle that is spectrally similar to, but kinetically slower than, that of PYP. Ppr also regulates chalcone synthase gene expression in response to blue light with autophosphorylation inhibited in vitro by blue light. Phylogenetic analysis demonstrates that R. centenum Ppr may be ancestral to cyanobacterial and plant phytochromes.

In photosynthetic light-harvesting systems carotenoids and chlorophylls jointly absorb light and transform its energy within about a picosecond into electronic singlet excitations of the chlorophylls only. This paper investigates this process for the light-harvesting complex II of the purple bacterium Rhodospirillum molischianum, for which a structure and, hence, the exact arrangement of the participating bacteriochlorophylls and carotenoids have recently become known. Based on this structure and on CI expansions of the electronic states of individual chromophores ~bacteriochlorophylls and carotenoids! as well as on an exciton description of a circular aggregate of bacteriochlorophylls, the excitation transfer between carotenoids and bacteriochlorophylls is described by means of Fermi’s golden rule. The electronic coupling between the various electronic excitations is determined for all orders of multipoles ~Coulomb mechanism! and includes the electron exchange ~Dexter mechanism! term. The rates and efficiencies for different pathways of excitation transfer, e.g., 1 1 B u (carotenoid)!bacteriochlorophyll aggregate and 2 1 A g (carotenoid)! bacteriochlorophyll aggregate, are compared. The results show that in LH-II the Coulomb mechanism is dominant for the transfer of singlet excitations. The 1 1 B u !Qx pathway appears to be partially efficient, while the 2 1 A g !Qy pathway, in our description, which does not include vibrational levels, is inefficient. An improved treatment of the excitation transfer from the 2 1 Ag state is required to account for observed transfer rates. Exciton splitting of bacteriochlorophyll Qy excitations slightly accelerates the excitation transfer from the 2 1 Ag state, while it plays a crucial role in accelerating the transfer from the B800 BChl Qy state. Photoprotection of bacteriochlorophylls through triplet quenching is investigated, too. The results suggest that eight of the 16 B850 bacteriochlorophylls in LH-II of Rhodospirillum molischianumare protected well by eight carotenoids observed in the x-ray structure of the protein. The remaining eight B850 bacteriochlorophylls can transfer their triplet excitation energy efficiently to their neighboring protected bacteriochlorophylls. Eight B800 bacteriochlorophylls appear not to be protected well by the observed carotenoids. @S1063-651X~98!11007-3#

The mid-point potential (Em7.0) of the primary quinone acceptor (Qa) and the biochemical features (Em7.0 and apparent molecular mass, MM) of the membrane bound c-type cytochromes (cyt) involved in photosynthetic electron transfer of the halophilic phototrophic bacterium Rhodospirillum (Rs.) salinarum were determined. A tetrahemic RC bound cytochrome was found (MM of 39.8 kDa) with Em7.0 of the hemes equal to +304, +98, +21, −134 (± 8) mV as determined by dark equilibrium redox titrations in the isolated purified form. The highest potential heme (Em7.0 = +304 mV, α band at 556 nm) was able to reduce the photo-oxidized reaction center (P+) in a sub-millisecond (≤ 20 μs) time scale reaction, acting most likely as the direct electron donor to P+). The midpoint potential of the primary electron donor (Em7.0 = + 455 mV) was found to be close to that reported for the primary donor of the non-halophilic Rhodospirillum species Rs. rubrum, whereas the quinone primary electron acceptor (Qa) was different showing the spectral features of a menaquinone molecule with Em7.0 at −128 (± 5) mV. A membrane bound c-type heme with Em7.0 of 259 (± 1) and MM of 40 kDa was also isolated and referred to an orthodox cytochrome c1). The present data on the photosynthetic apparatus, along with the previous results on the respiratory system [Moschettini et al. (1997) Arch Microbiol 168: 302-309], suggest that Rs. salinarum is biochemically distinct from Rs. rubrum, the most representative specie of the genus.

The light-harvesting complex I (LH-I) of Rhodobacter sphaeroides has been modeled computationally as a hexadecamer of αβ-heterodimers, based on a close homology of the heterodimer to that of light-harvesting complex II (LH-II) of Rhodospirillum molischianum. The resulting LH-I structure yields an electron density projection map that is in agreement with an 8.5-Å resolution electron microscopic projection map for the highly homologous LH-I of Rs. rubrum. A complex of the modeled LH-I with the photosynthetic reaction center of the same species has been obtained by a constrained conformational search. This complex and the available structures of LH-II from Rs. molischianum and Rhodopseudomonas acidophila furnish a complete model of the pigment organization in the photosynthetic membrane of purple bacteria.

We have performed low-temperature Stark spectroscopy on a variety of different LH2 complexes from four photosynthetic bacteria, with the aim of characterizing the electric field response of the B800 and B850 absorption properties as a function of the protein environment. The following LH2 complexes were investigated: B800-850 and B800-820 of Rhodopseudomonas (Rps) acidophila; B800-850, B800-840 (αTyr+13→Phe), and B800-826 (αTyr+13→Phe, αTyr+14→Leu) of Rhodobacter (Rb.) sphaeroides; B800-850 and B800-830 (obtained at high LDAO) of Ectothiorhodospira sp.; and B800-850 of Rhodospirillum (Rsp.) molischianum. For all these cases the spectral blue shift of B850 has been assigned to the loss hydrogen-bonding interaction with the acetyl carbonyl of bacteriochlorophyll a. |Δμ| values for the 850 nm bands as well as for the blue-shifted bands are all on the order of 3-4.5 D/f. The loss of hydrogen-bonding interactions has only small effects on |Δμ| in these complexes. The values of the difference polarizability, Tr(Δαa), are large (600-1400 Å3/f2). The results are discussed in terms of crystal-structure-based models for LH2, in which pigment-pigment and pigment-protein interactions are considered; strong pigment-pigment interactions were found to be especially important. The values of |Δμ| for the 800 nm band are small, 1.0-1.5 D/f for LH2 complexes from Rb. sphaeroides and Rps. acidophila. However, in Rsp. molischianum and Ectothiorhodospira sp. |Δμ| values are much larger, of the order of 3 D/f. The difference in the B800 band is assigned to the difference in orientation of the B800 pigments in Rsp. molischianum and Ectothiorhodospira sp., as compared to the Rps. acidophila and Rb. sphaeroides. Due to the difference in orientation, the interactions of the Bchl a with the surrounding protein and neighboring carotenoid pigments are also not identical.

CMP-ketodeoxyoctonate (CMP-KDO) and analogs, including CMP-5-deoxy-5-fluoro-KDO, CMP-5-deoxy-KDO, and CMP-5-epi-KDO, were prepared from CTP and the corresponding KDO sugars catalyzed by CMP-KDO synthetase. These analogs were found to be much more stable than CMP-KDO (t1/2 = 0.57 h) yet less stable than CMP-sialic acid (t1/2 = 151 h). Fluorination at the 5-position of CMP-KDO has a 200-fold enhanced stability compared to the 156-fold enhancement for the 3R-fluoro analog, probably due to the loss of H-bonding interactions (for the 5-F derivative) and the cause of remote inductive effect (for the 3- and the 5-F analogs) on the glycosidic cleavage. Hydrolysis of CMP-KDO is perhaps facilitated by an intramolecular hydrogen bond from the 5-OH group with the phosphate oxygen as demonstrated by the 3-5-fold enhanced stability of CMP-5-epi-KDO and CMP-5-deoxy-KDO compared to CMP-KDO and by molecular modeling studies of water-solvated CMP-KDO. Hydrolysis of CMP-KDO also was found to be subject to a substantial solvent isotope effect (kH/kD = 2.7), which is significantly different from the reported solvent isotope effect for the hydrolysis of sialyglycosides (kH/kD = 0.86) and dependent on both buffer and magnesium ion concentrations. Considering these results and molecular modeling studies, it is proposed that the hydrolysis of CMP-KDO under neutral conditions proceeds through a glycosidic cleavage which occurs at the electronically favorable twist-boat conformation, facilitated by intramolecular H-bonding interaction of the 4-, 5- and 7- (or 8-) OH groups and the phosphate oxygen and by the leaving group magnesium ion complexation.

We model the spectra (absorption and circular dichroism) and excitation dynamics in the B800 ring of the LH2 antenna complex from Rs. molischianum using different theoretical approaches, i.e., Förster theory, standard and modified versions of the Redfield theory, and the more versatile nonperturbative approach based on hierarchically coupled equations for the reduced density operator. We demonstrate that, although excitations in the B800 ring are localized due to disorder, thermal effects, and phonons, there are still sizable excitonic effects producing shift, narrowing, and asymmetry of the spectra. Moreover, the excitation dynamics reveals the presence of long-lived (up to 1 ps) non-oscillatory coherences between the exciton states maintained due to nonsecular population-to-coherence transfers. The sub-ps decay of the coherences is followed by slow motion of the excitation around the ring, producing equilibration of the site populations with a time constant of about 3-4 ps, which is slower than the B800 → B850 transfer. The exact solution obtained with the hierarchical equations is compared with other approaches, thus illustrating limitations of the Förster and Redfield pictures. © 2013 American Chemical Society.

The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl-CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C(10-14) fatty acyl-CoA unit, one malonyl-CoA unit and two methylmalonyl-CoA units. We identified these products as 4-hydroxy-3-methyl-6-(1-methyl-2-oxoalkyl)pyran-2-ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl-CoA as the extender substrate. In addition, in vitro reactions with (13)C-labeled malonyl-CoA revealed that RpsA produced tetraketide 6-alkyl-4-hydroxy-1,5-dimethyl-2-oxocyclohexa-3,5-diene-1-carboxylic acids from C(14-20) fatty acyl-CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.

Photoactive yellow protein (PYP) is a photoreceptor that has been isolated from three halophilic phototrophic purple bacteria. The PYP from Ectothiorhodospira halophila BN9626 is the only member for which the sequence has been reported at the DNA level. Here we describe the cloning and sequencing of the genes encoding the PYPs from E.halophila SL-1 (type strain) and Rhodospirillum salexigens. The latter protein contains, like the E.halophila PYP, the chromophore trans p-coumaric acid, as we show here with high performance capillary zone electrophoresis. Additionally, we present evidence for the presence of a gene encoding a PYP homolog in Rhodobacter sphaeroides, the first genetically well-characterized bacterium in which this photoreceptor has been identified. An ORF downstream of the pyp gene from E.halophila encodes an enzyme, which is proposed to be involved in the biosynthesis of the chromophore of PYP. The pyp gene from E.halophila was used for heterologous overexpression in both Escherichia coli and R.sphaeroides, aimed at the development of a holoPYP overexpression system (an intact PYP, containing the p-coumaric acid chromophore and displaying the 446 nm absorbance band). In both organisms the protein could be detected immunologically, but its yellow color was not observed. Molecular genetic construction of a histidine-tagged version of PYP led to its 2500-fold overproduction in E.coli and simplified purification of the heterologously produced apoprotein. HoloPYP could be reconstituted by the addition of p-coumaric anhydride to the histidine-tagged apoPYP (PYP lacking its chromophore). We propose to call the family of photoactive yellow proteins the xanthopsins, in analogy with the rhodopsins.

The amino acid sequences of the B800/850 light-harvesting proteins from Rhodospirillum molischianum were determined by Edman degradation. On the basis of these amino acid sequences, two degenerated oligonucleotides were synthesized and used for PCR of genomic DNA. The resulting 150 bp DNA fragment was cloned, sequenced and used for subsequent Southern blot analysis of digested genomic DNA. A 2.3 kbp EcoRI fragment strongly hybridized to the probe and a size selected genomic library from genomic DNA was constructed. One clone scored positive during screening of the library with the PCR-fragment and subsequent DNA sequence analysis of the clone revealed the presence of three A-genes (A1A2A3) encoding alpha-polypeptides and of two B-genes (B1B2) encoding beta-polypeptides of the B800/850 complex. The arrangement of the different genes are B1A1, B2A2 and A3 where only B1 and B2 are preceded by typical Shine-Dalgarno sequences. In addition, typical nucleotide sequences for a rho-independent termination of transcription are located downstream of the genes A1 and A2. The deduced amino acid sequences revealed that the alpha-genes encoded for identical polypeptides, whereas the deduced beta-polypeptides differed in their amino acid sequence at four positions. Transcriptional operon analysis revealed that the genes A1B1 and A2B2 are both dicistronically transcribed, whereas the gene A3 is not.

Photoactive yellow protein (PYP) is a photoreceptor that has been isolated from three halophilic phototrophic purple bacteria. The PYP from Ectothiorhodospira halophila BN9626 is the only member for which the sequence has been reported at the DNA level. Here we describe the cloning and sequencing of the genes encoding the PYPs from E.halophila SL-1 (type strain) and Rhodospirillum salexigens. The latter protein contains, like the E.halophila PYP, the chromophore trans p-coumaric acid, as we show here with high performance capillary zone electrophoresis. Additionally, we present evidence for the presence of a gene encoding a PYP homolog in Rhodobacter sphaeroides, the first genetically well-characterized bacterium in which this photoreceptor has been identified. An ORF downstream of the pyp gene from E.halophila encodes an enzyme, which is proposed to be involved in the biosynthesis of the chromophore of PYP. The pyp gene from E.halophila was used for heterologous overexpression in both Escherichia coli and R.sphaeroides, aimed at the development of a holoPYP overexpression system (an intact PYP, containing the p-coumaric acid chromophore and displaying the 446 nm absorbance band). In both organisms the protein could be detected immunologically, but its yellow color was not observed. Molecular genetic construction of a histidine-tagged version of PYP led to its 2500-fold overproduction in E.coli and simplified purification of the heterologously produced apoprotein. HoloPYP could be reconstituted by the addition of p-coumaric anhydride to the histidine-tagged apoPYP (PYP lacking its chromophore). We propose to call the family of photoactive yellow proteins the xanthopsins, in analogy with the rhodopsins.

The structural elucidation of lipid A of the cell wall lipopolysaccharide (LPS) of Rhodospirillum salinarum 40 by chemical methods and laser desorption mass spectrometry revealed the presence of a mixed lipid A composed of three different 1,4'bisphosphorylated beta (1 --6)-linked backbone hexosaminyl-hexosamine disaccharides, i.e. those composed of GlcN --GlcN, 2,3-diamino-2,3-dideoxy-D-Glc-(DAG --DAG, and DAG --GlcN. Lipid A of R. salinarum contained preferentially 3-OH-18:0 and 3-OH-14:0 as amide-linked and cis delta 11-18:1 and c19:0 as ester-linked fatty acids. The mass spectra of the liberated acyl-oxyacyl residues proved the concomitant presence of 3-O-(cis delta 11-18:1)-18:0 and 3-O-(c19:0)-14:0 as the predominating diesters in this mixed lipid A. The glycosidically linked and the ester-linked phosphate groups of the backbone disaccharide were neither substituted by ethanolamine, phosphorylethanolamine, nor by 4-amino-4-deoxy-L-arabinose, in contrast to most of the enterobacterial lipid As. In the core oligosaccharide fraction, a HexA (1 --4)HexA(1 --5)Kdo-trisaccharide was identified by methylation analysis. The terminal HexA (hexuronic acid) is possibly 4-OMe-GalA, a component described here as an LPS constituent for the first time. LPS of R. salinarum showed a lethality in C57BL/10 ScSN (LPS-responder)-mice) of an order of 10(-1)-10(-2) of that reported for Salmonella abortus equi LPS, and it was also capable of inducing TNF alpha and IL6 in macrophages of C57BL/10ScSN mice.

Electronic ground nature of ferric cytochromes c ′ isolated from five photosynthetic bacteria, Chromatium vinosum ATCC 17899, Rhodobacter capsulatus ATCC 11166, Rhodopseudomonas palustris ATCC 17001, Rhodospirillum molischianum ATCC 14031, and Rhodospirillum rubrum ATCC 11170 has been investigated by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra indicate that the electronic ground state of five ferric cytochromes c ′ is a quantum mechanical admixed-spin state of a high spin ( S = 5 2 ) and an intermediate spin ( S = 3 2 ) at pH 7.2 and is high-spin state at pH 11.0. At physiological pH, however, the content of an intermediate spin state differs with the bacterial source of the protein: ≈ 50%, Chromatium vinosum ; ≈ 40%, Rhodobacter capsulatus and Rhodopseudomonas palustris ; ≈ 10%, Rhodospirillum molischianum and Rhodospirillum rubrum . Computer simulation of the spectra supports this diversity of the contribution of an intermediate spin state. Model studies of the ferric porphyrin complexes suggest that the correlation between content of an intermediate spin state and heme iron displacement from the mean heme plane. Therefore, the variation of the content of an intermediate spin state observed in the present study reflects the subtle difference in the degree of heme iron displacement among the proteins.

The mechanisms employed by purple photosynthetic bacteria to convert light to utilizable chemical energy have been a major focus of research over the past 50 years. Utilization of light by photosynthetic bacteria for other purposes, however, has received relatively little attention. The recent discovery of phototaxis by Rhodospirillum centenum provides new opportunities for biochemical and molecular biological analysis of sensory processes in purple bacteria.

The 16S rDNA of 17 strains of Azospirillum , 14 assigned to one of the known species A. amazonense A. brasilense A. halopraeferens A. irakense and A. lipoferum , and the other three of uncertain taxonomic position, was sequenced after polymerase chain reaction amplification and analysed in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. The phylogenetic analysis confirms that the genus Azospirillum constitutes a phylogenetically separate entity within the a subclass of Proteobacteria and that the five species are well defined. A. brasilense and A. lipoferum are closely related species and form one cluster together with A. halopraeferens ; the pair of species A. amazonense and A. irakense forms a second cluster in which Rhodospirillum centenum is also placed.

Many oxidoreductases are constructed from (a) local sites of strongly coupled substrate-redox cofactor partners participating in exchange of electron pairs, (b) electron pair/single electron transducing redox centers, and (c) nonadiabatic, long-distance, single-electron tunneling between weakly coupled redox centers. The latter is the subject of an expanding experimental program that seeks to manipulate, test, and apply the parameters of theory. New results from the photosynthetic reaction center protein confirm that the electronic-tunneling medium appears relatively homogeneous, with any variances evident having no impact on function, and that control of intraprotein rates and directional specificity rests on a combination of distance, free energy, and reorganization energy. Interprotein electron transfer between cytochrome c and the reaction center and in lactate dehydrogenase, a typical oxidoreductase from yeast, are examined. Rates of interprotein electron transfer appear to follow intraprotein guidelines with the added essential provision of binding forces to bring the cofactors of the reacting proteins into proximity.

Spectroscopic properties, including low-temperature absorbance, linear and circular dichroism and site-selection fluorescence of the antenna complexes from Rhodospirillum molischianum have been determined. The unique ‘LH1-like’ character of the amino acid sequence from LH2 of this bacterium is reflected in the circular dichroism of the B850 band of this complex. The wavelength dependence of the polarization of the LH2 complex shows an unusual shape that is attributed to the octameric state of this complex. The complete amino acid sequence for the LH1 α-polypeptide and most of the β-polypeptides are presented. These conform to the general features of other LH1 polypeptides. This result, in combination with spectroscopic data for LH1 imply that the organisation of the core in this bacterium is not much different from that in other purple non-sulphur bacteria.

Membranes isolated from light- and dark-grown cells of the recently established new taxon of the purple nonsulfur bacteria, Rhodoferax fermentans , gen. nov., sp. nov., have been examined. The results have been interpreted to show that the oxidative electron transport chain is branched at the ubiquinone level and does not involve rhodoquinone. Dark-grown membranes contain four b -type and three c -type membrane-bound cytochromes with E m7.0 of +371, +315, +76 and −18 mV and +298, +201 and +44 mV, respectively. No significant amount of soluble c was found in aerobic cells. Conversely, photosynthetically grown cells contain a soluble c -type haem ( α max at 551 nm, E m7.0 = +287 mV), four membrane-bound c -type haems with E m7.0 of +358, +296, +78 and −1 mV and three cytochromes b with E m7.0 of +320, +30 and −50 mV. Notably, the absence of cyt b -371 from light-grown membranes parallels the very low rate of cyt c oxidase activity catalyzed by this type of membrane. Oxido-reduction kinetics demonstrated that most of the c -type haems detected in light-grown membranes are not involved in respiration. These data suggest that the facultative phototroph Rf. fermentans is endowed with an electron transport system different rrom that of typical facultative phototrophs, e.g., Rhodobacter and Rhodospirillum species, but similar to that of green- and purple-nonsulfur genera such as Chloroflexus, Rhodocyclus and Rhodopseudomonas .

To ascertain the minimal structural requirements for formation of the subunit and core light-harvesting complex (LH1), the alpha- and beta-polypeptides of the LH1 from three purple photosynthetic bacteria were enzymatically or chemically truncated or modified. These polypeptides were then used in reconstitution experiments with bacteriochlorophyll a (BChla), and the formation of subunit and LH1 complexes was evaluated using absorbance and circular dichroism spectroscopies. Truncation or modification outside of the conserved core sequence region of the polypeptides had no effect on subunit or LH1 formation. However, the extent of formation and stability of the subunit and LH1 decreased as the polypeptide was shortened inside the core region within the N-terminal domain. This behavior was suggested to be due to the loss of potential ion-pairing and/or hydrogen-bonding interactions between the polypeptides. While the spectroscopic properties of the subunit complexes generated using truncated polypeptides were analogous to those obtained using native polypeptides, in some cases the resulting LH1 complex absorption was blue-shifted relative to the control. Thus, truncation within the N-terminal domain may have long-range effects on the immediate BChla binding environment, since the putative BChla binding site resides near the C-terminal end of the polypeptides. It was also demonstrated that the His located within the membrane-spanning domain on the N-terminal end of the beta-polypeptide is not participating in ligation of the BChla in the reconstituted subunit and therefore probably not in LH1.

Summary Laser desorption mass spectrometry of free lipid A of the phototrophic bacterium Rhodospirillum fulvum revealed a glucosamine disaccharide backbone, where the reducing glucosamine was substituted at C-1 by galacturonic acid (substituted by an unknown compound) and the nonreducing glucosamine at C-4’ by L -glycero- D-manno -heptose. The mass spectra of the liberated acyl-oxyacyl residues showed 3-O-(14:0)-16:0 and 3-O-(16:0)-16:0 as amide-linked and 3-O-(12:0)-14:0 as ester-linked diesters in the lipid A. Lipid A was free of phosphate, as indicated by a colorimetric test and and confirmed by 31 P NMR. The lipopolysaccharide of Rhodospirillum fulvum contained xylose, glucose, glucuronic and galacturonic acids, L -glycero- and D -glycero- D-manno -heptoses as well as 2-keto-3-deoxyoctonate. It had a lethality of about 1/10 from that reported for Salmonella abortus equi lipopolysaccharide and was able to induce TNFα and IL6 in macrophages of endotoxin responder C57BL/10 ScSN-mice.

Here we present the draft genome sequence of the versatile and adaptable purple photosynthetic bacterium Phaeospirillum molischianum DSM120. This study advances the understanding of the adaptability of this bacterium, as well as the differences between the Phaeospirillum and Rhodospirillum genera.

Here, we present the shotgun genome sequence of the purple photosynthetic bacterium Rhodospirillum photometricum DSM122. The photosynthetic apparatus of this bacterium has been particularly well studied by microscopy. The knowledge of the genome of this oversize bacterium will allow us to compare it with the other purple bacterial organisms to follow the evolution of the photosynthetic apparatus.

Three phylogenetically distinct groups within the alpha-proteobacteria which differ in trifolitoxin sensitivity are described. Trifolitoxin sensitivity was found in strains of Agrobacterium, Brucella, Mycoplana, Ochrobactrum, Phyllobacterium, Rhodobacter, Rhodopseudomonas, Rhodospirillum, and Rhizobium. Strains of Agrobacterium, Brucella, Phyllobacterium, Rhizobium, and Rhodospirillum were capable of producing trifolitoxin upon conjugal transfer of tfxABCDEFG.

Four peptides corresponding to alpha-helical regions delimited by residues 63-73 and 97-112 of cytochrome c2 (Rhodospirillum) and residues 24-36 and 45-55 of bovine calcium binding protein are predicted to be alpha-helical by a recently developed method [Rooman, M., Kocher, J.P., & Wodak, S.J. (1991) J. Mol. Biol. 221, 961-979], synthesized by solid phase methods, and purified by HPLC, and their solution conformations are determined by NMR and CD. The observed conformational properties of these peptides in solution confirmed prediction results: in water/TFE (60/40, v/v) at room temperature, these peptides adopt an alpha-helical conformation, as shown by an extended pattern of strong, sequential dNN(i,i + 1) NOE cross-peaks, d alpha N(i,i + 1) NOEs of reduced intensity, several medium-range [d alpha N(i,i + 3), d alpha N(i,i + 4), d alpha beta-(i,i + 3)] NOE connectivities, small 3JH alpha N values, and more upfield alpha-proton chemical shifts. CD studies at different TFE concentrations and at room temperature provide further evidence of the propensity of these peptides to adopt an alpha-helical conformation in solution, as determined by the ellipticity values at 222 nm, and by deconvolution of the CD spectra. According to the method used, helicities in the range 34-50% and 55-75% are found for the 63-73 and 97-112 fragments of cytochrome c2, respectively, and in the range 53-80% and 42-65% for the fragments 24-36 and 45-55 of calcium binding protein in water/TFE (60/40, v/v) at 298 K. In addition, the experiments and predictions agree for those residues that are more flexible. Finally, the relevance of our results for the protein folding pathways is discussed.

The kinetics of electron transfer of cytochrome c2 from Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodospirillum centenum to reaction centers from Rb. sphaeroides and Rb. capsulatus have been measured. Observed in the kinetics of decay of the oxidized donor are a rapid first-order rate and one or more slower rates that are due to diffusion-limited complex formation. For reaction centers from Rb. sphaeroides, the fast component had time constants of 1.0 and 0.5 microsecond for cytochrome c2 from Rb. sphaeroides and Rb. capsulatus, respectively, but only a slow component was observed for cytochrome c2 from Rs. centenum. For reaction centers from Rb. capsulatus, the kinetics from all three cytochromes had a fast component with time constants of 1.0, 0.7, and 1.9 microseconds for cytochrome c2 from Rb. sphaeroides, Rb. capsulatus, and Rs. centenum, respectively, although the dissociation constant for cytochrome c2 from Rs. centenum was approximately 20 times larger than that of the other cytochromes. The observation of the fast component for cytochrome c2 from Rs. centenum in reaction centers from Rb. capsulatus but not Rb. sphaeroides demonstrates that the binding interactions for the two reaction centers differ, and the involvement of amino acid residues in the binding is discussed. The kinetics of electron transfer from cytochrome c2 to reaction centers of Rb. sphaeroides from wild type and three mutant strains that have altered carboxyl-terminal regions of the M subunit of the reaction center have also been measured. For cytochrome c2 from Rb. sphaeroides, the kinetics are very similar between the mutants and wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

The structure of two types of isolated light-harvesting antenna complexes from Rhodospirillum molischianum was studied by electron microscopy and image analysis. The B870 reaction center complex forms an almost circular particle with a diameter in the plane of the membrane of about 10.7–11.2 nm. A complex consists of a reaction center surrounded most likely by 12 B870 antennae units. An asymmetrical protrusion, consisting of the cytochrome subunit, gives the reaction center a height of 11.5 nm. The peripheral B800–820 complex is cylindrical, with a diameter of 5.3 nm and a small central indentation. At low resolution, it is structurally very similar to the B800–850 complex of Rhodobacter sphaeroides and is considered to be most likely of the α 6 β 6 type.

Both reaction centers containing and lacking cytochrome c were obtained from a new halophilic photosynthetic bacterium Rhodospirillum (R.) sodomense. The cytochrome c has a very low reduction potential not to be reduced by Na-ascorbate. The special pair bands in the both reaction centers exists at around 850 nm, some 20 nm to the blue of that of a nonhalophilic species, R. rubrum.

Rhodospirillum salexigens is a moderately halophilic purple phototrophic bacterium which grows optimally in 8% NaCl. The amino acid sequences of the two principal soluble cytochromes c have been determined. One of these is a cytochrome c2, similar in size to mitochondrial cytochrome c. While clearly of the same sequence class as mitochondrial cytochrome c and the proteins from several other Gram-negative bacteria, it does not show particular affinity to any already known sequence in terms of the percentage sequence identity. The other protein is a cytochrome c', but is also a divergent member of this widespread group. The lack of appreciable sequence identity to other species is probably due to a limit of divergence which has been reached for the majority of purple bacterial species. However, the numbers of insertions and deletions and their locations in cytochromes c2 and c' suggest that R salexigens may be related to Rhodospirillum molischianum. Like other electron transport proteins from halophiles, both of these cytochromes are notable for their high content of arginine as compared with lysine and both are acidic. However, they do not show any particular sequence homology to electron transport proteins that have been characterized from the extremely halophilic phototrophes of the genus Ectothiorhodospira. Thus, it appears that adaptation to halophilic habitats has independently occurred more than once in purple bacteria.

The crystallographic structure of cytochrome c' from the purple phototrophic bacterium Chromatium vinosum (CVCP) has been determined at 1.8 A resolution using multiple isomorphous replacement. The molecule is a dimer, with each 131-residue chain folding as a four-helical bundle incorporating a covalently bound heme group at the core. This structure is the third of the ubiquitous cytochromes c' to be solved and is similar to the known structures of cytochrome c' from R. molischianum (RMCP) and R. rubrum (RRCP). CVCP is unique in exhibiting ligand-controlled dimer dissociation while RMCP and RRCP do not. The Tyr16 side-chain, which replaced Met16 in RMCP and Leu14 in RRCP, is parallel to the heme plane and located directly above the sixth ligand site of the heme Fe. Any ligand binding to this site, such as CO or CN-, must move the Tyr16 side-chain, which would be expected to cause other conformational changes of helix A, which contributes to the dimer interface, and consequently disrupting the dimer. Thus, the crystallographic structure of CVCP suggests a mechanism for dimer dissociation upon ligand binding. The dimer interface specificity is due to a lock and key shape complementarity of hydrophobic residues and not to any charge complementarity or cross-interface hydrogen bonds as is common in other protein-protein interfaces. The co-ordinates have been deposited in the Brookhaven Data Bank (entry P1BBH).

The phylogenetic structure of non-sulfur purple bacteria in the Proteobacteria α group was elucidated by the comparative analysis of 16S rRNA sequences from 29 strains of phototrophs and 14 strains of related non-phototrophs. The sequences of 12 strains including 7 isolates were determined in this study. The phototrophs in the α group were found to be extremely diversified and intermingled with non-phototrophs. Rhodopseudomonas species were dispersed into 3 lines of descent and Rhodospirillum species were dispersed into 5 lines. Marine organisms were composed of 4 lineages which were independent of each other and of freshwater lineages. Rhodospirillum fulvum, Rhodospirillum molischianum and the genus Magnetospirillum were found to be monophyletic.

Homogeneous detergent-solubilized B873 light-harvesting complexes from a carotenoid-less mutant of the purple non-sulfur bacterium, Rhodospirillum rubrum G9, were reassembled spontaneously into two-dimensional (2D) hexagonal arrays during extensive and controlled dialysis. As the complexes contain only 1 to 2 mol phospholipid per mol alpha beta dimer, the arrays formed by a self assembly process are primary due to protein-protein interactions. The hexagonal lattices were analyzed by negative stain electron microscopy and digital image processing. They exhibited a unit cell size of 12.3 nm, in close agreement with the particle diameter of the active photo-unit in native chromatophore membranes. The unit cell contains a central 5 nm stain-filled depression, embraced by a ring with an outer diameter of 10 nm.

Light-induced and diffusion-potential-induced absorption changes of carotenoids were studied in two species of photosynthetic bacteria in the genus Rhodospirillum. In Rs. photometricum, illumination or inside-negative diffusion potentials elicited a red shift of the carotenoid spectrum. However, in Rs. molischianum the spectral shift was in the opposite direction: i.e., a blue shift. The major carotenoids in both species were lycopene and rhodopin. The opposite shift direction can be explained by an opposite polarity of the permanent fields around the carotenoid molecules, which probably originates from the surrounding polypeptides.

Most of the currently known light-harvesting complexes 2 (LH2) rings are formed by 8 or 9 subunits. As of now, questions like "what factors govern the LH2 ring size?" and "are there other ring sizes possible?" remain largely unanswered. Here, we investigate by means of molecular dynamics (MD) simulations and stochastic modeling the possibility of predicting the size of an LH2 ring from the sole knowledge of the high resolution crystal structure of a single subunit. Starting with single subunits of two LH2 rings with known size, that is, an 8-ring from Rs. moliscianum (MOLI) and a 9-ring from Rps. acidophila (ACI), and one with unknown size (referred to as X), we build atomic models of subunit dimers corresponding to assumed 8-, 9-, and 10-ring geometries. After inserting each of the dimers into a lipid-water environment, we determine the preferred angle between the corresponding subunits by three methods: (1) energy minimization, (2) free MD simulations, and (3) potential of mean force calculations. We find that the results from all three methods are consistent with each other, and when taken together, it allows one to predict with reasonable level of confidence the sizes of the corresponding ring structures. One finds that X and ACI very likely form a 9-ring, while MOLI is more likely to form an 8-ring than a 9-ring. Finally, we discuss both the merits and limitations of all three prediction methods.

Light-harvesting bacteria Rhodospirillum photometricum were recently found to adopt strikingly different architectures depending on illumination conditions. We present analytic and numerical calculations which explain this observation by quantifying a dynamical interplay between excitation transfer kinetics and reaction center cycling. High light-intensity membranes exploit dissipation as a photoprotective mechanism, thereby safeguarding a steady supply of chemical energy, while low light-intensity membranes efficiently process unused illumination intensity by channeling it to open reaction centers. More generally, our analysis elucidates and quantifies the trade-offs in natural network design for solar energy conversion.