Phytofluene as a Highly Efficient UVA Photosensitizer of Singlet Oxygen Generation.

Phytoene and phytofluene - uncolored C40 carotenoids with short chain of conjugated double bonds (3 and 5, respectively) - are known to be universal precursors in biosynthesis of colored carotenoids in photosynthesizing organisms. It is commonly recognized that C40 carotenoids are photoprotectors of cells and tissues. We have shown that phytofluene is an exception to this rule. By measuring photosensitized phosphorescence of singlet oxygen (1O2) we found out that phytofluene was very effective photosensitizer of 1O2 formation in aerated solutions under UVA irradiation (quantum yield of 85 ± 5%), whereas phytoene was almost inactive in this process. It was demonstrated that both carotenoids quench singlet oxygen in the dark. The obtained quenching rate constants [(4 ± 1) × 106 M-1·s-1 for phytoene and (2 ± 0.5) × 107 M-1·s-1 for phytofluene] were smaller than the rate constant of the diffusion-controlled reactions by 3-4 orders of magnitude. Thus, both carotenoids displayed rather weak protector properties. Moreover, phytofluene due to its high photosensitizing activity might be considered as a promoter of cell photodamage and a promising UVA photosensitizer for medical purposes.

Excitation energy transfer from the bacteriochlorophyll Soret band to carotenoids in the LH2 light-harvesting complex from Ectothiorhodospira haloalkaliphila is negligible.

Pathways of intramolecular conversion and intermolecular electronic excitation energy transfer (EET) in the photosynthetic apparatus of purple bacteria remain subject to debate. Here we experimentally tested the possibility of EET from the bacteriochlorophyll (BChl) Soret band to the singlet S2 level of carotenoids using femtosecond pump-probe measurements and steady-state fluorescence excitation and absorption measurements in the near-ultraviolet and visible spectral ranges. The efficiency of EET from the Soret band of BChl to S2 of the carotenoids in light-harvesting complex LH2 from the purple bacterium Ectothiorhodospira haloalkaliphila appeared not to exceed a few percent.

The genetic basis of anoxygenic photosynthetic arsenite oxidation.

'Photoarsenotrophy', the use of arsenite as an electron donor for anoxygenic photosynthesis, is thought to be an ancient form of phototrophy along with the photosynthetic oxidation of Fe(II), H2 S, H2 and NO2-. Photoarsenotrophy was recently identified from Paoha Island's (Mono Lake, CA) arsenic-rich hot springs. The genomes of several photoarsenotrophs revealed a gene cluster, arxB2AB1CD, where arxA is predicted to encode for the sole arsenite oxidase. The role of arxA in photosynthetic arsenite oxidation was confirmed by disrupting the gene in a representative photoarsenotrophic bacterium, resulting in the loss of light-dependent arsenite oxidation. In situ evidence of active photoarsenotrophic microbes was supported by arxA mRNA detection for the first time, in red-pigmented microbial mats within the hot springs of Paoha Island. This work expands on the genetics for photosynthesis coupled to new electron donors and elaborates on known mechanisms for arsenic metabolism, thereby highlighting the complexities of arsenic biogeochemical cycling.

The spirilloxanthine-pathway carotenoid incorporation into light-harvesting complexes of Ectothiorhodospira haloalkaliphila in vitro.

Carotenoidless light-harvesting complexes (DPA-complexes) LH1-RC and LH2 were isolated from the purple sulfur bacterium Ectothiorhodospira haloalkaliphila in which carotenoid biosynthesis was suppressed with diphenylamine (DPA). Carotenoids of the spirilloxanthine series, which were isolated from the same bacterium, were incorporated into the DPA-complexes in vitro with an efficiency of 95-100%. The comparison of characteristics of the complexes with the incorporated carotenoids and the control complexes showed that the LH2 complexes with the incorporated carotenoids restored their absorption spectra, circular dichroism signals, and energy transfer from carotenoids to bacteriochlorophyll, which indicates that carotenoids were correctly incorporated into the structure of this complex.

Distribution of colored carotenoids between light-harvesting complexes in the process of recovering carotenoid biosynthesis in Ectothiorhodospira haloalkaliphila cells.

The processes of recovering colored-carotenoid (Car) biosynthesis in Car-less cells of the purple sulfur bacterium Ectothiorhodospira haloalkaliphila grown with diphenylamine (DPA-cells) have been studied. It has been found that (1) the rate of recovering colored-Car biosynthesis in the lag-phase is far ahead of the growth rate of the cells themselves; (2) several Cars (ζ-carotene, neurosporene etc.) act as intermediates in Car biosynthesis; (3) because filling the "empty" Car pockets in the LH1-RC complexes is faster than in LH2, available spirilloxanthin is preferentially incorporated into the nascent LH1-RC core particles; (4) as a consequence of the resulting lack of spirilloxanthin availability, the biosynthetic intermediates (anhydrorhodovibrin, rhodopin and lycopene) fill the empty nascent LH2 Car pockets. In the present report, we further discuss the process of colored Car incorporation into LH complexes during the recovery of Car biosynthesis in the DPA-cells of Ect.haloalkaliphila.

Transformation of monothioarsenate by haloalkaliphilic, anoxygenic photosynthetic purple sulfur bacteria.

Thioarsenates are the dominant arsenic species in arsenic-rich, alkaline, and sulfidic waters, but bacterial interactions with these compounds have only recently been examined. Previous studies have shown that microorganisms play a role in the transformation of monothioarsenate to arsenate, including use of monothioarsenate as a chemolithotrophic electron donor coupled with oxygen as an electron acceptor. We obtained enrichment cultures from two saline, alkaline lakes (Mono Lake, CA and Big Soda Lake, NV) that are able to use monothioarsenate as the sole electron donor for anoxygenic photosynthesis. These anoxic cultures were able to convert a 1 mM mixture of thioarsenates completely to arsenate in c. 13 days and 4 mM monothioarsenate to arsenate in c. 17 days. This conversion was light dependent; thus, monothioarsenate can be used as the sole electron donor for anoxygenic photosynthesis. Both of the Mono Lake and Big Soda Lake enrichment cultures were dominated by an organism closely related to Ectothiorhodospira species. We tested additional strains of purple sulfur bacteria and found widespread ability to use monothioarsenate as an electron donor. The ability of bacteria to transform thioarsenates directly via anoxygenic photosynthesis adds a new perspective to the well-studied arsenic and sulfur cycles.

The LH2 complexes are assembled in the cells of purple sulfur bacterium Ectothiorhodospira haloalkaliphila with inhibition of carotenoid biosynthesis.

The effect of the inhibitor of carotenoid (Car) biosynthesis, diphenylamine (DPA), on the cells of the purple sulfur bacterium Ectothiorhodospira (Ect.) haloalkaliphila has been studied. There occurs an inhibition of the biosynthesis of colored Cars (≥99 %) at 71 µM DPA. Considering "empty" Car pockets (Moskalenko and Makhneva 2012) the content of Cars in the DPA-treated samples is first calculated more correctly. The total content of the colored Cars in the sample at 71 µM DPA does not exceed 1 % of the wild type. In the DPA-treated cells (membranes) a complete set of pigment-protein complexes is retained. The LH2 complex at 71 µM DPA is isolated, which is identical to the LH2 complex of the wild type in near IR absorption spectra. This suggests that the principles for assembling this LH2 complex in vivo in the absence of colored Cars remain the same. These results are in full agreement with the data obtained earlier for Allochromatium (Alc.) minutissimum (Moskalenko and Makhneva 2012). They are as follows: (1) DPA almost entirely inhibits the biosynthesis of the colored Cars in Ect. haloalkaliphila cells. (2) In the DPA-treated samples non-colored Cars are detected at 53.25 µM DPA (as traces) and at 71 µM DPA. (3) DPA may affect both phytoene synthase (at ≤71 µM DPA) and phytoene desaturase (at ≥53.25 µM DPA). (4) The assembly of LH2 complex does occur without any colored Cars.

Ultrafast hydrogen-bonding dynamics in the electronic excited state of photoactive yellow protein revealed by femtosecond stimulated Raman spectroscopy.

The ultrafast structural dynamics in the electronic excited state of photoactive yellow protein (PYP) is studied by femtosecond stimulated Raman spectroscopy. Stimulated Raman spectra in the electronic excited state, S(1), can be obtained by using a Raman pump pulse in resonance with the S(1)-S(0) transition. This is confirmed by comparing the experimental results with numerical calculations based on the density matrix treatment. We also investigate the hydrogen-bonding network surrounding the wild-type (WT)-PYP chromophore in the ground and excited states by comparing its stimulated Raman spectra with those of the E46Q-PYP mutant. We focus on the relative intensity of the Raman band at 1555 cm(-1), which includes both vinyl bond C═C stretching and ring vibrations and is sensitive to the hydrogen-bonding network around the phenolic oxygen of the chromophore. The relative intensity for the WT-PYP decreases after actinic excitation within the 150 fs time resolution and reaches a similar intensity to that for E46Q-PYP. These observations indicate that the WT-PYP hydrogen-bonding network is immediately rearranged in the electronic excited state to form a structure similar to that of E46Q-PYP.

Use of Raman spectroscopy for identification of compatible solutes in halophilic bacteria.

We explored the use of Raman spectroscopy to detect organic osmotic solutes as biomarkers in the moderately halophilic heterotrophic bacterium Halomonas elongata grown in complex medium (accumulation of glycine betaine) and in defined medium with glucose as carbon source (biosynthesis of ectoine), and in the anoxygenic phototrophic Ectothiorhodospira marismortui known to synthesize glycine betaine in combination with minor amounts of trehalose and N-α-carbamoyl glutamineamide. We tested different methods of preparation of the material: lyophilization, two-phase extraction of water-soluble molecules, and perchlorate extraction. Raman signals of glycine betaine and ectoine were detected; perchlorate extraction followed by desalting the extract on an ion retardation column gave the best results. Lyophilized cells of E. marismortui showed strong signals of carotenoid pigments, and glycine betaine could be detected only after perchlorate extraction and desalting. The data presented show that Raman spectroscopy is a suitable tool to assess the mode of osmotic adaptation used by halophilic microorganisms.

Description of Ectothiorhodospira salini sp. nov.

Strain JA430(T) is a Gram-negative, vibrioid to spiral shaped phototrophic purple sulfur bacterium isolated from anoxic sediment of a saltern at Kanyakumari in a mineral salts medium that contained 2% NaCl (w/v). Strain JA430(T) grows optimally at 5-6% NaCl and tolerates up to 12% NaCl. Intracellular photosynthetic membranes were of the lamellar type. Bacteriochlorophyll a and carotenoids of the spirilloxanthin series are present as photosynthetic pigments. Major cellular fatty acids are C(18:1)ω7c, C(16:0), C(19:0)cycloω8c and C(16:1)ω7c/C(16:1)ω6c. Strain JA430(T) exhibits photoorganoheterotrophy and chemoorganoheterotrophy and requires para-aminobenzoic acid, pantothenate and pyridoxal phosphate for growth. Phylogenetic analysis on the basis of 16S rRNA gene sequence analysis showed that strain JA430(T) forms monophyletic group in the genus Ectothiorhodospira. The highest sequence similarity for strain JA430(T) was found with the type strains of Ectothiorhodospira variabilis DSM 21381(T) (96.1%) and Ectothiorhodospira haloalkaliphila ATCC 51935(T) (96.2%). Morphological and physiological characteristics discriminate strain JA430(T) from other species of the genus Ectothiorhodospira, for which we describe this as a novel species, Ectothiorhodospira salini sp. nov. ( = NBRC 105915(T) = KCTC 5805(T)).

Arsenic(III) fuels anoxygenic photosynthesis in hot spring biofilms from Mono Lake, California.

Phylogenetic analysis indicates that microbial arsenic metabolism is ancient and probably extends back to the primordial Earth. In microbial biofilms growing on the rock surfaces of anoxic brine pools fed by hot springs containing arsenite and sulfide at high concentrations, we discovered light-dependent oxidation of arsenite [As(III)] to arsenate [As(V)] occurring under anoxic conditions. The communities were composed primarily of Ectothiorhodospira-like purple bacteria or Oscillatoria-like cyanobacteria. A pure culture of a photosynthetic bacterium grew as a photoautotroph when As(III) was used as the sole photosynthetic electron donor. The strain contained genes encoding a putative As(V) reductase but no detectable homologs of the As(III) oxidase genes of aerobic chemolithotrophs, suggesting a reverse functionality for the reductase. Production of As(V) by anoxygenic photosynthesis probably opened niches for primordial Earth's first As(V)-respiring prokaryotes.

Arsenite-dependent photoautotrophy by an Ectothiorhodospira-dominated consortium.

Over the past decade numerous lineages of bacteria have been shown to obtain energy for growth through redox transformations of arsenic. However, phototrophic growth using reduced arsenic as an electron donor has not been described. Here we report the light-dependent oxidation of arsenite to arsenate, coupled with autotrophic growth, by an Ectothiorhodospira-dominated consortium of bacteria from alkaline, hypersaline Mono Lake, California. Pure cultures of the Mono Lake Ectothiorhodospira were not capable of phototrophic arsenite oxidation under the culture conditions tested. Electron micrographs of the culture showed a close association between consortia members, although the specific contribution of the individual bacteria is currently unknown. This report extends the list of compounds known to support anoxygenic photosynthesis and documents a previously unknown pathway in arsenic geochemistry.

Conservation and specialization in PAS domain dynamics.

The PAS (Per-ARNT-Sim) superfamily is presented as a well-suited study case to demonstrate how comparison of functional motions among distant homologous proteins with conserved fold characteristics may give insight into their functional specialization. Based on the importance of structural flexibility of the receptive structures in anticipating the signal-induced conformational changes of these sensory systems, the dynamics of these structures were analysed. Molecular dynamics was proved to be an effective method to obtain a reliable picture of the dynamics of the crystal structures of HERG, phy3, PYP and FixL, provided that an extensive conformational space sampling is performed. Other reliable sources of dynamic information were the ensembles of NMR structures of hPASK, HIF-2alpha and PYP. Essential dynamics analysis was successfully employed to extract the relevant information from the sampled conformational spaces. Comparison of motion patterns in the essential subspaces, based on the structural alignment, allowed identification of the specialized region in each domain. This appears to be evolved in the superfamily by following a specific trend, that also suggests the presence of a limited number of general solutions adopted by the PAS domains to sense external signals. These findings may give insight into unknown mechanisms of PAS domains and guide further experimental studies.

Structure of photoactive yellow protein (PYP) E46Q mutant at 1.2 A resolution suggests how Glu46 controls the spectroscopic and kinetic characteristics of PYP.

Photoactive yellow protein from Ectothiorhodospira halophila is a photoreceptor protein involved in the negative phototaxis of this bacterium. Its chromophore (p-coumaric acid) is deprotonated in the ground state, which is stabilized by a hydrogen-bond network between Tyr42, Glu46 and Thr50. Glu46 is a key residue as it has been suggested that the proton at Glu46 is transferred to the chromophore during its photoconversion from the dark state to the signalling state. The structure of E46Q mutant protein was determined at 1.2 A resolution, revealing that the phenolic O atom of p-coumaric acid is hydrogen bonded to NH(2) of Gln46 in E46Q with a longer distance (2.86 +/- 0.02 A) than its distance (2.51 A) to Glu46 OH in the wild type. This and the decreased thermal stability of E46Q relative to the wild type show that this hydrogen bond is weakened in the E46Q mutant compared with the corresponding bond in the wild type. Several characteristic features of E46Q such as an alkali shift in the pK(a) and the rapid photocycle can be explained by this weakened hydrogen bond. Furthermore, the red shift in the absorption maximum in E46Q can be explained by the delocalization of the electron on the phenolic oxygen of p-coumaric acid owing to the weakening of this hydrogen bond.

PAS domains. Common structure and common flexibility.

PAS (PER-ARNT-SIM) domains are a family of sensor protein domains involved in signal transduction in a wide range of organisms. Recent structural studies have revealed that these domains contain a structurally conserved alpha/beta-fold, whereas almost no conservation is observed at the amino acid sequence level. The photoactive yellow protein, a bacterial light sensor, has been proposed as the PAS structural prototype yet contains an N-terminal helix-turn-helix motif not found in other PAS domains. Here we describe the atomic resolution structure of a photoactive yellow protein deletion mutant lacking this motif, revealing that the PAS domain is indeed able to fold independently and is not affected by the removal of these residues. Computer simulations of currently known PAS domain structures reveal that these domains are not only structurally conserved but are also similar in their conformational flexibilities. The observed motions point to a possible common mechanism for communicating ligand binding/activation to downstream transducer proteins.

Kinetics of proton uptake and dye binding by photoactive yellow protein in wild type and in the E46Q and E46A mutants.

We studied the kinetics of proton uptake and release by photoactive yellow protein (PYP) from Ectothiorhodospira halophila in wild type and the E46Q and E46A mutants by transient absorption spectroscopy with the pH-indicator dyes bromocresol purple or cresol red in unbuffered solution. In parallel, we investigated the kinetics of chromophore protonation as monitored by the rise and decay of the blue-shifted state I(2) (lambda(max) = 355 nm). For wild type the proton uptake kinetics is synchronized with the fast phase of I(2) formation (tau = 500 micros at pH 6.2). The transient absorption signal from the dye also contains a slower component which is not due to dye deprotonation but is caused by dye binding to a hydrophobic patch that is transiently exposed in the structurally changed and partially unfolded I(2) intermediate. This conclusion is based on the wavelength, pH, and concentration dependence of the dye signal and on dye measurements in the presence of buffer. SVD analysis, moreover, indicates the presence of two components in the dye signal: protonation and dye binding. The dye binding has a rise time of about 4 ms and is coupled kinetically with a transition between two I(2) intermediates. In the mutant E46Q, which lacks the putative internal proton donor E46, the formation of I(2) is accelerated, but the proton uptake kinetics remains kinetically coupled to the fast phase of I(2) formation (tau = 100 micros at pH 6.3). For this mutant the protein conformational change, as monitored by the dye binding, occurs with about the same time constant as in wild type but with reduced amplitude. In the alkaline form of the mutant E46A the formation of the I(2)-like intermediate is even faster as is the proton uptake (tau = 20 micros at pH 8.3). No dye binding occurred in E46A, suggesting the absence of a conformational change. In all of the systems proton release is synchronized with the decay of I(2). Our results support mechanisms in which the chromophore of PYP is protonated directly from the external medium rather than by the internal donor E46.

Structure and characterization of Ectothiorhodospira vacuolata cytochrome b(558), a prokaryotic homologue of cytochrome b(5).

A soluble cytochrome b(558) from the purple phototropic bacterium Ectothiorhodospira vacuolata was completely sequenced by a combination of automated Edman degradation and mass spectrometry. The protein, with a measured mass of 10,094.7 Da, contains 90 residues and binds a single protoheme. Unexpectedly, the sequence shows homology to eukaryotic cytochromes b(5). As no prokaryotic homologue had been reported so far, we developed a protocol for the expression, purification, and crystallization of recombinant cytochrome b(558). The structure was solved by molecular replacement to a resolution of 1.65 A. It shows that cytochrome b(558) is indeed the first bacterial cytochrome b(5) to be characterized and differs from its eukaryotic counterparts by the presence of a disulfide bridge and a four-residue insertion in front of the sixth ligand (histidine). Eukaryotes contain a variety of b(5) homologues, including soluble and membrane-bound multifunctional proteins as well as multidomain enzymes such as sulfite oxidase, fatty-acid desaturase, nitrate reductase, and lactate dehydrogenase. A search of the Mycobacterium tuberculosis genome showed that a previously unidentified gene encodes a fatty-acid desaturase with an N-terminal b(5) domain. Thus, it may provide another example of a bacterial b(5) homologue.

Light-induced generation of electric potential difference in membranes of purple and green sulfur bacteria.

When associated with a planar phospholipid membrane, chromatophores isolated from photosynthetic sulfur bacteria Chromatium minutissimum, Ectothiorhodospira shaposhnikovii, and Chlorobium limicola f. thiosulfatophilum were shown to generate a light-induced transmembrane electric potential difference measured by a direct method using macroelectrodes and a voltmeter. The maximal photoelectric responses were observed upon the addition of 1,4-naphthoquinone in combination with phenazine methosulfate (or TMPD) and ascorbate. The photoeffects were inhibited by CCCP and gramicidin. The data demonstrate that similar mechanisms of photoelectric generation function in membranes of the different bacteria studied.