Chemolithotrophic growth of the phototrophic sulfur bacterium Thiocapsa roseopersicina

Chemotrophic growth capacities of the purple sulfur bacterium Thiocapsa roseopersicina strain M1 were studied in continuous culture under thiosulfate limitation.Pigment synthesis was completely inhibited upon a shift from anaerobic to semi-aerobic conditions (52 μM O₂) in the light, but no active breakdown occurred. During the transient state, the cells grew in a mixed photo- and chemolithotrophic mode; the specific respiration rate gradually increased with a concomitant drop in the bacteriochlorophyll a content. Photolithotrophically grown cells have the ability to respire. It was concluded that photosynthesis and respiration compete for electrons, but that photosynthesis is preferred under electron donor-limiting conditions, when the cells still contain large amounts of pigments. Eventually, a fully chemolithotrophic steady state was attained.The chemolithotropic growth of T. roseopersicina was studied in the dark under semiaerobic conditions at various dilution rates. The maximum specific growth rate was 68% of the maximum attainable growth rate under photolithotrophic conditions. The growth affinity for thiosulfate was high (K_m = 1.5 μM). The yield on thiosulfate under chemolithotrophic conditions exceeded that of thiobacilli. Oxygen uptake was studied in short-term experiments. It was shown that respiration in T. roseopersicina has a K_m of approx. 1 μM O₂. the ecological importance for T. roseopersicina of chemolithotrophic growth and pigment content is discussed with respect to the occurrence of T. roseopersicina in laminated microbial ecosystems and its possible competition with colorless sulfur bacteria.     

Modular Broad-Host-Range Expression Vectors for Single-Protein and Protein Complex Purification

A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC₂ protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.     

Experimental Study of Interactions between Purple and Green Sulfur Bacteria in Sandy Sediments Exposed to Illumination Deprived of Near-Infrared Wavelengths

Sedimentary biofilms of the green sulfur bacterium Prosthecochloris aestuarii strain CE 2404, the purple sulfur bacterium Thiocapsa roseopersicina strain 5811, and a mixed culture of both were cultured in fine sand (100- to 300-μm grain size) within counter gradients of oxygen and sulfide. The artificial sediments were exposed to illumination deprived of near-infrared light (NIR) by filtering out the wavelengths longer than 700 nm to simulate the critical light conditions in submerged aquatic sediments. A 16 h of visible light-8 h of dark regimen was used. We studied the effects of these light conditions on the metabolisms of and interactions between both species by comparing the single species biofilms with the mixed biofilm. The photosynthesis rates of P. aestuarii were shown to be highly limited by the imposed light conditions, because the sulfide photooxidation rates were strongly stimulated when NIR was added. T. roseopersicina performed both aerobic chemosynthesis and photosynthesis, but the photosynthesis rates were low and poorly stimulated by the addition of NIR. This species decreased the penetration depth of oxygen in the sediment by about 1 mm by actively respiring oxygen. This way, the strict anaerobe P. aestuarii was able to grow closer to the surface in the mixed culture. As a result, P. aestuarii benefited from the presence of T. roseopersicina in the mixed culture, which was reflected by an increase in the biomass. In contrast, the density of the latter species was almost completely unaffected by the interaction. Both species coexisted in a layer of the same depth in the mixed culture, and the ecological and evolutionary implications of coexistence are discussed.     

Interaction of HydSL hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina BBS with methyl viologen and positively charged polypeptides

The effect of polypeptides having different charge on the activity of Thiocapsa roseopersicina HydSL hydrogenase was studied. Strong inhibition was shown for poly-L-lysine bearing positive charge. The inhibition was reversible and competitive to methyl viologen, an electron acceptor, in the reaction of hydrogen oxidation catalyzed by the hydrogenase. Peptides carrying less positive charge had weaker inhibiting effect, while neutral and negatively charged peptides did not inhibit the hydrogenase. Molecular docking of poly-L-lysine to T. roseopersicina hydrogenase showed strong affinity of this polypeptide to the acceptor-binding site of the enzyme. The calculated binding constant is close to the experimentally measured value (K _i = 2.1 μM).     

Optical parameters of leaves of 30 plant species

Optical parameters (absorption coefficient k, infinite reflectance R∞, scattering coefficient 8) are tabulated for seven wavelengths and analyzed for statistical differences for 30 plant species. The wavelengths are: 550 nm (green reflectance peak), 650 nm (chlorophyll absorption band), 850 nm (infrared reflectance plateau), 1450 nm (water absorption band), 1650 nm (reflectance peak following water absorption band at 1450 nm), 1950 nm (water absorption band), and 2200 nm (reflectance peak following water absorption band at 1950 nm).     

Isolation and characterization of sulfur globule proteins from Chromatium vinosum and Thiocapsa roseopersicina

Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope. The proteins associated with sulfur globules of Chromatium vinosum and Thiocapsa roseopersicina were isolated by extraction into 50% aqueous acetonitrile containing 1% trifluoroacetic acid and 10 mM dithiothreitol. The extracted proteins were separated by reversed-phase HPLC, revealing three major proteins from C. vinosum and two from T. roseopersicina. All of these proteins have similar, rather unusual amino acid compositions, being rich in glycine and aromatic amino acids, particularly tyrosine. The molecular masses of the C. vinosum proteins were determined to be 10,498, 10,651, and 8,479 Da, while those from T. roseopersicina were found to be 10,661 and 8,759 Da by laser desorption time-of-flight mass spectrometry. The larger T. roseopersicina protein is N-terminally blocked, probably by acetylation, but small amounts of the unblocked form (mass = 10,619) were also isolated by HPLC. Protein sequencing showed that the two larger C. vinosum proteins are homologous to each other and to the large T. roseopersicina protein. The 8,479 Da C. vinosum and 8,759 Da T. roseopersicina proteins are also homologous, indicating that sulfur globule proteins are conserved between different species of purple sulfur bacteria.Abbreviations BNPS-skatole 2 (2-Nitrophenylsulfenyl)-3-methyl-3-bromoindolenine - CNB Cyanogen bromide - Cv1, Cv2, and Cv3 Chromatium vinosum sulfur globule proteins - SGP and SGPs Sulfur globule protein(s) - TFA Trifluoroacetic acid - Tr0, Tr1, and Tr2 Thiocapsa roseopersicina sulfur globule proteins     

Organic Thiols as Organolithotrophic Substrates for Growth of Phototrophic Bacteria

Rhodopseudomonas sp. strain BB1, isolated from a coastal marine sediment, immediately metabolized mercaptomalate when grown on mercaptomalate. Sulfide was detected as an intermediate. Extracts of cells grown on mercaptomalate converted mercaptomalate or 3-mercaptopropionate to equimolar amounts of sulfide and either fumarate or acrylate, respectively. Rhodopseudomonas sp. strain BB1 gave higher growth yields on mercaptomalate than on sulfide or malate, consistent with metabolism of the carbon chain of the thiol and the liberated sulfide; i.e., the organic thiol was an organolithotrophic substrate. In contrast, Thiocapsa roseopersicina, isolated previously from a marine microbial mat, had similar growth yields on sulfide, mercaptomalate, or 3-mercaptopropionate, with fumarate or acrylate accumulation from the thiols. T. roseopersicina did not grow photoorganotrophically on fumarate or acrylate, and the thiols were only a source of sulfide for photolithoautotrophic growth.     

Optical characteristics of the phototroph Thiocapsa roseopersicina and implications for real-time monitoring of the bacteriochlorophyll concentration.

Optical characteristics of a Thiocapsa roseopersicina culture and environmental samples containing T. roseopersicina were investigated in the spectral range of 400 to 1,100 nm (absorption coefficient, diffuse attenuation coefficient, and reflectance). Specific absorption coefficients of T. roseopersicina at wavelengths of 480, 520, 550, 580, 805, 860, and 880 nm were determined. It is suggested that the optical properties of T. roseopersicina in the near-infrared range of 800 to 930 nm, confirmed in this study, may be used for development of remote sensing techniques for real-time monitoring of T. roseopersicina and other bacteriochlorophyll a-containing microbes.     

Localization of hydrogenase in the purple sulphur bacterium Thiocapsa roseopersicina

The localization of hydrogenase in the phototrophic bacterium Thiocapsa roseopersicina was investigated by subcellular fractionations, and transmission electron microscopic immunocytochemistry. By using sonicated cells and measuring in vitro hydrogenase activities in soluble and membrane fractions, respectively, a weak hydrophobic interaction between the hydrogenase enzyme and the T. roseopersicina membranes was observed. Polyclonal antisera directed against the purified hydrogenase were raised in rabbits and exhibited one band in native-PAGE/Western immunoblot analysis. Native-PAGE/activity stain confirmed the identity of this band as being hydrogenase. Immunocytolocalization experiments using ultrathin sections showed an internal localization of the hydrogenase enzyme. A higher specific labeling was associated with chromatophores, indicating a possible coupling of hydrogenase with the photosynthetic membranes in the T. roseopersicina cells.     

Utilization of nitrogen compounds and ammonia assimilation by chromatiaceae

Chromatium vinosum strain D, Thiocapsa roseopersicina strain 6311 and Ectothiorhodospira mobilis strain 8112 were grown anaerobically in the light with various single nitrogen sources. When substituted for NH₄Cl only glutamine and casamino acids supported good growth of all strains tested. Peptone and urea were utilized by C. vinosum and T. roseopersicina, glutamate, asparagine and nitrate only by C. vinosum. The strains were able to grow with molecular nitrogen; complete inhibition of this growth was observed in the presence of alanine with E. mobilis, and of alanine or asparagine with T. roseopersicina.Glutamate dehydrogenase, requiring either NADH or NADPH, NADH-linked glutamate synthase, and glutamine synthetase were demonstrate in the above organisms grown on NH₄Cl.     

Isolation and some properties of components of nuclear polyhedra from the cabbage looper, Trichoplusia ni

Nuclear polyhedra obtained from diseased cabbage looper, Trichoplusia ni, were digested with sodium carbonate-saline buffer, pH 11.0. The dissolved polyhedra formed 3 general zones when subjected to density gradient centrifugation. The slowest sedimenting component (Zone 1) had an ultraviolet absorption curve typical of protein and a sedimentation coefficient of 11 S. Capsids, 310 × 40 nm, were located in Zone 2. Virus particles were found in 1–3 bands (Zone 3); those with envelopes measured 300 × 72 nm, and those without envelopes measured 300 × 33 nm. Virus preparations stained with phosphotungstic acid at pH 7.0 exhibited extensive disruption whereas preparations stained at pH 3.0 did not. Virus particles in the sodium carbonate-saline-digested polyhedra had a sedimentation coefficient of 1228 S. Virus particles isolated by high speed centrifugation had a sedimentation coefficient of 1530 S.     

Chromophoric dissolved organic matter (CDOM) absorption characteristics in relation to fluorescence in Lake Taihu,China, a large shallow subtropical lake

Absorption measurements from chromophoric dissolved organic matter (CDOM) and their relationships with dissolved organic carbon (DOC) and fluorescence were studied in Lake Taihu, a large, shallow, subtropical lake in China. Absorption spectra of lake water samples were measured from 240 nm to 800 nm. Highest values of a(λ), DOC and F _n (355) occurred near the river inflow to Meiliang Bay and decreased towards the central lake basin. A significant spatial difference was found between Meiliang Bay and the central lake basin in absorption coefficient, DOC-specific absorption coefficient, exponential slope coefficient, DOC concentration and fluorescence value. The spatial distribution of CDOM suggested that a major part of CDOM in the lake was from river input. CDOM absorption coefficients were correlated with DOC over the wavelength range 280–500 nm, and a(355) was also correlated with F _n (355), which showed that CDOM absorption could be inferred from DOC and fluorescence measurement. The coefficient of variation between a(λ) and DOC concentration decreased with increase in wavelength from 240 nm to 800 nm. Furthermore, a significant negative linear relationship was recorded between S value and CDOM absorption coefficient, as well as DOC-specific absorption coefficient. S value and DOC-specific absorption coefficient were used as a proxy for CDOM composition and source. Accurate CDOM absorption measurements are very useful in explaining UV attenuation and in developing, validating remote sensing model of water quality in Lake Taihu.     

Optical properties of the monomeric red fluorescent protein mRFP1

For the expression in E. coli, the PCR-amplified fragment encoding mRFP1 from vector pMT-mRFP1 (Fungal Genetic Stock Center) was placed in the pQE-60 vector. Chemically competent E. coli ER1821 were transformed and grown overnight at 37°C. The protein was purified by Ni-NTA chromatography and dialyzed against 67mM Na₂HPO₄, 67mM KH₂PO₄, pH 7.5. There are two peaks (at 503 and 584 nm) in the mRFP1 absorption spectrum. The green component (503 nm) may correspond to a green fraction of the protein (a fraction that never matures beyond the green intermediate or a fraction which is trapped as a dead-end product such as the nonproductive trans conformation for the F65-Q66 peptide bond). The mRFP1’s extinction coefficient is equal to 42 mM^?1 cm^?1 at 584 nm; the emission maximum is at 607 nm; the excitation maximum is at 584–586 nm; the Stokes shift is equal to 23 nm; the fluorescence lifetime is about 1.8 ns; the quantum yield is 0.27; pKa is 4.0. Analysis of the mRFP1 absorption spectrum by high-order derivative spectroscopy shows that electron transition systems of both the fully matured form (absorption maximum at 584 nm) and the green fraction of the protein (absorption maximum at 503 nm) are practically identical.     

p-Toluenesulfonyldiazoacetates: Reagents for photoaffinity labeling

p-Toluenesulfonyldiazoacetyl chloride and p-nitrophenyl p-toluenesulfonyldiazoacetate have been prepared and offer potential advantages as reagents for photoaffinity labeling. (i) The extinction coefficient for the sulfonyldiazo compounds at 370 nm is about 10 times that for the long wavelength absorption of other diazoesters; this absorption permits reasonably rapid photolysis in the presence of compounds that are destroyed by short wavelength uv radiation. (ii) The two derivatives named above are stable thermally; furthermore, since sulfonyldiazoesters are stable to acid and to weak base, photoaffinity labeling can be conducted over a wide range of pH. (iii) Photolysis of ordinary (i.e., oxygen) esters of sulfonyldiazo compounds in methanol or cyclohexane leads to insertion into the solvent to the exclusion of Wolff rearrangement; photolysis of thioesters at 350 nm in methanol gives about 25% insertion into solvent, accompanied by about 75% Wolff rearrangement; in contrast, photolysis of most thioesters of diazo derivatives leads exclusively to Wolff rearrangement     

The effect of eye colour pigments on the action spectrum of Drosophila

In vivo absorption spectra for Drosophila melanogaster eye colour pigment classes (drosopterins and ommatins) were constructed by subtracting the whole eye electroretinographic (ERG) spectral sensitivities of cn and bw respectively from the sensitivities of white-eyed strains. In situ microspectrophotometric (MSP) absorption spectra were also obtained. Both the ERG and MSP drosopterin spectra show a visible peak at 500 nm compared to the 480 nm peak of in vitro drosopterins. For the ommatins, the ERG absorption spectrum peaks at 450 nm while the MSP spectrum peaks at 400 and 525 nm. The ERG spectrum is similar to the in vitro absorption spectrum of xanthommatin while the MSP spectrum is similar to the in vitro absorption spectrum of reduced xanthommatin. The ERG absorption spectra for the drosopterins and the ommatins yield an accurate prediction of the effect of the combined pigments in wild-type eyes. Newly emerged and 7 day post-emergence bw flies show quantitatively similar pigment absorption effects while the drosopterins depress the sensitivity of newly emerged cn flies to a greater extent than that of cn flies 7 days after emergence.     

A Microsensor Study of the Interaction between Purple Sulfur and Green Sulfur Bacteria in Experimental Benthic Gradients

Abstract The interaction between the purple sulfur bacterium Thiocapsa roseopersicina and the green sulfur bacterium Prosthecochloris aestuarii was studied in a gradient chamber under a 16-hours light-8-hours dark regime. The effects of interaction were inferred by comparing the final outcome of a mixed culture experiment with those of the respective axenic cultures using the same inoculation densities and experimental conditions. Densities of bacteria were deduced from radiance microprofiles, and the chemical microenvironment was investigated with O₂, H₂S, and pH microelectrodes. P. aestuarii always formed a biofilm below the maximal oxygen penetration depth and its metabolism was strictly phototrophic. In contrast, T. roseopersicina formed a bilayer in both the mixed and the axenic culture. The top layer formed by the latter organism was exposed to oxygen, and chemotrophic sulfide oxidation took place during the dark periods, while the bottom layer grew phototrophically during the light periods only. In the mixed culture, the relative density of P. aestuarii was lower than in the axenic culture, which reflects the effects of the competition for sulfide. However, the relative density of T. roseopersicina was actually higher in the mixed culture than in the corresponding axenic culture, indicating a higher growth yield on sulfide in the mixed culture experiment. Several hypotheses are proposed to explain the effects of the interaction. Received: 15 June 1998; Accepted: 18 January 1999     

The relationship of the quaternary structure of allophycocyanin to its spectrum

Allophycocyanin II in its trimer form (α₃β₃) at pH 7.0 has an absorption maximum at 652 nm. This band is selectively reduced in intensity at pH 7.0 when various salts are added. The loss of 652 nm absorption follows the order: NaClO₄ ? NaNO₃ > NaBr > NaCl. When the NaClO₄ concentration is in the range 0.6-1.0 m the 652-nm band is entirely lost, and sedimentation equilibrium and velocity studies suggest that the trimer is completely dissociated to monomers (αβ). Hydrophobic interactions appear to be important in maintaining the trimer. The monomer absorption maximum is at 616 nm. A series of experiments using these salts demonstrated at intermediate 652-nm intensities and the two extrema that an isobestic point at 626 nm is present which indicates an equilibrium between two species. Corresponding to the loss of 652 nm absorption is the disappearance of 661 nm fluorescence emission and the appearance of a new band at 642 nm. Removal of the NaClO₄ by dialysis essentially restores the 652-nm absorption and 661-nm emission and the trimeric protein structure. The near ultraviolet region is only slightly perturbed during the loss of 652 nm absorption. In the absence of any additional salts these spectral changes also occur in pH 7.0 buffer at very low protein concentrations.     

Action Spectrum between 260 and 800 Nanometers for the Photoinduction of Carotenoid Biosynthesis in Neurospora crassa

An action spectrum for light-induced carotenoid biosynthesis in Neurospora crassa was determined in 4 to 20 nm steps from 260 to 800 nm. Four-day, dark-grown mycelial pads of N. crassa were exposed to varying amounts of monochromatic radiant energy and time. After a 48-hour incubation period at 6 C, carotenoid content was assayed spectrophotometrically in vivo. The action spectrum has maxima at 450 and 481 nm in the visible range and at 280 and 370 nm in the ultraviolet. A pigment synthesized by Neurospora whose absorption spectrum resembles the action spectrum is β-carotene.     

One-electron reactions in biochemical systems as studied by pulse radiolysis. VI. Stages in the reduction of ferricytochrome c

When ferricytochrome c at pH about 9 is reduced by hydrated electrons and/or CO₂^?, it gives rise to an unstable form of ferrocytochrome c whose absorption spectrum, particularly in the Soret region, differs from that of normal ferrocytochrome c. This form changes intramolecularly (life-time about 0.1 s at ambient temperature) to yield normal ferrocytochrome c, and by 0.5 s the change in absorption spectrum in the range 225–600 nm produced by e^?_aq and/or CO₂^? is identical to the final change produced by reduction with an equivalent amount of sodium dithionite. This shows that both e^?_aq and CO^?₂ reduce cytochrome c with practically 100% efficiency. In the range 600–800 nm the spectrum of the unstable form is the same as that of normal ferrocytochrome c, both having small absorptions at 695 nm as compared with ferricytochrome c. As the unstable form disappears however a further loss of absorption at 695 nm occurs. This is taken to imply that the unstable form decays to a second unstable form which then rapidly donates an electron to the unchanged neutral form of ferricytochrome c, so reducing absorption in the 695 nm band. Subsequent to this process the absorption in the 695 nm band increases over a period of minutes owing to re-equilibration between the neutral and alkaline formes of ferricytochrome c. Between pH 7 and 10 the effect of pH on the absorption changes is consistent with the hypothesis of a second unstable form of ferrocytochrome c. Additional phenomena arise in more alkaline solutions. The rates of the various unimolecular processes are thought to be determined by the rates of change of conformation of the protein parts of the molecule following the change in oxidation state.