Growth of the phototrophic purple sulfur bacterium Thiocapsaroseopersicina under oxic/anoxic regimens in the light

Abstract The phototrophic purple sulfur bacterium Thiocapsa roseopersicina was grown in sulfide-limited continuous cultures exposed to oxic/anoxic regimens in continuous light. Synthesis of bacteriochlorophyll a (BChl a ) did not occur during the oxic periods, but started immediately upon the creation of anoxic conditions. In contrast, protein synthesis continued during both oxic and anoxic periods. Consequently, the specific content of BChla fluctuated. Despite the presence of oxygen and the fluctuating BChl a content, growth occurred predominantly in a phototrophic mode and respiration was virtually zero.
BChl a synthesis continued at high rates during anoxic periods, thus compensating for the lack of synthesis during oxic periods. When cultivated under regimens with oxic periods shorter than 12 h the highest specific BCh a content was 27 μg·mg⁻ protein. In contrast, when cultivated under regimens with oxic periods longer than 12 h the specific BChl a content was always lower than 27μg·mg⁻ length of the oxic periods. During the anoxic periods, BChl a synthesis occurred at the maximal velocity of 1.2 μg·mg⁻¹ protein·h⁻, but the length of the anoxic periods was not sufficient to allow the BChl a content to reach the maximum level.
Cultivation under continuously oxic conditions eventually resulted in pigmentless cells growing chemolithotrophically. The BChl a synthesizing ability was not lost during prolonged exposure to oxygen.
It was concluded that T. roseopersicina is very well adapted to oxic/anoxic cycles.     

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.     

Simultaneous phototrophic and chemotrophic growth in the purple sulfur bacterium Thiocapsa roseopersicina M1

Abstract The anoxygenic phototrophic purple sulfur bacterium Thiocapsa roseopersicina was grown in illuminated continuous cultures with thiosulfate as growth limiting substrate. Aeration resulted in completely colorless cells growing chemotrophically, whereafter the conditions were changed to a 23 h oxic/1 h anoxic regime. After 11 volume changes at a dilution rate of 0.031 h⁻¹ (35% of μ_max) a time dependent equilibrium was established. During the 23 h oxic periods bacteriochlorophyll a synthesis (BChl a ) was not observed, whereas during the 1 h anoxic periods synthesis was maximal (i.e. 1.1 μg (mg protein)⁻¹ h⁻¹). As a result the BChl a concentration gradually increased from zero to an average value over 24 h of 1.9 μg (mg protein)⁻¹. Concomitantly, the protein concentration increased from 13.9 mg 1⁻¹ during continuous oxic conditions to 28.8 mg 1⁻¹. For comparison, the protein concentration during fully phototrophic growth at an identical thiosulfate concentration in the inflowing medium was 53.7 mg 1⁻¹. The specific respiration rate was 8 μmol O₂ (mg protein)⁻¹ h⁻¹ during full chemotrophic growth and gradually decreased to 3.5 μmol O₂ (mg protein)⁻¹ h⁻¹ after 11 volume changes at the regime employed. These data show that T. rosepersicina is able to simultaneously utilize light and aerobic respiration of thiosulfate as sources of energy. The ecological relevance of the data is discussed.     

Evidence of covalent modification of glutamine synthetase in the purple sulfur bacterium Thiocapsa roseopersicina

Abstract It was shown that glutamine synthetase of purple sulfur bacterium Thiocapsa roseopersicina is regulated by covalent modification. This conclusion is made on the basis of results showing that: (i) incubation of cells under conditions of nitrogen deprivation in the light lead to an increase of glutamine synthetase activity; (ii) addition of ammonium to nitrogen-starved cell suspensions caused a rapid decrease of glutamine synthetase activity; (iii) inhibition of glutamine synthetase by feedback modifiers was higher in ammonium-treated cells than in those starved for a nitrogen source; (iv) treatment of purified glutamine synthetase and cell-free extracts with phosphodiesterase was accompanied by an increase of glutamine synthetase activity, indicating the cleavage of modifying residues covalently bound to glutamine synthetase molecules.     

Thiocapsa halophila sp. nov., a new halophilic phototrophic purple sulfur bacterium

A new phototrophic sulfur bacterium has been isolated from a red layer in a laminated mat occurring underneath a gypsum crust in the mediterranean salterns of Salin-de-Giraud (Camargue, France). Single cells were coccus-shaped, non motile, without gas vacuoles and contained sulfur globules. Bacteriochlorophyll a and okenone were present as major photosynthetic pigments. These properties and the G+C content of DNA (65.9–66.6 mol% G+C) are typical characteristics of the genus Thiocapsa. However, the new isolate differs from known species in the genus, particularly in NaCl requirement (optimum, 7% NaCl; range, 3–20% NaCl) and some physiological characteristics. Therefore, a new species is proposed, Thiocapsa halophila, sp. nov.Dedicated to Prof. Dr. Norbert Pfennig in occasion of his 65th birthday     

The capacity of phototrophic sulfur bacterium Thiocapsa roseopersicina for chemosynthesis

Purple sulfur bacterium Thiocapsa roseopersicina strain BBS requiring vitamin B₁₂ may grow in the dark in media containing no other organic compounds. Under such conditions the cells oxidize sulfide and thiosulfate with the use of O₂ and assimilate carbon dioxide. After 10–30 s assimilation of NaH¹⁴CO₃ about 60% of radioactivity is found in phosphorylated compounds characteristic for the reductive pentose phosphate cycle. The possibility of the function of this cycle in the dark in the presence of O₂ is confirmed by the capacity of cells grown under such conditions to synthesize ribulose-1,5-diphosphate carboxylase. All this evidence suggests the ability of T. roseopersicina to change from phototrophy to aerobic chemolithoautotrophy.     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

<Emphasis Type="Italic">Thiocapsa imhoffii</Emphasis>, sp. nov., an alkaliphilic purple sulfur bacterium of the family <Emphasis Type="Italic">Chromatiaceae</Emphasis> from Soap Lake,Washington (USA)

An alkaliphilic purple sulfur bacterium, strain SC5, was isolated from Soap Lake, a soda lake located in east central Washington state (USA). Cells of strain SC5 were gram-negative, non-motile, and non-gas vesiculate cocci, often observed in pairs or tetrads. In the presence of sulfide, elemental sulfur was deposited internally. Liquid cultures were pink to rose red in color. Cells contained bacteriochlorophyll a and spirilloxanthin as major photosynthetic pigments. Internal photosynthetic membranes were of the vesicular type. Optimal growth of strain SC5 occurred in the absence of NaCl (range 0–4%), pH 8.5 (range pH 7.5–9.5), and 32°C. Photoheterotrophic growth occurred in the presence of sulfide or thiosulfate with only a limited number of organic carbon sources. Growth factors were not required, and cells could fix N₂. Dark, microaerobic growth occurred in the presence of both an organic carbon source and thiosulfate. Sulfide and thiosulfate served as electron donors for photoautotrophy, which required elevated levels of CO₂. Phylogenetic analysis placed strain SC5 basal to the clade of the genus Thiocapsa in the family Chromatiaceae with a 96.7% sequence similarity to its closest relative, Thiocapsa roseopersicina strain 1711^T (DSM217^T). The unique assemblage of physiological and phylogenetic properties of strain SC5 defines it as a new species of the genus Thiocapsa, and we describe strain SC5 herein as Tca. imhoffii, sp. nov.     

A new purple sulfur bacterium from saline littoral sediments,Thiorhodovibrio winogradskyi gen. nov. and sp. nov.

Two strains of a new purple sulfur bacterium were isolated in pure culture from the littoral sediment of a saline lake (Mahoney Lake, Canada) and a marine microbial mat from the North Sea island of Mellum, respectively. Single cells were vibrioid-to spirilloid-shaped and motile by means of single polar flagella. Intracellular photosynthetic membranes were of the vesicular type. As photosynthetic pigments, bacteriochlorophyll a and the carotenoids lycopene, rhodopin, anhydrorhodovibrin, rhodovibrin and spirilloxanthin were present.Hydrogen sulfide and elemental sulfur were used under anoxic conditions for phototrophic growth. In addition one strain (06511) used thiosulfate. Carbon dioxide, acetate and pyruvate were utilized by both strains as carbon sources. Depending on the strain propionate, succinate, fumarate, malate, tartrate, malonate, glycerol or peptone may additionally serve as carbon sources in the light. Optimum growth rates were obtained at pH 7.2, 33 °C, 50 mol m^-2 s^-1 intensity of daylight fluorescent tubes and a salinity of 2.2–3.2% NaCl. During growth on sulfide, up to ten small sulfur globules were formed inside the cells. The strains grew microaerophilic in the dark and exhibited high specific respiration rates. No vitamins were required for growth. The DNA base composition was 61.0–62.4 mol% G+C.The newly isolated bacterium belongs to the family chromatiaceae and is described as a member of a new genus and species, Thiorhodovibrio winogradskyi gen. nov. and sp. nov. with the type strain SSP1, DSM No. 6702.     

Characterization of a new platelet-forming purple sulfur bacterium,Amoebobacter pedioformis sp. nov.

The dominant purple sulfur bacterium of a reddish-colored waste water pond near Taichung, Taiwan, was isolated in pure culture, strain CML2. Individual cells were nearly spherical, nonmotile, and contained in their peripheral parts was vacuoles that appeared like elongated, curved tubes. Four to sixteen cells formed platelet-like aggregates reminiscent of Thiopedia rosea. The intracellular photosynthetic membrane system of the cells was of vesicular type; the photosynthetic pigments consisted of bacteriochlorophyll a and spirilloxanthin as the major carotenoid. The color of cell suspensions was pink to rosered. Under anaerobic conditions photolithoautotrophic growth occurred with sulfide, elemental sulfur or thiosulfate; sulfur globules were stored as an intermediary oxidation product. In the presence of sulfide, acetate, lactate and pyruvate were photoassimilated; strain CML2 lacked assimilatory sulfate reduction. Fastest photoautotrophic growth (11 h doubling time) was obtained at pH 7.5, 35°C and a light intensity of about 1000 lux (tungsten lamp). Chemolithoautotrophic growth in the dark was possible under reduced oxygen partial pressure with reduced sulfur compounds as respiratory substrates. The DNA base composition of strain CML2 was 65.5 mol% G+C. Strain CML2 is described as type strain of a new species, Amoebobacter pedioformis sp. nov., in the family Chromatiaceae.     

Urease formation in purple sulfur bacteria (Chromatiaceae) grown on various nitrogen sources

Batch cultures of Thiocapsa roseopersicina strain 6311, Thiocystis violacea strain 2311 and Chromatium vinosum strain 1611, grown anaerobically in the light on sulfide with urea, ammonia, N₂ or casein hydrolysate as nitrogen source exhibited urease activity, while Chromatium vinosum strain D neither showed any degradation of urea nor urease activity on any of the nitrogen sources tested.In T. violacea and C. vinosum strain 1611 urease was little affected by the nitrogen source and seemed to be constitutive. In T. roseopersicina, however, the enzyme was repressed by ammonia (although a low basal level of activity remained) and, to a lesser degree, induced by urea: The presense of urea stimulated a temporary increase in urease activity in the early exponential growth phase. The highest activities, however, were found after growth on N₂, and especially on 0.1% casein hydrolysate (in the absence or after exhaustion of external ammonia), but not before the stationary growth phase was reached. Derepressed urease synthesis required an efficient external source of nitrogen.In cultures of T. roseopersicina urease activity showed a periodic oscillation which depended on the repeated feeding with sulfide and subsequent variation in the sulfur content of the cells. The possible reasons of this oscillation are discussed.     

Phylogenic status of a purple sulfur bacterium and its bloom in Lake Kaiike

Two bacterial species at the upper boundary of the H₂S-containing lower layer of Lake Kaiike, a purple sulfur bacterium and Macromonas sp., markedly changed their population densities in a single year (maximum cell numbers ranged between 10⁶ and <10³ cells ml^–1), although neither species ever entirely disappeared from the lake over at least the past 30 years. Genetic characteristics based on the sequence of the 16S rDNA of the purple sulfur bacterium showed it to be a new species of the Chromatiaceae family. This bloom of purple sulfur bacterium occurred when the H₂S layer was disturbed by an external intrusion of seawater.     

Iron sulfur proteins of the purple sulfur bacterium Ectothiorhodospira shaposhnikovii

In addition to several cytochromes three iron sulfur proteins were detected in mixotrophically grown cells of Ectothiorhodospira shaposhnikovii, a member of the Chromatiaceae. They were identified as a bacterial ferredoxin and two high potential iron sulfur proteins (HIPIPs). The two HIPIPs were purified and characterized. They were named according to their differing retention times on a DEAE-cellulose column using a continuous NaCl gradient: early and late HIPIP. The HIPIPs contain 4 mol of non-heme iron and 4 mol of acid labile sulfur per mol protein. Under the conditions of purification the early HIPIP (E _{m, 7}+270 mV) was present in a semi-reduced state. Using ion-exchange chromatography the early HIPIP could be split into a reduced green-brown (pI=3.7) and an oxidized red-brown (pI=3.9) fraction. The late HIPIP (pI=3.8) showed a midpoint potential of only+155 mV, the lowest redox potential of a HIPIP described so far.Non-common abbreviations HIPIP high potential iron sulfur protein - MOPS 3(N-morpholino)propane sulfonate - SDS sodium dodecylsulfate     

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     

Oxygen and temperature-dependent structural and redox changes in a novel cytochrome c(4) from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina

A novel cytochrome c₄, the first of this type in purple phototrophic bacteria has been discovered in Thiocapsa roseopersicina. The fact that cytochrome c₄ has been found in an anaerobic organism puts in question the up hereto suggested role of cytochromes c₄ in the aerobic respiratory metabolism. The structure of cytochrome c₄ was studied under both aerobic and anaerobic conditions, using differential scanning calorimetry and a combination of redox potentiostatic measurements with CD and UV-Vis absorption techniques. Cytochrome c₄ maintained its functional capability at high temperature (60 °C) if it was kept under anaerobic conditions. With increasing temperature under aerobic conditions, however, there are dramatic conformational changes in the protein and coordination changes on the iron side. Presumably oxygen binds to the iron at the position left vacant by the methionine and facilitates conformational changes with low reversibility.     

Phototrophic purple sulfur bacteria as heat engines in the South Andros Black Hole

Photosynthetic organisms normally endeavor to optimize the efficiency of their light-harvesting apparatus. However, here we describe two bacterial isolates belonging to the genera Allochromatium and Thiocapsa that demonstrate a novel adaptation by optimizing their external growth conditions at the expense of photosynthetic efficiency. In the South Andros Black Hole, Bahamas, a dense l-m thick layer of these anoxygenic purple sulfur bacteria is present at a depth of 17.8 m. In this layer the water temperature increases sharply to 36°C as a consequence of the low-energy transfer efficiency of their carotenoids (ca. 30%). These include spirilloxanthin, and related polyene molecules and a novel chiral carotenoid identified as spirilloxanthin-2-ol, not previously reported in purple bacteria. To our knowledge, this study presents the first evidence of such a bacterial mass significantly increasing the ambient water temperature. The transduction of light to heat energy to excess heat may provide these anoxygenic phototropic bacteria with a competitive advantage over non-thermotolerant species, which would account for their predominance within the microbial layer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ectothiorhodospira marismortui sp. nov., an obligately anaerobic,moderately halophilic purple sulfur bacterium from a hypersaline sulfur spring on the shore of the Dead Sea

A novel type of purple sulfur bacterium was isolated from a hypersaline sulfur spring on the shore of the Dead Sea. The cells of the isolate are irregularly rod-shaped or curved, and motile by means of a tuft of polar flagella. The photosynthetic system, containing bacteriochlorophyll a and carotenoids of the spirilloxanthin series, is located on stacks of lamellar membranes in the cell cytoplasm. The organism can grow either photoautotrophically with sulfide as electron donor, which is oxidized via extracellular sulfur to sulfate, or photoheterotrophically, using acetate, succinate, fumarate, malate or pyruvate as carbon sources. The bacterium is obligately anaerobic, and requires a source of reduced sulfur for growth. The isolate is moderately halophilic, and grows optimally at NaCl concentrations between 3 and 8%, temperatures between 30 and 45°C, and neutral pH. 16S ribosomal RNA oligonucleotide cataloging suggests a close relationship to purple sulfur bacteria of the genus Ectothiorhodospira. As the isolate differs greatly from the described members of the genus Ectothiorhodospira, we describe the isolate as a new species, and propose the name Ectothiorhodospira marismortui sp. nov.     

Growth yields of green sulfur bacteria in mixed cultures with sulfur and sulfate reducing bacteria

1. Dry weight yields from mixed cultures ofProsthecochloris aestuarii orChlorobium limicola with the sulfur reducingDesulfuromonas acetoxidans were determined on different growth limiting amounts of acetate, ethanol or propanol. The obtained yields agreed well with values predicted from stoichiometric calculations. 2. From mixed cultures of twoChlorobium limicola strains withDesulfovibrio desulfuricans orD. gigas on ethanol as the growth limiting substrate, dry weight yields were obtained as calculated for the complete utilization of the ethanol by the mixed cultures. 3. Dry weight yield determinations for two pure cultures ofChlorobium limicola with different growth limiting amounts of sulfide in the absence and presence of excess acetate confirmed that acetate is incorporated byChlorobium in a fixed proportion to sulfide; compared to the yield in the absence of acetate the yield is increased two to threefold in the presence of acetate. 4. The lowest possible sulfide concentrations necessary for optimal growth of mixed cultures of eitherProsthecochloris orChlorobium withDesulfuromonas on acetate were 7–8 mg H₂S per liter of medium. 5. Doubling times at the growth rate limiting light intensities of 5, 10, 20, 50, 100 and 200 lux were determined under optimal growth conditions for the following phototrophic bacteria:Prosthecochloris aestuarii, Chlorobium phaeovibriodes, Chromatium vinosum andRhodopseudomonas capsulata. Reasonably good growth was still obtained withProsthecochloris at 10 and 5 lux light intensity at which no growth of the purple bacteria could be observed.     

Production of elemental sulfur by green and purple sulfur bacteria

The utilization of sulfide by phototrophic sulfur bacteria temporarily results in the accumulation of elemental sulfur. In the green sulfur bacteria (Chlorobiaceae), the sulfur is deposited outside the cells, whereas in the purple sulfur bacteria (Chromatiaceae) sulfur is found intracellularly. Consequently, in the latter case, sulfur is unattainable for other individuals. Attempts were made to analyze the impact of the formation of extracellular elemental sulfur compared to the deposition of intracellular sulfur.According to the theory of the continuous cultivation of microorganisms, the steady-state concentration of the limiting substrate is unaffected by the reservoir concentration (S _R).It was observed in sulfide-limited continuous cultures ofChlorobium limicola f.thiosulfatophilum that higherS _R values not only resulted in higher steady-state population densities, but also in increased steady-state concentrations of elemental sulfur. Similar phenomena were observed in sulfide-limited cultures ofChromatium vinosum.It was concluded that the elemental sulfur produced byChlorobium, althouth being deposited extracellularly, is not easily available for other individuals, and apparently remains (in part) attached to the cells. The ecological significance of the data is discussed.Non-standard abbreviations RP reducing power - BChl bacteriochlorophyll - N_cell cell material - specific growth rate - {ie52-1} maximal specific growth rate - D dilution rate - K _s saturation constant - s concentration of limiting substrate - S _R same ass but in reservoir bottle - Y yield factor - iS^o intracellular elemental sulfur - eS^o extracellular elemental sulfur - PHB poly-beta-hydroxybutyric acid