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.     

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.     

Growth and metabolism of the purple sulfur bacterium Thiocapsa roseopersicina under combined light/dark and oxic/anoxic regimens

The dominant purple sulfur bacterium of laminated sediment ecosystems in temperate environments, Thiocapsa roseopersicina, was cultivated in sulfide-limited continuous cultures (D=0.03 h^-1) subjected to various combined diel regimen of aeration and illumination in order to simulate environmental conditions in microbial mats. For comparison, cultures were grown under similar illumination regimens but continuously anoxic conditions.Bacteriochlorophyll a (BChla) and carotenoid synthesis was restricted to anoxic-dark periods and did not occur during oxic-light periods. An increase in the length of the oxic-light periods resulted in decreased pigment contents. However, phototrophic growth remained possible even at 20 h oxic-light/4 h anoxic-dark regimens. When anoxic conditions were maintained throughtout, BChla synthesis occurred both during light and dark periods.Glycogen was synthesized in the light and degraded in the dark. Calculations showed that degradation of 1/4–1/5 of the glycogen is sufficient to account for the BChla and carotenoid synthesis in the dark.The data showed that T. roseopersicina is very well adapted to cope with the combined oxygen and light regimes as they occur in microbial mats, which may explain the dominance of this bacterium in the purple layer of these sediment ecosystems.Non-standard abbreviations BChl bacteriochlorophyll - specific growth rate - D dilution rate - S_R concentration of limiting substrate in reservoir bottle     

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.     

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.     

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     

Enhanced aniline degradation by Desulfatiglans anilini in a synthetic microbial community with the phototrophic purple sulfur bacterium Thiocapsa roseopersicina

Desulfatiglans anilini is a sulfate-reducing bacterium (SRB) capable of oxidizing aniline, although growth and aniline turnover rates are slow, making it difficult to analyze the metabolism of the strain. Therefore, this study was designed to investigate the effect of sulfide on growth of D. anilini cultures, in order to improve its growth and aniline turnover rates, and study the biochemical mechanisms of sulfide inhibition. Hydrogen sulfide was found to inhibit growth of D. anilini, regardless of whether the strain was grown with aniline or phenol, and complete inhibition was observed at 20 mM hydrogen sulfide. For improving the growth of D. anilini with aniline, the sulfide-consuming phototrophic bacterium Thiocapsa roseopersicina was co-cultured in a synthetic microbial community with D. anilini using a co-cultivation device that continuously removed hydrogen sulfide from the culture. The doubling time of D. anilini with aniline was 15 days in the co-cultivation device, compared to 26 days in the absence of a sulfide-oxidizing partner. Moreover, the aniline degradation rate was significantly increased by a factor of 2.66 during co-cultivation of D. anilini with T. roseopersicina. The initial carboxylation reaction during aniline degradation was measured in cell-free extracts of D. anilini with carbon dioxide (CO₂) as a co-substrate in the presence of aniline and ATP. The effects of hydrogen sulfide on this aniline carboxylating system and on phenylphosphate synthase activity for phenol activation were studied, and it was concluded that hydrogen sulfide severely inhibited these enzyme activities.     

Continuous chemotrophic growth and respiration of Chromatiaceae species at low oxygen concentrations

Endogenous and maximum respiration rates of nine purple sulfur bacterial strains were determined. Endogenous rates were below 10 nmol O₂ · (mg protein · min)^-1 for sulfur-free cells and 15–35 nmol O₂ · (mg protein · min)^-1 for cells containg intracellular sulfur globules. With sulfide as electron-donating substrate respiration rates were considerably higher than with thiosulfate. Maximum respiration rates of Thiocystis violacea 2711 and Thiorhodovibrio winogradskyi SSP1 (254.8 and 264.2 nmol O₂ · (mg protein · min)^-1, respectively) are similar to those of aerobic bacteria. Biphasic respiration curves were obtained for sulfur-free cells of Thiocystis violacea 2711 and Chromatium vinosum 2811. In Thiocystis violacea the rapid and incomplete oxidation of thiosulfate was five times faster than the oxidation of stored sulfur. A high affinity of the respiratoty system for oxygen (K _m =0.3–0.9 M O₂, V _max=260 nmol O₂ · (mg protein · min)^-1 with sulfide as substrate, K _m =0.6–2.4 M O₂, V _max=14–40 nmol O₂ · (mg protein · min)^-1 with thiosulfate as substrate), for sulfide (K _m =0.47 M, V _max=650 nmol H₂S · (mg protein × min)^-1, and for thiosulfate (K _m =5–6 M, V _max =24–72 nmol S₂O ₃ ^2- · (mg protein · min)^-1 was obtained for different strains. Respiration of Thiocystis violacea was inhibited by very low concentrations of NaCN (K _i =1.7 M) while CO concentrations of up to 300 M were not inhibitory. The capacity for chemotrophic growth of six species was studied in continuous culture at oxygen concentrations of 11 to 67 M. Thiocystis violacea 2711, Amoebobacter roseus 6611, Thiocapsa roseopersicina 6311 and Thiorhodovibrio winogradskyi SSP1 were able to grow chemotrophically with thiosulfate/acetate or sulfide/acetate. Chromatium vinosum 2811 and Amoebobacter purpureus ML1 failed to grow under these conditions. During shift from phototrophic to chemotrophic conditions intracellular sulfur and carbohydrate accumulated transiently inside the cells. During chemotrophic growth bacteriochlorophyll a was below the detection limit.     

Isolation and growth of the phototrophic bacterium Rhodopseudomonas palustris strain B1 in sago-starch-processing wastewater

An indigenous strain of the purple non-sulphur phototrophic bacterium, Rhodopseudomonas palustris strain B1, was selected for the utilization and treatment of wastewater from a sago-starch-processing decanter. Growth of Strain B1 under anaerobic–light conditions in the carbohydrate-rich effluent was optimized by using 50% (v/v) effluent diluted in a basal minimal mineral medium with the addition to 0.1% (w/v) yeast extract. The optimum level of nitrogen source supplement, ammonium sulphate, was 1.0g/l. Highest cell mass concentration was achieved by using tungsten lamps as the light source with a light intensity of 4 klux. Under these optimal conditions, a maximum biomass of about 2.5g dry cell/l with a pigment content of about 1.1mg carotenoid/g dry weight cell was achieved after 96h of anaerobic cultivation. There was a 77% reduc n the chemical oxygen demand (COD) of the effluent. A cell yield of about 0.59g dry weight cell/g COD was obtained.     

Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum

Sulfide oxidation in the phototrophic purple sulfur bacterium Chromatium vinosum D (DSMZ 180^T) was studied by insertional inactivation of the fccAB genes, which encode flavocytochrome c, a protein that exhibits sulfide dehydrogenase activity in vitro. Flavocytochrome c is located in the periplasmic space as shown by a PhoA fusion to the signal peptide of the hemoprotein subunit. The genotype of the flavocytochrome-c-deficient Chr. vinosum strain FD1 was verified by Southern hybridization and PCR, and the absence of flavocytochrome c in the mutant was proven at the protein level. The oxidation of thiosulfate and intracellular sulfur by the flavocytochrome-c-deficient mutant was comparable to that of the wild-type. Disruption of the fccAB genes did not have any significant effect on the sulfide-oxidizing ability of the cells, showing that flavocytochrome c is not essential for oxidation of sulfide to intracellular sulfur and indicating the presence of a distinct sulfide-oxidizing system. In accordance with these results, Chr. vinosum extracts catalyzed electron transfer from sulfide to externally added duroquinone, indicating the presence of the enzyme sulfide:quinone oxidoreductase (EC 1.8.5.-). Further investigations showed that the sulfide:quinone oxidoreductase activity was sensitive to heat and to quinone analogue inhibitors. The enzyme is strictly membrane-bound and is constitutively expressed. The presence of sulfide:quinone oxidoreductase points to a connection of sulfide oxidation to the membrane electron transport system at the level of the quinone pool in Chr. vinosum. Received: 5 November 1997 / Accepted: 30 March 1998     

The sulfide affinity of phototrophic bacteria in relation to the location of elemental sulfur

Seventeen strains of phototrophic bacteria (4 strains of Chromatium spp., 2 strains of Thiocapsa sp., 4 strains of Ectothiorhodospira spp., 2 strains of Rhodopseudomonas sp., and 5 strains of Chlorobium spp.) have been grown in sulfide-limited continuous cultures to assess the affinity for sulfide. It was found that the affinity (calculated as the initial slope of the specific growth rate versus the concentration of sulfide) is higher in those phototrophic bacteria that deposit elemental sulfur outside the cells, than in those bacteria that store the sulfur inside the cells. A hypothesis is presented to explain this correlation.Dedicated to Prof. Dr. Hans G. Schlegel on the occasion of his 60th birthday     

Occurrence of plant hormones in cells of the phototrophic purple bacterium Rhodospirillum rubrum 1R

Abstract Plant hormones from biomass of the purple non-sulfur bacterium Rhodospiririllum rubrum were isolated for the first time. These compounds show high physiological activities (300–330%) in the cytokinin bioassay. All three detected cytokinins are adenine derivatives, according to spectral analysis. One of them was identified as 6-(4-hydroxy-3-methyl-2-trans-2-bytenylamino)-9-ß-D-ribofuranosylpurine (zeatinriboside) as shown by thin-layer chromatography and reversed-phase high-performance liquid chromatography. The possible functions of bacterial cytokinins are also discussed.     

Expression and characterization of the assimilatory NADH-nitrite reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1

A nas gene region from Rhodobacter capsulatus E1F1 containing the putative nasB gene for nitrite reductase was previously cloned. The recombinant His₆-NasB protein overproduced in E. coli showed nitrite reductase activity in vitro with both reduced methyl viologen and NADH as electron donors. The apparent K _m values for nitrite and NADH were 0.5 mM and 20 μM, respectively, at the pH and temperature optima (pH 9 and 30°C). The optical spectrum showed features that indicate the presence of FAD, iron-sulfur cluster and siroheme as prosthetic groups, and nitrite reductase activity was inhibited by sulfide and iron reagents. These results indicate that the phototrophic bacterium R. capsulatus E1F1 possesses an assimilatory NADH-nitrite reductase similar to that described in non-phototrophic organisms.     

Occurrence and dynamics of phototrophic purple nonsulphur bacteria compared with other asymbiotic nitrogen fixers in ricefields of Egypt

The occurrence and the dynamics of phototrophic purple nonsulphur bacteria (PPNSB) as well as Azospirillum, Azotobacter, Clostridium, and cyanobacteria at different rice growth stages were studied in two ricefields, at Kafr-El-Shiekh and Al-Fayoum in Egypt.The PPNSB existed in the both rice fields examined, but their numbers varied according to field conditions, habitat and rice growth stage. After transplanting, the number of PPNSB increased gradually, reached its maximum at maximum tillering stage, and thereafter declined toward harvest time. Numbers of PPNSB were generally comparable with that of the heterotrophic N₂-fixers namely Azospirillum, Azotobacter, Clostridium and cyanobacteria, while that of phototrophic purple and green sulphur bacteria were relatively lower.The highest PPNSB numbers were generally found in rhizosphere (10³–10⁶ per g^–1 dw soil) followed by soil (10³–10⁵ per g^–1 dw soil) and floodwater (10–10² per ml). Rice plants showed a positive rhizosphere effect on PPNSB, clostridia, Azotobacter and Azospirillum, negative rhizosphere effect on cyanobacteria and green sulphur bacteria, and no effect on purple sulphur bacteria.     

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.     

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.     

Diversity of anoxygenic phototrophic sulfur bacteria in the microbial mats of the Ebro Delta: a combined morphological and molecular approach

The diversity of purple and green sulfur bacteria in the multilayered sediments of the Ebro Delta was investigated. Specific oligonucleotide primers for these groups were used for the selective amplification of 16S rRNA gene sequences. Subsequently, amplification products were separated by denaturing gradient gel electrophoresis and sequenced, which yielded a total of 32 sequences. Six of the sequences were related to different cultivated members of the green sulfur bacteria assemblage, whereas seven fell into the cluster of marine or halophilic Chromatiaceae. Six sequences were clustered with the family Ectothiorhodospiraceae, three of the six being closely related to chemotrophic bacteria grouped together with Halorhodospira genus, and the other three forming a group related to the genus Ectothiorhodospira. The last thirteen sequences constituted a cluster where no molecular isolate from microbial mats has so far been reported. Our results indicate that the natural diversity in the ecosystem studied has been significantly underestimated in the past and point out the presence of novel species not related to all known purple sulfur bacteria. Furthermore, the detection of green sulfur bacteria, after only an initial step of enrichment, suggests that -- with the appropriate methodology -- several genera, such as Prosthecochloris, could be established as regular members of marine microbial mats.     

The genes coding for the L,M and cytochrome subunits of the photosynthetic reaction center from the thermophilic purple sulfur bacterium t Chromatium tepidum

The complete nucleotide sequences of the genes coding for L, M protein subunits and part of cytochrome subunit of the photosynthetic reaction center were determined for the thermophilic purple sulfur bacterium t Chromatium tepidum (t Chr. tepidum) which belongs to the subclass. The DNA fragments with 860 bp and 1900 bp were amplified by the Polymerase Chain Reaction (PCR) with the primers designed on the basis of amino acid sequences according to chemical sequence analysis of the proteins. The deduced amino acid sequences of these genes showed a significantly high degree of homology with those from purple non-sulfur bacteria. The L subunit consisted of 280 amino acids and had a molecular mass of 31,393. The M subunit consisted of 324 amino acids and had a molecular mass of 36,299. The aligned sequences of the L subunits of other purple bacterial reaction center polypeptides, showed the insertion of 8 amino acids in t Chr. tepidum in the connection of the first and second membrane-spanning helices different from those of purple non-sulfur bacteria. The aligned sequences of the L, M and cytochrome subunits were compared with other species and discussed in terms of phylogenetic trees.