Pigment production and isotopic fractionations in continuous culture: okenone producing purple sulfur bacteria Part II

Okenone is a carotenoid pigment unique to certain members of Chromatiaceae, the dominant family of purple sulfur bacteria (PSB) found in euxinic photic zones. Diagenetic alteration of okenone produces okenane, the only recognized molecular fossil unique to PSB. The in vivo concentrations of okenone and bacteriochlorophyll a (Bchl a) on a per cell basis were monitored and quantified as a function of light intensity in continuous cultures of the purple sulfur bacterium Marichromatium purpuratum (Mpurp1591). We show that okenone‐producing PSB have constant bacteriochlorophyll to carotenoid ratios in light‐harvesting antenna complexes. The in vivo concentrations of Bchl a, 0.151 ± 0.012 fmol cell^?1, and okenone, 0.103 ± 0.012 fmol cell^?1, were not dependent on average light intensity (10–225 Lux) at both steady and non‐steady states. This observation revealed that in autotrophic continuous cultures of Mpurp1591, there was a constant ratio for okenone to Bchl a of 1:1.5. Okenone was therefore constitutively produced in planktonic cultures of PSB, regardless of light intensity. This confirms the legitimacy of okenone as a signature for autotrophic planktonic PSB and by extrapolation water column euxinia. We measured the δ¹³C, δ¹⁵N, and δ³⁴S bulk biomass values from cells collected daily and determined the isotopic fractionations of Mpurp1591. There was no statistical relationship in the bulk isotope measurements or stable isotope fractionations to light intensity or cell density under steady and non‐steady‐state conditions. The carbon isotope fractionation between okenone and Bchl a with respect to overall bulk biomass (¹³ε_{pigment – biomass}) was 2.2 ± 0.4‰ and ?4.1 ± 0.9‰, respectively. The carbon isotopic fractionation () for the production of pigments in PSB is more variable than previously thought with our reported values for okenone at ?15.5 ± 1.2‰ and ?21.8 ± 1.7‰ for Bchl a.     

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     

Mathematical simulation of the interactions among cyanobacteria, purple sulfur bacteria and chemotrophic sulfur bacteria in microbial mat communities

Abstract: A deterministic one-dimensional reaction diffusion model was constructed to simulate benthic stratification patterns and population dynamics of cyanobacteria, purple and colorless sulfur bacteria as found in marine microbial mats. The model involves the major biogeochemical processes of the sulfur cycle and includes growth metabolism and their kinetic parameters as described from laboratory experimentation. Hence, the metabolic production and consumption processes are coupled to population growth. The model is used to calculate benthic oxygen, sulfide and light profiles and to infer spatial relationships and interactions among the different populations. Furthermore, the model is used to explore the effect of different abiotic and biotic environmental parameters on the community structure. A strikingly clear pattern emerged of the interaction between purple and colorless sulfur bacteria: either colorless sulfur bacteria dominate or a coexistence is found of colorless and purple sulfur bacteria. The model predicts that purple sulfur bacteria only proliferate when the studied environmental parameters surpass well-defined threshold levels. However, once the appropriate conditions do occur, the purple sulfur bacteria are extremely successful as their biomass outweighs that of colorless sulfur bacteria by a factor of up to 17. The typical stratification pattern predicted closely resembles the often described bilayer communities which comprise a layer of purple sulfur bacteria below a cyanobacterial top-layer; colorless sulfur bacteria are predicted to sandwich in between both layers. The profiles of oxygen and sulfide shift on a diel basis similarly as observed in real systems.     

Susceptibility of various purple and green sulfur bacteria to different antimicrobial agents

Abstract Several purple and green sulfur bacteria (genera Chromatium, Thiocapsa and Chlorobium ) were tested for their sensitivity to different antimicrobial agents by a disc diffusion assay, using thioacetamide as a source of hydrogen sulfide for plate growth. Chlorobium limicola strains were more sensitive to amoxicillin, erythromycin and nalidixic acid, whereas gentamicin and netilmicin were more active against the purple bacteria tested. None of the organisms were sensitive to oxacillin and trimethoprim + sulfamethoxazole. The critical concentrations at the edge of the inhibition zone were also calculated for three organisms and the antimicrobials colistin, mitomycin C, penicillin G, rifampicin, and streptomycin. The results obtained suggest that colistin, mitomycin C, penicillin G would provide selective conditions against the growth of Chlorobium limicola strains, while streptomycin and other aminoglycoside antibiotics would select against purple bacteria.     

Physiology of purple sulfur bacteria forming macroscopic aggregates in Great Sippewissett Salt Marsh, Massachusetts

Abstract Purple bacterial aggregates found in tidal pools of Great Sippewissett Salt Marsh (Falmouth, Cape Cod, MA) were investigated in order to elucidate the ecological significance of cell aggregation. Purple sulfur bacteria were the dominant microorganisms in the aggregates which also contained diatoms and a high number of small rod-shaped bacteria. Urea in concentrations of ≥ 1 M caused disintegration of the aggregates while proteolytic enzymes, surfactants or chaotropic agents did not exhibit this effect. This suggests that polysaccharides in the embedding slime matrix stabilize the aggregate structure. In addition cell surface hydrophobicity is involved in aggregate formation. The concentration of dissolved oxygen decreased rapidly below the surface of aggregates while sulfide was not detected. The apparent respiration rate in the aggregates was high when the purple sulfur bacteria contained intracellular sulfur globules. In the presence of DCMU, respiration remained light-inhibited. Light inhibition disappeared in the presence of KCN. These results demonstrated that respiration in the aggregates is due mainly to purple sulfur bacteria. The concentration of bacteriochlorophyll (Bchl) a in the aggregates (0.205 mg Bchl a cm⁻³) was much higher than in the pool sediments but comparable to concentrations in microbial mats of adjacent sand flats. Purple aggregates may therefore originate in the microbial mats rather than in the pools themselves. Rapid sedimentation and high respiration rates of Chromatiaceae in the aggregates would prevent the inhibition of Bchl synthesis if aggregates were lifted off the sediment and up into the oxic pool water by tidal currents.     

C and N stable isotope signatures reveal constraints to nutritional modes in orchids from the Mediterranean and Macaronesia

We compared the nutritional modes and habitats of orchids (e.g., autotrophic, partially or fully mycoheterotrophic) of the Mediterranean region and adjacent islands of Macaronesia. We hypothesized that ecological factors (e.g., relative light availability, surrounding vegetation) determine the nutritional modes of orchids and thus impose restrictions upon orchid distribution. Covering habitats from dark forests to open sites, orchid samples of 35 species from 14 genera were collected from 20 locations in the Mediterranean and Macaronesia to test for mycoheterotrophy. Mycorrhizal fungi were identified via molecular analyses, and stable isotope analyses were applied to test whether organic nutrients are gained from the fungal associates. Our results show that orchids with partial or full mycoheterotrophy among the investigated species are found exclusively in Neottieae thriving in light-limited forests. Neottioid orchids are missing in Macaronesia, possibly because mycoheterotrophy is constrained by the lack of suitable ectomycorrhizal fungi. Furthermore, most adult orchids of open habitats in the Mediterranean and Macaronesia show weak or no N gains from fungi and no C gain through mycoheterotrophy. Instead isotope signatures of some of these species indicate net plant-to-fungus C transfer.     

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 bacteriaChromatium minutissimum, Ectothiorhodospira shaposhnikovii, andChlorobium 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.     

Fractionation of stable carbon isotopes by photoautotrophically growing anoxygenic purple and green sulfur bacteria

Fractionation of stable carbon isotopes ¹²C and ¹³C by three pure cultures of photoautotrophic purple sulfur bacteria (Ectothiorhodospira shaposhnikovii, Lamprocystis purpureus, and Thiocapsa sp.) (PSB) and the green sulfur bacterium Prosthecochloris sp. (GSB) was investigated in 13–15-day experiments. The cultivation was carried out in a luminostat (2000 lx) on mineral media with 1–1.5 g/l NaHCO₃ (inoculum) with the subsequent transfer to the medium with up to 10 g/l NaHCO₃. For PSB, the difference in the quantitative characteristics of the isotopic composition of suspended carbon (including bacterial cells) and mineral carbon of the medium (Δ¹³C = δ¹³C_substrate − δ¹³C_biomass) changed from 15.0 to 34.3‰. For GSB, the range of Δ¹³C changes was significantly less (18.3–22.7‰). These data suggested the possibility of a pool of soluble mineral carbon in PSB cells. The pool of intracellular mineral carbon was calculated; depending on the PSB species and growth stage, it varied from 0 to 68% of the total cell carbon. The α coefficients reflecting the carbon isotope fractionation by PSB and GBS and calculated from the changes of the bicarbonate carbon isotopic composition in the medium depending on its consumption were 1.029 ± 0.003 and 1.019 ± 0.001, respectively. These α values did not depend on the growth rate. CO₂ fixation on ribulose-bisphosphate was shown to be the major factor determining the carbon isotope fractionation by PSB; at the stage of CO₂ penetration into the cell, fractionation was insignificant. In GSB, fractionation occurred mostly at CO₂ penetration into the cell, while it was insignificant at the stage of carbon dioxide fixation in the reverse TCA cycle. Analysis of the isotopic data of the photosynthesis by PSB and GSB in meromictic lakes also revealed that in PSB-dominated natural communities suspended organic matter was more enriched with light ¹³C (Δ¹³C = 23.4−24.6‰) than in the communities with more active GSB (Δ¹³C = 10.2−14.0‰)     

Coexistence of organisms competing for the same substrate: An example among the purple sulfur bacteria

The purpose of this study was to find a possible explanation for the coexistence of large and small purple sulfur bacteria in natural habitats. Experiments were carried out withChromatium vinosum SMG 185 andChromatium weissei SMG 171, grown in both batch and continuous cultures. The data may be summarized as follows: (a) In continuous light, with sulfide as growth rate-limiting substrate, the specific growth rate ofChr. vinosum exceeds that ofChr. weissei regardless of the sulfide concentration employed. Consequently,Chr. weissei is unable to compete successfully and is washed out in continuous cultures. (b) With intermittant light-dark illumination, the organisms showed balanced coexistence when grown in continuous cultures. The “steady-state” abundance ofChr. vinosum was found to be positively related to the length of the light period, and that ofChr. weissei to the length of the dark period. (c) Sulfide added during darkness is rapidly oxidized on subsequent illumination, resulting in the intracellular storage of reserve substances, which are later utilized for growth. The rate of sulfide oxidation/mg cell N/hr was found to be over twice as high inChr. weissei as inChr. vinosum. The observed coexistence may be explained as follows. In the light, with both strains growing, most of the sulfide will be oxidized byChr. vinosum [see (a)]. In the dark, sulfide accumulates. On illumination, the greater part of the accumulated sulfide will be oxidized byChr. weissei [see (c)]. A changed light-dark regimen should then have the effect as observed [see (b)]. These observations suggest that intermittant illumination may, at least in part explain the observed coexistence of both types of purple sulfur bacteria in nature.     

Effect of temperature on the phototransformation of purple sulfur bacteria bacteriochlorophylls and isolated chromatophores]

Photobleaching of P890 was shown to be independent of temperature within the range of +20 to -160 degrees C in purple sulphur bacteria and isolated chromatophores under oxidative conditions; therefore changes in the absorption at 890 nm are due to the primary photoact. No changes were detected in the absorption at 850 nm upon a slight decrease of temperature, which suggested the absorption at 850 nm upon a slight decrease of temperature, which suggested the conformation nature of these changes. The effect of temperature, which suggested the conformation nature of these changes. The effect of temperature on the photoinduced changes of absorption under reductive conditions seems to be due to the electron transport and the accompanying processes being blocked. The effect of temperature on the kinetics of P890+ reduction in the darkness under conditions when the cytochromes are preliminarily oxidized is determined by the participation of the secondary electron acceptors in this process. A decrease in temperature leads to blocking the transport of electrons from the primary acceptor to the secondary acceptors, which is expressed by a gradual disappearance of a slow component in the kinetics of p890+ reduction in the disappearance of a slow component in the kinetics of P890+ reduction in the darkness and by the intensification of a fast component resulting from the darkness and by the intensification of a fast component resulting from the interaction between the primary acceptor and P890+. Methodical aspects of absorption differential spectrophotometry of photosynthesizing organisms at low temperatures are discussed.     

In situ specific loss and growth rates of purple sulfur bacteria in Lake Cisó

Abstract Growth rates and population dynamics of phototrophic bacteria in Lak Cisó were analysed by measuring bacterial abundances and determining specific rates of growth and loss. Net growth rates were calculated from actual changes in biomass assuming exponential growth. Values ranged between −0.072 and 0.037 per day (d⁻¹) for Chromatium , and between −0.043 and 0.022 d⁻¹ for Amoebobacter . Exponential loss rates through sedimentation, decomposition and washout were determined independently. Values ranged between 0 and 0.025 d⁻¹ in the case of Chromatium and between 0 and 0.015 d⁻¹ in the case of Amoebobacter . Finally, gross growth rates were calculated by adding net growth to losses. Maximal values were 0.063 d⁻¹ for Chromatium and 0.037 d⁻¹ for Amoebobacter . In the case of Chromatium , population growth rates were found to be correlated with the amount of light available per unit of growing biomass. It was concluded that growth of phototrophic bacteria in Lake Cisó was limited by light availability. Altogether, purple sulfur bacteria seemed to maintain a very large biomass with very slow growth, thanks to very slow losses during stratification. During holomixis the situation was more dynamic. Washout of cells and disappearance of algal cells allowed more light to reach the bacteria. Therefore, high growth rates were found towards the end of the winter. A similar pattern repeated itself from year to year. These are the first estimates of in situ growth rates for populations of phototrophic bacteria.     

Filamentous sulfur bacteria of activated sludge: characterization of Thiothrix, Beggiatoa, and Eikelboom type 021N strains

Seventeen strains of filamentous sulfur bacteria were isolated in axenic culture from activated sludge mixed liquor samples and sulfide-gradient enrichment cultures. Isolation procedures involved plating a concentrated inoculum of washed filaments onto media containing sulfide or thiosulfate. The isolates were identified as Thiothrix spp., Beggiatoa spp., and an organism of uncertain taxonomic status, designated type 021N. All bacteria were gram negative, reduced nitrate, and formed long, multicellular trichomes with internal reserves of sulfur, volutin, and sudanophilic material. Thiothrix spp. formed rosettes and gonidia, and four of six strains were ensheathed. Type 021N organisms utilized glucose, lacked a sheath, and differed from Thiothrix spp. in several aspects of cellular and cultural morphology. Beggiatoa spp. lacked catalase and oxidase, and filaments were motile. Biochemical and physiological characterization of the isolates revealed important distinguishing features between the three groups of bacteria. Strain differences were most evident among the Thiothrix cultures. A comparison of the filamentous sulfur bacteria with freshwater strains of Leucothrix was made also.     

Filamentous sulfur bacteria of activated sludge: characterization of Thiothrix, Beggiatoa, and Eikelboom type 021N strains.

Seventeen strains of filamentous sulfur bacteria were isolated in axenic culture from activated sludge mixed liquor samples and sulfide-gradient enrichment cultures. Isolation procedures involved plating a concentrated inoculum of washed filaments onto media containing sulfide or thiosulfate. The isolates were identified as Thiothrix spp., Beggiatoa spp., and an organism of uncertain taxonomic status, designated type 021N. All bacteria were gram negative, reduced nitrate, and formed long, multicellular trichomes with internal reserves of sulfur, volutin, and sudanophilic material. Thiothrix spp. formed rosettes and gonidia, and four of six strains were ensheathed. Type 021N organisms utilized glucose, lacked a sheath, and differed from Thiothrix spp. in several aspects of cellular and cultural morphology. Beggiatoa spp. lacked catalase and oxidase, and filaments were motile. Biochemical and physiological characterization of the isolates revealed important distinguishing features between the three groups of bacteria. Strain differences were most evident among the Thiothrix cultures. A comparison of the filamentous sulfur bacteria with freshwater strains of Leucothrix was made also.     

Distribution of iron in Lake Banyoles in relation to the ecology of purple and green sulfur bacteria

The distribution of iron both in suspended sediment and in the water column has been studied during summer stratification in Lake Banyoles. In this lake, near bottom springs, a very fine material suspended sediment remains in suspension. Dissolved Fe²⁺ in interstitial water of this suspended sediment, is related to redox potential and to the bottom water inflow. In the water column, soluble iron is present in the hypolimnion of the six different basins forming Lake Banyoles. Under those conditions Fe²⁺ is partially removed by sulfide produced in the anoxic sediment. In addition, a peak of Fe²⁺ found at the density gradient level in basins C-III, C-IV and C-VI. A three compartment model on the dynamics of the processes involving iron in Lake Banyoles is proposed. The bottom springs supply oxygen to the anoxic hypolimnion affecting chemical processes of the iron cycle. The presence of phototrophic sulfur bacteria in the anoxic monimolimnion of basins C-III and C-IV can be related to the kinetics of Fe²⁺ and sulfide. In C-III sulfide concentration exceeds Fe²⁺ concentration whereas in C-IV sulfide is not detectable and iron reached values up to 60 mM. The presence of phototrophic sulfur bacteria in iron-containing environments with no detectable sulfide is explained by the ability of such microorganisms to use FeS as electron donor instead of H₂S.     

Dihydroxylycopene diglucoside diesters: a novel class of carotenoids from the phototrophic purple sulfur bacteria Halorhodospira abdelmalekii and Halorhodospira halochloris

The carotenoids in Halorhodospira abdelmalekii and Halorhodospira halochloris were analyzed by spectroscopic methods. The carotenoid composition of the two species was almost the same. Both species contained substantial amounts of unusual carotenoid glycoside fatty acid esters, which have been found for the first time in phototrophic purple bacteria. Methoxy-hydroxylycopene glucoside was a major component, and dihydroxylycopene diglucoside and dihydroxylycopene diglucoside diester were also found. Lycopene, rhodopin, and 3,4,3',4'-tetrahydrospirilloxanthin were present in very small amounts. Methoxy, glucosyl, and glucosyl ester groups were observed as substituents at the positions of the two original hydroxyl groups of dihydroxylycopene and made up approximately 20, 50, and 20%, respectively, of the total end groups (100%). The fatty acid components of the three carotenoid glucoside esters were the same (C12:0 and C14:1) and were rare in the cellular lipids of the two species.     

Dynamics of C,N, P and S in grassland soils: a model

We have developed a model to simulate the dynamics of C, N, P, and S in cultivated and uncultivated grassland soils. The model uses a monthly time step and can simulate the dynamics of soil organic matter over long time periods (100 to 10,000 years). It was used to simulate the impact of cultivation (100 years) on soil organic matter dynamics, nutrient mineralization, and plant production and to simulate soil formation during a 10,000 year run. The model was validated by comparing the simulated impact of cultivation on soil organic matter C, N, P, and S dynamics with observed data from sites in the northern Great Plains. The model correctly predicted that N and P are the primary limiting nutrients for plant production and simulated the response of the system to inorganic N, P, and S fertilizer. Simulation results indicate that controlling the C:P and C:S ratios of soil organic matter fractions as functions of the labile P and S levels respectively, allows the model to correctly simulate the observed changes in C:P and C:S ratios in the soil and to simulate the impact of varying the labile P and S levels on soil P and S net mineralization rates.     

Hydrogenase of purple bacteria: properties and regulation of synthesis

Physico-chemical properties of homogeneous preparations of soluble and membrane-bound hydrogenases from the purple sulfur bacterium Thiocapsa roseopersicina BBS and membrane-bound hydrogenase of Rhodopseudomonas capsulata, strain B10 have been studied. Compared to the enzymes from other sources, the hydrogenase of Thiocapsa roseopersicina is more stable to O₂ and products of its reduction (O ₂ ^- , H₂O₂), temperature and a number of other factors of the medium. A natural electron donor for T. roseopersicina hydrogenase is a low-potential cytochrome C₃, while the natural electron acceptors for hydrogenases of R. capsulata, T. roseopersicina, Ectothiorhodospira shaposhnikovii and Anabaena cylindrica are cytochromes of groups c and b.In different phototrophs, synthesis of hydrogenase can be inhibited by the presence of high concentrations of O₂. In some microorganisms (e.g. Rhodopseudomonas capsulata, strain B10) the repressing effect on hydrogenase formation is also exhibited by organic compounds. H₂ may not necessarily be present for hydrogenase synthesis by purple bacteria, but its presence may considerable increase the level of the enzyme.Abbreviations SDS sodium dodecylsulfate - Hipip high-potential iron — sulfur protein - R Rhodopseudomonas - T Thiocapsa - Rh Rhodospirillum - C Chromatium This paper is dedicated to Professor Dr. H.G. Schlegel in honour of his sixtieth birthday and in recognition of his great contribution in the field of physiology and biochemistry of microorganisms capable of using H₂. Professor H.G. Schlegel had a profound and most fuitful influence on the progress in the research of the laboratory headed by the author