Growth and respiratory oxidation of reduced sulfur compounds by intact cells ofThiobacillus novellus (type strain) grown on thiosulfate

Thiobacillus novellus (type strain) was grown chemolithoautrophically on thiosulfate in batch cultures under microaerophilic conditions. Under these conditions,T. novellus grew exponentially (=0.05–0.06 h^–1). The respiratory oxidation rates of tetrathionate, thiosulfate, elemental sulfur (S^o), and sulfite were measured respirometrically with an oxygen electrode, with exponentially growing cells. Cells growing on thiosulfate as the unique energy source retain thiosulfate-oxidizing activity, S^o-oxidizing activity (SOA), and very high sulfite-oxidizing activity, but lack respiratory tetrathionate-oxidizing activity. HQNO (50 m), an inhibitor of the quinone-cytochrome b region, strongly inhibited the SOA (70%), moderately the sulfite-oxidizing activity (45%), and poorly the thiosulfate-oxidizing activity (15%), 1mm KCN totally inhibited (>89%) all respiratory activities. This study confirms that inThiobacillus novellus, as well as in otherThiobacilli, SOA is present in cells grown with thiosulfate as sole electron donor. SOA appears not to be an oxygenase; it is linked to the respiratory chain, and the electrons are probably released in the quinone-cytochrome b region.     

Carbon assimilation pathways in sulfate-reducing bacteria II. Enzymes of a reductive citric acid cycle in the autotrophic Desulfobacter hydrogenophilus

The strict anaerobe Desulfobacter hydrogenophilus is able to grow autotrophically with CO₂, H₂, and sulfate as sole carbon and energy sources. The generation time at 30°C under autotrophic conditions in a pure mineral medium was 15 h, the growth yield was 8 g cell dry mass per mol sulfate reduced to H₂S. Enzymes of the autotrophic CO₂ assimilation pathway were investigated. Key enzymes of the Calvin cycle and of the acetyl CoA pathway could not be found. All enzymes of a reductive citric acid cycle were present at specific activities sufficient to account for the observed growth rate. Notably, an ATP-citrate lyase (1.3 mol · min^-1 · mg cell protein^-1) was present both in autotrophically and in heterotrophically grown cells, which was rapidly inactivated in the absence of ATP. The data indicate that in D. hydrogenophilus a reductive citric acid cycle is operating in autotrophic CO₂ fixation. Since other autotrophic sulfate reducers possess an acetyl CoA pathway for CO₂ fixation, two different autotrophic pathways occur in the same physiological group.Dedicated to Prof. H. G. Wood on the occasion of his 80th birthday     

Ammonium uptake by cowpea Rhizobium strain MNF 2030 and Rhizobium trifolii MNF 1001

Free-living Rhizobium trifolii MNF 1001 and cowpea Rhizobium MNF 2030 grown in chemostat culture under nitrogen limitation had high activities of an ammonium permease. In phosphate-limited, nitrogen-excess conditions, strains MNF 1001 and MNF 2030 retained 20% and 50%, respectively, of the ammonium uptake activity found in nitrogen-limited cells. Uptake in both strains was sensitive to azide, cyanide, carbonyl cyanide m-chlorophenyl hydrazone and 2,4-dinitrophenol. A gradient of ammonium concentration greater than 150-fold developed across the membrane within 20 min in cells of strain MNF 1001 grown under ammonia limitation. The pH optimum for ammonium uptake by N-limited cells of both MNF 1001 and MNF 2030 was around pH 7. The apparent K _m values for the ammonium permease in strains MNF 2030 and MNF 1001 were 3.9±1.6 M and 2.0±1.6 M respectively, and the V _max was 47±2.6 nmol min^-1 (mg protein)^-1 for MNF 2030 and 101±5.1 nmol min^-1 (mg protein)^-1 for MNF 1001. Isolated snake bean bacteroids of strain MNF 2030 capable of transporting succinate and l-glutamate had no detectable ammonium uptake activity. It therefore appears that the ammonium permeases in cells of these two strains are not as tightly regulated as in R. leguminosarum MNF 3841.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrzone - HEPES N-Hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Transport and catabolism of proline betaine in salt-stressed Rhizobium meliloti

Exogenous proline betaine (N,N-dimethylproline or stachydrine) highly stimulated the growth rate of Rhizobium meliloti, in media of inhibitory concentration of NaCl whereas proline was ineffective. High levels of proline betaine uptake occurred in cells grown in media of elevated osmotic strength; on the contrary, only low activity was found in cells grown in minimal medium. The apparent K _m was 10 M with a maximal transport rate of 25 nmol min^-1 mg^-1 of protein in 0.3 M NaCl-grown cells. The concentrative transport was totally abolished by KCN (2 mM), 2,4-dinitrophenol (2 mM), and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP 10 M) but was insensitive to arsenate (5 mM). Glycine betaine was a very potent inhibitor of proline betaine uptake while proline was not. Proline betaine transport was not reduced in osmotically shocked cells and no proline betaine binding activity was detected in the crude periplasmic shock fluid. In the absence of salt stress, Rhizobium meliloti actively catabolized proline betaine but this catabolism was blocked by increasing the osmotic strength of the medium. The osmolarity in the growth medium regulates the use of proline betaine either as a carbon and nitrogen source or as an osmoprotectant.Abbreviations LAS lactate-aspartate-salts - MSY mannitol-salts-yeast - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DCCD dicyclohexylcarbodiimide - KCN potassium cyanide - Hepes 4-(2-hydroxyethyl)-1-piperzine-ethanesulphonic acid     

Significance of phosphoglucose isomerase for the shift between heterolactic and mannitol fermentation of fructose by Oenococcus oeni

The bacterium Oenococcus oeni employs the heterolactic fermentation pathway (products lactate, ethanol, CO₂) during growth on fructose as a substrate, and the mannitol pathway when using fructose as an electron acceptor. In this study, [U-¹³C]glucose, [U-¹³C]fructose, HPLC, NMR spectroscopy, and enzyme analysis were applied to elucidate the use of both pathways by the hexoses. In the presence of glucose or pyruvate, fructose was metabolized either by the mannitol or the phosphoketolase pathways, respectively. Phosphoglucose isomerase, which is required for channeling fructose into the phosphoketolase pathways, was inhibited by a mixed-type inhibition composed of competitive (K _i=180 M) and uncompetitive (K_i=350 M) inhibition by 6-phosphogluconate. Erythrose 4-phosphate inhibited phosphoglucose isomerase competitively (K _i=1.3 M) with a low contribution of uncompetitive inhibition (K_i=13 M). The cellular 6-phosphogluconate content during growth on fructose plus pyruvate (<75 M) was significantly lower than during growth on fructose alone or fructose plus glucose (550 and 480 M). We conclude that competitive inhibition of phosphoglucose isomerase by 6-phosphogluconate (and possibly erythrose 4-phosphate) is responsible for exclusion of fructose from the phosphoketolase pathway during growth on fructose plus glucose, but not during growth on fructose plus pyruvate.     

Transport of glycine-betaine by Listeria monocytogenes

Uptake of [¹⁴C]glycine-betaine by Listeria monocytogenes was stimulated by NaCl with optimal stimulation at 0.4–0.5 M. The glycine-betaine transport system had a K _m of 22 M and a V _max of 11.7 nmol^-1 min^-1 mg^-1 protein when grown in the absence of NaCl. When grown in the presence of 0.8 M NaCl the V _max increased to 27.0 nmol^-1 min^-1 mg^-1 protein in 0.8 M NaCl. At NaCl concentrations above 0.5 M the uptake rate of glycine-betaine was reduced. Measurement of intracellular K⁺ concentrations and fluorescent dye quenching indicated that higher NaCl concentrations also led to a decrease in the electrochemical potential difference across the cytoplasmic membrane. Uptake of glycine was also observed, but this was not stimulated by NaCl.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Photosynthetic acclimation of higher plants to growth in fluctuating light environments

This paper describes a study into the potential of plants to acclimate to light environments that fluctuate over time periods between 15 min and 3 h. Plants of Arabidopsis thaliana (L.) Heynh., Digitalis purpurea L. and Silene dioica (L.) Clairv. were grown at an irradiance 100 mol m^-2 s^-1. After 4–6 weeks, they were transferred to light regimes that fluctuated between 100 and either 475 or 810 mol m^-2 s^-1, in a regular cycle, for 7 days. Plants were shown, in most cases, to be able to undergo photosynthetic acclimation under such conditions, increasing maximum photosynthetic rate. The extent of acclimation varied between species. A more detailed study with S. dioica showed that this acclimation involved changes in both Rubisco protein and cytochrome f content, with only marginal changes in pigment content and composition. Acclimation to fluctuating light, at the protein level, did not fully reflect the acclimation to continuous high light - Rubisco protein increased more than would be expected from the mean irradiance, but less than expected from the high irradiance; cytochrome f increased when neither the mean nor the high irradiance would be expected to induce an increase.This revised version was published online in October 2005 with corrections to the Cover Date.     

Uptake of phosphate by symbiotic and free-living dinoflagellates

Uptake of phosphate in the light by Amphidinium carterae, Amphidinium klebsii, cultured and symbiotic Gymnodinium microadriaticum conformed to Michaelis-Menten type saturation kinetics with all organisms showing similar K _m values, namely 0.005 to 0.016 M phosphorus. V _max values were 0.009–0.32 nmol phosphorus · 10⁵ cells^-1 · 10 min^-1. Phosphate uptake by all the dinoflagellates was greater in the dark than in the light. The metabolic inhibitor 3-(3,4-dichlorophenyl) 1,1-dimethylurea stimulated phosphate uptake in the light by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. Carbonylcyanide 3-chlorophenylhydrazone (CCCP) inhibited phosphate uptake by A. carterae and A. klebsii under both light and dark conditions. Uptake of phosphate by cultured and symbiotic G. microadriaticum in the light, but not in the dark, was inhibited by CCCP. Low concentrations of arsenate (5 g As · l^-1) stimulated phosphate by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. High concentrations of arsenate (100 g As · l^-1) did not affect uptake of phosphate by A. carterae and A. klebsii.     

Purification and characterization of phenylacetate-coenzyme A ligase from a denitrifying Pseudomonas sp., an enzyme involved in the anaerobic degradation of phenylacetate

The enzyme catalysing the first step in the anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses the reaction phenylacetate+CoA+ATP phenylacetyl-CoA+AMP+PP_i and requires Mg²⁺. Phenylacetate-CoA ligase (AMP forming) was found in cells grown anaerobically with phenylacetate and nitrate. Maximal specific enzyme activity was 0.048 mol min^-1 x mg^-1 protein in the mid-exponential growth phase. After 640-fold purification with 18% yield, a specific activity of 24.4 mol min^-1 mg^-1 protein was achieved. The enzyme is a single polypeptide with Mr of 52 ±2 kDa. The purified enzyme shows high specificity towards the aromatic inducer substrate phenylacetate and uses ATP preferentially; Mn²⁺ can substitute for Mg²⁺. The apparent K _m values for phenylacetate, CoA, and ATP are 60, 150, and 290 M, respectively. The soluble enzyme has an optimum pH of 8.5, is insensitive to oxygen, but is rather labile and requires the presence of glycerol and/or phenylacetate for stabilization. The N-terminal amino acid sequence showed no homology to other reported CoA-ligases. The expression of the enzye was studied by immunodetection. It is present in cells grown anaerobically with phenylacetate, but not with mandelate, phenylglyoxylate, benzoate; small amounts were detected in cells grown aerobically with phenylacetate.     

Properties of an enzymatic complex active in sulfite and thiosulfate oxidation by Rhodotorula sp.

An enzymatic complex from Rhodotorula was characterized and it was indicated that it possessed thiosulfate-oxidizing activity, forming tetrathionate as well as sulfite oxidase activity. Both activities coupled with ferricyanide and native cytochrome c but no with mammalian cytochrome c. Activities of these enzymes were inhibited by thiol inhibitors. Chelating agents did not affect thiosulfate oxidizing activity and only moderately inhibited sulfite oxidase. Both activities disappeared after treatment with proteolytic enzymes or sodium deoxycholate which indicates an essential role played not only by protein but also by phospholipids in the enzymatic activity of the complex. Thiosulfate oxidizing enzyme had a K _m for thiosulfate of 0.16 mM with ferricyanide as electron acceptor and of 14 M with native cytochrome c and of 0.34 mM for ferricyanide. Optimum pH for this activity was 7.8. Other properties of this enzyme were similar to those of thiobacilli and heterotrophic bacteria. The activity of sulfite oxidase was inhibited by 50% with 10 M AMP. The K _m values of this enzyme were 1 mM with ferricyanide as electron acceptor and 60 M with native cytochrome c for sulfite and 0.42 mM for ferricyanide. The enzyme did not show a specific optimum pH value with ferricyanide as electron acceptor. However, with native cytochrome c optimum pH was 7.8 for its activity. In many properties the sulfite oxidase from Rhodotorula was similar to the enzyme from Thiobacillus ferrooxidans, T. concretivorus, T. thioparus and T. novellus.Abbreviations CSH reduced glutathion - APS reductase, adenosine-S-phosphosulfate reductase - pHMB p-hydroxymercuribenzoate - NEM N-ethylmalcimide - TCA trichloroacetic acid - PPO 2,5-diphenyloxazole - POPOP 2,2-p-phenylen-bis 5-phenyloxazol     

Establishment of hairy root cultures of Psoralea species

Eight Psoralea species (Leguminosae) were inoculated with Agrobacterium rhizogenes, strains 8196 and 9402. Hairy roots were only induced by strain 9402. Attention was focussed on Psoralea lachnostachys. Transformed roots grew very rapidly in Gamborg B5 liquid medium with a doubling time of the culture of 38 hours. Whatever the culture conditions, the two furanocoumarins usually found in roots of Psoralea plants, psoralen and angelicin, were not detected in cultured transformed and non transformed roots even when some chitosan was added to the medium. However, 669 g.g^–1 dry matter of psoralen and 215 g.g^–1 dry matter of angelicin were found in roots from soil grown plants. A possible translocation of these compounds from the aerial parts to the roots is suggested.Abbreviations B5 medium Gamborg's medium (Flow laboratories's formulation) - NAA Naphthaleneacetic acid     

Polysulfide utilization by Thiocapsa roseopersicina

The purple sulfur bacterium Thiocapsa roseopersicina, being the dominant anoxygenic phototroph in microbial mats, was tested for growth on polysulfide as the electron donor for carbon dioxide fixation. Data collected in continuous cultures revealed _max to be 0.065 h^-1 and the saturation affinity constant K _s to be 6.7 M. The value of the inhibition constant K _i was estimated in batch cultures and was found to be approximately 1100 M. When grown on monosulfide, the organism was capable of trisulfide utilization without lag. Monosulfide-limited growth was established to have a _max of 0.091 h^-1 and K _s of 8.0 M. Field observations revealed polysulfide, present at supra-optimal concentrations, as a major pool of reduced sulfur in a laminated marine sediment ecosystem.Non-standard abbreviations DLP Direct Linear Plot - TS Total Sugar - SS Structural Sugar - P Protein - R _R concentration of growth limiting nutrient in reservoir vessel - S _nutrient residual concentration of growth-limiting nutrient in the culture vessel - S _{sulfur compound} concentration of sulfur in the corresponding compound - D dilution rate - _max maximum specific growth rate - K _s saturation constant - K _i inhibition constant Dedicated to Prof. Dr. Norbert Pfennig on the occasion of his 65th birthday     

Simultaneous use of 14C and 3H to determine autotrophic production and bacterial protein production in periphyton

A method of simultaneously quantifying photoautotrophic (algae and cyanobacteria) and bacterial production in periphyton communities by ¹⁴C-bicarbonate and ³H-leucine incorporation was investigated and applied to communities subjected to specific intensities of photosynthetically active radiation (400–700 nm). Maximum photosynthetic output (2.23 ± 0.29 (SE) g C cm^-2 h^-1) and bacterial production (0.07 ± 0.006 g C cm^-2 h^-1) occurred at the highest photon flux density (400 mol m^-2 s^-1). Over a photon flux density range of 20–400 mol m^-2 s^-1, bacterial and autotroph productivity were significantly and positively correlated (r = 0.89). Furthermore, application of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, a photosystem 11 inhibitor, to periphyton films reduced bacterial production by 46%, but it had no such effect on bacteria-only cultures. Therefore, the magnitude of bacterial production in periphyton was coupled to the photosynthesis/metabolism of algae and/or cyanobacteria.     

Comparison of photobioreactors for cultivation of Undaria pinnatifida gametophytes

Undaria pinnatifida gametophytes were grown in 2.5 l bubble column and airlift reactor at 25 °C and light intensity of 40 mol m^–2 s^–1 for 6 days. With aeration at 1 l min^–1, the airlift reactor yielded higher growth rate (0.12 mg DW ml^–1 d^–1) than a bubble column (0.08 mg DW ml^–1 d^–1). The advantages were related to the more homogeneous fluid dynamic characteristics of the airlift reactor.     

Electron-transport chain and coupled oxidative phosphorylation in methanol-grown Paracoccus denitrificans

Methanol dehydrogenase of Paracoccus denitrificans was shown to be very similar to the enzyme of Pseudomonas sp, M. 27. The K _m value for methanol with excess activator (ammonium ions) is 35 M. The pH optimum for enzyme activity with 2,6-dichlorophe-nolindophenol as electronacceptor was at 9.0 A CO-binding type of cytochrome c was present only in cells grown with methanol as carbon and energy source.It has been shown that methanol-oxidation involves electron-transport via cytochrome c and an a-type cytochrome to oxygen. Antimycin A did not inhibit this electron transport and 90% inhibition was obtained by 375 M potassium cyanide. Electron transport from endogenous substrates is possible via cytochrome b and possibly cytochrome o to oxygen. Potassium cyanide inhibited 90% of the electron transport via this pathway at a concentration of 1.42 mM. Measurement of respiration-driven proton translocation proved that during oxidation of methanol to formaldehyde by oxygen one mole of adenosine triphosphate is synthesized in the site 3 region of the electron transport chain. The H⁺/O value found confirmed the H⁺/site ratio of 3–4 found in heterotrophic grown cells. During electron transport from endogenous substrates to oxygen there is a possible synthesis of 3 moles of adenosine triphosphate.In heterotrophically grown cells electron transfer to oxygen follows almost only the branch of the respiratory chain containing cytochrome o. In methanol-grown cells the pathway via the a-type cytochrome seems more important.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - EPR electron paramagnetic resonance - S.D. standard deviation - ATP adenosine triphosphate     

Relationship between substrate inhibition and maintenance energy ofChlamydomonas reinhardtii in heterotrophic culture

The relationship between substrate inhibition and maintenance energy ofChlamydomonas reinhardtii grown heterotrophically on acetate was investigated. At low acetate concentrations (<0.4 g l^–1), where no inhibition of cell growth was observed, the cell growth yield and specific growth rate could be represented by the Pirt model, 1/Y=1/Y _g +m/ with a constant value of maintenance energy coefficient m. However, at high acetate concentrations (>0.4 g l^–1), inhibition of cell growth occurred, in which m became variable and dependent on the acetate concentration. A simple mathematical model was proposed to predict the actual maintenance energy coefficient m in the inhibited cultures and experimentally validated.Author for correspondence     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Effect of salinity on photosynthetic activity of Nodularia spumigena

The aim of the study was to determine the influence of total dissolvedsolids/salinity (mg L^-1 TDS) on photosynthetic activity of Nodularia spumigena strain 001E isolated from Lake Alexandrina, SouthAustralia, using photosynthesis-irradiance (PI) curves. N. spumigena001E cultures were grown in ASM medium at a range of TDSconcentrations (360, 6,600, 13,200, 19,800, 26,400 mg L^-1)at an irradiance of 30 mol m^-2 s^-1 (PAR, 400–700 nm) at 25 °C. The PI relationship was determined at 25 °Cfor irradiances between 0 and 500 mol photon m^-2s^-1 (PAR). The initial slope of PI curve, , a function of lightharvesting efficiency and photosynthetic energy conversion, decreasedproportionally with an increase in salinity from 360 to 26,400 mgL^-1 TDS. The maximum rate of photosynthesis (P_max),occurred at 6,600 mg L^-1 TDS. No influence of salinity onI_k, the irradiance at which P_max was measured, or on R_d, the dark respiration rate, was identified.     

Rapid in vitro multiplication of jujube through mature stem explants

Stem explants obtained from a mature tree of Ziziphus mauritiana Lamk were grown on modified Murashige and Skoog medium containing 3800 mg l^-1 potassium nitrate, 2475 mg l^-1 ammonium nitrate, 11 M benzyladenine and 0.5 M indole-3-acetic acid. During successive subcultures 15–20 shoots per inoculum were produced. Rooting was induced by pretreatment with 50 M indolebutyric acid or 1-naphthaleneacetic acid for 24 h followed by transfer to auxin-free White's medium. Plantlets grew well in a soil and vermiculite mixture.Abbreviations IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog