<Emphasis Type="SmallCaps">l</Emphasis>-Tryptophan catabolism by <Emphasis Type="Italic">Rubrivivax benzoatilyticus</Emphasis> JA2 occurs through indole 3-pyruvic acid pathway

Rubrivivax benzoatilyticus JA2 utilizes l-tryptophan as the sole source of nitrogen for growth, and it has a doubling time of ~11 h (compared to 8 h with ammonium chloride). With cell free extracts in the presence of 2-oxoglutarate, indole-3-pyruvic acid, indole-3-acetaldehyde, indole-3-acetic acid, isatin, benzaldehyde, gallic acid and pyrogallol were identified using high performance liquid chromatography (HPLC) and liquid chromatography–mass spectroscopy (LC–MS) analysis. The conversion of l-tryptophan into indole 3-pyruvic acid and glutamate by an enzyme aminotransferase was confirmed and the catabolism of indole-3-pyruvic acid via side chain oxidation followed by ring oxidation, gallic acid and pyrogallol were confirmed as metabolites. In addition, the proposed pathway sequential conversion of indole-3-pyruvic acid to the end product of pyrogallol was identified, including an enzymatic step that would convert isatin to benzaldehyde by an enzyme yet to be identified. At this stage of the study, the enzyme tryptophan aminotransferase in R. benzoatilyticus JA2 was demonstrated.     

Production of indole-3-acetic acid and related indole derivatives from L-tryptophan by <Emphasis Type="Italic">Rubrivivax benzoatilyticus</Emphasis> JA2

Rubrivivax benzoatilyticus JA2 produces indoles with simultaneous utilization of L-tryptophan. Fifteen chromatographically distinct indole derivatives were detected from the L-tryptophan-supplemented cultures of R. benzoatilyticus JA2. Nine of these were identified as, indole 3-acetamide, Methoxyindole-3-aldehyde, indole 3-aldehyde, methoxyindole-3-acetic acid, indole 3-acetic acid, indole-3-carboxylic acid, indole-3-acetonitrile, indole, and trisindoline. Tryptophan stable isotope feeding confirmed the indoles produced are from the supplemented L-tryptophan. Indole 3-acetic acid is one of the major products of L-tryptophan catabolism by R. benzoatilyticus JA2 and its production was influenced by growth conditions. Identification of indole 3-acetamide and tryptophan monooxygenase activity suggests indole 3-acetamide routed IAA biosynthesis in R. benzoatilyticus JA2. The study also indicated the possible multiple pathways of IAA biosynthesis in R. benzoatilyticus JA2.     

Carbon Catabolite Repression-Independent and pH-Dependent Production of Indoles by Rubrivivax benzoatilyticus JA2

Rubrivivax benzoatilyticus JA2 produces indole derivatives (indoles) from aniline, anthranilate or l-tryptophan. Glucose repressed indole production in R. benzoatilyticus JA2, while malate had no effect. Growth of R. benzoatilyticus JA2 on glucose resulted in decrease in culture pH (6.4) compared with malate (8.4). Growth of R. benzoatilyticus JA2 on sugar carbon sources decreased culture pH (6.4–6.6) and indole production. Further, culture pH of 6.4 repressed the indole production, and pH 8.4 promoted the production irrespective of carbon sources used for growth. Moreover, correlation between indole production and culture pH was observed, where acidic pH inhibited indole production, while alkaline pH promoted the production, suggesting the role of pH in indole production. Tryptophan-catabolizing enzyme activities are significantly high in malate-grown cultures (pH 8.4) compared with that of the glucose (pH 6.4)-grown cultures and corroborated well with indole production, indicating their role in indole production. These results confirm that indole production in R. benzoatilyticus JA2 is pH dependent rather than carbon catabolite repression.     

Femtosecond charge separation in dry films of reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> and <Emphasis Type="Italic">Chloroflexus aurantiacus</Emphasis>

In this work, the influence of the crystallographic water on electron transfer between primary donor P and acceptor B_A was studied in reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides and the green bacterium Chloroflexus aurantiacus. For this purpose, time constants and oscillations of charge separation kinetics are compared between dry film RCs and RCs in glycerol-water buffer at 90 K. A common result of the drying of Rba. sphaeroides and Cfx. aurantiacus RCs is slowing of the charge separation process, decrease in amplitude of the oscillatory components of the kinetics, and the depletion of its spectrum. Thus, the major time constant of stimulated emission decay of P* bacteriochlorophyll dimer at 940 nm is increased from 1.1 psec for water-containing Rba. sphaeroides RCs to 1.9 psec for dry films of Rba. sphaeroides RCs. An analogous increase from 3.5 to 4.2 psec takes place in Cfx. aurantiacus RCs. In dry films of Rba. sphaeroides RCs, the amplitude of coherent oscillations of the absorption band of monomeric bacteriochlorophyll B_A⁻ at 1020 nm is 1.8 times less for the 130-cm⁻¹ component and 2.3 times less for the 32-cm⁻¹ component than the analogous amplitudes for water-containing RCs. Measurements in the analogous band of Cfx. aurantiacus RCs show that strong decrease (∼5-10 times) of the B_A⁻ absorption band and strong slowing (from ∼0.8 to ∼3 psec) of B_A⁻ accumulation together with ∼3-fold decrease in oscillation amplitude occurs on drying of these RCs. The overtones of the 32-cm⁻¹ component disappeared from the oscillations of the kinetics at 940 and 1020–1028 nm after drying of the Rba. sphaeroides and Cfx. aurantiacus RCs. The results are in agreement with the results for GM203L mutant of Rba. sphaeroides, in which the HOH55 water molecule is sterically removed, and with the results for dry films of pheophytin-modified RCs of Rba. sphaeroides R-26 and for YM210W and YM210L Rba. sphaeroides mutant RCs. The data are discussed in terms of the influence (or participation) of the HOH55 water molecule on electron transfer along the chain of polar atomic groups N-Mg(P_B)-N-C-N(HisM202)-HOH55-O=(B_A) connecting P_B and B_A in Rba. sphaeroides RCs.     

l-Phenylalanine catabolism and l-phenyllactic acid production by a phototrophic bacterium,Rubrivivax benzoatilyticus JA2

A phototrophic bacterium (Rubrivivax benzoatilyticus JA2) grows at the expense of l-phenylalanine as sole source of nitrogen but not as carbon source. Near stoichiometric yields of l-phenylpyruvic acid (0.4 mM) and l-phenyllactate (0.4 mM) were observed from l-phenylalanine (0.9 mM consumed). Aminotransfarase and dehydrogenase activities involved in the formation of l-phenylpyruvic acid and l-phenyllactate were demonstrated unequivocally in Rubrivivax benzoatilyticus JA2. Growth conditions and carbon sources had an influence on l-phenyllactate production. The process yielded a maximum of 0.92 mM l-phenyllactate from l-phenylalanine (1 mM) when fructose served as carbon source for R. benzoatilyticus JA2.     

Aerobic chemolithoautotrophic growth and RubisCO function in Rhodobacter capsulatus and a spontaneous gain of function mutant of Rhodobacter sphaeroides

Photosynthetic prokaryotes that assimilate CO₂ under anoxic conditions may also grow chemolithoautotrophically with O₂ as the electron acceptor. Among the nonsulfur purple bacteria, two species (Rhodobacter capsulatus and Rhodopseudomonas acidophilus), exhibit aerobic chemolithoautotrophic growth with hydrogen as the electron donor. Although wild-type strains of Rhodobacter sphaeroides grow poorly, if at all, with hydrogen plus oxygen in the dark, we report here the isolation of a spontaneous mutant (strain HR-CAC) of Rba. sphaeroides strain HR that is fully capable of this mode of growth. Rba. sphaeroides and Rba. capsulatus fix CO₂ via the reductive pentose phosphate pathway and synthesize two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). RubisCO levels in the aerobic-chemolithoautotrophic-positive strain of Rba. sphaeroides were similar to those in wild-type strains of Rba. sphaeroides and Rba. capsulatus during photoheterotrophic and photolithoautotrophic growth. Moreover, RubisCO levels of Rba. sphaeroides strain HR-CAC approximated levels obtained in Rba. capsulatus when the organisms were grown as aerobic chemolithoautotrophs. Either form I or form II RubisCO was able to support aerobic chemolithoautotrophic growth of Rba. capsulatus strain SB 1003 and Rba. sphaeroides strain HR-CAC at a variety of CO₂ concentrations, although form II RubisCO began to lose the capacity to support aerobic CO₂ fixation at high O₂ to CO₂ ratios. The latter property and other facets of the physiology of this system suggest that Rba. sphaeroides and Rba. capsulatus strains may be effectively employed for the biological selection of RubisCO molecules of altered substrate specificity. Received: 8 August 1997 / Accepted: 26 December 1997     

Production of Phenols and Alkyl Gallate Esters by Rhodobacter sphaeroides OU5

Rhodobacter sphaeroides OU5 grows phototrophically with generation doubling time of 18 h on l-phenylalanine when used as sole source of nitrogen. Phenols accumulated in the medium and gallate (0.5 mM) was identified as one of the major product. The others namely protocatechuate (0.2 mM) and caffeate (0.1 mM) and also three putative phenol alkyl esters were identified using Liquid chromatography–mass spectroscopy (LC–MS) analysis from the culture supernatant. Rhodobacter sphaeroides OU5 strain could explain its capability to produce the bioactive compounds during the growth. This study sheds production of bioactive and their biological exploring molecules.     

A Study of the Mechanism of Acetate Assimilation in Purple Nonsulfur Bacteria Lacking the Glyoxylate Shunt: Acetate Assimilation in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The mechanism of acetate assimilation in the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate shunt, has been studied. It has been found that the growth of this bacterium in batch and continuous cultures and the assimilation of acetate in cell suspensions are not stimulated by bicarbonate. The consumption of acetate is accompanied by the excretion of glyoxylate and pyruvate into the medium, stimulated by glyoxylate and pyruvate, and inhibited by citramalate. The respiration of cells in the presence of acetate is stimulated by glyoxylate, pyruvate, citramalate, and mesaconate. These data suggest that the citramalate cycle may function in Rba. sphaeroides in the form of an anaplerotic pathway instead of the glyoxylate shunt. At the same time, the low ratio of fixation rates for bicarbonate and acetate exhibited by the Rba. sphaeroides cells (approximately 0.1), as well as the absence of the stimulatory effect of acetate on the fixation of bicarbonate in the presence of the Calvin cycle inhibitor iodoacetate, suggests that pyruvate synthase is not involved in acetate assimilation in the bacterium Rba. sphaeroides.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 313–318.Original Russian Text Copyright © 2005 by Filatova, Berg, Krasil’nikova, Tsygankov, Laurinavichene, Ivanovsky.     

Annatto pigment production in root cultures of Achiote (<Emphasis Type="Italic">Bixa orellana</Emphasis> L.)

Bixa orellana L. is a tree native to South America known for its reddish orange pigment ‘annatto’ produced only on the aril portion of its seeds. It is the most preferred natural food colorant next to saffron, having wide applications in the dairy industry and also as a cosmeceutical. Normal root cultures of B. orellana were established under in vitro conditions on Murashige and Skoog (MS) medium containing α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) at 0.05–0.2 mg l⁻¹. The annatto pigment from in vitro-raised normal roots was extracted with chloroform, and later the ethanol-dissolved extract was analyzed both qualitatively by thin-layer chromatography (TLC) and spectrophotometrically quantified followed by High Performance Liquid Chromatography (HPLC) confirmation. The maximum amount of annatto pigment (346 ± 3.8 mg/100 g dry wt.) and maximum root biomass (152 ± 2.5 mg dry wt.) were recorded after 45 and 60 days of growth, respectively, on MS medium containing 0.1 mg l⁻¹ indole-3-butyric acid (IBA). Producing annatto pigment from normal root cultures under in vitro conditions is a novel approach when compared to the natural annatto pigment that is produced only on the aril portion of seeds. This allows the production of fresh pigment throughout the year.     

Carbohydrate metabolism of the phase variants of purple photosynthetic bacteria

The R and M phase variants of Rhodobacter sphaeroides and Rhodobacter capsulatus were isolated. The growth rates in the dark and in the light in glucose-containing media were much higher for the Rba. sphaeroides R variant than for the M variant. For the Rba. capsulatus R and M variants, growth rates in the dark and in the light in fructose- or glucose-containing media differed insignificantly. The cells of Rba. sphaeroides and Rba. capsulatus phase variants growing in media with glucose and fructose exhibited differences in activity of the key enzymes of the Embden–Meyerhof–Parnas (EMP) and Entner–Doudoroff (ED) pathways. The oxidative pentose phosphate pathway (PPP) does not participate in glucose and fructose metabolism in the studied bacteria. Specific activity of the ED pathway enzymes was higher in dark-grown R and M variants of both Rba. sphaeroides and Rba. capsulatus than in the cells grown under light. Specific activity of the EMP enzymes was higher for the R and M variants of both cultures grown in the light than for those grown in the dark. Activities of the 2-keto-3-deoxy-6-phosphogluconate and fructose bisphosphate aldolases, the key enzymes of the ED and EMP pathways in Rba. sphaeroides M variant grown in the medium with glucose in the light or in the dark, were approximately twice those of the R variant. In the medium with fructose activities of these enzymes in both R and M variants did not change significantly depending on growth conditions. Activities of the enzymes of the EMP and ED pathways in the extracts of the Rba. capsulatus R and M cells grown with glucose or fructose did not change significantly. Cultivation of Rba. sphaeroides and Rba. capsulatus phase variants in the medium with fructose resulted in a considerably increased synthesis of 1-phosphofructokinase. Induction of 1-phosphofructokinase synthesis in Rba. sphaeroides occurred only in the light, while in Rba. capsulatus induction of this enzyme in the medium with fructose was observed both in the dark and in the light. Thus, under aerobic conditions in the dark the phase variants of both bacteria probably assimilated glucose and fructose via the ED pathway, while in the light the EMP pathway was active.     

Differences in the control of bacteriochlorophyll formation by light and oxygen

Control of bacteriochlorophyll formation was studied with continuous cultures of Rhodospirillum rubrum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas capsulata. Oxygen controlled specific bacteriochlorophyll contents of the three species in a hyperbolical fashion irrespective of the presence of light. In Rps. sphaeroides, this applied to oxygen concentrations above 16% air saturation of the medium while at lower oxygen concentrations control followed a kinetics with negative cooperativity. Cell protein formation of R. rubrum and Rsp. sphaeroides was independent of oxygen concentrations while protein formation of Rps. capsulata increased at lower concentrations. Light controlled bacteriochlorphyll contents of R. rubrum and Rps. sphaeroides in a sigmoidal fashion. When growing at a constant low oxygen concentration cell protein formation increased with light energy flux in Rps. sphaeroides but remained unaffected in R. rubrum. Protein formation of R. rubrum increased with light energy flux only under anaerobic conditions. Two factor analyses were performed with R. rubrum and Rps. sphaeroides to study the combined effects of light and oxygen on bacteriochlorophyll formation. The results showed that both factors act independent of each other.Abbreviations ALA 5-aminolevulinic acid - R Rhodospirillum - Rsp. Rhodopseudomonas     

A Study of the Mechanism of Acetate Assimilation in Purple Nonsulfur Bacteria Lacking the Glyoxylate Shunt: Enzymes of the Citramalate Cycle in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The mechanism of acetate assimilation by the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate shunt, has been studied. In a previous work, proceeding from data on acetate assimilation by Rba. sphaeroides cell suspensions, a suggestion was made regarding the operation, in this bacterium, of the citramalate cycle. This cycle was earlier found in Rhodospirillum rubrum in the form of an anaplerotic reaction sequence that operates during growth on acetate instead of the glyoxylate shunt, which is not present in the latter bacterium. The present work considers the enzymes responsible for acetate assimilation in Rba. sphaeroides. It is shown that this bacterium possesses the key enzymes of the citramalate cycle: citramalate synthase, which catalyzes condensation of acetyl-CoA and pyruvate and, as a result, forms citramalate, and 3-methylmalyl-CoA lyase, which catalyzes the cleavage of 3-methylmalyl-CoA to glyoxylate and propionyl-CoA. The regeneration of pyruvate, which is the acetyl-CoA acceptor in the citramalate cycle, involves propionyl- CoA and occurs via the following reaction sequence: propionyl-CoA (+CO₂) å methylmalonyl-CoA å succinyl-CoA å succinate å fumarate malate å oxaloacetate (−CO₂) å phosphoenolpyruvate å pyruvate. The independence of the cell growth and the acetate assimilation of CO₂ is due to the accumulation of CO₂/HCO ₃ ⁻ (released during acetate assimilation) in cells to a level sufficient for the effective operation of propionyl-CoA carboxylase.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 319–328.Original Russian Text Copyright © 2005 by Filatova, Berg, Krasil’nikova, Ivanovsky.     

Light-Dependent Transformation of Aniline to Indole Esters by the Purple Bacterium Rhodobacter sphaeroides OU5

In an attempt to understand the aromatic hydrocarbon metabolism by purple bacteria that do not grow at their expense, we earlier reported 2-aminobenzoate transformation by a purple non-sulfur bacterium, Rhodobacter sphaeroides OU5 (Sunayana et al., 2005, J Ind Microbiol Biotech 32:41–45), which is extended in the present study with aniline, a major environmental pollutant. Aniline did not support photo (light anaerobic) or chemo (dark aerobic) heterotrophic growth of Rhodobacter sphaeroides OU5 either as a sole source of carbon or nitrogen. However, light-dependent aniline transformation was observed in the culture supernatants and the products were identified as indole derivatives. The transformation was dependent on a tricarboxylate intermediate, fumarate. Five intermediates of the aniline biotransformation pathway were isolated and identified as indole esters having a mass of 443, 441, 279, 189, and 167 with unstoichiometric total indole yields of 0.16 mM from 5 mM of aniline consumed. The pathway proposed based on these intermediates suggest a novel xenobiotic detoxification process in bacteria.     

Efficient production of ectoine using ectoine-excreting strain

Halophilic bacteria strain Halomonas salina DSM 5928 was found to excrete ectoine, suggesting its potential in the development of a new method of ectoine production. We performed HPLC and LC–MS analyses that showed that Halomonas salina DSM 5928 excreted ectoine under constant extracellular osmolarity. Medium adopting monosodium glutamate as a sole source of carbon and nitrogen was beneficial for ectoine synthesis. The total concentration of ectoine was not affected by NaCl concentration in the range 0.5–2 mol l⁻¹. The total concentration of ectoine and productivity in a 10-l fermentor with 0.5 mol l⁻¹ NaCl were 6.9 g l⁻¹ and 7.9 g l⁻¹ d⁻¹, respectively. These findings show that Halomonas salina DSM 5928 efficiently produces ectoine at relatively low NaCl concentration. This research also indicates the potential application of free or immobilized cells for continuous culture to produce ectoine.     

Simultaneous quantification of phytohormones in fermentation extracts of Botryodiplodia theobromae by liquid chromatography–electrospray tandem mass spectrometry

Fermentation broth and biomass from three strains of Botryodiplodia theobromae were characterized by high performance liquid chromatography–electrospray tandem mass spectrometry (HPLC–ESI–MS/MS) method, in order to quantify different phytohormones and to identify amino acid conjugates of jasmonic acid (JA) present in fermentation broths. A liquid–liquid extraction with ethyl acetate was used as sample preparation. The separation was carried out on a C18 reversed-phase HPLC column followed by analysis via ESI–MS/MS. The multiple reaction monitoring mode was used for quantitative measurement. For the first time, indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and JA were identified and quantified in the ethyl acetate extracts from the biomass, after the separation of mycelium from supernatant. The fermentation broths showed significantly higher levels of JA in relation to the other phytohormones. This is the first report of the presence of gibberellic acid, abscisic acid, salicylic acid and the cytokinins zeatin, and zeatin riboside in fermentation broths of Botryodiplodia sp. The presence of JA-serine and JA-threonine conjugates in fermentation broth was confirmed using HPLC-ESI tandem mass spectrometry in negative ionization mode, while the occurrence of JA-glycine and JA-isoleucine conjugates was evidenced with the same technique but with positive ionization. The results demonstrated that the used HPLC–ESI–MS/MS method was effective for analysing phytohormones in fermentation samples.     

In vitro morphogenesis of organogenic nodules derived from <Emphasis Type="Italic">Sclerocarya birrea</Emphasis> subsp. <Emphasis Type="Italic">caffra</Emphasis> leaf explants

Sclerocarya birrea (marula) is an indigenous South African tree with highly valued medicinal and nutritional properties. Induction of nodular meristemoids from leaf explants was achieved on Murashige and Skoog (MS) and woody plant medium (WPM) supplemented with 6-benzyladenine (BA) in combination with naphthalene acetic acid (NAA), indole-3-butryric acid (IBA) and indole-3-acetic acid (IAA). Induction of nodular meristemoids from 86% of the leaf cultures was achieved on MS medium with 4.0 μM BA and 1.0 μM NAA. High levels (78–100%) of induction were also achieved on WPM with different concentrations of BA (1.0–4.0 μM) and IBA (1.0–4.0 μM). The highest conversion of meristemoids into shoots was only 22% for 4.0 μM BA and 1.0 μM NAA on MS initiation medium. This was improved to 62% when nodular clusters were cultured in a MS liquid medium. Histological studies revealed the globular stage of the nodular meristemoids. This protocol has potential for application in mass micropropagation and plant breeding of S. birrea.     

Chlorate and nitrate reduction in the phototrophic bacteriaRhodobacter capsulatus andRhodobacter sphaeroides

Chlorate or trimethylamine-N-oxide (TMAO) added to phototrophic cultures ofRhodobacter sphaeroides DSM 158 increased both the growth rate and the growth yield although this stimulation was not observed in the presence of tungstate. This strain, exhibited basal activities of nitrate, chlorate, and TMAO reductases independently of the presence of these substrates in the culture medium, and nitrate reductase (NR) activity was competitively inhibited by chlorate. Phototrophic growth ofRhodobacter capsulatus B10, a strain devoid of NR activity, was inhibited only by 100 mM chlorate. However, growth of the nitrate-assimilatingR. capsulatus strains E1F1 and AD2 was sensitive to 10mm chlorate, and their NR activities were not inhibited by chlorate. Both NR and chlorate reductase (CR) activities of strain E1F1 were induced in the presence of nitrate or chlorate respectively, whereas strain AD2 showed basal levels of these activities in the absence of the substrates. A basal TMAO reductase (TR) activity was also observed when these strains ofR. capsulatus were cultured in the absence of this electron acceptor. These results suggest that chlorate and TMAO can be used as ancillary oxidants byRhodobacter strains and that a single enzyme could be responsible for nitrate and chlorate reduction inR. sphaeroides DSM 158, whereas these reactions are catalyzed by two different enzymes inR. capsulatus E1F1 and AD2.     

Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems

A 3.0-kb genomic fragment has been isolated from Rhodospirillum rubrum (ATCC 25903) that contains an open reading frame (ORF) with strong homology to other known polyhydroxyalkanoate (PHA) synthase genes. This ORF has lower homology to the R. rubrum strain Ha PHA synthase than would be expected within the same species. We have conducted a series of heterologous expression studies evaluating the in vivo substrate specificity of PHA synthase genes from Rhodobacter sphaeroides, Ralstonia eutropha (formerly Alcaligenes eutrophus), Thiocystis violacea, and Nocardia corrallina, within the PHA-synthase-negative hosts, Ralstonia eutropha DSM541 and Pseudomonas putida GpP104. The N. corrallina PHA synthase incorporated the highest percentage of C5 monomers in the polymer when fermented in medium supplemented with 0.1% heptanoate as the sole carbon source. When the T. violacea and R. sphaeroides were expressed in the PHA-negative host DSM541, a greater percentage of C5 monomer was observed in the polymer as compared to the expression of the PHA synthase of R. eutropha, when the transconjugants were fermented in medium supplemented with 0.4% propionate. Evaluation for preference of medium-chain-length monomers demonstrated the flexibility of the N. corrallina, T. violacea, and R. eutropha synthase genes to polymerize a copolyester composed of short- and medium-chain-length monomers when the respective transconjugants were fermented in medium supplemented with 0.5% octanoate. These studies demonstrate that the PHA synthase from N. corrallina, T. violacea, and R. eutropha are able to polymerize a copolyester composed of short- and medium-chain-length monomers, while the PHA synthase from R. sphaeroides lacks this ability and only produces a short-chain-length polymer. These observations suggest that the composition of the PHA from the PHA-producing organisms does not necessarily reflect the inherent specificity of the PHA synthase. Received: 16 March 1999 / Received revision: 24 August 1999 / Accepted: 24 September 1999     

Reduction of molybdate by sulfate-reducing bacteria

Molybdate is an essential trace element required by biological systems including the anaerobic sulfate-reducing bacteria (SRB); however, detrimental consequences may occur if molybdate is present in high concentrations in the environment. While molybdate is a structural analog of sulfate and inhibits sulfate respiration of SRB, little information is available concerning the effect of molybdate on pure cultures. We followed the growth of Desulfovibrio gigas ATCC 19364, Desulfovibrio vulgaris Hildenborough, Desulfovibrio desulfuricans DSM 642, and D. desulfuricans DSM 27774 in media containing sub-lethal levels of molybdate and observed a red-brown color in the culture fluid. Spectral analysis of the culture fluid revealed absorption peaks at 467, 395 and 314 nm and this color is proposed to be a molybdate–sulfide complex. Reduction of molybdate with the formation of molybdate disulfide occurs in the periplasm D. gigas and D. desulfuricans DSM 642. From these results we suggest that the occurrence of poorly crystalline Mo-sulfides in black shale may be a result from SRB reduction and selective enrichment of Mo in paleo-seawater.     

A simple and efficient protocol for cryopreservation of embryogenic calli of the medicinal plant <Emphasis Type="Italic">Anemarrhena asphodeloides</Emphasis> Bunge by vitrification

Somatic embryogenesis and whole plant regeneration was achieved in callus cultures derived from immature zygotic embryos of Prosopis laevigata (Humb. & Bonpl. ex Willd.) M.C. Johnst., recently identified as chromium (Cr), cadmium (Cd), lead (Pb) and nickel (Ni) accumulator. Embryogenic calli were induced on Murashige and Skoog (MS) medium added with a mixture of organic components plus N-6 benzyladenine (BA) (6.62 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (2.26 μM) or thidiazuron (4.54–9.08 μM) and indole-3-acetic acid (1.42 μM). Embryogenic calli transferred onto half-strength MS medium without plant growth regulators developed globular embryos, of which 20% matured when treated with 3.75% (w/v) polyethylene glycol (PEG), and of these 50% fully differentiated into plantlet embryo. Regenerated plants were successfully acclimatized (90%), while in vitro seedlings transferred to MS medium containing 0.5 mM Cd, Cr, Ni or Pb, exhibited high heavy metals accumulation (627 mg Cr kg⁻¹, 5,688 mg Cd kg⁻¹, 1,148 mg Ni kg⁻¹, and 3,037 mg Pb kg⁻¹ dry weight) and efficient roots to shoots translocation (42–73%).