Exploring potential applications of a novel extracellular polymeric substance synthesizing bacterium (<Emphasis Type="Italic">Bacillus licheniformis</Emphasis>) isolated from gut contents of earthworm (<Emphasis Type="Italic">Metaphire posthuma</Emphasis>) in environmental remediation

The aim was to isolate, characterize, and explore potentials of gut bacteria from the earthworm (Metaphire posthuma) and imply these bacteria for remediation of Cu(II) and Zn(II). An extracellular polymeric substance (EPS) producing gut bacteria (Bacillus licheniformis strain KX657843) was isolated and identified based on 16S rRNA sequencing and phylogenetic analysis. The strain showed maximum tolerance of 8 and 6 mM for Cu(II) and Zn(II) respectively. It removed 34.5% of Cu(II) and 54.4% of Zn(II) at 25 mg L^?1 after 72 and 96 h incubation respectively. The bacteria possessed a great potential to produce indole acetic acid (38.49 μg mL^?1) at 5 mg mL^?1 l-tryptophan following 12 days incubation. The sterilized seeds of mung beans (Vigna radiata) displayed greater germination and growth under bacterium enriched condition. We observed that the bacterial strain phosphate solubilization ability with a maximum of 204.2 mg L^?1 in absence of Cu(II) and Zn(II). Endowed with biosurfactant property the bacterium exhibited 24% emulsification index. The bacterium offered significant potential of plant growth promotion, Cu(II) and Zn(II) removal, and as such this study is the first report on EPS producing B. licheniformis KX657843 from earthworm which can be applied as powerful tool in remediation programs of Cu(II) and Zn(II) contaminated sites.     

Effects of carbon sources on growth and extracellular polysaccharide production of Nostoc flagelliforme under heterotrophic high-cell-density fed-batch cultures

Dissociated cells separated from a natural colony of Nostoc flagelliforme were cultivated heterotrophically in the darkness on glucose under fed-batch culture conditions. The effects of carbon sources (glucose, fructose, xylose, and sucrose) and concentrations on cell growth and extracellular polysaccharide (EPS) production were investigated. At harvest, the culture contained 1.325 g L^?1 of biomass and 117.2 mg L^?1 of EPS, respectively. The gravimetric EPS production rate was 16.7 mg g^?1 cell dry weight day^?1, which was 2.1 times higher than previously reported. Using sigmoid model, batch fermentation of N. flagelliforme was kinetically simulated to obtain equations including substrate consumption, biomass growth, and EPS accumulation. Results from a simulation correlated well with the experimental ones, indicating that this method could be useful in studying EPS production from batch and fed-batch cultures.     

Cytogenetics, geographical distribution, pollen stainability and fecundity of five diploid taxa of Santolina rosmarinifolia L. aggregate (Asteraceae: Anthemideae)

The chromosome analysis of Santolina rosmarinifolia subsp. rosmarinifolia, S. oblongifolia, S. semidentata subsp. semidentata, S. semidentata subsp. melidensis, S. canescens and the hybrid complex (S. rosmarinifolia subsp. rosmarinifolia, S. oblongifolia and their putative hybrids) shows that all the taxa are diploids (2n = 2x = 18; 18 + 1 or more B chromosomes, with 2n = 19, 20 only in the hybrid complex). The results show a conserved general structure of the karyotype (14m + 2sm + 2st), but in S. semidentata subsp. melidensis it is variable, with 14m + 2sm + 2st in ten individuals, 14m + (1m ? 1sm) + (1 m ? 1st) in nine individuals and 12m + (1m ? 1sm) + (1m ? 1st) + 2st + 1B in five individuals. Tetraploid individuals occurred in the diploid populations of S. rosmarinifolia subsp. rosmarinifolia and S. canescens, and their autopolyploid origin is discussed. Multivalent configurations at diakinesis, simple and double chromosome bridges and delayed disjunction of homologous and non-homologous chromosomes at anaphase I have negative effects on pollen stainability. The mean fructification percentage is moderate. The results suggest that the complex is a mosaic of introgressive hybrids.     

Kinetic Study and Mathematical Modeling of Chromium(VI) Reduction and Microorganism Growth Under Mixed Culture

The kinetics of chromium(VI) reduction by Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) was studied under both pure and mixed cultures. Initially, the study of kinetics was performed in pure culture. It was observed that the growth of the two bacteria was both inhibited in the presence of chromium(VI). The maximum specific growth rate (μ _m ) of P. aeruginosa decreased from 2.3942 h^?1 (without Cr(VI)) to 1.8551 h^?1 (with Cr(VI)). Under the mixed culture, the growth of E. coli was inhibited by P. aeruginosa. The maximum specific growth rate (μ _m ) of E. coli decreased form 0.871 h^?1 (in pure culture) to 0.153 h^?1 (in mixed culture). When the concentration of each bacterium was 4.5 × 10⁸ cells ml^?1, the half-velocity reduction rate constant (K _C) and the maximum specific reduction rate constant (v _max) of chromium(VI) were 80.05 mg chromium(VI) l^?1 and 3.674 mg chromium(VI) cells^?1 h^?1, respectively. The results showed that the simulation appeared in good agreement with the experimental data, supporting the series of mathematical models represented the bacteria growth and chromium(VI) reduction in both pure and mixed cultures usefully.     

Culture and exopolysaccharide production from sugarcane molasses by Gordonia polyisoprenivorans CCT 7137, isolated from contaminated groundwater in Brazil

Gordonia polyisoprenivorans CCT 7137 was isolated from groundwater contaminated with leachate in an old controlled landfill (São Paulo, Brazil), and cultured in GYM medium at different concentrations of sugarcane molasses (2%, 6%, and 10%). The strain growth was analyzed by monitoring the viable cell counts (c.f.u. mL^?1) and optical density and EPS production was evaluated at the end of the exponential phase and 24 h after it. The analysis of the viable cell counts showed that the medium that most favored bacterial growth was not the one that favored EPS production. The control medium (GYM) was the one that most favored the strain growth, at the maximum specific growth rate of 0.232 h^?1. Differences in bacterial growth when cultured at three different concentrations of molasses were not observed. Production of EPS, in all culture media used, began during the exponential phase and continued during the growth stationary phase. The highest total EPS production, after 24 h of stationary phase, was observed in 6% molasses medium (172.86 g L^?1) and 10% (139.47 g L^?1) and the specific total EPS production was higher in 10% molasses medium (39.03 × 10^?11 g c.f.u.^?1). After the exponential phase, in 2%, 6%, and 10% molasses media, a higher percentage of free exopolysaccharides (EPS) was observed, representing 88.4%, 62.4%, and 64.2% of the total, respectively. A different result was observed in pattern medium, which presented EPS made up of higher percentage of capsular EPS (66.4% of the total). This work is the first study on EPS production by G. polyisoprenivorans strain in GYM medium and in medium utilizing sugarcane molasses as the sole nutrient source and suggests its potential use for EPS production by G. polyisoprenivorans CCT 7137 aiming at application in biotechnological processes.     

Effect of different media on exopolysaccharide and biomass production by the green microalga Botryococcus braunii

Botryococcus braunii is a colonial green microalga with recognized potential to synthesize lipids and hydrocarbons for biofuel production. Besides this ability, this microalga also produces exopolysaccharides (EPS). Nevertheless, there are few reports about their biotechnological aspects and industrial applications. In this study, the effect of the nutritional conditions was examined by using two different culture media (BG11 and D medium). To our knowledge, the latter has not been reported before for culturing B. braunii. After 49 days of incubation, the final production of EPS was found to be statistically higher (P < 0.05) in the D medium (0.549?±?0.044 g L^?1) than in BG11 (0.336?±?0.009 g L^?1). On the contrary, the biomass production was found to be higher in BG11 (1.019?±?0.051 g L^?1) than in the D medium (0.953?±?0.056 g L^?1). However, this difference was not statistically significant. The difference in salinity and nitrogen concentration between both media is suggested as the main factor involved in the EPS and biomass results. FTIR spectra of B. braunii EPS from both media revealed presence of uronic acids and absence of amino and sulfate groups. Despite the similarity between both spectra, there were some different signals (at 1,921.52 and 720.60 cm^?1) which may mean a difference in glycosyl composition.     

Photosynthetic characteristics and UV stress tolerance of Antarctic seaweeds along the depth gradient

The photosynthetic characteristics through P-E curves and the effect of UV radiation on photosynthesis (measured as rapid adjustment of photochemistry, F _v/F _m) and DNA damage (as formation of CPDs) were studied in field specimens of green, red and brown algae collected from the eulittoral and sublittoral zone of Fildes Peninsula (King George Island, Antarctic). The content of phenolic compounds (phlorotannins) and the antioxidant activity were also studied in seven brown algae from 0 to 40 m depth. The results indicated that photosynthetic efficiency (α) was high and did not vary between different species and depths, while irradiances for saturation (E _k) averaged 55 μmol m^?2 s^?1 in subtidal and 120 μmol m^?2 s^?1 in eulittoral species. The studied species exhibited notable short-term UV tolerance along the vertical zonation. In intertidal and shallow water species, decreases in F _v/F _m by UV radiation were between 0 and 18 %, while in sublittoral algae, decreases in F _v/F _m varied between 3 and 35 % relative to PAR treatment. In all species, recovery was high averaging 84–100 %. The formation of CPDs increased (15–150 %) under UV exposure, with the highest DNA damage found in some subtidal species. Phlorotannin content varied between 29 mg g^?1 DW in Ascoseira mirabilis from 8 m depth and 156 mg g^?1 DW in Desmarestia menziesii from 17 m depth. In general, phlorotannin concentrations were constitutively high in deeper sublittoral brown algae, which were correlated with higher antioxidant activities of algal extracts and low decreases in photosynthesis. UV radiation caused a strong decrease in phlorotannin content in the deep-water Himantothallus grandifolius, whereas in D. menziesii and Desmarestia anceps, induction of the synthesis of phlorotannins by UV radiation was observed. The antioxidant activity was in general less affected by UV radiation.     

Agrobacterium-mediated transformation of embryogenic cell suspension cultures and plant regeneration in Lilium tenuifolium oriental × trumpet ‘Robina’

A method has been developed for embryogenic cell suspension cultures, plant regeneration and transformation of the important ornamental lily genotype (Lilium tenuifolium oriental × trumpet ‘Robina’). Bulb scales, filaments, ovaries and stem axis tissues were used as explants for callus induction in Murashige and Skoog (MS) medium with additions of growth regulators: picloram on its own, or in combination with 1-naphthaleneacetic acid (NAA), and thidiazuron (TDZ). The results show that the optimum medium for callus induction in bulb scale and filament tissue is MS + picloram 1.0 mg L^?1, and for the ovary, it is MS + picloram 1.5 mg L^?1. The stem axis had the highest rate (89.2 %) of callus induction with MS + NAA 2.2 mg L^?1 + TDZ 0.1 mg L^?1. The suspension cultures were established with the combination of NAA and TDZ with 2–5 mm cell clusters. These took a long time compared with suspension cultures established by picloram with 1–3 mm cell clusters. In three suspension cultures induced by picloram, the best callus from the point of view of proliferation and regeneration was derived from filaments. For plant regeneration, the growth rate of suspension cultures from the stem axis was higher than from the other three suspension culture induced by picloram. Vector pCAMBIA1301 with the β-glucuronidase (GUS) gene as reporter was transformed by Agrobacterium mediation into suspension cultures initiated from filament and stem axis material. After co-cultivation, the numbers of blue spots in material from the two sources were 26.8 ± 4.3 and 24.0 ± 4.7, respectively (difference not significant). Hygromycin-resistant callus was successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were also confirmed by the GUS histochemical assay, polymerase chain reaction.     

Mechanisms associated to impaired activity of cardiac P-type ATPases in endothelial nitric oxide synthase knockout mice

The effect of long-lasting in vivo restriction of nitric oxide (NO) bioavailability on cardiac and renal P-type ATPases critical for intracellular ion homeostasis is controversial. Previous work has shown in eNOS knockout (eNOS^?/?) mice hearts that Na⁺/K⁺- and Ca²⁺-ATPase activities were depressed but the underlying mechanisms are still unclear. The goal of this study was to characterize potential alterations responsible for impaired enzyme activity in eNOS^?/? mice. Na⁺/K⁺-ATPase activity from crude preparations of adult male eNOS^?/? mice hearts and kidneys was reduced compared with wild-type animals (32 %, p?<?0.05 and 16 %, p?<?0.0001, respectively). Immunoblot analysis showed that although the expression of the predominant (or exclusive, for the kidney) Na⁺/K⁺-ATPase α1 isoform was not significantly changed, there was an important downregulation of the less abundant α2 isoform in the heart (57 %, p?<?0.0001). In addition, although cardiac Ca²⁺-ATPase activity was unaltered, the expression of sarco/endoplasmic reticulum Ca²⁺-ATPase 2 protein in eNOS^?/? mice was very high (290 % compared with wild-type animals, p?<?0.0001) without any significant change in phospholamban expression. Consistent with these findings, the content of cardiac and renal free sulfhydryl groups, essential for the catalytic function of such ATPases, was decreased (23 %, p?<?0.01 and 35 %, p?<?0.05, respectively). Altogether, the present results suggest that the absence of eNOS promotes a compartmentalized altered redox balance that affects the activity and expression of ion transport ATPases.     

Development of a Polygonum minus cell suspension culture system and analysis of secondary metabolites enhanced by elicitation

Polygonum minus has been reported to contain valuable metabolites and to date, there is no report on using cell culture technique for metabolite production in P. minus. Naphthalene acetic acid (NAA) concentrations in the range of 2–6 mg L^?1 were used in a matrix of combinations with dichlorophenoxyacetic acid (2,4-D) concentrations in the range of 2–10 mg L^?1 as plant growth regulators (PGRs) to induce callus cultures. Media that were supplemented with 2 mg L^?1 2,4-D + 4 mg L^?1 NAA, 2 mg L^?1 2,4-D + 6 mg L^?1 NAA and 6 mg L^?1 2,4-D + 8 mg L^?1 NAA were effective for callus induction (93.3 % of the explants produced callus). To establish cell culture, the best growth was obtained from medium that was supplemented with 1 mg L^?1 2,4-D + 2 mg L^?1 NAA. From a 1-g inoculum size, the fresh weight increases exponentially after 5–10 days of culture, and a 26.71 g maximum fresh weight was obtained after 25 days of culture. The cell culture medium was then analyzed using gas chromatography–mass spectrometry (GC–MS). Jasmonic acid (100, 50, 25 and 5 μM), salicylic acid (100, 50, 25 and 5 μM), yeast extract (500, 250 and 100 mg L^?1) and glass beads were used in this research as elicitors. The cell cultures were then incubated with the different elicitors for 1, 2, 3 and 4 days. Several compounds with high peak area percentages were detected, including 2-furancarboxaldehyde, 5-hydroxymethyl, furfural, and 2-cyclopenten-1-one, 2-hydroxy. These results show the diversity of metabolites released by P. minus cell into the culture medium under control conditions.     

Influence of thidiazuron on callus induction and crocin production in corm and style explants of <Emphasis Type="Italic">Crocus sativus</Emphasis> L.

Crocus sativus L., mostly famous as saffron, has gained more attention due to its crocin (crocetin ester) pigment responsible for its extensive pharmaceutical properties. In this study, we established two different callus cultures from corm and style explants of saffron to find out the best explant as a suitable source for crocin production. Comparative analyses of total phenolic, flavonoid, carotenoid and anthocyanin contents were also performed in the two callus cultures. For callus induction, different combinations of MS medium with name thidiazuron (TDZ), benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination were tested. Of the used media, all the combinations containing TDZ and NAA gave 100% callus induction. HPLC-DAD and HPLC–ESI-MS analysis were used for identification of crocin esters in established callus cultures. The highest amount of 0.35 mg g^?1 DW crocin was detected in style originated calli grown on the medium containing 3 mg L^?1 NAA?+?1 mg L^?1 TDZ while the corm calli showed the most abundant total carotenoid (0.73 mg g^?1 DW), phenolic (15.04 mg gallic acid equivalent g^?1 DW) and flavonoid (0.76 mg rutin equivalent g^?1 DW) contents. In general, style-derived calli showed longer time survival with a fine texture and good quality compared to corm-derived calli.     

PAR modulation of the UV-dependent levels of flavonoid metabolites in Arabidopsis thaliana (L.) Heynh. leaf rosettes: cumulative effects after a whole vegetative growth period

Long-term effects of ultraviolet (UV) radiation on flavonoid biosynthesis were investigated in Arabidopsis thaliana using the sun simulators of the Helmholtz Zentrum München. The plants, which are widely used as a model system, were grown (1) at high photosynthetically active radiation (PAR; 1,310 µmol m^?2?s^?1) and high biologically effective UV irradiation (UV-B_BE 180 mW m^?2) during a whole vegetative growth period. Under this irradiation regime, the levels of quercetin products were distinctively elevated with increasing UV-B irradiance. (2) Cultivation at high PAR (1,270 µmol m^?2?s^?1) and low UV-B (UV-B_BE 25 mW m^?2) resulted in somewhat lower levels of quercetin products compared to the high-UV-B_BE conditions, and only a slight increase with increasing UV-B irradiance was observed. On the other hand, when the plants were grown (3) at low PAR (540 µmol m^?2?s^?1) and high UV-B (UV-B_BE 180 mW m^?2), the accumulation of quercetin products strongly increased from very low levels with increasing amounts of UV-B but the accumulation of kaempferol derivatives and sinapoyl glucose was less pronounced. We conclude (4) that the accumulation of quercetin products triggered by PAR leads to a basic UV protection that is further increased by UV-B radiation. Based on our data, (5) a combined effect of PAR and different spectral sections of UV radiation is satisfactorily described by a biological weighting function, which again emphasizes the additional role of UV-A (315–400 nm) in UV action on A. thaliana.     

Accumulation of rosmarinic, chlorogenic and caffeic acids in in vitro cultures of Eryngium planum L.

Eryngium planum L. cell and organ cultures were maintained on Murashige and Skoog media (MS), supplemented with exogenous hormones of different types and various concentrations for high biomass growth. The callus and cell suspension cultures were treated with increased sucrose concentration and/or elicited by methyl jasmonate for the enhancement of selected phenolic acids accumulation. Three phenolic acids, rosmarinic acid (RA), chlorogenic acid (CGA) and caffeic acid (CA), were detected by HPLC-DAD in those cultures. The sum of their content in the dry material was found to be higher in the shoot culture (3.95 mg g^?1), root culture (7.05 mg g^?1), callus (6.20 mg g^?1) and cell suspension (2.04 mg g^?1) than in the leaves (1.87 mg g^?1) and roots (0.76 mg g^?1) of intact plants. The major compound of in vitro cultures was always rosmarinic acid. The content of RA could be increased approximately threefold (16.24 mg g^?1) in the callus culture and approximately twofold (3.91 mg g^?1) in the cell suspension culture by elicitation with 100 μM methyl jasmonate (MeJA). The higher concentration of sucrose (S) in the medium (5, 6 %) led to over a twofold increase of CGA content in the callus culture (2.54 mg g^?1). The three mentioned phenolic acids have been found in E. planum undifferentiated and differentiated in vitro cultures for the first time.     

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg^?1, K _0.5 = 0.10 ± 0.01 mmol L^?1. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg^?1, K _0.5 = 1.30 ± 0.03 mmol L^?1), Mg²⁺ (V _M = 115.0 ± 4.6 U mg^?1, K _0.5 = 0.96 ± 0.03 mmol L^?1), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg^?1, K _0.5 = 1.90 ± 0.04 mmol L^?1), and K⁺ (V _M = 120.0 ± 2.4 U mg^?1, K _M = 2.74 ± 0.08 mmol L^?1) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L^?1. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.     

The effect of sodium bicarbonate supplementation on growth and biochemical composition of marine microalgae cultures

The addition of bicarbonate (NaHCO₃; 0, 1, or 2 g L^?1) to microalgal cultures has been evaluated for two species (Tetraselmis suecica and Nannochloropsis salina) in respect of growth and biochemical composition. In batch cultures, addition of bicarbonate (1 g L^?1) resulted in significantly (P?<?0.05) higher final mean cell abundances for both species. No differences in specific growth rates (SGRs) were recorded for T. suecica between treatments; however, increasing bicarbonate addition decreased SGR values in N. salina cultures. Bicarbonate addition (1 g L^?1) significantly improved nitrate utilisation from the external media and photosynthetic efficiency (F _v /F _m ) in both species. For both T. suecica and N. salina, bicarbonate addition significantly increased the cellular concentrations of total pigments (3,432–3,587 and 19–37 fg cell^?1, respectively) compared to cultures with no additional bicarbonate (1,727 and 11 fg cell^?1, respectively). Moreover, final concentrations of total cellular fatty acids in T. suecica and N. salina cultures supplemented with 2 g L^?1 bicarbonate (7.6?±?1.2 and 1.8?±?0.1 pg cell^?1, respectively) were significantly higher than those cells supplemented with 0 or 1 g L^?1 bicarbonate (3.2–3.5 and 0.9–1.0 pg cell^?1, respectively). In nitrate-deplete cultures, bicarbonate addition caused species-specific differences in the rate of cellular lipid production, rates of change in fatty acid composition and final lipid levels. In summary, the addition of sodium bicarbonate is a viable strategy to increase cellular abundance and concentrations of pigments and lipids in some microalgae as well as the rate of lipid accumulation in nitrate-deplete cultures.     

Influence of salicylic acid and methyl jasmonate elicitation on anthocyanin production in callus cultures of Rosa hybrida L.

This study was undertaken to investigate the effects of salicylic acid (SA) and methyl jasmonate (MeJA) on anthocyanin induction, biomass accumulation, and color value (CV) indices for both pigment content (PC) and pigment production (PP) in callus cultures of Rosa hybrida cv. Pusa Ajay. A concentration-dependent response was exhibited by cultures on SA and MeJA at different concentrations individually or in combinations to Euphorbia millii medium supplemented with 204.5 mM sucrose, 2.45 μM indole butyric acid and 2.33 μM kinetin. There was positive influence on both callus biomass and anthocyanin accumulation. Treatment with 0.5 μM MeJA was most effective in inducing anthocyanin biosynthesis in callus cultures. Anthocyanin accumulation in callus cultures was enhanced with the addition of SA and MeJA, but these did not differ significantly from control for the number of days required for pigment initiation and for color intensification. Moreover, the addition of 0.5 μM MeJA alone resulted in a higher frequency of color response (97.25 %), PC (3.48 ± 0.07 CV g^?1 FW), and PP (1.56 ± 0.03 CV test tube^?1) over control. In contrast, the presence of higher levels of SA (400 μM) and MeJA (5.0 μM) reduced frequency of color response, as well as levels of PC and PP. MeJA did not increase biomass accumulation but promoted frequency of color response, PC and PP. Hence, it was suggested that 0.5 μM MeJA promoted anthocyanin production in rose callus cultures. Significant correlation was found between frequency of response and each of the PC (r = 0.988) and PP (r = 0.990). Furthermore, PC and PP were also highly correlated (r = 0.998).     

Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

A Novel EPS-Producing Strain of Bacillus licheniformis Isolated from a Shallow Vent Off Panarea Island (Italy)

A haloalkaliphilic, thermophilic Bacillus strain (T14), isolated from a shallow hydrothermal vent of Panarea Island (Italy), produced a new exopolysaccharide (EPS). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain T14 was highly related (99 % similarity) to Bacillus licheniformis DSM 13^T and Bacillus sonorensis DSM 13779^T. Further DNA–DNA hybridization analysis revealed 79.40 % similarity with B. licheniformis DSM 13^T and 39.12 % with B. sonorensis DSM 13779^T. Sucrose (5 %) was the most efficient carbon source for growth and EPS production. The highest EPS production (366 mg l^?1) was yielded in fermenter culture at 300 rpm after 48 h of incubation. The purified fraction EPS1 contained fructose/fucose/glucose/galactosamine/mannose in a relative proportion of 1.0:0.75:0.28:tr:tr and possessed a molecular weight of 1,000 kDa displaying a trisaccharide unit constituted by sugars with a β-manno-pyranosidic configuration. Screening for biological activity showed anti-cytotoxic effect of EPS1 against Avarol in brine shrimp test, indicating a potential use in the development of novel drugs.     

Temporal and spectral characterization of the photosynthetic reaction center from Heliobacterium modesticaldum

A time-resolved spectroscopic study of the isolated photosynthetic reaction center (RC) from Heliobacterium modesticaldum reveals that thermal equilibration of light excitation among the antenna pigments followed by trapping of excitation and the formation of the charge-separated state P₈₀₀ ⁺A₀ ^– occurs within ~25 ps. This time scale is similar to that reported for plant and cyanobacterial photosystem I (PS I) complexes. Subsequent electron transfer from the primary electron acceptor A₀ occurs with a lifetime of ~600 ps, suggesting that the RC of H. modesticaldum is functionally similar to that of Heliobacillus mobilis and Heliobacterium chlorum. The (A₀ ^– ? A₀) and (P₈₀₀ ⁺ ? P₈₀₀) absorption difference spectra imply that an 8¹-OH-Chl a _F molecule serves as the primary electron acceptor and occupies the position analogous to ec3 (A₀) in PS I, while a monomeric BChl g pigment occupies the position analogous to ec2 (accessory Chl). The presence of an intense photobleaching band at 790 nm in the (A₀ ^– ? A₀) spectrum suggests that the excitonic coupling between the monomeric accessory BChl g and the 8¹-OH-Chl a _F in the heliobacterial RC is significantly stronger than the excitonic coupling between the equivalent pigments in PS I.     

Carotenoid Production by a Novel Isolate of <Emphasis Type="Italic">Microbacterium paraoxydans</Emphasis>

This study reports extraction and characterization of carotenoid pigments from Microbacterium paraoxydans, a non-photosynthetic bacterium, cultivated in Luria–Bertani (LB) medium. The isolate was identified to be moderately halo- and osmo-tolerant capable of withstanding high (~ 6%) salt and sugar (30% w/v sucrose, 20% w/v glucose) concentrations after a brief period of adaptation. The pigments were characterized using a combination of UV–Vis spectral analysis with the λ_max at 407, 436 and 466 nm and ESI–MS with an m/z value at 536.44. The absorption profile of the pigments and their nature was influenced by carbon, nitrogen source and presence of salt in the growth medium. Highest level of pigment (~ 16 g kg dry wt cells^?1) was produced in NH₄Cl supplemented LB medium. The pigment displayed free radical scavenging, anticancer activity, characteristic of the plant carotenoids. Based on the accumulation of pigments under different conditions, a biochemical pathway for synthesis of neurosporene was proposed.