Ectoine accumulation in Brevibacterium epidermis

As a halotolerant bacterial species, Brevibacterium epidermis DSM 20659 can grow at relatively high salinity, tolerating up to 2 M NaCl. It synthesizes ectoine and the intracellular content increases with the medium salinity, with a maximum of 0.14 g ectoine/g CDW at 1 M NaCl. Sugar-stressed cells do not synthesize ectoine. Ectoine synthesis is also affected by the presence of external osmolytes. Added betaine is taken up and completely replaced ectoine, while L-proline is only temporarily accumulated after which ectoine is synthesized. The strain can metabolize ectoine; L-glutamate is a better carbon source for ectoine synthesis than L-aspartate.     

Operational stability of Brevibacterium imperialis CBS 489-74 nitrile hydratase

Brevibacterium imperialis CBS 489-74 was grown in broths prepared with yeast and malt extract, bacteriological peptone and 2% glucose or differently modified with the addition of Na-phosphate buffer, FeSO₄, MgSO₄ and CoCl₂. The peak production of nitrile hydratase (NHase) did not change significantly. At the stationary growth phase, the units per milliliter of broth (60 units ml⁻¹) were more important than those at the exponential growth phase.

The NHase operational stability of whole resting cells was monitored following the bioconversion of acrylonitrile to acrylamide in continuous and stirred UF-membrane reactors. The rate of inactivation was independent on buffer molarity from 25 to 75 mM and on pH from 5.8 to 7.4. Enzyme stability and activity remained unchanged in distilled water. The initial reaction rate increased from 12.8 to 23.8 g acrylamide/g dry cell/h, but NHase half-life dropped from 33 to roughly 7 h when temperature was varied from 4°C to 10°C. The addition of butyric acid up to 20 mM did not improve enzyme operational stability, and largely reduced (94%) enzyme activity. Acrylonitrile caused an irreversible damage to NHase activity. High acrylonitrile conversion (86%) was attained using 0.23 mg cells/ml in a continuously operating reactor.     

Identification, characterization, and chromosomal organization of the ftsZ gene from Brevibacterium lactofermentum

The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the previously cloned and sequenced ftsZ genes from other microorganisms. ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes). The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ. The gene was expressed in E. coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50 kDa. Expression of the B. lactofermentum ftsZ gene in E. coli inhibited cell division and led to filamentation. The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E. coli, when cloned on low or high-copy-number vectors. Received: 14 January 1998 / Accepted: 31 March 1998     

Cadmium accumulation and tolerance in Populus nigra and Salix alba

Rooted cuttings of Populus nigra L. clone Poli and Salix alba L. clone SS5 were treated for three weeks with 50 μM CdSO₄ in nutrient solution. The willow showed a far higher Cd tolerance, expressed as tolerance index (Ti), than the poplar in both roots and leaves. The root Cd content was higher in poplar than in willow, whereas in leaves the opposite was found. As a consequence, the translocation factor (Tf) revealed a greater ability of Cd transport in willow than in poplar. Cd treatment enhanced cysteine, γ-glutamylcysteine and reduced glutathione contents in roots of both species, whereas in leaves they were only enhanced in poplar. Furthermore, only poplar leaves showed an enhanced content of phytochelatins, whereas malic and citric acids rose in response to Cd only in the willow leaves. Cd treatment increased putrescine, spermidine and spermine contents in both roots and leaves of the willow, whereas in poplar only the putrescine content was enhanced in roots.     

Metal accumulation by Halodule wrightii populations

Metal concentrations and population parameters of the seagrass Halodule wrightii were determined at three locations at Rio de Janeiro State, Brazil. The possible increase of metal availability in one of these areas, Sepetiba Bay, as a result of dredging of contaminated bottom sediments which ocurred, was evaluated by analyses of Al, Cd, Cr, Cu, Fe, Ni, Pb and Zn in root, rhizome and shoots. In addition, analyses were carried out in H. wrightii populations from non-contaminated areas located at northwestern (Cabo Frio) and southeastern (Angra do Reis) regions of Rio de Janeiro State. Concurrently, abundance and density data of the seagrass populations were obtained. It was found that concentration from Sepetiba Bay samples up to 1.6 ± 0.4 μg g⁻¹ of Cd, 12 ± 1.0 μg g⁻¹ of Cr, 27 ± 2.4 μg g⁻¹ of Pb, 291 ± 47 μg g⁻¹ of Mn, 128 ± 23 μg g⁻¹ of Zn were significantly higher than that from two other collection sites. An increase in Cd and Zn concentration was observed in H. wrightii from Sepetiba Bay indicating that metal mobilization from contaminated sediments through dredging activities were, at least in part, transferred to the biotic compartment via accumulation by the seagrass. The populations of seagrass within the region demonstrated quite substantial changes in biomass data but not in shoot or rhizome density during the study. Such changes in biomass are to be expected, as these dynamics are typical of the small, isolated monospecific populations of H. wrightii along the Rio de Janeiro coast.     

Regulation of 3-hydroxypropionaldehyde accumulation in Klebsiella pneumoniae by overexpression of dhaT and dhaD genes

3-Hydroxypropionaldehyde (3-HPA), an important intermediary metabolite of 1,3-propanediol (PDO) production, would be toxic to the cell growth and led to the abnormal cessation of the fermentation process. In this study, the dhaD gene encoding glycerol dehydrogenase (GDH) and dhaT gene encoding 1,3-propanediol oxidoreductase (PDOR) were overexpressed in Klebsiella pneumoniae ACCC 10082 to decrease the 3-HPA accumulation and increase the coenzyme NADH supply. By the construction of pTD plasmid, GDH and PDOR were both overexpressed and their enzyme activities were increased by 2.6- and 3.2-fold, respectively. The enzyme activity ratio of PDOR/GDHt (glycerol dehydratase) also was increased. On the other hand, NADH production was enhanced and the ratio of NADH/NAD⁺ exceeded 1 after the inducement of IPTG for the constructed strain. The two factors enhanced the transformation of 3-HPA to PDO. In the batch and fed-batch fermentation by the constructed strain, the peak of 3-HPA accumulation reduced by 52.2% and 33.3%, respectively, compared with the control. The PDO concentration and yield reached 59.2 g/L and 0.48 mol/mol, respectively. Furthermore, the fed-batch fermentation process appeared easier to be regulated. This work is considered helpful for the further understanding on the PDO metabolic mechanism of K. pneumoniae and also useful for the PDO fermentation in a large-scale bioreactor.     

Trehalose accumulation in Baudoinia compniacensis following abiotic stress

Baudoinia compniacensis is a microfungus recently described as the principal agent of fouling known as “warehouse staining”, affecting building exteriors, fixtures and vegetation surfaces in areas proximate to distillery aging warehouses, commercial bakeries and other areas subject to low-level ethanol vapour exposure. The surfaces most affected tend to be highly exposed and undergo extreme diurnal temperature fluctuations. In previous work, we have demonstrated the existence of heat-inducible putative chaperone proteins that may also be induced by low-level exposures to ethanol vapour (e.g., <10 ppm). The present study investigated the cellular accumulation of trehalose, a disaccharide identified in some microorganisms to be important in the protection of cell components during adverse stress conditions, such as thermal stress. Following heat shock at 45 °C, we observed a 2.5-fold accumulation of trehalose relative to unheated controls maintained at 26 °C. Peak trehalose concentrations of 10 mg g⁻¹ dry wt were seen at 90 min after heat treatment, followed by a gradual return to post-treatment by 150 min. Exposure of B. compniacensis cells to ethanol resulted in a similar increased accumulation of trehalose compared to unexposed controls. These findings imply that trehalose may be important in the tolerance of this fungus to abiotic stresses, such as heat and solvent exposure, and suggest future research directions for the control and prevention of warehouse staining.     

Cell type-specific protoberberine alkaloid accumulation inThalictrum flavum

Berberine is a common benzylisoquinoline alkaloid with potent antimicrobial properties, which suggest it functions to protect some plants from pathogen challenge. Berberine was identified as the major alkaloid in meadow rue (Thalictrum flavum ssp. glaucum), a medicinal member of the Ranunculaceae, and was detected in seeds and all organs of the plant. The high level of berberine in roots, rhizomes, and older petioles is mainly responsible for the intense yellow color of these organs. In rhizomes, protoberberine alkaloids were detected throughout the pith and, to a lesser extent, the cortex, but were absent from the vascular tissues. Similarly, protoberberine alkaloids were detected in the rib parenchyma of older petioles. In roots, alkaloid accumulation was detected only in the endodermis at the onset of secondary growth. Rather than being sloughed off, the endodermis was found to undergo extensive anticlinal division leading to an expanding cellular cylinder that ultimately displaced all external tissues. Endodermal-specific protoberberine alkaloid accumulation continued throughout root development, but was extended to include 3 to 4 layers of smaller pericycle cells in the oldest roots near the base of the stem. The cell type-specific accumulation of antimicrobial alkaloids and the unusual development of the endodermis and pericycle in T. flavum roots support the putative role of berberine in plant defense.     

Ferritin accumulation under iron scarcity in Drosophila iron cells

Ferritins are highly stable, multi-subunit protein complexes with iron-binding capacities that reach 4500 iron atoms per ferritin molecule. The strict dependence of cellular physiology on an adequate supply of iron cofactors has likely been a key driving force in the evolution of ferritins as iron storage molecules. The insect intestine has long been known to contain cells that are responsive to dietary iron levels and a specialized group of “iron cells” that always accumulate iron-loaded ferritin, even when no supplementary iron is added to the diet. Here, we further characterize ferritin localization in Drosophila melanogaster larvae raised under iron-enriched and iron-depleted conditions. High dietary iron intake results in ferritin accumulation in the anterior midgut, but also in garland (wreath) cells and in pericardial cells, which together filter the circulating hemolymph. Ferritin is also abundant in the brain, where levels remain unaltered following dietary iron chelation, a treatment that depletes ferritin from the aforementioned tissues. We attribute the stability of ferritin levels in the brain to the function of the blood-brain barrier that may shield this organ from systemic iron fluctuations. Most intriguingly, our dietary manipulations demonstrably iron-depleted the iron cells without a concomitant reduction in their production of ferritin. Therefore, insect iron cells may constitute an exception from the evolutionary norm with respect to iron-dependent ferritin regulation. It will be of interest to decipher both the physiological purpose served and the mechanism employed to untie ferritin regulation from cellular iron levels in this cell type.     

Evidence for demand-regulation of ribosome accumulation in E coli

We have determined the relative concentrations of ribosomes accumulated under different growth conditions for a number of translational mutants as well as for some natural isolates of Escherichia coli. The mutants are a tRNA modification mutant (miaA), a streptomycin resistant (SmR) and a streptomycin pseudodependent (SmP) mutant as well as two ribosome ambiguity (ram) mutants. The natural isolates used in this study are known to function with submaximal ribosome kinetics. The data show that for all the ribosome mutants the concentration of ribosomes relative to that in wild type bacteria increases when the growth rate decreases. A small increase is also seen in the natural isolates. In contrast, the miaA mutant shows no increase in ribosome concentration under the same slow growth conditions. The results suggest that bacteria with kinetically impaired ribosomes can to some extent increase the number of ribosomes accumulated under poor growth conditions in order to compensate for their slower function. We use this observation to explain in part how bacteria growing in natural environments can escape the strong selection for maximized growth rates and for optimized ribosomes that are characteristic of laboratory strains.     

RAPD markers associated with quercetin accumulation in Psidium guajava

We used a random amplified polymorphic DNA (RAPD) amplification method to identify molecular markers associated with high quercetin accumulation in the leaves of Psidium guajava L. trees, selected from four different Mexican agronomic regions. We identified six polymorphic RAPD fragments of 620, 590, 370, 690, 480 and 460 bp among individuals of P. guajava. Genetic linkage disequilibrium analysis revealed that three RAPD profiles considered as DNA markers (620/590 bp, 370 bp and 480/460 bp) had a positive, direct association with quercetin content. These informative molecular markers can be used for selective identification of plants with the highest accumulation of flavonoids.     

Ectoine Biosynthesis in Mycobacterium smegmatis

Mycobacterium smegmatis is a commonly used mycobacterial model system. Here, we show that M. smegmatis protects itself against elevated salinity by synthesizing ectoine and hydroxyectoine and characterize the phenotype of a nonproducing mutant. This is the first analysis of M. smegmatis halotolerance and of the molecular mechanism that supports it.     

Evaluation of zinc accumulation potential of Hydrilla verticillata

Biofortification of foods with essential micronutrients and phytoremediation of the contaminated sites are the two sides of the same coin for metals like zinc. In the present study, Zn accumulation potential, growth and antioxidant status of Hydrilla verticillata (L.f.) Royle plants were studied upon supplementation of Zn (0–5 000 μM) for 2 and 7 d. At 5000 μM Zn, plants accumulated about 7.60 and 18.07 mg(Zn) g⁻¹(d.m.) after 2 and 7 d, respectively. Plants exposed to Zn concentrations up to 500 μM showed significantly increased contents of low molecular mass antioxidants and activities of antioxidant enzymes in comparison with controls. Only upon exposure of plants to 5 000 μM Zn, toxicity was observed after 7 d. Therefore, owing to their high Zn accumulation capacity, Hydrilla plants may be used both as a Zn source (via culturing in ca. 100 μM Zn supplemented nutrient medium) or as a phytoremediator.     

Growth and isoflavonoid accumulation of Pueraria candollei var. candollei and P. candollei var. mirifica cell suspension cultures

We established cell suspension cultures derived from leaf, stem, and root calli of Pueraria candollei var. candollei and P. candollei var. mirifica using liquid Murashige and Skoog (MS) medium supplemented with 0.56 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Growth of the cell suspension cultures progressed to the stationary phase within 15–24 days. Methanolic extracts of cell suspension cultures of both varieties of P. candollei were analyzed using a validated HPLC protocol. All cell lines derived from leaf, stem, and root explants produced four major isoflavonoids: daidzein, daidzin, genistein, and genistin; these isoflavonoids were detected only in the roots of intact plants. Furthermore, the isoflavonoid contents of the cell suspension cultures were higher than those of intact plants. Thus, cell suspension culture of both varieties of P. candollei may be an effective tool for isoflavonoid production.     

ATP increases chemoattractant induced cyclic GMP accumulation in Dictyostelium discoideum

Changes in guanosine cyclic 3′,5′-monophosphate associated with adenosine cyclic 3′,5′-monophosphate and folic acid addition in the presence of ATP have been examined in Dictyostelium discoideum. Preincubation with 1 mM ATP had no effect on the basal cyclic GMP level but increased the cycli GMP accumulation in response to cylci AMP (5·10⁻⁸ M) or folic acid (5·10⁻⁶ M) 40–50%. ATP could not be replaced by ADP of 5′-adenylyliminodiphosphate. Because ATP has no effect on cyclic AMP receptor binding these results indicate that structural membrane alterations (e.g. membrane phosphorylation) may control the transduction of a chemotactic signal.     

Phytosterols accumulation in the seeds of Linum usitatissimum L.

A comparative study was performed to determine the free sterols content and composition during the development of three varieties of linseed (H52, O116 and P129). Seed samples were collected at regular intervals from 7 to 60 days after flowering (DAF). Ten compounds were identified: cholesterol, campesterol, brassicasterol, stigmasterol, β-sitosterol, Δ5-avenasterol, cycloartenol; 24-methylene cycloartanol, obtusifoliol, citrostadienol. The maximum level of 4-desmethylsterols (1515 mg/100 g oil) was reached at 7 DAF in P129 variety. H52 had the highest level of 4-4 dimethylsterols (355 mg/100 g oil) at 28 DAF. The greatest amount of 4-monomethylsterols (35 mg/100 g oil) was detected in H52 at 14 DAF. During linseed development, β sitosterol (830 mg/100 g oil) was the major 4-desmethylsterols, followed by campesterol (564 mg/100 g oil) and stigmasterol (265 mg/100 g oil). Some of these compounds followed nearly the same accumulation pattern during linseed maturation.