The structure of the O-specific polysaccharide from a mutant of nitrogen-fixing rhizobacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 with an altered plasmid content

The rhizobacteria Azospirillum brasilense Sp245 produce immunochemically different lipopolysaccharides LPSI and LPSII, both containing identical pentasaccharides built from D-rhamnose residues as the repeating units of O-specific polysaccharides (OPS). In this study, we report the structure of the OPS from A. brasilense LPSI⁻LPSII⁻ mutant Sp245.5, which spontaneously lost the p85 and p120 plasmids upon the formation of a new 300-MDa megaplasmid after the long-term storage of the bacteria in a rich medium. The repeating unit of the OPS of A. brasilense Sp245.5 appeared to be a disaccharide consisting of residues of N-acetyl-D-galactosamine and N-acetyl-D-mannosaminuronic acid:

Hemagglutinating activity and motility of the bacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> in the presence of various nitrogen sources

Hemagglutinating activity of the Azospirillum brasilense strain Sp245 grown in liquid media and the swarming motility of those bacteria grown in semisolid media vary significantly depending on the nitrogen source. In media with nitrate or nitrite, an increase in the hemagglutinating activity and a decrease in the swarming circles’ diameter of Sp245 were observed, compared to bacteria grown in the presence of ammonium or N₂. A ∼67-kDa hemagglutinin exhibiting affinity to the O-specific polysaccharide, an acidic D-rhamnan (OPS-I), was isolated from the surface of Sp245 cells. Introduction of the hemagglutinin into the media resulted in a decrease in the Sp245 cell motility while not affecting its mutants lacking the acidic D-rhamnan or the Sp245.5 mutant with a different OPS structure. Cells of strain Sp245.5 demonstrated hemagglutinating activity two times higher than that of the parent Sp245 strain and formed “diffuse” colonies, rather than distinct swarming circles Sp245 formed when grown in a semisolid medium. The data obtained demonstrate that intercellular contacts mediated by the interaction between the surface hemagglutinin and OPS-I, which is sensitive to environmental factors, affect the collective motility of cells.     

Added salt helps sunbirds and honeyeaters maintain energy balance on extremely dilute nectar diets

Nectar-feeding birds ingest excess water and risk loss of solutes when they excrete it. Previous work has shown that white-bellied sunbirds (Cinnyris talatala) are unable to maintain energy balance on extremely dilute sucrose diets without salts (e.g. <0.25 mol l⁻¹), and that they lose more electrolytes (i.e. Na⁺ and K⁺) via cloacal fluid on these diets than on more concentrated diets. Using white-bellied sunbirds and New Holland honeyeaters (Phylidonyris novaehollandiae) we tested the effect of adding electrolytes to a 0.1 mol l⁻¹ sucrose diet, by including equimolar NaCl and KCl at concentrations from 5 to 40 mmol l⁻¹ and the individual salts at 20 mmol l⁻¹. Addition of salts enabled both species to drink significantly more of the 0.1 mol l⁻¹ sucrose diet than in the absence of salts, and mass loss during the experiment was reduced when salt was included. The larger honeyeaters may be more susceptible to electrolyte depletion than the smaller sunbirds. On 20 mmol l⁻¹ combined salts, both sunbirds and honeyeaters consumed eight times their body mass in fluid daily. KCl alone had no effect. Birds are thus limited in their consumption of extremely dilute diets by increasing losses of Na⁺. This was confirmed by measuring plasma Na⁺ levels, which decreased in both species in the absence of dietary Na⁺. In addition, sucrose assimilation efficiencies were slightly, but significantly lower when sunbirds were fed salt-free diet, while glucose levels in ureteral urine remained extremely low. It is concluded that Na⁺ depletion on very dilute salt-free diets does not affect Na⁺-glucose transport activity in the kidney, but interferes with sugar digestion and/or assimilation in the intestine.     

Effect of aeration strategy on the metabolic flux of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> producing 1,3-propanediol in continuous cultures at different glycerol concentrations

The microbial production of 1,3-propaneidol (1,3-PD) by Klebsiella pneumoniae in continuous fermentation was investigated under low, medium and high glycerol concentrations in the absence and presence of oxygen. The production of 1,3-PD increased with increasing glycerol concentrations, reaching a maximum (266 mmol l⁻¹) under high glycerol concentration (760 mmol l⁻¹) with air sparging at 0.04 vvm. The yield of 1,3-PD, however, decreased gradually with increasing glycerol concentrations, with the highest yield (0.52 mol mol⁻¹) obtained for low glycerol concentration (270 mmol l⁻¹) under anaerobic condition. Enzyme activity assays showed that the specific activity of glycerol dehydratase was highest (0.04 U mg⁻¹) for culture sparged with 0.04 vvm air under high glycerol concentration. The specific activities of glycerol dehydrogenase and 1,3-propanediol oxidoreductase were also improved for all glycerol concentrations and in the presence of oxygen, implying that the dha operon was not repressed under microaerobic conditions. Analysis of metabolic fluxes showed that more carbon flux was shifted to the oxidative pathway with increasing glycerol concentrations, resulting in a reduced flux to 1,3-PD formation. However, the increases in carbon fluxes were not evenly distributed among the oxidative branches of the pathway. Furthermore, ethanol and acetic acid levels were slightly increased whereas 2,3-butanediol and lactic levels were greatly enhanced.     

In vivo evolutionary engineering of a boron-resistant bacterium: <Emphasis Type="Italic">Bacillus boroniphilus</Emphasis>

Boron is an industrially and biologically important element. However, the mechanisms of boron tolerance and its transport in bacteria and many other living systems are still not clearly understood. In this study, the boron resistance level of a boron-tolerant bacterium, Bacillus boroniphilus DSM 17376, was improved up to 300 mmol l⁻¹ boron, by employing an in vivo evolutionary engineering strategy based on batch selection under continuous exposure to gradually increasing boron stress levels. The resistance was heterogeneous within the final mutant population which ranged from about 1- to 16-fold of the wild type resistance at 150 mmol l⁻¹ boron stress level. Boron-resistant mutants had significant cross-resistance to iron and copper stresses, and were also cross-resistant to salt (NaCl) stress, suggesting a common resistance mechanism between these stress types. Additionally, highly boron-resistant mutants had up to 2.8-fold higher boron contents than the wild-type, when exposed to high levels of (150 mmol l⁻¹) continuous boron stress throughout their cultivation. It was shown that evolutionary engineering is a successful approach to significantly increase bacterial boron resistance and investigate the complex mechanism of boron tolerance and transport in microbial systems.     

Epipelic and pelagic primary production in Alaskan Arctic lakes of varying depth

We compared on eight dates during the ice-free period physicochemical properties and rates of phytoplankton and epipelic primary production in six arctic lakes dominated by soft bottom substrate. Lakes were classified as shallow ( < 2.5 m), intermediate in depth (2.5 m <  < 4.5 m), and deep ( > 4.5 m), with each depth category represented by two lakes. Although shallow lakes circulated freely and intermediate and deep lakes stratified thermally for the entire summer, dissolved oxygen concentrations were always >70% of saturation values. Soluble reactive phosphorus and dissolved inorganic nitrogen (DIN = NO₃ ⁻–N + NH₄ ⁺–N) were consistently below the detection limit (0.05 μmol l⁻¹) in five lakes. However, one lake shallow lake (GTH 99) periodically showed elevated values of DIN (17 μmol l⁻¹), total-P (0.29 μmol l⁻¹), and total-N (33 μmol l⁻¹), suggesting wind-generated sediment resuspension. Due to increased nutrient availability or entrainment of microphytobenthos, GTH 99 showed the highest average volume-based values of phytoplankton chlorophyll a (chl a) and primary production, which for the six lakes ranged from 1.0 to 2.9 μg l⁻¹ and 0.7–3.8 μmol C l⁻¹ day⁻¹. Overall, however, increased resulted in increased area-based values of phytoplankton chl a and primary production, with mean values for the three lake classes ranging from 3.6 to 6.1 mg chl a m⁻² and 3.2–5.8 mmol C m⁻² day⁻¹. Average values of epipelic chl a ranged from 131 to 549 mg m⁻² for the three depth classes, but levels were not significantly different due to high spatial variability. However, average epipelic primary production was significantly higher in shallow lakes (12.2 mmol C m⁻² day⁻¹) than in intermediate and deep lakes (3.4 and 2.4 mmol C m⁻² day⁻¹). Total primary production (6.7–15.4 mmol C m⁻² day⁻¹) and percent contribution of the epipelon (31–66%) were inversely related to mean depth, such that values for both variables were significantly higher in shallow lakes than in intermediate or deep lakes. Handling editor: L. Naselli-Flores     

Responses of Azospirillum brasilense to Nitrogen Deficiency and to Wheat Lectin: A Diffuse Reflectance Infrared Fourier Transform (DRIFT) Spectroscopic Study

For the rhizobacterium Azospirillum brasilense, the optimal nutritional range of C:N ratios corresponds to the presence of malate (ca. 3 to 5 g l⁻¹ of its sodium salt) and ammonium (ca. 0.5 to 3 g l⁻¹ of NH₄Cl) as preferred carbon and nitrogen sources, respectively. This microaerophilic aerotactic bacterium is known to have a narrow optimal oxygen concentration range of ca. 3 to 5 μM, which is 1.2% to 2% of oxygen solubility in air-saturated water under normal conditions. In this work, the effects of stress conditions (bound-nitrogen deficiency related to a high C:N ratio in the medium; excess of oxygen) on aerobically grown A. brasilense Sp245, a native wheat-associated endophyte, were investigated in the absence and presence of wheat germ agglutinin (WGA, plant stress protein and a molecular host-plant signal for the bacterium) using FTIR spectroscopy of whole cells in the diffuse reflectance mode (DRIFT). The nutritional stress resulted in the appearance of prominent spectroscopic signs of poly-3-hydroxybutyrate (PHB) accumulation in the bacterial cells; in addition, splitting of the amide I band related to bacterial cellular proteins indicated some stress-induced alterations in their secondary structure components. Similar structural changes were observed in the presence of nanomolar WGA both in stressed A. brasilense cells and under normal nutritional conditions. Comparative analysis of the data obtained and the relevant literature data indicated that the stress conditions applied (which resulted in the accumulation of PHB involved in stress tolerance) and/or the presence of nanomolar concentrations of WGA induced synthesis of bacterial cell-surface (glyco)proteins rich in β-structures, that could be represented by hemagglutinin and/or porin.     

Active calcium ion transport in Xenopuslaevis skin

The skin of intact, free-swimming Xenopus laevis transports Ca²⁺ inwardly in a manner that is proportional to the external [Ca²⁺] up to about 0.3 mmol · l⁻¹, saturates above 0.3 mmol · l⁻¹, and is opposed to the electrochemical gradient. Efflux is relatively constant at external concentrations between 0.016 and 0.6 mmol · l⁻¹; net flux which is negative below 0.125 mmol · l⁻¹ becomes positive above this external [Ca²⁺]. Allometric analysis suggests that both Ca²⁺ influx and efflux scale to the 2/3 power approximately like surface area. There were no significant differences in influx between summer and fall animals; however, efflux was greater in the fall and this resulted in a change from positive balance in the summer to negative balance in the fall. Isolated skins were shown to support a Ca²⁺ uptake rate of nearly 30 nmol · cm⁻² · h⁻¹. The phenylalkylamine verapamil in the apical bathing solution significantly inhibited this at 25 μmol · l⁻¹. The benzothiazepine diltiazem was also effective at 50 μmol · l⁻¹ while the dihydropyradine nifedipine was ineffective up to 100 μmol · l⁻¹. The inorganic ion La³⁺ was effective at blocking Ca²⁺ uptake at 300 μmol · l⁻¹; Ni²⁺ was also effective at 500 μmol · l⁻¹ but Co²⁺ was ineffective up to 500 μmol · l⁻¹. These results suggest that apical calcium channels in Xenopuslaevis skin have properties similar to mammalian L-channels and fish gill Ca²⁺ channels. Accepted: 23 January 1997     

Adventitious bud regeneration from leaf explants of <Emphasis Type="Italic">Platanus occidentalis</Emphasis> L. and genetic stability assessment

The aim of this work is to develop a method of plant regeneration from leaf explants of Platanus occidentalis L. successfully. Woody plant medium (HortScience 16:453–459, 1981) and Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium were used as induced and rooted basal medium, respectively. The effects of combinations of 6-BA, IBA, NAA and KT with different concentrations on adventitious bud regeneration from P. occidentalis leaf explants were compared. The results showed that the highest shoot regeneration frequency (90%) and maximum number (13.72 ± 0.44) of shoots per explant was recorded on WPM medium supplemented with 22.20 mmol l⁻¹ 6-BA and 0.49 mmol l⁻¹ IBA. A 40-day-old explants were much more productive for shoot formation than others in this study. The regenerated shoots were cultured on MS medium supplemented with 1.33 mmol l⁻¹ 6-BA, 0.16 mmol l⁻¹ NAA and 2% (w/v) adenine, after 2-week shoots were transferred to 1/2 MS medium supplemented with 0.49 mmol l⁻¹ IBA for rooting. Hardened plantlets via acclimatization were transferred to pots and transplanted to the soil finally. To ascertain whether tissue culture had effects on the genetic stability of plantlets regenerated, the genetic diversity was assessed using RAPD marker. A total of 96 bands ranging from 0.5 to 2.2 kb with an average of 6.4 bands per primer, were obtained using 15 primers. Amplified products exhibited few of polymorphic patterns across all the plants of P. occidentalis and the overall frequency of detection of somaclonal polymorphisms was lower than 0.0104%. Yuehua Sun, Yanling Zhao, and Xiaojuan Wang contributed equally to this work.     

Significant changes in VLDL-Triacylglycerol and glucose tolerance in obese subjects following ten days of training

We characterized the effect of ten days of training on lipid metabolism in 6 [age 37.2 (2.3) years] sedentary, obese [BMI 34.4 (3.0) kg · m⁻²] males with normal glucose tolerance. An oral glucose tolerance test was performed prior to and at the end of the 10 d of training period. The duration of each daily exercise session was 40 min at an intensity equivalent to ˜75% of the age predicted maximum heart rate. Blood measurements were performed after an overnight fast, before and at the end of the 10 d period. Plasma triacylglycerol was significantly (p < 0.05) reduced following exercise training (2.15 ± 0.29 vs. 1.55 ± 0.28 mmol · l⁻¹). Very low density lipoprotein-triacylglycerol was also significantly (p < 0.05) reduced (1.82 ± 0.3 vs. 1.29 ± 0.29 mmol · l⁻¹). No significant changes in high density lipoprotein-cholesterol were observed as a result of training. Following training fasting plasma glucose and fasting plasma insulin were significantly reduced [Glucose: 5.9 (0.2) mmol · l⁻¹ vs. 5.3 (0.22) mmol · l⁻¹ (p < 0.05); Insulin 264.3 (53.8) ρ · mol · l⁻¹ vs. 200.9 (30.1) ρ · mol · l⁻¹, p = 0.05]. The total area under the glucose curve during the OGTT decreased significantly (p < 0.05). These preliminary data suggest that short-term exercise, without concomitant loss of body mass, induces favorable changes in plasma triacylglycerol, and very low density lipoprotein-triacylglycerol and glucose tolerance but has no effect on high density lipoproteincholesterol. Accepted: 7 January 1998     

Phenotypic variability in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains Sp7 and Sp245: Association with dormancy and characteristics of the variants

The state of metabolic dormancy in diazotrophic bacteria Azospirillum brasilense Sp7 (non-endophytic strain) and Sp245 (endophytic strain) was found to be associated with phenotypic variability. The latter manifested itself in the extension of the spectrum of A. brasilense phenotypic variants resulting from plating of cyst-like resting cells (CRC) on solid media and was more pronounced in strain Sp7. The major colony’s morphological variants of strain Sp7 were (1) the dominant S type; (2) the highly pigmented Pg type; (3) the R type; (4) the Sm type, forming small colonies; and (5) the Sg type, forming segmented colonies. In addition to their colony morphology, the variants differed in the phenotype stability during transfers on the standard solid medium and in their motility in semisolid agar. The occurrence frequency of the phenotypic variants depended on the conditions and duration of incubation (storage) of the CRC of strain Sp7, as well as on heat treatment (at 55 and 60°C for 10 min) of the cells prior to inoculation. The maximum frequency of S → Pg transitions (up to 74%) was observed during the germination of CRC stored in a spent culture medium at −20°C for 4 months; the maximum frequency (up to 100%) of S → Sm transitions was observed after inoculation of the CRC subjected to heat treatment. The Pg variants were the most stable, whereas other types reverted rapidly to the S or Pg variant. The S variant grown in semisolid agar exhibited the mixed type of motility (Swa⁺Gri⁺, swarming and migration in the form of microcolonies); the Pg and Sg variants showed the Swa⁺Gri⁻ (swarming) phenotype and the Sm variant was nonmotile (Swa⁻Gri⁻ phenotype). The spectrum of phenotypic variants of the endophytic strain Sp245 was narrower than that of strain Sp7 and was represented by S, Sm, and M (mucoid) variants that differed in the patterns of cell motility: the dominant S type displayed the swarming pattern (Swa⁺Gri⁻), the mucoid M type showed the mixed type (Swa⁺Gri⁺) of motility, and the Sm variant was nonmotile. The differences between the nonendophytic strain Sp7 and the endophytic strain Sp245 in their capacity for phenotypic dissociation and cell motility in semisolid media may reflect their ability to adapt to changing ambient conditions and specificity of plant-microbial interactions.     

The effects of copper on the photosynthetic response of <Emphasis Type="Italic">Phaeocystis cordata</Emphasis>

We investigated the effects of limiting (1.96 × 10⁻⁹ mol l⁻¹ total Cu, corresponding to pCu 14.8; where pCu = −log [Cu²⁺]) and toxic Cu concentrations up to 8.0 × 10⁻⁵ mol l⁻¹ total Cu (equivalent to pCu 9.5) on growth rates and photosynthetic activity of exponentially grown Phaeocystis cordata, using batch and semi-continuous cultures. With pulse amplitude modulated (PAM) fluorometry, we determined the photochemical response of P. cordata to the various Cu levels, and showed contrasting results for the batch and semi-continuous cultures. Although maximum photosystem II (PSII) quantum yield (Φ_M) was optimal and constant in the semi-continuous P. cordata, the batch cultures showed a significant decrease in Φ_M with culture age (0–72 h). The EC50 for the batch cultures was higher (2.0 × 10⁻¹⁰ mol l⁻¹, pCu9.7), than that for the semi-continuous cultures (6.3 × 10⁻¹¹ mol l⁻¹, pCu10.2). The semi-continuous cultures exhibited a systematic and linear decrease in Φ_M as Cu levels increased (for [Cu²⁺] < 1.0 × 10⁻¹² mol l⁻¹, pCu12.0), however, no effect of high Cu was observed on their operational PSII quantum yield (Φ′_M). Similarly, semi-continuous cultures exhibited a significant decrease in Φ_M, but not in Φ′_M, because of low-Cu levels. Thus, Cu toxicity and Cu limitation damage the PSII reaction centers, but not the processes downstream of PSII. Quenching mechanisms (NPQ and Q _n) were lower under high Cu relative to the controls, suggesting that toxic Cu impairs photo-protective mechanisms. PAM fluorometry is a sensitive tool for detecting minor physiological variations. However, culturing techniques (batch vs. semi-continuous) and sampling time might account for literature discrepancies on the effects of Cu on PSII. Semi-continuous culturing might be the most adequate technique to investigate Cu effects on PSII photochemistry.     

Production and characterization of esterase and lipase from <Emphasis Type="Italic">Haloarcula marismortui</Emphasis>

The present study was conducted to investigate the capability of Haloarcula marismortui to synthesize esterases and lipases, and the effect of physicochemical conditions on the growth and the production of esterases and lipases. Finally, the effect of NaCl concentration and temperature on esterase and lipase activities was studied using intracellular crude extracts. In order to confirm the genomic prediction about the esterase and lipase synthesis, H. marismortui was cultured on a rich medium and the crude extracts (intra- or extracellular) obtained were assayed for both activities using p-nitrophenyl esters and triacylglycerides as substrates. Studies on the kinetics of growth and production of esterase and lipase of H. marismortui were performed, reaching a maximum growth rate of 0.053 h⁻¹ and maximal productions of intracellular esterase and lipase of 2.094 and 0.722 U l⁻¹ using p-nitrophenyl valerate and p-nitrophenyl laurate, respectively. Both enzymes were produced as growth-associated metabolites. The effects of temperature, pH, and NaCl concentration on the growth rate and production of enzymes were studied by using a Box–Behnken response surface design. The three response variables were significantly influenced by the physicochemical factors and an interaction effect between temperature and NaCl concentration was also evidenced. The surface response method estimated the following maximal values for growth rate and productions of esterase and lipase: 0.086 h⁻¹ (at 42.5°C, pH 7.4, and 3.6 mol l⁻¹ NaCl), 2.3 U l⁻¹ (at 50°C, pH 7.5, and 4.3 mol l⁻¹ NaCl), and 0.58 U l⁻¹ (at 50°C, pH 7.6, and 4.5 mol l⁻¹ NaCl), respectively. Esterases were active at different salt concentrations, showing two optimal activities (at 0.5 and 5 mol l⁻¹ NaCl), which suggested the presence of two different esterases. Interestingly, in the absence of salt, esterase retained 50% residual activity. Esterases and lipase activities were maximal at 45°C and inactive at 75°C. This study represents the first report evidencing the synthesis of esterase and lipase by H. marismortui.     

The Use of Fragments of the 85- and 120-MDa Plasmids of Azospirillum brasilense Sp245 to Study the Plasmid Rearrangement in This Bacterium and to Search for Homologous Sequences in Plasmids of Azospirillum brasilense Sp7

A spontaneous loss of the 85- (p85) and 120-MDa (p120) replicons and simultaneous generation of a plasmid of more than 300 MDa were associated with defects in synthesis of O-specific and Calcofluor-binding polysaccharides and had no effect on flagellation and motility of theAzospirillum brasilenseSp245.5 mutant. The plasmid rearrangement was studied by hybridization of DNAs from the wild-type Sp245 strain and the Sp245.5 mutant with p85 and p120 fragments that contained loci involved in formation of the polar (fla) and lateral (laf) flagella, synthesis of O-specific and Calcofluor-binding polysaccharides (lps/cal), swimming (mot), and swarming (swa) of bacteria. Hybridization with the p120 fragments revealed incorporation of the intact fla/swa loci and the altered lps/cal loci into a new megaplasmid. Two EcoRI fragments homologous to the fla/laf/mot/swa loci of p85 were found in A. brasilense Sp245 DNA, whereas only one copy was preserved in the Sp245.5 mutant. Hybridization of the p120 and p85 fragments of Sp245 to the A. brasilenseSp7 DNA for the first time revealed regions of substantial homology to these fragments in the 90- and 115-MDa Sp7 plasmids, respectively.     

Phototropic H<Subscript>2</Subscript> production by a newly isolated strain of <Emphasis Type="Italic">Rhodopseudomonas</Emphasis><Emphasis Type="Italic">palustris</Emphasis>

Rhodopseudomonas palustris TN1 was isolated from Songkhla Lake, Thailand. It phototrophically generates H₂ from the predominant volatile fatty acids (VFAs) produced from microbial dark-fermentations of palm oil milling effluent; yields from 20 mM butyrate, acetate and propionate were 4.7, 2.5, and 1.7 mol H₂ mol VFA⁻¹ with light efficiencies of 1.8, 1, and 0.2%, respectively. Optimum conditions were pH 7 and 3000 lux, although production was reduced by only 33% at 1000 lux. CO₂ evolution never exceeded 9 mmol mol VFA⁻¹.     

Active excretion of ammonia across the gills of the shore crab Carcinus maenas and its relation to osmoregulatory ion uptake

The mechanism of transbranchial excretion of total ammonia of brackish-water acclimated shore crabs, Carcinus maenas was examined using isolated, perfused gills. Applying physiological gradients of NH₄Cl (100–200 μmol · l⁻¹) directed from the haemolymph space to the bath showed that the efflux of total ammonia consisted of two components. The saturable component (excretion of NH₄ ⁺) greatly exceeded the linear component (diffusion of NH₃). When an outwardly directed gradient (200 μmol · l⁻¹) was applied, total ammonia in the perfusate was reduced by more than 50% during a single passage of saline through the gill. Effluxes of ammonia along the gradient were sensitive to basolateral dinitrophenol, ouabain, and Cs⁺ and to apical amiloride. Acetazolamide (1 mmol · l⁻¹ basolateral) or Cl⁻-free conditions had no substantial effects on ammonia flux, which was thus independent of both carbonic anhydrase mediated pH regulation and osmoregulatory NaCl uptake. When an inwardly directed gradient (200 μmol · l⁻¹) was employed, influx rates were about 10-fold smaller and unaffected by basolateral ouabain (5 mmol · l⁻¹) or dinitrophenol (0.5 mmol · l⁻¹). Under symmetrical conditions (100 μmol · l⁻¹ NH₄Cl on both sides) ammonia was actively excreted against the gradient of total ammonia, which increased strongly during the experiment and against the gradient of the partial pressure of NH₃. The active excretion rate was reduced to 7% of controls by basolateral dinitrophenol (0.5 mmol · l⁻¹), to 44% by basolateral ouabain (5 mmol · l⁻¹), to 46% by Na⁺-free conditions and to 42% by basolateral Cs⁺ (10 mmol · l⁻¹), indicating basolateral membrane transport of NH₄ ⁺ via the Na⁺/K⁺-ATPase and K⁺-channels and a second active, apically located, Na⁺ independent transport mechanism of NH₄ ⁺. Anterior gills, which are less capable of active ion uptake than posterior gills, exhibited even increased rates of active excretion of ammonia. We conclude that, under physiological conditions, branchial excretion of ammonia is a directed process with a high degree of effectiveness. It even allows active extrusion against an inwardly directed gradient, if necessary. Accepted: 11 March 1998     

Enhanced <Emphasis Type="SmallCaps">d</Emphasis>-ribose biosynthesis in batch culture of a transketolase-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain by citrate

In this study, the effects of citrate addition on d-ribose production were investigated in batch culture of a transketolase-deficient strain, Bacillus subtilis EC2, in shake flasks and bioreactors. Batch cultures in shake flasks and a 5-l reactor indicated that supplementation with 0.2–0.5 g l⁻¹ of citrate enhanced d-ribose production. When B. subtilis EC2 was cultivated in a 15-l reactor in a complex medium, the d-ribose concentration was 70.9 g l⁻¹ with a ribose yield of 0.497 mol mol⁻¹. When this strain was grown in the same medium supplemented with 0.3 g l⁻¹ of citrate, 83.4 g l⁻¹ of d-ribose were obtained, and the ribose yield was increased to 0.587 mol mol⁻¹. Addition of citrate reduced the activities of pyruvate kinase and phosphofructokinase, while it increased those of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Metabolic flux distribution in the stationary phase indicated that citrate addition resulted in increased fluxes in the pentose phosphate pathway and TCA cycle, and decreased fluxes in the glycolysis and acetate pathways.     

Production of ectoine through a combined process that uses both growing and resting cells of <Emphasis Type="Italic">Halomonas salina</Emphasis> DSM 5928<Superscript>T</Superscript>

Using ectoine-excreting strain Halomonas salina DSM 5928^T, we developed a new process for high-efficiency production of ectoine, which involved a combined process of batch fermentation by growing cells and production by resting cells. In the first stage, batch fermentation was carried out using growing cells under optimal fermentation conditions. The second stage was the production phase, in which ectoine was synthesized and excreted by phosphate-limited resting cells. Optimal conditions for synthesis and excretion of ectoine during batch fermentation in a 10 l fermentor were 0.5 mol l⁻¹ NaCl and an initial monosodium glutamate concentration of 80 g l⁻¹ respectively. The pH was adjusted to 7.0 and the temperature was maintained at 33°C. In phosphate-limited resting cells medium, monosodium glutamate and NaCl concentration was 200 g l⁻¹ and 0.5 mol l⁻¹, respectively, as well as pH was 7.0. The total concentration of ectoine produced was 14.86 g l⁻¹, the productivity and yield of ectoine was 7.75 g l⁻¹ day⁻¹ and 0.14 g g⁻¹, respectively, and the percentage of ectoine excreted was 79%. These levels of ectoine production and excretion are the highest reported to date.     

Indirect regeneration of the Cancer bush (<Emphasis Type="Italic">Sutherlandia frutescens</Emphasis> L.) and detection of <Emphasis Type="SmallCaps">l</Emphasis>-canavanine in <Emphasis Type="Italic">in vitro</Emphasis> plantlets using NMR

The present study reports a simple protocol for indirect shoot organogenesis and plant regeneration of Sutherlandia using rachis and stem segments. Different concentrations (0.0–68.08 μmol l⁻¹) of thidiazuron (TDZ) were used for callus induction and shoot organogenesis. The highest percentage of callus formation (97.5%) and the highest percentage of explants forming shoots (88.8%) were obtained from rachis explants cultured onto Murashige and Skoog (MS) medium (Murashige and Skoog, Physiol. Plant. 15:473–495, 1962) supplemented with 45.41 μmol l⁻¹ TDZ. Scanning electron microscopy demonstrated the early development of adventitious shoots derived from callus cultures. Shoot clusters were further developed and grown in MS hormone-free medium. The presence of l-canavanine was determined by thin-layer chromatography and confirmed after column fractionation using silica gel and nuclear magnetic resonance spectroscopy. Individual shoots were rooted on different concentrations and combinations of MS salt strength and IBA. Half-strength MS salt medium supplemented with 24.6 μmol l⁻¹ IBA was optimal for root induction in which 78% of shoots were rooted. The in vitro plants were successfully acclimatized in a growth chamber with a 90% survival rate.     

Paclobutrazol and plant-growth promoting bacterial endophyte Pantoea sp. enhance copper tolerance of guinea grass (Panicum maximum) in hydroponic culture

As most gramineous plants, guinea grass (Panicum maximum) comprise cellulosic biomass, which may be used as a feedstock for bioenergy. In order to develop such potential energy plants on copper-polluted lands, the hydroponic experiments with Cu, Paclobutrazol (PP333, a kind of antigibberellin) and plant growth-promoting bacterial endophyte (PGPB) treatments were carried out in a greenhouse. The seedlings of two cultivars of guinea grass, GG1 (P. maximum var. Natsukomaki) and GG2 (P. maximum var. Natsukaze) in 3 weeks old were treated, respectively, with different Cu treatments [0(CK), 100, 200, 300, 400, 500 μM l⁻¹ Cu] for estimating Cu toxicity. The results showed that elevated Cu restrained plant growth and reduced biomass. According to the EC50 value [the Cu concentration when the relative gain in fresh weight ratio was 50% of control] of two tested cultivars, the concentration of Cu for further experiments was decided as 300 μM l⁻¹. Both pretreatments of PP333 (200, 400, 600 mg l⁻¹) and PGPB (Pantoea sp.) significantly alleviated the negative affect caused by stress of 300 μM l⁻¹ Cu. The pretreatment of 400 mg l⁻¹ PP333 promoted both two cultivars in biomass, compared to 300 μM l⁻¹ Cu treat. The inoculation of Pantoea sp. Jp3-3 increased shoot dry weight, compared to Cu treat. The results suggested that the main reason for both PP333 and Pantoea sp. Jp3-3 enhanced Cu tolerance in guinea grass was that their pretreatments significantly decreased Cu absorption and accumulation under excessive Cu stress. The present study has provided a new insight into the exploitation of energy plant in heavy metal polluted condition by the way of plant growth regulation for increasing heavy metal tolerance.