Microcystin production by Microcystis aeruginosa in a phosphorus-limited chemostat

The production of microcystins (MC) from Microcystis aeruginosa UTEX 2388 was investigated in a P-limited continuous culture. MC (MC-LR, MC-RR, and MC-YR) from lyophilized M. aeruginosa were extracted with 5% acetic acid, purified by a Sep-Pak C(18) cartridge, and then analyzed by high-performance liquid chromatography with a UV detector and Nucleosil C(18) reverse-phase column. The specific growth rate (mu) of M. aeruginosa was within the range of 0.1 to 0.8/day and was a function of the cellular P content under a P limitation. The N/P atomic ratio of steady-state cells in a P-limited medium varied from 24 to 15 with an increasing mu. The MC-LR and MC-RR contents on a dry weight basis were highest at mu of 0.1/day at 339 and 774 microg g(-1), respectively, while MC-YR was not detected. The MC content of M. aeruginosa was higher at a lower mu, whereas the MC-producing rate was linearly proportional to mu. The C fixation rate at an ambient irradiance (160 microeinsteins m(-2) s(-1)) increased with mu. The ratios of the MC-producing rate to the C fixation rate were higher at a lower mu. Accordingly, the growth of M. aeruginosa was reduced under a P limitation due to a low C fixation rate, whereas the MC content was higher. Consequently, increases in the MC content per dry weight along with the production of the more toxic form, MC-LR, were observed under more P-limited conditions.     

Cellular Microcystin Content in N-Limited Microcystis aeruginosa Can Be Predicted from Growth Rate

Cell quotas of microcystin (Q_MCYST; femtomoles of MCYST per cell), protein, and chlorophyll a (Chl a), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship between Q_MCYST and specific growth rate (μ), from which we propose a generalized model that enables Q_MCYST at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts Q_MCYST from μ, μ_max (maximum specific growth rate), Q_MCYSTmax (maximum cell quota), and Q_MCYSTmin (minimum cell quota). Under the conditions examined in this study, we predict a Q_MCYSTmax of 0.129 fmol cell⁻¹ at μ_max and a Q_MCYSTmin of 0.050 fmol cell⁻¹ at μ = 0. Net MCYST production rate (R_MCYST) asymptotes to zero at μ = 0 and reaches a maximum of 0.155 fmol cell⁻¹ day⁻¹ at μ_max. MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with μ, whereas the MCYST/protein ratio reached a maximum at intermediate μ. In contrast, the MCYST/Chl a ratio remained constant. Cell volume correlated negatively with μ, leading to an increase in intracellular MCYST concentration at high μ. Taken together, our results show that fast-growing cells of N-limited M. aeruginosa are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components.     

Biodegradation of Chloromethane by Pseudomonas aeruginosa Strain NB1 under Nitrate-Reducing and Aerobic Conditions

Pseudomonas aeruginosa strain NB1 uses chloromethane (CM) as its sole source of carbon and energy under nitrate-reducing and aerobic conditions. The observed yield of NB1 was 0.20 (±0.06) (mean ± standard deviation) and 0.28 (±0.01) mg of total suspended solids (TSS) mg of CM⁻¹ under anoxic and aerobic conditions, respectively. The stoichiometry of nitrate consumption was 0.75 (±0.10) electron equivalents (eeq) of NO₃⁻ per eeq of CM, which is consistent with the yield when it is expressed on an eeq basis. Nitrate was stoichiometrically converted to dinitrogen (0.51 ± 0.05 mol of N₂ per mol of NO₃⁻). The stoichiometry of oxygen use with CM (0.85 ± 0.21 eeq of O₂ per eeq of CM) was also consistent with the aerobic yield. Stoichiometric release of chloride and minimal accumulation of soluble metabolic products (measured as chemical oxygen demand) following CM consumption, under anoxic and aerobic conditions, indicated complete biodegradation of CM. Acetylene did not inhibit CM use under aerobic conditions, implying that a monooxygenase was not involved in initiating aerobic CM metabolism. Under anoxic conditions, the maximum specific CM utilization rate (k) for NB1 was 5.01 (±0.06) μmol of CM mg of TSS⁻¹ day⁻¹, the maximum specific growth rate (μ_max) was 0.0506 day⁻¹, and the Monod half-saturation coefficient (K_s) was 0.067 (±0.004) μM. Under aerobic conditions, the values for k, μ_max, and K_s were 10.7 (±0.11) μmol of CM mg of TSS⁻¹ day⁻¹, 0.145 day⁻¹, and 0.93 (±0.042) μM, respectively, indicating that NB1 used CM faster under aerobic conditions. Strain NB1 also grew on methanol, ethanol, and acetate under denitrifying and aerobic conditions, but not on methane, formate, or dichloromethane.     

3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation

A modified 3-hydroxypropionate cycle has been proposed as the autotrophic CO₂ fixation pathway for the thermoacidophilic crenarchaeon Metallosphaera sedula. The cycle requires the reductive conversion of 3-hydroxypropionate to propionyl-coenzyme A (propionyl-CoA). The specific activity of the 3-hydroxypropionate-, CoA-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.023 μmol min⁻¹mg protein⁻¹. The reaction sequence is catalyzed by at least two enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the following reaction: 3-hydroxypropionate + ATP + CoA → 3-hydroxypropionyl-CoA + AMP + PP_i. The enzyme was purified 95-fold to a specific activity of 18 μmol min⁻¹ mg protein⁻¹ from autotrophically grown M. sedula cells. An internal peptide sequence was determined and a gene encoding a homologous protein identified in the genome of Sulfolobus tokodaii; similar genes were found in S. solfataricus and S. acidocaldarius. The gene was heterologously expressed in Escherichia coli, and the His-tagged protein was purified. Both the native enzyme from M. sedula and the recombinant enzyme from S. tokodaii not only activated 3-hydroxypropionate to its CoA ester but also activated propionate, acrylate, acetate, and butyrate; however, with the exception of propionate, the affinities for these substrates were reduced. 3-Hydroxypropionyl-CoA synthetase is up-regulated eightfold in autotrophically versus heterotrophically grown M. sedula, supporting its proposed role during CO₂ fixation in this archaeon and possibly other members of the Sulfolobaceae family.     

Kinetic Parameters of Denitrification in a River Continuum

Kinetic parameters for nitrate reduction in intact sediment cores were investigated by using the acetylene blockage method at five sites along the Swale-Ouse river system in northeastern England, including a highly polluted tributary, R. Wiske. The denitrification rate in sediment containing added nitrate exhibited a Michaelis-Menten-type curve. The concentration of nitrate for half-maximal activity (K_map) by denitrifying bacteria increased on passing downstream from 13.1 to 90.4 μM in the main river, but it was highest (640 μM) in the Wiske. The apparent maximal rate (V_maxap) ranged between 35.8 and 324 μmol of N m⁻² h⁻¹ in the Swale-Ouse (increasing upstream to downstream), but it was highest in the Wiske (1,194 μmol N m⁻² h⁻¹). A study of nitrous oxide (N₂O) production at the same time showed that rates ranged from below the detection limit (0.05 μmol of N₂O-N m⁻² h⁻¹) at the headwater site to 27 μmol of N₂O-N m⁻² h⁻¹ at the downstream site. In the Wiske the rate was up to 570 μmol of N₂O-N m⁻² h⁻¹, accounting for up to 80% of total N gas production.     

Active Transport and Accumulation of Iodide by Newly Isolated Marine Bacteria

Iodide (I⁻)-accumulating bacteria were isolated from marine sediment by an autoradiographic method with radioactive ¹²⁵I⁻. When they were grown in a liquid medium containing 0.1 μM iodide, 79 to 89% of the iodide was removed from the medium, and a corresponding amount of iodide was detected in the cells. Phylogenetic analysis based on 16S rRNA gene sequences indicated that iodide-accumulating bacteria were closely related to Flexibacter aggregans NBRC15975 and Arenibacter troitsensis, members of the family Flavobacteriaceae. When one of the strains, strain C-21, was cultured with 0.1 μM iodide, the maximum iodide content and the maximum concentration factor for iodide were 220 ± 3.6 (mean ± standard deviation) pmol of iodide per mg of dry cells and 5.5 × 10³, respectively. In the presence of much higher concentrations of iodide (1 μM to 1 mM), increased iodide content but decreased concentration factor for iodide were observed. An iodide transport assay was carried out to monitor the uptake and accumulation of iodide in washed cell suspensions of iodide-accumulating bacteria. The uptake of iodide was observed only in the presence of glucose and showed substrate saturation kinetics, with an apparent affinity constant for transport and a maximum velocity of 0.073 μM and 0.55 pmol min⁻¹ mg of dry cells⁻¹, respectively. The other dominant species of iodine in terrestrial and marine environments, iodate (IO₃⁻), was not transported.     

Primary and Bacterial Secondary Production in a Southwestern Reservoir

Rates of primary and bacterial secondary production in Lake Arlington, Texas, were determined. The lake is a warm (annual temperature range, 7 to 32°C), shallow, monomictic reservoir with limited macrophyte development in the littoral zone. Samples were collected from six depths within the photic zone from a site located over the deepest portion of the lake. Primary production and bacterial production were calculated from NaH¹⁴CO₃ and [methyl-³H]thymidine incorporation, respectively. Peak instantaneous production ranged between 14.8 and 220.5 μg of C liter⁻¹ h⁻¹. There were two distinct periods of high rates of production. From May through July, production near the metalimnion exceeded 100 μg of C liter⁻¹ h⁻¹. During holomixis, production throughout the water column was in excess of 100 μg of C liter⁻¹ h⁻¹ and above 150 μg of C liter⁻¹ h⁻¹ near the surface. Annual areal primary production was 588 g of C m⁻². Bacterial production was markedly seasonal. Growth rates during late fall through spring were typically around 0.002 h⁻¹, and production rates were typically 5 μg of C liter⁻¹ h⁻¹. Growth rates were higher during warmer parts of the year and reached 0.03 h⁻¹ by August. The maximum instantaneous rate of bacterial production was approximately 45 μg of C liter⁻¹ h⁻¹. Annual areal bacterial production was 125 g of C m⁻². Temporal and spatial distributions of bacterial numbers and activities coincided with temporal and spatial distributions of primary production. Areal primary and bacterial secondary production were highly correlated (r = 0.77, n = 15, P < 0.002).     

Impact of Different In Vitro Electron Donor/Acceptor Conditions on Potential Chemolithoautotrophic Communities from Marine Pelagic Redoxclines

Anaerobic or microaerophilic chemolithoautotrophic bacteria have been considered to be responsible for CO₂ dark fixation in different pelagic redoxclines worldwide, but their involvement in redox processes is still not fully resolved. We investigated the impact of 17 different electron donor/acceptor combinations in water of pelagic redoxclines from the central Baltic Sea on the stimulation of bacterial CO₂ dark fixation as well as on the development of chemolithoautotrophic populations. In situ, the highest CO₂ dark fixation rates, ranging from 0.7 to 1.4 μmol liter⁻¹ day⁻¹, were measured directly below the redoxcline. In enrichment experiments, chemolithoautotrophic CO₂ dark fixation was maximally stimulated by the addition of thiosulfate, reaching values of up to 9.7 μmol liter⁻¹ CO₂ day⁻¹. Chemolithoautotrophic nitrate reduction proved to be an important process, with rates of up to 33.5 μmol liter⁻¹ NO₃⁻ day⁻¹. Reduction of Fe(III) or Mn(IV) was not detected; nevertheless, the presence of these potential electron acceptors influenced the development of stimulated microbial assemblages. Potential chemolithoautotrophic bacteria in the enrichment experiments were displayed on 16S ribosomal complementary DNA single-strand-conformation polymorphism fingerprints and identified by sequencing of excised bands. Sequences were closely related to chemolithoautotrophic Thiomicrospira psychrophila and Maorithyas hadalis gill symbiont (both Gammaproteobacteria) and to an uncultured nitrate-reducing Helicobacteraceae bacterium (Epsilonproteobacteria). Our data indicate that this Helicobacteraceae bacterium could be of general importance or even a key organism for autotrophic nitrate reduction in pelagic redoxclines.     

Effect of Tungstate on Nitrate Reduction by the Hyperthermophilic Archaeon Pyrobaculum aerophilum

Pyrobaculum aerophilum, a hyperthermophilic archaeon, can respire either with low amounts of oxygen or anaerobically with nitrate as the electron acceptor. Under anaerobic growth conditions, nitrate is reduced via the denitrification pathway to molecular nitrogen. This study demonstrates that P. aerophilum requires the metal oxyanion WO₄²⁻ for its anaerobic growth on yeast extract, peptone, and nitrate as carbon and energy sources. The addition of 1 μM MoO₄²⁻ did not replace WO₄²⁻ for the growth of P. aerophilum. However, cell growth was completely inhibited by the addition of 100 μM MoO₄²⁻ to the culture medium. At lower tungstate concentrations (0.3 μM and less), nitrite was accumulated in the culture medium. The accumulation of nitrite was abolished at higher WO₄²⁻ concentrations (<0.7 μM). High-temperature enzyme assays for the nitrate, nitrite, and nitric oxide reductases were performed. The majority of all three denitrification pathway enzyme activities was localized to the cytoplasmic membrane, suggesting their involvement in the energy metabolism of the cell. While nitrite and nitric oxide specific activities were relatively constant at different tungstate concentrations, the activity of nitrate reductase was decreased fourfold at WO₄²⁻ levels of 0.7 μM or higher. The high specific activity of the nitrate reductase enzyme observed at low WO₄²⁻ levels (0.3 μM or less) coincided with the accumulation of nitrite in the culture medium. This study documents the first example of the effect of tungstate on the denitrification process of an extremely thermophilic archaeon. We demonstrate here that nitrate reductase synthesis in P. aerophilum occurs in the presence of high concentrations of tungstate.     

Steady-State Effects of 2,5,2′,5′-Tetrachlorobiphenyl on Growth, Photosynthesis, and P Uptake in Selenastrum capricornutum

The steady-state effect of 2,5,2′,5′-tetrachlorobiphenyl (TCBP) on the green alga Selenastrum capricornutum was investigated in a P-limited two-stage chemostat system. The partition coefficient of this polychlorinated biphenyl congener was 5.9 × 10⁴ in steady-state cultures. At a cellular TCBP concentration of 12.2 × 10⁻⁸ ng · cell⁻¹, growth rate was not affected. However, photosynthetic capacity (P_max) was significantly enhanced by TCBP (56 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ versus 34 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ in the control). Photosynthetic efficiency, or the slope of the photosynthesis-irradiance curve, was also significantly higher. There was little difference in the cell chlorophyll a content, and therefore the difference in these photosynthetic characteristics was the same even when they were expressed on a per-chlorophyll a basis. Cell C content was higher in TCBP-containing cells than in TCBP-free cells, but approximately 36% of the C fixed by cells with TCBP was not incorporated as cell C. The maximum P uptake rate was also enhanced by TCBP, but the half-saturation concentration appeared to be unaffected.     

Biphasic Behavior of Anammox Regulated by Nitrite and Nitrate in an Estuarine Sediment

The production of N₂ gas via anammox was investigated in sediment slurries at in situ NO₂⁻ concentrations in the presence and absence of NO₃⁻. With single enrichment above 10 μM ¹⁴NO₂⁻ or ¹⁴NO₃⁻ and ¹⁵NH₄⁺, anammox activity was always linear (P < 0.05), in agreement with previous findings. In contrast, anammox exhibited a range of activity below 10 μM NO₂⁻ or NO₃⁻, including an elevated response at lower concentrations. With 100 μM NO₃⁻, no significant transient accumulation of NO₂⁻ could be measured, and the starting concentration of NO₂⁻ could therefore be regulated. With dual enrichment (1 to 20 μM NO₂⁻ plus 100 μM NO₃⁻), there was a pronounced nonlinear response in anammox activity. Maximal activity occurred between 2 and 5 μM NO₂⁻, but the amplitude of this peak varied across the study (November 2003 to June 2004). Anammox accounted for as much as 82% of the NO₂⁻ added at 1 μM in November 2003 but only for 15% in May 2004 and for 26 and 5% of the NO₂⁻ added at 5 μM for these two months, respectively. Decreasing the concentration of NO₃⁻ but holding NO₂⁻ at 5 μM decreased the significance of anammox as a sink for NO₂⁻. The behavior of anammox was explored by use of a simple anammox-denitrification model, and the concept of a biphasic system for anammox in estuarine sediments is proposed. Overall, anammox is likely to be regulated by the availability of NO₃⁻ and NO₂⁻ and the relative size or activity of the anammox population.     

Effect of Sarcoplasmic Reticulum (SR) Calcium Content on SR Calcium Release Elicited by Small Voltage-Clamp Depolarizations in Frog Cut Skeletal Muscle Fibers Equilibrated with 20 mM EGTA

Cut muscle fibers from Rana temporaria (sarcomere length, 3.5–3.9 μm; 14–16°C) were mounted in a double Vaseline-gap chamber and equilibrated with an external solution that contained tetraethyl ammonium– gluconate and an internal solution that contained Cs as the principal cation, 20 mM EGTA, and 0 Ca. Fibers were stimulated with a voltage-clamp pulse protocol that consisted of pulses to −70, −65, −60, −45, and −20 mV, each separated by 400-ms periods at −90 mV. The change in total Ca that entered into the myoplasm (Δ[Ca_T]) and the Ca content of the SR ([Ca_SR]) were estimated with the EGTA/phenol red method (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259–336). Fibers were stimulated with the pulse protocol, usually every 5 min, so that the resting value of [Ca_SR] decreased from its initial value of 1,700–2,300 μM to values near or below 100 μM after 18–30 stimulations. Three main findings for the voltage pulses to −70, −65, and −60 mV are: (a) the depletion-corrected rate of Ca release (release permeability) showed little change when [Ca_SR] decreased from its highest level (>1,700 μM) to ∼1,000 μM; (b) as [Ca_SR] decreased below 1,000 μM, the release permeability increased to a maximum level when [Ca_SR] was near 300 μM that was on average about sevenfold larger than the values observed for [Ca_SR] > 1,000 μM; and (c) as [Ca_SR] decreased from ∼300 μM to <100 μM, the release permeability decreased, reaching half its maximum value when [Ca_SR] was ∼110 μM on average. It was concluded that finding b was likely due to a decrease in Ca inactivation, while finding c was likely due to a decrease in Ca-induced Ca release.     

Kinesin-2 KIF3AB Exhibits Novel ATPase Characteristics

KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. There has been significant interest in KIF3AB in defining the key principles that underlie the processivity of KIF3AB in comparison with homodimeric processive kinesins. To define the ATPase mechanism and coordination of KIF3A and KIF3B stepping, a presteady-state kinetic analysis was pursued. For these studies, a truncated murine KIF3AB was generated. The results presented show that microtubule association was fast at 5.7 μm⁻¹ s⁻¹, followed by rate-limiting ADP release at 12.8 s⁻¹. ATP binding at 7.5 μm⁻¹ s⁻¹ was followed by an ATP-promoted isomerization at 84 s⁻¹ to form the intermediate poised for ATP hydrolysis, which then occurred at 33 s⁻¹. ATP hydrolysis was required for dissociation of the microtubule·KIF3AB complex, which was observed at 22 s⁻¹. The dissociation step showed an apparent affinity for ATP that was very weak (K_½,ATP at 133 μm). Moreover, the linear fit of the initial ATP concentration dependence of the dissociation kinetics revealed an apparent second-order rate constant at 0.09 μm⁻¹ s⁻¹, which is inconsistent with fast ATP binding at 7.5 μm⁻¹ s⁻¹ and a K_d,ATP at 6.1 μm. These results suggest that ATP binding per se cannot account for the apparent weak K_½,ATP at 133 μm. The steady-state ATPase K_m,ATP, as well as the dissociation kinetics, reveal an unusual property of KIF3AB that is not yet well understood and also suggests that the mechanochemistry of KIF3AB is tuned somewhat differently from homodimeric processive kinesins.     

In glucose-limited continuous culture the minimum substrate concentration for growth,smin, is crucial in the competition between the enterobacterium Escherichia coli and Chelatobacter heintzii,an environmentally abundant bacterium

The competition for glucose between Escherichia coli ML30, a typical copiotrophic enterobacterium and Chelatobacter heintzii ATCC29600, an environmentally successful strain, was studied in a carbon-limited culture at low dilution rates. First, as a base for modelling, the kinetic parameters μ_max and K_s were determined for growth with glucose. For both strains, μ_max was determined in batch culture after different precultivation conditions. In the case of C. heintzii, μ_max was virtually independent of precultivation conditions. When inoculated into a glucose-excess batch culture medium from a glucose-limited chemostat run at a dilution rate of 0.075 h⁻¹ C. heintzii grew immediately with a μ_max of 0.17±0.03 h⁻¹. After five transfers in batch culture, μ_max had increased only slightly to 0.18±0.03 h⁻¹. A different pattern was observed in the case of E. coli. Inoculated from a glucose-limited chemostat at D=0.075 h⁻¹ into glucose-excess batch medium E. coli grew only after an acceleration phase of ∼3.5 h with a μ_max of 0.52 h⁻¹. After 120 generations and several transfers into fresh medium, μ_max had increased to 0.80±0.03 h⁻¹. For long-term adapted chemostat-cultivated cells, a K_s for glucose of 15 μg l⁻¹ for C. heintzii, and of 35 μg l⁻¹ for E. coli, respectively, was determined in ¹⁴C-labelled glucose uptake experiments. In competition experiments, the population dynamics of the mixed culture was determined using specific surface antibodies against C. heintzii and a specific 16S rRNA probe for E. coli. C. heintzii outcompeted E. coli in glucose-limited continuous culture at the low dilution rates of 0.05 and 0.075 h⁻¹. Using the determined pure culture parameter values for K_s and μ_max, it was only possible to simulate the population dynamics during competition with an extended form of the Monod model, which includes a finite substrate concentration at zero growth rate (s_min). The values estimated for s_min were dependent on growth rate; at D=0.05 h⁻¹, it was 12.6 and 0 μg l⁻¹ for E. coli and C. heintzii, respectively. To fit the data at D=0.075 h⁻¹, s_min for E. coli had to be raised to 34.9 μg l⁻¹ whereas s_min for C. heintzii remained zero. The results of the mathematical simulation suggest that it is not so much the higher K_s value, which is responsible for the unsuccessful competition of E. coli at low residual glucose concentration, but rather the existence of a significant s_min.     

Spatiotemporal Variations in Microcystin Concentrations and in the Proportions of Microcystin-Producing Cells in Several Microcystis aeruginosa Populations

With the aim of explaining the variations in microcystin (MC) concentrations during cyanobacterial blooms, we studied several Microcystis aeruginosa populations blooming in different freshwater ecosystems located in the same geographical area. As assessed by real-time PCR, it appeared that the potentially MC-producing cells (mcyB⁺) were predominant (70 to 100%) in all of these M. aeruginosa populations, with the exception of one population in which non-MC-producing cells always dominated. Apart from the population in the Grangent Reservoir, we found that the proportions of potentially MC-producing and non-MC-producing cells varied little over time, which was consistent with the fact that according to a previous study of the same populations, the intergenic transcribed spacer (ITS) genotype composition did not change (38). In the Grangent Reservoir, the MC-RR variant was the dominant microcystin variant throughout the bloom season, despite changes in the ITS composition and in the proportions of mcyB⁺ cells. Finally, the variations in total MC concentrations (0.3 to 15 μg liter⁻¹) and in the MC cellular quotas (0.01 to 3.4 pg cell⁻¹) were high both between and within sites, and no correlation was found between the MC concentrations and the proportion of mcyB⁺ cells. All of these findings demonstrate that very different results can be found for the proportions of potentially MC-producing and non-MC-producing cells and MC concentrations, even in M. aeruginosa populations living in more or less connected ecosystems, demonstrating the importance of the effect of very local environmental conditions on these parameters and also the difficulty of predicting the potential toxicity of Microcystis blooms.Microcystins (MCs) are the most common cyanotoxins and have been involved in several animal and human poisoning episodes (8). These hepatotoxic cyclic heptapeptides are synthesized by a multifunctional enzyme complex (10, 40), and the discovery of the gene cluster encoding this complex has made it possible in recent years to develop molecular tools for studying the relative proportions of MC-producing and non-MC-producing cells in natural cyanobacterial populations. Potentially MC-producing and non-MC-producing cells can coexist in these populations, but the factors and processes governing the dynamics of these subpopulations remain unclear.Some recent papers on the Microcystis genus have shown that the proportions of potentially MC-producing cells can differ considerably from lake to lake. For example, in Lake Wannsee, Germany, this proportion was between 0 and 40% (28), as it was in Lake Oneida, United States (18), and in Lake Mikata, Japan (48). In contrast, large variations over time (6 to 93%) of potentially MC-producing cells were found in the Grangent Reservoir, France (4). Major variations (30 to 80%) were also found in a natural French population of Planktothrix agardhii (3), and the variations in the proportions of potentially MC-producing cells reflected those of the MC concentrations. However, only 54% of the variation in MC concentrations could be explained by changes in the proportion of MC-producing cells, suggesting that a considerable part of the MC concentrations was also due to variations in MC cell quotas. These findings suggest that the toxic risks during cyanobacterial proliferations are due to variations in both the proportion of MC-producing cells and the production of MC by the toxic cells.Numerous papers have already investigated the impact of various biotic and abiotic environmental factors on microcystin production by toxic cells. These studies demonstrate that MC production can be influenced by temperature (35), light (46), nutrients such as nitrogen and phosphorus (12, 32), pH (39), iron (42), xenobiotics (17, 34, 45), and predators (22, 23, 47). Despite inconsistent results, the production of microcystins by the cells does seems to be linked to their growth rate (11, 31, 33), which is itself affected by environmental conditions. On the other hand, several studies of variations in the proportions of MC-producing cells have demonstrated the potential influence of nutrient concentrations (9, 48) and light and temperature (5), and two papers (3, 5) have suggested that there is a negative correlation between the proportions of MC-producing cells and the abundance of cyanobacterial cells. These findings are consistent with the data of Kardinaal and Visser (26), showing that in Dutch lakes there is a negative relationship between the densities of cyanobacterial cells and the mean MC concentration in the cells.In an overall attempt to explain the variations of toxicity during cyanobacterial blooms, we studied the spatiotemporal variations in MC concentrations and in the proportions of MC-producing and non-MC-producing cells in several Microcystis aeruginosa populations blooming in different freshwater ecosystems located in the same geographical area. The point of this study was to analyze these variations in terms of the characteristics of these ecosystems and the population dynamics of the M. aeruginosa populations. In addition, these data were compared to the variations in the intergenic transcribed spacer (ITS) composition of the same populations recently reported by Sabart et al. (38). The proportion of potentially MC-producing cells was estimated by a real-time quantitative PCR approach, the change in threshold cycle (ΔC_T) method recently developed by Briand et al. for Planktothrix (3) and Microcystis (4) and targeting the mcyB (mcyA for Planktothrix) and phycocyanin (PC) genes.     

Transition from Anaerobic to Aerobic Growth Conditions for the Sulfate-Reducing Bacterium Desulfovibrio oxyclinae Results in Flocculation

A chemostat culture of the sulfate-reducing bacterium Desulfovibrio oxyclinae isolated from the oxic layer of a hypersaline cyanobacterial mat was grown anaerobically and then subjected to gassing with 1% oxygen, both at a dilution rate of 0.05 h⁻¹. The sulfate reduction rate under anaerobic conditions was 370 nmol of SO₄²⁻ mg of protein⁻¹ min⁻¹. At the onset of aerobic gassing, sulfate reduction decreased by 40%, although viable cell numbers did not decrease. After 42 h, the sulfate reduction rate returned to the level observed in the anaerobic culture. At this stage the growth yield increased by 180% compared to the anaerobic culture to 4.4 g of protein per mol of sulfate reduced. Protein content per cell increased at the same time by 40%. The oxygen consumption rate per milligram of protein measured in washed cell suspensions increased by 80%, and the thiosulfate reduction rate of the same samples increased by 29% with lactate as the electron donor. These findings indicated possible oxygen-dependent enhancement of growth. After 140 h of growth under oxygen flux, formation of cell aggregates 0.1 to 3 mm in diameter was observed. Micrometer-sized aggregates were found to form earlier, during the first hours of exposure to oxygen. The respiration rate of D. oxyclinae was sufficient to create anoxia inside clumps larger than 3 μm, while the levels of dissolved oxygen in the growth vessel were 0.7 ± 0.5 μM. Aggregation of sulfate-reducing bacteria was observed within a Microcoleus chthonoplastes-dominated layer of a cyanobacterial mat under daily exposure to oxygen concentrations of up to 900 μM. Desulfonema-like sulfate-reducing bacteria were also common in this environment along with other nonaggregated sulfate-reducing bacteria. Two-dimensional mapping of sulfate reduction showed heterogeneity of sulfate reduction activity in this oxic zone.     

Chromosomal Gene Inactivation in the Green Sulfur Bacterium Chlorobium tepidum by Natural Transformation

Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40°C on agar plates could be completely inhibited by 100 μg of gentamicin ml⁻¹, 2 μg of erythromycin ml⁻¹, 30 μg of chloramphenicol ml⁻¹, or 1 μg of tetracycline ml⁻¹ or a combination of 300 μg of streptomycin ml⁻¹ and 150 μg of spectinomycin ml⁻¹. Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10⁻⁷ were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10⁻³ were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.     

Valuable ingredients and feed toxicity evaluation of Microcystis aeruginosa acidolysis product in mice

This research studied the extraction from Microcystis aeruginosa using hydrochloric acid method as a potentially valuable protein resource from eutrophic lakes. Amino acid composition, residual algal toxins, and heavy metals of the acidolysis product were studied. After 18 h of hydrochloric acid treatment, the product of M. aeruginosa contained 17 amino acids, 51.34% of total amino acid requirements, and 30.25% of the livestock and poultry essential amino acid (Eaa). The residual microcystin-LR (MC-LR) was 0.94 µg kg⁻¹, which was less than WHO drinking water limit of microcystins. The removal ratio of microcystins was higher than 99.99% during the process of hydrolysis. The concentration of heavy metals of the product was in compliance with feed standards. Furthermore, using Horn’s method, Mouse Micronucleus Test and Sperm Shape Abnormality Test were conducted to study the forage safety of the product. Half lethal dose (LD₅₀) of acidolysis product in mice was >9.09 g kg⁻¹ body weight, actually belonging to non-toxic grade. Every dose treatment did not significantly increase activities of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and γ-glutamyltransferase (γ-GT). The results of both micronucleus test and sperm shape abnormality test were negative, which suggested the product with no mutagenicity and sperm malformation effects. This study indicated that the acidolysis product of M. aeruginosa was safe to be used as a feed ingredient.     

Chlorophyll a Fluorescence and Photosynthetic and Growth Responses of Pinus radiata to Phosphorus Deficiency, Drought Stress, and High CO(2)

Needles from phosphorus deficient seedlings of Pinus radiata D. Don grown for 8 weeks at either 330 or 660 microliters CO₂ per liter displayed chlorophyll a fluorescence induction kinetics characteristic of structural changes within the thylakoid chloroplast membrane, i.e. constant yield fluorescence (F_O) was increased and induced fluorescence ([F_P-F_I]/F_O) was reduced. The effect was greatest in the undroughted plants grown at 660 μl CO₂ L⁻¹. By week 22 at 330 μl CO₂ L⁻¹ acclimation to P deficiency had occurred as shown by the similarity in the fluorescence characteristics and maximum rates of photosynthesis of the needles from the two P treatments. However, acclimation did not occur in the plants grown at 660 μl CO₂ L⁻¹. The light saturated rate of photosynthesis of needles with adequate P was higher at 660 μl CO₂ L⁻¹ than at 330 μl CO₂ L⁻¹, whereas photosynthesis of P deficient plants showed no increase when grown at the higher CO₂ concentration. The average growth increase due to CO₂ enrichment was 14% in P deficient plants and 32% when P was adequate. In drought stressed plants grown at 330 μl CO₂ L⁻¹, there was a reduction in the maximal rate of quenching of fluorescence (R_Q) after the major peak. Constant yield fluorescence was unaffected but induced fluorescence was lower. These results indicate that electron flow subsequent to photosystem II was affected by drought stress. At 660 μl CO₂ L⁻¹ this response was eliminated showing that CO₂ enrichment improved the ability of the seedlings to acclimate to drought stress. The average growth increase with CO₂ enrichment was 37% in drought stressed plants and 19% in unstressed plants.     

Methane and Trichloroethylene Degradation by Methylosinus trichosporium OB3b Expressing Particulate Methane Monooxygenase

Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on Methylosinus trichosporium OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For M. trichosporium OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (V_max) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (K_s) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane, V_max/K_s, doubled from 4.9 × 10⁻³ to 9.9 × 10⁻³ liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by M. trichosporium OB3b was also examined with varying copper and formate concentrations. M. trichosporium OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the V_max for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the V_max to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in K_s from 36 to 7.9 μM. V_max/K_s for TCE degradation by pMMO also increased from 6.9 × 10⁻⁵ to 5.2 × 10⁻⁴ liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO.