THE EFFECT OF IRON NUTRITION ON PHOTOSYNTHESIS AND NITROGEN FIXATION IN CULTURES OF TRICHODESMIUM (CYANOPHYCEAE)1

Cultures of Trichodesmium NIBB 1067 were grown in the synthetic medium AQUIL with a range of iron added from none to 5 × 10^?7 M Fe for 15 days. Chlorophyll-a, cell counts, and total cell volume were two or three times higher in medium with 10^?7 M Fe than with no added Fe. Oxygen production rate per chlorophyll-a was over 60% higher with higher iron. Increased iron stimulated photosynthesis at all irradiances from about 12–250 μE · m^?2· s^?1. Nitrogen fixation rate, estimated from acetylene reduction, for 10^?7 and 10^?8 M Fe cultures was approximately twice that of the cultures with no added Fe. The range of rates of O₂ production and N₂ fixation in cultures at the iron concentrations we used were similar to the rates from natural samples of Trichodesmium from both the Atlantic, and the Pacific oceans. This similarity may allow this clone to be used, with some caution, for future physiological ecology studies. This study demonstrates the importance of iron to photosynthesis and nitrogen fixation and suggests that Trichodesmium plays a central role in the biogeochemical cycles of iron, carbon and nitrogen.     

IRON STIMULATION OF PHOTOSYNTHESIS AND NITROGEN FIXATION IN ANABAENA 7120 AND TRICHODESMIUM (CYANOPHYCEAE)1

Iron availability may limit carbon and nitrogen fixation in the oceans. The freshwater cyanobacterium, Anabaena, was used as a laboratory model for the biochemical and physiological effects of iron. Increased iron nutrition, in the range of 10^?8 M to 10^?6 M resulted in increases of approximately four fold in carbon and nitrogen fixation rates. Chlorophyll concentration increased, and the relative amount of in vivo fluorescence was reduced with more iron. Natural samples of Trichodesmium, collected off Barbados and incubated with increased iron for two days, showed similar effects. Trichodesmium responded to iron additions indicating that it may be Fe limited in its natural environment. These responses to iron are consistent with the biochemical roles of iron in photosynthesis and nitrogen fixation. The results are discussed in the geochemical context of the sporadic total iron input to tropical oceans and possible implications to spatial and temporal patterns of productivity.     

DIFFERENT PHYSIOLOGICAL RESPONSES OF FOUR MARINE SYNECHOCOCCUS STRAINS (CYANOPHYCEAE) TO NICKEL STARVATION UNDER IRON‐REPLETE AND IRON‐DEPLETE CONDITIONS1

Synechococcus species are important primary producers in coastal and open‐ocean ecosystems. When nitrate was provided as the sole nitrogen source, nickel starvation inhibited the growth of strains WH8102 and WH7803, while it had little effect on two euryhaline strains, WH5701 and PCC 7002. Nickel was required for the acclimation of Synechococcus WH7803 to low iron and high light. In WH8102 and WH7803, nickel starvation decreased the linear electron transport activity, slowed down Q_A reoxidation, but increased the connectivity factor between individual photosynthetic units. Under such conditions, the reduction of their intersystem electron transport chains was expected to increase, and their cyclic electron transport around PSI would be favored. Nickel starvation decreased the total superoxide dismutase (SOD) activity of WH8102 and WH7803 by 30% and 15% of the control, respectively. The protein‐bound ⁶³Ni of the oceanic strain WH8102 comigrated with SOD activity on nondenaturing gels and thus provided additional evidence for the existence of active NiSOD in Synechococcus WH8102. In WH7803, it seems likely that nickel starvation affected other metabolic pathways and thus indirectly affected the total SOD activity.     

Chemotaxis toward Nitrogenous Compounds by Swimming Strains of Marine Synechococcus spp.

Many of the open-ocean isolates of the marine unicellular cyanobacterium Synechococcus spp. are capable of swimming motility, whereas coastal isolates are nonmotile. Surprisingly, the motile strains do not display phototactic or photophobic responses to light, but they do demonstrate positive chemoresponses to several nitrogenous compounds. The chemotactic responses of Synechococcus strain WH8113 were investigated using blind-well chemotaxis chambers fitted with 3.0-μm-pore-size Nuclepore filters. One well of each chamber contained cells suspended in aged Sargasso Sea water, and the other well contained the potential chemoattractant in seawater. The number of cells that crossed the filter into the attractant-seawater mixture was measured by direct cell counts and compared with values obtained in chambers lacking gradients. Twenty-two compounds were tested, including sugars, amino acids, and simple nitrogenous substrates, at concentrations ranging from 10⁻⁵ to 10⁻¹⁰ M. Strain WH8113 responded positively only to ammonia, nitrate, β-alanine, glycine, and urea. Typically, there was a 1.5- to 2-fold increase in cell concentrations above control levels in chambers containing these compounds, which is comparable to results from similar experiments using enteric and photoheterotrophic bacteria. However, the threshold levels of 10⁻⁹ to 10⁻¹⁰ M found for Synechococcus spp. chemoresponses were lower by several orders of magnitude than those reported for other bacteria and fell within a range that could be ecologically significant in the oligotrophic oceans. The presence of chemotaxis in motile Synechococcus spp. supports the notion that regions of nutrient enrichment, such as the proposed microzones and patches, may play an important role in picoplankton nutrient dynamics.     

EFFECT OF SPECTRAL QUALITY ON GROWTH AND PIGMENTATION OF PICOCYANOBACTERIA1

The influence of spectral quality on growth and pigmentation was compared among five strains of marine and freshwater picocyanobacteria grown under the same photon flux density (28 μE · m^?2·s^?1). Growth and phycoerythrin (PE) concentration per unit carbon increased when marine Synechococcus WH7803 was grown under green light as compared to red light, but no change in phycocyanin concentration occurred. Marine Synechococcus strain 48B66 also showed greater levels of PE when grown under green light than under red light, but no concomitant growth increase occurred. Both strains thus exhibited Group II chromatic adaptation. Additionally, strain 48B66 increased the relative level of phycourobilin compared to phycoerythrobilin when grown under red light. In contrast, both marine and freshwater Synechococcus strains containing no PE showed decreased growth under green light. Chlorophyll a concentrations were greatest or among the greatest in all strains grown under green light. These results suggest that light quality, through its effects on growth rate, may be an important factor controlling the distribution and abundance of the various pigment types of Synechococcus.     

THE EFFECT OF SELENITE ON THE PHYSIOLOGICAL AND MORPHOLOGICAL PROPERTIES OF THE BLUE-GREEN ALGA PHORMIDIUM LURIDUM VAR. OLIVACEA1

Phormidium luridum cultures were treated with sodium selenite in concentrations ranging from 10^?6 M to 10^?2 M. In contrast to the increasing culture turbidity of control and 10^?6 M selenite cultures, the turbidity of the other selenite cultures declined in proportion to time and selenite concentration. Chlorophyll extraction revealed similar results. Photosynthetic activity was inhibited within 6 hr in all cultures except control and 10^?6 M selenite. Phormidium at concentrations greater than 10^?6 M selenite showed a gradual loss of the bright green color and turned semitransparent. Cell-associated granules of reduced selenium were observed at higher selenite concentrations. Other structural changes observed were the presence of intracellular and intercellular spaces, spheroplast formation, and gradual cell lysis. Protein analyses of total cell samples and supernatant fractions confirmed cellular breakdown of selenite-treated algal cells.     

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance ¹³C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 ± 1.5 to 62.3 ± 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His•bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and ¹H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.     

Comparative membrane proteomics reveal contrasting adaptation strategies for coastal and oceanic marine Synechococcus cyanobacteria

Marine cyanobacteria genus Synechococcus are among the most abundant and widespread primary producers in the open ocean. Synechococcus strains belonging to different clades have adapted distinct strategies for growth and survival across a range of marine conditions. Clades I and IV are prevalent in colder, mesotrophic, coastal waters, while clades II and III prefer warm, oligotrophic open oceans. To gain insight into the cellular resources these unicellular organisms invest in adaptation strategies we performed shotgun membrane proteomics of four Synechococcus spp. strains namely CC9311 (clade I), CC9605 (clade II), WH8102 (clade III) and CC9902 (clade IV). Comparative membrane proteomes analysis demonstrated that CC9902 and WH8102 showed high resource allocation for phosphate uptake, accounting for 44% and 38% of overall transporter protein expression of the species. WH8102 showed high expression of the iron uptake ATP-binding cassette binding protein FutA, suggesting that a high binding affinity for iron is possibly a key adaptation strategy for some strains in oligotrophic ocean environments. One protein annotated as a phosphatase 2c (Sync_2505 and Syncc9902_0387) was highly expressed in the coastal mesotrophic strains CC9311 and CC9902, constituting 14%–16% of total membrane protein, indicating a vital, but undefined function, for strains living in temperate mesotrophic environments.     

Speciation of copper in relation to its bioavailability

Abstract

Copper speciation and bioavailability for Scenedesmus quadricauda has been studied in natural waters and in synthetic culture media. Other elements were studied simultaneously. When phosphorus and nitrogen limitation were excluded by adding these elements, copper was limiting algal growth in some natural waters. In the toxic range, growth inhibition by copper was highly correlated with copper detected by electrochemical methods and with calculated free copper.

Copper was toxic to S. quadricauda when free copper concentrations roughly exceeded 10^?10.5 M, and was limiting for values somewhere lower than 10^?12.5 M. Because we found copper limitation in some natural water samples, free copper concentration in those water samples therefore must have been lower than 10^?12.5 M.

The hypothesis that the free metal concentration rather than the total concentration determines bioavailability was confirmed for copper, cobalt and zinc.     

Effect of Gibberellic Acid on the Elongation Rate of Agrostis alba Root Hairs

The influence of gibberellic acid over a wide range of concentrations on the rate of elongation of root hairs of redtop grass was investigated. The rate of root hair elongation was increased by GA over the concentration range of 10^?7 to 10^?12 M inclusive, with peak stimulation occurring at 10^?6 M. Although root hair growth was slightly accelerated by 10^?6 M GA, this concentration damaged many root hairs and caused some to stop growing altogether. Rate of root hair elongation was reduced to less than 84% of the control by 10^?5 M GA.     

SELENIUM STIMULATES GROWTH OF MARINE MACROALGAE IN AXENIC CULTURE1

The marine brown alga Fucus spiralis L. and the red alga Goniotrichum alsidii (Zanard) increase their growth upon the, addition of SeO₃^2- or SeO₄^2- when cultivated axenically in the artificial seawater ASP6 F2. In the concentration range 1 · 10^?10-1 · 10^?7 M there are two optima, one at 3.3 · 10^?10 M and another at 3.3 · 10^?8 M. α-To-copherol, often administered together with selenium to mammals suffering from selenium deficiency, gives no additive effect with selenium, but α-tocopherol in the concentration range 1 × 10^?7-1 × 10^?6 M does influence the morphology of the Fucus plants. Organically bound selenium has no effect.     

Lutein-chlorophyll-a energy transfer in detergent micelles

Absorption, fluorescence and fluorescence excitation spectra were determined for equimolar mixed micellar detergent solutions of lutein and chlorophyll-a in the concentration range from 9·10^?6 to 1.8·10^?4 M, with detergent (triton-X100) concentrations from 3·-10^?4 to 7·10^?3 M. In the range of detergent concentrations studied the pigments incorporated into the detergent micelles attained a high local concentration (0.1 to 0.01 M), reminiscent of pigment concentration within the chloroplast. A lutein → chlorophyll-a energy transfer with an efficiency of about 15% was found in these systems. In dilute (9·10^?6 M) pigment solution with concentrated (7·10^?3 M) detergent practically no transfer is observed. The extent of aggregation and the efficiency of transfer depend on the composition of the system. The aggregation of chlorophyll-a is partly inhibited by lutein molecules. It is shown that the energy transfer efficiency as function of distance follows anr ^?3 relationship,R ₀ being 22 å.     

GROWTH REGULATION OF rRNA CONTENT IN PROCHLOROCOCCUS AND SYNECHOCOCCUS (MARINE CYANOBACTERIA) MEASURED BY WHOLE‐CELL HYBRIDIZATION OF rRNA‐TARGETED PEPTIDE NUCLEIC ACIDS1

The cyanobacteria Synechococcus and Prochlorococcus are important primary producers in marine ecosystems. Because currently available approaches for estimating microbial growth rates can be difficult to apply in the field, we have been exploring the feasibility of using quantitative rRNA measurements as the basis for making such estimates. In this study we examined the relationship between rRNA and growth rate in several Synechococcus and Prochlorococcus strains over a range of light‐regulated growth rates. Whole‐cell hybridization with fluorescently labeled peptide nucleic acid (PNA) probes was used in conjunction with flow cytometry to quantify rRNA on a per cell basis. This PNA probing technique allowed rRNA analysis in a phycoerythrin‐containing Synechococcus strain (WH7803) and in a non–phycoerythrin‐containing strain and in Prochlorococcus. All the strains showed a qualitatively similar tri‐phasic relationship between rRNA·cell^?1 and growth rate, involving relatively little change in rRNA·cell^?1 at low growth rates, linear increase at intermediate growth rates, and a plateau and/or decrease at the highest growth rates. The onset of each phase was associated with the relative, rather than absolute, growth rate of each strain. In the Synechococcus strains, rRNA normalized to flow cytometrically measured forward angle light scatter (an indicator of size) was well‐correlated with growth rate across strains. These findings support the idea that cellular rRNA may be useful as an indicator of in situ growth rate in natural Synechococcus and Prochlorococcus populations.     

The Effect of Environmental Conditions on the Expression of Disease in Cultured Tissues of Brassica napus ssp. oldfera Infected with Leptosphaeria maculans

As a basis for devising an in vitro screening programme, culture conditions were optimized so that tissue cultures from two resistant cultivars of Brassica napus ssp. oleifera (Mikado, Bienvenu) and two susceptible cultivars (Lesira, Ceres) could be differentiated using a disease scoring scheme, when inoculated with Leptosphaeria maculans. Tissues inoculated included thin cell layer explants from soil-grown plants and in vitro-grown shoot cultures and callus tissue formed on such explants. The period of incubation and the incubation temperature were of importance in the development of differential disease reactions. Increasing temperature generally resulted in an increase in infection and too great an incubation period resulted in total overgrowth of the tissue. Increasing concentrations (1 × 10^?6 M-1 ×10^?4 M) of the auxins 1-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and mdole-3-acetic acid (IAA) in the culture medium, resulted in a decrease in disease score of the thin cell layer (TCL) explants from soil-grown plants. The cytokinins examined 6-benzyl-aminopurine (BAP) and 6-4-hydroxy-3-methyl-2-enylaminopurine (zeatin), reduced the extent of infection of the TCL explants when used in combination with the auxin NAA. Medium containing NAA at a concentration of 1 × 10^?6 M in combination with BAP at a concentration of 1× 10^?6 or 1 × 10^?4 M allowed differentiation of the disease reactions of the resistant and susceptible cultivars, when the explants were incubated for 10 days at 20 °C after inoculation. Similar conditions of incubation and the addition of NAA (1 × 10^?6 M) combined with BAP (1 × 10^?6 M) to the medium also allowed the differentiation of the disease reactions on TCL explants from stems of in vitro shoot cultures of the cultivars Mikado and Lesira. Increasing concentrations of the auxin NAA and the cytokinin BAP resulted in a reduction in the mean disease score of the callus tissue produced on TCL explants from soil-grown plants, and NAA (1 × 10^?5 M) combined with BAP (1 × 10^?6 or 1 × 10^?5 M) allowed differentiation of resistance and susceptibility in callus tissues when incubated for 5 days at 20 °C. 2,4-D did not allow differentiation of the cultivars. This was in contrast to the inoculation of callus tissue attached to TCL explants of in vitro shoot cultures, where combinations of 2,4-D and BAP at concentrations of 1 × 10^?6 M allowed differentiation of the resistant and susceptible cultivars. These findings provide a basis for designing selection protocols of value in both traditional as well as in vitro breeding programmes to select lines of oilseed rape with resistance/novel resistance to L. maculans.     

Effect of iron on growth and toxin production byClostridium botulinum type A

The kinetics of growth and toxin production by the Hall strain ofClostridium botulinum type A was examined in the presence of various concentrations of iron (0.1 to 10.1 g/ml, 1.8 to 182 M) in a chemically defined medium. At concentrations below 0.5 g/ml, iron insufficiency limited the growth of the organism. The maximum amount of toxin produced varied by only twofold (6×10⁵ to 1.2×10⁶ mouse median lethal doses/ml per A₅₄₀ unit) over the 100-fold range of iron concentrations used. High concentrations of iron did not reduce the elaboration of botulinum toxin, in contrast with its marked inhibitory effects on the production of many bacterial toxins. Iron is unlikely to be a regulatory effector for the formation of botulinum toxin by the Hall strain of type A.     

Effects of iron on the growth and minimal fluorescence yield of three marine Synechococcus strains (Cyanophyceae)

Two coastal Synechococcus stains PCC 7002 and CC9311 and one oceanic strain WH8102 were cultured with 4–1000 nM Fe in Aquil medium. Compared with those under iron‐replete conditions, their growth rates were significantly decreased by 59% for WH8102 at 15 nM Fe, by 37% for CC9311 at 15 nM Fe and by 57% for PCC 7002 at 4 nM Fe. Among these three strains, PCC 7002 was the most tolerant to iron limitation while WH8102 was the most sensitive to iron limitation. For each strain under the same iron concentration, the growth rates calculated from the minimal fluorescence yield and cell concentration showed no significant difference. The linear correlation was established between the minimal fluorescence yield and cell concentration although the minimal fluorescence yield per cell varied depending on the strains and iron levels. Under iron‐replete conditions, the minimal fluorescence yield per cell was 100‐fold higher for the phycoerythrin‐lacking strain PCC 7002 than two phycoerythrin‐containing strains WH8102 and CC9311. Under iron‐deplete conditions, it was increased respectively by 128% and 7% for WH8102 and CC9311 but was decreased by 30% for PCC 7002. Furthermore, the minimal fluorescence yield per cell for PCC 7002 and CC9311 showed little difference throughout the light and dark diel cycle. However, it was significantly higher for WH8102 in the daytime than in the dark.     

Copper complexation by natural organic matter in contaminated and uncontaminated ground water

Abstract

Ground-water samples were collected from an uncontaminated and a contaminated site. Copper complexation was characterized by ion- selective electrode (ISE), fluorescence quenching (FQ), and cathodic stripping voltammetric (CSV) titrations. All of the samples were titrated at their natural pH values and some of the samples were also titrated at other pH values. For a total Cu concentration of 10^?6 M, the free Cu²⁺ concentrations in the samples from the uncontaminated site were all less than 10^?7 M, while free Cu²⁺ in the samples from the contaminated site were all less than 10^?8 M. For a particular sample and total Cu concentration, the free Cu²⁺ concentration decreased as the pH increased. Relative to ISE, FQ underestimated and CSV overestimated the degree of Cu²⁺ binding. The Cu²⁺ -complexing properties of the ground waters are similar to many published results for the same pH and for ligand concentrations normalized to T.O.C. Chemical equilibrium computations indicate that organic complexes would dominate Cu speciation in the uncontaminated ground waters for 10^?7 to 10^?5 M total Cu. In the contaminated ground waters, sulfide complexes would be the predominant Cu species for total Cu less than the total S(?11) concentration. Organic complexes would dominate Cu speciation for total Cu greater than total S(?11).     

Kinetics of Potassium and Magnesium Uptake by Intact Soybean Roots

The kinetics of uptake of K⁺ and Mg²⁺ were studied by using intact soybean [Glycine max (L.) Merr. cv. Amsoy] roots. Uptake of K⁺ in the concentration range 1.29 × 10^?5 to 1.82 × 10^?3 M can be represented by two phases of a single, multiphasic mechanism. Similarly, uptake of Mg²⁺ in the concentration range 4.10 × 10^?6 to 2.49 × 10^?4M was biphasic.     

Effect of 4-hydroxynonenal on superoxide anion production from primed human neutrophils

HNE (4-hydroxy-2,3-trans-nonenal), an aldehydic product of lipid peroxidation, has been reported to modulate different functional parameters of human and rat neutrophils (PMNs), such as chemiluminescence, migration and some enzymatic activities, thus exerting effects that varied according to the concentration tested. Experiments were done to evaluate the effects of HNE on superoxide anion (O₂^?.) production from human PMNs, isolated from healthy volunteers. After having tested that HNE by itself was not able to activate the cells, comparisons were made between its effects on PMNs, stimulated by either a single stimulus, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or a combination of stimuli, such as FMLP and the neuropeptide substance P (SP; primed PMNs). In the concentration range tested (10^?12–10^?4 M ), HNE inhibited FMLP-evoked O₂^?. production with an IC₅₀ of 11·6 ± 1·5 × 10^?6 M ; at concentrations ≤10^?6 M , HNE enhanced O₂^?. production elicited by FMLP + SP, while higher concentrations were inhibitory. There was a bell-shaped dose–response curve to the enhancing effects of HNE, depending on the incubation time being recorded after only short periods (≤5 min) of the exposure of the cells to HNE; this was not shown by structurally-related aldehydes, such as 2-nonenal and nonanal. These results suggest that low concentrations of HNE may participate in the evolution of the inflammatory process, by contributing to the activation of PMNs. The effects of high concentrations of the aldehyde may represent a mechanism which contributes to the regulation of the extent of the inflammatory response.     

Analyzing nitrogen and sulphide elimination from leachate by coulometric continuous flow titration

The process of leachate denitrification by populations of nitrifying and denitrifying bacteria was investigated. Leachate, derived from a local municipal landfill site, was nitrified in a continuously operating packed-bed biofilm reactor and thereafter denitrified in an activated sludge bioreactor. To follow the progress of nitrogen elimination, ammonium, nitrite and nitrate concentrations were determined at all stages of the process. While the nitrite and nitrate concentrations were measured by conventional colorimetric methods, computer controlled coulometric titration with in situ generated hypobromite was used for ammonium determination, which had previously been selectively separated from the sample matrix by gas dialysis. The detection range of the method was from 1 × 10^?6 to 1 × 10^?3 M ammonium (relative standard deviation (RSD) = 2%, n = 6). No interference of the complex sample matrix was found in ammonium determination. The average ammonium concentration in the leachate was 409 mg/l (standard deviation (SD) = 142 mg/l, n = 55). The ammonium concentrations decreased to 1–5 mg/l during nitrification under continuous operating conditions. Increased ammonium concentrations after nitrification correlated with a decrease in the efficiency of nitrogen elimination by up to 45% due to the build-up of high concentrations of nitrite. The concentration of sulphides, another source of pollution of the leachate, was also determined by triangle programmed coulometric titration. The average concentration of sulphides in the leachate was 221 mg/l (SD = 374 mg/l; n = 55). The sulphide concentrations decreased to concentrations below the detection limit of the coulometric titration (2 × 10^?6M) during nitrification.