Development of an ELISA approach for the determination of flavodoxin and ferredoxin as markers of iron deficiency in phytoplankton

Quantification of the iron-nutritional status of phytoplankton is of great interest not only for the study of oceans but also for fresh waters. Flavodoxin is a small flavoprotein proposed as a marker for iron deficiency, since it is induced as a consequence of iron deprivation, replacing the iron-sulphur protein ferredoxin. Flavodoxin and ferredoxin have been frequently used as markers for determination of iron deficiency in phytoplankton. Using purified flavodoxin and ferredoxin from Scenedesmus vacuolatus and polyclonal antibodies against both proteins, individual ELISA tests have been developed. The assays have a linear response in the range of 30-600 ng/ml of protein.     

Electron acceptor specificity of ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli

Reduced flavodoxin I (Fld1) is required in Escherichia coli for reductive radical generation in AdoMet-dependent radical enzymes and reductive activation of cobalamin-dependent methionine synthase. Ferredoxin (Fd) and flavodoxin II (Fld2) are also present, although their precise roles have not been ascertained. Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) was discovered in E. coli as an NADPH-dependent reductant of Fld1 that facilitated generation of active methionine synthase in vitro; FNR and Fld1 will also supply electrons for the reductive cleavage of AdoMet essential for generating protein or substrate radicals in pyruvate formate-lyase, class III ribonucleotide reductase, biotin synthase, and, potentially, lipoyl synthase. As part of ongoing efforts to understand the various redox pathways that will support AdoMet-dependent radical enzymes in E. coli, we have examined the relative specificity of E. coli FNR for Fd, Fld1, and Fld2. While FNR will reduce all three proteins, Fd is the kinetically and thermodynamically preferred partner. Fd binds to FNR with high affinity (K(d)相似文献   

Ferredoxin-NADP+ reductase uses the same site for the interaction with ferredoxin and flavodoxin

The enzyme ferredoxin-NADP(+) reductase (FNR) forms a 1 : 1 complex with ferredoxin (Fd) or flavodoxin (Fld) that is stabilised by both electrostatic and hydrophobic interactions. The electrostatic interactions occur between acidic residues of the electron transfer (ET) protein and basic residues on the FNR surface. In the present study, several charge-reversal mutants of FNR have been prepared at the proposed site of interaction of the ET protein: R16E, K72E, K75E, K138E, R264E, K290E and K294E. All of these mutants have been assayed for reactivity with Fd and Fld using steady-state and stopped-flow kinetics. Their abilities for complex formation with the ET proteins have also been tested. The data presented here indicate that the mutated residues situated within the FNR FAD-binding domain are more important for achieving maximal ET rates, either with Fd or Fld, than those situated within the NADP(+)-binding domain, and that both ET proteins occupy the same region for the interaction with the reductase. In addition, each individual residue does not appear to participate to the same extent in the different processes with Fd and Fld.     

Microbiota within the perennial ice cover of Lake Vida, Antarctica

Lake Vida, located in the McMurdo Dry Valleys, Antarctica, is an 'ice-sealed' lake with approximately 19 m of ice covering a highly saline water column (approximately 245 ppt). The lower portions of the ice cover and the lake beneath have been isolated from the atmosphere and land for circa 2800 years. Analysis of microbial assemblages within the perennial ice cover of the lake revealed a diverse array of bacteria and eukarya. Bacterial and eukaryal denaturing gradient gel electrophoresis phylotype profile similarities were low (<59%) between all of the depths compared (five depths spanning 11 m of the ice cover), with the greatest differences occurring between surface and deep ice. The majority of bacterial 16S rRNA gene sequences in the surface ice were related to Actinobacteria (42%) while Gammaproteobacteria (52%) dominated the deep ice community. Comparisons of assemblage composition suggest differences in ice habitability and organismal origin in the upper and lower portions of ice cover. Specifically, the upper ice cover microbiota likely reflect the modern day transport and colonization of biota from the terrestrial landscape, whereas assemblages in the deeper ice are more likely to be persistent remnant biota that originated from the ancient liquid water column of the lake that froze.     

Microbial loop malfunctioning in the annual sea ice at Terra Nova Bay (Antarctica)

We investigated organic carbon quantity and biochemical composition, prokaryotic abundance, biomass and carbon production in the annual and platelet sea ice of Terra Nova Bay (Antarctica), as well as the downward fluxes of organic matter released by melting ice during early spring. Huge amounts of biopolymeric C accumulated in the bottom layer of the ice column concomitantly with the early spring increase in sympagic algal biomass. Such organic material, mostly accounted for by autotrophic biomass, was characterised by a high food quality and was rapidly exported to the sea bottom during sea ice melting. Prokaryote abundance (up to 1.3 × 10⁹ cells L⁻¹) and extracellular enzymatic activities (up to 24.3 μM h⁻¹ for amino-peptidase activity) were extremely high, indicating high rates of organic C degradation in the bottom sea ice. Despite this, prokaryote C production values were very low (range 5–30 ng C L⁻¹ h⁻¹), suggesting that most of the degraded organic C was not channelled into prokaryote biomass. In the platelet ice, we found similar organic C concentrations, prokaryote abundance and biomass values and even higher extracellular enzymatic activities, but values of prokaryote C production (range 800–4,200 ng C L⁻¹ h⁻¹) were up to three orders of magnitude higher than in the intact bottom sea ice. Additional field and laboratory experiments revealed that the dissolved organic material derived from algae accumulating in the bottom sea ice significantly reduced prokaryote C production, suggesting the presence of a potential allopathic control of sympagic algae on prokaryote growth. This article belongs to a special topic: Five articles on Sea-ice communities in Terra Nova Bay (Ross Sea), coordinated by L. Guglielmo and V. Saggiomo, appear in this issue of Polar Biology. The studies were conducted in the frame of the National Program of Research in Antarctica (PNRA) of Italy.     

Effect of iron deficiency on ferredoxin levels in Anabaena variabilis PCC 6309

Cultures of the nitrogen-fixing cyanobacterium Anabaena variabilis PCC 6309 were grown under several iron concentrations, and changes in growth rates and chlorophyll and phycocyanin concentrations were determined. The total amount of ferredoxin present in the cells was found to be dependent on the concentration of iron in the media, as was the pattern distribution of the two different forms of the iron-sulfur protein described in this organism. Flavodoxin was not found to be present in these cells even when they were grown in the absence of added iron, indicating that this flavoprotein does not replace ferredoxin in this particular strain.     

The oxidant-responsive diaphorase of Rhodobacter capsulatus is a ferredoxin (flavodoxin)-NADP(H) reductase

Challenge of Rhodobacter capsulatus cells with the superoxide propagator methyl viologen resulted in the induction of a diaphorase activity identified as a member of the ferredoxin (flavodoxin)-(reduced) nicotinamide adenine dinucleotide phosphate (NADP(H)) reductase (FPR) family by N-terminal sequencing. The gene coding for Rhodobacter FPR was cloned and expressed in Escherichia coli. Both native and recombinant forms of the enzyme were purified to homogeneity rendering monomeric products of approximately 30 kDa with essentially the same spectroscopic and kinetic properties. They were able to bind and reduce Rhodobacter flavodoxin (NifF) and to mediate typical FPR activities such as the NADPH-driven diaphorase and cytochrome c reductase.     

Structure of the summer under fast ice microbial community near Syowa Station,eastern Antarctica

Temporal variations in the microbial community structure of plankton, which is composed of autotrophic and heterotrophic pico-, nano- and microplankton, were investigated during the austral summer of 2005/2006 under fast ice near Syowa Station, eastern Antarctica. Autotrophic algal populations were composed almost entirely of diatoms followed by phytoflagellates such as autotrophic dinoflagellates and cryptophytes. Among the microbial community, heterotrophic biomass was dominated by heterotrophic dinoflagellates and naked ciliates and finally exceeded autotrophic biomass. Qualitative microscopic analysis revealed that heterotrophic dinoflagellates were ingesting large number of diatoms. Synchronizing fluctuation of naked ciliates with phytoflagellates suggested a predator–prey relationship between them. Our results suggest that the pelagic food webs under the extensive ice-covered areas in coastal Antarctic regions are not short but complex.     

Ferredoxin degradation in growing Clostridium pasteurianum during periods of iron deprivation

Clostridium pasteurianum was grown in batch cultures on media with an initial iron concentration of 10 M. The uptake of iron and the synthesis of ferredoxin was followed. All the iron present in the medium was taken up by the cells before 50% of the final cell density was attained. The bacteria then continued to grow in the complete absence of exogenous iron. Ferredoxin was synthesized during growth until the exogenous iron concentration dropped below 1 M. During growth in the absence of iron ferredoxin was degraded with the result that at the end of growth the cells did not contain ferredoxin. The specific activity of the iron sulfur protein, pyruvate synthase (E.C. 1.2.7.1), remained constant during growth of C. pasteurianum in the absence of exogenous iron. This finding suggests that ferredoxin was used as an endogenous source of iron for the synthesis of essential iron proteins during periods of iron deprivation.The term ferredoxin degradation is used here to indicate that the ferredoxin content in the growing cells decreased more than could be accounted for by repeated cell division. Ferredoxin = holoferredoxin = protein containing iron and sulfide; apoferredoxin = protein free of iron and sulfide     

Backbone dynamics of oxidized and reduced D. vulgaris flavodoxin in solution

Recombinant Desulfovibrio vulgaris flavodoxin was produced inEscherichia coli. A complete backbone NMR assignment for the two-electronreduced protein revealed significant changes of chemical shift valuescompared to the oxidized protein, in particular for the flavinemononucleotide (FMN)-binding site. A comparison of homo- and heteronuclearNOESY spectra for the two redox states led to the assumption that reductionis not accompanied by significant changes of the global fold of the protein.The backbone dynamics of both the oxidized and reduced forms of D. vulgarisflavodoxin were investigated using two-dimensional¹⁵N-¹H correlation NMR spectroscopy.T₁, T₂ and NOE data are obtained for 95%of the backbone amide groups in both redox states. These values wereanalysed in terms of the model-free approach introduced by Lipari andSzabo [(1982) J. Am. Chem. Soc., 104, 4546-;4559, 4559-;4570]. Acomparison of the two redox states indicates that in the reduced speciessignificantly more flexibility occurs in the two loop regions enclosing FMN.Also, a higher amplitude of local motion could be found for the N(3)H groupof FMN bound to the reduced protein compared to the oxidized state.     

Distribution and characterization of dissolved and particulate organic matter in Antarctic pack ice

Distribution and composition of organic matter were investigated in Antarctic pack ice in early spring and summer. Accumulation of organic compounds was observed with dissolved organic carbon (DOC) and particulate organic carbon (POC) reaching 717 and 470 μM C, respectively and transparent exopolymeric particles (TEP) up to 3,071 μg Xanthan gum equivalent l⁻¹. POC and TEP seemed to be influenced mainly by algae. Particulate saccharides accounted for 0.2–24.1% (mean, 7.8%) of POC. Dissolved total saccharides represented 0.4–29.6% (mean, 9.7%) of DOC, while dissolved free amino acids (DFAA) accounted for only 1% of DOC. Concentrations of TEP were positively correlated with those of saccharides. Monosaccharides (d-MCHO) dominated during winter–early spring, whereas dissolved polysaccharides did in spring–summer. DFAA were strongly correlated with d-MCHO, suggesting a similar pathway of production. The accumulation of monomers in winter is thought to result from limitation of bacterial activities rather than from the nature of the substrates.     

Tuning of the FMN binding and oxido-reduction properties by neighboring side chains in Anabaena flavodoxin

Contribution of three regions (phosphate-binding, 50’s and 90’s loops) of Anabaena apoflavodoxin to FMN binding and reduction potential was studied. Thr12 and Glu16 did not influence FMN redox properties, but Thr12 played a role in FMN binding. Replacement of Trp57 with Glu, Lys or Arg moderately shifted E_ox/sq and E_sq/hq and altered the energetic of the FMN redox states binding profile. Our data indicate that the side chain of position 57 does not modulate E_ox/sq by aromatic stacking or solvent exclusion, but rather by influencing the relative strength of the H-bond between the N(5) of the flavin and the Asn58-Ile59 bond. A correlation was observed between the isoalloxazine increase in solvent accessibility and less negative E_sq/hq. Moreover, E_sq/hq became less negative as positively charged residues were added near to the isoalloxazine. Ile59 and Ile92 were simultaneously mutated to Ala or Glu. These mutations impaired FMN binding, while shifting E_sq/hq to less negative values and E_ox/sq to more negative. These effects are discussed on the bases of the X-ray structures of some of the Fld mutants, suggesting that in Anabaena Fld the structural control of both electron transfer steps is much more subtle than in other Flds.     

A device for remote sampling of benthic algae under ice

A benthic algae sampling device designed for use through surface ice, uses the principle of pivoting arms. It is designed to be activated below ice sheets and is capable of passing easily through a hole of 11 cm diameter bored by a SIPRE ice auger.     

Copepods in summer platelet ice in the eastern Weddell Sea,Antarctica

Copepods in platelet-ice layers underlying fast ice and in the water column below were studied at Drescher Inlet, eastern Weddell Sea in February 1998. Three copepod species were found: Drescheriella glacialis and Paralabidocera antarctica occurred in platelet-ice layers, while Stephos longipes was only present in the water column. The distribution of all species varied considerably between station and depth. D. glacialis dominated the platelet-ice community and occurred at all five platelet-ice sampling sites, except one, with numbers of up to 26 ind. l^–1. In contrast, P. antarctica was only found in low numbers (up to 2 ind. l^–1) at one site. The total copepod abundance in the platelet ice was not associated with algal biomass, although it was strongly correlated with high ammonium concentrations (up to 9 M) in the interstitial water between the platelets. This is the first indirect evidence to support the hypothesis that zooplankton excretion can partly account for the high ammonium values often found in platelet-ice layers.     

Movements and dive behaviour of two leopard seals (<Emphasis Type="Italic">Hydrurga leptonyx</Emphasis>) off Queen Maud Land,Antarctica

Two adult female leopard seals (Hydrurga leptonyx) were tagged with satellite-linked dive recorders off Queen Maud Land, Antarctica, just after moulting in mid-February. The transmitters transmitted for 80 and 220 days, respectively. Both seals remained within the pack ice relatively close to the Antarctic Continent until early May, when contact was lost with one seal. The one remaining seal then migrated north, to the east side of the South Sandwich Islands in 3 weeks, whereafter it headed east, until contact was lost at 55°S in early September. From mid-May to late September this animal always stayed close to the edge of the pack ice. Both seals made mostly short (<5 min) dives to depths of 10–50 m and only occasionally dove deeper than 200 m, the deepest dive recorded being 304 m. A nocturnal diving pattern was evident in autumn and early winter, while day-time diving prevailed in mid-winter. Haul out probability was highest at mid-day (about 40% in late February and more than 80% in March and April). From May till September the remaining animal mainly stayed at sea, in the vicinity of the pack ice, with only occasional haul outs. These data suggest that a portion of the adult leopard seals may spend the winter mainly in open water, off the edge of the pack ice, where they primarily hunt near the surface. In that case, it is likely that krill (Euphausia superba), as well as penguins, young crabeater seals (Lobodon carcinophaga) and a variety of fish are important prey items.     

An r-strategist in Antarctic pack ice

Summary The Antarctic copepod Drescheriella glacialis, an inhabitant of sea ice, is the first polar invertebrate metazoan to have been cultured throughout its life cycle. We describe its demographic characteristics on the basis of a laboratory cohort study and correlative field data. When compared to its closest temperate-zone relatives, D. glacialis shows temperature compensation of developmental and reproductive rates. A genuine r-strategist in every respect, it does not fit established trends for Antarctic invertebrates but appears well adapted to the peculiar spatio-temporal variability of the sea ice habitat.     

Negative feedback in the cold: ice retreat produces new carbon sinks in Antarctica

Feedbacks on climate change so far identified are predominantly positive, enhancing the rate of change. Loss of sea‐ice, increase in desert areas, water vapour increase, loss of tropical rain forest and the restriction of significant areas of marine productivity to higher latitude (thus smaller geographical zones) all lead to an enhancement of the rate of change. The other major feedback identified, changes in cloud radiation, will produce either a positive feedback, if high level clouds are produced, or a negative feedback if low level clouds are produced. Few significant negative feedbacks have been identified, let alone quantified. Here, we show that the loss of ice shelves and retreat of coastal glaciers around the Antarctic Peninsula in the last 50 years has exposed at least 2.4 × 10⁴ km² of new open water. We estimate that these new areas of open water have allowed new phytoplankton blooms containing a total standing stock of ～5.0 × 10⁵ tonnes of carbon to be produced. New marine zooplankton and seabed communities have also been produced, which we estimate contain ～4.1 × 10⁵ tonnes of carbon. This previously unquantified carbon sink acts as a negative feedback to climate change. New annual productivity, as opposed to standing stock, amounts to 3.5 × 10⁶ tonnes yr^?1 of carbon, of which 6.9 × 10⁵ tonnes yr^?1 deposits to the seabed. By comparison the total aboveground biomasses of lowland American tropical rainforest is 160–435 tonnes ha^?1. Around 50% of this is carbon. On this basis the carbon held in new biomass described here is roughly equivalent to 6000–17 000 ha of tropical rainforest. As ice loss increases in polar regions this feedback will become stronger, and eventually, over thousands to hundreds of thousands of years, over 50 Mtonnes of new carbon could be fixed annually in new coastal phytoplankton blooms and over 10 Mtonnes yr^?1 locked in biological standing stock around Antarctica.     

Identification of Global Ferredoxin Interaction Networks in Chlamydomonas reinhardtii

Ferredoxins (FDXs) can distribute electrons originating from photosynthetic water oxidation, fermentation, and other reductant-generating pathways to specific redox enzymes in different organisms. The six FDXs identified in Chlamydomonas reinhardtii are not fully characterized in terms of their biological function. In this report, we present data from the following: (a) yeast two-hybrid screens, identifying interaction partners for each Chlamydomonas FDX; (b) pairwise yeast two-hybrid assays measuring FDX interactions with proteins from selected biochemical pathways; (c) affinity pulldown assays that, in some cases, confirm and even expand the interaction network for FDX1 and FDX2; and (d) in vitro NADP⁺ reduction and H₂ photo-production assays mediated by each FDX that verify their role in these two pathways. Our results demonstrate new potential roles for FDX1 in redox metabolism and carbohydrate and fatty acid biosynthesis, for FDX2 in anaerobic metabolism, and possibly in state transition. Our data also suggest that FDX3 is involved in nitrogen assimilation, FDX4 in glycolysis and response to reactive oxygen species, and FDX5 in hydrogenase maturation. Finally, we provide experimental evidence that FDX1 serves as the primary electron donor to two important biological pathways, NADPH and H₂ photo-production, whereas FDX2 is capable of driving these reactions at less than half the rate observed for FDX1.     

Observations on the relationship between the Antarctic coastal diatoms Thalassiosira antarctica Comber and Porosira glacialis (Grunow) Jørgensen and sea ice concentrations during the late Quaternary

The available ecological and palaeoecological information for two sea ice-related marine diatoms (Bacillariophyceae), Thalassiosira antarctica Comber and Porosira glacialis (Grunow) Jørgensen, suggests that these two species have similar sea surface temperature (SST), sea surface salinity (SSS) and sea ice proximity preferences. From phytoplankton observations, both are described as summer or autumn bloom species, commonly found in low SST waters associated with sea ice, although rarely within the ice. Both species form resting spores (RS) as irradiance decreases, SST falls and SSS increases in response to freezing ice in autumn. Recent work analysing late Quaternary seasonally laminated diatom ooze from coastal Antarctic sites has revealed that sub-laminae dominated either by T. antarctica RS, or by P. glacialis RS, are nearly always deposited as the last sediment increment of the year, interpreted as representing autumn flux. In this study, we focus on sites from the East Antarctic margin and show that there is a spatial and temporal separation in whether T. antarctica RS or P. glacialis RS form the autumnal sub-laminae. For instance, in deglacial sediments from the Mertz Ninnis Trough (George V Coast) P. glacialis RS form the sub-laminae whereas in similar age sediments from Iceberg Alley (Mac.Robertson Shelf) T. antarctica RS dominate the autumn sub-lamina. In the Dumont d'Urville Trough (Adélie Land), mid-Holocene (Hypsithermal warm period) autumnal sub-laminae are dominated by T. antarctica RS whereas late Holocene (Neoglacial cool period) sub-laminae are dominated by P. glacialis RS. These observations from late Quaternary seasonally laminated sediments would appear to indicate that P. glacialis prefers slightly cooler ocean–climate conditions than T. antarctica. We test this relationship against two down-core Holocene quantitative diatom abundance records from Dumont d'Urville Trough and Svenner Channel (Princess Elizabeth Land) and compare the results with SST and sea ice concentration results of an Antarctic and Southern Ocean Holocene climate simulation that used a coupled atmosphere–sea ice–vegation model forced with orbital parameters and greenhouse gas concentrations. We find that abundance of P. glacialis RS is favoured by higher winter and spring sea ice concentrations and that a climatically-sensitive threshold exists between the abundance of P. glacialis RS and T. antarctica RS in the sediments. An increase to > 0.1 for the ratio of P. glacialis RS:T. antarctica RS indicates a change to increased winter sea ice concentration (to >80% concentration), cooler spring seasons with increased sea ice, slightly warmer autumn seasons with less sea ice and a change from ~ 7.5 months annual sea ice cover at a site to much greater than 7.5 months. In the East Antarctic sediment record, an increase in the ratio from <0.1 to above 0.1 occurs at the transition from the warmer Hypsithermal climate into the cooler Neoglacial climate (~ 4 cal kyr) indicating that the ratio between these two diatoms has the potential to be used as a semi-quantitative climate proxy.     

Biogeochemistry and microbial community composition in sea ice and underlying seawater off East Antarctica during early spring

Pack ice, brines and seawaters were sampled in October 2003 in the East Antarctic sector to investigate the structure of the microbial communities (algae, bacteria and protozoa) in relation to the associated physico-chemical conditions (ice structure, temperature, salinity, inorganic nutrients, chlorophyll a and organic matter). Ice cover ranged between 0.3 and 0.8 m, composed of granular and columnar ice. The brine volume fractions sharply increased above −4°C in the bottom ice, coinciding with an important increase of algal biomass (up to 3.9 mg C l⁻¹), suggesting a control of the algae growth by the space availability at that period of time. Large accumulation of NH₄ ⁺ and PO₄ ³⁻ was observed in the bottom ice. The high pool of organic matter, especially of transparent exopolymeric particles, likely led to nutrients retention and limitation of the protozoa grazing pressure, inducing therefore an algal accumulation. In contrast, the heterotrophs dominated in the underlying seawaters.