Siderophore-mediated iron uptake in different strains of Anabaena sp.

Anabaena sp. strain 6411, which produces the dihydroxamate siderophore schizokinen to facilitate iron uptake, is also capable of using the related siderophore aerobactin. The two siderophores compete for the same iron transport system, but there is a markedly higher affinity for ferric schizokinen than for ferric aerobactin. The trihydroxamate siderophore ferrioxamine B is far less effective as an iron donor in this organism. Anabaena sp. strain 7120 appears to be closely related to strain 6411. It synthesizes schizokinen as its major siderophore and shows rates of iron uptake from ferric schizokinen, ferric aerobactin, and ferrioxamine B which are similar to those observed with strain 6411. Anabaena cylindrica Lemm. 7122 and 1611, on the other hand, differ from strain 6411. In contrast to schizokinen, the hydroxamate which they produce in response to iron starvation cannot be extracted with water from the organic layer and does not support the growth of the siderophore auxotroph Arthrobacter flavescens JG-9. Strain 7122 can use its endogenous siderophore or schizokinen to promote iron uptake, but at 50-fold-lower rates than are observed with Anabaena sp. strain 6411 or 7120.     

Siderophore-mediated uptake of iron in Azotobacter vinelandii

Azotobacter vinelandii produces two siderophores, N,N'-bis-(2,3-dihydroxybenzoyl)-L-lysine (azotochelin) and a yellow-green fluorescent peptide (azotobactin), under iron-limited growth conditions. 55Fe uptake was not observed until the substantial nonspecific binding of 55Fe to the cell surface was eliminated by the addition of 10 mM sodium citrate to the uptake medium. Citrate alone did not promote rapid 55Fe uptake in A. vinelandii, nor did it induce Fe-repressible outer membrane proteins. Siderophore-mediated 55Fe uptake appeared biphasic, with both the initial rapid and ensuing slower uptake being energy dependent. The purified siderophores demonstrated the same uptake pattern as the Fe-limited culture supernatant fluid, but either individually or in combination accounted for less than the total 55Fe uptake activity found in the latter. The purified siderophores appeared to be sensitive to acid, but the inhibition of 55Fe uptake was in fact caused by salt generated during neutralization. Similar 60% inhibition of 55Fe uptake activity was caused by the addition of 40 mM Na+, K+, Li+, or Mg2+ salts to the uptake medium. Ammonium was less inhibitory than the latter ions. 55Fe uptake mediated by azotobactin was more sensitive to added NaCl than was that mediated by azotochelin. Neither the chelation of iron nor the stability of the ferrisiderophore was affected by added NaCl.     

Siderophore-mediated iron uptake in fluorescent Pseudomonas: characterization of the pyoverdine-receptor binding site of three cross-reacting pyoverdines.

Two Pseudomonas fluorescens and one Pseudomonas aeruginosa strains, although producing structurally different pyoverdines, demonstrated highly efficient cross-reactions when tested for pyoverdine-mediated iron uptake. A ferripyoverdine receptor-deficient mutant of the P. aeruginosa strain was unable to use any of the three pyoverdines. Moreover, the three strains presented each a specific outer membrane siderophore-receptor pattern. Thus, the capacity of using heterologous pyoverdines was related not to the presence of supplementary specific ferripyoverdine receptors but to the existence within the respective pyoverdine-peptide chains of a common dipeptide motif which should act as the receptor-binding site for the three pyoverdines. Other pyoverdines sharing the same motif but at another position within the peptide chain were not efficient in iron transport, demonstrating the importance of the spatial position of the binding site.     

Siderophore-mediated iron uptake in Alcaligenes eutrophus CH34 and identification of aleB encoding the ferric iron-alcaligin E receptor.

Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E. Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups. Alcaligin E promoted the growth of siderophore-deficient A. eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by iron-limited cells. However, the growth of the Sid- mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E. AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E. This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated. The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins. Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake. Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A. eutrophus CH34. Genetic studies of different Sid- mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A. eutrophus CH34 chromosome in the proximity of the cys-232 locus.     

Phylogenetic diversity and specificity of bacteria closely associated with Alexandrium spp. and other phytoplankton

While several studies have suggested that bacterium-phytoplankton interactions have the potential to dramatically influence harmful algal bloom dynamics, little is known about how bacteria and phytoplankton communities interact at the species composition level. The objective of the current study was to determine whether there are specific associations between diverse phytoplankton and the bacteria that co-occur with them. We determined the phylogenetic diversity of bacterial assemblages associated with 10 Alexandrium strains and representatives of the major taxonomic groups of phytoplankton in the Gulf of Maine. For this analysis we chose xenic phytoplankton cultures that (i) represented a broad taxonomic range, (ii) represented a broad geographic range for Alexandrium spp. isolates, (iii) grew under similar cultivation conditions, (iv) had a minimal length of time since the original isolation, and (v) had been isolated from a vegetative phytoplankton cell. 16S rRNA gene fragments of most Bacteria were amplified from DNA extracted from cultures and were analyzed by denaturing gradient gel electrophoresis and sequencing. A greater number of bacterial species were shared by different Alexandrium cultures, regardless of the geographic origin, than by Alexandrium species and nontoxic phytoplankton from the Gulf of Maine. In particular, members of the Roseobacter clade showed a higher degree of association with Alexandrium than with other bacterial groups, and many sequences matched sequences reported to be associated with other toxic dinoflagellates. These results provide evidence for specificity in bacterium-phytoplankton associations.     

A molecular assessment of the iron stress response in the two phylogenetic clades of Trichodesmium

Trichodesmium spp. play key roles in global carbon and nitrogen budgets and thus defining what controls their productivity is important for understanding climate change. While iron availability has been shown to be an important chemical factor for controlling both growth and nitrogen fixation rates in Trichodesmium , all culture experiments to date have focused solely on representatives from one clade of Trichodesmium . Genomic sequence analysis determined that the Trichodesmium erythraeum (IMS101) genome contains many of the archetypical genes involved in the prokaryotic iron stress response. Focusing on three of these genes, isiB , idiA and feoB , we found that all three showed an iron stress response in axenic T. erythraeum (IMS101), and their sequences were well conserved across four species in our Trichodesmium culture collection [consisting of two T. erythraeum strains (IMS101 and GBRTRLI101), two Trichodesmium tenue strains (Z-1 and H9-4), Trichodesmium thiebautii and Trichodesmium spiralis ]. With clade-specific quantitative PCR (qPCR) primers for one of these genes, isiB , we found that high isiB expression at low Fe levels corresponded to specific reductions in N₂ fixation rates in both major phylogenetic clades of Trichodesmium (the T. erythraeum clade and T. tenue clade). With regard to the two clades, the most significant difference determined was temperature optima, while more subtle differences in growth, N₂ fixation rate and gene expression responses to Fe stress were also observed. However the apparent conservation of the Fe stress response in the Trichodesmium genus suggests that it is an important adaptation for their niche in the oligotrophic ocean.     

Siderophore-mediated iron transport correlates with the presence of specific iron-regulated proteins in the outer membrane of Rhizobium meliloti.

A universal chemical assay used to detect the production of siderophores in a range of Rhizobium strains showed that production is strain specific. Iron nutrition bioassays carried out on Rhizobium meliloti strains to determine cross-utilization of their siderophores showed that R. meliloti 2011, 220-5, and 220-3 could each use the siderophores produced by the other two but not the siderophore produced by R. meliloti DM4 (and vice versa). Mutants of R. meliloti 2011 and 220-5 defective in siderophore production were isolated by Tn5-mob mutagenesis. The Tn5-mob-containing EcoRI fragment of mutant R. meliloti 220-5-1 was cloned into pUC19. By using this fragment as a probe, the presence of a homologous region was observed in R. meliloti 2011 and 220-3 but not in R. meliloti DM4. A complementing cosmid from a gene bank of R. meliloti 2011 was identified by using the same probe. Introduction of this cosmid into R. meliloti 102F34, a strain not producing a siderophore, resulted in the ability of this strain to produce a siderophore and also in the ability to utilize the siderophores produced by R. meliloti 2011, 220-5, and 220-3 but not the siderophore produced by R. meliloti DM4. A comparative analysis of the outer membrane proteins prepared from iron-deficient cultures of R. meliloti 102F34 and 102F34 harboring the cosmid revealed the presence, in the latter, of a low-iron-induced outer membrane protein corresponding to a low-iron-induced protein in R. meliloti 2011, 220-5, and 220-3. This protein is not present in R. meliloti DM4. The results suggest that R. meliloti 2011, 220-5, and 220-3 produce siderophores that are identical or sufficiently similar in structure to be transported by the membrane transport system of each strain while also indicating that utilization of a particular siderophore is correlated with the presence of specific outer membrane proteins.     

Siderophore-mediated iron (III) transport in the mycelia of the cultivated fungus, Agaricus bisporus.

Three structurally diverse iron (III) sequestering compounds (siderophores) were isolated from the supernatants of early stationary phase iron-deficient cultures of vegetative mycelia of the cultivated mushroom, Agaricus bisporus (ATCC 36416). The compounds were purified as their ferric chelates to homogeneity by gel permeation, cation exchange, and low-pressure reversed phase C18 chromatographies, and characterized as trihydroxamic acids. The chelates were identified as ferrichrome, ferric fusarinine C, and an unusual compound, des (diserylglycyl) ferrirhodin (DDF) by HPTLC cochromatography and electrophoresis against authentic samples, hydrolysis and amino acid analysis, and FAB-MS and 1H NMR spectroscopy. The iron transport activities of the three compounds (and of some structurally similar exogenous compounds) in young mycelial cells were determined by time- and concentration-dependent kinetic assays and inhibition experiments (CN-, N3-) using 55Fe(3+)-labeled chelates. 55Iron (III) uptake mediated by all three compounds was found to be via high affinity, energy-dependent processes; transport effectiveness was in the order: ferrichrome > DDF > ferric fusarinine C. The relative uptake of iron by lambda-cis ferrichromes was: ferrichrome > ferrirhodin > ferrichrome A; transport activity by the delta-cis fusarinines was: ferric fusarinine C > tris cis-(and trans-) fusarinine iron (III) > ferric N1-triacetylfusarinine C.     

Seasonality of Dinophysis spp. and Prorocentrum lima in Black Sea phytoplankton and associated shellfish toxicity

Plankton surveys, between 2001 and 2005 along the Russian Caucasian Black Sea Coast, revealed Dinophysis rotundata, D. caudata and Prorocentrum lima as the most ubiquitous of the known dinoflagellates associated with diarrhetic shellfish poisoning (DSP). Dinophysis spp. were first observed during the spring phytoplankton succession and persist throughout the late summer phytoplankton peak. The highest total concentration, 3000 cells/L, of D. rotundata and D. caudata was observed in April 2001. Unlike Dinophysis, P. lima was rarely observed in plankton samples but closely followed storm events with maximum cell counts of P. lima occurred in July 2002.The presence of Dinophysis in mussel (Mytilus galloprovincialis) hepatopancreas correlated with concentration with Dinophysis observed in the plankton samples. Conversely, P. lima could be found in most hepatopancreas samples collected during the May to October period. Therefore, planktonic concentration of P. lima does not reflect its availability for and consumption by shellfish.Samples of mussel hepatopancreas, from August 2002, with a corresponding Dinophysis concentration of 250 cells/L and no observable P. lima, were found to contain 0.03 ng OAE/g. This sample analyses by LC-MS/MS displayed okadaic acid (OA) and related congeners (DTX1) along with the pectinotoxins (PTX2 and PTX2sa). Highest observed levels of P. lima-induced DSP-toxicity in hepatopancreas was 0.41 g OA-equivalents/g corresponded to the highest observed planktonic cell counts of P. lima, 300 cell/L in August 2001. Cultures isolated from this sample were found to produce OA, DTX1 and their related diol esters.These data reveal a threat, represented by DSP-toxic species, at Black Sea coasts, and provide grounds for the introduction of phycotoxin control measures in the region.     

The role of phytoplankton in determining the underwater light climate in Lake Constance

At all seasons, the underwater light field of meso-eutrophic large (480 km²) deep (mean: 100 m) Lake Constance was studied in conjunction with the assessments of vertical distributions of phytoplankton chlorophyll concentrations. Vertical profiles of scalar, downwelling and upwelling fluxes of photosynthetically available radiation, as well as fluxes of spectral irradiance between 400 and 700 nm wavelength were measured.The overall transparency of the water for PAR is highly dependent on chlorophyll concentration. However, the spectral composition of underwater light is narrowing with water depth regardless of phytoplankton biomass.Green light is transmitted best, even at extremely low chlorophyll concentrations. This is explained by the selective absorption of blue light by dissolved organic substances and red light by the water molecules. Nevertheless, significant correlations were found between vertical attenuation coefficients of downwelling spectral irradiance and chlorophyll concentrations at all wavelengths. The slopes of the regression lines were used as estimates of chlorophyll-specific spectral vertical light attenuation coefficients (K _c()).The proportions of total upwelling relative to total downwelling irradiance (reflectance) increased with water depth, even when phytoplankton were homogeneously distributed over the water column. Under such conditions, reflectance of monochromatic light remained constant. Lower reflectance of PAR in shallow water is explained by smaller bandwidths of upwelling relative to downwelling light near the water surface. In deeper water, by contrast, the spectra of both upwelling and downwelling irradiance are narrowed to the most penetrating components in the green spectral range. Reflectance of PAR was significantly correlated with chlorophyll concentration and varied from 1% and 1-% at low and high phytoplankton biomass, respectively. Over the spectrum, reflectance exhibited a maximum in the green range. Moreover, in deeper layers, a red maximum was observed which is attributed to natural fluorescence by phytoplankton chlorophyll.     

Reduction-based iron uptake revisited: On the role of secreted iron-binding compounds

With the exception of the grasses, plants rely on a reduction-based iron (Fe) uptake system that is compromised by high soil pH, leading to severe chlorosis and reduced yield in crop plants. We recently reported that iron deficiency triggers the production of secondary metabolites that are beneficial for Fe uptake in particular at high external pH when iron is present but not readily available. The exact function of these metabolites, however, remains enigmatic. Here, we speculate on the mechanism by which secondary metabolites secreted by roots from Fe-deficient plants improve Fe acquisition. We suggest that the production and excretion of Iron Binding Compounds (IBCs) constitute an integrative, pH-insensitive component of the reduction-based iron uptake strategy in plants.     

Characterization of phosphonate uptake in two Phytophthora spp. and its inhibition by phosphate

The uptake of the phosphonate ion, the active breakdown product in plant tissues of the systemic anti-Oomycete compound Fosetyl-Al (aluminium tris-Oethylphosphonate), was investigated in two Phytophthora spp. of differential sensitivity. Uptake was due to the simultaneous operation of two transport systems, one of low affinity (high K _m) and one of high affinity (low K _m). The relative contribution of each transport system varied with the external concentration of phosphonate, suggesting that phosphonate was a potent regulator of both systems. Phosphate was a partial competitive inhibitor with respect to phosphonate. Phosphate competed with phosphonate for uptake with a K _i of 105 M for P. cryptogea and 68 M for P. citrophthora. Uptake was sensitive to pH, showing a maximum at pH 5.0 to 5.5. P. cryptogea was more efficient in phosphonate uptake, although it was less sensitive to inhibition by phosphonate in vitro, than P. citrophthora. This implied that the selective activity of phosphonate was not due to differential rates of uptake of this oxyanion. These results were discussed in relation to the mode of action of phosphonate towards Oomycetes.     

Molecular genetics of fungal siderophore biosynthesis and uptake: the role of siderophores in iron uptake and storage

To acquire iron, all species have to overcome the problems of iron insolubility and toxicity. In response to low iron availability in the environment, most fungi excrete ferric iron-specific chelators--siderophores--to mobilize this metal. Siderophore-bound iron is subsequently utilized via the reductive iron assimilatory system or uptake of the siderophore-iron complex. Furthermore, most fungi possess intracellular siderophores as iron storage compounds. Molecular analysis of siderophore biosynthesis was initiated by pioneering studies on the basidiomycete Ustilago maydis, and has progressed recently by characterization of the relevant structural and regulatory genes in the ascomycetes Aspergillus nidulans and Neurospora crassa. In addition, significant advances in the understanding of utilization of siderophore-bound iron have been made recently in the yeasts Saccharomyces cerevisiae and Candida albicans as well as in the filamentous fungus A. nidulans. The present review summarizes molecular details of fungal siderophore biosynthesis and uptake, and the regulatory mechanisms involved in control of the corresponding genes.     

Ferredoxin containing bacteriocins suggest a novel mechanism of iron uptake in Pectobacterium spp

In order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium Pectobacterium carotovorum carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of Pectobacterium carotovorum and Pectobacterium atrosepticum with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that Pectobacterium spp. carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of Pectobacterium carotovorum and atrosepticum that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells.     

The role of chlorophyllase and light in the decomposition of chlorophyll from marine phytoplankton

In a study of the biochemical aspects of the decomposition of chlorophyll, measurement of the activity of the enzyme chlorophyllase in marine phytoplankton was attempted in vitro by observing the conversion of chlorophyll to chlorophyllide. Our experiments indicate that in darkness and at room temperatures any enzymatic activity, if present, is immeasurable by the techniques employed.As a consequence, we have considered the photo-bleaching of chlorophyll as a possible means of decomposition. This has been identified as a photo-oxidation and the decomposition of the pigment is extremely rapid. Our experiments show that a similar photo-oxidation exists in fractured cells and degraded pigments found in the feces of marine herbivores. Although the rate of photo-bleaching is slower in vivo than in vitro, we postulate that this is a principle mechanism for pigment decomposition.