Thalassiosira oceanica, a marine centric diatom, possesses an extracellular reductase that reduces iron (Fe(III)) bound to organic complexes as part of a high-affinity Fe transport mechanism. A number of Fe(III) organic complexes are reduced, including siderophores---effective Fe chelates produced by microorganims in response to Fe stress. Reduction rates are inversely related to the relative stability constants of the oxidized and reduced Fe chelates (log Kox/Kred), and vary by a factor of 2.4 in accordance with theoretical predictions. Under Fe-limiting conditions, reduction rates increase and the ability of T. oceanica to transport Fe from siderophores is enhanced. Iron bound to the siderophore desferrioxamine B (DFB) is reduced 2 times faster than it is taken up, suggesting that the reductase is well coupled to the Fe transporter, and can provide all the inorganic Fe to account for the measured Fe uptake rates in the presence of excess DFB. The efficacy of the reductase in providing inorganic Fe for uptake and growth is ultimately dependent on the relative concentrations of excess ligands in solution and cell surface Fe transporters competing for inorganic Fe. The rates of Fe reduction and uptake are twice as fast in cells grown in NO3- compared to those grown in NH 4+, suggesting a link with cellular N metabolism and with NO3- utilization in particular. Enhanced Fe reductase activity in NO3--grown cells enables them to maintain a 1.6-fold higher cellular Fe concentration under low Fe conditions. / Experiments conducted in the subarctic Pacific, an Fe-limited oceanic region, demonstrated that even indigenous plankton (both prokaryotic and eukaryotic plankton) have the ability to acquire Fe bound to strong organic chelates. Large phytoplankton species (>3 mum) reduce Fe bound to siderophores extracellularly. Because the predominant form of dissolved Fe in the sea is bound to strong organic complexes, a reductive mechanism as described here may be a critical step in Fe acquisition by phytoplankton.

Recent studies demonstrate that the dissolved iron in seawater is bound to strong organic complexes that have stability constants comparable to those of microbial iron chelates (siderophores). We investigated iron acquisition by 7 strains of heterotrophic marine bacteria using siderophores as a model for the natural iron-binding ligands. Siderophores were detected in the supernatants of 4 strains. All strains utilized iron bound to siderophores regardless of whether they produced their own. The majority took up iron bound to the fungal siderophore desferrioxamine B (dfoB). Over half also utilized iron bound to strain Neptune's siderophore, nep-L, while iron bound to pwf-L was available solely to the producing strain, Pwf3. Uptake rates of Fe-siderophores were similar among iron-limited strains and among ligands. Transport of Fe-dfoB in Neptune was enhanced 20 times by iron limitation. The half-saturation constant of Fe-dfoB transport was 15 nM, the lowest reported for Fe-siderophore transport in microorganisms. In contrast, uptake of inorganic iron (Fe' ) by iron-limited Neptune did not saturate at the highest concentration tested and was not upregulated under iron stress. This suggests that Fe ' uptake occurs by simple diffusion through the outer membrane. / Strain Lmg1, the sole catechol producer, did not take up iron bound to exogenous siderophores (dfoB, pwf-L, or nep-L). However, it utilized iron bound to its own ligand and, possibly, iron bound to the synthetic chelator EDTA. Transport of Fe' by iron-limited Lmg1 was 10 times higher than in the other strains and was upregulated 46 times in low iron conditions. The results suggest iron transport in Lmg 1 may be mediated by surface-associated catechol siderophores that scavenge inorganic ferric species as well as iron bound to weaker complexes, such as EDTA. This study elucidates the importance of siderophores in iron transport by heterotrophic marine bacteria. (Abstract shortened by UMI.)

Despite being abundant in the earth's crust, the concentration of Fe in many oceanic regions is so low that it is limiting to the growth of photosynthetic plankton. Heterotrophic bacteria play key roles in the oceanic cycling of carbon and nutrients, but it is unclear whether they can be Fe-deficient in nature, or what possible effects Fe-deficiency might have on their ecology and physiology. In chapter 1, I investigated the response of a natural bacterial community to a mesoscale Fe-enrichment experiment in the northeast subarctic Pacific. The addition of Fe to surface waters caused a rapid stimulation of bacterial growth and production, and induced the organic Fe uptake systems of bacteria. These findings suggest that bacteria responded directly to increased Fe availability, and may be Fe-deficient in situ. In chapter 2, I examined the effects of Fe-deficiency on the coupled processes of carbon catabolism and adenosine triphosphate (ATP) production in cultures of the marine bacterium Pseudoalteromonas haloplanktis. In Fe-limited cells, Fe-dependent oxidative pathways of ATP production were downregulated, leading to an intracellular energy deficit. Thus, by altering carbon metabolism and energy acquisition of heterotrophic bacteria, Fe may affect the cycling of carbon in parts of the sea.

Three species of photosynthetic flagellates capable of phagotrophy (mixotrophic species) were tested for their abilities to use inorganic iron colloids for growth. Ochromonas sp., Chrysochromulina ericina (a coastal strain) and C. ericina (an oceanic strain) were grown in iron-free seawater supplemented with 1 muM goethite, hematite, magnetite/maghemite or ferrihydrite (90°) in the presence and absence of desferrioxamme B, an iron-binding siderophore. Both strains of Chrysochromulina grew at 35--70% of their maximum rates with goethite, hematite, and magnetite/maghemite, but were unable to use ferrihydrite. Ochromonas, however, grew well with ferrihydrite, but could not use any of the other forms. All the flagellates were able to acquire iron from ingested bacteria. Diatoms that were known only to take up dissolved forms of iron, Thalassiosira oceanica (clone 1003) and T. pseudonana (clone 3H), were unable to use any of the colloids tested. The mechanism of iron acquisition by the flagellates appeared to involve ingestion of the iron colloids as DFB had no effect on colloidal iron availability and bacteria resident in the cultures were unable to use the iron contained in the colloids. Variations in the size of the colloids were hypothesized to account for differences in their availability, independent of colloid chemical stability. The results provide the first strong evidence for direct utilization (i.e. without prior dissolution) of colloidal iron by mixotrophic phytoplankton and document a new pathway of iron acquisition that may be important for their survival in low-iron waters of the sea.

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published_or_final_version / Biological Sciences / Master / Master of Philosophy

[Truncated abstract] Iron is essential to most biological systems. Under pathological conditions affecting the iron metabolic pathway, iron can be deposited in the tissue in various forms. The work presented in this thesis has exploited the relationship between the magnetic and the chemical properties of tissue iron deposits to further understanding of two major pathologies, the haemoglobinopathies termed thalassaemias and the malaria parasite Plasmodium falciparum, both amongst the most common health concerns in tropical countries. The iron-specific magnetic susceptibilities ¿Fe for spleen tissue samples from 7 transfusion dependent ß-thalassaemia (ß-thal) patients and 11 non-transfusion dependent ß-thalassaemia/Haemoglobin E (ß/E) patients were measured at 37°C. Both groups of patients were iron loaded with no significant difference in the distribution of spleen iron concentrations between the two groups. There was a significant difference between the mean ¿Fe of the spleen tissue from each group. The ß/E patients had a higher mean (± standard deviation) spleen ¿Fe (1.55 ± 0.23 × 10-6 m3.kgFe -1) than the ß-thal patients (1.16 ± 0.25 × 10-6 m3.kgFe -1). Correlations were observed between ¿Fe of the spleen tissue and the fraction of magnetic hyperfine split sextet in the 57Fe Mössbauer spectra of the tissues at 78 K (Spearman rank order correlation ¿ = -0.54, p = 0.03) and between ¿Fe of the spleen tissue and the fraction of doublet in the spectra at 5 K (¿ = 0.58, p = 0.02) indicating that ¿Fe of the spleen tissue is related to the chemical speciation of the iron 2 deposits in the tissue. The biological variability of the iron-specific magnetic susceptibility of the tissue iron examined would contribute a random uncertainty of 19% to magnetic susceptibility based non-invasive measurements of tissue iron concentration. ... Magnetic susceptibility measurements were also performed on malaria parasitised red blood cells. In vitro cultures of P. falciparum were magnetically enriched up to 61-fold using high field gradient magnetic separation columns, and the magnetic susceptibility of cell contents was directly measured. Forms of haem iron were quantified spectroscopically. Further fractionations were performed such that, by controlling the fluid velocity through the column, cells with more than a critical amount of paramagnetic 3 iron were preferentially extracted. A chloroquine-sensitive (CQS) laboratory strain of parasites converted approximately 60% of host cell haem iron to haemozoin and this product was the primary source of the increase in cell magnetic susceptibility. The volumetric magnetic susceptibility of the magnetically enriched cells was found to be 0.15 ± 0.03 × 10-7 relative to the suspension medium, accounting for the enrichment of mature parasites. Comparisons of fractionation samples of two pairs of CQS and chloroquine resistant (CQR) strains showed enrichment of mature parasites was significantly greater in the CQS than the CQR strains. The results suggest the possibility of using magnetic separation columns in identifying CQR strains of P. falciparum, potentially in a diagnostic or research setting. The study also underlines the need to identify and quantify the forms of iron in CQR and CQS parasite strains as the fate of haem iron will have implications in understanding the mechanisms of chloroquine resistance.