Cécile E. Mioni

Characterization and field trials of a bioluminescent bacterial reporter of iron bioavailability

To better understand Fe cycling in marine and freshwater systems, we have developed a biomolecular tool to track the perceived bioavailability of Fe to heterotrophic bacteria. Bioluminescent reporters, constructed by fusing the fepA–fes promoter of Escherichia coli (an Enterobactin biosynthesis gene regulated by the ferric uptake regulatory [Fur] system) to a luxCDABE cassette, were integrated into the chromosome of a halotolerant Pseudomonas putida, which uses the Fur system to regulate high-affinity Fe uptake. The resultant P. putida bioreporter has been successfully tested both in lab and field studies. Laboratory cultures were maintained at a range of concentrations of total Fe (0–25 nM) or limited by the addition of concentrations of wellcharacterized siderophores (desferrioxamine B [DFB], ferrichrome, 2,2V-dipyridyl [DP] and Rhodotorulic acid [RA], 0–200 nM) and used to establish the dynamic range of this reporter system. Analysis of sample incubations after only 4 h suggest that both of the trihydroxamate-type siderophores DFB and ferrichrome efficiently reduced Fe availability, resulting respectively in a 1.77- and 1.88-fold increase in luminescence relative to Fe-replete conditions. In contrast, additions of the dihydroxamate-type siderophore RA and the synthetic chelator DP resulted in no response from the system, suggesting that cells could access Fe complexed to these compounds without activating high-affinity Fe transport systems. Field studies were performed in the central basin of Lake Erie, which has previously been shown to undergo sporadic Fe limitation during summer stratification. DFB concentrations were titrated across a range of 0–50 nM into unfiltered water to manipulate Fe availabilities. Bioreporters expressed Fe stress (ca. a 2-fold increase in luminescence) at concentrations of DFB equivalent to the total (dissolved+particulate) Fe in the system (c30 nM), indicative of the concentration of bioavailable Fe. In a similar experiment with 0.2-Am pre-filtered water (2.25–5.24 nM Fe), a 6-fold increase in luminescence (relative to controls) was observed at the lowest (15 nM) concentration of chelators. The results of this study demonstrate the validity of bioreporters as a complimentary tool to measurements of total Fe. Moreover, these results suggest that a significant source of

Impact of Phytoplankton on the Biogeochemical Cycling of Iron in Subantarctic Waters Southeast of New Zealand During FeCycle

[1] During austral summer 2003, we tracked a patch of surface water infused with the tracer sulfur hexafluoride, but without addition of Fe, through subantarctic waters over 10 days in order to characterize and quantify algal Fe pools and fluxes to construct a detailed biogeochemical budget. Nutrient profiles characterized this patch as a highnitrate, low-silicic acid, low-chlorophyll (HNLSiLC) water mass deficient in dissolved Fe. The low Fe condition was confirmed by several approaches: shipboard iron enrichment experiments and physiological indices of Fe deficiency (Fv/Fm 40% of total chlorophyll. Whereas the picophytoplankton accounted for � 50% of total primary production, they were responsible for the majority of community iron uptake in the mixed layer. Thus ratios of 55 Fe: 14 C uptake were highest for picophytoplankton (median: 17 mmol:mol) and declined to � 5 mmol:mol for the larger algal size fractions. A pelagic Fe budget revealed that picophytoplankton were the largest pool of algal Fe (>90%), which was consistent with the high (� 80%) phytoplankton Fe demand attributed to them. However, Fe regenerated by herbivory satisfied only � 20% of total algal Fe demand. This iron regeneration term increased to 40% of algal Fe demand when we include Fe recycled by bacterivory. As recycled, rather than new, iron dominated the pelagic iron budget (Boyd et al., 2005), it is highly unlikely that the supply of new Fe would redress the imbalance between algal Fe demand and supply. Reasons for this imbalance may include the overestimation of algal iron uptake from radiotracer techniques, or a lack of consideration of other iron regeneration processes. In conclusion, it seems that algal Fe uptake cannot be supported solely by the recycling of algal iron, and may require an Fe ‘‘subsidy’’ from that regenerated by heterotrophic pathways.