Michael R. Twiss

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

Mapping cyanobacterial blooms in the Great Lakes using MODIS

ABSTRACT Toxin-producing Cyanobacteria have been documented in Lake Erie and Ontario in the last several years. We developed algorithms to discriminate potentially toxic cyanobacterial blooms from other harmless phytoplankton blooms and to extract relative phycocyanin abundances from Moderate Resolution Imaging Spectrometer (MODIS) satellite data. Lees quasi-analytical algorithm was used to calculate total absorption and backscatter from the 250 m, 500 m and 1 km bands of MODIS scenes. A non-negative least square algorithm was then utilized to discern relative concentrations of Chlorophyta (green algae), phycocyanin-rich Cyanobacteria (blue-green algae), and colored dissolved organic matter and suspended sediments combined in lake waters using published absorption spectra for these components. MODIS-derived cyanobacterial concentrations and/or bloom distributions from 10 scenes acquired in the summers of 2004 and 2005 were successfully verified against contemporaneous calibrated measurements of pigments that were acquired from measurements made using continuous fluorimetric measurements of surface water (1 m depth) from six cruises, and three additional cyanobacterial blooms reported in the scientific literature between 2002 and 2006. These results demonstrate that this methodology could be used to develop a cost-effective practical screening method for rapid detection and warning of potentially toxic cyanobacterial blooms in the lower Great Lakes.

Electrochemically stimulated release of lysozyme from an alginate matrix cross-linked with iron cations

An electrochemically generated alginate matrix cross-linked with Fe3+ cations was used to entrap lysozyme and then release it upon application of an electrochemical signal. The switchable behavior of the alginate hydrogel was based on the different interaction of Fe3+ and Fe2+ cations with alginate. The oxidized Fe3+ cations strongly interact with alginate resulting in its cross-linking and formation of the hydrogel, while the reduced Fe2+ cations weakly interact with alginate and do not keep it in the hydrogel state. Thus, the electrochemical oxidation of iron cations at +0.8 V (Ag/AgCl) in the presence of alginate and lysozyme resulted in the Fe3+-cross-linked alginate hydrogel thin-film on the electrode surface with the physically entrapped lysozyme. On the other hand, application of reductive potentials (e.g. −1.0 V) converted the iron cations to the Fe2+ state, thus resulting in dissolution of the alginate thin-film and lysozyme release. The bactericidal effect of the electrochemically released lysozyme was tested on the Gram-positive bacterium Micrococcus luteus demonstrating the same activity as the unadulterated lysozyme commercially supplied by Sigma-Aldrich. The present result represents the first step towards drug delivering systems (exemplified by the lysozyme release) based on alginate hydrogels and activated by electrochemical stimuli.

Ratios of Community Respiration to Photosynthesis and Rates of Primary Production in Lake Erie Via Oxygen Isotope Techniques

Abstract To evaluate levels of primary production and community metabolism in Lake Erie, we conducted incubations with 18 O-labelled water and determined the ratio of respiration to primary production (R:P) during the summer and early fall of 2002. The epilimnion of Lake Erie was characterized by δ 18 O values less than 0.7‰ at all times that reflects a strong contribution of O 2 from primary production. High δ 18 O values (maximum of 6.6‰) were common in the O 2 depleted waters of the hypolimnion as a consequence of isotope fractionation during respiration. Hypoxic conditions were evident in the central basin in August when the fractional abundance of O 2 reached a minimum value of 0.04. Rates of primary production varied from 0.23 to 1.76 mmol-O 2 .m –3 .h –1 , decreased from west to east in August, and were at a minimum in the central basin in September. Ratios of respiration to photosynthesis (R:P) (determined from the abundance and isotopic composition of dissolved O 2 ) in the epilimnion as low as 0.45 in July were strong evidence of net autotrophy and preceded the development of hypoxia in August. Net heterotrophy prevailed in August and September. The absolute values of R:P ratios were not indicative of trophic state, however, the wide range of R:P ratios (0.45 to 20.40), fraction of O 2 saturation (0.04 to 1.36), and δ 18 O-O 2 (−6.0 to 7.5‰) values were all indicative of a eutrophic system. An average isotope fractionation factor for respiration of 7.9‰ was determined from samples in the hypolimnion in July and August. Based on unique fractionation factors for respiration in the water column (23.5‰) and in sediments (3‰) we calculate that 61% of hypolimnion O 2 respiration occurs within sediments and 39% occurs within the water column.

Application of the biotic ligand model to explain potassium interaction with thallium uptake and toxicity to plankton

Competitive interaction between TI(I) and K was successfully predicted by the biotic ligand model (BLM) for the microalga Chlorella sp. (Chlorophyta; University of Toronto Culture Collection strain 522) during 96-h toxicity tests. Because of a greater affinity of T1(I) (log K = 7.3-7.4) as compared to K (log K = 5.3-6.3) for biologically sensitive sites, an excess of 40- to 160-fold of K is required to suppress T1(I) toxic effects on Chlorella sp., regardless of [T1(I)] in solution. Similar excess of K is required to suppress T1(I) toxicity to Synechococcus leopoliensis (Cyanobacteria; University of Texas Culture Collection strain 625) and Brachionus calyciflorus (Rotifera; strain AB-RIF). The mechanism for the mitigating effect of K on T1(I) toxicity was investigated by measuring 204T1(I) cellular uptake flux and efflux in Chlorella sp. Potassium shows a competitive effect on T1(I) uptake fluxes that could be modeled using the BLM-derived stability constants and a Michaelis-Menten relationship. A strong T1 efflux dependent only on the cellular T1 concentration was measured. Although T1 efflux does not explain the effect of K on T1(I) toxicity and uptake, it is responsible for a high turnover of the cellular T1 pool (intracellular half-life = 12-13.5 min). No effect of Na+, Mg2+, or Ca2+ was observed on T1+ uptake, whereas the absence of trace metals (Cu, Co, Mo, Mn, Fe, and Zn) significantly increased T1 uptake and decreased the mitigating effect of K+. The importance of K+ in determining the aquatic toxicity of T1+ underscores the use of ambient K+ concentration in the establishment of T1 water-quality guidelines and the need to consider K in predicting biogeochemical fates of T1 in the aquatic environment.

Ice cover extent drives phytoplankton and bacterial community structure in a large north‐temperate lake: implications for a warming climate

Mid-winter limnological surveys of Lake Erie captured extremes in ice extent ranging from expansive ice cover in 2010 and 2011 to nearly ice-free waters in 2012. Consistent with a warming climate, ice cover on the Great Lakes is in decline, thus the ice-free condition encountered may foreshadow the lakes future winter state. Here, we show that pronounced changes in annual ice cover are accompanied by equally important shifts in phytoplankton and bacterial community structure. Expansive ice cover supported phytoplankton blooms of filamentous diatoms. By comparison, ice free conditions promoted the growth of smaller sized cells that attained lower total biomass. We propose that isothermal mixing and elevated turbidity in the absence of ice cover resulted in light limitation of the phytoplankton during winter. Additional insights into microbial community dynamics were gleaned from short 16S rRNA tag (Itag) Illumina sequencing. UniFrac analysis of Itag sequences showed clear separation of microbial communities related to presence or absence of ice cover. Whereas the ecological implications of the changing bacterial community are unclear at this time, it is likely that the observed shift from a phytoplankton community dominated by filamentous diatoms to smaller cells will have far reaching ecosystem effects including food web disruptions.

Field Investigations of Trace Metal Effects on Lake Erie Phytoplankton Productivity

Abstract Responses of phytoplankton to trace metal and phosphate enrichments were made in pelagic Lake Erie surface waters over the time period of 1999–2003. All experiments employed trace metal clean sampling protocols. Bioassays were incubated over a 0.75–4 d period. Response was evaluated by measures of biomass (chlorophyll-a; chl-a), photosynthesis (using the carbon-14 technique), and dilution assays used to measure chl-a specific growth and grazing rates. Metals assayed were Cd, Co, Zn (5–50 nM) and Fe over the range of (20–100 nM). Phosphorus was added singly (0.1–1 μM) or in addition with Zn or Fe. The principle finding from this study was that the frequency of observed trace metal limitation in pelagic Lake Erie phytoplankton was low. Picoplankton (0.2–2 μm) responded most frequently to the metal enrichment; metals were as frequently toxic as they were stimulatory. Nanoplankton (2–20 μm) were nearly insensitive to metal enrichment as were the microplankton (20–210 μm). An EDTA chelated mixture of Fe, Cu, Zn, Co, Mn, and Mo did stimulate picoplankton chl-a production over 3 days and the growth and grazing rate of this important size fraction. Toxicity of Zn at 50 nM was observed; the presence of phosphate reduced inhibition by Zn at this concentration. The results suggest that trace metals are not as important over the short term as the availability of phosphorus in controlling phytoplankton productivity; however, trace metal enrichment can periodically have a stimulatory effect, particularly on the picoplankton size class.

Consideration of the bioavailability of iron in the North American Great Lakes: Development of novel approaches toward understanding iron biogeochemistry

There is increasing recognition that iron distribution and availability is significant in terms of global oceanic production. Low availability of iron and other nutritive trace metals may also constrain productivity in the North American Great Lakes. Despite its importance, the biogeochemistry of iron in the water column of lacustrine systems remains poorly characterized. In addressing the current state of iron biogeochemistry, a workshop organized a decade ago at the Bermuda Biological Station for Research brought together a cross-disciplinary team of chemists and biologists who sought to synthesize current knowledge and identify research priorities in this field. Key among goals identified during the workshop, and one that remains today for the most part unfulfilled, was to ‘develop techniques to quantify those fractions of Fe that are accessible to phytoplankton.’ Here we review recent progress toward meeting this objective, drawing on specific examples from Lake Superior where these approaches have been applied.

Optimization of iron-dependent cyanobacterial (Synechococcus, Cyanophyceae) bioreporters to measure iron bioavailability

Complex chemistry and biological uptake pathways render iron bioavailability particularly difficult to assess in natural waters. Bioreporters are genetically modified organisms that are useful tools to directly sense the bioavailable fractions of solutes. In this study, three cyanobacterial bioreporters derived from Synechococcus PCC 7942 were examined for the purpose of optimizing the response to bioavailable Fe. Each bioreporter uses a Fe‐regulated promoter (isiAB, irpA and mapA), modulated by distinct mechanisms under Fe deficiency, fused to a bacterial luciferase (luxAB). In order to provide a better understanding of the way natural conditions may affect the ability of the bioreporter to sense iron bioavailability, the effect of relevant environmental parameters on the response to iron was assessed. Optimal conditions (and limits of applicability) for the use of these bioreporters on the field were determined to be: a 12 h (12–24 h) exposure time, temperature of 15°C (15°C–22°C), photon flux density of 100 μmol photons·m−2·s−1 (37–200 lmol photons·m−2·s−1), initial biomass of 0.6–0.8 lg chlorophyll a (chl a)·L−1 (0.3–1.5 lg chl a·L−1) or approximately 105 bioreporter cells·mL−1, high phosphate (10 lM), and low micronutrients (absent). The measured luminescence was optimal with an exogenous addition of 60 lM aqueous decanal substrate allowing a 5 min reaction time in the dark before analysis. This study provides important considerations relating to the optimization in the use of bioreporters under field conditions that can be used for method development of other algal and cyanobacterial bioreporters in aquatic systems.

Assessment of Phosphorus-microbe Interactions in Lake Ontario by Multiple Techniques

ABSTRACT Conventional and newly-developed techniques to determine the phosphorus (P) status of Lake Ontario phytoplankton were employed in September 2003, immediately after the passage of the storm system associated with Hurricane Isabel. Surface water (1–5 m) was collected at 29 stations, with selected stations sampled throughout the water column. Chemical estimates of total P concentrations were compared with proxies of P bioavailability: P enrichment bioassays of lake water, alkaline phosphatase activity (APA), and P-dependent bioreporter assays. Average total P (314 nM) and total chlorophyll-α (2.12 μg/L) concentrations measured in pelagic surface waters from throughout Lake Ontario suggest an oligotrophic status prevailed across much of this lake during the sample period. Autotrophic picoplankton (0.2–2 μm) displayed the highest growth rates and were grazed at the highest rate, whereas P-enrichment bioassays favored the production of autotrophic nanoplankton (2–20 μm) and autotrophic microplankton (> 20 μm) biomass. Average concentrations of bacteria (2.61 ×1010 cells/L) were higher than those measured during summer in a similar lake (Erie), whereas the average viral density (1.38 ×1010 virus particles/L) was similar. Pelagic stations exhibited higher APA than coastal stations; cyanobacterial bioreporter responses did not show high correlation with APA suggesting that proxies of P-demand based on residual effects (e.g., enzyme production) were not indicative of shorter-term biological responses related to planktonic growth (bioreporter genetic response). The combination of traditional chemical, biochemical (APA), and cutting-edge biological methods (bioreporter) provided information on nutrient concentrations and primary productivity throughout Lake Ontario, while concurrently allowing real-time assessment of P bioavailability.