Marine coccolithophorid phytoplankton are major producers of biogenic calcite, playing a significant role in the global carbon cycle. Predicting the impacts of ocean acidification on coccolithophore calcification has received much recent attention and requires improved knowledge of cellular calcification mechanisms. Uniquely amongst calcifying organisms, coccolithophores produce calcified scales (coccoliths) in an intracellular compartment and secrete them to the cell surface, requiring large transcellular ionic fluxes to support calcification. In particular, intracellular calcite precipitation using HCO3 − as the substrate generates equimolar quantities of H+ that must be rapidly removed to prevent cytoplasmic acidification. We have used electrophysiological approaches to identify a plasma membrane voltage-gated H+ conductance in Coccolithus pelagicus ssp braarudii with remarkably similar biophysical and functional properties to those found in metazoans. We show that both C. pelagicus and Emiliania huxleyi possess homologues of metazoan Hv1 H+ channels, which function as voltage-gated H+ channels when expressed in heterologous systems. Homologues of the coccolithophore H+ channels were also identified in a diversity of eukaryotes, suggesting a wide range of cellular roles for the Hv1 class of proteins. Using single cell imaging, we demonstrate that the coccolithophore H+ conductance mediates rapid H+ efflux and plays an important role in pH homeostasis in calcifying cells. The results demonstrate a novel cellular role for voltage gated H+ channels and provide mechanistic insight into biomineralisation by establishing a direct link between pH homeostasis and calcification. As the coccolithophore H+ conductance is dependent on the trans-membrane H+ electrochemical gradient, this mechanism will be directly impacted by, and may underlie adaptation to, ocean acidification. The presence of this H+ efflux pathway suggests that there is no obligate use of H+ derived from calcification for intracellular CO2 generation. Furthermore, the presence of Hv1 class ion channels in a wide range of extant eukaryote groups indicates they evolved in an early common ancestor.

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The apparent sensitivities of several bacterial pathogens to tetracyclines varied by up to 128-fold with the medium content of Fe, but not of other metals. The effect of Fe was independent of superoxide dismutase activity and of intracellular Fe, but accumulation of tetracyclines was blocked in high-Fe medium. Thus, synergistic suppression of bacterial growth in the presence of a low Fe concentration and tetracyclines arises because of elevated antibiotic accumulation.The tetracyclines (TCs) are broad-spectrum bacteriostatic antibiotics that inhibit the binding of aminoacyl-tRNA to the ribosomal A site, blocking protein synthesis (8). Environmental parameters may modulate the activity of the TCs. For example, MICs are influenced by pH, oxygen concentration, and external cations (1,11,16,19,21,23). Such factors are an important consideration when predicting MICs for bacteria isolated from human infections: laboratory-derived MICs may not faithfully reflect antibiotic concentrations that will be effective in vivo. There have been almost no studies to explore the mechanisms by which certain external parameters affect the activity of TCs.It has been established recently that Cu,Zn-superoxide dismutase (Sod1p)-defective mutants of the yeast Saccharomyces cerevisiae are susceptible to oxytetracycline (OTC) via a mechanism that is dependent on oxidative damage (2, 3, 5). This susceptibility could be suppressed with Cu in a Sod1p-independent manner (2). We initiated the present work to investigate whether similar effects occur in prokaryotic cells that are normally sensitive to TCs.To test whether the sensitivity of Escherichia coli to TCs is copper dependent, the MICs of OTC and TC against E. coli were determined at a range of Cu(NO 3 ) 2 concentrations by the agar dilution method (17). The MICs (ϳ4 g ml Ϫ1 for both antibiotics) were unaffected at up to 3 mM Cu, the highest Cu concentration tested (data not shown).Since OTC can promote oxidative stress (3) and Cu is a Fenton catalyst, we also tested whether Fe influences the sensitivity of E. coli to TCs. The MICs of TC, OTC, and minocycline (MC) were increased markedly by increasing concentrations of FeSO 4 in the medium (Fig. 1). Thus, 25 M FeSO 4 rescued E. coli growth at 4 g of OTC ml Ϫ1 and the MIC was increased to 64 g ml Ϫ1 at 400 M Fe and to 128 g ml Ϫ1 at 1.6 mM Fe (Fig. 1A). The effect of Fe supplementation on the MICs of TC and MC was less marked than for OTC. Nevertheless, eightfold higher TC and MC concentrations were required to inhibit E. coli growth at 1.6 mM Fe than in Feunsupplemented Luria-Bertani (LB) medium (Յ3 M Fe) (Fig. 1A). In medium supplemented with the Fe chelator ferrozine (0.5 mM), the MIC of OTC was lowered to 1 g ml Ϫ1(giving a 128-fold full-range variation in the MIC of OTC in this study) and the MIC of TC was lowered to 2 g ml Ϫ1 (data not shown). Furthermore, ferrozine at 0.5 mM reversed the protective effect of 25 M FeSO 4 against OTC, and protection with 100 M FeSO 4 was reversed at 2.5 mM ferrozine. The effects were relatively spec...