Zinc homeostasis was investigated in Nostoc punctiforme. Cell tolerance to Zn 2؉ over 14 days showed that ZnCl 2 levels above 22 M significantly reduced cell viability. After 3 days in 22 M ZnCl 2 , ca. 12% of the Zn 2؉ was in an EDTA-resistant component, suggesting an intracellular localization. Zinquin fluorescence was detected within cells exposed to concentrations up to 37 M relative to 0 M treatment. Radiolabeled 65 Zn showed Zn 2؉ uptake increased over a 3-day period, while efflux occurred more rapidly within a 3-h time period. Four putative genes involved in Zn 2؉ uptake and efflux in N. punctiforme were identified: (i) the predicted Co/Zn/Cd cation transporter, putative CDF; (ii) the predicted divalent heavy-metal cation transporter, putative Zip; (iii) the ATPase component and Fe/Zn uptake regulation protein, putative Fur; and (iv) an ABC-type Mn/Zn transport system, putative zinc ZnuC, ZnuABC system component. Quantitative real-time PCR indicated the responsiveness of all four genes to 22 M ZnCl 2 within 3 h, followed by a reduction to below basal levels after 24 h by putative ZIP, ZnuC, and Fur and a reduction to below basal level after 72 h by putative CDF efflux gene. These results demonstrate differential regulation of zinc transporters over time, indicating a role for them in zinc homeostasis in N. punctiforme.Cyanobacteria are photosynthetic prokaryotes, many of which accumulate heavy metals. Consequently, they have attracted interest as a tool for the removal of metals from wastewater. Cyanobacteria, including Anabaena nodosum (10), Nostoc linkia (12), Microcystis aeruginosa (39), and Synechococcus sp. strain PCC7942 (16), are able to accumulate the heavy metals cadmium, zinc, copper, and chromium, respectively. Heavy metal accumulation involves an initial rapid, passive adsorption of the metal to components of the cell wall over seconds or minutes, followed by a slower process that results in the sequestration of the metal to an EDTA-resistant compartment, such as the cytoplasm, within hours (for reviews, see references 7 and 29). Cell wall components that have a high affinity for metals account for the bulk of the adsorbed metals (17). In M. aeruginosa, metal accumulation was only marginally decreased in cells treated with metabolic inhibitors and in heat-killed cells compared to the total metal uptake (34). Substantial data exist showing sorption of metals in cyanobacteria; however, the capacity of live cells to tolerate or accumulate metals and the molecular mechanisms underlying these processes are relatively unknown. In contrast to many bacterial species, cyanobacteria possess a metallothionein, SmtA, which may store Zn 2ϩ and prevent intracellular Zn 2ϩ toxicity (8). Polyphosphate granules may sequester intracellular metal ions, including zinc ions (4). These cellular characteristics make cyanobacteria unique organisms in regard to their ability to accumulate metals.Genomic analysis indicated that Nostoc punctiforme, a filamentous cyanobacterium, contains transporters that may function in...