IRON-DEPENDENT CYANOBACTERIAL BIOREPORTER FOR MEASURING FE BIOAVAILABILITY IN FRESHWATER ENVIRONMENTS

Iron is one of the essential nutrient elements and exists in many forms in aquatic environments, and only free ferric ion can be utilized by phytoplankton. Physicochemical analysis can provide information on total iron content in natural water samples, but bioassay approach which uses living organisms as bioreporters can tell us the bioavailable fraction of dissolved iron. Anabaena sp. PCC 7120 is a filamentous and nitrogen-fixing cyanobacterium. Alr2581 is an outer membrane transporter associated with Fe uptake in Anabaena sp. PCC 7120 and its expression is enhanced under iron-limiting conditions. In this study, an Anabaena sp. PCC 7120 Fe-responsive bioreporter strain Palr2581-luxAB was constructed by fusing the promoter of gene alr2581 to luxAB reporter gene. Our results showed that growths were no difference in the bioreporter Palr2581-luxAB grown in Fe-replete and Fe-deplete BG11 medium. However, contents of chlorophyll α of cells cultured in Fe-replete medium were much higher than those in Fe-deplete medium. LuxAB activities were significantly induced in Fe-depleted growth medium in each growth phase, indicating continuous transcription from the alr2581 promoter. A quantitative assessment of bioluminescent was made by incubating cells for 0-48h in trace metal-buffered Fraquil medium containing various additions of Fe. An ideal dose-response curve was generated relating bioreporter luminescent to the pFe (pFe=lg Fe3+ free ferric) between 19.6 and 21.7 after 12h incubation time and was best represented by sigmoidal curve (y=41852.4-31891.8/1+exp(x-20.69)/0.16, R2=0.9875) having a linear component extending between pFe 20.7 (Fe3+=10-20.7 mol/L) and pFe 21.2 (Fe3+=10-21.2 mol/L) (y=35472x-707426, R2=0.9988). In addition, the applicability of using this Fe bioreporter to assess Fe availability in the natural environment has been tested by using water sample from Lake Donghu (contained pFe 20.56, Fe3+=10-20.56 mol/L). It indicated that the bioreporter could serve as a quantitative tool to assess Fe-nutrient status in natural freshwater environments.