Construction and characterization of luminescent Synechococcus PCC 7942 cyanobacterial iron-dependent bioreporters

It has been well documented that the low availability of iron constrains phytoplankton growth and productivity in many marine and lacustrine environments. Ferric ion (Fe3+) is conventionally recognized as the relevant form for direct uptake by phytoplankton, however in the presence of oxygen, iron forms insoluble inorganic complexes or alternatively is solubilized by organic ligands. There are several techniques that can determine the amount and speciation of iron in any given water sample, however a key question that remains unanswered pertains to the amount of iron that is actually bioavailable to phytoplankton. This research focused on constructing iron-dependent bioreporters capable of endogenously detecting and quantifying those fractions of iron accessible to phytoplankton in natural aquatic environments. The fresh water cyanobacterium, Synechococcus PCC 7942 was the organism chosen for this project. Promoters of three iron responsive Synechococcus genes, isiAB, irpA and dpsA were fused to the Vibrio harveyi luxAB genes and integrated into the Synechococcus chromosome at a genetically inert site. luxAB-dependent luminescence, monitored through the growth curve under iron replete and iron deplete conditions, indicated transcriptional activity of the promoters. Due to only the transient and overall low level expression of dpsA during transition into iron deplete medium, this promoter was considered an inappropriate choice to use as a bioreporter. Transcription from the isiAB promoter was induced in mid exponential phase of growth followed by rapid repression as cells progressed into late log phase. isiAB activation corresponded to intracellular iron stores of ∼12 attomoles Fe per cell. Constitutive isiAB-dependent transcription was observed in cells grown in Fe-depleted media. Initial field trials conducted with the isiAB Fe-dependent bioreporter, using water sampled from Lake Erie, demonstrated that the I bioreporter could serve to detect Fe deficiency in natural freshwater environments. Further characterization to ascertain the quantitative nature of isiAB luminescence response is currently being done. Initial studies completed on the irpA promoter demonstrated transcriptional activation during mid to late log phase however, unlike the isiAB Fe-dependent bioreporter, luminescence is sustained well into stationary phase. In iron deplete medium, constitutive irpA-dependent transcription was observed. Further characterization of this promoter will be required to determine not only its independent value as an iron-dependent bioreporter, but also the potential of using it in conjunction with the isiAB bioreporter to more accurately measure iron availability to phytoplankton in natural aquatic environments.