Construction and characterization of a cyanobacterial bioreporter capable of assessing nitrate assimilatory capacity in freshwaters

Abstract

The use of cyanobacterial whole-cell luminescent bioreporters has enhanced our ability to monitor nutrient availability in aquatic ecosystems. We have constructed a Synechocystis sp. strain PCC6803 bioluminescent reporter for the assessment of nitrate bioavailability. Specifically, a 380—base pair DNA fragment containing the NtcA/B-dependent nitrate/nitrite-activated nirA promoter (regulating expression of genes encoding nitrite reductase) was fused to the bacterial luciferase genes, luxAB, and introduced into Synechocystis by genetic transformation. Characterization of this strain, designated AND100, yielded dose-dependent increased bioluminescence coincident with increased nitrate added to the growth medium from 1 to 100 µM. Bioluminescence in response to nitrate addition was light dependent up to 50 µmol quanta m−2 s−1. Assessing environmental samples collected from oligotrophic Lake Superior, we demonstrated that the onset of luminescence coincided with the drawdown of nitrate by simultaneously monitoring nitrate depletion from reaction vessels. Nitrate in the Lake Superior samples was consistently underestimated by the bioreporter. Only by following amendment of these samples with phosphate and iron was total nitrate accurately reflected by the cyanobacterial bioreporter. Thus, strain AND100 can be used to elucidate factors that constrain use of nitrate in freshwaters. This is pertinent to a system such as Lake Superior where the concentration of nitrate has increased 6-fold in the last century. Indeed, pilot experiments with the bioreporter suggest that nutrient co-limitation (P and Fe), as well as low light, may reduce the capacity for nitrate assimilation in field samples from Lake Superior.