Differential Effects of Varying Concentrations of Phosphorus, Iron, and Nitrogen in N₂-Fixing Cyanobacteria

Diazotrophs or N₂-fixers are one of the most ecologically significant groups in marine ecosystems (pelagic and benthic). Inorganic phosphorus (PO₄ ^3-) and iron (Fe) can limit the growth and N₂-fixing capacities of cyanobacteria. However, studies investigating co-limitation of these factors are lacking. Here, we added different concentrations of PO₄ ^3- and Fe in two cyanobacterial species whose relatives can be found in seagrass habitats: the unicellular Halothece sp. (PCC 7418) and the filamentous Fischerella muscicola (PCC 73103), grown under different nitrate (NO₃ ^-) concentrations and under N₂ as sole N source, respectively. Their growth, pigment content, N₂-fixation rates, oxidative stress responses, and morphological and cellular changes were investigated. Our results show a serial limitation of NO₃ ^- and PO₄ ^3- (with NO₃ ^- as the primary limiting nutrient) for Halothece sp. Simultaneous co-limitation of PO₄ ^3- and Fe was found for both species tested, and high levels of Fe (especially when added with high PO₄ ^3- levels) inhibited the growth of Halothece sp. Nutrient limitation (PO₄ ^3-, Fe, and/or NO₃ ^-) enhanced oxidative stress responses, morphological changes, and apoptosis. Furthermore, an extensive bio-informatic analysis describing the predicted Pho, Fur, and NtcA regulons (involved in the survival of cells to P, Fe, and N limitation) was made using the complete genome of Halothece sp. as a model, showing the potential of this strain to adapt to different nutrient regimes (P, Fe, or N).