Heterocyst-specific flavodiiron protein Flv3B enables oxic diazotrophic growth of the filamentous cyanobacterium Anabaena sp. PCC 7120

Abstract

Flavodiiron proteins are known to have crucial and specific roles in photoprotection of photosystems I and II in cyanobacteria. The filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 contains, besides the four flavodiiron proteins Flv1A, Flv2, Flv3A, and Flv4 present in vegetative cells, two heterocyst-specific flavodiiron proteins, Flv1B and Flv3B. Here, we demonstrate that Flv3B is responsible for light-induced O2 uptake in heterocysts, and that the absence of the Flv3B protein severely compromises the growth of filaments in oxic, but not in microoxic, conditions. It is further demonstrated that Flv3B-mediated photosynthetic O2 uptake has a distinct role in heterocysts which cannot be substituted by respiratory O2 uptake in the protection of nitrogenase from oxidative damage and, thus, in an efficient provision of nitrogen to filaments. In line with this conclusion, the Δflv3B strain has reduced amounts of nitrogenase NifHDK subunits and shows multiple symptoms of nitrogen deficiency in the filaments. The apparent imbalance of cytosolic redox state in Δflv3B heterocysts also has a pronounced influence on the amounts of different transcripts and proteins. Therefore, an O2-related mechanism for control of gene expression is suggested to take place in heterocysts.

Keywords: nitrogen fixation; oxygen protection; photosynthesis.

Fig. 1.

Growth phenotype of Anabaena WT and the flv mutants. (A) Filaments were grown with combined N (+N) for 3 d followed by adjustment of OD₇₅₀ to 0.1 and shifting to N₂-fixing conditions (-N). ■, WT; mutant strains: ○, Δflv1B; ●, Δflv3B; ∆, Δflv1B/3B. (B) Growth of the WT and Δflv3B in O₂-depleted atmosphere after combined N stepdown: ■, WT; ○, Δflv3B. (C) Filaments of the WT and mutants after 4 d in N₂-fixing conditions visualized with bright field microscopy. (D) Chl a autofluorescence of the WT and Δflv3B in N₂-fixing conditions. (A and B) Values are mean ± SD, n = 3; Insets demonstrate amount of the NifH protein in the growth conditions. (Scale bars: 10 μm.)

Fig. 2.

O₂ uptake by N₂-fixing filaments and heterocysts of the WT and mutant strains. (A) MIMS measurements of O₂ consumption during the dark-to-light transition by whole filaments of the WT (solid line) and mutants: ∆flv1B (dotted line), ∆flv3B (dashed line), ∆flv1B/3B (dash-dot line). (B) O₂ uptake by WT heterocysts with or without inhibitors. (C) Light-induced O₂ uptake by WT and ∆flv3B heterocysts; dark O₂ uptake rates were subtracted for better legibility. Arrows indicate the beginning of illumination. Chl a content of samples was adjusted to 15 μg⋅ml⁻¹.

Fig. 3.

RT-q-PCR analysis of gene expression in whole filaments of the WT and mutants incubated in the absence of combined nitrogen. The fold change of relative transcript abundance of the selected genes in mutant cells compared with the WT is shown: white bars, ∆flv1B; light gray bars, ∆flv3B; dark gray bars, ∆flv1B/3B. Mean ± SD, n = 3. Asterisks indicate statistically significant differences with the WT (*P < 0.05).