FIG 1.

Representation of the NifB library and in vivo complementation of A. vinelandii strain UW140 (ΔnifB) with TS-NifB variants. (a) Plot of the organisms from where the NifB protein variants were selected. Four features are indicated: (i) growth temperature (x axis); (ii) nitrogen fixing capacity (y axis), where confirmed diazotrophs are above the x axis, organisms with no published data regarding N-fixing capacity are in line with the x axis, and nondiazotrophs are below the x axis; (iii) phylum, represented by color (see color scheme at the bottom of the panel); and (iv) presence (circle) or absence (square) of the NifX-like domain at each NifB protein. The size of each symbol corresponds to the number of representatives sharing same features. Black stars indicate NifB variants shown soluble in tobacco organelles. (b) Growth comparison of A. vinelandii UW140 (ΔnifB) strain complemented with some ts-nifB on plates with ammonium (+NH₄) or N₂ (-NH₄) as sole nitrogen source. A. vinelandii DJ is the wild-type strain. The stars indicate complemented strains with phenotype of slow diazotrophic growth on solid medium. (c) Acetylene reduction activity measured at 18 h (blue bars) or 42 h (grey bars) after nitrogen source removal from the media. Red crosses represent individual biological replicates. ARA activities are presented as nanomoles of ethylene produced per minute and the OD₆₀₀. UW482 is the wild-type strain DJ transformed with the parental empty vector pN2SB51. Cyanothece sp. strain PCC 7425 (nifB) restored the Nif⁺ phenotype, but no acetylene reduction activity could be measured at 18 h or 42 h (bdl, below detection limit). The activity of the A. vinelandii DJ wild type was 21.1 ± 0.2 U. Error bars indicate means ± the standard deviations (SD; n ≥ 2 biological replicates).