Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum

Abstract

Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase-dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization of glnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P(II)) were constructed. While P(II)-Y51F showed a lower nitrogenase activity than that of wild type, a P(II) deletion mutant showed very little nif expression. This effect of P(II) on nif expression is apparently the result of a requirement of P(II) for NifA activation, whose activity is regulated by NH(4)(+) in R. rubrum. The modification of glutamine synthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P(II) might exist in R. rubrum and regulate the modification of GS. P(II) also appears to be involved in the regulation of DRAT activity, since an altered response to NH(4)(+) was found in a mutant expressing P(II)-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH(4)(+) and darkness treatments.

FIG. 1

Regulation of nitrogenase activity by NH₄⁺ (A) and darkness (B) in R. rubrum UR2 (wild type) (○), UR659 (P_II-Y51F) (●), and UR687 (GS-Y398F) (■). (A) NH₄Cl was added at t = 0 to derepressed cultures UR2, UR659, and UR687 to a final concentration of 10 mM. (B) Derepressed cultures of UR2, UR659, and UR687 were shifted to darkness at t = 0, and cells were returned to light at 60 min. At the times indicated, 1-ml portions of cells were withdrawn and assayed for nitrogenase activity anaerobically under illumination conditions for 2 min. Initial nitrogenase activities (100%) in UR2, UR659, and UR687 were about 1,200, 200, and 1,000 nmol of ethylene produced per h per ml of cells, respectively, at an optical density of 1.0 at 600 nm. Each point represents an average of at least three replicate assays, with about 10% deviation.

FIG. 2

Physical map of nifA region from R. rubrum. Plasmids used for cloning and expression of nifA were indicated.

FIG. 3

Alignment of amino acid sequences of NifA from R. rubrum (Rr), A. brasilense (Ab), R. capsulatus (Rc), Rhizobium meliloti (Rm), A. vinelandii (Av), and K. pneumoniae (Kp). Four cysteine residues (★) located in interdomain linker might involve in sensing O₂.

FIG. 4

Western immunoblot of GS in UR2 (wild type), UR659(P_II-Y51F), UR687 (GS-Y398F), and UR717 (no P_II). Samples of GS were from cultures grown in the presence of a high concentration of NH₄⁺ (MN) or in the absence of NH₄⁺ (MG). MG-grown cells were also treated with 10 mM NH₄Cl (MN+N) or shifted from light to dark (MN+D) for 60 min. Similar amounts of total protein were loaded on SDS-PAGE gels and immunoblotted with antibody against R. rubrum GS. Arrow M indicates the position of the modified subunit, and arrow U indicates the position of the unmodified subunit.