Effect of point mutations on Herbaspirillum seropedicae NifA activity

Abstract

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

Figure 1. Scheme of Herbaspirillum seropedicae NifA domains. Numbers indicate the amino acid position on the primary structure. The N-terminal GAF domain, central domain, and C-terminal domain are indicated as open rectangles. The ATP-binding motifs, located in the central domain, and the helix-turn-helix (HTH) motif in the C-terminal domain are indicated as gray rectangles, and were predicted using the ScanProsite tool (http://prosite.expasy.org/scanprosite) (29) and Gym2.0 (30), respectively. Point-mutations (K22V, G25E, T160E, M161V, L172R, A215D, Q216I, and S220I) are indicated by gray triangles in the N-terminal GAF and central domains of NifA.

Figure 2. Transcriptional activity of NifA variant proteins in Escherichia coli JM109 (DE3) carrying pRT22 (nifH::lacZ). (-) indicates cells carrying pET-29a. Full-length NifA was expressed from pRAM1. ΔN-NifA indicates an N-truncated form of NifA expressed from pRAM2. Full-length NifA mutants, as indicated, were expressed from pET-29a-based plasmids. β-galactosidase expression experiments were performed in NFDM medium (31) with 20 mM of ammonium chloride (+N) or 0.2% casamino acids (-N), in the absence of O₂. Data are reported as the mean±SD of 3 independent assays. β-galactosidase activity is reported as Miller units.

Figure 3. Transcriptional activity of ΔN-NifA mutant proteins in Escherichia coli JM109 (DE3) carrying pRT22 (nifH::lacZ). ΔN-NifA indicates NifA lacking 203 amino acid residues at the N-terminal GAF domain. ΔN-215D, ΔN-Q216I, and ΔN-S220I indicate the N-truncated forms of NifA mutants. β-galactosidase expression experiments were performed in NFDM medium supplemented with 20 mM ammonium chloride (+N) or 0.2% casamino acids (-N) in the presence (+O) or absence (-O) of O₂. Data are reported as the mean±SD of 3 independent assays. β-galactosidase activity is reported as Miller units.

Figure 4. Transcriptional activity of indicated Herbaspirillum seropedicae strains carrying a chromosomal nifH:lacZ fusion. +G25E indicates cells carrying a pLAFR3.18-based plasmid expressing the G25E NifA mutant; (-) indicates absence of plasmid. Cells were grown in NFbHP medium supplemented with 10 mM NH₄Cl under aerobic conditions at 30°C. Cells were then centrifuged (1700 g for 2 min), resuspended in NFbHP (nitrogen-free) medium, and de-repressed for 7 h under 1.5% oxygen. β-galactosidase was determined as described. Data are reported as the mean±SD of at least 3 independent experiments. β-galactosidase activity is reported as Miller units.