Fig 1.
GlnR controls the nitrogenase activity and nif transcription in P. polymyxa WLY78.
A. Nitrogenase activities of WT (the wild-type), ΔglnR (deletion mutant), ΔglnR/glnR (complementation strain) and WT/glnR (overexpression strain). These strains were grown anaerobically in nitrogen-deficient medium containing 2 mM glutamate supplemented with different concentration of NH4Cl at 0, 1, 5, 10, 50 and 100 mM. The nitrogenase activities of these strains were assayed by C2H4 reduction method and expressed at nmol C2H4/mg protein/hr. B. qRT-PCR analysis of the relative mRNA levels of the nifHDK genes in the WT and ΔglnR strains grown in nitrogen-limited and -excess media. N-: nitrogen-limited condition (2 mM glutamate as the only nitrogen source). N+: nitrogen-excess condition (2 mM glutamate + 100 mM NH4+). The relative expression level was calculated using ΔΔCt method.The transcription levels of genes in the WT strain under nitrogen-excess condition were arbitrarily set to 1.0. C. β-galactosidase activity of a Pnif-lacZ fusion in the WT and ΔglnR strains grown in nitrogen-limited (N-) and -excess (N+) conditions. D. qRT-PCR analysis of the relative transcription levels of glnR gene under nitrogen-limited (N-) and -excess (N+) conditions. The transcription levels of glnR gene under nitrogen-excess condition were arbitrarily set to 1.0. E. qRT-PCR analysis of the transcription profiles of glnR and nifH under nitrogen limitation. The transcription levels of genes at time 0 hr were arbitrarily set to 1.0. Results are representative of at least three independent experiments. Error bars indicate SD. **P < 0.01; *P < 0.05.
Fig 2.
The functions of GlnR and GS proteins in repressing nitrogenase activity and nif expression.
A. Nitrogenase activities of WT, deletion mutants ΔglnA1, ΔglnA and ΔglnRA and complementary strains ΔglnA/glnA and ΔglnRA/ glnRA under nitrogen-limited and -excess conditions. B. qRT-PCR analysis of the relative transcription levels of the nifH gene in different strains under both nitrogen-limited and–excess conditions. The transcription levels of nifH in the WT strain under nitrogen-excess condition were arbitrarily set to 1.0. Results are representative of at least three independent experiments. Error bars indicate SD. **P < 0.01; *P < 0.05.
Fig 3.
SPR analysis of interaction of GlnR with GS proteins.
A. Interaction between GS and GlnR. B. Interaction between FBI-GS (GS and 1 mM glutamine) and GlnR. C. Interactions between GS1 and GlnR (red dashed line), FBI-GS1 (GS1 and 1 mM glutamine) and GlnR (red continuous line), and interaction between FBI-GS and GlnRΔ25 (blue continuous line). 100 nM GS1 alone or with 1 mM glutamine was injected onto the chip surface-immobilized GlnR. For the interaction between FBI-GS and GlnRΔ25, 100 nM GS with 1 mM glutamine was injected onto the chip surface-immobilized GlnRΔ25.
Fig 4.
The in vitro and in vivo binding of GlnR protein to two GlnR-binding sites in the nif promoter region of P. polymyxa WLY78.
A. Prediction of two GlnR-binding sites in the nif promoter region. B. EMSA revealed the in vitro binding of GlnR to the two GlnR-binding sites. Two DNA fragments: a 59 bp DNA fragment harboring the GlnR-binding site Ⅰ and a 53 bp DNA fragment carrying GlnR-binding site Ⅱ, were synthesized and biotin-labeled. The biotin-labeled DNA fragments were incubated with His-GlnR supplemented without or with FBI-GS (5 mM glutamine and 500 nM His-GS). Lane 1 contained no GlnR. Lanes 2–10 contained increasing concentrations of His-tagged GlnR (4, 8, 16, 32, 64, 128, 256, 512, 1024 nM). C. ChIP-qPCR assays revealed in vivo biding of GlnR to both GlnR-binding sites under both nitrogen-limited (N-) and -excess (N+) conditions. The binding levels of control (ΔglnR) were arbitrarily set to 1.0. Error bars indicate SD from three independent experiments. **P < 0.01; *P < 0.05. D. SPR analysis of GlnR binding to both GlnR-binding sites. 500 nM GlnR alone or with FBI-GS (GS + 1 mM glutamine) was injected onto the chip surface-immobilized DNA fragments harboring GlnR-binding site Ⅰ or site Ⅱ. GlnR (Site Ⅰ) indicates the binding of GlnR alone to site Ⅰ; GlnR (Site Ⅱ) indicates the binding of GlnR alone to site Ⅱ; GlnR+FBI-GS (Site Ⅰ) indicates the binding of GlnR plus FBI-GS to site Ⅰ; GlnR+FBI-GS (Site Ⅱ) indicates the binding of GlnR plus FBI-GS to site Ⅱ. E. SPR analysis of the binding affinity of the truncated GlnR (GlnRΔ25) protein to the two GlnR-binding sites. 500 nM GlnR or GlnRΔ25 alone or with FBI-GS (GS + 1 mM glutamine) was injected onto the chip surface-immobilized DNA fragments harboring GlnR-binding site Ⅰ or site Ⅱ. In comparison with wild-type GlnR, GlnRΔ25 protein has the increased binding affinity to both sites, especially site Ⅱ. The addition of FBI-GS does not obviously increase the binding affinity of GlnR.
Fig 5.
Binding affinity of GlnR to GlnR-binding site Ⅰ and GlnR-binding site Ⅱ.
A. SPR titration analysis of GlnR binding to GlnR-binding site Ⅰ. B. SPR titration analysis of GlnR binding to GlnR-binding site Ⅱ. C. Binding affinity of GlnR to GlnR-binding site Ⅰ and GlnR-binding site Ⅱ.
Fig 6.
Mutation analysis of the GlnR-binding sites in P. polymyxa WLY78.
A. A schematic site-specific mutation or deletion of the GlnR-binding sites. B. EMSA verification of the binding affinity of GlnR to the mutated GlnR-biding site Ⅰ and GlnR-binding site Ⅱ. Site Ⅰ indicates wild-type GlnR-binding site Ⅰ; Site Ⅱ indicates wild-type GlnR-binding site Ⅱ.–and + indicate that the absence and presence of GlnR protein, respectively. C. Nitrogenase activity in wild-type (WT) and four mutants MPnif1, MPnif2, MPnif3 and MPnif97 under both nitrogen-limited and -excess conditions. D. qPCR analysis of the relative transcription levels of the nifHDK genes in the wild-type and mutant strains under both nitrogen-limited and -excess conditions. The transcription levels of nifH in the WT strain under nitrogen-excess condition were arbitrarily set to 1.0. Results are representative of at least three independent experiments. Error bars indicate SD. **P < 0.01; *P < 0.05.
Fig 7.
Regulatory model of GlnR involved in nitrogen fixation in P. polymyxa WLY78.
A. GlnR protein exists as a mixture of dimer and monomer. Monomer of GlnR is an autoinhibitory form whose C-terminal region folds back and inhibits dimer formation. During nitrogen limitation, dimer of GlnR binds to GlnR-binding site Ⅰ in a weak and transient association way and activates nif transcription. B. During excess nitrogen, glutamine (Gln) is produced by GS and GS1 catalyzing glutamate and NH4+. Gln binds to and feedback inhibits GS by forming the complex FBI-GS. FBI-GS interacts with the C-terminal tail of GlnR and relieves its autoinhibition, shifting the monomer to the DNA-binding active form. The FBI-GS further stabilizes the binding affinity of GlnR to GlnR-binding site Ⅱ and thus represses nif transcription.