PII protein is essential for transcriptional regulation of anf gene cluster for iron-only nitrogenase in Rhodopseudomonas palustris

Abstract

In addition to catalyzing the biological nitrogen fixation, iron-only (Fe-only) nitrogenase is also able to reduce carbon dioxide (CO₂) to formate (HCOO^-) and methane (CH₄). AnfA is responsible for the transcriptional activation of the anf gene cluster for Fe-only nitrogenase, whose expression is repressed by fixed nitrogen. However, it remains unclear how AnfA is regulated to control the expression of Fe-only nitrogenase. Here, we found that in Rhodopseudomonas palustris, P_II proteins play a critical role in regulating the expression of Fe-only nitrogenase genes via AnfA. We hypothesize that the deuridylylated P_II protein GlnK1, which was upregulated in the presence of ammonium (NH₄⁺), could inhibit AnfA activity by forming a potential AnfA-GlnK1 complex. This likely serves as a fail-safe mechanism to prevent R. palustris from expressing Fe-only nitrogenase when AnfA is accidentally expressed under nitrogen-excess conditions. The uridylylated P_II protein GlnK2^UMP, which was upregulated in response to nitrogen starvation, stimulated the expression of an active AnfA hexamer that further activated the expression of Fe-only nitrogenase under nitrogen-fixing and Mo-free conditions. This study provides new insights into the regulation of Fe-only nitrogenase in R. palustris.IMPORTANCEThe expression and maturation of nitrogenase are tightly regulated by ambient nitrogen levels, which limits the persistence and efficiency of biological nitrogen fixation. This study offers new insights into the regulatory mechanism of AnfA by P_II proteins in Rhodopseudomonas palustris. Understanding the regulation of AnfA, the transcriptional activator of the Fe-only nitrogenase gene cluster, could provide strategies to better control the expression of iron-only nitrogenase. Nitrogen-fixing bacteria that constitutively express iron-only nitrogenase have the potential to be developed into promising biofertilizers, as their nitrogen-fixing activity is enhanced and independent of molybdenum availability in the soil.

Keywords: Fe-only nitrogenase; PII proteins; Rhodopseudomonas; gene regulation; photosynthetic bacteria.

Fig 1

Transcriptomic and proteomic analyses indicate P_II proteins may regulate the expression of Fe-only nitrogenase. (A) The operons of all the P_II genes and the anf gene cluster in R. palustris CGA009. (B) For transcriptome and proteome sequencing, R. palustris CGA009 was grown with nitrogen gas (N₂) as the sole nitrogen source for diazotrophic growth. In contrast, CGA009 grown with NH₄⁺ for non-diazotrophic growth was used as the control. Mo was removed from both nitrogen-excess (NH₄⁺) and nitrogen-fixing (N₂) media. In addition to anfHDGK and anfA genes coding for Fe-only nitrogenase and its transcriptional activator, the P_II genes (glnK1, glnK2, and glnB) and their protein products were also differentially expressed, suggesting that P_II proteins may play pivotal roles in Fe-only nitrogenase regulation. The volcano plots were generated by the ggplot2 R package. The thresholds of |log₂FC| ≥ 1 (FC, fold change) and |log₂FC| ≥ 0.585 were used to define differentially expressed genes and proteins, respectively.

Fig 2

P_II proteins are involved in the transcriptional and posttranslational regulation of AnfA. (A) An RFP reporter system in plasmid with the mCherry gene expressed from the promoter (P_anfH) of Fe-only nitrogenase genes (anfHDGK) was created to monitor the Fe-only nitrogenase expression by fluorescence intensity. When necessary, AnfA was overexpressed from a strong constitutive promoter P_J23119. (B) The effect of P_II proteins on the expression and activity of AnfA. R. palustris CGA009-7 (WT), CGA3033-13 (ΔP_II), CGA009-8 (WT-AnfA), and CGA3033-14 (ΔP_II-AnfA) strains expressing mCherry from P_anfH promoter were grown with NH₄⁺ (red bars) or N₂ gas (blue bars) under Mo-free conditions. The fluorescence intensity of mCherry was indicated by the relative fluorescence unit (RFU) normalized to optical density of R. palustris at 660 nm (OD₆₆₀). R. palustris strains: CGA009-7, GGA009 expressing mCherry from P_anfH; CGA009-8, GGA009 expressing mCherry from P_anfH and anfA from P_J23119; CGA3033-13, a glnK1 glnK2 glnB triple deletion mutant (ΔP_II) expressing mCherry from P_anfH; CGA3033-14, ΔP_II expressing mCherry from P_anfH and anfA from P_J23119. (C) H₂/CH₄ production and C₂H₂ reduction were determined by gas chromatography to examine the activity of Fe-only nitrogenase. These data are the average of three independent experiments, and the error bars represent the SD.

Fig 3

Uridylylated GlnK2 stimulates the expression of anfA, and deuridylylated GlnK1 inhibits the activity of AnfA. (A) An RFP reporter system was created to examine the influence of P_II proteins or their variants P_II^Y51F on the expression of the anfA gene and the activity of AnfA protein, respectively. When necessary, AnfA, P_II, or P_II^Y51F was overexpressed from a strong constitutive promoter (P_J23119 or P_GAPDH). (B and C) The effect of P_II proteins on the activity of AnfA protein (B) and the expression of anfA gene (C). GlnK1, GlnK2, and GlnB were overexpressed in ΔP_II mutant grown to the logarithmic phase under nitrogen-fixing and Mo-free conditions. The ΔP_II mutant complemented with plasmid carrying the glnK2 gene (CGA3033-16) greatly enhanced the expression of anfHDGK and anfA genes, which were determined by fluorescence intensity of RFP reporter and qRT-PCR, respectively. (D) Deuridylylated P_II proteins inhibit AnfA activity in vivo. The deuridylylated GlnK1, GlnK2, GlnB, GlnK1^Y51F, GlnK2^Y51F, and GlnB^Y51F were expressed in R. palustris strains grown with NH₄⁺ or N₂ under Mo-free conditions. As the uridylylation site of GlnK1, GlnK2, and GlnB, the amino acid substitution Y51F makes GlnK1, GlnK2, and GlnB unable to be uridylylated even under nitrogen-fixing conditions. NG, no growth. These data are the average of three independent experiments, and the error bars represent the SD.

Fig 4

Interactions between AnfA and GlnK1 predicted by AlphaFold 3. (A and B) The predicted structures of dimeric and hexameric AnfA by AlphaFold 3. The top-ranked models for AnfA dimer (ipTM = 0.31, pTM = 0.42) and AnfA hexamer (ipTM = 0.40, pTM = 0.43) were used in the study. The GAF, Q-linker, AAA+, and HTH domains are colored in slate/marine, cyan, salmon/deep salmon, and pink/light pink, respectively. ipTM, interface predicted template modeling; pTM, predicted template modeling. (C and D) The docking model of AnfA-GlnK1 complex predicted by AlphaFold3 and Gluspro, respectively. The deuridylylated GlnK1 (yellow) shows potential interactions with the GAF domain of AnfA.

Fig 5

Deuridylylated GlnK1 interacts with the GAF domain of AnfA. (A) Schematic diagrams of the truncated AnfA lacking the GAF-Q domain. (B) Identification of the AnfA domain responsible for potential interactions with P_II proteins. The AnfA and its truncated variant lacking GAF/Q-linker domain (AnfA-ΔGAFQ) were expressed using the pBBR1MCS6-P_anfH-RFP plasmid in R. palustris CGA009 (WT, red bar) and CGA3033 (ΔP_II, blue bar), respectively. R. palustris CGA009 and CGA3033 harboring pBBR1MCS6-P_nifH-RFP were used as negative controls. AnfA showed activity when its GAF-Q domain was removed. These data are the average of three independent experiments, and the error bars represent the SD.

Fig 6

A model for the genetic regulation of Fe-only nitrogenase in response to nitrogen availability in R. palustris. Under nitrogen-replete (NH₄⁺) conditions, no AnfA expression is observed. Additionally, AnfA activity can be inhibited by deuridylylated GlnK1, likely serving as a fail-safe mechanism. When R. palustris is exposed to nitrogen-limited (N₂) and Mo-free conditions, uridylylated GlnK2 stimulates the expression of active AnfA, which further activates the expression of Fe-only nitrogenase.