The Novel PII-Interacting Protein PirA Controls Flux into the Cyanobacterial Ornithine-Ammonia Cycle

Abstract

Among prokaryotes, cyanobacteria have an exclusive position as they perform oxygenic photosynthesis. Cyanobacteria substantially differ from other bacteria in further aspects, e.g., they evolved a plethora of unique regulatory mechanisms to control primary metabolism. This is exemplified by the regulation of glutamine synthetase (GS) via small proteins termed inactivating factors (IFs). Here, we reveal another small protein, encoded by the ssr0692 gene in the model strain Synechocystis sp. PCC 6803, that regulates flux into the ornithine-ammonia cycle (OAC), the key hub of cyanobacterial nitrogen stockpiling and remobilization. This regulation is achieved by the interaction with the central carbon/nitrogen control protein P_II, which commonly controls entry into the OAC by activating the key enzyme of arginine synthesis, N-acetyl-l-glutamate kinase (NAGK). In particular, the Ssr0692 protein competes with NAGK for P_II binding and thereby prevents NAGK activation, which in turn lowers arginine synthesis. Accordingly, we termed it P_II-interacting regulator of arginine synthesis (PirA). Similar to the GS IFs, PirA accumulates in response to ammonium upshift due to relief from repression by the global nitrogen control transcription factor NtcA. Consistent with this, the deletion of pirA affects the balance of metabolite pools of the OAC in response to ammonium shocks. Moreover, the PirA-P_II interaction requires ADP and is prevented by P_II mutations affecting the T-loop conformation, the major protein interaction surface of this signal processing protein. Thus, we propose that PirA is an integrator determining flux into N storage compounds not only depending on the N availability but also the energy state of the cell.IMPORTANCE Cyanobacteria contribute a significant portion to the annual oxygen yield and play important roles in biogeochemical cycles, e.g., as major primary producers. Due to their photosynthetic lifestyle, cyanobacteria also arouse interest as hosts for the sustainable production of fuel components and high-value chemicals. However, their broad application as microbial cell factories is hampered by limited knowledge about the regulation of metabolic fluxes in these organisms. Our research identified a novel regulatory protein that controls nitrogen flux, in particular arginine synthesis. Besides its role as a proteinogenic amino acid, arginine is a precursor for the cyanobacterial storage compound cyanophycin, which is of potential interest to biotechnology. Therefore, the obtained results will not only enhance our understanding of flux control in these organisms but also help to provide a scientific basis for targeted metabolic engineering and, hence, the design of photosynthesis-driven biotechnological applications.

Keywords: PII protein; cyanobacteria; nitrogen metabolism; small inhibitory proteins.

FIG 1

N-regulated gene pirA and its occurrence among cyanobacteria. (A) Amino acid alignment of randomly selected cyanobacterial PirA homologs. The alignment was made using ClustalW and visualized by using Jalview. (B) Phylogenetic tree of selected cyanobacteria based on 16S rRNA gene sequences. The tree was generated with the MEGA7 (83) software package and the neighbor-joining method. Note that we reused a calculated tree from our previous publication (38) and assigned the presence of genes in the corresponding genomes manually. Gene presence (illustrated by filled rectangles) was investigated using the BLASTP algorithm (84). As a reference, the amino acid sequences of PirA, IF7 (GifA, Ssl1911), and IF17 (GifB, Sll1515) from Synechocystis were used. (C) Overview of the promoter region upstream of the pirA gene in Synechocystis. Two putative NtcA binding sites are highlighted. The transcriptional start site (TSS; +1) and the location of the −10 element were extracted from differential transcriptome sequencing data (85). (D) Changes of mRNA levels for several Synechocystis genes in response to N limitation. Data were extracted and plotted from previously published microarray data (86). (E) Northern blot showing transcript accumulation of pirA in nitrate-grown Synechocystis cells upon addition of 10 mM ammonium chloride. 16S rRNA was used as a loading control. (F) Western blot showing changes in PirA protein levels in response to ammonium upshifts. For this, a specific, customized antibody against PirA was raised in rabbit. An antibody against thioredoxin (TrxA) was used to verify equal loading.

FIG 2

Properties and expression profiles in ΔpirA and pirA⁺ recombinant strains. (A) Schematic view of the pirA locus in the WT and in the ΔpirA knockout strain as well as of a pVZ322 plasmid derivative harboring a pirA gene copy under the control of the Cu²⁺-inducible promoter PpetE that is present in the pirA⁺ overexpression strain. In the ΔpirA knockout strain, pirA was replaced by a kanamycin resistance cassette (Km^r) via homologous recombination. The plasmid enabling ectopic pirA expression was introduced into Synechocystis WT. The arrows labeled with asterisks indicate the binding sites for primers used to verify the mutants. (B) PCR verification of the genotype of independently obtained mutant strains. In each case three clones were tested using primer combinations Ssr0692_KO-seg_fw/Ssr0692_KO-seg_rev (in case of ΔpirA strain) or PpetE_fw(XhoI) and Toop_rev(AseI) (in case of pirA⁺ strain). M, marker; bp, base pairs; cl., clone; −, negative control (water as the template); +, positive control (purified plasmid as the template). (C) Relative abundance of the pirA mRNA, measured via Northern blotting using sequence-specific ³²P-labeled ssRNA probes. In all cases, RNA was isolated from cells grown in the presence of 1 μM CuSO₄. (D) Western blot showing PirA protein levels in cells of the WT and pirA⁺ strains, treated with 1 μM Cu²⁺ for 3 h and afterwards with 10 mM ammonium. Thioredoxin (TrxA) levels verify equal loading. (E) PirA levels relative to WT. Data were obtained by densitometric evaluation of respective bands using the ImageJ software (87). Data are mean ± standard deviation (SD) values obtained from two independent Western blots, i.e., two biological replicates (independent clones). (F) PirA accumulation in a ΔpirA strain that was complemented with a pirA gene fused to the petE promoter. Note that the data shown here were obtained using a mutant in which the PpetE-pirA construct was integrated into the chromosome, i.e., this strain does not harbor the plasmid derivative shown in panel A.

FIG 3

Growth and pigmentation of the WT and the ΔpirA and pirA⁺ mutant strains when N is oscillating. (A and B) Growth under standard conditions and when ammonium is consecutively added to N-starved cultures. Arrows indicate time points at which 1 mM NH₄Cl was added. Data are the means ± SD from three independent cultures (including three independent clones of each mutant). (C) Representative photographs of cultures used in the experiment. Ammonium was added after day 3 and again after days 5 and 6. (D) Whole-cell absorption spectra. Values were normalized to A₇₅₀ values.

FIG 4

Kinetics of metabolites linked to the OAC cycle in response to ammonium addition. (A) Simplified overview of metabolic pathways associated with ammonium assimilation and a possible regulatory impact of PirA on certain enzymatic reactions. 2-OG, 2-oxoglutarate; CP, carbamoyl phosphate; GS, glutamine synthetase; GOGAT, glutamine oxoglutarate aminotransferase; NAG, N-acetyl-glutamate; NAGK, N-acetyl glutamate kinase; OAC, ornithine-ammonia cycle. (B to G) Kinetics of selected metabolites after adding 10 mM ammonium to nitrate-grown cells in the exponential phase. Metabolites were determined by ultrahigh-performance liquid chromatography-tandem mass spectrometry after ethanol extraction from cells of the WT, ΔpirA, and pirA⁺ strains. Data are the means ± SD from two independent experiments, each conducted with three biological replicates (independent clones). Significant differences in the mutant strains compared to WT at each time point are labeled and were revealed by one-way analysis of variance (ANOVA; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 5

Determination of complex formation between PirA and the P_II protein, measured by biolayer interferometry (BLI). (A) Schematic view of the measuring principle. (B) Representation of the maximum binding response of P_II(WT)-His and GST-PirA interaction in the presence of different concentrations of ADP, ATP, or ATP/2-OG. (C) The maximum binding response at different protein concentrations of GST-PirA, tag-free PirA, or free GST in the presence of 2 mM ADP. As the binding response is a function of the mass of bound interactor, the response with GST-tagged PirA is correspondingly higher than that with isolated PirA peptide. (D) Representation of the maximum binding response at increasing concentrations of GST-PirA in the absence of effector molecules or in the presence of 2 mM ADP with three different T-loop variants of P_II. Data are the means ± SD from triplicate measurements.

FIG 6

Impact of PirA on P_II-dependent NAGK activity in vitro. (A) Inhibition of NAGK by arginine in the presence or absence of 2.4 μg P_II. (B) NAGK activity as a function of increasing PirA concentration in the presence or absence of P_II and 1 mM ADP. The assay otherwise contained 0.1 mM arginine and 1 mM ATP. Data are the means ± SD from triplicate measurements.

FIG 7

Anticipated model of PirA function. Metabolite kinetics have been approximated based on available literature data (34, 38). Upon shifts in the ammonium concentration, PirA accumulates via 2-OG-dependent derepression of the pirA gene. The gene product is presumably required to slow down ATP-consuming synthesis of arginine. This could be achieved by ADP-dependent sequestration of P_II protein bound to NAGK, which is required to diminish feedback inhibition of the enzyme and, in turn, activate arginine synthesis. The sequestration of P_II results in stronger arginine feedback inhibition of NAGK, diminishing energy consumption and flux into arginine. After metabolic reorganization (e.g., by inactivating glutamine synthetase activity and decreasing ATP consumption), ADP levels may fall below a critical level, preventing interaction between PirA and P_II. Accordingly, a higher fraction of the P_II pool will again interact with and activate NAGK, which in turn results in elevated arginine synthesis.