Substrate specificity and ecological significance of PstS homologs in phosphorus uptake in marine Synechococcus sp. WH8102

Abstract

Phosphorus, a vital macronutrient, often limits primary productivity in marine environments. Marine Synechococcus strains, including WH8102, rely on high-affinity phosphate-binding proteins (PstS) to scavenge inorganic phosphate in oligotrophic oceans. However, WH8102 possesses three distinct PstS homologs whose substrate specificity and ecological roles are unclear. The three PstS homologs were heterologously expressed and purified to investigate their substrate specificity and binding kinetics. Our study revealed that all three PstS homologs exhibited a high degree of specificity for phosphate but differed in phosphate binding affinities. Notably, PstS1b displayed nearly 10-fold higher binding affinity (K_D = 0.44 µM) compared to PstS1a (K_{D =} 3.3 μM) and PstS2 (K_{D =} 4.3 μM). Structural modeling suggested a single amino acid variation in the binding pocket of PstS1b (threonine instead of serine in PstS1a and PstS2) likely contributed to its higher Pi affinity. Genome context data, together with the protein biophysical data, suggest distinct ecological roles for the three PstS homologs. We propose that PstS1b may be involved in scavenging inorganic phosphorus in oligotrophic conditions and that PstS1a may be involved in transporting recycled phosphate derived from organic phosphate cleavage. The role of PstS2 is less clear, but it may be involved in phosphate uptake when environmental phosphate concentrations are transiently higher. The conservation of three distinct PstS homologs in Synechococcus clade III strains likely reflects distinct adaptations for P acquisition under varying oligotrophic conditions.IMPORTANCEPhosphorus is an essential macronutrient that plays a key role in marine primary productivity and biogeochemistry. However, intense competition for bioavailable phosphorus in the marine environment limits growth and productivity of ecologically important cyanobacteria. In oligotrophic oceans, marine Synechococcus strains, like WH8102, utilize high-affinity phosphate-binding proteins (PstS) to scavenge inorganic phosphate. However, WH8102 possesses three distinct PstS homologs, with unclear substrate specificity and ecological roles, creating a knowledge gap in understanding phosphorus acquisition mechanisms in picocyanobacteria. Through genomic, functional, biophysical, and structural analysis, our study unravels the ecological functions of these homologs. Our findings enhance our understanding of cyanobacterial nutritional uptake strategies and shed light on the crucial role of these conserved nutrient uptake systems in adaptation to specific niches, which ultimately underpins the success of marine Synechococcus across a diverse array of marine ecosystems.

Keywords: Synechococcus; differential scanning fluorimetry (DSF); high-affinity; phosphate binding protein (PstS); phosphate stress.

Fig 1

Phylogenetic analyses of Synechococcus PstS sequences. Amino acid sequences of Synechococcus PstS1 and PstS2, belonging to cluster CK_00043821 and CK_00000023, were aligned using MAFFT (28). The phylogenetic tree was inferred using IQ-Tree (29) and visualized using iTOL (30). Cyanorak cluster designations (27) are used to denote the gene numbers. Protein clusters are labeled with Synechococcus clade designation along with PstS1 and PstS2 lineages. Synechococcus PstS1 with threonine residue in the binding pocket are highlighted in yellow (see Fig. 4 for further details).

Fig 2

Genomic context analysis of PstS homologs in WH8102 and other selected Synechococcus clade representatives. Visualization of conservation of (A) pstS1a, (B) pstS1b, and (C) pstS2 in representative Synechococcus strains. Synechococcus clade information for each representative strain is shown in a bracket. Genes are highlighted by color. The numbers above represent the gene locus tag in WH8102, for which respective gene product details are shown next to color legends. Each pstS homolog in WH8102 and the features noted in the text are shown in a box. The gene-neighborhood diagram was made using Clinker (34).

Fig 3

Thermal shift assay and binding affinity measurement of PstS proteins. DSF thermal melt assay for (A) PstS1a, (B) PstS1b, and (C) PstS2 with increasing concentration of phosphate and sodium tripolyphosphate and (D) binding affinity of PstS proteins calculated based on isothermal analysis of the DSF data (39, 40). The confidence interval [marginal asymmetric confidence interval at 95% confidence level (CI)] was estimated as suggested by Paketuryte et al. (41).

Fig 4

Comparison of the predicted structures of WH8102 PstS1a, PstS1b, and PstS2 with the E. coli PstS crystal structure. (A) An overlay of WH8102 PstS1b (pink) with the E. coli PstS crystal structure (PDB: 1ixH; marine blue) is shown as cartoon representatives. (B) An overlay of ribbon structures of E. coli PstS (marine blue) with WH8102 PstS1a (RMSD 1.274 Å, pink), PstS1b (RMSD 1.373 Å, green), and PstS2 (RMSD 1.479 Å, yellow). Details of the ligand binding residues for (C) E. coli, (D) PstS1a, (E) PstS1b, and (F) PstS2, with phosphate overlaid from the E. coli crystal structure showing the T10 residue of the E. coli crystal structure conserved in PstS1b but replaced with a serine residue at position S10 and S15 in Pst1a and PstS2, highlighted in yellow. (G) Sequence logo representation of the amino acid multiple sequence alignment of (i) PstS1a, (ii) PstS1b, and (iii) PstS2 proteins in the region of E. coli PstS T10 generated using WebLogo 3.7.12 (43). Binding residues are numbered according to amino acid residue per E. coli PstS. Binding residues involved in hydrogen bonding with phosphate are highlighted in green (the rest of the alignment is included as Fig. S3). The star above the amino acid alignments shows the T residue corresponding to T10 in E. coli PstS and its replacement as S in PstS1a and PstS2.

Fig 5

Ocean metatranscript distribution of Synechococcus WH8102 PstS homologs. Environmental abundance of (A) PstS1a, (B) PstS1b, and (C) PstS2 homologs extracted from the Tara Oceans MetaT data set (46, 47). Transcript abundance (expression data) is plotted for surface waters. The blue-filled circle size denotes measured abundance at a particular sampling site. Sampling sites are represented by “X.” The red circle highlights NAO stations (TARA 141-151), and the green circle highlights MS stations (TARA 011-025). The corresponding bubble plot for identified transcripts across surface water sampling depth (SRF) is shown as a function of phosphate concentration. The Krona plot shows the taxonomic distribution of PstS homolog hits selected to analyze meta transcriptome abundance.