Functional Characterization of Synechocystis sp. Strain PCC 6803 pst1 and pst2 Gene Clusters Reveals a Novel Strategy for Phosphate Uptake in a Freshwater Cyanobacterium

Abstract

ABSTRACT Synechocystis sp. strain PCC 6803 possesses two putative ABC-type inorganic phosphate (P i ) transporters with three associated P i -binding proteins (PBPs), SphX (encoded by sll0679 ), PstS1 (encoded by sll0680 ), and PstS2 (encoded by slr1247 ), organized in two spatially discrete gene clusters, pst1 and pst2 . We used a combination of mutagenesis, gene expression, and radiotracer uptake analyses to functionally characterize the role of these PBPs and associated gene clusters. Quantitative PCR (qPCR) demonstrated that pstS1 was expressed at a high level in P i -replete conditions compared to sphX or pstS2 . However, a P i stress shift increased expression of pstS2 318-fold after 48 h, compared to 43-fold for pstS1 and 37-fold for sphX . A shift to high-light conditions caused a transient increase of all PBPs, whereas N stress primarily increased expression of sphX . Interposon mutagenesis of each PBP demonstrated that disruption of pstS1 alone caused constitutive expression of pho regulon genes, implicating PstS1 as a major component of the P i sensing machinery. The pstS1 mutant was also transformation incompetent. 32 P i radiotracer uptake experiments using pst1 and pst2 deletion mutants showed that Pst1 acts as a low-affinity, high-velocity transporter ( K s , 3.7 ± 0.7 μM; V max , 31.18 ± 3.96 fmol cell −1 min −1 ) and Pst2 acts as a high-affinity, low-velocity system ( K s , 0.07 ± 0.01 μM; V max , 0.88 ± 0.11 fmol cell −1 min −1 ). These P i ABC transporters thus exhibit differences in both kinetic and regulatory properties, the former trait potentially dramatically increasing the dynamic range of P i transport into the cell, which has potential implications for our understanding of the ecological success of this key microbial group.