Functional characterization of Synechocystis sp. strain PCC 6803 pst1 and pst2 gene clusters reveals a novel strategy for phosphate uptake in a freshwater cyanobacterium.
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 + or - 0.7 microM; V(max), 31.18 + or - 3.96 fmol cell(-1) min(-1)) and Pst2 acts as a high-affinity, low-velocity system (K(s), 0.07 + or - 0.01 microM; V(max), 0.88 + or - 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.
Pitt
Frances D
FD
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
Mazard
Sophie
S
Humphreys
Lee
L
Scanlan
David J
DJ
D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D001692:Biological Transport / Q000235:genetics / Q000502:physiology; D019295:Computational Biology; D005434:Flow Cytometry; D005618:Fresh Water / Q000382:microbiology*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D007700:Kinetics; D005810:Multigene Family / Q000235:genetics / Q000502:physiology*; D016296:Mutagenesis; D010710:Phosphates / Q000378:metabolism*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D046939:Synechocystis / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D010710:Phosphates