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

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 M; 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 M; 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.Phosphorus input into aquatic systems is largely in the form of poorly soluble, eroded mineral phosphate, which enters these systems via runoff from land, making P i a key growthlimiting nutrient, particularly in freshwater environments (13,23,41). A recent survey of 34 inland lakes from three (physiographic) regions of Canada (25) revealed total P i concentrations ranging between 0.058 and 7.64 M. Thus, organisms occupying such environments invariably need to make key biochemical and regulatory adaptations to their P i uptake system in order to sustain growth. One such group is the cyanobacteria, one of the largest, most diverse, and most widely distributed prokaryotic lineages (42). Their ability to acclimate to a varying-light environment as well as their ability to acquire nutrients present at low ambient concentrations has led to their present-day dominance in vast tracts of oligotrophic open ocean waters (40) and in freshwater systems (14).Studies of bacterial P i acquisition have largely focused on model organisms such as Escherichia coli (52) and Bacillus subtilis (26). In E. coli, uptake utilizes both a low-affinity permease, the Pit system (54) [with uptake of P i being reliant on cotransport with divalent metal cations such as Mg(II) or Ca(II) through the formation of a soluble...

Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation in Synechocystis

“…The transport of various nutrients, like potassium and metal ions, amino acids, and polysaccharides (Table III), demonstrated marked down-regulation in the DndbC mutant except for the HCO 3 2 transporter BCT1 (Omata et al, 1999), which was up-regulated in the DndbC mutant compared with the wild type. The strongest down-regulation was observed for both ABC-type inorganic phosphate transporters (Table III), the constitutive low-affinity phosphate transporter Pst1 and the inducible, highaffinity phosphate transporter Pst2 (Pitt et al, 2010;Burut-Archanai et al, 2011). Furthermore, in the DndbC mutant, the levels of enzymes involved in nitrogen uptake, NRT transporters responsible for nitrate and nitrite uptake, as well as urea and ammonium transporters were down-regulated (Table III).…”

Comparative genomic, proteomic and exoproteomic analyses of three Pseudomonas strains reveals novel insights into the phosphorus scavenging capabilities of soil bacteria

“…The existence of two distinct Pst systems in both BIRD‐1 and SBW25 is similar to that of Synechocystis , Vibrio cholerae and the Archaeon, Halobacterium salinarium R1 (Furtwängler et al ., 2010; Pitt et al ., 2010; Mudrak and Tamayo, 2012). As Pst2 is present in all Pseudomonas strains, while Pst1 appears in only a few, we hypotheize that Pst2 is essential for efficient uptake of Pi in Pseudomonas .…”

“…Furthermore, although disruption of PstS1 in SBW25 did not affect growth on low Pi in isolation, it did confer a fitness reduction in the presence of the wild type (Zhang et al ., 2007). In Synechocystis, Vibrio and H. salinarium , Pst1 and Pst2 either have different kinetic parameters for the uptake of Pi (Furtwängler et al ., 2010; Pitt et al ., 2010) or are expressed during different growth phases (planktonic v biofilm) (Pratt et al ., 2010; Mudrak and Tamayo, 2012). The data presented in this study favours the hypothesis that they have different kinetic parameters as both were expressed in BIRD‐1 and SBW25 during planktonic growth.…”

Rapid regulation of phosphate uptake in freshwater cyanobacterial blooms

“…8, Case 3) (Falkner et al 1989(Falkner et al , 1993. This complex regulation of cellular uptake systems can be explained in single cyanobacteria by concurrently operating binding sites of membrane proteins with different phosphate affinity , Pitt et al 2010). The absence of 1 of the high-affinity phosphate-binding proteins (sphX) disables the capacity of a Synechococcus mutant to adapt to phosphate pulses, although it can still incorporate phosphate down to nanomolar levels (Falkner et al 1998).…”

“…However, other studies have addressed the complex features of phosphate uptake regulation by cyanobacteria. These organisms sense phosphate availability in the environment by histidine kinases and response regulators, which induce the expression of Pho regulon genes that encode high affinity P-binding proteins, alkaline phosphatase and other relevant proteins (Suzuki et al 2004, Schwarz & Forchhammer 2005, Juntarajumnong et al 2007, Pitt et al 2010. Additional complexities in phosphate regulation are related to changes in the kinetics and energetics of uptake systems during responses to sudden phosphate fluctuations , Falkner & Falkner 2003, Falkner et al 2006.…”