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. ³²P_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.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 growth-limiting 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 μM. 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, neutral metal-phosphate complex, which is then the transported species (28, 49)] and a high-affinity Pst transport system (52). The Pst transporter comprises a periplasmic P_i-binding protein (PstS), two integral membrane proteins (PstA and PstC), and an ATP-binding protein (PstB) (10, 44). Regulation of this complex is dependent on a two-component system encoded by the phoBR operon (31). In addition, the Pst system itself seems to play a role in regulation, with mutations in genes of the pst operon leading to constitutive expression of the pho regulon (52). Thus, the periplasmic PstS, which binds P_i with high affinity, could potentially act as the primary sensor of external P_i. Once loaded with P_i, PstS interacts with membrane components of the Pst system, causing a conformational change which is sensed by the PhoU protein, not involved in P_i transport (51). However, increased activity of the Pit transporters PitA and PitB can alleviate constitutive expression of the pho regulon and restore P_i regulation of the regulon (24).In the freshwater cyanobacterium Synechocystis sp. strain PCC 6803 (herein, Synechocystis), while orthologs of the PhoB/R two-component system have been identified and the system has been shown to be exclusively responsible for the specific P_i limitation response (45), there are several features of the P_i acquisition system which are unusual and warrant further investigation. Firstly, Synechocystis, like several other freshwater strains (43) and most marine picocyanobacteria (40), contains no identifiable Pit transporter. In contrast, there are two gene clusters encoding potential ABC transporters for P_i (Fig. ​(Fig.1),1), which we designate here pst1 and pst2, with three associated P_i-binding proteins (PBPs) (2, 32). sll0540, which encodes a fourth PBP, has also been identified in the Synechocystis genome, but its PBP is not colocalized with either pst1 or pst2. Indeed, pho regulon predictions of several cyanobacterial genomes showed that 50% of freshwater strains contain “duplicate” pst transporters (43), while many freshwater and marine strains contain multiple associated PBPs (40, 43). However, despite clear evidence of multiple P_i transport elements in cyanobacteria, little is known of the functional significance of individual, and apparently redundant, components of the cyanobacterial pho regulon.Open in a separate windowFIG. 1.Schematic representation of the two ABC P_i transporters and the phoA-nucH gene clusters.Here, we assessed the role of multiple P_i transporter elements in Synechocystis by creating both mutants with complete deletions of the pst1 and pst2 gene clusters and single interposon mutants with mutations of the associated pstS and sphX genes. We generated gene expression profiles using quantitative PCR (qPCR) to analyze both wild-type (WT) and specific interposon mutants, under P_i-replete, P_i stress, and nitrate (N) stress conditions, as well as following a shift to high light. We show that disruption of pstS1 (sll0680) leads to constitutive pho regulon gene expression consistent with PstS1 as a primary component of the P_i sensor. Such a phenotype is not observed in the pstS2 (slr1247) and sphX (sll0679) mutants. Moreover, using radiotracer incorporation studies with pst gene cluster deletion mutants, we show that while both systems transport P_i, there are dramatic differences in their maximum uptake rates (V_max) and half-saturation constants (K_s) for P_i. These data demonstrate a novel strategy for P_i acquisition in a freshwater cyanobacterium.     

An ABC transporter is essential for resistance to the antitumor agent mithramycin in the producerStreptomyces argillaceus

The organization of the phosphate-specific transport (pst) operon inPseudomonas aeruginosa has been determined. The gene order of thepst operon ispstC, pstA, pstB, phoU, and a well-conserved Pho box sequence (16/18 bases identical) exists in the promoter region. The most striking difference from the knownEscherichia coli pst operon is the lack of thepstS gene encoding a periplasmic phosphate (P_i)-binding protein. Even though the threepst genes were absolutely required for P_i-specific transport, expression of thepst operon at high levels did not increase P_i uptake inP. aeruginosa. DNA sequences for thepstB andphoU genes have been determined previously. The newly identifiedpstC andpstA genes encode possible integral membrane proteins of 677 amino acids (M _r 73 844) and 513 amino acids (M _r 56 394), respectively. The amino acid sequences of PstC and PstA predict that these proteins contain a long hydrophilic domain not seen in theirE. coli counterparts. A chromosomal deletion of the entirepst operon renderedP. aeruginosa unable to repress P_i taxis under conditions of P_i excess. ThephoU andpstB genes are essential for repressing P_i taxis. However, mutants lacking either PstC or PstA alone were able to repress P_i taxis under conditions of P_i excess.     

The Integration Host Factor (IHF) Affects the Expression of the Phosphate-Binding Protein and of Alkaline Phosphatase in Escherichia coli

The genes encoding alkaline phosphatase (phoA) and the inducible inorganic phosphate transport system Pst (pstS,C,A,B,U) belong to the PHO regulon. Mutants of Escherichia coli lacking the global regulatory protein integration host factor (IHF) show an increased level of alkaline phosphatase and a decreased level of Pst. IHF binds weakly but specifically to a DNA fragment containing the promoter region of the pst operon but does not bind to a fragment that includes the promoter region of phoA. It is proposed that IHF is a positive regulator of the pst operon and as such controls indirectly the expression of phoA. Received: 4 May 1998 / Accepted: 19 August 1998     

Proton-coupled high-affinity phosphate transport revealed from heterologous characterization in Xenopus of barley-root plasma membrane transporter, HvPHT1;1

High‐affinity phosphate transporters mediate uptake of inorganic phosphate (P_i) from soil solution under low P_i conditions. The electrophysiological properties of any plant high‐affinity P_i transporter have not been described yet. Here, we report the detailed characterization of electrophysiological properties of the barley P_i transporter, HvPHT1;1 in Xenopus laevis oocytes. A very low K_m value (1.9 µm ) for phosphate transport was observed in HvPHT1;1, which falls within the concentration range observed for barley roots. Inward currents at negative membrane potentials were identified as nH⁺:P_i^‐ (n > 1) co‐transport based on simultaneous P_i radiotracer uptake, oocyte voltage clamping and pH dependence. HvPHT1;1 showed preferential selectivity for P_i and arsenate, but no transport of the other oxyanions SO₄^2? and NO₃^‐. In addition, HvPHT1;1 locates to the plasma membrane when expressed in onion (Allium cepa L.) epidermal cells, and is highly expressed in root segments with dense hairs. The electrophysiological properties, plasma membrane localization and cell‐specific expression pattern of HvPHT1;1 support its role in the uptake of P_i under low P_i conditions.     

Duplication of genes encoding the immunodominant 38 kDa antigen in Mycobacterium intracellulare

Abstract Mycobacterium avium is a causative agent of mycobacterioses in systemically immunocompromised individuals, whereas Mycobacterium intracellulare is responsible for causing infections in relatively immunocompetent hosts. In an attempt to identify components that could be involved in virulence, we characterised the 38 kDa-encoding gene of M. intracellulare that is absent in M. avium . This antigen cross-reacts immunologically with a major 38 kDa antigen of M. tuberculosis , and both antigens are homologues of the phosphate transport subunit S (PstS) of the pst complex of Escherichia coli . Unlike the M. tuberculosis complex the M. intracellulare coding gene was found to be duplicated. We also identified and characterised other pst genes that may constitute an operon. Considering that multiple isoforms of PstS are present in mycobacteria the possible role of pstS1 genes for pathogenesis is discussed.     

The pho regulon of Shigella flexneri

Growth of Escherichia coli K-12 in low-phosphate conditions results in the induction of the synthesis of many proteins, including the outer membrane porin PhoE, alkaline phosphatase, and the Pst system for the transport of phosphate (P_i). This response is controlled by a two-component regulatory system of which PhoB and PhoR are the response-regulator and the sensor/kinase, respectively. When Shigella flexneri was starved for P_i, neither PhoE nor alkaline phosphatase was produced. However, induction of the synthesis of the PstS protein was observed, indicating that S. flexneri contains a functional PhoB/PhoR regulatory system. Consistent with this notion, the introduction of the B. coli phoA gene in S. flexneri resulted in the induction of alkaline phosphatase synthesis under phosphate limitation. However, introduction of phoE on a plasmid did not lead to the expression of PhoE protein, indicating that S. flexneri PhoB does not recognize the phoE promoter region. The phoB gene was cloned and sequenced and in the deduced amino acid sequence two deviations from that of E. coli PhoB were detected. Site-directed mutagenesis revealed that one of these deviations, i.e. Leu-172, which is Arg in E. coli PhoB, is responsible for the lack of expression of the PhoE protein in S. flexneri.     

Involvement of a natural transport system in the process of efflux-mediated drug resistance in Mycobacterium smegmatis

The phosphate-specific transporter (Pst) in bacteria is a multi-subunit system which belongs to the ABC family of transporters. The gene forms part of an operon and it is involved in phosphate uptake in prokaryotes. Its import function is known to be operative only under conditions of phosphate starvation. However, we found overexpression of this transporter in a Mycobacterium smegmatis strain selected for ciprofloxacin resistance (CIPr) which was grown under conditions in which the phosphate-scavenging function of this operon was inoperative. In CIPr cells, active efflux of the drug plays a predominant role in conferring high levels of fluoroquinolone resistance. We therefore investigated the role of this transporter in the process of efflux-mediated drug resistance by inactivating the pst operon in the CIPr strain. Phenotypic characterization of the resulting strain, CIPrd, showed a striking reduction in the minimal inhibitory concentration (MIC) of ciprofloxacin and in the drug extrusion profile as well. Genotype analysis, on the other hand, revealed partial disruption of the pst operon in CIPrd as a consequence of transporter gene amplification. Furthermore, disruption of this operon in wild-type cells resulted in hypersensitivity to ciprofloxacin and other xenobiotics to which CIPr cells exhibited cross-resistance. Thus our results provide strong evidence that Pst is a natural membrane transport system that has the ability to promote drug efflux in addition to its phosphate-scavenging function in the CIPr strain.     

The extended N-terminal region of SphS is required for detection of external phosphate levels in Synechocystis sp. PCC 6803

A novel 47 amino acid extension at the N-terminus of the SphS histidine kinase has been identified in the cyanobacterium Synechocystis sp. PCC 6803. Here, we demonstrate this region is required for activation of the SphS-SphR phosphate-sensing two-component system under phosphate-limiting conditions and mutants lacking this extension do not show constitutive alkaline phosphatase activity when the negative regulator SphU is inactivated. We have also identified a putative membrane-associated domain within this region involved in control of the Pho regulon. In addition, there are two high-affinity ABC-type phosphate uptake systems in this organism. Our results demonstrate that the Pst1 system, but not the Pst2 system, is required for suppression of the Pho regulon under phosphate-sufficient conditions. Deletion of the pst1 operon and disruption of the membrane-spanning domain may both target the same control mechanism since constitutive alkaline phosphatase activity is similar in the double and single mutants.     

Uptake of Selenite by Saccharomyces cerevisiae Involves the High and Low Affinity Orthophosphate Transporters

Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low P_i concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity P_i transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low P_i medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both P_i transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.     

Role of an ABC Importer in Mycobacterial Drug Resistance

Phosphate specific transporter (Pst) in bacteria is involved in phosphate transport. Pst is a multisubunit system which belongs to the ABC family of transporters. The import function of this transporter is known to be operative at media phosphate concentrations below the millimolar range. However, we found amplification of this transporter in a laboratory generated ciprofloxacin resistant Mycobacterium smegmatis colony (CIP^r) which was grown in a condition when phosphate scavenging function of this operon was inoperative. Our results therefore argue the role of this ABC importer in conferring high level of fluoroquinolone resistance in CIP^r.