Characterization of a monomeric heat-labile classical alkaline phosphatase from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

Alkaline phosphatases (APs), known inducible enzymes of the Pho regulon and poorly characterized in cyanobacteria, hydrolyze phosphomonoesters to produce inorganic phosphate (P_i) during P_i starvation. In this study, two predicted alkaline phosphatase genes in the genome of Anabaena sp. PCC 7120, all2843 and alr5291, were apparently induced during P_i starvation. Sequence analysis showed that alr5291 encodes a protein that is an atypical alkaline phosphatase like other cyanobacteria PhoAs, but the protein encoded by all2843 is very similar to the classical PhoAs, such as Escherichia coli alkaline phosphatase (EAP). To date, there have been no reports about classical phoA in cyanobacterial genomes. The alkaline phosphatase AP^A, coded by all2843, is characterized as a metalloenzyme containing Mg²⁺ and Zn²⁺ with molar ratio of 1: 2. Site-directed mutagenesis analysis indicated that, though the active center of AP^A is highly conserved in comparison with EAP, differences do exist between AP^A and EAP in metal ion coordination. Besides, biochemical analysis revealed that AP^A is a monomeric protein and inactivated rapidly at 50°C. These results suggest that AP^A is the first monomeric heat-labile classical PhoA found in cyanobacteria.     

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.     

Phylogenomics Uncovers Evolutionary Trajectory of Nitrogen Fixation in Cyanobacteria

Biological nitrogen fixation (BNF) by cyanobacteria is of significant importance for the Earth’s biogeochemical nitrogen cycle but is restricted to a few genera that do not form monophyletic group. To explore the evolutionary trajectory of BNF and investigate the driving forces of its evolution, we analyze 650 cyanobacterial genomes and compile the database of diazotrophic cyanobacteria based on the presence of nitrogen fixation gene clusters (NFGCs). We report that 266 of 650 examined genomes are NFGC-carrying members, and these potentially diazotrophic cyanobacteria are unevenly distributed across the phylogeny of Cyanobacteria, that multiple independent losses shaped the scattered distribution. Among the diazotrophic cyanobacteria, two types of NFGC exist, with one being ancestral and abundant, which have descended from diazotrophic ancestors, and the other being anaerobe-like and sparse, possibly being acquired from anaerobic microbes through horizontal gene transfer. Interestingly, we illustrate that the origin of BNF in Cyanobacteria coincide with two major evolutionary events. One is the origin of multicellularity of cyanobacteria, and the other is concurrent genetic innovations with massive gene gains and expansions, implicating their key roles in triggering the evolutionary transition from nondiazotrophic to diazotrophic cyanobacteria. Additionally, we reveal that genes involved in accelerating respiratory electron transport (coxABC), anoxygenic photosynthetic electron transport (sqr), as well as anaerobic metabolisms (pfor, hemN, nrdG, adhE) are enriched in diazotrophic cyanobacteria, representing adaptive genetic signatures that underpin the diazotrophic lifestyle. Collectively, our study suggests that multicellularity, together with concurrent genetic adaptations contribute to the evolution of diazotrophic cyanobacteria.     

Response of Alkaline Phosphatases in the Cyanobacterium Anabaena sp. FACHB 709 to Inorganic Phosphate Starvation

Alkaline phosphatases (APases) play a crucial role in phosphorus (P) metabolism and regulation, but their physiological functions largely remain unclear in cyanobacteria. Here, we identified four putative APase genes, designated as phoA-709, phoD1-709, phoD2-709, and phoS-709, in the cyanobacterium Anabaena sp. FACHB 709, and investigated their response to inorganic phosphate (P_i) starvation. With the exception of phoD2-709, three other APase genes were expressed at a constant and relative low level in P_i-replete medium, whereas the expression of all four APase genes was elevated in response to P_i starvation but phoA-709 significantly. However, disruption of phoA-709 did not affect the total APase activity but caused the expressional up-regulation of phoD1-709 and phoS-709 under P_i-sufficient and P_i-limiting conditions. These suggest that, the four APases of Anabaena sp. FACHB 709 are involved in P metabolism and regulation, and PhoA-709 is the main, yet dispensable, APase.     

Inorganic Polyphosphate in Escherichia coli: the Phosphate Regulon and the Stringent Response

Escherichia coli transiently accumulates large amounts of inorganic polyphosphate (polyP), up to 20 mM in phosphate residues (P_i), in media deficient in both P_i and amino acids. This transient accumulation is preceded by the appearance of nucleotides ppGpp and pppGpp, generated in response to nutritional stresses. Mutants which lack PhoB, the response regulator of the phosphate regulon, do not accumulate polyP even though they develop wild-type levels of (p)ppGpp when subjected to amino acid starvation. When complemented with a phoB-containing plasmid, phoB mutants regain the ability to accumulate polyP. PolyP accumulation requires high levels of (p)ppGpp independent of whether they are generated by RelA (active during the stringent response) or SpoT (expressed during P_i starvation). Hence, accumulation of polyP requires a functional phoB gene and elevated levels of (p)ppGpp. A rapid assay of polyP depends on its adsorption to an anion-exchange disk on which it is hydrolyzed by a yeast exopolyphosphatase.     

Regulation of the phosphate regulon of Escherichia coli: Characterization of the promoter of the pstS gene

Summary The pstS gene belongs to the phosphate regulon whose expression is induced by phosphate starvation and regulated positively by the PhoB protein. The phosphate (pho) box is a consensus sequence shared by the regulatory regions of the genes in the pho regulon. We constructed two series of deletion mutations in a plasmid in vitro, with upstream and downstream deletions in the promoter region of pstS, which contains two pho boxes in tandem, and studied their promoter activity by connecting them with a promoterless gene for chloramphenicol acetyltransferase. Deletions extending into the upstream pho box but retaining the downstream pho box greatly reduced promoter activity, but the remaining activity was still regulated by phosphate levels in the medium and by the PhoB protein, indicating that each pho box is functional. No activity was observed in deletion mutants which lacked the remaining pho box or the-10 region. Therefore, the pstS promoter was defined to include the two pho boxes and the-10 region. The PhoB protein binding region in the pstS regulatory region was studied with the deletion plasmids by a gelmobility retardation assay. The results suggest the protein binds to each pho box on the pstS promoter. A phoB deletion mutant was constructed, and we demonstrated that expression of pstS was strictly dependent on the function of the PhoB protein.     

Phosphorus strategy in bloom-forming cyanobacteria (Dolichospermum and Microcystis) and its role in their succession

Dolichospermum (formerly Anabaena) and Microcystis cause harmful cyanobacterial blooms in freshwater ecosystems worldwide. Input reduction of both nitrogen (N) and phosphorus (P) are commonly recognized as basic ways of controlling blooms, but little is known about the roles of nutrients and their using strategy among cyanobacteria in triggering the succession of diazotrophic to non-diazotrophic cyanobacteria. In this study, we investigated in situ responses of cyanobactria to ambient P status during the transition from Dolichospermum flos-aquae to Microcystis spp. in Lake Taihu and Lake Chaohu. While dominant in phytoplankton community, D. flos-aquae experienced P deficiency as evidenced by qualitative detection of extracellular phosphatase via enzyme labeled fluorescence (ELF). The percentage of ELF-labelled D. flos-aquae cells was 33% when it dominated the phytoplankton community, and was 78% when it co-dominated with Microcystis spp., indicating an increase in P deficiency. Meanwhile, no ELF-labelled Microcystis cells were observed while polyphosphate body (PPB) were present, suggesting that Microcystis spp. were not P deficient. Additionally, the percentages of Microcystis cells containing PPB showed an inverted “U-shaped” relationship with concentrations on soluble reactive phosphorus (SRP). To validate the field observation, a laboratory study of the monocultures of the dominant cyanobacteria was conducted. Extracellular alkaline phosphatase activity (APA) and PPB accumulation were regulated by P availability in monocultures of D. flos-aquae. Interestingly, no cell bound extracellular phosphatase was found on Microcystis aeruginasa even in the culture without P supply. Consistently, the expressions of phosphatase encoding gene phoX showed no differences among the treatments. The way in which PPB accumulation occurred in Microcystis spp. in response to P availability in the cultures was similar to that observed in the field, demonstrating a strategy of energy conservation over P accumulation. The competitive advantage of Microcystis spp. was displayed at low P concentrations: where it could rapidly uptake and store inorganic P, which also increased the P deficiency of the coexisting phytoplankton species. Responses of P-transport gene pstS confirmed this hypothesis. The physiological and molecular mechanisms mentioned above enable Microcystis to survive and proliferate in environment with low available P supply more efficiently. In conclusion, different cyanobacterial species have distinct ways of responding to P availability, suggesting that the control of cyanobacterial blooms by targeted nutrient reduction is largely dependent upon the dominant species. P reduction is more effective in controlling diazotrophic cyanobacteria than non-diazotrophic cyanobacteria.     

Extracellular phosphomannan as a phosphate reserve in the yeast Kuraishia capsulata

We have found that extracellular phosphomannan is the main phosphate reserve in the yeast Kuraishia capsulata, in contrast to other yeast species effectively absorbing P_i. Under nitrogen starvation, K. capsulata absorbed essentially all P_i from the medium containing 240 mM glucose, 2.5 mM MgSO₄, and 11 mM KH₂PO₄. Inorganic polyphosphate level in the cells was about 14% of the P_i absorbed. Most of the P_i (～60%) was found in the fraction of extracellular phosphomannan that can be used as a carbon and phosphorus source by this yeast in deficient media.     

In vitro construction of the tufB-lacZ fusion: analysis of the regulatory mechanism of tufB promoter

Summary Genetic studies suggest that the so-called phosphorus-family of enzymes inN. crassa are controlled by a complex system of regulatory genes which are responsive to the level of phosphorus in the growth medium. The intracellular metabolite(s) that interact with this system to signal changes in the external phosphorus concentration has not been identified. In this study the pools of acid-soluble, phosphorus-containing, compounds are measured in wild-type and phosphorus-family enzyme regulatory mutant strains ofN. crassa before and during phosphorus starvation.Prolonged phosphorus starvation of wild-typeN. crassa failed to alter significantly the pre-starvation level of intracellular orthophosphate, suggesting that intracellular P_i would be a poor effector signal for the control of the phosphorus family enzymes. However, inorganic pyrophosphate (PP_i) decreased 15-fold, and tri- and tetrapolyphosphate (PPP_i and PPPP_i) increased 3- to 5-fold within 15 minutes after transfer of the wild-type strain to phosphorus-free medium. Phosphate starvation of seven different regulatory gene mutant strains resulted in a rapid decrease in the PP_i pool similar to that which occurred in the wild-type. However, only two of these seven strains showed increased PPP_i and PPPP_i pools following phosphate starvation. Additional experiments demonstrated that PP_i pools, but not PPP_i and PPPP_i pools, were unaffected by several starvation regimens other than phosphorus starvation. Metabolic studies employing H₃ ³²PO₄ showed that the pool of PP_i was labeled to steady-state levels after two minutes of continuous labeling of a phosphate-sufficient culture. Furthermore, long-term steady-state labeling showed that the intracellular PP_i pool was directly responsive to the decrease in the extracellular P_i concentration of the medium resulting from cell growth. Growth on phosphoethanolamine, a phosphorus source that allows a modest degree of derepression even in growing cells, resulted in lower levels of PP_i than were seen in phosphate-grown cells. These observations suggest that PP_i may be involved in the mechanism responsible for the control of phosphorus-family enzyme regulatory gene product activity.     

The phosphotransferase protein EIIA(Ntr) modulates the phosphate starvation response through interaction with histidine kinase PhoR in Escherichia coli

Many Proteobacteria possess the paralogous PTS^Ntr, in addition to the sugar transport phosphotransferase system (PTS). In the PTS^Ntr phosphoryl‐groups are transferred from phosphoenolpyruvate to protein EIIA^Ntr via the phosphotransferases EI^Ntr and NPr. The PTS^Ntr has been implicated in regulation of diverse physiological processes. In Escherichia coli, the PTS^Ntr plays a role in potassium homeostasis. In particular, EIIA^Ntr binds to and stimulates activity of a two‐component histidine kinase (KdpD) resulting in increased expression of the genes encoding the high‐affinity K⁺ transporter KdpFABC. Here, we show that the phosphate (pho) regulon is likewise modulated by PTS^Ntr. The pho regulon, which comprises more than 30 genes, is activated by the two‐component system PhoR/PhoB under conditions of phosphate starvation. Mutants lacking EIIA^Ntr are unable to fully activate the pho genes and exhibit a growth delay upon adaptation to phosphate limitation. In contrast, pho expression is increased above the wild‐type level in mutants deficient for EIIA^Ntr phosphorylation suggesting that non‐phosphorylated EIIA^Ntr modulates pho. Protein interaction analyses reveal binding of EIIA^Ntr to histidine kinase PhoR. This interaction increases the amount of phosphorylated response regulator PhoB. Thus, EIIA^Ntr is an accessory protein that modulates the activities of two distinct sensor kinases, KdpD and PhoR, in E. coli.     

Distribution of alkaline phosphatase genes in cyanobacteria and the role of alkaline phosphatase on the acquisition of phosphorus from dissolved organic phosphorus for cyanobacterial growth

Phosphorus is a vital nutrient for cyanobacterial growth. Aside from dissolved inorganic phosphorus, dissolved organic phosphorus (DOP) is used by cyanobacterial species via the activity of alkaline phosphatase (APase), which likely plays an important role in acquiring phosphorus for algal growth in the same manner as it does in other bacteria. In this work, APase genes phoA, phoD, and phoX were found distributed in the cyanobacterial strains included in the algal genome collection of the NCBI database. PhoX has a wider distribution than the classical phoA and phoD. Furthermore, multiple types of APase genes were simultaneously identified in a single strain or genome. Anabaena flos-aquae FACHB-245 was selected as a typical strain to study the performance of cyanobacteria growing on DOP. In algal growth involving AMP or lecithin, APase regulates the release of phosphorus from DOP as confirmed by the relative quantification of phoD and phoX expression levels. Our results confirmed that the distribution of APase is prevalent in cyanobacteria and thus provides a new insight into the potential role of cyanobacterial APase on phosphorus acquisition in natural environment.