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.     

An alkaline phosphatase/phosphodiesterase, PhoD, induced by salt stress and secreted out of the cells of Aphanothece halophytica, a halotolerant cyanobacterium

Alkaline phosphatases (APases) are important enzymes in organophosphate utilization. Three prokaryotic APase gene families, PhoA, PhoX, and PhoD, are known; however, their functional characterization in cyanobacteria largely remains to be clarified. In this study, we cloned the phoD gene from a halotolerant cyanobacterium, Aphanothece halophytica (phoD(Ap)). The deduced protein, PhoD(Ap), contains Tat consensus motifs and a peptidase cleavage site at the N terminus. The PhoD(Ap) enzyme was activated by Ca(2+) and exhibited APase and phosphodiesterase (APDase) activities. Subcellular localization experiments revealed the secretion and processing of PhoD(Ap) in a transformed cyanobacterium. Expression of the phoD(Ap) gene in A. halophytica cells was upregulated not only by phosphorus (P) starvation but also under salt stress conditions. Our results suggest that A. halophytica cells possess a PhoD that participates in the assimilation of P under salinity stress.     

Acid phosphatase isozymes secreted under phosphate-deficient conditions inPholiota nameko

We previously reported the purification of an acid phosphatase (APase52) secreted from the mycelia ofPholiota nameko under phosphate-deficient conditions. In the present study, two other isozymes (APase47 and APase48) were found and their structures were compared with that of APase52. Thirteen amino acid residues at theN-terminus of APase47 were completely identical with those of APase48 and had partial homology with those of APase52. The deglycosylation of proteins indicated that three APase isozymes differ in theN-linked oligosaccharide content. The protease-generated peptide maps of the APases differed from one another in the band pattern. These results suggest that the APases are the products of different genes.     

Role of the all1549 (ana-rsh) gene, a relA/spoT homolog, of the Cyanobacterium Anabaena sp. PCC7120

The role of a single relA/spoT homolog all1549 (designated hereafter as ana-rsh) of the cyanobacterium Anabaena sp. PCC7120 was investigated. The complementation test in Escherichia coli showed that the protein encoded by ana-rsh possesses guanosine tetraphosphate (p)ppGpp-synthase/hydrolase activity. Under laboratory growth conditions, a low level of ppGpp was detected in Anabaena sp. PCC7120 and the loss of ana-rsh was lethal. Amino acid starvation induced ppGpp accumulation to an appropriate level, and nitrogen deficiency did not alter the ppGpp concentration in Anabaena cells. These data suggest that ana-rsh is required for cell viability under normal growth conditions and involved in the (p)ppGpp-related stringent response to amino acid deprivation, but not related to heterocyst formation and nitrogen fixation of Anabaena sp. PCC7120.     

Carbohydrate uptake genes inEscherichia coli are induced by carbon starvation

Escherichia coli produces several membrane-associated and periplasmic proteins in response to deprivation for a carbon source. The carbon starvation response involves a two- to fourfold, cAMP-dependent induction of operons involved in carbohydrate uptake and utilization, including thelac operon. Threecarbon-starvation-inducible (sci) gene fusions to aphoA reporter sequence were characterized. ThephoA-fusions werecya ⁺/crp ⁺-dependent and located in three previously characterized genes involved in high-affinity uptake of alternative carbon sources:mglB, encoding the periplasmic galactose binding protein;rbsB, encoding the periplasmic ribose binding protein; andlamB, encoding the maltodextrin-specific outer membrane porin.     

Effect of aluminum on the in vitro activity of acid phosphatases of four potato clones grown in three growth systems

The aim of this study was to assess the effect of aluminum on the in vitro activity of acid phosphatases (APases) of four potato clones, Macaca and Dakota Rose (Al-sensitive), and SMIC148-A and Solanum microdontum (Al-tolerant), grown in vitro, in hydroponics or in a greenhouse. The enzyme was assayed in vitro in the presence of 0, 1.85, 3.70, 5.55 and 7.40 mM Al. In plantlets grown in vitro, root APases were inhibited by Al in all clones, while shoot APases were inhibited by Al in S. microdontum and Dakota Rose and increased in Macaca at all Al concentrations. In plantlets grown in hydroponics, root APases increased in Macaca at 1.85 mM Al, whereas decreased at all Al levels in S. microdontum. In greenhouse plantlets, root APases decreased at 7.40 mM Al in S. microdontum and SMIC148-A, and at 3.70, 5.55 and 7.40 mM Al in Dakota Rose. Shoot APases decreased in Macaca and SMIC148-A. Conversely, in Dakota Rose, APases increased at 1.85 and 3.70 mM Al. These results show that the effect of Al toxicity on in vitro APase activity depends not only on Al availability but also on the plant organ, genetic background, and the growth conditions. Therefore, it suggests that acid phosphatases activity assessed in vitro might not be a good parameter to validate the screening for adaptation of potato clones to Al toxicity.     

Comparative genetic analysis of,Arabidopsis purple acid phosphatases AtPAP10, AtPAP12, and AtPAP26 provides new insights into their roles in plant adaptation to phosphate deprivation

Induction and secretion of acid phosphatases （APases） is thought to be an adaptive mechanism that helps plants survive and grow under phosphate （Pi） deprivation, in Arabidopsis, there are 29 purple acid phosphatase （AtPAP） genes. To systematically investigate the roles of different AtPAPs, we first identified knockout or knock-down T-DNA lines for all 29 AtPAP genes. Using these atpap mutants combined with in-gel and quantitative APase enzyme assays, we demonstrated that AtPAP12 and AtPAP26 are two major intracellular and secreted APases in Arabidopsis while AtPAPlo is mainly a secreted APase. On Pi-deficient （P-） medium or P- medium supplemented with the organophosphates ADP and fructose-6-phosphate （Fru-6-P）, growth of atpaplo was significantly reduced whereas growth of atpap12 was only moderately reduced, and growth of atpap26 was nearly equal to that of the wild type （WT）. Overexpression of the AtPAP12 or AtPAP26 gene, however, caused plants to grow better on P- or P- medium supplemented with ADP or Fru-6-P. Interest-ingly, Pi levels are essentially the same for the WT and overexpressing lines, although these two types of plants have significantly different growth phenotypes. These results suggest that the APases may have other roles besides enhancing internal Pi recycling or releasing Pi from external organophosphates for plant uptake.     

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.     

Regulation of bacterial phosphate taxis and polyphosphate accumulation in response to phosphate starvation stress

Phosphorus (P) is an essential constituent in all types of living organisms. Bacteria, which use inorganic phosphate (P_i), as the preferred P source, have evolved complex systems to survive during P_i starvation conditions. Recently, we found thatPseudomonas aeruginosa, a monoflagellated, obligately aerobic bacterium, is attracted to P_i. The evidence that the chemotactic response to P_i (P_i taxis) was observed only with cells grown in P_i-limiting medium suggests that P_i taxis plays an important role in scavenging P_i residues under conditions of P_i starvation. Many bacteria also exhibit rapid and extensive accumulation of polyphosphate (polyP), when P_i is added to cells previously subjected to P_i starvation stress. Since polyP can serve as a P source during P_i starvation conditions, it is likely that polyP accumulation is a protective mechanism for survival during P_i starvation. In the present review, we summarize our current knowledge on regulation of bacterial P_i taxis and polyP accumulation in response to P_i starvation stress.     

Enzyme promiscuity in natural environments: alkaline phosphatase in the ocean

Alkaline phosphatase (APase) is one of the marine enzymes used by oceanic microbes to obtain inorganic phosphorus (P_i) from dissolved organic phosphorus to overcome P-limitation. Marine APase is generally recognized to perform P-monoesterase activity. Here we integrated a biochemical characterization of a specific APase enzyme, examination of global ocean databases, and field measurements, to study the type and relevance of marine APase promiscuity. We performed an in silico mining of phoA homologs, followed by de novo synthesis and heterologous expression in E. coli of the full-length gene from Alteromonas mediterranea, resulting in a recombinant PhoA. A global analysis using the TARA Oceans, Malaspina and other metagenomic databases confirmed the predicted widespread distribution of the gene encoding the targeted PhoA in all oceanic basins throughout the water column. Kinetic assays with the purified PhoA enzyme revealed that this enzyme exhibits not only the predicted P-monoester activity, but also P-diesterase, P-triesterase and sulfatase activity as a result of a promiscuous behavior. Among all activities, P-monoester bond hydrolysis exhibited the highest catalytic activity of APase despite its lower affinity for phosphate monoesters. APase is highly efficient as a P-monoesterase at high substrate concentrations, whereas promiscuous activities of APase, like diesterase, triesterase, and sulfatase activities are more efficient at low substrate concentrations. Strong similarities were observed between the monoesterase:diesterase ratio of the purified PhoA protein in the laboratory and in natural seawater. Thus, our results reveal enzyme promiscuity of APase playing potentially an important role in the marine phosphorus cycle.Subject terms: Microbial biooceanography, Biogeochemistry, Microbial ecology     

The type II secretion system (Xcp) of Pseudomonas putida is active and involved in the secretion of phosphatases

The genome of the Gram‐negative bacterium Pseudomonas putida harbours a complete set of xcp genes for a type II protein secretion system (T2SS). This study shows that expression of these genes is induced under inorganic phosphate (P_i) limitation and that the system enables the utilization of various organic phosphate sources. A phosphatase of the PhoX family, previously designated UxpB, was identified, which was produced under low P_i conditions and transported across the cell envelope in an Xcp‐dependent manner demonstrating that the xcp genes encode an active T2SS. The signal sequence of UxpB contains a twin‐arginine translocation (Tat) motif as well as a lipobox, and both processing by leader peptidase II and Tat dependency were experimentally confirmed. Two different tat gene clusters were detected in the P. putida genome, of which one, named tat‐1, is located adjacent to the uxpB and xcp genes. Both Tat systems appeared to be capable of transporting the UxpB protein. However, expression of the tat‐1 genes was strongly induced by low P_i levels, indicating a function of this system in survival during P_i starvation.     

Differential responses of oat cultivars to phosphate deprivation: plant growth and acid phosphatase activities

The effect of phosphate starvation on growth and acid phosphatases (APases) localization and activity in oat tissues was investigated. Oat cultivars (Avena sativa L.??Arab, Polar, Szakal) were grown for 1?C3?weeks in complete nutrient medium (+P) and without phosphate (?P). Pi concentration in plant tissues decreased strongly after culturing on ?P medium. Pi deficit reduced shoot growth, stimulated root elongation and increased ratio of root/shoot in all oat cultivars. Pi deficit had a greater impact on growth of oat cv. Polar than other varieties. A decrease in the internal Pi status led to an increase of acid phosphatase activities in extracts from shoots and roots, and in root exudates. The highest activity of secreted APases was observed for oat cv. Arab, during the third week of growth under Pi-deficient conditions. The activity of extracellular APase was high in young, growing zones of roots of ?P plants. Histochemical visualization indicated high activity of APases in the epidermis and vascular tissues of ?P plants. Pi deficiency increased intracellular APase activity in shoot mainly in oat cv. Polar, whereas APase activity in roots was the highest in oat cv. Szakal. Protein extracts from roots and shoots were run on native discontinuous PAGE to determine which isoform(s) may be affected by Pi deficiency. Three major APase isoforms were detected in all oat plants; one was strongly induced by Pi deficit. The studied oat cultivars differed in terms of acclimation to deficiency of phosphate??used various pools of APases to acquire Pi from external or internal sources.     

Iron deficiency influences NtcA‐dependent regulation of fatty acid desaturation and heterocyte envelop formation in Anabaena sp. PCC 7120

In Anabaena sp. PCC 7120, iron is an essential trace element and its availability determines proper functioning of several kinds of metabolisms. Iron deficiency leads to several unavoidable consequences including membrane damage. In the present study, we dealt with the impact of iron deficiency on NtcA (global nitrogen regulator)‐dependent regulation of two important processes, i.e. fatty acid desaturation and heterocyte envelop formation in cyanobacterium Anabaena sp. PCC 7120. In Anabaena sp. PCC 7120, NtcA regulates fatty acid desaturation by regulating enzyme fatty acid desaturases. The NtcA‐based regulation of fatty acid desaturation may be direct or indirect. Furthermore, the expression of genes involved in the heterocyte envelope polysaccharide (HEP) layer formation (hepABCK) and heterocyte‐specific glycolipids (HGLs) synthesis (devH, hglE_A, prpJ and devB) were also under the control of NtcA and reduced under iron deficiency background. The enhanced expression of furA and early downregulation of ntcA under iron deficiency is responsible for reduction in fatty acid desaturation as well as decrease in the expression of genes involved in HEP layer formation and HGL synthesis. Overall results confirmed that iron deficiency influences the NtcA‐based regulation of fatty acid desaturation and heterocyte envelop formation in Anabaena sp. PCC 7120.