3 Biotech (2021) 11:218 https://doi.org/10.1007/s13205-021-02774-z ORIGINAL ARTICLE Identification and characterization of alkaline phosphatase gene phoX in Microcystis aeruginosa PCC7806 Sujuan Hong1 · Qianhui Pan1 · Siyu Chen1 · Yao Zu1 · Chongxin Xu2 · Jianhong Li1 Received: 4 November 2020 / Accepted: 3 April 2021 © King Abdulaziz City for Science and Technology 2021 Abstract PhoX is an extracellular alkaline phosphatase that is widely found in cyanobacteria and plays an important role in the conversion of extracellular organophosphorus into soluble inorganic phosphorus. However, the phoX gene has not yet been experimentally confirmed to exist in bloom-forming Microcystis species. In this study, we identified a putative phoX gene (GenBank accession no. ARI79942.1) in M. aeruginosa PCC7806 and overexpressed it in Escherichia coli 21 (DE3). The expressed PhoX protein displayed phosphodiesterase and phosphomonoesterase activities. In contrast to other bacterial PhoX proteins, which are activated mainly by C ­ a2+, Microcysits PhoX was most strongly activated by M ­ g2+, followed by 2+ 2+ 2+ 2+ 2+ ­Co , ­Ca , ­Zn and M ­ n , but it was inhibited by N ­ i . Sequence analysis showed that phoX was highly conserved in the Microcystis genus (DNA similarity > 96% between species). phoX expression responded significantly to different environmental phosphorus levels. When PCC7806 cells were cultured in phosphorus-deficient medium (BG11-P), phoX expression reached its highest level at 2 h and then decreased to a low level at 4 h. Organophosphate induced the expression of phoX; its expression reached the highest level at 4 h and was maintained at a high level at 6 h. Our results confirmed a putative phoX gene and demonstrated that the phoX gene of Microcystis is conserved. Keywords PhoX · Alkaline phosphatase · Microcystis · Phosphorus metabolism · Cyanobacterial bloom Introduction Cyanobacterial blooms have become commonplace in many freshwater ecosystems (Benayache et al. 2019; Chorus and Bartram 1999; Hudnell et al. 2008). The typical cyanobacterial genus Microcystis can proliferate rapidly in a short time under suitable environmental conditions, forming visible blue-green oil-like films or floating foams on the water surface (Kannan and Lenca 2012). Microcystis blooms not only destroy hydroecosystems, but also produce microcytin peptide toxins. Microcystis blooms are a very concerning environmental problem worldwide (Butler et al. 2009; Duy et al. 2000; Harke et al. 2016). * Jianhong Li lijianhong@njnu.edu.cn; 516389186@qq.com 1 School of Life Sciences, Nanjing Normal University, Nanjing 210023, China 2 Laboratory for Food Quality, Safety‑State Key Laboratory Cultivation Base of Ministry of Science and Technology, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China Phosphorus (P) is an essential nutrient for Microcystis growth and bloom occurrence (Jankowiak et al. 2019; Vuorio et al. 2020). Orthophosphate is a form of P that is directly used by phytoplankton (Correll 1998; Lee et al. 1980; Lin et al. 2016). However, the concentration of orthophosphate is very low in most water bodies, and dissolved organic phosphorus (DOP) is the main source of P for phytoplankton (Krom et al. 2005; Pulido-Villena et al. 2010). Alkaline phosphatase (APase) plays an important role in the absorption and utilization of organophosphorus by cyanobacteria and can convert organophosphorus into orthophosphate (Ammerman 1991; Shun et al. 1994; Siuda and Chrost 2001). There are three families of prokaryotic APases, PhoA, PhoD and PhoX (Luo et al. 2009; Ragot et al. 2017), which correspond to three organophosphorus metabolism pathways in bacteria (White 2009). First, small molecular phospholipids are transported to the cytoplasm via uptake by the glycerol phosphate system in the cytoplasm and are decomposed by intracellular APase PhoA and/or PhoD. Second, the small organic phospholipid molecules enter the periplasmic space of the cell and are then broken down by APase 13 Vol.:(0123456789) 218 Page 2 of 12 in the periplasm. Third, organophosphates in the ambient environment are hydrolysed by extracellular APase (PhoX). PhoX proteins are distinct from the well-characterized PhoA family (Zaheer et al. 2009) and are more widely distributed in marine bacteria than classical PhoA proteins (Sebastian and Ammerman 2009). phoX is also more widely distributed algae and cyanobacteria than phoA and phoD (Ammerman 1991; Dai et al. 2016; Lin et al. 2016); however, only a few phoX genes have been identified in these species, including genes from Prochlorococcus, Synechococcus (Kathuria and Martiny 2011), Volvox carteri (Hallmann 1999) and Chlamydomonas reinhardtii (Quisel et al. 1996). In the best-studied harmful cyanobacterial genus, Microcystis, although the putative phoX gene has been studied (Harke et al. 2012; Harke and Gobler 2013; Lin et al. 2018), it has not been experimentally verified, and little is currently known about its detailed characteristics to date. Based on sequence analysis, we identified a putative phoX gene (accession no. ARI79942.1) in the Microcystis aeruginosa PCC7806 (PCC7806) genome. To reveal its function, we expressed the gene in E. coli and determined its enzyme properties. We also investigated the expression pattern of phoX in PCC7806 cells in response to different P conditions in the environment. Materials and methods Culture of the Microcystis strain and bacteria Microcystis aeruginosa PCC7806 (PCC7806) was obtained from the Pasteur Culture Collection of Cyanobacteria, France. The strain was grown in BG11 medium (Waterbury 2006) illuminated by a bank of fluorescent lights that provided a light intensity of 3,000 lx and was shaken several times a day at 28 ± 2 °C. E. coli DH5α, which was used for gene cloning, and E. coli BL21 (DE3), which was used for recombinant protein overexpression (Goldman et al. 1990), were purchased from Novagen (Germany) and grown at 37 °C in Luria–Bertani (LB) medium supplied with appropriate antibiotics. BLAST search for a putative phoX gene in M. aeruginosa PCC 7806 The amino acid sequence of a verified PhoX APase (accession no. WP_138072907) from Synechococcus sp. PCC 11,901 (Kathuria and Martiny 2011) was used subjected to a BLAST search in the GenBank database (https://www. ncbi.nlm.nih.gov/), and a putative phoX gene (accession no. ARI79942.1) with 68% similarity was found in PCC7806. 13 3 Biotech (2021) 11:218 Bioinformatics analysis of putative PhoX The properties of the putative PhoX sequence, including its basic physical and chemical properties, conserved domains, transmembrane domains, signal peptides, secondary structures, N-glycosylation, O-glycosylation and phosphorylation, were analysed using the Expasy Proteomics Server (http://www.expasy.org/). Gene expression vector construction Two primers, phoX-F (5′-GTTT AAC TTT AAG AAG GAG AT ATCAAC ATGAGTATTTCTCGCCGTAATTTC-3′) and phoX-R (5′-AGTGATGGTGATGGTGATGTGTACATT TTGACTGGATTCGGCGAA-3′), were used to amplify the DNA fragment containing the entire phoX open reading frame (accession no. ARI79942.1), and the length of the PCR fragment was 2202 bp. The PCR products were cloned into the pBRT7 vector to generate the pBRT7-7806 expression plasmid. The plasmid was confirmed by enzyme digestion and gene sequencing. The pBRT7-7806 and pBRT7 vectors were transformed into E. coli DH5α (1:50). Transformants were screened on plates with 100 mg/L ampicillin (Amp) and checked by PCR. Positive colonies checked by PCR were incubated in 250 mL LB medium with 100 mg/L Amp for 8 h at 37 °C and 250 rpm. Then, the plasmids were extracted with an extraction kit (TIANGEN, DP103) and checked by digestion with the endonuclease Eco32 I and sequencing. Protein expression and purification The recombinant plasmid pBRT7-7806 and the control plasmid pBRT7 were transformed into E. coli BL21 (DE3). To overexpress the protein, bacteria carrying the plasmids were incubated in LB (with Amp) liquid medium and cultured for 3–4 h at 37 °C until the ­OD720 was approximately 0.6. Then, 0.4 mM isopropyl β-d-thiogalactoside (IPTG) was added to the culture, which was incubated overnight at 16 °C. The liquid cultures were centrifuged at 7000 g for 15 min to collect the bacteria. The bacteria were disrupted by sonication in 5 mL of 50 mM phosphate buffer with 1 mM phenylmethylsulphonyl fluoride (PMSF). The cell lysates were centrifuged at 10,000 g for 30 min at 4 °C, and the supernatant was used for protein purification. The target protein was recovered with a His-Trap affinity column, and the purification protocol was provided by Smart Life Science (Ni NTA Beads 6FF). The eluted proteins were analysed by SDS-PAGE and Western blotting. The target protein was immunochemically detected using a mouse antiHis antibody (GenScript, A00186). In the Western blotting Page 3 of 12 74 91 −0.415 80,557.3 733 Theoretical pI Formula Microcystis aeruginosa PCC 7806 To investigate the expression of phoX and phoA under different P supply conditions, PCC7806 cells were cultured in BG11, P-deficient BG11 (-P) and organic-P BG11 (-P + AMP) media, and gene expression was measured at 0, 2, 4 and 6 h by qPCR. Total RNA was extracted from the cells with an RNAprep Plank Kit (TIANGEN, Beijing, China), and the RNA was Molecular weight Expression of phoA and phoX in Microcystis under different P conditions Number of amino acids To examine the metal dependence of PhoX, apoenzymes were prepared by dialyzing purified PhoX against elution buffer (50 mM ­NaH2PO4, 300 mM NaCl, pH 8.0) containing 100 mM EDTA. The resulting apoenzymes were then dialyzed against the elution buffer to remove EDTA. Apoenzyme activity was measured in the presence of 2 mM concentrations of different divalent metal ions (­ Ca2+, ­Mn2+, ­Co2+, ­Ni2+, ­Mg2+, ­Zn2+); the control treatment lacked metal ions. Table 1 Predicted properties of PhoX protein in Microcystis aeruginosa PCC7806 Metalloenzyme analysis Instability index To better understand the characteristics of the APase deduced from the phoX sequence, the optimum temperature and pH, substrate specificity and metals required for enzyme activity were studied. Substrate Specificity: Different possible substrates, including glucose-1-phosphate (G-1-P), disodium 4-nitrophenyl phosphate (PNPP), adenosine monophosphate (AMP), adenosine triphosphate (ATP), sodium pyrophosphate and glucose-6-phosphate (G-6-P), were tested by examining inorganic phosphate (Pi) release during incubation in the presence of phosphatase. 75.87 Analysis of enzymatic characteristics 31.33 (stable) Aliphatic index Grand average of hydropathicity (GRAVY) Total number of negatively charged residues APase activity was measured according to the release of paranitrophenol from paranitrophenol phosphate (pNPP) (Roy et al. 1982). One unit of enzyme was defined as the amount required to release 1 nmol of paranitrophenol per min at 37 °C. C3613H5553N 965O1106S11 Measurement of APase activity 5.23 Total number of positively charged residues The N-terminal of the sequence considered assay, the mouse anti-His antibody or the secondary antimouse antibody (BioWord, catalogue number AA66182) was diluted with dilution buffer at a ratio of 1:5000 (20 mM Tris–HCl, pH 6.8, 150 mM NaCl, 0.1% [w/v] Tween-20). Next, the immunoblotting band signals were visualized by enhanced chemiluminescence (ELC) (Tanon, catalogue number 180-500), and images were obtained with a cooled charge-coupled device (CCD) camera (Tanon-4100). 218 Met (2021) 11:218 Organism/ Name 3 Biotech 13 218 Page 4 of 12 3 Biotech (2021) 11:218 Fig. 1 Prediction of signal peptide in PhoX of Microcystis aeruginosa PCC7806. A Tat-way signal peptide is at N-terminal reverse-transcribed with HiScript 1­ 32® II Q RT SuperMIX for qPCR (Vazyme, Nanjing, China). qPCR was performed with ­AceQ® qPCR ­SYBR® Green Master Mix (Vazyme, Nanjing, China) on a Roche ­LightCycle® 96 real-time PCR machine using the rnpB gene as a control (Yoshida et al. 2010). Primers for the qPCR amplification of phoX (accession no. ARI79942.1) and phoA (accession no. ARI81755.1) were designed according to their respective gene sequences. Primers for the rnpB gene were designed with reference to the research of Yoshida et al. (2010). The gene-specific primers used for phoX were phoX-1F (5-GAGGGGAAC CGACCGAGA-3′) and phoX-1R (5′-TGGCAAATACCC AAGCGC-3′); those for phoA were phoA-1F (5′-CAGGCG CAAC AGG AAA GTA C-3′) and phoA-1R (5′-CCAG AAC TT TGCCATCTTGCT-’); and those for rnpB were rnpB-F (5′TGCCACAGAAAAATACCGCC-3′) and rnpB-R (5′-CTC CACCTTGCTCCCCAC-3′). Results Bioinformatic characteristics of the putative PhoX of PCC7806 The putative PhoX (accession no. ARI79942.1) of PCC7806 consisted of 735 amino residues (Table 1), and alpha helixes were mainly distributed at its N-terminus. 13 Predictions of the posttranslational modification of the protein indicated that it contained numerous glycosylation and phosphorylation sites. The protein was a hydrophilic protein without a transmembrane domain but included an N-terminal Tat-way signal peptide (Fig. 1), which conformed to the characteristics of bacterial PhoX (Wu et al. 2007). To compare the similarity of phoX genes in cyanobacteria, based on the PhoX protein sequence of PCC7806, 106 similar proteins were found by Blastp searches in GenBank. A phylogenetic tree was constructed with the MEGA program. The tree showed that Microcystis PhoXs formed an independent branch (Fig. 2). Amino acid sequence analysis showed that phoX was conserved in Microcystis, and the identity was more than 98%. However, the conservation of phoX in cyanobacteria is low, even in unicellular cyanobacteria with close genetic relationships. For example, the identity of PCC7806 with Gloeocapsa sp. PCC 73,106, Synechococcus sp. PCC 7336, and Synechocystis sp. PCC6803 is only 57%, 52% and 52%, respectively. Expression and purification of the putative phoX An expression vector (pBRT7-7806) carrying the putative phoX gene and a control plasmid (pBRT7) were constructed successfully and verified by colony PCR, plasmid digestion, and plasmid sequencing (Fig. 3). 3 Biotech Page 5 of 12 (2021) 11:218 218 Fig. 2 The maximum likelihood tree of alkaline phosphatase protein PhoX in Cyanobacteria. The red part is Microcystis Figure 4 shows the results of Western blotting and SDSPAGE electrophoresis of the expressed protein. The results showed that the His-Trap eluent contained the target protein, which showed a size of approximately 80 kDa (Fig. 5), in accord with the theoretical value. The measurement of APase activities showed a value of approximately 57 U/L for the eluent with the expressed protein (Fig. 6), which was three times higher than the value for the control, and the catalytic activity was completely abolished by incubation at 100 °C for 15 min. These data confirmed that the expressed protein was an APase. Enzymatic property of the PhoX The measurement of APase activities showed that the optimal temperature and pH for PhoX were 37 °C and 10, respectively (Fig. 7). The substrate specificity analysis showed that PhoX presented phosphodiesterase and phosphomonoesterase activities (Table 2). Divalent cations were necessary for the catalytic activity of PhoX, and the order of catalytic activity was M ­ g2+, ­Co2+, ­Ca2+, ­Zn2+ and ­Mn2+. In 2+ contrast, ­Ni inhibited PhoX activity (Table 3). 13 218 Page 6 of 12 3 Biotech (2021) 11:218 Fig. 3 Colony identification by PCR and plasmid pBRT7-7806 identification by restriction. A Colony PCR, M marker; C PCR product of a colony. B Restriction identification, M marker; R restriction map of plasmid pBRT7-7806 by Eco32 I Fig. 4 Western-bolt (up) and SDS-PAGE (down) of expression protein of the putative phoX gene in E. coli strain BL21(DE3). The target protein is about 88 KDa. Lane M: protein marker. A Bacteria with control plasmid (pBRT7); B bacteria with expression plasmid (pBRT7-7806); Lane A1 and B1, total proteins of bacteria without IPTG. Lane A2 and B2, cytoplasmic supernatant without IPTG. Lane A3 and B3, total proteins of bacteria with 0.4 mM IPTG. Lane A4 and B4, cytoplasmic supernatant with 0.4 mM IPTG. Lane B4 show the target protein PhoX is overexpressed induced by IPTG in E. coli with pBRT7-7806 Expression of Microcystis APase genes under different P conditions Based on BLAST searches of alignment in NCBI, a putative phoX gene (accession no. ARI79942.1) and a putative phoA gene (accession no. ARI81755.1) were found in PCC7806, but no phoD gene was found. 13 In P-sufficient BG11 medium, the expression of phoX in PCC7806 cells was very low, while in P-deficient BG11(-P) medium, phoX expression increased almost ten-fold at 2 h and then decreased to a level similar to that in BG11 cells at 4 h (Fig. 8A). phoX expression was also induced by AMP, and its activity in AMP medium reached the highest level at 4 h, when it was 2.5 times as 3 Biotech Page 7 of 12 (2021) 11:218 Fig. 5 SDS-PAGE of the purified protein of the putative phoX gene in E. coli strain BL21 (DE3). Lane M, protein marker. C, E. coli with control plasmid (pBRT7); D, E. coli with expression plasmid (pBRT7-7806); Lane C1 and D1, cytoplasmic supernatant. Lane C2 and D2, proteins recovered by His-tag affinity chromatography from C1 and D1, respectively. D2 shows the target protein (square box). 218 Fig. 7 The enzyme properties of expressed PhoX. A The optimum temperature, B optimum pH. Error bars indicate SD of three independent biological replicates Table 2 Substrate specificity of Microcystis PhoX Substrates Pi liberated relative to that from ­PNPPa PNPP Sodium pyrophosphate β-Glycerophosphate ATP AMP G-6-P 100 261 ± 0.29 107 ± 0.24 100 ± 0.13 46 ± 0.10 39 ± 0.09 a The Pi released from PNPP is set at 100%. All results are the mean of three experiments. The SEMs are also provided Fig. 6 Effect of high temperature on the enzyme activity of purified proteins from E. coli with control (pBRT7) and expression plasmid (pBRT7-7806). A Room temperature; B the purified proteins were denatured at 100 ℃ for 15 min. Error bars indicate SD of three independent biological replicates. Asterisks indicate t-test significant differences (NS no significant; **, P < 0.01). APA alkaline phosphatase activity high as that in BG11, and a higher level was maintained than in BG11 and BG11(-P). The expression pattern of the putative phoA gene (intracellular APase) was very different from that of extracellular APase phoX, and a high expression level of phoA appeared after 4 h in BG11(-P) cells (Fig. 8B). The total APase activity of Microcystis varied depending on the P nutrition supply and time duration (Fig. 8C). In p-deficient medium, total APase activity was high, while in organophosphorus medium, APase activity was induced at 13 218 Page 8 of 12 3 Biotech (2021) 11:218 Table 3 The ability of various divalent metal ions to restore the enzymatic activity to the apoenzyme of PhoX Metal ion (2 mM) APase activity restored (%)a Ca2+ Mn2+ Co2+ Ni2+ Mg2+ Zn2+ None 221.64 ± 2.14 112.98 ± 2.25 427.70 ± 6.43 −162.77 ± 7.42 585.71 ± 1.46 123.81 ± 2.07 100.00 ± 5.86 a Results are expressed as the percentage of activity restored when compared with the enzyme that is not added mental ions. All results are the means of three experiments. The SEMs are provided 4 h and then decreased to a lower level within 2 h, possibly due to the hydrolysis of organophosphorus to compensate for the shortage of P. These results imply that Microcystis could quickly respond to the supply of organophosphorus in the environment. Discussion PhoX in Microcystis and other microorganisms APase plays a key role in the P metabolism of cyanobacteria and has attracted the attention of researchers. Harke et al. (Harke et al. 2012; Harke and Gobler 2013) found a putative phoX gene in M. aeruginosa and showed that higher expression of the gene was induced by P stress, but the authors did not provide the gene sequence. We searched sequences similar to those of Harke’s phoX primers in GenBank using BLAST and found penicillin-binding proteins in M. aeruginosa NIES843 (accession no. BAG00368.1) and PCC7806 (accession no. ARI82939.1). Disappointingly, neither of the two genes matched the characteristics of phoX. Lin et al. (2018) analysed 32 cyanobacterial strains according to gene similarity (> 60%) and found that phoX-like genes were common in cyanobacteria, including three strains of M. aeruginosa. However, these putative phoX genes were not confirmed by experiments. It is worth mentioning that the PCR primers in Lin’s article did not match any phoX gene sequence found in 19 Microcystis genomes in GenBank. Therefore, if these primers are used to measure PhoX levels in Microcystis blooms, the levels would be seriously underestimated. In an analysis based on the identified phoX gene of PCC7806, we found conservation of the phoX gene in all 19 sequenced Microcystis strains. In particular, two M. wesenbergii strains (Fig. 2) showed very conserved phoX genes, which have not been previously reported. There have been some reports on phoX in bacteria and cyanobacteria (Hallmann 1999; Kathuria and Martiny 2011; Quisel et al. 1996; White 2009), and a putative phoX gene 13 Fig. 8 Relative expression of phoX and phoA, and total alkaline phosphatase activity. A The expression of phoX gene in medium, BG11(P), BG11, and BG11(-P + AMP). B The expression of the phoA gene in medium, BG11(-P), BG11,and BG11(-P + AMP). C Total alkaline phosphatase activity of Microcystis in different medium, BG11(-P), BG11, BG11(-P + AMP). Error bars indicate SD of three independent biological replicates. Asterisks indicate t test significant differences. (NS no significant; *, P < 0.05; **, P < 0.01). APA alkaline phosphatase activity 3 Biotech (2021) 11:218 Page 9 of 12 218 Fig. 9 The model of PhoX by swissmodel. B The enlarged version of A, in which the green site is the C ­ a2+ binding site Fig. 10 The model result of Microcystis PhoX is also found in Microcystis (Harke et al. 2012; Harke and Gobler 2013; Lin et al. 2018). However, in research on the abundance of phoX in the environment, conserved bacterial sequences have generally been used to design PCR primers, which results in the omission of Microcystis phoX, and some results have shown that there is no correlation between the abundance of phoX and Microcystis blooms in lakes (Cao et al. 2005; Dai et al. 2014). Our results should provide useful data for the accurate evaluation of phoX in Microcystis blooms occurring in water. 13 218 Page 10 of 12 The specificity of PhoX in Microcystis Studies have shown that extracellular Apases in different species require different divalent cations. In the archaebacterium Haloarcula marismortui, the complete inhibition of extracellular APase by EDTA can be reversed by adding ­Ca2+ ions but not by adding Z ­ n2+, ­Mn2+, or ­Mg2+ (Goldman et al. 1990). Similarly, in the green alga Volvox carteri and the azotobacter Sinorhizobium meliloti, only ­C a 2+ can reactivate EDTA-inactivated APase, whereas the other divalent metal cations cannot (Hallmann 1999; Zaheer et al. 2009). In Pasteurella multocida X-73, although ­Ca2+ was shown to be the best metal ion for restoring PhoX apoenzyme activity, C ­ o 2+ and N ­ i 2+ resulted in approximately equal activities (reactivation: ­Ca2+ 132%, C ­ o2+ 126% and N ­ i2+ 122%, respectively) (Wu et al. 2007). The PhoX proteins of bacteria usually contain a conserved calcium-binding domain (Wu et al. 2007; Zaheer et al. 2009), and Microcystis PhoX also contains a nonconserved calcium-binding site (data not listed, see Appendix), as shown by SwissModel (https://swissmodel. expasy.org). However, its apoenzyme produced the highest activity when combined with M ­ g 2+ and displayed different levels of dependence on the other divalent cations, which demonstrated that PhoX in Microcystis is unique. Phosphorus metabolism Cyanobacteria have developed several strategies for responding to a low P supply, including compensating for their need for P using cellular phosphates and/or synthesizing phosphate transporters to enhance P acquisition (Orchard et al. 2009; Tetu et al. 2009; Van Mooy et al. 2006) and increasing the affinity and rate of P uptake (Mazard et al. 2012). The P-specific transport (Pst) system includes a periplasmic P-binding protein (PstS) that sensitively reacts to a lack of P. The ability to use the DOP pool, mediated by APase, is an additional strategy for P acquisition. When P (Pitt et al. 2010) is abundant, microbes store excess P in the DOP pool as polyphosphate (poly P), and poly P is decomposed when microbes are in a P-deficient environment (Garbisu et al. 1993). Harke et al. (2012) and Lin et al. (2018) studied the responses of APase genes to environmental P in Microcystis. In their studies, samples were taken every other day, and their results showed the responses over a longer period of time. In this study, we took samples every 2 h to observe the more detailed responses of APase genes to P conditions in the environment. Our results showed that in organophosphorus medium, phoX expression was obviously higher than that of phoA (Fig. 8A, B), which 13 3 Biotech (2021) 11:218 implied that Microcystis increased extracellular APase secretion to decompose organic substrates in the environment. In P-deficient medium, phoA could be induced to decompose the poly-P substrates stored in cells. 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