Identification and characterization of alkaline phosphatase gene phoX in Microcystis aeruginosa PCC7806.

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 Ca2+, Microcysits PhoX was most strongly activated by Mg2+, followed by Co2+, Ca2+, Zn2+ and Mn2+, but it was inhibited by Ni2+. 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.

Microcystin-LR heterologous genetically engineered antibody recombinant and its binding activity improvement and application in immunoassay.

Microcystin-LR (MC-LR) is a high-toxic biohazard that pollutes ecological environment and agroproducts. In this study, a newly recombined genetically engineered antibody (AVHH-MVH) with higher thermal stability and binding activity was designed by chain shuffling and based on our previously obtained anti-MC-LR scFv and nanobody. Based on AVHH-MVH template, a capacity of 8.99 × 105 CFU/mL of phage display AVHH-MVH mutagenesis library was constructed by site-directed mutagenesis in MVH-CDR3 region, and then used for ultrasensitive mutants screening. Afterwards, a total of five positive AVHH-MVH mutants were isolated from the mutagenesis library, and their binding activity was higher than AVHH-MVH for MC-LR. The AVHH-MVH mutant 3 was cloned into pET-25b vector for soluble expression, and the concentration of target protein expressed in culture system was 43.5 mg/L. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was established based on purified AVHH-MVH mutant 3 protein, and it showed ultrasensitive binding activity for MC-LR with the detection limit of 0.0075 µg/L, which was far below the maximum residue limit standard of 1.0 µg/L in drinking water proposed by World Health Organization. The established IC-ELISA shows good accuracy, repeatability, stability and applicability for MC-LR spiked samples, and it is promising for MC-LR ultrasensitive monitoring.

Response of extracellular and intracellular alkaline phosphatase in Microcystis aeruginosa to organic phosphorus.

Cyanobacterial blooms caused by Microcystis have become a menace to public health and water quality in the global freshwater ecosystem. Alkaline phosphatases (APases) produced by microorganisms play an important role in the mineralization of dissolved organic phosphorus (DOP) into orthophosphate (Pi) to promote cyanobacterial blooms. However, the response of extracellular and intracellular alkaline phosphatase activity (APA) of Microcystis to different DOP sources is poorly understood. In this study, we compared the growth of M. aeruginosa on two DOP substrates (ß-glycerol-phosphate (ß-GP) and lecithin (LEC)) and monitored the changes of P fractions and the extra- and intracellular APA under different P sources and concentrations. M. aeruginosa can utilize both ß-GP and LEC to sustain its growth, and the bioavailability of LEC was greater than ß-GP. For the ß-GP treatment, there was no significant difference in the algal growth at different concentrations (P > 0.05), while the algal growth in the LEC treatment groups was significantly affected by concentrations (P < 0.05). The results showed that intracellular APA of M. aeruginosa could be detected in all DOP treatment groups and generally higher than extracellular APA. In addition, the intracellular APA per cell increased first and then decreased in all DOP treatment groups. Compared with the ß-GP treatment, M. aeruginosa in the LEC groups could secret more extracellular APA.

Cell wall surface layer (S-layer) promotes colony formation in Microcystis: comparison of S-layer characteristics between colonial and unicellular forms of Microcystis and function conformation.

Colony is a key to Microcystis becoming a dominant population and forming blooms. To find the mechanism of colony formation, we investigated cell wall structures of colonial and unicellular strains. Results showed that colonial strains had significant surface layer protein (S-layer) on the surface of cells than unicellular strains by transmission electron microscopy. Western blot showed colonial strains had more S-layer than the unicellular strains. When the S-layer gene (GenBank accession number CAO89090.1) of Microcystis aeruginosa PCC7806 was expressed in Synechocystis sp. PCC6803, PCC6803 aggregated into colonial morphology. The results indicated that the S-layer could promote colony formation in Microcystis. Based on the S-layer sequences of PCC6803 and PCC7806, nine S-layer genes in other Microcystis strains were screened from the GenBank. Sequence comparing showed that the S-layers conserved regions were all located in N-terminal. The S-layers contain repeats-in-toxin (RTX) sequences with Ca2+-binding site, and their amino acid composition, hydrophobicity, isoelectric point, etc. were consistent with the characteristics of RTX-type S-layer in bacteria.