| Phosphate fertilizers are immobilized as calcium/magnesium phosphate (Ca-P and Mg-P) in calcareous soils and aluminum/iron phosphate (Al-P and Fe-P) in acidic soils, with plant use ratio below20%. Long-term and excess application of chemical phosphate fertilizers cause rock phosphate (RP) resource exhausted and non-point source pollution. Phosphate fertilizer pollution and discharge of all kinds of phosphate-containing sewage derived from production and living give rise to destructive eutrophication of water bodies and algae bloom. The phosphate solubilizing microorganisms (PSMs) and polyphosphate (polyP) accumulating organisms (PAOs) have great potentials in activating immobilized phosphate in soils and in removal of phosphate in water respectively. At present, however, most of the strains have been observed to solubilize Ca-P well, but they often have a low or even no capacity to solubilize other phosphates, such as Mg-P, Al-P, Fe-P and RP. And mechanisms for microbial solubilization of different phosphates are not known well. Meanwhile, few typical pure culture PAOs are reported, and little attention has been paid to their polyP accumulating features and metabolic regulation mechanisms. Strain An2capable of efficiently solubilizing various phosphates and strain PAO19of a typical PAO were obtained in this study. Characteristics and mechanisms for phosphate solubilization by strain An2and features and mechanisms for polyP accumulating metabolism regulation were investigated to lay the foundation for efficient utilization of phosphate fertilizers and control of eutrophication with microbial approaches.Strain An2was isolated from Chinese cabbage rhizosphere soil and identified as Aspergillus niger. At optimum phosphate concentration and incubation time, up to1722,2066,2356,215and179mg L-1of phosphorus (P) was release by An2from Ca-P, Mg-P, Al-P, Fe-P and RP, respectively. HPLC assay demonstrated that An2mainly secreted oxalic acid to solubilize Ca-P, Mg-P, Al-P and Fe-P, whereas secreted tartaric acid to solubilize RP. During efficient phosphate solubilization, An2could be satisfied with low concentrations of diverse carbon and nitrogen sources, and tolerate salinity, acidity and metal ions. Furthermore, An2could produce indole-3-acetic acid and antagonize Fusarium phytopathogens. The soil experiment proved that An2could efficiently activate the immobilized phosphates in general calcareous, acidic as well as saline-alkali soils. Therefore, An2is an efficient, stress-tolerant and multifunctional PSM, with great value in development and application.Strain PAO19was isolated from maize rhizosphere soil and identified as Arthrobacter nicotianae. Incubated in synthetic wastewater medium for12h, PAO19was characterized by5.2%P content on dry weight and7mg P L-1of polyP. PAO19had the most identity to A. arilaitensis Re117in polyP kinase1(PPK1) gene and its amino acid sequences. Phosphate concentration, temperature and salinity were discovered to be key environmental factors affecting polyP accumulating capacity of PAO19. The genome size of PAO19was3.64M, with59.5%GC content, including3451open reading frames (ORFs),2336ORFs annotated in Kyoto Encyclopedia of Genes and Genomes (KEGG) database. PAO19was discovered to possess two sets of phosphate limitation two-component regulatory system PhoR-PhoB and SenX3-RegX3.Regulation mechanisms for polyP accumulation metabolism of PAO19in response to phosphate concentration and temperature were explored with transcriptome sequencing (RNA-seq) method. Setting the high concentration of phosphate-normal temperature condition (10mg P L-1,30℃) in favor of polyP accumulation as the control, differentially expressed genes of PAO19under low concentration of phosphate (2mg P L-1,30℃) and high temperature (10mg P L-1,37℃) conditions adverse to polyP accumulation were studied, respectively. Under low concentration of phosphate condition, PhoR-PhoB and SenX3-RegX3were both activated, with the core component alkaline phosphatase PhoA and PhoD expressing at high level, in response to low Pi stress; Meanwhile, Pi transporter PstSCAB was upregulated significantly, in favor of Pi transport. Under high temperature condition, PhoR-PhoB and SenX3-RegX3were both inhibited, with PhoA and PhoD expressing at very low level, in response to high Pi environment; Meanwhile, Pi transporter PstSCAB was downregulated significantly, adverse to Pi transport. Under two conditions of low concentration of phosphate and high temperature, limited intracellular Pi gave rise to lack of ATP and polyP, and their synthesis enzymes including F-type ATPase PPK1and polyP-glucokinase (PPGK) were upregulated and the degradation enzymes including exopolyphosphatase PPX and PPK2were downregulated for positive feedback metabolism regulation. Under two conditions, most enzymes involved in glycolysis, TCA cycle and oxidative phosphorylation were upregulated significantly, for positive feedback regulation of ATP production. Meanwhile, most enzymes and proteins involved in pentose phosphate pathway, DNA replication and transcription and protein synthesis were upregulated significantly, demonstrating that limited Pi preferentially satisfied cell growth. Mechanisms for polyP accumulating metabolism regulated by Pi concentration and temperature of strain PAO19were preliminarily illuminated in this study. |
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