The Response Of Alkaline Phosphatase Genes In The Cyanobacterium Anabeana Sp.FACHB 709 To Inorganic Phosphorus Starvation

Cyanobacteria are an ancient and diverse group of microorganisms found in many different ecosystems. In a long history of adaptation to the extreme or variable environments, cyanobacteria have developed more complicated regulatory mechanisms than those of other prokaryotes. In recent years, due to the eutrophication of water bodies, cyanobacteria often form blooms, even produce toxin. It has been a worldwide environmental problem that causes public attention. The extremely strong bioavailability of cyanobacterial cells for inorganic phosphorus plays a key role in the ecological success of cyanobacteria which causes occurance of cyanobacterial blooms. Phosphorus sources are extremely rich in eutrophic waters, but inorganic phosphorus, which can be directly used by cyanobacterial and other microorganism cells is limited. Studies on the phosphorus metabolic regulation in cyanobacteria are very important to reveal the mechanism for and to monitor the outbreak of algal blooms.An additional filamentous nitrogen-fixing cyanobacterium, Anabeana sp. FACHB 709 (hereinafter FACHB 709), was isolated from a freshwater lake in Wuhan, China (East lake). It is closely related to Anabeana sp. PCC 7120 and able to be genetically manipulated by using conjugal transfer system. In this research, characters of growth and alkaline phosphatases activity of FACHB 709 were investigated, and then the response of the FACHB 709 alkaline phosphatases to inorganic phosphorus starvation was discovered. So, genes encoding alkaline phosphatase were identified in FACHB 709. In order to elucidate their action mechanism in phosphorus metabolic regulation, the expression, regulation and physiological function of the identified alkaline phosphatase genes in FACHB 709 were analyzed under the conditions of different phosphorus sources.This research mainly obtained the following results:(1) The growth characteristics and response of the FACHB 709 to different phosphorus were studied. The results show that inorganic phosphorus starvation induces growth cessation but not cell death. FACHB 709 increase alkaline phosphatase activity in response to the inorganic phosphorus-limiting stress. Phosphorus-starved cells can utilize G-6-P and pNPP to support some growth in the absence of inorganic phosphorus. At the same time, the absorption of organic phosphorus will partially suppress the alkaline phosphatase activity of starved cultures.(2) The analysis of phylogenetic tree based on the 16S rDNA sequences of FACHB 709 and several other cyanobacteria prompts that FACHB 709 is very closed to Anabeana sp. PCC 7120 and the blooms-forming alga Anabaena variabilis ATCC 29413. The DNA sequences of putative alkaline phosphatase genes in FACHB 709 were amplified and cloned, then sequenced and analyzed. The results indicate that there are four alkaline phosphatase genes, phoA-709, phoD 1-709, phoD2-709 and phoS-709 exist in FACHB 709 cells.(3) The recombinant plamids with lacZ transcriptional fusions were constructed and introduced into FACHB 709 via conjugal transfer, producing strains FACHB 709 (pJS795), FACHB 709 (pTH70), FACHB 709 (pTH67), FACHB 709 (pTH68) and FACHB 709 (pTH64), respectively. By determining P-galactosidase enzyme activity of these strains, transcriptional regulation of four alkaline phosphatase genes was analyaed in the presence of different concentrations of inorganic or organic phosphorus conditions. The results indicate that under inorganic phosphorus-sufficient conditions, except phoD2-709, three other alkaline phosphatase genes are functionally expressed, but all four alkaline phosphatase genes, phoA-709, phoDl-709, phoD2-709 and phoS-709 were upregulated transcription to be involved in phosphorus metabolic regulation during phosphate starving, and the performance of phoA-709 was the best.(4) Using the immunological methods, the expression at the protein level and location of four alkaline phosphatase genes from FACHB 709 were studied. The results show that the four alkaline phosphatase genes were upregulated and their products were secreted to the extracellular medium in the absence of inorganic phosphorus.(5) The phoA-709 and phoS-709-inactivated mutants, DRTH65 and DRTH61of FACHB 709 were constructed, and the investigations into the growth and physiological phenotype of mutants were focused on. The results indicate that under inorganic phosphorus-sufficient and -limiting conditions, no difference was observed in the growth rate and the alkaline phosphatase activity between the wild-type strain and the mutant DRTH65 or DRTH61. So, the lacZ transcription fusion plasmids pTH67, pTH68 and pTH64 were introduced into the mutant strain DRTH65 to obtain the strains DRTH65 (pTH67), DRTH65 (pTH68) and DRTH65 (pTH64), respectively. And similarly, the lacZ transcription fusion plasmids pTH67, pTH70 and pTH64 were introduced into the mutant strain DRTH61 to obtain the strains DRTH61 (pTH67), DRTH61 (pTH70) and DRTH61 (pTH64), respectively. By detecting theβ-galactosidase enzyme activity of these strains, the transcriptional regulation of other three active alkaline phosphatase genes was studied. The results show that to some extent, the increased expression of phoD 1-709 and phoS-709 could counteract the phoA-709 inactivation in DRTH65 under experimental conditions, and similarly, expression of phoD1-709 and phoA-709 to some extent could counteract the phoS-709 inactivation in DRTH61 under experimental conditions.On the basis of above results, we conclude as follows:(1) Inorganic phosphorus is the major form of phosphorus source and a key growth factor for the Anabaena sp. FACHB 709 cells. Cyanobacterial cells increase alkaline phosphatase activity in response to the growth inhibition of phosphate starvation-induced cells.(2) Anabaena sp. FACHB 709 is closely related to Anabaena sp. PCC 7120. And this strain also has the same composition and highly homologous sequences of the alkaline phosphatase genes, phoA-709, phoDl-709, phoD2-709 and phoS-709 with Anabaena sp. PCC 7120.(3) Inorganic phosphorus starvation induced the upregulation of four alkaline phosphatase genes, phoA-709,phoDl-709, phoD2-709 and phoS-709 from Anabaena sp. FACHB 709 at the transcription and translation levels, but the increases of their transcription levels were different from one another. phoA-709 plays a crucial role in the phosphorus metabolic regulation of the FACHB 709 cells.(4) The alkaline phosphatase of Anabaena sp. FACHB 709, PhoA-709,PhoD1-709,PhoD2-709 and PhoS-709 all are secreted proteins. They are induced by inorganic phosphorus starvation and released out of the cells into the medium. (5) The mutational analysis showed that phoA-709 and phoS-709 play an important role in phosphate metabolism of cyanobacteria cells but not essential genes, and the function of alkaline phosphatases encoded by the genes, phoA-709, phoDl-709 and phoS-709 probably overlap or complement each other.