Cloning And Characterization Of Genes Related To Polyphosphorus Accumulation From Strain Pseudomonas Putida GM6

Eutrophication of water bodies is one of the ten serious global environmental problems.Nitrogen and phosphate, especially phosphate is the key contributor to eutrophication ofwater bodies. Discharge of untreated domestic sewage is one of the major sources of thephosphorus in the water bodies. In order to eliminate the phosphorus pollution, it isessential to treat the sewage to reduce its P concentration before it is discharged into rivers.Currently most large scale wastewater treatment plants use enhanced biological phosphorusremoval(EBPR) process characterized by less sludge generation, freedom of chemicals andeconomical operation, which is extensively used in countries all over the world. PAOs playkey roles in EBPR system to remove phosphorus. Therefore, it is quite necessary tocharacterize the regulation of related genes in PAOs.Pi-accumulation-related ppk gene, ppx gene, phoU gene, phaZ gene of strain GM6,which has high capability of accumulating phosphorus, were cloned and analyzed by geneknock-out in this study.â… . Cloning and characterization ofppk gene from Pseudomonas putida GM6The polyphosphate kinase gene (ppk) with a length of 2 220 bps was cloned fromGM6 by SEFA-PCR. The deduced amino acid sequence of PPK showed a significanthomology to that of Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1,Acinetobacter sp. ADP1 and Escherichia coli K12-MG1655 with 89%, 80%, 58% and 33%identities, respectively. Through homologous recombination, appk- mutant with deletion of1000 bps segments was obtained after dual exchange.The mutants and wild type strains were cultured under aerobic conditions onlow-phosphate medium and synthetic wastewater, respectively. The results showed that theamount of phosphorus assimilated by per milligram of cell dry weight decreased along withthe increase of original OD_600nm. Poly-P grannuals were checked in both mutant and wild strains by staining. Accumulation of poly-P could be observed in wild strain while not inmutant strain. Since ppk gene cloned in this study catalyzed the synthesis oflong-chain-length poly-P, and the short-chain-length poly-P synthesized by a second ppkgene (ppk2) might also be an important cause for cellular phosphorus accumulation.â…¡. Cloning and characterization ofppx gene from Pseudomonas putida GM6Based on method of ppk gene cloning, the downstream of ppk gene encodingexopolyphosphatase gene (ppx) was amplified. The sequence of this gene showed thepresence of an open reading frame (ORF) encoding a 690-amino-acid protein. The ORFbegan with an ATG start codon and ended with a UGA terminate codon. The translatedamino acid sequence of this ORF showed 88%, 84%, and 83% identities with that of PPXfrom P. fluorescens PfO-1, P. putida KT2440, and P. syringae pv. syringae B728a,respectively, according to an alignment performed with BLAST. A mutant with 500 bpsdeletion inppx gene was obtained by homologous recombination.The mutant and wild strains were cultured under aerobic conditions on low-phosphatemedium and synthetic wastewater, respectively. There was no essential difference betweenthe amount of phosphorus assimilated by per milligram of cell dry weight in two strains, aswell as the efficiency of phosphorus removing and growth. When the original OD_600nm wasunequally, the amount of phosphorus assimilated by per milligram of cell dry weight turnedinto inverse related. Poly-P granules were observed in the wild-type strain and mutants bypoly-P staining and transmission electron microscopy in the experiment of phosphatereleasing under anaerobic conditions, and the number of poly-P granules in wild type strainwas more than that in the mutant. Moreover, the amounts of PHAs in the two strainsincreased gradually along with the increase of phosphorus amount released in the strain, butboth the rate of phosphorus releasing and the proportion of synthesized PHAs to the drycell weight in strain GM6 were much higher than that in the mutant 114.Thus, we could presume that there was another ppx gene (ppx2) in strain GM6, whichcould also catalyze hydrolyzing poly-P into phosphate, and the hydrolyzed poly-P wasshort-chain-length.â…¢. Cloning, characterization of phoU gene and alkaline phosphatase activityfrom Pseudomonas putida GM6 and KT2440We constructed phoU mutants of KT2440 using the same method of ppk geneknocking out. The mutant and KT2440 were inoculated onto the agarplates of MOPS(X-Pi)with Pi-limitation and Pi-excess, and the mutant formed blue-colored colonies instead of white-colored under Pi-excess conditions. It revealed that after strain KT2440 lost functionof phoU gene, alkaline phosphatase could be expressed under high phosphate conditions.This result proved that the phoU gene was one of the negative regulatory genes of the phoregulon of P. putida GM6. By measuring the growth and alkaline phosphatase activity ofwild-type strain and the mutant, we could find that the growth of mutant was lower thanthat of KT2440 in LB medium, and the alkaline phosphatase activity of the mutant inPi-limitation and Pi-excess mediums was higher than that of wild-type strain.Then the genes ofpstSCAB -phoU from KT2440 were cloned, and expressed vectorwas constructed which was then transferred into strain GM6 formed a expression strain.However, the expression strain still showed blue-colored colonies on agar plates ofMOPS(X-Pi) with Pi-excess medium. The results suggested that phoU gene evenphoU-pstSCAB genes could not restrain the expression of alkaline phosphatase in strainGM6.According to the conservative sequences of phoU gene from the reportedPseudomonas strains, we obtained phosphate transport regulating system genes aboutpstSCA -phoU. And the translated amino acid sequence of phoU gene showed 92%identity with P. putida KT244 PhoU.â…£. Cloning and characterization ofphaZ gene from Pseudomonas putida GM6PHA related genes phaC1-phaZ-phaC2 was cloned from GM6, two PHA synthasegenes, phaC1 and phaC2, which were separated by the PHA depolymerase encoding thephaZ gene. The length of phaZ gene was about 852 bps, and the translated amino acidsequence of phaZ gene showed 84% identity with R putida PhaZ, 87% identity withPseudomonas sp. PC17 PhaZ, and 88% identity with P. fluorescens Pro-1 PhaZ. In courseof constructing homologous recombination vector and exchanging two homologous arms,we obtained phaZ^- mutant named D7.When the mutant and wild-type strain were inoculated into LB and low-phosphatemediums, we could find that the growth of the mutant was less than the wild-type strain inthe two mediums. PHA granules could be observed in the two strains by Sudan-blackstaining method during the experiment of phosphate releasing under anaerobic conditions.There were also many PHA granules in the cells of the two strains observed bytransmission electron microscopy. It indicated that the strain once lost phaZ gene, it wouldlead to more PHA production. After measuring the concentration of phosphorus in solution,the results were that the rate of releasing phosphorus in strain GM6 was more significant than in the mutant D7 and after the same time the two strains all achieved the maximum ofthe phosphorus release under the condition of maintaining the original OD_600nm Of the twostrains. The amounts of PHAs in the two strains increased gradually along with the increaseof phosphorus release amount in the strain, but the ratio of synthesized PHAs to the dry cellweight in strain GM6 was lower than in strain D7.After strain GM6 and D7 were grown in synthetic wastewater under aerobicconditions, we found that the growth of them was similar to that in LB medium. Moreover,although the amount of phosphorus accumulated by per cell dry weight of both strains wasincreased gradually, the maximum of accumulated phosphorus of wild type strain wasabout double of that of the mutant. In addition, electron micrographs of thin sections ofGM6 and D7, grown for 10 h in synthetic wastewater, showed that the number of poly-Pgranules in wild type strain cells was more than that in the mutant cells.