Phosphate-Absorbing Characterization Of A Marine Polyphosphate-Accumulating Bacterium, Halomonas YSR-3

Halomonas sp. YSR-3 not only belongs to genus Halomonas, but also can accumulate polyphosphate (polyP) in its cells. This trait is not reported in other strains of the genus Halomonas and this special trait may be important in the research of the phosphorus circulation in the marine system. The ability of strain YSR-3 phosphate-absorbing may be applied in the process of the biological phosphorus removal of seawater. We also researched on the optimum conditions for forming intracellular granules, the phosphate-absorbing characterization, cloning and analyze of D-hydantoinase gene sequence and nucleoside diphosphate kinase (NDPK) gene sequence as the template of strain YSR-3 total DNA.A polyphosphate-accumulating bacterium YSR-3 was isolated from the Yellow Sea. The cell is motile and rod-shape, 3.5μm×1μm, Gram negative, aerobic. Phosphate in culture media is absorbed and stored as the form of polyP polymer in cells. On the basis of 16S rDNA analyze, strain YSR-3 belongs to genus Halomonas ofγ- Proteobacteria. The cells are susceptible to antibiotics Kn and Cl. The reaction of amylase is positive, but denitrification is negative. The cells can use glucose as sole carbon source and energy source but can not degrade chitin. The cells can be cultivated well as following conditions: 3% (w/v) NaCl, 24℃, 180 rpm, and initial pH 6.5.Under various culture conditions the growth and phosphorus absorption of the marine bacterium Halomonas YSR-3 was studied. In phosphorus-free medium, theΔOD480 of the cells was almost invariable and cells didn't store polyP granules. YSR-3 grew better and had higher ratio of accumulating-polyP cells in medium containing sodium phosphate than potassium phosphate. When the concentration of KH2PO4 was 1 mmol/L, YSR-3 grew well and absorbed more phosphate with more intracellular polyP granules. When the medium pH value was 5, the stored intracellular polyP granules had not been metabolized. However, when the value was 6, 7 or 8, YSR-3 grew well and beyond 95% cells accumulated polyP, with decreasing phosphorus concentration in supernatant. When YSR-3 was incubated in high-phosphorus medium, the phosphorus concentration in supernatant decreased from 1.84 mmol/L to 1.14 mmol/L, absorbing 0.7 mmol/L phosphorus with the 37.5% phosphorus-absorbing ratio; when YSR-3 was incubated in low-phosphorus medium, the phosphorus concentration in supernatant decreased from 0.028 mmol/L to 0.008 mmol/L, absorbing 0.02 mmol/L phosphorus with the 72.2% phosphorus-absorbing ratio.D-hydantoinase gene sequence and NDPK gene sequence were amplified respectively by PCR technique as the template of YSR-3 total DNA and then inserted into pGM-T vector. The recombinant plasmids were transformed into E. coli TOP10 strain. The positive transformants with D-hydantoinase gene sequence or NDPK gene sequence were obtained by blue-white selection and clony PCR method, and then the positive ones were sequenced. The sequenced fragments of D-hydantoinase gene including 1510 bp and NDPK gene including 420 bp were analyzed by NCBI database. The similarity of D-hydantoinase gene sequences between YSR-3 and Pseudomonas entomophila L4 is 77%. The similarity of YSR-3 translatedD-hydantoinase gene sequence to the sequences of Pseudomonas fluorescens Pf-5, Marinomonas sp. MED121, Burkholderia vietnamiensis G4 is 75%, 73%, 70% respectively. The similarity of YSR-3 translated NDPK gene sequence to the sequences of Loktanella vestfoldensis SKA53, Jannaschia sp. CCS1, Roseobacter sp. CCS2 is 89%, 86%, 85% respectively.