HASEGAWA Yoshie Kansai Univ., Faculty of Eng., Biotech., Associate Prof, 工学部, 助教授 (30156327)
OBATA Hitoshi Kansai Univ., Faculty of Eng., Biotech., Prof, 工学部, 教授 (00067646)
|Budget Amount *help
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1992: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1991: ¥1,100,000 (Direct Cost: ¥1,100,000)
A cosmic library containing Pseudomonas viridiflava KUIN-2 DNA was constructed in the vector Homer III and used to transform E. coli HB101. Five colonies (INAC-1-INAC-5) with ice-nucleating activity (INA) were obtained and all these transformants had in common an approximately 13.2-kb Eco RI fragment (pNVR-1). Among them, the transformant INAC-2, which harbored a single plasmid (pCH-1) and had an excellent INA, was selected for studying the characteristics of the INA^+ transformant. The INA in culture was not detectable until the bacterial titer reached 10^5 to 10^6 cells/ml.
A novel plasmid pNVR-1, a self-circularized DNA segment which consists of 13.2 kb-Eco RI fragment, was introduced into both E. coli JM109 and P. aeruginosa PAO1161 by transformation. Both of the strains harboring the pNVR-1 plasmid showed good INA and resistance to ampicillin. In addition, the optimum growth temperatures for these strains shifted to temperatures lower than those of strains without pNVR-1.
lava KUIN-2 originally had a single plasmid, called pINA, and three Eco RI fragments 13.2, 8.8, and 6.8 kb long were detected in pINA. The size of an EcoRI-digested fragment from pINA (13.2 kb) agreed with that of EcoRI-digested pNVR-1. From the results of Southern blotting with the probe pNVR-1, it was found that an EcoRI-digested fragment about 13.2 kb long from pINA hybridized with pNVR-1. Furthermore, E. coli JM109 harboring pINA had ice-nucleation activity, and the ice-nucleation temperatures, (T_<50>), of the transformations were -4.9 to -5.2. These results suggest that both the ampicillin-resistance gene and the ice-nucleation gene of P. viridiflava KUIN-2 were coded on pINA.
A mini-plasmid, pNVR-1012 (approximately 2.7 kb), was constructed from a novel plasmid pNVR-1 encoded ice-nicleation gene. This plasmid encoded an ampicillin resistance gene, and had unique restriction sites as EcoRI, Hind III, PstI, and SphI. To confirm the eligibility of pNVR-1012 as cloning vector, a 3.1 kb- Hind III fragment encoded for the salicylate hydroxylase (nahG) gene from NAH plasmid was ligated to pNVR-1012. The nahG gene was expressed in both E. coli and P. aeruginosa harboring this recombinant plasmid under the culture without salicylate. From the level of nahG expression in the recombinant strains, it is suggested that the origin of the expression of the nahG gene appear to derive from NAH plasmid promoter, and recombinant plasmid functions efficiently in P. aeruginosa rather than in E. coli. From these results, pNVR-1012 seems to be very useful for gene cloning vector in E. coli and P. aeruginosa. Less