[Cloning and orientation of a promoter of thermophilic Thiobacillus sp].

Thiobacillus sp. is an obligate autotrophic thermophilic bacterium which was isolated from an acidic hot spring in Yunnan Province. Its optimum growth temperature is 45-50 degrees C and its optimum pH is 2.0-3.0. Using DNA recombinant technique, we inserted the HindIII fragments of the Thiobacillus sp. chromosomal DNA into the HindIII site of promoter-probe plasmid pSDSI (AprTcs, 5.65 kb). Transformants resistant to tetracycline were obtained on Tc plates (12 micrograms/ml). Of these, twenty transformants were able to grow on 120 micrograms/ml Tc plates, and two of them, designated pSDH7 and pSDH11, were able to grow on plates containing Tc at concentration up to 360 micrograms/ml. With HindIII, pSDH11 produced a 0.95kb fragment which had the function of promoter and a PstI site besides the 5.65 kb fragment of pSDSI. Southern blot hybridization showed that the 0.95 kb insert was from the Thiobacillus sp. chromosomal DNA. After restriction mapping, a 2.85 kb fragment of pSDH11 (which contained 0.7 kb of the inserted fragment) was removed with the aid of Pst1, and the remained fragment was used to construct a 3.75 kb plasmid (named pSDH114) which was resistant to a higher level of tetracycline (360 micrograms/ml) than for pBR322 (120 micrograms/ml). The remained 0.25 kb foreign fragment in pSDH114 still retained full function of the promoter contained in the original 0.95 kb. Thus we could orient the cloned promotor function fragment (0.25 kb) from Thiobacillus sp. in pSDH114.