CONSTRUCTION AND CHARACTERIZATION OF THE B2-LEVEL HOST OF THE BACILLUS SUBTILIS HOST-VECTOR SYSTEM

Abstract

B2-level host strains, HV514 (trpC2 metB5 leuA8 thr-5 sir^d spo0C9V uvrA114 dal-1 nonA nonB) and HV553 (trpC2 metB5 leuA8 thr-5 hisA1 argC4 sir^d spo0C9V uvrA114 dal-1 nonA nonB recE4), of Bacillus subtilis were constructed from the B. subtilis Marburg strain PS9, which has no restriction-modification enzyme system and requires six amino acids and one base. Genetic markers dal-1, uvrA114, spo0C9V, str^d and recE4 were introduced sequentially into strain PS9 by congression with purB⁺ and amino acid prototroph.
Survival of HV514 and HV553 in nonpermissive conditions was 10^-4 to 10^-5 lower than that of the wild type strain 168Trp. The sporulation frequency of HV514 and HV553 was less than 10spores/ml, while that of 168Trp was more than 10⁸spores/ml. The sensitivity of these strains to ultraviolet light was 10⁵ to 10⁶ higher than that of 168Trp. These strains have normal viability under permissive conditions and high protoplast transforming activity. These properties make the strains highly safe and useful hosts for recombinant DNA experiments.