OVERPRODUCTION OF THE ATP-DEPENDENT NUCLEASE ADDAB IMPROVES THE STRUCTURAL STABILITY OF A MODEL PLASMID SYSTEM IN BACILLUS-SUBTILIS

被引:13
作者
MEIMA, R [1 ]
HAIJEMA, BJ [1 ]
VENEMA, G [1 ]
BRON, S [1 ]
机构
[1] UNIV GRONINGEN, GRONINGEN BIOMOLEC SCI & BIOTETECHNOL INST, DEPT GENET, 9751 NN HAREN, NETHERLANDS
来源
MOLECULAR AND GENERAL GENETICS | 1995年 / 248卷 / 04期
关键词
STRUCTURAL PLASMID INSTABILITY; DELETION FORMATION; ADDAB; DELETION MUTAGENESIS; GENE AMPLIFICATION;
D O I
10.1007/BF02191638
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The effect of the ATP-dependent exonuclease AddAB complex on the structural stability of plasmid pGP1 in Bacillus subtilis was studied. Using deletion mutagenesis and gene amplification techniques, B. subtilis strains were constructed either lacking or overproducing the AddAB complex, a key enzyme in homologous recombination. The deletion mutant possessed no residual ATP-dependent nuclease activity; in contrast, the nuclease activity was up to 30 times higher in lysates of strains carrying multiple copies of the addAB genes in the chromosome. Southern blot analyses of these strains indicated that a linear relationship exists between the number of chromosomal gene copies and the level of AddAB activity. The structural stability of pGP1 was analyzed in the AddAB-deficient and over-producing backgrounds. Frequencies of deletion formation in the plasmid, as monitored by the expression of the pGP1-encoded penP-lacZ fusion on media containing X-gal, were shown to be increased at least 25-fold in the addAB knock-out mutant, whereas the stability of pGP1 was improved up to 15-fold in strains overproducing the AddAB enzyme. A possible explanation for these findings is that interactions between AddAB and plasmid molecules prevent the formation of secondary structures that constitute potential deletion target sites, and thereby enhance the structural stability of plasmids.
引用
收藏
页码:391 / 398
页数:8
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