Functional Analysis of Plasmid pSAl and Its Derivatives in Streptomyces azureus

azureus链霉菌ATCC 14921 （PK0）携带1个或更少的质粒pSA1拷贝（8.8kb），其原始序列也处于整合状态，在无质粒的菌株上以0.1 ~ 1%的频率诱发斑点。菌株PK100携带20 ~ 30个pSA1.1拷贝（8.8kb）， pSA1.1是pSA1的衍生物，并以100%的频率诱发痘。染色PK10携带两个质粒（pSA1.1, pSA1.2），以10 - 50%的频率诱发痘。质粒pSA1.2 （7.6kb，拷贝大小5 ~ 10）缺失了pSA1.1的两个片段（约1.2kb和30b长），没有形成袋的能力。其中一个或两个缺失片段似乎对麻袋的形成至关重要。PK100的孢子和硫链蛋白产量很低，PK0和PK10的孢子和硫链蛋白产量正常，而无质粒菌株的孢子和硫链蛋白产量丰富。这些结果表明，抑制二次产物和高麻袋形成是由于高拷贝的pSA1.1。来自PK10的质粒pSA1.1在转化子和PK100中扩增了20 - 30个拷贝，抑制了它们的孢子和硫链蛋白的产生，而在PK10中，拷贝大小较低（10 - 20），没有观察到抑制作用。这些结果表明，缺陷pSA1.2似乎会抑制pSA1.1对宿主细胞的作用。半胱氨酸也抑制了pSA1.1的表达能力。在具有pSA1.1的菌株中发现了一种特异性蛋白（分子量约为70,000）。从pSA1.2和大肠杆菌质粒pBR322或pUC13中利用所有独特的核酸内切酶位点很容易获得杂交质粒。但是，利用pSA1.1和pBR322的BamHI位点，只获得了一个杂种。这些结果表明，缺陷pSA1.2序列在大肠杆菌宿主-载体系统中容易扩增，而形成袋状的pSA1.1则不容易扩增。pSA1.1和pSA1.2之间的这些不同特征可能是由于pSA1.2中30b缺失片段靠近BamHI位点。

Streptomyces azureus ATCC 14921 (PK0) harbored one copy or less of plasmid pSA1 (8.8kb), also its primal sequences in an integrated state, and elicited pocks at 0.1 to 1% frequency on the plasmid-free strains. Strain PK100 carried 20 to 30 copies of pSA1.1 (8.8kb), a derivative of pSA1, and elicited pocks at 100% frequency. Stain PK10 carried two plasmids (pSA1.1, pSA1.2), and elicited pocks at 10 to 50% frequency. Plasmid pSA1.2 (7.6kb; copy size, 5 to 10) had two deleted portions (ca. 1.2kb and 30b long) of pSA1.1 together with no pock-forming ability. Either one or both of the deletion segments seem to be essential to the pock formation. The production of spores and thiostrepton was very low in PK100, normal in PK0 and PK10, and abundant in plasmid-free strains. These results show that the inhibition on the secondary products and the high pock formation are due to pSA1.1 in high copies. Plasmid pSA1.1 from PK10 amplified 20 to 30 copies in transformants as well as from PK100 and inhibited their spore and thiostrepton production, whereas, in PK10, copy size was lower (10 to 20) and no inhibition was observed. These results show that the defective pSA1.2 appears to depress the ability of pSA1.1 on the host cells. Cysteine also depressed the ability of pSA1.1. A specific protein (MW ca. 70,000) was found in the strains having pSA1.1.The hybrid plasmids were easily obtained from pSA1.2 and E. coli plasmid pBR322 or pUC13 using all the unique sites of endonucleases. But, only one hybrid was obtained from pSA1.1 and pBR322 using their BamHI sites. These results indicate that the sequence of defective pSA1.2 is easily amplified in the host-vector system of E. coli but not that of pock-forming pSA1.1. These different features between pSA1.1 and pSA1.2 may be due to the 30b-deletion segments in pSA1.2 near by BamHI site.