Functional Analysis of Plasmid pSAl and Its Derivatives in Streptomyces azureus

天青链霉菌14921(PK0)含有一个或更少的pSA1(8.8kb)及其原始序列，在不含pSA1的菌株上产生麻点的频率为0.1%~1%。菌株PK100携带20-30个拷贝的pSA1.1(8.8kb)，是pSA1的衍生物，以100%的频率诱发花斑。菌株PK10携带双质粒(pSA1.1、pSA1.2)，并以10%~50%的频率诱发麻点。PSA1.2(7.6kb；拷贝大小为5~10)含有pSA1.1的两个缺失部分(约1.2kb和30b)，但不具有麻点形成能力。其中一个或两个缺失片段似乎对麻点的形成是必不可少的。PK100菌株产孢量和硫链菌素含量很低，PK0和PK10产生量正常，无质粒菌株产生量丰富。这些结果表明，pSA1.1对二次产物的抑制和高POCK的形成是由于高拷贝的pSA1.1所致。来自PK10的pSA1.1在转化子和PK100中扩增了20~30个拷贝，并抑制了它们的孢子和硫链霉菌的产生，而PK10的拷贝大小较小(10~20)，没有观察到抑制作用。这些结果表明，缺陷的pSA1.2似乎抑制了pSA1.1在宿主细胞上的能力。半胱氨酸也抑制了pSA1.1的活性。在含有pSA1.1的菌株中发现了一种相对分子质量约为7万的特异性蛋白。利用pSA1.2和pBR322或pUC13的所有独特酶切位点，可以很容易地从pSA1.2和pBR322或pUC13中获得杂交质粒。但是，利用它们的BamHI位点从pSA1.1和pBR322中只获得了一个杂种。这些结果表明，缺陷型pSA1.2的序列在宿主-载体系统中很容易被扩增，而形成麻点的pSA1.1则不能。PSA1.1和pSA1.2的这些不同特征可能是由于pSA1.2中靠近BamHI位点的30b缺失片段所致。

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.