Keggins K M, Duvall E J, Lovett P S
J Bacteriol. 1978 May;134(2):514-20. doi: 10.1128/jb.134.2.514-520.1978.
Plasmid pSL103 was previously constructed by cloning a Trp fragment (approximately 2.3 X 10(6) daltons) from restriction endonuclease EcoRI-digested chromosome DNA of Bacillus pumilus using the neomycin-resistance plasmid pUB110 (approximately 2.8 X 10(6) daltons) as vector and B. subtilis as transformation recipient. In the present study the EcoRI Trp fragment from pSL103 was transferred in vitro to EcoRI fragments of the Bacillus plasmid pPL576 to determine the ability of the plasmid fragments to replicate in B. subtilis. Endonuclease EcoRI digestion of pPL576 (approximately 28 X 10(6) daltons) generated three fragments having molecular weights of about 13 X 13(6) (the A fragment), 9.5 X 10(6) (B fragment, and 6.5 X 10(6) (C fragment). Trp derivatives of pPL576 fragments capable of autonomous replication in B. subtilis contained the B fragment (e.g., pSL107) or both the B and C fragments (e.g., pSL108). Accordingly, the B fragment of pPL576 contains information essential for autonomous replication. pSL107 and pSL108 are compatible with pUB110. Constructed derivatives of the compatible plasmids pPL576 and pUB110, harboring genetically distinguishable EcoRI-generated Trp fragments cloned from the DNA of a B. pumilus strain, exhibited relatively high frequency recombination for a trpC marker when the plasmid pairs were present in a recombination-proficient strain of B. subtilis. No recombination was detected when the host carried the chromosome mutation recE4. Therefore, the recE4 mutation suppresses recombination between compatible plasmids that contain homologous segments.
质粒pSL103先前是通过使用新霉素抗性质粒pUB110(约2.8×10⁶道尔顿)作为载体,以短小芽孢杆菌经限制性内切酶EcoRI消化的染色体DNA中的一个色氨酸片段(约2.3×10⁶道尔顿)进行克隆构建而成,枯草芽孢杆菌作为转化受体。在本研究中,将pSL103中的EcoRI色氨酸片段体外转移至芽孢杆菌质粒pPL576的EcoRI片段中,以确定质粒片段在枯草芽孢杆菌中复制的能力。用EcoRI对pPL576(约28×10⁶道尔顿)进行酶切产生了三个片段,分子量分别约为13×10⁶(A片段)、9.5×10⁶(B片段)和6.5×10⁶(C片段)。能够在枯草芽孢杆菌中自主复制的pPL576片段的色氨酸衍生物含有B片段(如pSL107)或B和C片段(如pSL108)。因此,pPL576的B片段包含自主复制所必需的信息。pSL107和pSL108与pUB110兼容。构建的兼容质粒pPL576和pUB110的衍生物,含有从一株短小芽孢杆菌DNA中克隆的、在遗传上可区分的经EcoRI产生的色氨酸片段,当质粒对存在于枯草芽孢杆菌的重组能力强的菌株中时,对于trpC标记表现出相对较高频率的重组。当宿主携带染色体突变recE4时未检测到重组。因此,recE4突变抑制了含有同源片段的兼容质粒之间的重组。