Espinosa M, López P, Lacks S A
Gene. 1984 Jun;28(3):301-10. doi: 10.1016/0378-1119(84)90147-1.
The pneumococcal mal recombinant plasmid pLS70, which carries two strong promoters for transcription, could not be transferred and maintained intact in Bacillus subtilis. Although it could be established at low frequency, pLS70 was unstable and was rapidly replaced by deleted forms of the plasmid. A deleted derivative plasmid, pLS69, could be transferred at high frequency and maintained intact. In pLS69 the deletion reduces function of both the malM (amylomaltase) and malX (X-fragment) promoters. This mutant mal plasmid still codes for an intact amylomaltase, and the enzyme is produced in both S. pneumoniae and B. subtilis. The amylomaltase, which is inducible by maltose in S. pneumoniae, is synthesized constitutively in B. subtilis and is localized in the cytosol. Although pLS69 enables S. pneumoniae to grow with maltose, the plasmid did not enhance the ability of B. subtilis to use this sugar, presumably because the latter does not transport free maltose into the cell. Minicells of B. subtilis containing pLS69 synthesized the amylomaltase polypeptide but no X-fragment. In S. pneumoniae carrying pLS69, production of the X-fragment is also reduced more than the amylomaltase, when compared to cells carrying pLS70, which produce equal amounts of the two proteins. Inasmuch as the down promoter mutation leaves unchanged both structural genes, their ribosome-binding sites and -10 and -35 promoter sequences, the unequal effect is attributed to differential reduction in AT composition proximal to the promoters. Vector proteins were revealed in minicells as several bands, all located in the cytosol except for an Mr 35000 polypeptide located in the membrane.
携带两个强转录启动子的肺炎球菌麦芽糖重组质粒pLS70无法在枯草芽孢杆菌中转移并完整维持。尽管它能以低频率建立，但pLS70不稳定，很快被质粒的缺失形式所取代。一种缺失衍生质粒pLS69能高频转移并完整维持。在pLS69中，缺失降低了malM（淀粉麦芽糖酶）和malX（X片段）启动子的功能。这种突变的麦芽糖质粒仍编码完整的淀粉麦芽糖酶，该酶在肺炎链球菌和枯草芽孢杆菌中均有产生。在肺炎链球菌中由麦芽糖诱导产生的淀粉麦芽糖酶，在枯草芽孢杆菌中组成型合成并定位于胞质溶胶中。尽管pLS69使肺炎链球菌能够利用麦芽糖生长，但该质粒并未增强枯草芽孢杆菌利用这种糖的能力，推测是因为后者不将游离麦芽糖转运到细胞中。含有pLS69的枯草芽孢杆菌微小细胞合成了淀粉麦芽糖酶多肽，但没有合成X片段。与携带产生等量两种蛋白质的pLS70的细胞相比，在携带pLS69的肺炎链球菌中，X片段的产生也比淀粉麦芽糖酶减少得更多。由于启动子下调突变使两个结构基因、它们的核糖体结合位点以及 -10和 -35启动子序列均未改变，这种不等效作用归因于启动子近端AT组成的差异减少。微小细胞中的载体蛋白显示为几条带，除了位于膜上的一条分子量为35000的多肽外，所有带均位于胞质溶胶中。
