Schoenlein P V, Gallman L M, Ely B
Department of Biology, University of South Carolina, Columbia 29208.
Gene. 1988 Oct 30;70(2):321-9. doi: 10.1016/0378-1119(88)90204-1.
Cloning vectors for studies of Caulobacter crescentus genes should be transferrable between Escherichia coli and C. crescentus since a transformation system has not been developed for C. crescentus. We have tested a large number of vectors containing IncP or IncQ replicons and found that many of the vectors containing IncQ replicons, and all but one of the vectors containing IncP replicons, are readily transferred by conjugation into C. crescentus. All of the plasmids tested were maintained in C. crescentus at 1 to 5 copies per cell, but plasmids containing IncP replicons were more stable than plasmids containing IncQ replicons. Further studies with a derivative of the IncQ plasmid R300B showed that when a promoterless kanamycin (Km)-resistance gene (npt2) was inserted into the intercistronic region of the sul-aphC (SuR-SmR) operon, Km resistance was expressed only when the npt2 gene was inserted such that it would be transcribed from the sul promoter. These data indicate that R300B does not contain sequences which would provide promoter function in C. crescentus in the orientation opposite to that of the sul operon and that any genes cloned in this orientation would require native promoters for expression. To provide greater versatility for cloning into R300B, additional vectors were constructed by the addition of multiple cloning sites in the intercistronic region of the sul-aphC operon. In addition, chromosomal DNA libraries were constructed in R300B and in the cosmid vector pLAFR1-7. Specific clones from these libraries containing genes of interest were identified by complementation of the appropriate C. crescentus mutants.
由于尚未开发出针对新月柄杆菌的转化系统，用于研究新月柄杆菌基因的克隆载体应能在大肠杆菌和新月柄杆菌之间进行转移。我们测试了大量含有IncP或IncQ复制子的载体，发现许多含有IncQ复制子的载体，以及除一个之外的所有含有IncP复制子的载体，都能通过接合轻易转移到新月柄杆菌中。所有测试的质粒在新月柄杆菌中以每个细胞1至5个拷贝的数量维持，但含有IncP复制子的质粒比含有IncQ复制子的质粒更稳定。对IncQ质粒R300B的衍生物进行的进一步研究表明，当一个无启动子的卡那霉素（Km）抗性基因（npt2）插入到sul-aphC（SuR-SmR）操纵子的基因间区域时，只有当npt2基因以能从sul启动子转录的方向插入时，Km抗性才会表达。这些数据表明，R300B不包含在与sul操纵子相反方向上能在新月柄杆菌中提供启动子功能的序列，并且以这种方向克隆的任何基因都需要天然启动子来表达。为了在克隆到R300B时提供更大的通用性，通过在sul-aphC操纵子的基因间区域添加多克隆位点构建了额外的载体。此外，还在R300B和粘粒载体pLAFR1-7中构建了染色体DNA文库。通过对适当的新月柄杆菌突变体进行互补，从这些文库中鉴定出了包含感兴趣基因的特定克隆。
