Cobley J G, Zerweck E, Reyes R, Mody A, Seludo-Unson J R, Jaeger H, Weerasuriya S, Navankasattusas S
Department of Chemistry, University of San Francisco, California 94117.
Plasmid. 1993 Sep;30(2):90-105. doi: 10.1006/plas.1993.1037.
In some strains of cyanobacteria the composition of the light-harvesting antennae is determined by the color of available light. The mechanism of this chromatic adaptation involves the regulation of gene expression by red and green light and has been most studied in Fremyella diplosiphon (Calothrix sp. PCC 7601), a filamentous cyanobacterium for which there has been no reported means of genetic manipulation. We have constructed shuttle plasmids which can be efficiently mobilized by RP4 from Escherichia coli into F. diplosiphon and which can be recovered from transconjugant F. diplosiphon and returned to E. coli by transformation. The ability of these plasmids to replicate in F. diplosiphon is conferred by an 8.0-kb DNA fragment isolated from pFDA, a plasmid native to F. diplosiphon. To create these shuttle plasmids the 8.0-kb fragment was cloned into pJCF22, a mobilizable plasmid constructed from oriV and bom from pBR322, cat from pACYC184 and aphA from pACYC177.pJCF22 lacks sites for the restriction enzymes FdiI and II. Transconjugant F. diplosiphon containing shuttle plasmid pJCF62 are resistant to chloramphenicol and highly resistant to the aminoglycosides, G418 and neomycin. When aadA from the omega interposon was incorporated into a shuttle plasmid transconjugant F. diplosiphon could also be selected with streptomycin or spectinomycin. In F. diplosiphon shuttle plasmid pJCF62 replicates with a minimum copy number of seven. The oriV for replication in F. diplosiphon was localized to a 2.8-kb region within the cyanobacterial part of pJCF62. The presence on a shuttle plasmid of a single recognition site for FdiI reduced the efficiency of mobilization into F. diplosiphon by 5- to 10-fold. Restriction at this site was prevented when the E. coli donor strain in the mating contained the enzyme Eco47II methylase.
在某些蓝藻菌株中，光捕获天线的组成由可用光的颜色决定。这种色适应机制涉及红光和绿光对基因表达的调控，并且在双歧鱼腥藻（集胞藻属PCC 7601）中得到了最多的研究，双歧鱼腥藻是一种丝状蓝藻，尚未有关于其遗传操作方法的报道。我们构建了穿梭质粒，这些质粒可以被RP4从大肠杆菌高效转移到双歧鱼腥藻中，并且可以从转接合子双歧鱼腥藻中回收，并通过转化回到大肠杆菌中。这些质粒在双歧鱼腥藻中复制的能力由从双歧鱼腥藻天然质粒pFDA分离的一个8.0 kb DNA片段赋予。为了构建这些穿梭质粒，将8.0 kb片段克隆到pJCF22中，pJCF22是一个可转移质粒，由pBR322的oriV和bom、pACYC184的cat以及pACYC177的aphA构建而成。pJCF22缺乏限制性内切酶FdiI和II的识别位点。含有穿梭质粒pJCF62的转接合子双歧鱼腥藻对氯霉素有抗性，并且对氨基糖苷类抗生素G418和新霉素具有高度抗性。当来自ω插入序列的aadA被整合到穿梭质粒中时，转接合子双歧鱼腥藻也可以用链霉素或壮观霉素进行筛选。在双歧鱼腥藻中，穿梭质粒pJCF62以至少七个拷贝数进行复制。双歧鱼腥藻中复制的oriV定位于pJCF62蓝藻部分内的一个2.8 kb区域。穿梭质粒上单个FdiI识别位点的存在使转移到双歧鱼腥藻中的效率降低了5至10倍。当交配中的大肠杆菌供体菌株含有Eco47II甲基化酶时，该位点的限制作用被阻止。
