Hedstrom R C, Funk C R, Kaper J B, Pavlovskis O R, Galloway D R
Infect Immun. 1986 Jan;51(1):37-42. doi: 10.1128/iai.51.1.37-42.1986.
We have cloned a gene from Pseudomonas aeruginosa that stimulates the expression of exotoxin A. A recombinant library of genomic DNA from strain PA103 constructed with a broad-host-range plasmid vector containing chromosomal insert fragments generated by Sau3A was used to transform the hypotoxigenic mutant strain PA103-29. A recombinant plasmid, pFHK6, was isolated from a PA103-29 transformant which displayed increased toxin production. From pFHK6, which contained a 20-kilobase-pair chromosomal insert, a 3-kilobase-pair XhoI fragment was isolated and subcloned into the plasmid cloning vector pVK101 to give pFHK10. In toxigenic P. aeruginosa strains containing pFHK10, toxin expression was increased 10-fold and high levels of iron in the culture medium only partially inhibited the overproduction. Expression studies suggested that pFHK10 did not contain the toxin structural gene. In addition, Southern analysis with the 3-kilobase-pair XhoI fragment suggested that the putative toxin regulatory gene is common among different strains of P. aeruginosa including previously reported nontoxigenic strains.
我们从铜绿假单胞菌中克隆了一个能刺激外毒素A表达的基因。用一种广泛宿主范围的质粒载体构建了来自PA103菌株的基因组DNA重组文库，该载体含有由Sau3A产生的染色体插入片段，用于转化低毒突变菌株PA103 - 29。从一个毒素产生增加的PA103 - 29转化子中分离出一个重组质粒pFHK6。从含有一个20千碱基对染色体插入片段的pFHK6中，分离出一个3千碱基对的XhoI片段，并亚克隆到质粒克隆载体pVK101中，得到pFHK10。在含有pFHK10的产毒铜绿假单胞菌菌株中，毒素表达增加了10倍，并且培养基中的高铁水平仅部分抑制了过量产生。表达研究表明pFHK10不包含毒素结构基因。此外，用3千碱基对的XhoI片段进行的Southern分析表明，推定的毒素调节基因在包括先前报道的无毒菌株在内的不同铜绿假单胞菌菌株中是常见的。
