Smith C J, Rogers M B, McKee M L
Department of Microbiology and Immunology, East Carolina University, Greenville, North Carolina 27858.
Plasmid. 1992 Mar;27(2):141-54. doi: 10.1016/0147-619x(92)90014-2.
Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.
脆弱拟杆菌和其他胃肠道拟杆菌是不同寻常的革兰氏阴性真细菌，因为当将来自其他革兰氏阴性真细菌的基因导入这些生物体时，这些基因不会表达。为了分析拟杆菌中的基因表达，开发了表达载体和启动子探针（检测）载体系统。表达载体的基本特征是并入了拟杆菌插入序列元件IS4351，其具有从其末端向外定向的启动子活性。插入到IS4351 DNA片段下游多克隆位点的基因很容易在脆弱拟杆菌中表达。对来自Tn9的氯霉素乙酰转移酶（cat）结构基因进行了测试，并赋予了脆弱拟杆菌氯霉素抗性。氯霉素抗性和CAT活性均显示依赖于IS4351启动子。用大肠杆菌β-葡萄糖醛酸酶基因（uidA）也获得了类似的结果，但活性仅为cat基因表达水平的30%。还检测了两个四环素抗性决定簇，即来自无乳链球菌的tetM和来自大肠杆菌的tetC。tetC未导致可检测到的四环素抗性，但革兰氏阳性tetM基因赋予了拟杆菌宿主对四环素和米诺环素的高水平抗性。基于cat基因的结果，构建了含有无启动子cat基因的启动子探针载体。这些载体用于从部分基因组文库中克隆随机的脆弱拟杆菌启动子，重组体在脆弱拟杆菌宿主中表现出一系列的CAT活性和氯霉素最低抑菌浓度（MIC）。此外，还测试了已知的大肠杆菌启动子（Ptet、Ptac、Ptrc、Psyn和P1P2rrnB）在脆弱拟杆菌中的活性。在脆弱拟杆菌中用这些启动子未观察到氯霉素抗性或CAT活性。
