Nassif X, Puaoi D, So M
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
J Bacteriol. 1991 Apr;173(7):2147-54. doi: 10.1128/jb.173.7.2147-2154.1991.
The ability to study the virulence of pathogenic Neisseria spp. has been greatly limited by the absence of genetic tools which allow the construction of defined mutants. We have engineered a transposon system which allows random mutagenesis of the Neisseria genome at relatively high frequency. Tn1545-delta 3 is a 3.4-kb derivative of the gram-positive transposon Tn1545 encoding resistance to kanamycin. Tn1545-delta 3 was subcloned into an erythromycin-resistant derivative of the mobilizable shuttle vector pLES2 to yield the plasmid pMGC20. This latter plasmid was introduced by conjugation from Escherichia coli S17-1 into Neisseria meningitidis 8013N and Neisseria gonorrhoeae 15063G. Kanamycin-resistant 8013N and 15063G transconjugants appeared at frequencies of 10(-5) and 10(-6), respectively. Restriction enzyme analysis and Southern blot hybridization of these transconjugants showed that, in Neisseria spp., the transposon excised spontaneously from pMGC20 and integrated into chromosomal DNA. Our studies revealed that (i) transposition of Tn1545-delta 3 was in numerous, apparently distinct sites, (ii) in most cases, for each transconjugant a single copy of Tn1545-delta 3 was integrated into the chromosome, and (iii) several passages on selective media did not induce secondary transposition. The kanamycin resistance marker expressed by the transconjugants was subsequently transformed into a parental background without change in the chromosomal location of the transposon. To assess the role of the general recombination system in the transposition of Tn1545-delta 3, the recA gene of N. meningitidis has been cloned and a rec derivative of 8013N has been engineered. Similar results were obtained when this latter strain was used as recipient, suggesting that recA function were not required for Tn1545-delta 3 transposition in N. meningitidis. Transposition with Tn1545-delta 3 may be an important technique for mutagenesis of the pathogenic neisseriae.
由于缺乏构建特定突变体的遗传工具，对致病性奈瑟菌属细菌毒力的研究能力受到了极大限制。我们构建了一个转座子系统，该系统能够以相对较高的频率对奈瑟菌基因组进行随机诱变。Tn1545 - delta 3是革兰氏阳性转座子Tn1545的一个3.4 kb衍生物，编码对卡那霉素的抗性。Tn1545 - delta 3被亚克隆到可移动穿梭载体pLES2的一个耐红霉素衍生物中，产生质粒pMGC20。通过从大肠杆菌S17 - 1进行接合转移，将后一种质粒导入脑膜炎奈瑟菌8013N和淋病奈瑟菌15063G。耐卡那霉素的8013N和15063G接合子出现的频率分别为10^(-5)和10^(-6)。对这些接合子进行的限制性内切酶分析和Southern印迹杂交表明，在奈瑟菌属中，转座子从pMGC20上自发切除并整合到染色体DNA中。我们的研究表明：(i) Tn1545 - delta 3的转座发生在众多明显不同的位点；(ii) 在大多数情况下，每个接合子中Tn1545 - delta 3的单个拷贝整合到染色体中；(iii) 在选择性培养基上传代几次并未诱导二次转座。接合子表达的卡那霉素抗性标记随后被转化到亲本背景中，转座子的染色体位置没有改变。为了评估通用重组系统在Tn1545 - delta 3转座中的作用，脑膜炎奈瑟菌的recA基因已被克隆，并且构建了8013N的rec缺陷衍生物。当使用后一种菌株作为受体时获得了类似的结果，这表明recA功能对于脑膜炎奈瑟菌中Tn1545 - delta 3的转座不是必需的。用Tn1545 - delta 3进行转座可能是对致病性奈瑟菌进行诱变的一项重要技术。
