Boos W, Steinacher I, Engelhardt-Altendorf D
Mol Gen Genet. 1981;184(3):508-18. doi: 10.1007/BF00352531.
The tetracycline resistance transposon Tn10 was inserted into the E. coli chromosome near mglB550, a structural gene for the galactose-binding protein. P1 transductions established the position of these Tn10 insertions (zee-700, 701, 702::Tn10) close to the genes ptsF, fpk, cdd, mglB550, his, and gatA with 85%-95%, 85%, 36%, 20%-40%, 12%-15%, and 0.5% cotransduction frequency. Three factor crosses revealed the relative sequence of the genes as: mglB550, zee-700::Tn10, ptsF, fpk, cdd, his, gatA was found to be 1.3% cotransducible with mglB550. Two Tn10 insertions near gatA were isolated and characterized. One, zef-704::Tn10, was 3% cotransducible with fpk, 8% with mglB550, and 42% with gatA. The other, zef-703::Tn10, was 98% cotransducible with gatA but not with mglB550 or fpk. Neither of these two Tn10 insertions was cotransducible with cdd. Four factor crosses revealed the sequence gatA, zef-704::Tn10, mglB550, fpk. Neither zee-700::Tn10 nor zef-703::Tn10 showed an (0/300) cotransduction with either glpT or gyrA. The clockwise order of genes is then: his, cdd, fpk, ptsF, zee-700::Tn10, mglB550, zef-704::Tn10, gatA. With a fix-point for his at 44 min, fpk would be placed at 45 min and mglB550 at 45.5 min. During the course of this work we noticed that the cotransduction frequency between Tn10 insertions and nearby markers tended to increase when new P1 lysates were prepared from freshly reisolated strains. This may indicate loss of nonessential genes adjacent to Tn10 insertions. Using insertion zee-703::Tn10, we isolated deletions extending into an mgl gene other than mglB. Crosses between such a deletion mutant and an mglB550 mutant were done. The analysis of the periplasmic proteins of these as well as other transductants or recombinants involving the mglB550 or the mglB551 gene revealed the existence of strains synthesizing both the wild-type as well as the corresponding mutant protein. Strains containing both proteins exhibit either wild-type or mutant phenotype. These strains appeared unstable. Upon reisolation from purified stock cultures kept in glycerol at -20 degrees C, colonies could be isolated that carried only mutant or wild-type protein.
四环素抗性转座子Tn10插入到大肠杆菌染色体中靠近mglB550（一种半乳糖结合蛋白的结构基因）的位置。P1转导确定了这些Tn10插入位点（zee - 700、701、702::Tn10）的位置，它们与ptsF、fpk、cdd、mglB550、his和gatA基因相邻，共转导频率分别为85% - 95%、85%、36%、20% - 40%、12% - 15%和0.5%。三因子杂交揭示了基因的相对顺序为：mglB550、zee - 700::Tn10、ptsF、fpk、cdd、his、gatA，发现zee - 700::Tn10与mglB550的共转导率为1.3%。分离并鉴定了gatA附近的两个Tn10插入位点。一个是zef - 704::Tn10，与fpk的共转导率为3%，与mglB550的共转导率为8%，与gatA的共转导率为42%。另一个是zef - 703::Tn10，与gatA的共转导率为98%，但与mglB550或fpk不共转导。这两个Tn10插入位点与cdd均不共转导。四因子杂交揭示了基因顺序为gatA、zef - 704::Tn10、mglB550、fpk。zee - 700::Tn10和zef - 703::Tn10与glpT或gyrA均未显示共转导（0/300）。基因的顺时针顺序为：his、cdd、fpk、ptsF、zee - 700::Tn10、mglB550、zef - 704::Tn10、gatA。以his位于44分钟为固定点，fpk将位于45分钟，mglB550位于45.5分钟。在这项工作过程中，我们注意到当从新重新分离的菌株制备新一批P1裂解物时，Tn10插入位点与附近标记之间的共转导频率往往会增加。这可能表明Tn10插入位点附近非必需基因的丢失。利用插入位点zee - 703::Tn10，我们分离出了延伸到除mglB之外的另一个mgl基因的缺失片段。进行了这种缺失突变体与mglB550突变体之间的杂交。对这些以及其他涉及mglB550或mglB551基因的转导子或重组体的周质蛋白分析表明，存在同时合成野生型和相应突变蛋白的菌株。含有这两种蛋白的菌株表现出野生型或突变型表型。这些菌株似乎不稳定。从保存在 - 20℃甘油中的纯化原种培养物中重新分离时，可以分离出仅携带突变型或野生型蛋白的菌落。
