Reimmann C, Rella M, Haas D
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
J Gen Microbiol. 1988 Jun;134(6):1515-23. doi: 10.1099/00221287-134-6-1515.
R68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2.1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a 'cut-and-paste' mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely (less than 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.
R68.45及其他携带2.1 kb插入元件IS21串联重复序列的类似广宿主范围（IncP）质粒可动员许多不同革兰氏阴性细菌的染色体。为了分析R68.45 - 染色体共整合体的结构（此前已推测其参与了转移过程），我们选择使R68.45样质粒稳定整合到铜绿假单胞菌染色体中。选择了两种质粒：pME28，一种转移缺陷型、可动员的RP1衍生物，其复制控制（trfA）基因无活性；以及pME487，一种带有trfA（ts）突变导致温度敏感复制的R68.45衍生物。发现染色体整合的pME28和pME487两侧各有一个IS21元件。这种结构与R68.45转座的“剪切粘贴”模式一致。pME28和pME487显示出低插入特异性，但很少（小于0.1%）诱导营养缺陷型突变。通过pME487或pME28的染色体整合可获得铜绿假单胞菌的高频重组（Hfr）供体；在后一种情况下，pME28缺乏的转移功能必须由自主质粒反式提供。与R68.45供体相比，Hfr供体具有更高的接合连锁性，并且转移的铜绿假单胞菌染色体片段更长。这表明在铜绿假单胞菌中，R68.45整合到供体染色体中的情况是短暂的。
