Barrett J T, Croft R H, Ferber D M, Gerardot C J, Schoenlein P V, Ely B
J Bacteriol. 1982 Aug;151(2):888-98. doi: 10.1128/jb.151.2.888-898.1982.
Chromosomal insertions of Tn5 in Caulobacter crescentus displayed complete stability upon transduction and proved useful in strain building on complex media. RP4-primes constructed in vitro containing C. crescentus genomic sequences in the HindIII site of the kanamycin resistance gene failed to show enhanced or directed chromosome mobilization abilities. One of these kanamycin-sensitive RP4 derivatives, pVS1, was used as a mobilization vector in conjugation experiments on complex media where chromosomal Tn5 transfer to the recipient was selected. pVS1-mediated transfer of Tn5-induced auxotrophic mutations occurred at frequencies of 10(-6) to 10(-8) per donor cell. During conjugation with Tn5-encoded kanamycin resistance as the selected marker, Tn5 remained in its donor-associated locus in 85 to 100% of the transconjugants. A collection of eight temperature-sensitive donor strains bearing Tn5 insertion mutations from various regions of the C. crescentus genetic map were used to provide a rapid means for the determination of the map location of a new mutation. Use of the techniques described in this paper allowed an expansion of the C. crescentus genetic map to include the relative locations of 32 genes.
在新月柄杆菌中，Tn5的染色体插入在转导后表现出完全稳定性，并证明在复杂培养基上构建菌株时很有用。在体外构建的RP4引物，其卡那霉素抗性基因的HindIII位点含有新月柄杆菌基因组序列，但未显示出增强的或定向的染色体转移能力。这些对卡那霉素敏感的RP4衍生物之一，pVS1，在复杂培养基的接合实验中用作转移载体，其中选择将染色体Tn5转移到受体中。pVS1介导的Tn5诱导的营养缺陷型突变的转移频率为每个供体细胞10^(-6)至10^(-8)。在以Tn5编码的卡那霉素抗性作为选择标记进行接合期间，在85%至100%的转接合子中，Tn5保留在其与供体相关的位点。使用来自新月柄杆菌遗传图谱各个区域的八个携带Tn5插入突变的温度敏感供体菌株的集合，为确定新突变的图谱位置提供了一种快速方法。使用本文所述的技术使新月柄杆菌遗传图谱得以扩展，包括32个基因的相对位置。
