Gliesche C G, Hirsch P
Institut für Allgemeine Mikrobiologie, Universität Kiel, Germany.
Can J Microbiol. 1992 Nov;38(11):1167-74. doi: 10.1139/m92-191.
Spontaneously derived antibiotic-resistant mutants of Hyphomicrobium facilis B-522, a restricted facultative methylotroph, occurred at a high frequency on agar plates with low antibiotic concentrations. Mutants specifically defective in methanol oxidation have been obtained using an allyl alcohol direct selection technique. By chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine in the presence of chloramphenicol several stable auxotrophic mutants could be isolated: three leucine auxotrophs, two threonine auxotrophs, and two leucine-methionine double auxotrophic mutants. Optimal conditions for transposon mutagenesis have been developed by comparing several transposon delivery vectors. With the suicide plasmid pRK2013 as a vector, the tetracycline resistance conferring transposon Tn5-132 was introduced into the genome of H. facilis B-522. The following insertion mutants have been obtained: leu-3::Tn5-132, ilv-1::Tn5-132, and pur-1::Tn5-132. Broad host range IncP-1 plasmids could be successfully transferred by interspecific matings. Chromosome mobilization was demonstrated with the conjugative IncP-1 plasmids RP1, R68.45, pMO60, and H. facilis 2189 (leu-2, met-1, mox-1, nfs-1, str-12) as recipient strain. Transconjugants occurred at frequencies ranging from 10(-6) to 10(-8) for each marker.
易变迟缓根瘤菌B - 522是一种受限的兼性甲基营养菌，其自发产生的抗生素抗性突变体在低抗生素浓度的琼脂平板上高频出现。利用烯丙醇直接筛选技术获得了甲醇氧化存在特异性缺陷的突变体。通过在氯霉素存在的情况下用N - 甲基 - N'-硝基 - N - 亚硝基胍进行化学诱变，可分离出几个稳定的营养缺陷型突变体：三个亮氨酸营养缺陷型、两个苏氨酸营养缺陷型以及两个亮氨酸 - 蛋氨酸双营养缺陷型突变体。通过比较几种转座子传递载体，已开发出转座子诱变的最佳条件。以自杀质粒pRK2013为载体，将赋予四环素抗性的转座子Tn5 - 132导入易变迟缓根瘤菌B - 522的基因组。已获得以下插入突变体：leu - 3::Tn5 - 132、ilv - 1::Tn5 - 132和pur - 1::Tn5 - 132。通过种间交配可成功转移广泛宿主范围的IncP - 1质粒。以接合型IncP - 1质粒RP1、R68.45、pMO60以及易变迟缓根瘤菌2189（leu - 2、met - 1、mox - 1、nfs - 1、str - 12）作为受体菌株，证明了染色体转移。每个标记的接合子出现频率在10^(-6)到10^(-8)之间。
