Locus determining the synthesis of delta-aminolevulinic acid in Escherichia coli K-12
Locus determining the synthesis of delta-aminolevulinic acid in Escherichia coli K-12
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确定大肠杆菌 K-12 中 δ-氨基乙酰丙酸合成的基因座
DOI：
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发表时间：
1968
影响因子：
3.2
通讯作者：
T. Horodniceanu
中科院分区：
文献类型：
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作者：
A. Sasarman;M. Surdeanu;T. Horodniceanu
通过使用青霉素进行选择，Wulff(9)最近能够分离出大肠杆菌K-12的一些需要6-氨基乙酰丙酸的突变体。在分离的1500个营养不良突变体中，只有一个被证明是5-ALA-。对该突变位点的初步研究表明，该突变位点位于染色体前苏段(9)。大肠杆菌K-12的6-氨基乙酰丙酸需要突变体也已通过新霉素分离出来(5)，并显示出Ncfmutants（正常集落形成缺陷；4）的特征。据推测，这些突变体在血红素合成的第一步有缺陷；因此，他们可以被归类为Hemmutants。与其他Hemmutants中受影响的位点不同，参与5-氨基乙酰丙酸合成的hemA位点不能与lac位点共转导。对分离的5-ALAmutants之一（SHSP8）的遗传研究表明，受影响的位点靠近trp和cysB位点，hemA位点被证明是可共转导的。这种定位与Wulff(9)指出的不同。为了研究SHSP8突变体，我们进行了交配和转导实验。所用菌株列于表1。根据Wollman和Jacob(8)描述的方法进行偶联实验，并用同一研究者描述的方法(7)中断交配。Plkc噬菌体按照Lennox(3)的方法进行转导。这些配对实验的结果见表2。如果hemA位点位于大肠杆菌K-12染色体的前3段，那么Hfr P4x6菌株可能会在近端注射hemA位点。同时，对照实验表明，Hfr P4x6菌株确实在近端注射了前-thr段。使用不同的供体（Hfr SHSH1） re1现地址：蒙特利尔大学微生物与免疫学系，Case Postale 6128, Montreal 3, Canada。揭示了在对照交配表明供体菌株正常行为的情况下，hemA和trp标记之间存在连锁。为了明确hemA和trp标记的空间关系，将hemA8突变转移到trpstrain中获得了另一个hemA突变体（SHSP18），并进行了交配实验。在这样的实验中，与位于相同距离的远端标记相比，已知近端标记与未选择标记之间的连锁频率更大。通过使用供体近端注射trp标记，选择trp+和hemnA+重组并分析未选择标记的频率（表2）。虽然这些结果似乎表明了染色体上标记的a (gal), hemA, trp, （cysB）序列，但它们并不是非常确定的。Plkc噬菌体的转导结果见表3。同样，在trp和cysB标记上，同样类型的连锁也很明显（共转导频率分别为6.4和2.8%）。尽管在hemA、trp和cysB标记物的序列上，trp和cysB标记物的共转导频率的差异并不是特别具有决定性的，但对这些类型的转导剂的分析证明在这方面是非常显著的（表4）。在选择trp+时，假设序列II，第2类出现的概率（4次交叉）低于第1类出现的概率（2次交叉）。然而，序列II似乎不太可能，因为第2类记录的频率为4.5%，第1类记录的频率为1.8%。在选择cysB时，在序列I的假设下，第6类（2次交叉）出现的概率高于第5类（4次交叉）；同样，所记录的频率，即第6类(1)为4.4%，第5类为1.5%，与序列1一致。互导的结果，选择hemA+标记，指向相同的方向。在序列I的假设下，类9（4次交叉）的出现概率低于类10（2次交叉），而在序列II的假设下，类9（4次交叉）的出现概率低于类10（2次交叉）
By using penicillin for selection, Wulff (9) was recently able to isolate some 6-aminolevulinic acid-requiring mutants of Escherichia coli K-12. Of 1,500 auxotrophic mutants isolated, only one proved to be 5-ALA-. Preliminary investigations concerning the locus involved in this mutation resulted in its localization in the pro-thr segment of the chromosome (9). 6-Aminolevulinic acid-requiring mutants of E. coli K-12 have been isolated also by means of neomycin (5) and have displayed the characters of Ncfmutants (normal colony formation deficient; 4). Presumably, these mutants are defective in the first step of heme synthesis; consequently, they may be classed as Hemmutants. In contradistinction to the loci affected in the other Hemmutants, the hemA locus, involved in the synthesis of 5-aminolevulinic acid, could not, however, be cotransduced with the lac locus. A genetic study of one of the 5-ALAmutants isolated (SHSP8) showed that the locus affected lies close to the trp and cysB loci, with which the hemA locus proved to be cotransducible. This localization differs from that indicated by Wulff (9). To study the SHSP8 mutant, we performed mating and transduction experiments. The strains used are listed in Table 1. Conjugation experiments were carried out according to the method described by Wollman and Jacob (8), and mating was interrupted by the method described by the same investigators (7). Transduction by Plkc phage was carried out according to the method of Lennox (3). The results of these mating experiments are given in Table 2. The hemA locus was not injected proximally by the Hfr P4x6 strain, as might have been expected if it were situated in the pro-thr segment of the chromosome of E. coli K-12. At the same time, control experiments showed that the Hfr P4x6 strain does inject the pro-thr segment proximally. The use of a different donor (Hfr SHSH1) re1 Present address: Departement de Microbiologie et d'Immunologie, Universite de Montreal, Case Postale 6128, Montreal 3, Canada. vealed the existence of linkage between the hemA and trp markers under circumstances in which control matings indicated a normal behavior of the donor strain. To specify the spatial relationship of the hemA and trp markers, mating experiments were carried out with another hemAmutant (SHSP18) obtained by transfer of the hemA8 mutation to a trpstrain. In such experiments, the frequency of linkage between an unselected marker and the selected marker is known to be greater for proximal markers, compared with that of distal markers situated at the same distance. By using a donor injecting the trp marker proximally, trp+ and hemnA+ recombinants were selected and analyzed for the frequency of the unselected marker (Table 2). Although, these results seem to indicate a (gal), hemA, trp, (cysB) sequence of the markers on the chromosome, they are not very conclusive. The results of transduction by Plkc phage are given in Table 3. Again, the same type of linkage becomes apparent with the trp and cysB markers (frequencies of cotransduction 6.4 and 2.8%, respectively). Although the difference in cotransduction frequency between the trp and cysB markers was not particularly conclusive with regard to the sequence of the hemA, trp, and cysB markers, an analysis of the classes of transductants proved highly significant in this respect (Table 4). In selection for trp+, class 2 has a lower probability of appearance (four crossovers) than class 1 (two crossovers) on the assumption of sequence II. However, sequence II seems unlikely, since the frequencies recorded are 4.5% for class 2 and 1.8% for class 1. In selection for cysB, the probability of appearance is higher for class 6 (two crossovers) than for class 5 (four crossovers) on the assumption of sequence I; again, the frequencies recorded, i.e., 4.4% for class 6 (1) and 1.5% for class 5, are in agreement with sequence I. The result of reciprocal transduction, with selection for the hemA+ marker, points in the same direction. On the assumption of sequence I, class 9 (four crossovers) has a lower probability of appearance than class 10 (two crossovers), whereas on the assumption of sequence II class