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
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