The temperate bacteriophage P22 mediates both generalized and specialized transduction in Salmonella typhimurium. Specialized transduction by phage P22 is different from, and less restricted than, the well characterized specialized transduction by phage lambda, due to differences in the phage DNA packaging mechanisms. Based on the properties of the DNA packaging mechanism of phage P22, a model for the generation of various types of specialized transducing particles is presented that suggests generation of substantial numbers of specialized transducing genomes which are heterogeneous but only some of which have terminally redundant ends. The primary attachment site, ataA, for phage P22 in S. typhimurium is located between the genes proA,B and supQ newD. (The newD gene is a substitute gene for the leuD gene, restoring leucine prototrophy of leuD mutant strains.) The proA,B and supQ newD genes are very closely linked and thus cotransducible by generalized transducing particles. Specialized transducing particles can carry either proA,B or supQ newD but not both simultaneously, and thus cannot give rise to cotransduction of the proA,B and supQ newD genes. This difference is used to calculate the frequency of generalized and specialized transducing particles from the observed cotransduction frequency in phage lysates. By this method, very high frequencies of supQ newD (10-2/PFU)-and proA,B (10-/PFU)-specialized transducing particles were detected in lysates produced by induction of lysogenic strains. These transducing particles, most of which would have been produced by independent aberrant excision events (which include in situ packaging), were of various types.
The gene order and orientation in the leu-pyrA region of the Salmonella typhimurium linkage map was established by phage P22-mediated transductions. The gene order, in counterclockwise orientation, is leuO-leuA-leuB-leuC-leuD-ara-fol-pyrA . The fol locus is co-transducible with either the ara and leu loci or the pyrA locus, whereas no co-transduction for the ara and pyrA loci can be found.
Wfld type K. Sanderson; 45 araB9 P. Margolin; 27 JK433 srl-2::tet-517 strAr recAl a J. Roth JK245 leuD798ara gal-205proA575 P. Margolin JK74 leuD798ara fol-101 proAB47b J. Kemperc JK486 leuD798ara fol-101 proAB47 (P22sieAl h21) Lysogenaof JK74 JK79 leuD798ara fol-101 proB25 J. Kemperc JK447 leuD798ara fol-101 proB25 (P22tsc230 sieABI m3) Lysogenaof JK79 JK436 leuD798ara fol-101 proB25 srl-2::tet-517 strA' recAl Transductiona of JK79 by phage on JK433 JK450 leuD798ara fol-101 proB25 srl-2::tet-517 strAr recAl Lysogen' of JK436 (P22tsc230 sieAB1 m3) JK80 leuD798ara fol-101 proCiIOb J. Kemper' JK446 leuD798ara fol-101 proCIO srl-2::tet-517 strAr recAl Transductiona of JK80by phage on JK433 JK1394 leuD798ara supQ394 J. Kemper, 22 JK367 leuD798ara supQ394 (P22tsc229) Lysogen' of JK1394 JK480 leuD798ara supQ394 (P22tsc229 int3) Lysogen" of JK1394 a For origin and construction of these strains, see the text. bproAB47 and proCIO mutant strains were isolated by Demerec and obtained through K. E. Sanderson. "The construction of recombinant strains, integrating the leuD798ara mutation into the genome of different complex strains, utilized the fol-101 marker (conferring resistance to trimethoprim), which is cotransducible with the ara and leu operons (22).
Alpha-isopropylmalate isomerase, the second specific enzyme in the biosynthesis of leucine, is coded for by two genes, leuC and leuD. Leucine auxotrophs, harboring leuD mutations including a deletion of the entire leuD gene, revert to leucine prototrophy owing to mutations at a locus, supQ, substantially distant to the leucine operon. A large number of independently isolated supQ mutations were characterized. A significant increase in the spontaneous frequency of supQ mutations was found after mutagenesis with 2-aminopurine, N-methyl-N'-nitro-N-nitrosoguanidine, diethyl sulfate, and nitrous acid. The supQ function in most of these strains is temperature sensitive, resulting in more efficient suppression with decreasing temperature. At higher temperatures, the supQ limits the growth rate of leuD supQ mutant strains. All supQ mutations are co-transducible with proA and proB, with co-transduction frequencies ranging from 5.4 to 99.9% for different supQ mutations. Many supQ mutations were isolated, especially after nitrous acid mutagenesis, that had acquired a simultaneous proline requirement. The data support the idea of two genes, supQ and newD, whose protein products form a complex. The newD gene product, without any genetic alteration, is capable of substituting for the missing leuD protein. However, mutations in the supQ gene (point mutations or deletions) are necessary to make the newD protein available, which is normally tied up in a complex with the supQ protein.ductions have been described previously (9). For some experiments, a special complete medium was used that contains no leucine, but contains all other amino acids, all bases for deoxyribonucleic acid and ribonucleic acid synthesis, and six vitamins. This medium is similar to an arginine-free medium described by R. P. 937 on July 31, 2020 by guest http://jb.asm.org/ Downloaded from 938 KEMPER on July 31, 2020 by guest
The isopropylxnalate isomerase in Salmonella typhimurium is the second enzyme specific for leucine biosynthesis. It is a complex enzyme composed of two subunits which are coded for by two genes of the leucine operon, leuC and leuD. The two polypeptides have been shown to copurify through successive ammonium sulfate fractionations and have been identified on sodium dodecyl sulfate-polyacrylamide gels as having molecular weights of 51,000 (leuC gene product) and 23,500 (leuD gene product). They have also been shown to be fairly stable, since in vitro complementation of cell-free extracts of leuC and leuD mutant strains was demonstrated, with only a 40% loss of activity 16 h after preparation of the extracts. The native isopropylmalate isomerase was shown to have a Km for its substrate a-isopropyhnalate of 3 x 10-4 M. flr-19 leuD657 A(leuD-ara) 798 A(gpt-proBA-supQ-newD)47 fol-101IF' leu ara
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