Reciprocal Transduction Research Articles

Two-step method for curing Escherichia coli of ColE1-derived plasmids

To cure Escherichia coli for plasmids derived from the ColE1 replicon advantage is taken of the fact that maintenance of this replicon requires a wild-type allele of polA, encoding DNA polymerase I. Curing is achieved by cotransduction of a mutant polA allele with metE::Tn 10, fadAB::Tn 10 or other transposon insertions near polA. Reciprocal transduction to Met + Pol + or to Fad + Pol + ensures reestablishment of the original genotype except for loss of the plasmid. A set of useful bacterial strains is provided.

Polymorphism in Escherichia coli: rtl atl and gat regions behave as chromosomal alternatives.

Amongst forty wild strains of Escherichia coli, sixteen utilized galactitol (as did K12) and seven utilized ribitol (as did C) of which six utilized D-arabitol; none utilized all three polyols. Transduction of genes for ribitol utilization (rtl+) to strains able to utilize galactitol (gat+), whether K12 or wild strains, using wild strains and E. coli C as donors always resulted in loss of the galactitol phenotype. The genes for D-arabitol use (atl+) were always cotransduced with rtl+ in interstrain crosses. We confirm and extend the mapping of gat+ (Lengeler, 1977) and rtl+ atl+ (Reiner, 1975) in their respective hosts, K12 and C, by showing both regions to be 50% cotransducible with metG and 3% cotransducible with fpk. In reciprocal transductions, gat+ replaced rtl+ atl+. In partial diploids, rtl+ atl+ and gat+ regions did not interfere with each other's expression. Transfer of rtl+ from an Rtl+ Atl- donor by R plasmid (pE10)-mediated conjugation, gave Gat- transconjugants of K12 in which rtl+ and a kanamycin resistance gene were 100% cotransducible in the metG region of the chromosome. It is suggested that the rtl+ atl+ and gat+ genes (or parts of them) act as alternative, or mutually exclusive, regions in the chromosome. Possible reasons for the existence of alternative characters are discussed.

Arginine gene cluster of Serratia marcescens.

Biochemical and genetic studies on the arginine-requiring auxotrophs derived from a Serratia marcescens strain were carried out. The arg mutants were classified into seven biochemical groups based on their growth response to five precursors of arginine biosynthesis and enzyme deficiency. Reciprocal transduction tests among these arg mutants divided them into three linkage groups, and the fine mapping in each of the groups by two- or three-point crosses revealed the following arrangement of loci. (1) arg44-thy11-lys1; (2) met1-glt2-argE-(arg19-arg51)-arg120-argG-argH; (3) arg33-pyr4. Five of the seven biochemically distinct arg mutants belonged to the second linkage group, and they constituted an agr-gene cluster. A characteristic feature of the arg-gene cluster of S. marcescens is that it involves argG, which was previously reported only in the Proteus group of Enterobacteriaceae.

Mutants of Salmonella typhimurium defective in transport of branched-chain amino acids.

Five mutants, CD15, CD31, CE4, CE5, and CE14, defective in the transport of branched-chain amino acids were isolated as glycyl-l-isoleucine and glycyl-l-valine requirers from an isoleucine-valine-requiring mutant, KA931, of Salmonella typhimurium LT2. Although these transport mutants do not grow in minimal medium supplemented with isoleucine (10 mug/ml) and valine (20 mug/ml), where the parent strain, KA931, grows normally, they do when the supplements are increased 10-fold in concentration, and the growth rate becomes comparable to that of the parent strain. When the apparent K(m) and V(max) for uptake of isoleucine, valine, and leucine in the transport mutants were compared to those of KA931, the K(m) was generally lower in the mutants, and the V(max) for isoleucine and leucine decreased to one-fourth to one-seventh and for valine one-eighth to one-fifteenth of that in the parent. In all mutants except CE5, the transport of methionine, arginine, threonine, and glycine was normal. The transport of threonine in CE5 appeared to be slightly impaired. In addition to the original mutation in the ilvC locus, the transport mutant has a mutation at the ilvT locus, which is closely linked to proC and may be located on the side of proC proximal to purE. About 26-fold difference in values of the co-transduction is noted in reciprocal transductions between KA502 and CD15 strains.

Physiology and genetics of carbamoylphosphate synthesis in Escherichia coli K12.

76 mutants have been isolated in which the function of the single carbamoylphosphate synthetase of Escherichia coli K 12 is affected. A wide variety of phenotypes have been observed among these mutants, the most typical ones being: requirement for arginine and uracil, arginineless behaviour, sensitivity towards arginine and sensitivity towards uracil. The mutations have been localized by reciprocal transduction and deletion mapping; all are clustered in the same locus, car. The study of carbamoylphosphate synthesizing activities of these mutants and the combination of car mutations in various in vivo as well as in vitro complementation tests lead to the conclusion that car contains two genes: carA, covering the left part of the locus and coding for the “glutamine subunit” of the enzyme; carB, to the right, governing the synthesis of the heavy subunit of the enzyme.

ExrB: a malB-linked gene in Escherichia coli B involved in sensitivity to radiation and filament formation.

SUMMARYPAM 26, a radiation-sensitive mutant ofEscherichia colistrain B, is described. Its properties are attributable to a mutation in a gene,exrB, which is cotransducible withmalB. It differs fromuvrA(alsomalB-linked) derivatives of strain B in being sensitive to 1-methyl-3-nitro-1-nitroso-guanidine and γ-radiation, and in being able to reactivate UV-irradiated phage T3. It differs fromexrA(alsomalB-linked) derivatives of strain B in forming filaments during the course of normal growth as well as after irradiation. WhenexrBwas transduced into a K12 (lon+) strain, filaments did not form spontaneously. Three-point transductions established the order of markers asmet A malB exrB. Based on an analysis of the frequency of wild-type recombinants in a reciprocal transduction betweenexrAandexrBstrains, it was inferred that they are not isogenic and that the order of markers ismalB exrA exrB.

Genetic fine structure of the leucine operon of Escherichia coli K-12.

The order of mutational sites in 10 independently isolated leucine auxotrophys of Escherichia coli K-12 was determined by three-point reciprocal transductions. The sites of mutation mapped in linear sequence in a cluster; all leucine auxotrophic mutations were cotransducible with mutations in the arabinose operon. The mutations were assigned to four complementation groups by abortive transduction tests, designated D, C, B, and A, reading in a clockwise direction from the arabinose operon. Enzyme analyses showed that strains with a mutation in gene A lacked alpha-isopropylmalate synthetase activity (EC 4.1.3), and those with a mutation in gene B lacked beta-isopropylmalate dehydrogenase activity (EC 1.1.1). It is concluded that the gross structure of the leucine operon in E. coli is closely similar to, if not identical with, the gross structure of the leucine operon in Salmonella typhimurium.

Nature of the elimination of the penicillinase plasmid from Staphylococcus aureus by surface-active agents.

Growth of Stapylococcus aureus in various ionic surface-active agents resulted in loss of the ability to produce penicillinase, whereas growth in nonionic surface-active agents had no effect on penicillinase production. The curing effect of various alkyl sulfates was found to be dependent upon the chain length. Curing by surface-active agents could be inhibited by magnesium. Reciprocal transduction experiments showed that curing by a surface-active agent was a property of the plasmid, not of the bacterial strain in which the plasmic resides.

Genetic analysis of resistance to erythromycin and oleandomycin in Staphylococcus aureus.

Strains U9(pase, tet, ery, nov) and U9(pase, tet, ol, nov) of Staphylococcus aureus were used as donors of the ery and ol markers, respectively, to strain 152. Thirty-three erythromycin-sensitive (ery-s) mutants of strain 152(ery) and 19 oleandomycin-sensitive (ol-s) mutants of strain 152(ol) were isolated following diethyl sulfate mutagenesis. The results of reciprocal transductions involving these mutants are as follows: (a) the frequency at which the ery and ol markers were transduced into each mutant varied greatly, and was especially low in those mutants which yielded recombinants in reciprocal transductions between the mutants; (b) 13 of the 19 ol-s mutants yielded recombinants in reciprocal transductions between the ol-s mutants; (c) no recombinants were detected in reciprocal transductions between ery-s mutants; (d) antibiotic-resistant recombinants were obtained in reciprocal transductions between three ery-s mutants and three ol-s mutants. The results of this study demonstrate that there is only one genetic locus which controls resistance to erythromycin and oleandomycin in these strains, and that it exists in an inducible state (the ery marker) and a constitutive state (the ol marker).

Mapping the -aminolaevulinic acid synthetase locus in Bacillus subtilis.

SUMMARY: HemA mutants of Bacillus subtilis 168 requiring δ-aminolaevulinic acid for their growth were isolated either following selection with streptomycin or by exposure to copper sulphate, and studied in reciprocal transduction experiments. Both groups of mutants were found to be genetically identical and the hemA locus was unlinked to a streptomycin resistance marker; it was linked to the group III markers mapped by Dubnau, Goldthwaite, Smith & Marmur (1967) and is located between the ilvA1 and phe-1 loci.

A Chromosomal Locus Which Controls the Ability of Shigella flexneri to Evoke Keratoconjunctivitis

The primary step in the pathogenesis of bacillary dysentery is the penetration of intestinal epithelial cells by shigellae. Lacking this capacity, Shigella flexneri becomes avirulent. By means of intergeneric conjugation between various Escherichia coli K-12 Hfr strains and S. flexneri 2a virulent recipients and by reciprocal transduction analysis with phage P1 vir, we established a locus on the genome of S. flexneri 2a which is necessary for the ability of this strain to penetrate epithelial cells as measured by the Sereney test for keratoconjunctivitis. This locus, termed kcpA (in reference to its involvement in provoking keratoconjunctivitis), has been positioned between the lac and gal chromosomal markers and is contransducible with the purE allele.

Genetic recombination between alpha-toxin mutants of Staphylococcus aureus.

McClatchy, J. K. (The University of Texas Southwestern Medical School, Dallas), and E. D. Rosenblum. Genetic recombination between alpha-toxin mutants of Staphylococcus aureus. J. Bacteriol. 92:580-583. 1966.-A demonstration of genetic recombination between Staphylococcus aureus nonhemolytic mutants was attempted by means of transduction. The results of two-point reciprocal transductions placed the mutants into two genetic groups. Recombination within each group was not detectable within the limits of the method, but hemolytic recombinants were obtained in transductional crosses when donor and recipient were from different groups. At least two genetic loci are therefore involved in alpha-toxin production. The 11 mutants of group II were fibrinolysin-negative. The recombinants were always found to be restored to fibrinolysin production as well as to alpha-toxin production. These data suggest the existence of a pleiotropic gene simultaneously affecting the synthesis of both alpha toxin and fibrinolysin. The nine mutants of group I were fibrinolysin-positive. Group I members are postulated to be alpha-toxin structural mutants. Three mutants were also negative for bound coagulase, but no linkage was observed between the locus controlling bound coagulase and the loci for either fibrinolysin or alpha-toxin production.

TRANSDUCTION ANALYSIS OF THE HISTIDINE REGION IN STAPHYLOCOCCUS AUREUS.

SUMMARY The genetic control of histidine biosynthesis in Staphylococcus aureus was examined by an analysis of 66 histidine-dependent mutants by using stable and abortive transduction. These mutants, which previously had been differentiated into classes according to their biochemical characteristics, all occupied sites within a single linkage group, referred to as the histidine region. This region has been separated into six gene loci on the basis of complementation studies and the biochemical characteristics of the mutants. The order of genes within the histidine region, E, A, B, C, D, G, was determined from the results of reciprocal transductions to wild type and donor type. The order of mutant sites was also linearly related to the ability of these mutants to form histidine-independent transductants when infected with phage prepared on the parent strain. This observation, and the inequalities in reciprocal transduction frequencies obtained in inter-mutant transductions, support the hypothesis that all donor fragments which participate in transductions involving the histidine region are identical, and that the histidine region is located extremely close to one terminus of the donor fragment. Intergenic complementation occurred among mutants of all gene loci; in addition, three complementation units within the A gene and two complementation units within the G gene were detected.

TRANSDUCTION OF SPOROGENESIS IN BACILLUS SUBTILIS.

Takahashi, I. (McMaster University, Hamilton, Ontario, Canada). Transduction of sporogenesis in Bacillus subtilis. J. Bacteriol. 89:294-298. 1965.-A number of asporogenous mutants of Bacillus subtilis were isolated and genetic analyses with these mutants were carried out by means of transduction mediated by bacteriophage PBS 1. The ability to form spores could be restored by transduction in all mutants examined, with the exception of Sp(-)H12-3, although they were not transformable under my experimental conditions. The results of reciprocal transduction suggest that genetic loci at which a mutation can affect sporulation are numerous. A spore marker (sp 1) was transduced jointly with prototrophy and some antibiotic resistance markers (streptomycin, erythromycin, and neomycin). Other spore markers (sp N2-2, sp 170-2, and sp H12-4) were also linked to auxotrophic markers (ser, tyr, and phe) at a fairly high frequency. This linkage relationship indicates that genes controlling the formation of spores are located along the chromosome far apart from each other. The result also suggests that blocks in a variety of biochemical processes may result in the expression of the asporogenous phenotype.

NATURE OF THE DETERMINANT CONTROLLING PENICILLINASE PRODUCTION IN STAPHYLOCOCCUS AUREUS.

Harmon, Shirley A. (Ohio State University, Columbus), and Jack N. Baldwin. Nature of the determinant controlling penicillinase production in Staphylococcus aureus. J. Bacteriol. 87:593-597. 1964.-From three penicillinase-producing strains of Staphylococcus aureus 38 penicillin-sensitive mutants were isolated. With phages 80 and 53 of the International Typing Series, each of these mutants was a competent recipient of the penicillinase marker from the parent strains. Although numerous reciprocal transductions were performed between penicillin-sensitive mutants, no penicillinase-producing recombinants were observed. Reversion of the penicillinase-negative mutants to capacity to produce penicillinase was never observed. When tested for 50 distinctive physiological characteristics, the mutants generally were similar to the parent strains except for resistance to penicillin. One penicillin-sensitive mutant failed to produce detectable amounts of five exoenzymes. By treating resistant strains with acridine orange, it was possible to isolate sensitive strains at a frequency that was 60 times greater than the spontaneous mutation rate. Thus, the determinant controlling the capacity of strains of S. aureus to produce penicillinase appears to be a cytoplasmic element.

STUDY ON THE CROSS-RESISTANCE OF ESCHERICHIA COLI TO STREPTOMYCIN AND KANAMYCIN, WITH SPECIAL REFERENCE TO STREPTOMYCIN DEPENDENCE ANALYZED BY TRANSDUCTION

The cross resistance of intermediate resistant mutants to streptomycin (SM) and kanamycin (KM) was studied genetically by several investigators. The present author attempted to observe the cross resistance of one-step highly SM resistant mutants and SM dependent mutants against KM, and to analyze the genetics of the cross resistance using SM dependent mutants. Strains used were Escherichia coli, strain B/r and its mutants. Phage P 1 was used for transduction technique.One-step highly SM resistant mutants showed the slightly increased resistance to KM, but SM dependent mutants showed various resistant levels, increased or decreased, to KM.From the results of reciprocal transduction between SM dependent mutants and original strain, it was clearly shown that a resistance level to KM was controlled by SM dependent mutation itself or by a closely linked mutation not separable from SM dependent mutation through transduction.The analysis of non-dependent (suppressor) mutants derived from SM dependent mutants indicated that resistance level to KM was also controlled by the suppressor mutation.

Transductional studies of thiamine and nicotinic acid requiring streptomycin resistant mutants of Salmonella typhimurium.

Summary: By selection with concentrations of streptomycin from 25 to 250 μg./ml., auxotrophic mutants were obtained with high frequency from Salmonella typhimurium strain LT-2. All of them were found to require both thiamine and nicotinic acid for their growth. The requirement for thiamine can be satisfied by either 4-methyl-5-hydroxyethyl-thiazole, cystine or cysteine but not by methionine. Nicotinic acid can be replaced by nicotinamide. No other amino add, vitamin, purine or pyrimidine can replace these vitamins. In addition to the nutritional requirement, all of these mutants are streptomycin resistant slow growers. These four characters, i.e. thiamine and nicotinic acid- requirement, slow growth and streptomycin resistance are converted to wild-type in a single step by transduction with phage PLT-22, grown on wild-type bacteria as well as on prototrophic one-step intermediate streptomycin resistant mutants and streptomycin indifferent mutants. Spontaneous mutants with wild phenotype were also obtained from some unstable mutants of this group, apparently in a single mutational event. The transductional findings suggest that these auxotrophic mutants are probably due not to some chromosomal aberration but to mutation within a single locus. The data from reciprocal transductions between these mutants suggest that multiple sites of the same locus are responsible. The mutations at different sites seem to have different levels of mutability and some of them have different levels of streptomycin resistance.