mating types, to study the genetics of the arginine repression system. At one point, it became ..... This work was supported by grant RG-6048 from the U. S. Public Health Service. t U. S. Public ... 13 Gorini, L., W. Gundersen, andM. Burger, in ...
INTRODUCTION OF A GENE FROM ESCHERICHIA COLI B INTO HFR AND F- STRAINS OF ESCHERICHIA COLI K-12* BY RENATA MAASt AND WERNER K. MAAS4 DEPARTMENT OF MICROBIOLOGY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE
Communicated by B. L. Horecker, September 24, 1962
In this paper, we shall describe a general method for transferring genes from E. coli B into Hfr and F- strains of E. coli K-12. This method was developed during our studies on the mechanism of repression in the biosynthesis of arginine.1 We had used for this work strains of K-12 which permitted us, because of the availability of mating types, to study the genetics of the arginine repression system. At one point, it became necessary to have mutants blocked in the formation of one of the arginine enzymes, ornithine transcarbarmylase (arg5-) both in Hfr and F- strains. Such mutants had never been isolated in K-12, despite repeated attempts, although they had been found in other strains of E. coli, such as the B strain. We therefore undertook to transfer this gene from E. coli B to Hfr and F- strains of K-12. E. coli B has been reported to be F-.2 We adopted the following steps for the transfer of the arg6- allele into K-12. The arg5- mutant of B was converted into a genetic donor by introducing into it an Flac+ episome through mating with an Flac+ strain. Such Flac+ strains transfer the Flac+ particle with very high frequency and their chromosomal genes with a frequency somewhat lower than that of ordinary Hfr strains.3 The resulting arg5- Flac+ strain was mated with an Hfr strain of K-12, which had been converted by prolonged aeration into a temporary F- phenocopy. Hfr recombinants were isolated, some of which were args-. From these hybrids, the args- allele could easily be introduced into F- strains of K-12 by mating. We shall describe in detail the steps involved in this transfer and also the mapping of the arg5 gene. During these studies, we discovered an incompatibility phenomenon which brought to light a new type of regulatory system in the cell. In crosses between arg5- Flac+ and arg5+ Hfr strains, only non-Hfr recombinants carried the Flac+ episome, whereas Flac+ was excluded from Hfr recombinants. Besides its intrinsic interest, this exclusion phenomenon proved to be instrumental for the effectiveness of our transfer technique. Materials and Methods.-Media: The minimal medium used was medium A,4 with 0.2 per cent of the sugar specified as carbon source. Other additions were made as indicated. Amino acids were used at a concentration of 100 pg per ml and thiamin at one ,g per ml. Enriched medium was either Difco neopeptone broth (NPB), or NY medium, the latter being prepared by supplementing medium A with 0.2 per cent glucose, 0.2 per cent hydrolyzed casein (NZ Case, Sheffield), and 0.2 per cent yeast extract. For solid media, agar was added to a concentration of 2 per cent. Bacto MacConkey Agar (Difco) served as indicator agar for lactose fermentation. Strains: The pertinent properties of the strains used in the present paper are summarized in Table 1. Mating conditions: Exponentially growing cultures on NPB medium, at 109 cells/ml, were mixed as follows: into a 125-ml Erlenmeyer flask were added 4.5 ml of the female culture, 0.5 of the male culture, and 5 ml of NPB. Since the male was the minority parent, results are expressed in terms of per cent male input. The mating mixture was incubated at 370 with gentle shaking. In interrupted mating experiments, samples were withdrawn at the times specified, diluted in medium A-N (medium A without ammonium sulfate), and plated on the appropriate selective media. Whenever the frequency of recombination was so low that a dilution of less than 103 1887
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TABLE 1 SUMMARY OF STRAINS USED -
Auxotrophic Characters
arg + ura
---
-Energy Source-_ Mating lac Sm gly xyl mal val T6 type r r r "F-" + + + + + r r r Flac+ + + + + Flac+ + + Flac+ nt nt nt nt nt 200 PC Flac+ + + + s r Hfr AB-312 + + + + + 8 + nt r Hfr s 312R4 + + + 8 nt r r F PA260A + + + nt r s F P678AU + + + nt Hfr P4X 8 8 + + + + + + + + + nt Hfr 8 8 30SO + + + + + + + + + nt s Hfr 8 30SO-Lac + + + + + + + + + + nt r r F 260R9 + + - + + We are indebted to L. Gorini for strain B9OH, to F. Jacob for strain 200 PC from which all our Flac + strains were derived, and to E. A. Adelberg for strain AB 312. The following abbreviations are used to describe genetic markers: arg, arginine; arg + ura, arginine plus uracil; his, histidine; met, methionine; pro, proline; ser/gly, serine or glycine; thi, thiamin; thr-leu, threonine and leucine, which were always treated as a unit because of their proximity; lac, lactose; mal, maltose; xyl, xylose. Smr and valj refer to resistance to streptomycin and valine respectively, while Sms and vals refer to sensitivity to these agents. The subscript 5 below arg refers to the fifth reaction step in the arginine biosynthesis pathway (ornithine - citrulline). Genetic characters not tested are marked as nt. Hfr refers to strains that transfer their chromosome with high frequency and F- to female recipients. When F precedes lac it denotes that the lac operon is on the F episome.8 Strain No. B9OH B90H Flac
thrleu pro his + + + + + + + + + + + + + +
met thi
arg5
ser/
had to be plated, the cultures were washed twice with medium A-N prior to dilution and plating. For timed interruption of matings, either the phage 1T6 technique5 or the blender technique6 was used. For the former, a sample of the mating mixture was added to a T6 suspension to give approximately 500 phage per male bacterium and vigorously shaken at 370 for 30 min, prior to dilution and plating. For the latter technique, the mating mixture was diluted one hundredfold with iced A-N, blended for 30 sec in a Lourdes Multi-Mixer, speed 60-70, diluted further if necessary, and plated. The parent cultures were also plated on each kind of selective medium at the lowest dilution used for the selection of recombinants. In Hfr X F- crosses this was 10-3. In the T6 interruption experiments, the zero-time samples from the mating mixture after the phage treatment were plated on a medium selective for the male parent; this gave a measure of male survivors. For all the experiments reported in this paper, the control parent plates had no cublnies, there were no recombinants on the zero-time plates, and male survivors, after T6 killing, were at most 104 cells per ml. Tests for Flac +, maleness, femaleness, and unselected markers: To test a strain for the presence of an Flac+ episome, 109 cells of a logarithmically growing broth culture were mixed with 109 cells of a lac- female broth culture in a total volume of 3 ml NPB. Both strains carried auxotrophic mutations permitting differential selection of either parent or of recombinants. After incubation for 60 min at 37°, with gentle agitation, a 10-7 dilution of the mating mixture was spread on a medium selective for the female parent. As controls, the female parent alone was also plated on the same medium at the same dilution, and the male parent was plated at the same dilution on NY agar. When the colonies had grown to moderate size, they were replicated7 onto lactose indicator plates. The replicas from the female parent consisted of only lac- colonies; the replicas from the male parent were all lac +, except for an occasional lac - colony. The replicated colonies from the mating mixture, in case of a positive test, were 30-60% lac +. Test for maleness was carried out according to the method of Taylor and Adelberg.8 Test for femaleness was carried out in the same way except that isolated prospective F- colonies were replicated onto two lawns of the Hfr mutant cells. The lawns of the Hfr strain were prepared from exponentially growing cultures in order to avoid the conversion of the Hfr cells into phenotypically F- cells. To test for unselected markers among recombinants, colonies picked from the selective plates were purified by one single colony isolation and plated in patch form on a "master" plate. After the patches had grown, they were replicated onto various test plates.
Results.-1. Isolation of an Flac+ arg5- mutant of E. coli B: Strain B90H was crossed with Flac+ strain 200 PC (see Table 1 for genetic markers). B9OH cells that had received the Flac+ element were selected by plating a low dilution (10-7) on a medium containing histidine, arginine, and lactose as sole carbon source.
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At this low dilution, only cells receiving the Flac+ element, but none of the chromosomal genes of the donor, were recovered. Many colonies appeared on the selective plates. Several were isolated, purified, and checked for their nutritional requirements (arginine, histidine), maleness, and ability to transfer Flac+. Most of them were like the original B90H except that they were lac +, behaved as males, and were able to transfer the Flac + element. 2. Transfer of the argr- allele to an Hfr strain: The acceptor strain used with B9OH Flac + was Hfr AB-312 (see Fig. 1 for location of the Hfr gene). Hfr strains can be altered so as to behave temposee rarily as F- recipients (phenocopied) . Ace ro by aeration of a stationary-phase culIn our experiture for several hours.9
