Vol. 128, No. 2 Printed in U.S.A.

JOURNAL

OF BACTERIOLOGY, Nov. 1976, p. 681-682 Copyright C) 1976 American Society for Microbiology

New Types of Escherichia coli Recombination-Deficient Mutants' DAVID FREIFELDER Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154

Received for publication 20 August 1976

A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperaturesensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage X, filament formation, and mutation frequency. Konrad (unpublished data) has described a new method of isolating bacteria carrying mutations that affect genetic recombination. In brief, the method is as follows. An Escherichia coli strain was prepared that contained two copies of the lactose operon, each of which included a deletion such that the strain was lac-. These deletions do not overlap, and occasionally a lac+ cell arises by recombination; this recombination is manifested by the appearance of lac+ papillae on the surface of a lac- colony. After mutagenesis with ethyl methane sulfonate, Konrad found colonies that contained an abnormally large number of papillae. This was termed the hyper-Rec phenotype, and further study showed that these clones included a number of new and interesting mutants involved in the processing of deoxyribonucleic acid (DNA) (1; Konrad, unpublished data). It was noticed that on plates used to select hyper-Rec strains there were also colonies having no papillae (Pap-). Some of these were isolated from one of Konrad's mutagenized populations in the hope that new types of mutants involved in genetic recombination might be found. The properties of some of these Papmutants are described in this note. The mutants were isolated as follows. The parent strain KS391 (Hfr Hayes lacMS286 b80d1I lacBKI thi-) was mutagenized with ethyl methane sulfonate (2). Colonies free of papillae (a normal colony has one to ten papillae) were selected after 3 days of growth at 30°C on lactose-tetrazolium agar (2) and tested for a variety of properties. Papillation was retested by restreaking since some primary isolates failed to form papillae because they were small colonies. Those that either were highly I Publication no. 1098 from the Department of Biochemistry, Brandeis University. Reprints will not be available. 681

sensitive to ultraviolet light (UV) or methyl methane sulfonate (MMS) or failed to grow at 42°C were tested further. Of this class, four that reverted simultaneously to normal papillation and temperature resistance were retained. The Pap- phenotype has several possible causes. (i) One of the lac copies (e.g., the 480d1ac prophage) may have been lost. (ii) The cell may have acquired a lac- point mutation in the region of the lac operon covered by one of the deletions. (iii) The cells may have a lesion in a gene involved in some way with recombination. The four Pap- mutants described here cannot be of type (i) since they revert to Pap+ and are not of type (ii) since they exhibit growth defects in media in which lactose is not the carbon source. We assume that they are of type (iii), and we call this phenotype hypo-Rec. Preliminary characterization of these mutants (as well as a fifth mutant that does form papillae) indicated that they had novel properties, deserving additional study. However, further work on these mutants will probably not be carried out in this laboratory, and these mutants are available to anyone wishing to use them. Some characteristics of these mutants and their temperature-resistant revertants are shown in Table 1. These mutants have combinations of properties that seem interesting: (i) temperature sensitivity and lack of papilla formation without MMS sensitivity (e.g., strains 105 and 218); (ii) temperature sensitivity, lack of papilla formation, and MMS sensitivity (e.g., strain 130); and (iii) temperature and MMS sensitivity with normal papilla formation (e.g., strain 242). Strain 130 is a double mutant since its sensitivity to UV light does not co-revert with temperature sensitivity. We attempted to obtain revertants that were resistant to UV light by selecting survivors at the 10-10 level;

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NOTES

J. BACTERIOL.

TABLE 1. Properties of mutants lack Straina

KS391 105 130 169 218 242 Temperature-resistant revertants 105+ 130+ 169+ 218+ 242+

papil-

laebpil+ -

+

Colony Colony formaformation (423C)c

R S S S (37°C) S S

Fila-

Map posisynthe- frequen tion elimtion sensi formasis at t inated vitye c -e (n) 42C MMSdtin R R S R R S

UV

R R St R R S

DNA

ment

+ +

+ NT + NT NT +

Mutation

5 x 10-7 NT 8 x 10-' NT NT 6 x 10-7

1-40 60-61 1-15 1-80 1-80

Lyso-

Burst size ratio

(42°C/32°C)k

gLyszation

A

rd

+ NT + NT NT +

0.9 0.1 0.1 0.1 0.3 0.06

0.8 0.03 0.03 NT 0.2 0.006

R R NT NT R NT 0.3 0.1 5 x 10-7 R + + R NT R 0.8 0.8 R R R NT NT NT NT 0.3 R R NT NT R NT 0.2 0.3 9 x 10-7 R R NT R + 0.2 0.7 a The parent strain KS391 is an Hfr. Strains 105, 169, and 242 were shown to be of the Hfr type by the ability to transfer the threonine-leucine loci to an appropriate female strain; neither strain 130 nor 218 would do so. Strain 218 was made streptomycin resistant and shown to be a female in that it could receive the temperature resistance allele from a wild-type Hfr. Strain 130 was never tested for recipient ability because of the lack of a suitable counterselection. b +, Ability to form lac+ papillae; -, inability to form papillae. c R, Ability to form a colony at 42°C except where otherwise indicated; S, inability to do so. d R, Ability to form a colony on agar containing MMS; S, inability to do so. R, 37% survival dose is 7 a; S, 37% survival dose is 4 s; S', 37% survival dose is 0.25 s. '+, Forms filaments after 3 h of growth at 42°C; -, no filaments formed. 9 +, Normal rate of incorporation of [H3]thymidine at 42°C; NT, not tested. I Fraction of cells (derived from a single clone) capable of forming a colony at 32°C on an agar surface seeded with 109 particles of E. coli phage T4. i Large regions of the map were eliminated by the inability of an Hfr to cotransfer temperature sensitivity with either leucine, galactose, histidine, or arginine. Strain 169 would not transfer past galactose. The small interval for 130 was eliminated by P1 transduction. +, Frequency of lysogenization at 32°C by XcI857 is >50%. k Either Ac1857 or Ac857red3 was adsorbed at 32°C at a multiplicity of infection of 0.1 and then allowed to grow and lyse the cells at either 32 or 42°C. The value gives the ratio of phages produced at 42 and 32°C. + + + + +

however, all survivors retained high UV sensitivity. Strain 130 also has another unusual property: a streptomycin-resistant mutant could not be made either by selection of spontaneous mutants (frequency

New types of Escherichia coli recombination-deficient mutants.

Vol. 128, No. 2 Printed in U.S.A. JOURNAL OF BACTERIOLOGY, Nov. 1976, p. 681-682 Copyright C) 1976 American Society for Microbiology New Types of E...
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