Molec. gen. Genet. 147, 67-69 (1976) MG13 © by Springer-Verlag 1976

Phenotypic Instability in a tif-1 Mutant of Escherichia coli

II. Recessiveness of the tif-1 Mutation

William Hayes Genetics Department, Research School of Biological Sciences, The Australian National University, P.O. Box 475, Canberra, A.C.T. 2601, Australia

Summary. Merodiploids of the type t i f -1 /F ' t i f +, con- structed in E. coli K12 strain T44(2), show that the tif-1 mutat ion is recessive with respect to induction of phage 2 and thermolability. An additional manife- station of t/f-1 expression is increased tolerance to low levels of streptomycin (SM) and chloramphenicol (CM) and this, too, is abolished[ in the merodiploids.

impaired ribosomal function (Ephrati-Elizur, Luther- Davies and Hayes, 1976). The present communicat ion demonstrates that strain T4400 is additionally charac- terised by tolerance to chloramphenicol and shows, by means of a t i f -1 /F ' t i f + merodiploid, that the tif-1 mutat ion is recessive and is correlated with the va- rious phenotypes described.

Introduction

Strain T44(2) is a temperature-sensitive mutant (t/f- 1 : thermal induction andfi lamentat ion) of Escherichia coli K-12 C600 which induces prophage 2, and other inducible prophages, when the temperature is raised f rom 30 ° to 40 ° (Goldthwait and Jacob, 1964). In non-lysogenic derivatives cell division is inhibited so that the ceils become filamentous (Kirby, Jacob and Goldthwait , 1967). In addition, expression of the tif-1 mutat ion leads to repair of UV-irradiated phage 2 (Castellazzi, George and Buttin, 1972a) and to increa- sed mutability (Witkin, 1974; George, Castellazzi and Buttin, 1975). These pleiotropic effects are enhanced by the presence of adenine and prevented by guano- sine and cytidine (Kirby, Jacob and Goldthwait, 1967), and growth in rich media such as L broth (Castellazzi, George and Buttin, t972a). The tif-1 mu- tation maps very close to the recA and zab genes and is suppressed by mutat ions in these and in the lex gene (Castellazzi, George and Buttin, 1972b). A model system to explain these and other finding has been proposed, whereby the t i f-zab region of the E. coli chromosome acts as a repressible operator which controls the synthesis of various proteins involved in D N A repair (Gudas and Pardee, 1975).

It has recently been shown that expression of the tif-1 gene leads to increased tolerance to streptomycin, while growth on streptomycin suppresses the t t f phe- notype; in vitro experiments with crude cell extracts imply that t~f expression is directly associated with

Materials and Methods

1. Bacterial Strains. E. coli K12 C600 (thi, thr, leu), C600 tif-1 (2)=T44(2). Prototrophic recombinant derivatives of C600 and T44(2) were isolated by crossing with Hfr Cavalli (met, tel-l, str-s) for 45 rains., plating dilutions on unsupplemented minimal agar (Min. agar) and testing prototrophs for retention of thermal induci- bility. Thymine-requiring (thyA) derivatives (see 4. below); T44(,;) tif-1/F'tif + merodiploids (5. below).

2. Media. As in Ephrati-Elizur, Luther-Davies and Hayes (1976).

3. Isolation of thyA Mutants of T44(2). Selection was made for resistance to Trimethoprim (10 gg/ml) by T44(2) prototrophs (Mil- ler, 1972).

4. Construction of tif-1/ U tif + Merodiploids. E. coli K 12 strain KL259 is a multiple auxotroph (pyrD, trp, his, tyrA, recA, thyA, thO car- rying the F' factor 143 (~lysA +-thyA +-recA +-tyrA + --) which bears the wild-type thyA gene and also recA + which is very closely linked to t/f +. T44(2) thyA derivatives were crossed with KL259 (F'143) for one h in L broth at 35 ° and dilutions plated on Min. agar to select for inheritance of thyA +. Recombinants were purified on Min. agar and tested for t/f-1 expression. The merodiploids so constructed were maintained on Min. agar.

5. Assay of Streptomycin and Chloramphenicol Resistance. Resist- ance varies appreciably with population density, so that equivalent numbers of cells must be plated for comparison. 0.01 ml of dilu- tions of cultures or suspensions were plated on ca. 1.5 cm diam. areas of plates containing a critical concentration of the drug. Results were usually read after 48 h at 34 °.

6. Thermal Induction of T44(2). As in Kirby, Jacob and Goldth- wait, 1967.

68 W. Hayes: Phenotypic Instability in a t~'-I Mutant of Escherichia coli

Results

1. Thermal induction of prophage 2. Figure 1 compa- res the growth curves, in MinCA-adenine at 30 ° and following a shift to 42 °, of a T44()0 (F'143) merodi- ploid, the original T44(2) strain and one of its protro- phic derivatives. On shifting the temperature to 42 ° the diploid multiplies exponentially, while the two tif-1 strains begin to lyse after 40 rain.

2. Resistance to low concentrations of streptomy- cin (SM) and chloramphenicol (CM). It has been shown that strain T44(2) is characterised by increased resistance to low concentrations of streptomycin as compared with strain C600 and that, unlike C600, resistance may be strikingly increased by growth in the presence of adenine. In view of the evidence that this is due to involvement of the tif-1 mutation in ribosome function (Ephrati-Elizur, Luther-Davies and Hayes, 1976), it was of interest to test the effect of other antibiotics which affect ribosomal activity, and to use the merodiploid strain to explore the rela- tionship of u f to such resistance.

Table 1 shows that CM, at a critical concentration of 7.5 ~tg/ml in L agar, allows growth of virtually all T44(2) cells, while the number of C600 colony- formers is reduced by at least four orders of magni- tude. Moreover the increased resistance of T44(2) to both SM and CM is abolished in the merodiploid which displays the same degree of sensitivity as C600.

0.50

0,40 o/O Diploid

o/ 0.30 oJ

o /

I 0.20 / o o o ",

q ,~ " ',,, o ~ e\ 0 o...~" .

0.10 o.--~s j / . fa • '= ~ 'i f .a \ ,.,

i"oJZ°'° pro,o,roph °'°' F , , \i"o-,,o T.

' 8'0 0 40 120 160 Time (minutes]

Fig. 1. Thermal induction of strain T44(2) : comparison of growth curves of the original T44 strain, and of its prototrophic and mero- diploid derivatives, following a shift from 30 ° to 42 °. An overnight static culture of each strain in MinCA-adenine (100 gg/ml) at 30 ° was diluted 1 in 10 into 25 ml MinCA-adenine in each of two Ehrlenmyer flasks with side-arms. After 45 rain in a shaking water- bath at 30 °, one flask was transferred to a similar waterbath at 42 ° and O.D. readings (Unicam SP600 at 600 rag) taken at 10- 15 rain intervals thereafter

Table 1. The effect of the F'143 factor on the sensitivity of strain T44(~) to streptomycin and chloramphenicol

Strain Number colonies/106 cells plated on:

(1) (2) Min. aga r+SM 2.5 L-aga r+CM 7.5

T44(2) prototroph 103 2.4 x 105 T44(2) (F'143) 1.0 5.9 C600 prototroph 1.5 20

(1) and (2) are separate experiments

Table 2. The effect o f temperature on the sensitivity to chloramphe- nicol of strain T44(2) and its merodiploid derivative

Strain Number of colonies/ Temper- 106 cells plated on ature of L-agar ÷ C M 7.5 incubation

T44(2) prototroph 7.7 x 104 30 ° 1.7 x 103. 38 °

T44(~.) (F'143) 34 30 ° 39 38 °

Dilutions of overnight MinCA cultures at 30 ° were plated

Table 2 compares the effect of elevated tempera- ture on the resistance to CM of T44(2)and its merodi- ploid derivative. Raising the temperature from 30 ° to 38 ° reduces the number of colonies of T44(2), gro- wing on L-agar+CM (7.5 pg/ml), about 50-fold; the proportion of merodiploid cells growing on CM is more than 1,000-fold lower than that of T44(,~) at 30 ° , but this proportion remains unaffected by tem- perature.

Discussion

Expression of the tif-1 phenotype manifests itself in many central aspects of cellular activity. It has been shown to be involved in such diverse phenomena as the induction of prophage 2 in the absence of protein synthesis (Shinagawa and Itoh, 1973; West, Powell and Emmerson, 1975), cell division (Kirby, Jacob and Goldthwait, 1967), DNA repair (Castellazzi, George and Buttin, 1972a; Gudas and Pardee, 1975), mutage- nesis (Witkin, 1974; George, Castellazzi and Buttin, 1975) and the production of a particular protein, X, which is normally induced by inhibition of DNA syn- thesis (Gudas and Pardee, 1975). Moreover the sup- pression of the tif-1 phenotype by mutations in the recA and lex genes, which in t i f + cells inhibit functions induced by the tif-1 mutation, implies that the ttf + gene plays a key role in a co-ordinated system of

W. Hayes: Phenotypic Instability in a tif-1 Mutant of Escherichia coli 69

cell regulation. Such a system has recently been pro- posed in which the t/f region is an operator controlling the synthesis of a number of proteins, including X, involved in D NA repair. However, this model embo- dies several unproven assumptions of which one of the most important is that the tif-1 mutation should be cis-dominant (Gudas and Pardee, 1975). Studies of a tif-1/F'tif + merodiploid, reported here, show that this is not the case and that tif-1 is recessive for all the expressions of t if activity investigated.

In a companion paper Ephrati-Elizur, Luther-Da- vies and Hayes (1976) report an increased resistance to streptomycin of E. coli strains carrying the tif-1 mutation and demonstrate thai: t if expression results in altered translation by cell-free extracts, implying that ribosomal ~function is impaired. Resistance to streptomycin is associated with mutations involving the 30S ribosomal subunit (Cox, White and Flaks, 1964; Birge and Kurland, 1969).

This paper shows that the tiJ:l mutation also leads to increased resistance to chloramphenicol which acts on the 50S subunit (Vazquez, 1965), but this resist- ance, unlike that to streptomycin, is decreased at ele- vated temperatures which enhance the uf phenotype. Increased resistance to both streptomycin and chlor- amphenicol is abolished in the tif-1/F'tif + merodi- ploid. This F' factor carries about 6% of the E. coli chromosome, from 49 to 55 rain. on the map, cover- ing the tif-1 mutation which maps at about 51 rain. On the other hand, the streptomycin (str) locus maps at 62 rain. and the chloramphenicol (cml) loci at 19 and 21 rain. It is therefore clear that the increase in resistance to these two drugs is a function of the tif-1 phenotype and cannot be ascribed to secondary mutations at the streptomycin or chloramphenicol loci.

A direct demonstration of the recessiveness of the tif-1 mutation with respect to ribosomal function is the normality of in vitro extracts of the merodiploid in the efficiency of translation, the misincorporation of isoleucine and the optimal Mg ++ concentration (Ephrati-Elizur, Luther-Davies .and Hayes, 1976).

Acknowledgements. I am grateful to Dr. Barry Rolfe for providing strain T44(2) and to Mr. Graham Woodrow for strain KL259 (F'143).

References

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Communicated by B.A. Bridges

Received April 5, 1976