JOURNAL OF BACTERIOLOGY, Apr. 1979, p. 1-6 0021-9193/79/04-0001/06$02.00/0

Vol. 138, No. 1

Thermoresistant Revertants of an Escherichia coli Strain Carrying tif-1 and ruv Mutations: Non-Suppressibility of ruv by sfi NOZOMU OTSUJI* AND HIROAKI IYEHARA-OGAWAt Faculty of Pharmaceutical Sciences, Kyushu University 62, Maidashi, Higashi-ku, Fukuoka 812, Japan Received for publication 22 January 1979

Spontaneous thermoresistant revertants were isolated from Tifl Ruv- and Tifl Ruv+ strains of Escherichia coli K-12. They were divided into five groups; backmutants to tir and recA structural gene mutants accounted for at least two of these groups. Mutations with an unconditional RecA- phenotype were detected at a higher frequency in the Tifl Ruv- strains (65%) than in the Tifl Ruv+ strains (25%). A third group consisted of revertants exhibiting a RecA- phenotype at low temperature. Revertants with normal recombination ability and UV resistance, but with a thermosensitive defect in propagating Abioll phage, were also isolated (group 4). The alleles responsible for this property were cotransducible with the srl gene, suggesting that they are located at the recA locus. Other revertants, which might carry lexA, lexB, or zab mutations, were UV sensitive and were able to propagate Abioll phage (group 5). The sfi mutation, which suppresses filamentation in the Tifl and UV-sensitive Lon- strains, does not restore UV resistance of the Ruv- mutant. The Ruv- mutants of Escherichia coli K-12 are sensitive to UV and gamma ray irradiation, but have normal ability for host cell reactivation (Hcr+) and recombination (Rec+). They form nonseptate, multinucleate filaments during incubation on enriched medium after low doses of UV irradiation or after treatment with inhibitors of DNA synthesis (21, 33). The Lon- mutants of E. coli were first isolated as a class of UV-sensitive mutants that make extremely mucoid colonies (18, 29). They also form long nonseptate filaments after temporary inhibition of DNA synthesis (1, 18). Lon- cells are also derepressed for the gal operon and other enzymes in the pathway to mucopolysaccharide synthesis (20, 29). The Lon- mutants are defective in host-viral interactions. Phages P1 and XsusN are unable to form stable extrachromosomal plasmids in Lon- cells (35). Wild-type A phage lysogenizes Lon- cells with reduced frequency (36). In addition, lon mutations stabilize abnormal polypeptides (e.g., nonsense fragments and some classes of missense proteins) against degradation (14, 34). Among these pleiotropic effects of the lon mutation, the division defect is reversed by mutations in recA, lexA, and sul genes (8, 10, 13, 14, 22). The sul mutation suppresses radiation sensitivity and prophage lyso-

genization defects, but does not affect mucoidy or the polypeptide degradation defect (10, 14). A Tif- mutant of E. coli carries a mutation in the recA gene (9, 17, 26) which results in a variety of pleiotropic effects when grown at high temperature. These include: (i) inhibition of cell division (24), (ii) induction of prophage development (13, 24), (iii) increase in the synthesis or activity of DNA repair enzyme (4), (iv) mutator activity (4, 38, 39), and (v) increase in protein X synthesis (9, 16, 17, 26, 37). The expression of these pleiotropic properties of the tif mutation is suppressed by mutation in the recA, zab, or lexA genes (5, 11, 16, 17). The sfi mutations, which were detected among spontaneous thermoresistant revertants of a Tif- Lon- strain, suppress filamentation, but do not affect other Tif phenotypes (11). The sfi mutation is identical to sul, since they are located at the same locus on the chromosome map and suppress division defects mediated by tif and lon mutations (10, 11, 14,22). Since it has also been shown that tif-mediated filamentation is enhanced in Lon- strains, tif and lon genes may affect a common regulatory circuit of cell division (11). As reported in a previous paper, strains carrying ruv lon mutations are no more sensitive to UV irradiation than strains carrying a single mutation, suggesting that the ruv and lon genes t Present address: The Kitakyushu Municipal Institute of affect some steps in the common pathway in Environmental Health Sciences, Shinike, Tobata-Ku, Kita- septum formation after temporary inhibition of kyushu 804, Japan. DNA synthesis (21). However, Lon- mutants, 1

2

OTSUJI AND IYEHARA-OGAWA

but not Ruv-, form mucoid colonies (18, 29, 33), and Lon- cells are poorly lysogenized by phages P1 and A (35, 36), although they lysogenize Ruvcells normally (manuscript in preparation). To examine how the ruv and lon gene products participate in a process of cell division and to study whether the ruv gene products are involved in the expression of the tif mutation, we isolated thermoresistant revertants of Tif- Ruvstrains and analyzed their properties. This paper demonstrates that revertants are divided into five groups by their UV sensitivity and their ability to propagate Abioll phage and to carry out genetic recombination. We also describe that recA mutations are detected at higher frequency in revertants of Tif- Ruv- strains than in those of Tif Ruv+ strains, as in the case of Tif- Lonstrains (11). The sfi mutations, which suppress division defects by the tif and Ion mutations, do not suppress UV sensitivity of Ruv- mutants. Isolation of a new type of RecA- mutant is also described in this paper. MATERIALS AND METHODS Bacterial and phage strains. The bacterial strains of E. coli K-12 are listed in Table 1. Genetic symbols used are reported in Bachmann et al. (2). Abioll phage, which is unable to grow on a RecAstrain, was obtained from K. Matsubara. The red gam

J. BACTERIOL.

region of the phage is replaced with the bio region of the E. coli chromosome. Media. Compositions of nutrient broth (N broth), peptone broth (P broth), and M medium are described elsewhere (32). NB agar and M agar were solidified with 1.5% agar. P agar and soft P agar contained 1.2% and 0.6% agar in P broth, respectively, and were used for assaying A phage. Isolation of thermoresistant revertants of the TMl and Til Ruv- strains. Independent clones of the Tifl and Tifl Ruv- strains were grown in M medium to about 1 x 108 to 2 x 108 cells per ml at 28°C. A 0.1-ml portion of the culture was plated on M agar containing 75 jg of adenine per ml to express tif1 function, and incubated at 42°C for 2 days. One colony from each plate was picked up and purified. Characterization of the revertants. Each revertant was grown overnight in N broth at 28°C and streaked on two NB agar plates. The plates were irradiated with UV at a dose of 10 J/m2, which is enough for inhibition of the growth of RecA- mutant strains, but not for Ruv- strains. One plate was incubated at 28°C and the other at 42°C. Overnight cultures of each revertant were plated with Abiol l phage at 28 and 42°C. RecA- mutant strains are unable to propagate the phage (28). Genetic crosses. Conjugation experiments and transduction with Plvir phage were performed as described previously (32, 33). UV irradiation. A 15-W Toshiba germicidal lamp was used as the source of UV light. The dose rates at distances of 43 and 61 cm from the lamp were 0.68 and 0.34 J/m2, respectively.

TABLE 1. Bacterial strains used Strain

Genotype

Source or derivation

BE5024 BE5036 AB1899 AB2463 JM12 BE5554

F- thr-1 leu-6 proA2 his-4 argE2 thi-1 ara-14 lacYl gaiK2 xyl-5 mtl-l tsx-33 rpsL31 supE37 As AB1157, also ruv-4 As AB1157, also ruv-9 As AB1157, also lon-i As AB1157, also recA13 As AB1157, also tif-1 As JM12, but his' ruv-4 thyA554

BE5556

As JM12, but his' ruv-9 thyA556

BE5674 BE5690 BE1594 BE5700 BE5701 BE5702 GC579

As BE5554, also recA 701 As BE5556, also recA702 F- srl gal-i gal-2 lac rpsL As BE1594, but srl+ recA701 As BE1594, but srl+ recA702 As BE1594, but srl+ recA13 F- sifAII thr-I leu-6 proA2 his-4 gaiK2 rpsL31 thi-I? ara-14? xyl-5? mtl-l? tsx-33? supE37? F- capR9 sulB leu-6purE42 trpE38 thi-i ara-14 lacYI gaiK12 xyl-5 mtl-l tonA23 tsx-67 azi-6 rpsL109pon supE44 Hfr A'; injection order: dsdA his trp As KL98, but ruv-4 AAs KL98, but ruv-9 AHfrC metBI rel-i; injection order: purE lac leu

E. A. Adelberg P. Howard-Flanders (18) (21, 33); same as HI24 (21, 33); same as HI36 P. Howard-Flanders (18) P. Howard-Flanders (19) J. George (11) BE5468 x JM12, his+ strr, and trimethoprim selection BE5471 x JM12, his+ strr, and trimethoprim selection This paper; cold-sensitive RecAThis paper; cold-sensitive RecAT. Ogawa, W3350-594 srl P1 (BE5674) x BE1594, srl+ P1 (BE5690) x BE1594, srl+ P1 (AB2463) x BE1594, srl+ J. George (11)

AB1157

RGC103-9 KL98 BE5468 BE5471 W2252

A. Markovitz (10) K. B. Low (21) (21) (33)

THERMORESISTANT MUTANTS OF E. COLI tif ruv

VOL. 138, 1979

RESULTS Classification of thermoresistant revertants. Revertants from Tifl (JM12) and Tifl Ruv- (BE5554, BE5556) strains were divided into five groups by their UV sensitivity and their ability to propagate Xbioll phage (Table 2). Among 69 independent revertants from the Tifl strain, 44 (63.8%) revertants had the same resistance to UV as the parental strain, and were able to propagate Xbioll phage. These strains were assigned to class 1. Class 2 revertants were extremely sensitive to UV and did not permit the growth of Xbioll phage. These strains are probably RecA- mutants (7, 19, 28). The frequency of class 2 revertants of the Tifl strain was 26%, whereas about 60 to 70% of the revertants of the Tifl Ruv- strain were in class 2. Revertants of class 3 seem to be cold-sensitive RecA, since they exhibit the RecA- phenotype only at low temperature. Class 4 revertants had the same UV sensitivity as their parent Tifl Ruv- strains, but were unable to propagate Xbioll phage at high temperature. Class 5 revertants might carry lexA, lexB, or zab mutations, since they were UV sensitive and were able to support the growth of Xbioll phage. Precise genetic mapping of class 5 mutations has not been performed. Analysis of class 3 revertants. Revertants of class 3 were more sensitive to UV at 28 than at 42°C and did not permit the growth of Xbioll phage at 28°C. The mutations were cotransducible with the srl+ at an average frequency of 71%, suggesting that they are located in the recA region. Two Srl+ transductants from independent revertants were named BE5700 (recA701)

3

and BE5701 (recA 702), respectively, and used for further study. These two strains recombined with Hfr W2252 at 44 to 70% and 0.14 to 3.0% of the wild-type level at 42 and 28°C, respectively. The frequencies of recombination at 42 and 28°C were almost the same as those in the crosses of the same Hfr strain with the Rec+ strain (BE1594) and with the RecA13 (BE5702) strain, respectively. Abioll phage plated on strains BE5700 and BE5701 at 42°C with the same frequency as on the wild-type strain, whereas the efficiency of plating of the phage on these two strains at 28°C was less than lo-. UV doses to reduce survivors to 37% for the two strains at 28°C were about 3 J/m2, whereas the dose of 10 J/m2 was required to yield the same survival frequency at 42°C, indicating that the mutants are more sensitive to UV irradiation at low temperature than at high temperature. These results were summarized in Table 3. Spontaneous and UV-induced DNA degradation at 28 and 42°C in the revertants took place almost at the same level as those in the RecA13 and wild-type strains, respectively (data not presented). The cold-sensitive RecA- strain BE5701 reverts to mitomycin C resistance at a frequency of about 10'5. These revertants have properties characteristic of a Rec+ strain as to UV sensitivity, recombination ability, and propagation of Xbioll phage. Analysis of class 4 revertants. A class 4 revertant was as resistant to UV irradiation and as proficient in genetic recombination as its parent strains at both 28 and 42°C. However, plating efficiency of Xbioll phage on this revertant was less than i0-5 at 42°C. The mutation responsible for this property was cotransducible

TABLE 2. Classification of thermoresistant revertants of tif-1 and tif-1 ruv strains Class

UV sensitivity'

Growth of

___________

Xbioll

280C 42°C 420C 280C 420C 28°C 420C 28°C

1 1 2 3 4 5

JM12C

+

+

+

s

S

SS

Ss

Ss

S

+ + +

+d

s

s

Ss

s8

+

(s)d

+

BE5554c s

()

JM12 BE5554 (tif-1 ruv') (tif-1 ruv-4)

18 (26.1) 0 (0) 0 (0) 7 (10.1)

-

a

(tif-I ruv-9)

21 (16.4) 78 (60.9) 2 (1.6) 11 (8.6) 16 (12.5)

16 105 2 10 14

(10.9) (71.4) (1.4) (6.8) (9.5)

tif+ ruv+ tif+ ruv RecA-; tif-1 ruv+1 Cold sensitive RecA Abnormal recA? lexA? IexB? zab;

tif-1 ruv+1

ss, growth is inhibited

tif- 1 ruv by UV irradiation at 10 J/m2;

b Parentheses indicate percentage of each of the revertant classes. Properties of parent strains are quoted for comparison. d The Tifl strain can grow on NB and P agar at 42°C, when adenine is not supplied. c

Genotype or phenotype

tif- I ruv'

d

+ + (s)d s, Growth is inhibited by UV irradiation at 40 J/m2; +, growth is not inhibited by UV irradiation at 40 J/m2.

BE5556C

BE5556

44 (63.8)b

+

+ + +

No. of revertants from strains

4

OTSUJI AND IYEHARA-OGAWA

J. BACTERIOL.

TABLE 3. Properties of cold-sensitive recA mutant strains Recombination effiStrain

ciencya ___netic Relevant gemarker

280C

___

__

__

420C

UV resist-

A prophage

anceb

280C 420C

o

28°C

-bio

inductiond

42°C

28°C

420C

100 100 50 50 1 1 BE1594 + + recA+ 3.0 recA 701 43.8 3 10 10-7 BE5700 0.8 - + recA 702 0.14 70.1 3 10 10-7 BE5701 0.8 - + 0.05 0.1 3 3 recA13 BE5702 lo-7 1O-3 a In a cross, BE5702 (F-recA13) x W2252 (HfrC), as compared with BE1594 (F- recA+) x W2252 at each temperature, taken as 100%. Mating was interrupted at 40 min, and Lac' Strr recombinants were selected. b The dose of UV light (JWm2) that results in 37% survival of the irradiated bacteria. ' EOP, Efficiency of plating. d +, Prophage is induced by UV irradiation; -, not induced by UV irradiation.

with the srl' at about 77%, suggesting that the (11). In our experiment, 60 to 70% of the thermutation lies in the recA region. moresistant revertants of Tifl Ruv- strains were A sfi mutation does not suppress an ruv classifiable as RecA-, whereas 26% of the remutation. sifA and sfiB mutations suppress vertants of the Tif- strain were classifiable as filament formation produced in Tif or Lon- RecA- (Table 2). Preferential detection of mumutant strains under nonpermissive conditions tations in the recA locus can thus be ascribed to (11). We could not find a similar type of muta- the combination of Ion or ruv mutations with tion from the Tifl Ruv- strains. This result tif- 1. There were thermoresistant revertants that suggests that sfi mutations may not suppress ruv mutations. To test this possibility, two types had properties different from those of the stanof experiments were performed. In the first ex- dard RecA- mutant strain. Four revertants from periment, strain GC579 (F- his-4 sfiAll strr, Tifl Ruv- strains had stronger RecA- properties etc.) was crossed with Hfr BE5471, which trans- at low temperature (280C) than at high temperfers his' and ruv-9 as early markers. If sfiAll ature (42°0): lower recombination ability, lower suppresses the ruv mutation, then UV-sensitive plating efficiency of Abioll phage, higher UV strains should not be produced. Among 93 His' sensitivity, and higher level of DNA degradaStrr recombinants, 9 were UV sensitive. In the tion. These properties of the mutants are very cross between the same Hfr BE5471 and strain similar to those of the Zab-4 strain recently AB1157 (F- his-4 sfi+ strr), 10% of the His' Strr reported by Castellazzi et al. (6). Class 4 revertants were distinguishable from recombinants received the unselected donor ruv marker as indicated by their UV sensitivity. The RecA-, Zab-, and LexB- mutants by their propsimilarity of these frequencies suggests that erties. RecA- mutants are recombination defisfiAll does not suppress the UV sensitivity of cient, sensitive to UV, and not able to propagate the Ruv- mutant. Abioll phage (7, 19,28). Lysogenic RecA- strains In the second experiment, P1 phage grown on exhibit low spontaneous phage production and a strain (RGC103-9) carrying sulB (the same as are not inducible with various treatments such sfiB) was transduced into BE5036 (leu-6 ruv-9) as UV and X-ray irradiation (3). The LexB- and and AB1899 (leu-6 ion-i) strains, and Leu+ Zab- mutants, although they resemble RecA transductants were selected. None of 54 Ruv-9 mutants, are recombination proficient, but are strains inheriting the leu+ marker became UV able to propagate Abioll phage (5, 30). Revertresistant, whereas 14 of 54 Lonl strains inherit- ants of class 4 are proficient in genetic recombiing leu+ were UV resistant (26%). These two nation but are not able to permit the growth of experiments indicate that although sfi sup- Xbioll phage. Since these mutations lie in the recA region (6, 12, 31), they may alter in a presses Ion, it does not suppress ruv. different way the activities of the same recA + DISCUSSION product, protein X (9, 17, 25-27), which may Thermoresistant revertants of a Tifl strain consist of multifunctional structure (31). An can be divided into groups with mutations called analysis of protein X in the class 4 revertants tif+, recA, zab, and lexA, in which 70% of the and precise location of the mutation in the recA clones were true Tif+ revertants (5). However, locus are in progress. sfi mutations were isolated by George et al. only 3% of the revertants of the Tif- Lon- double mutant strains were classified as true Tif+, and (11) in spontaneous thermoresistant revertants 60% of them were classified as RecA- mutants of a Tif- Lon- double mutant strain, and may

THERMORESISTANT MUTANTS OF E. COLI tif ruv

VOL. 138, 1979

be identical to Sul- mutants isolated by Johnson and Greenberg (22) and Gayda et al. (10). These mutations suppress the division defects caused by either Ion or tif, but other phenotypic alterations caused by these mutations are not modified (10, 11, 14). Thus, the mutation sfi (sul) prevents radiation sensitivity, but not the overproduction of capsular polysaccharide in the Lonf strain. Although most of the effects of the ruv and the Ion mutations are very similar (they lead to UV sensitivity and formation of multinucleate filaments after temporary inhibition of DNA synthesis by DNA inhibitors [18, 33]), their roles in cell division differ, since sfi (sul) suppresses the inhibition of cell division in the Lon- and Tif mutant strains, but not in a Ruvmutant strain. The Ion and ruv mutations can also be distinguished by their other properties. The Lon- mutant produces excess capsular polysaccharide and forms mucoid colonies on minimal medium or nutrient broth agar at low temperature, whereas the Ruv- strain forms nonmucoid colonies under these conditions. Furthermore, A phage fails to lysogenize the Lonmutant efficiently, forming clear plaques on this strain (36), whereas it lysogenizes the Ruv- and the wild-type strains with the same frequency (manuscript in preparation). Thus, although the Ion and the ruv mutations may share a common phenotype with respect to UV sensitivity and septum formation, they clearly have distinct roles in the process of cell division when their DNA synthesis is temporarily impaired. ACKNOWiLEDGMENTS We are thankful to T. Horiuchi for helpful discussions during the course of study and to A. J. Clark for his critical reading of the manuscript. We thank J. George, K. B. Low, A. Markowitz, K. Matsubara, and T. Ogawa for supplying bacterial and phage strains.

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25. Little, J. W., and D. G. Kleid. 1977. Escherichia coli protein X is the recA gene product. J. Biol. Chem. 252: 6251-6252. 26. McEntee, K. 1977. Protein X is the product of the recA gene of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 74:5275-5279.

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27. McEntee, K., J. E. Hesse, and W. Epstein. 1976. Identification and radiochemical purification of the recA protein of Escherichia coli K12. Proc. Natl. Acad. Sci. U.S.A. 73:3973-3983. 28. Manly, K. F., E. R. Signer, and C. M. Radding. 1969. Nonessential functions of bacteriophage A. Virology 37: 177-188. 29. Markovitz, A. 1964. Regulatory mechanisms for synthesis of capsular polysaccharide in mucoid mutants of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 51:239246. 30. Morand, P., A. Goze, and R. Devoret. 1977. Characterization of lexB mutations in Escherichia coli K-12. J. Bacteriol. 131:572-582. 31. Morand, P., A. Goze, and R. Devoret. 1977. Complementation pattern of lexB and recA mutations in Escherichia coli K12: mapping tif-1, lexB and recA mutations. Mol. Gen. Genet. 157:69-82. 32. Otsuji, N. 1968. Properties of mitomycin C-sensitive mutants of Escherichia coli K-12. J. Bacteriol. 95:540-545. 33. Otauji, N., H. Iyehara, and Y. Hideshima. 1974. Isolation and characterization of an Escherichia coli ruv mutant which forms nonseptate filaments after low doses of ultraviolet light irradiation. J. Bacteriol. 117:

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