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Biochimica et Biophysiea Acta, 517 (1978) 531--534 Elsevier/North-Holland Biomedical Press

BBA Report BBA 91460

BACTERIOPHAGE ~X174 GROWTH IN AN ESCHERICHIA COLI dnaI ts MUTANT, KS810

HIROSHI SAKAI, KOUNOSUKE WATABE and TOHRU KOMANO

Laboratory of Biochemistry, Department of Agricultural Chemistry, Kyoto University, Kyoto (Japan) (Received October 19th, 1977)

Summary A bacteriophage ~bX174-sensitive Escherichia coli dnaI ts mutant, KS810, was constructed and growth of ~X174 in the cells was investigated. ¢X174 and ~X174am3trD could grow normally at 43°C as well as 27°C, therefore we conclude that the growth of bacteriophage ¢X174 is not dependent upon the host dnaI gene product.

Replication of small bacteriophage DNA such as ~X174, G4 and M13 in vivo and in vitro requires functional products of some host dna genes, and the dependency of phage growth on these gene products has also been investigated [1--6]. The investigation on this line is useful not only for analysis of process of phage DNA replication but for elucidation of the dna gene function in the Escherichia coli cells. ~X174 DNA replication requires dnaB, C, E, G, H and Z gene products, while dnaA and P functions are dispensable for ¢X174 DNA replication. Also the dnaI gene product has been demonstrated to be dispensable for ~X174 phage growth, but this demonstration was performed by the use of a transfection method [2]. E. coli KS810 (thy-, trp-, his-, Hcr-, ~X s, dnaltS), a mutant temperaturesensitive in the initiation of chromosome replication, was constructed in this laboratory by the cross of E. coil WM301-208, an original dnaI mutant from Dr. Messer [7], with E. coli H550 (Hfr313, EndI-, Str R, ilv-, leu-, ~X s) from Dr Hoffmann-Berling. A temperature-insensitive revertant of KS810 was also used. Bacteriophage ~X174 wild-type and ~X174am3trD, a temperatureinsensitive mutant isolated in our laboratory by mutagenesis of ~X174am3 with nitrous acid, were used. ~X174am3trD exhibited unusual resistance to heat inactivation at 56°C, and was, therefore, assumed to be a capsid mutant. Serological and electronmicrographic analyses of phage particles, and electrophoretic analysis of viral coat proteins in the presence of sodium dodecyl

532

sulfate could n o t distinguish CX174am3 and ¢X174am3trD (data not shown: detailed results will be presented elsewhere). KS810 strain constructed as described above exhibited a typical character for a DNA initiation mutant. As shown in Fig. 1, it was confirmed that DNA synthesis and the increase in viable counts of KS810 gradually ceased at 43°C, the restrictive temperature as reported by Beyersmann [ 12]. These results indicated that the dnaI function was inactivated at the restrictive temperature. E. coli KS810 and the revertant were grown to 1- 10s--2 • 108 cells/ml in L-broth medium containing 50 t~g/ml thymine at 27°C and divided into t w o portions. One was further incubated at 27°C for 70 min, and the other was transferred to 43°C and incubated for 70 min. The cells were infected with ~X174 wild-type at a multiplicity of infection of 0.3 and were allowed to stand for 10 min at 27 and 43°C, respectively. Then, aeration was started and a sample of 0.1 ml was pipetted o u t into ice-cold starvation buffer [8] at the indicated times. In order to test whether comparable results were obtained in different experimental conditions, a similar experiment was performed in a synthetic medium, TPG-CA [ 3]. It is reported [ 14,15] that some capsid mutants of ¢ X 1 7 4 can grow in the dna mutant cells in which wildt y p e phage cannot grow. It is probably because of some interaction between phage capsid protein molecules and the inactive dna product of host cells. In the present experiment, CX174am3trD may exhibit behavior different from ~X174 wild-type in dnaI mutant. So, the growth of ~X174am3trD was also examined in L-broth medium. Intracellular phages were assayed as described by Denhardt and Sinsheimer [8]. Normal growth of phage was observed at 27 and 43°C (Fig. 2). Phage yield was 2300 at 27°C and 3000 at 43°C in KS810 cells; 1500 at 27°C and 900 at 43°C in the revertant cells. The ratio of the increase at 43°C to that at 27°C was 1.3 in the KS810 and 0.6 in the revertant cells, respectively (Table I). These results suggested that the m u t a n t cells could support the growth of phage ¢ X 1 7 4 wild-type both at 27°C and at 43°C. In three cases of this study, the ratio of phage increase at 43°C to that at 27°C ranged from 1.3 to 1.6 in KS810 cells and from 0.6 to 0.8 in the revertant cells (Table I). A significant difference was n o t observed. It may be because lysis of KS810 cells was slightly delayed that the ratios of phage yield in KS810 cells were a little higher than those in the revertant cells. Thus, the present data show that the p r o d u c t of dnaI gene is n o t essential for growth of ¢X174. The present data also suggest that an interaction between the dnaI gene p r o d u c t and the phage capsid molecules is unlikely because CX174am3trD exhibited the same behavior in dnaI mutant cells as ¢ X 1 7 4 wild-type. Recently, Taketo has examined the dependency of ~X174 growth on the dnaI function by the transfection method, showing the same conclusion as ours [2]. His experimental results have suggested that at least the dnaI function is n o t required for ¢ X 1 7 4 DNA replication in dnaI mutant cells. In our study we have used the normal infection system of ¢X174. Therefore, in addition to the Taketo's evidence, the present results would suggest that the dnaI function is neither required for the growth of phage nor for the process of adsorption, eclipse and DNA penetration. The following interpretations

533

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Fig. 1. G r o w t h a n d D N A s y n t h e s i s o f K S 8 1 0 a f t e r a t e m p e r a t u r e s h i f t f r o m 27 t o 4 3 ° C . E. coli K S 8 1 0 a n d a t e m p e r a t u r e - i n s e n s i t i v e r e v e r t a n t w e r e g r o w n a t 2 7 ° C t o a c o n c e n t r a t i o n o f 7 • 107 e e l i s / m l in T P G - C A m e d i u m [ 3 ] c o n t a i n i n g 2 0 # g t h y m i n e / m 1 . F o r t h e m e a s u r e m e n t o f cell g r o w t h , h a l f t h e culture was transferred to 43°C and the other half was kept at 27°C and the incubation was continued. 0 . 1 m l o f t h e c u l t u r e w a s w i t h d r a w n a n d viable c o u n t s w e r e m e a s u r e d . T h e r e l a t i v e v a l u e o f 1 , 0 c o r r e sponds to 7 • 10 ~ celis/mL For the measurement of DNA synthesis, [3H]thymine was added to the c u l t u r e a t a c o n c e n t r a t i o n o f 1 . 6 # C i / 2 0 ~tg p e r m l a n d i n c u b a t i o n w a s c o n t i n u e d o v e r n i g h t a t 2 7 ° C . T h e c u l t u r e w a s d i l u t e d 1 0 0 f o l d i n t o t h e s a m e m e d i u m a t - - 2 h. T h e n , 0 . 2 m l w a s w i t h d r a w n a n d radioactivity incorporated into acid-precipitable materials was measured. Temperature shift to 43°C was performed at the zero time. The relative value of 1.0 corresponds to 2350 epm/0.2 ml. e, growth o f a t e m p e r a t u r e - i n s e n s i t i v e r e v e r t a n t (cells p e r m l ) ; o, g r o w t h o f K S S 1 0 (cells p e r rni); ~7, D N A s y n thesis of KS810.

Fig. 2. G r o w t h o f ~ X 1 7 4 w i l d - t y p e in K S 8 1 0 a n d t h e r e v e r t a n t a t 2 7 ° C a n d 4 3 ° C in L - b r o t h c o n t a i n i n g 5 0 # g / m l t h y m i n e . E. coli K S 8 1 0 a n d t h e r e v e r t a n t w e r e g r o w n t o l o g p h a s e in L - b r o t h a t 2 7 ° C . T h e cells w e r e i n f e c t e d w i t h (bX174 w i l d - t y p e a t a m u l t i p l i c i t y o f i n f e c t i o n o f 0 . 3 a t 2 7 ° C o r 7 0 r a i n a f t e r the temperature shift-up at 43°C. After 10 rain for adsorption, a 0.1 ml sample was pipctted out into ice-cold starvation buffer at the indicated times. The infective centers were assayed by the soft agar m e t h o d [ 2 4 ] a f t e r a p p r o p r i a t e d i l u t i o n w i t h s t a r v a t i o n b u f f e r , o, K S 8 1 0 a t 2 7 ° C ; [], t h e r e v e r t a n t a t 2 7 ° C . e , K S 8 1 0 a t 4 3 ° C ; m, t h e r e v e r t a n t a t 4 3 ° C .

TABLE I P H A G E Y I E L D IN d n a I + A N D d n a l ts C E L L S Phage ~X174 was infected at a multiplicity of infection.of 0.3 at 27 and 43°C after preincubation for 7 0 m i n . I n f e c t i v e c e n t e r s (a) a n d p l a q u e - f o r m i n g u n i t s (b) w e r e a s s a y e d b y t h e r e p o r t e d p r o c e d u r e [ 2 0 ] . B u r s t size

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43°C

43°C/27°C

27°C

43°C

43°C/27°C

27°C

43°C

43°C/27°C

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900 3000

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500 960

400 1300

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35 35

0.62 1.6

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1.5

3.3

1.9

3.3

0.4

1.0

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are possible for why the dnaI gene product is not essential for the growth of ¢X174; (1) the DNA replication of phage ¢X174 involves a functional substitute for the dnaI gene function; (2) the DNA replication of phage ¢X174 does not involve a process mediated by the dnaI gene function or by a substitute for it. This work was supported in part by a scientific grant from the Ministry of Education, Japan. References 1 2 3 4 5 6 7 S 9 10

Denhardt, D.T, (1975) CRC Critical Rev. MicrobioL 4. 161--223 Taketo, A. (1975) MoL Gen. Genet. 139, 285--291 Sakai, H. and Komano, T. (1975) Biochim. Biophys. Acta 395, 811--820 Wickner, S. and Hurwitz, J. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4 1 2 0 - - 4 1 2 4 Sckeckman, R.. Weiner~ J.H., Weiner, A. and Kornberg~ A. (1975) J. Biol. Chem. 250, 5859--5865 Wickner, S. and Hurwitz, J. (1976) Proc. Natl. Acad. Sci. U.S. 73, 1053--1057 Beyersmann, D., Messer, W. and Schlicht, M. (1974) J. Bacteriol. 1 1 8 , 7 8 3 - - 7 8 9 Denhardt, D.T. and Sinsheimer, R.L. (1965) J. Mol. Biol. 12, 641--646 Christine, C.V., Primrose, S.B. and Dowell, C.E. (1975) J, Virol. 15, 281--287 Christine, C.V. and Dowell, C.E. (1976) J. Virol. 18, 942--949