FEMS Microbiology Letters 44 (1987) 115-119 115 Published by Elsevier

FEM 02901

Bacteriophage MB78 DNA synthesis is specifically inhibited by the chelating agent ethylene diamine tetraacetic acid

Mukesh Verma and Maharani Chakravorty

Molecular Biology Unit, Institute of Medical Sciences. Banaras Hindu University, Varanasi, U.P. India

Received 27 April 1987 Accepted 21 May 1987

Key words: Bacteriophage; Salmonella phage MB78; Phage DNA synthesis

1. SUMMARY

The growth of bacteriophage MB78, a virulent phage of Salmonella typhimurium is extremely sen- sitive to the chelating agent EDTA. Other chelat- ing agents like EGTA, a specific chelator for Ca 2 + and orthophenanthroline which chelates Zn 2 + and Fe z+ have no effect. EDTA stops phage MB78 DNA synthesis while synthesis of host DNA and other Salmonella phage DNA are not affected in presence of such low concentrations of EDTA. The present report indicates that some early phage function(s) and most probably the phage DNA synthesis are sensitive to EDTA which is probably due to chelation of Mg 2+.

2. INTRODUCTION

of host-virus interaction [2,3]. In the course of experimentation with this phage is was observed that phage MB78 could not grow in minimal medium, MM [4] whereas it could grow in an other minimal medium, M9 [5]. Further experi- mentation revealed that citrate present in MM inhibited phage development. Citrate being a chelating agent, the effects of other chelating agents like EDTA (ethylenediamine tetraacetic acid), EGTA (ethylene glycolbis (amino ethyl ether) tetraacetic acid and orthophenanthroline on the phage development were tested. The present report indicates that growth of bacteriophage MB78 is extremely sensitive to EDTA unlike its host Salmonella typhimurium, phage P22 [6] and 9NA [7] and inhibition is at the level of DNA synthesis.

Bacteriophage MB78, isolated in our labora- tory, is a virulent phage of Salmonella typhimurium [1]. This phage possesses a number of interesting properties and is a suitable material for the study

Correspondence to: M. Chakravorty, Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221 005, U.P. India.

3. MATERIALS AND METHODS

[14C]Thymidine (60 mCi /m mole) was pur- chased from New England Nuclear Corp., Chicago, IL, U.S.A:, 2,5-diphenyl oxazole (PPO) and di- methyl 1,4-bis-(5-phenyl oxazolyl) benzene (POPOP) were products of the Amersham/Searle Corp., Arlington Heights, IL, U.S.A. Nitrocellu- lose filters (0.45 Fm) were obtained from Sigma

0378-1097/87/$03.50 © 1987 Federation of European Microbiological Societies

116

Chemicals, U.S.A. All other chemicals were com- mercial preparations and of analytical grade.

S. typhimurium (LT2, strain 18) and clear plaque-forming mutant (C1) of phage P22 were originally obtained from Prof. M. Levine, Depart- ment of Human Genetics, University of Michigan, Ann Arbor, MI, U.S.A. Phage MB78 was isolated in our laboratory [1]. Phage 9NA was a gift from Prof. B.A.D. Stocker, Stanford University School of Medicine, CA, U.S.A.

Growth and purification of phages were as described earlier [3]. S. typhimurium strain 18 was grown at 37°C in M9CAA to a cell density of 2.6 x 108 cells/ml. The cells were then infected with the phage at a multipficity of infection (m.o.i.) of 0.1 and incubated with aeration till lysis was completed. Chloroform was added to lyse the unlysed cells.

The lysate was concentrated by a two-phase system using 6.5% polyethylene glycol, 0.2% dex- tran sulfate and 0.3 M NaC1 [8]. The phage was finally purified through a stepwise gradient of CaC1 (= 1.4-1.6 g / m l ) as described by Botstein

[91. The rate of incorporation of [14C]thymidine

into the trichloracetic acid-insoluble fraction was followed as described by Smith and Levine [5].

4. RESULTS AND DISCUSSION

4.1. Phage MB78 is extremely sensitive to EDTA As the minimal medium MM which contains

citrate was found unsuitable for the development of phage MB78 the effect of citrate on the devel- opment of this phage in minimal medium [5] and LB medium [6] which do not contain any citrate was examined. Phage P22, a well studied tempera- ture phage [6] and phage 9NA, a virulent phage [7] which can grow in the same S. typhimurium host were used as controls for comparative purposes. Such experiments indicated that in the presence of sodium citrate the phage MB78 could not multiply whereas phage P22 and 9NA could (results not presented). As citrate is not a very strong chelat- ing agent, the effect of EDTA on the phage MB78 development was tested. It is evident from the results presented in Fig. 1 that MB78 is much

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Fig. 1. Effect of EDTA and EGTA on the development of MB78 and P22. LT2 cells were grown in M9 at 37 ° C. To various concentrations of EDTA or EGTA (as shown in the figure) were added 2 rain prior to infection. Multiplicity of infection was 5. The infected cells were allowed to grow for 120 min. A few drops of chloroform were added to the infected cell suspension to lyse the infected cells. Relative phage yield was calculated as percentage of plaque forming units (pfu) ob- tained in absence of chelating agent. Symbols: O, MB78 in presence of EDTA; O, P22 in presence of EDTA; zx, MB78 in presence of EGTA; A, P22 in presence of EGTA.

more sensitive to EDTA than the phage P22. In the presence of 0.5 mM EDTA, the growth of MB78 was almost completely inhibited whereas that of P22 was practically unaffected even at double the concentration of EDTA. Further, EGTA which specifically chelates Ca 2+ had no effect (up to a concentration of 5 mM) on the development of either P22 or MB78 (Fig. 1). As high as 100 mM EGTA reduces the burst size of phage MB78 to 70% only. Orthophenanthroline, a specific chelator for Zn 2+ (and Fe z+ also), also has no effect on the phage development.

In order to find out whether the chelating agents, EDTA and citrate, are interfering with early or late step(s) of phage development the effects of addition of chelating agents at different times after infection were studied. Both sodium citrate and EDTA affected the growth of MB78 if added within 20 min of infection (Fig. 2). The fact that both citrate and EDTA exhibited a similar type of effect with respect to time of addition suggested that these agents probably inhibited the same step.

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Fig. 2. Effect of addition of sodium citrate and EDTA at different times after infection. LT2 cells were grown in M9 till the cell density was 2.6x108 cells/ml. The cells were then divided into a number of batches and infected with phage at a multiplicity of infection of 5. Sodium citrate (2.6 mM) or EDTA (0.5 a multi-mM) was added at different times after infection as shown in the figure. The points represent time of addition of chelating agents. After 120 min chloroform was added to the infected cell suspension and the lysate was plated to determine the phage yield (pfu). Symbols: O, pfu in pres- ence of EDTA; O, pfu in absence of EDTA; zx, pfu in presence of sodium citrate; A, pfu in absence of sodium citrate.

117

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Fig. 3. Effect of EDTA on DNA synthesis of phage MB78. Cells growing exponentially in M9 (2.6 x l0 s cells/ml) were infected with phage MB78 at a multiplicity of infection of 5, and divided into a few batches. EDTA was added to different batches of infected cells at different times as indicated by an arrow mark in the figure. Rate of DNA synthesis was followed as described in Section 3. Symbols: e, uninfected cells; O, MB78-infected cells in absence of EDTA; zx, MB78-infected cells in presence of EDTA.

4.2. Effect of EDTA on phage DNA synthesis E D T A did neither affect adsorpt ion of MB78

phage to its host nor inject ion of D N A into the host (data not presented). No D N A synthesis was, however, observed when E D T A was added at the very time of the infect ion (Fig. 3). If E D T A was added 12 and 15 min after infection, when phage D N A synthesis had already started, D N A synthe- sis cont inued only for a very short period of t ime and then stopped. There was no effect of E D T A on the rate of D N A synthesis if added 20 min after infection. Similar results were obta ined when the experiment was carried out with cells growing in M9CAA. The results clearly indicate that E D T A is interfering with phage D N A synthesis. U n d e r similar experimental condi t ions neither host D N A synthesis nor the D N A synthesis of phage P22 and 9NA is inhibi ted (data not presented).

4.3. Comparison between Mg 2 + requirement of P22 and MB 78

Our results with E G T A and or thophenanthro- l ine suggest that the effect of E D T A or citrate is not due to deplet ion of Ca 2+, Z n 2+ or Fe 2+.

Hence the Mg 2+ requirements of the two phages were compared. Cells growing exponent ia l ly in M9 medium were collected when the concentra- t ion of the cells was 2.6 x 108 c e l l s / m l and washed twice with M9 med ium without Mg 2+. The cells

were then infected with either phage MB78 or P22 at m.o.i, of 10. After 10 min, i.e. after 5 min of antisera treatment, the cell suspension was divided into several batches and different amount s of MgSO 4 were added (as indicated in Fig. 4). The infected cells were incubated further and the burs t size was determined in all cases. It may be men- t ioned that the deplet ion of Mg 2+ did not affect

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Fig. 4. Mg 2+ requirement for growth of phages P22 and MB78. Growth of cells and infections were carried out as described in the legend to Fig. 3. Rest of the experimental conditions as described in the text. Symbols: zx, P22; ©, MB78.

neutralizing the negatively charged DNA. The in- activation of thermophilic bacteriophage T5 was reported to be due to dissociation of the phage into its structural components such as empty head, free tail and liberated DNA [16]. Such inactiva- tion, however, requires much higher concentra- tions of chelating agents than observed in case of MB78. The inactivation of the phage particles as a possible mode of action of EDTA was tested.

Purified phages at two different concentrations were suspended in 0.2 M Tris-HCl, pH 7.4, and incubated for 30 min in the presence of various concentrations of EDTA. A phage concentration of 1 0 9 p f u / m l was specifically chosen as phage particles were present at that concentration during physiological experiments described earlier. After treatment the phage samples were diluted and plated for counting of viable phage particles. Phage particles at a concentration of 1 0 9 p f u / m l were not much inactivated when incubated in the pres- ence of EDTA at 37 °C for 30 min (Fig. 5). The extent of inactivation of phage increased when the phage sample was more concentrated (1013 p f u / ml).

The interesting part of the present set of inves- tigations is that phage MB78 development is in- hibited by EDTA at a concentration as low as 0.5 mM of EDTA which is not found for other

the number of infective centres suggesting thereby that adsorption of the phage to the host is not dependent on Mg 2+. It is 'clear from the results (Fig. 4) that the Mg 2+ requirement of phage MB78 is higher than that of P22. The relationship be- tween Mg 2+ concentration and the burst size of MB78 shows a sigmoidal curve suggesting some kind of cooperative interaction.

4.4. Inactivation of phage particles as a possible mode of action of EDTA

Inactivation og phage by chelating agents and protection offered by divalent cations are not un- common phenomena [10-16]. Inactivation of phage by chelating agents is usually interpreted to be due to complexing of cations present in the phage with the chelating agents. The cations are required to maintain the stability of the phage by

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Fig. 5. Effect of chelating agents on the viability of phage MB78. Purified phage particles (1013 pfu/ml or 10 9 pfu/ml) in 0.02 M Tris-HC1, pH 7.4, were incubated at 37 °C for 30 min in the presence of various concentrations of EDTA. Sym- bols: e, Phage concentrations 1013 pfu/ml; e, Phage con- centration 10 9 pfu/ml.

bac te r iophages or bac te r ia and this is due to inter- ference with phage D N A synthesis. M a y be, some phage- induced enzyme or enzymes of the D N A biosynthe t ic pa thway get easily dep le ted of some cat ion, p r o b a b l y Mg 2+. Such enzyme or a subuni t thereof has to be phage MB78-specif ic as the growth and deve lopmen t of nei ther the host cell nor the o ther phages growing in the same host are inhib i ted under s imilar condi t ions . I t will be qui te interest ing to look for such a phage -coded meta l - loprotein.

A C K N O W L E D G E M E N T S

The f inancia l ass is tance f rom the Counci l of Scientific and Indus t r ia l Research, G o v e r n m e n t of India , the D e p a r t m e n t of Science and Technology, G o v e r n m e n t of Ind i a and the Univers i ty G r a n t s Commiss ion are gra teful ly acknowledged.

R E F E R E N C E S

[1] Joshi, A., Siddiqui, J.Z., Rao, G.R.K. and Chakravorty, M. (1982) J. Virol. 41, 1038-1043.

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[2] Verma, M. and Chakravorty, M. (1985) Biochem. Bio- phys. Res. Commun. 132, 42-48.

[3] Verma, M., Rao, A.S.M.K. and Chakravorty, M. (1986) Virology 151,274-285.

[4] Chakravorty, M. (1970) J. Virol. 5, 541-547. [5] Smith, H.O. and Levine, M. (1964) Proc. Natl. Acad. Sci.

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[10] Lark, K.G. and Adams, M.H. (1953) Cold Spring Harbor Symposium. Quant. Biol. 18, 171-183.

[11] Van Vunakis, H. and Herriott, R.M. (1962) J. Bacteriol. 83, 590-596.

[12] Shafia, F. and Thompson, T.L. (1964) J. Bacteriol. 88, 293-296.

[13] Villarejo, M., Hua, S. and Evans, Jr., E.A. (1967) J. Virol. 1, 928-934.

[14] Luftig, R. and Haselkorn, R. (1968) Virology, 11 664-674. [15] Yamamoto, N., Fraser, D. and Mahler, H.R. (1968) J.

Virol. 2, 944-950. [16] Bassel, A., Shaw, M. and Campbell, L (1971) Virology 7,

663-672.