Isolation and Characterization of a Bacillus ...

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May 16, 1977 - We thank David Lancki and Carl Cohen for the antisera, R. Slepecky for data before publication and for phage CS-I, and M. S. Hanlon for the ...

J. gen. ViroL (I977), 36, ~ Printed in Great Britain



Isolation and Characterization of a Bacillus megaterium QM B1551 Bacteriophage (Accepted 16 May 1977) SUMMARY

A bacteriophage specific for Bacillus megaterium QM Br55I was isolated. This phage (MP-7) was a lytic phage of typical morphology and distinct stability properties. The DNA was double-stranded, with a mole ~ G + C of 38"9 + 0"7 and a mol. wt. of 42 to 44 x io 6. Bacillus megaterium QM BI55I has been used extensively to study the properties of spores and the mechanism of germination. While there are considerable biochemical data and a variety of mutants available (Hyatt & Levinson, I964; Vary, I972; Setlow, 1975), there is no method to study the genetics of this strain (Vary, I975). Towards this end, we have isolated a bacteriophage that is specific for B. megaterium QM BI55I which may be used (a) to identify this strain, (b) as a source of DNA for transfection studies, and (c) as a potential helper phage in attempts at isolating transducing particles. The properties of this bacteriophage are reported here. Bacteriophage were isolated and purified from the soil by the method of Adams 0959). One of the clear plaques (MP-7) was arbitrarily chosen for further study. Lysates were made from single plaques and assayed for plaque-forming units (p.f.u.) by the overlay method of Adams 0959)- The indicator cells were grown from B. megaterium QM BI55 r by suspending lyophilized spores (approx. io 6 spores) in I ml sterile H~O followed by heat activation at 6o °C for Io min and dilution into 5o ml of supplemented nutrient broth (SNB; Vary, I97z). The culture was incubated on a rotary shaker at 3oo rev/min and 3o °C until the extinction at 66o nm was about o'5. The characteristics of MP- 7 were as follows. The phage produced clear plaques surrounded by a halo as has been reported for other B. megaterium phages (Murphy, I957; Cooney, Jacob & Slepecky, I975). Electron microscopy showed that MP-7 had a hexagonal head of 6o to 7o nm in diam. and a thin tail about ~5o nm long, not unlike other bacillus lytic and transducing phages. Host range studies showed that MP- 7 was specific for B. megaterium strains QM BI55I and ATCC 7o5I and did not infect strains ATCC 9885 or ATCC 19213, B. cereus T, B. licheniformis A5 or B. subtilis I68 at either low (< o.I) or high (> Iooo) multiplicities of infection. With those strains of B. megaterium that were insensitive to MP- 7, attempts at phage induction by ultraviolet light irradiation (Yelton & Thorne, I97o) were unsuccessful. In a one-step growth curve by the method of Adams (I959), the latent period was 6o to 65 min, similar to other bacillus phages. However, the eclipse time was 5o min and the burst size was I37O _+I9o (average + standard deviation from 7 experiments). This large burst size was probably not a result of multiple infections because the multiplicities of infection were < o.I. Some of the other properties of MP-7 (Table I) were its stability to heating at 5o °C but not 6o °C, relatively high stability in organic solvents and sensitivity to salt. The inhibition by salt was a result of inhibition of adsorption similar to that described for Eseherichia eoli T phages and B. subtilis ¢25 phages (Kozloff & Henderson, I955; Doyle et al. I974). 35-2

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548 Table L

Characteristics of MP-7 phage particles % decrease in viability

Heat stability* 50 °C for 2 h 60 °C for I5 min

1 76

Sensitivity to organic solvents :t Chloroform Toluene Benzene Hexane

o o 30 5

Sensitivity to 60 mM-salt::~ NazHPOa K~HPO~ K2SO4 KC1

90 90 90 90

* Samples of a lysate containing lO1° p.f.u./ml were placed in tubes at the indicated temperature and time and then cooled on ice. Samples were assayed for phage viability by the agar overlay method. t A mixture ofo.2 ml of each solvent was added to 1.o ml of lysate containing approx. IO9 p.f.u./ml. The suspension was mixed for I5 s on a Vortex mixer, incubated for 30 min at 30 °C and the aqueous layer was assayed for phage viability by the agar overlay method. :~ A lysate was assayed by the agar overlay technique on plates containing each of the above listed salts at a final concentration of 6o mM (pH 7). Table 2. Experiment Base composition by paper chromatography* Thermal denaturation in o'o15 MNaC1 to o'o15 M-sodium citrate (pH 7'2)* Analysis of hyperchromicity data Equilibrium sedimentation in CsCI* Mol. wt. by chemical analysis'~ Mol. wt. by velocity sedimentation:~

Properties of MP-7 DNA


Mole ~ G + C

G = C 39"3 4-o.6 A = T Tm = 70 °C 39"3

p = 1"6973 43"9 × IO6 41"5x IO~

41 38"1 -

Reference for method Sinsheimer & IZoerner (1952) Mandel & Marmur (I968) Hirschman & Felsenfeld (1966) Schildkraut, Marmur & Doty (1962)



Bruner & Vinograd (1965)

* DNA was extracted from a high titre lysate by phenol extraction, dialysed against o'I5 M-NaCI-o'I5 Msodium citrate (pH 7"2) and used in the indicated experiments. t A high titre lysate was assayed for the number of p.f.u, by the overlay method. The amount of DNA was estimated by the diphenylamine positive material that was soluble in hot 5 ~ (v/v) perchloric acid. The mol. wt. was calculated from the #g DNA/p.f.u., assuming double-stranded DNA with an average mol. wt. of 625/nucleotide pair. ~: The DNA was extracted from a high titre lysate with NaClO4, dialysed against o'I5 M-NaCI-o.I5 Msodium citrate, treated with 250 #g Pronase/ml at 30 °C for z h, dialysed again and the S value determined. In the course o f these studies, we learned o f the isolation a n d c h a r a c t e r i z a t i o n o f a lytic p h a g e ( C S - I ) f o r B. megaterium A T C C t 9 z i 3 (Cooney, J a c o b & Slepecky, 1975). These t w o phages were similar in b o t h m o r p h o l o g y a n d the p h y s i c o c h e m i c a l properties o f their D N A . H o w e v e r , the f o l l o w i n g properties distinguished the t w o phages. T h e CS-I p h a g e was sensitive to o r g a n i c solvents whereas M P - 7 was n o t ; CS-I was stable to heat at 6o °C whereas M P - 7 was not. T h e host range specificity o f M P - 7 was limited to 2 strains whereas C S - I infected all the B. megaterium strains tested. While the latent p e r i o d o f the two phages was the same, the eclipse p e r i o d o f M P - 7 was longer t h a n that o f C S - I a n d no burst size was m e a s u r e d f o r C S - I .

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The phage D N A was extracted and characterized. In order to concentrate the phage, lysates were centrifuged at 59000 g for 3 h and the phage pellet was suspended in phage buffer (Romig, 1962). The D N A was extracted from this high titre lysate by either phenol (Epstein, i967) or sodium perchlorate (Freifelder, I966). The D N A sample was assayed for deoxyribose (Burton, 1956) and phosphate (Ames & Dubin, I96o) and found to contain an average ofo.989 #mol ofphosphate//zmol of deoxyribose. The sample contained undetectable amounts of R N A and < 3 ~ protein by the methods of Keleti & Lederer (I974) and Lowry et al. (I95I). The phage D N A had a spectrum typical for D N A with a ratio of extinctions at 260 nm and 28o nm of 1.84 and a ratio of extinctions at 26o nm and 23o nm of 1.9. No unusual extinction peaks were observed. When phage D N A was denatured using alkali a typical melting profile was obtained for a double-stranded D N A . Table 2 summarizes the properties of MP- 7 phage DNA. The mole ~ G + C for the phage D N A calculated from thermal denaturation, buoyant density in CsCI, and base composition agreed closely. These studies suggest that the phage has a double-stranded D N A with a mole ~ G + C of 38"9 + o'7 with no unusual bases. Mol. wt. determinations were done by sedimentation velocity with sodium perchlorate extracted DNA. This D N A was first treated with pronase to digest the phage protein that tended to complex with the released DNA. The phage D N A had an S value of 39"95 corresponding to a mol. wt. of 4I"5 x lO6. This value agrees, within the error of the methods, with the mol. wt. obtained by chemical analysis of the amount of D N A per viable phage (Table 2). We thank David Lancki and Carl Cohen for the antisera, R. Slepecky for data before publication and for phage CS-I, and M. S. Hanlon for the use of her analytical instruments. This work was supported by grants from the National Science Foundation (GB-36987) and the National Institutes for Health (AI I2678-o 0.

Department of Biological Chemistry University of Illinois Medical Center Chicago~ Illinois 60612, U.S.A.

P . M . CARVALHO* J . C . VARY~"


ADAMS,M. H. (1959). In Bacteriophages. New York: Interscience Publishers. AMES,B. Iq. & DUBIN,D. T. (I960)- The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. Journalof Biological Chemistry235, 769-775. BRINER,~. &VlNOORAD,J. (1965)- The evaluation of standard sedimentation coefficients of sodium RNA and sodium DNA from sedimentation velocity data in concentrated NaCI and CsC1 solutions. Biochimica et Biophysica Acta xo8, I8-2 9.

BURTON,K. (1956). A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. BiochemicalJournal 62, 315-323. COONEY, P. H., JACOB,R. S. & SLEPECK.Y,R. A. (I975). Characteristics of a Bacillus megaterium bacteriophage. Journal of General Virology 26, 13I-I34. DOYLE, R. J., McDANNEL,M. L., STREIPS,LI. N., BIRDSEEL,D. C. & YOUNG, F. E. (1974). Polyelectrolyte nature of bacterial teichoic acids. Journal of Bacteriology xx8, 6o6-615. EPSTEIN, H. T. (1967). Transfection enhancement by ultraviolet irradiation. Biochemical and Biophysical Research Communications 27, 258-262. FREIFELDER,D. (1966). Effect of NaC104 on bacteriophage release of D N A and evidence of population heterogeneity. Virology 28, 742-750. HIRSCHMAN,S. Z. & FELSENFELD,G. (1966). Determination of D N A composition and concentration by spectral analysis. Journal of Molecular Biology I6, 347-358. HYATT, M. T. & LEVINSON, I-I.S. (1964). Effect of sugars and other carbon compounds on germination and postgerminative development of Bacillus megaterium spores. Journal of Bacteriology 88, 14o3-1415. * Present address: Department of Microbiology, University of Illinois Medical Center, Chicago, Illinois. t To whom correspondence should be addressed.



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KELETt, G. & LEDERER, W. H. (I974). In Handbook of Micromethods for the Biological Sciences. New York, London: Van Nostrand Reinhold Company. KOZLOFF, L. M. & r~ENDERSON, K. 0955). Action of complexes of the zinc group metals on the tail protein of bacteriophage T2r +. Nature, London x76, 1169-I17I. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (I95I). Protein measurement with the folin phenol reagent. Journal o f Biological Chemistry x93, 265-275. MANDEL, ra. & MARMUR, S. (I968). Use of ultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA. In Methods in Enzymotogy, vol. x~I, part B, pp. I95-2o6. Edited by L. Grossman and K. Moldave. New York and London: Academic Press. MURPHY, S. S. (1957). A phage associated enzyme of Bacillus megaterium which destroys the bacterial cell wall. Virology 4, 563-58I. ROMIG, W. R. (I962). infection of Bacillus subtilis with phenol-extracted bacteriophage. Virology x6, 452-459. SCHILDKRAUT, C. L., MARMUR, J. & DOTY, P. (I962). Determinations of the base composition of deoxyribonucleic acid from its buoyant density in CsC1. Journal o f Molecular Biology 4, 430-443. SETLOW, V. (1975). Energy and small-molecule metabolism during germination of Bacillus spores. In Spores, voL VI, pp. 443-450. Edited by P. Gerhardt, R. N. Costilow and H. L. Sadoff. Washington, D.C.: American Society for Microbiology. SINSHEIMER, R. L. & KOERNER, J. F. (1952). A purification of venom phosphodiesterase. Journal of Biological Chemistry x98, 293-295. VARY, J. (2. (I972). Spore germination of Bacillus megaterium QM B1551 mutants. Journal of Be cteriology x xz, 640-642. VARY, s. c. (I975). N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis during germination of Bacillus spores. Mutation Research 29, 493-496. YELTON, D. B. & TRORr~E, C.B. (t970). Transduction in Bacillus cereus by each of two bacteriophages. Journal o f Bacteriology xo2, 573-579-

(Received 29 March I 9 7 7 )

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