Large scale preparation of Rhizobium meliloti bacteriophages by fermenter culture

Journal of Virological Methods, 8 (1984) 155-160

155

Elsevier JVM 00297

LARGE SCALE PREPARATION OF RHIZOBIUMMELILOTl BACTERIOPHAGES BY FERMENTER CULTURE

MICHEL

WERQUIN,

CLAUDE

DEFIVES,

LAHCEN

HASSANI

and MONIQLJE

ANDRIANTSIMIAVONA-OTONIA Laboratoire de Microbiologic. Bdt. SN2, UniversitP des Sciences et Techniques de Lille, 59655 Villeneuve d’dscq Cedex, France

I December

(Accepted

A simple fermenter

method

for preparation

of Rhizobium melilofi bacteriophages

was established

employing

culture.

This technique ranging

1983)

allowed phage production

from 5. IOl2 to 1.2.10i3 PFU/ml

to be checked by dissolved oxygen measure.

were found after polyethylene

glycol precipitation

Phage suspensions and centrifuga-

tion in CsCI. Rhizobium meliloti

bacteriophages

fermenter

culture

INTRODUCTION

Bacteria of the genus Rhizobium are nitrogen-fixing

microorganisms

of importance

in agriculture. Recently, many genetic experiments have been carried out with them to locate nitrogen fixation genes (Ruvkun and Ausubel, 1981; Ruvkun et al., 1982; Corbin et al., 1983; Forrai et al., 1983). Bacteriophages might be used as vectors for introducing

DNA into their genome,

and transduction

was achieved in various strains

of Rhizobium melifoti (Kowalski, 1967; Sik and Orosz, 1971; Kowalski and Denarit, 1972; Sik et al., 1980). Preliminary characterization of several Rhizobium meliloti was undertaken in our laboratory

(Krsmanovic-Simic

and Werquin,

1973; Werquin

et al., 1977). Indeed,

physicochemical and genetic data on viruses require high titred bacteriophages in large amounts. However, the classical methods yield only small quantities of high titred phage stocks and such methods are time-consuming (Hershey et al., 1943; Swanstrom and Adams, 1951; Adams, 1959). Bacterial growth in shaked liquid medium has also been used for bacteriophage production but this procedure provides insufficient oxygenation for the cells, and therefore the yield of aerobic bacteria such as Rhizobium is relatively low. Concomitantly, an unsatisfactory yield of phages is obtained. Moreover, concentration and purification of the lysates could be achieved by several methods such as polyethylene 0 I66-0934/X4/$03.00

c, 1984 ElseTier Science Publishers

B.V

156

These glycol precipitation (Yamamoto et al., 1970) and high speed centrifugation. methods may have deleteriouseffects on bacteriophages. The good aeration obtained in allows Rhizobium

fermenter obtain

large amounts

MATERIALS

to be grown

quickly,

of high titred bacteriophages

and this procedure

was used to

before lysates treatment.

AND METHODS

Bacteriophages and bacterial strains These are listed in Table 1. Bacteriophages

were isolated

during

screening

of soil

samples for Rhizobium meliloti phages (Krsmanovic-Simic and Werquin, 1973). The four nitrogen-fixing Rhizobium meliloti wild type strains M&S, M,,S, MI&S, M,,S (originating from our laboratory) were used as sensitive strains. Media Rich medium (RC) used for bacterial growth contained per litre K,HPO,, 1 g; MgS0,.7H,O, 0.2 g; Difco yeast extract, 1 g; fructose, 10 g. pH was adjusted to 7.2. 1.5% Difco agar was added for solid medium. The overlayer agar medium used to titrate viable bacteriophages contained per litre Na,HPO,. 12H20, 0.45 g; Na,SO,. loH,O, 0.06 g; KNOJ, 0.06 g; FeCl,, 0.01 g; CaCl,.2H,O, 0.06 g; MgC12.6H,0, 0.1 g; mannitol, 10 g; Difco agar, 7.5 g; pH 7.2. For bacteriophage production, RC medium was supplemented with 2.5 g of Difco Bacto-Peptone, 0.06 g of CaC1,.2H,O and 0.1 g of MgCl,.6H,O per litre. Preparation of small quantities of high-titre bacteriophages Bacteriophages were grown using the agar layer method (1959). TABLE

as described

1

Bacteriophage

and bacterial

strains

Phages

Rhizobium

NM,

M,S

NM,

M,S

NM,

M8

NM,

MYS

NM,

M,,S

CM, CM,

M,& M,,S

CM,

M,,S

MM,

M,,S

M: Meliloti: Bradley’s

by Adams

N: non contractile

group

C.

tail: Bradley’s

group A; C: contractile

meliloti

host strams

tail: Bradley’s group B: M: mmus tall:

157

Fermentation process Host bacteria

were grown in 300 ml of RC medium at 30°C with vigorous

When the cells reached a concentration ture was inoculated containing following

into a fermenter

of about 2. lo9 bacteria/ml, of 4.5 1 capacity

2700 ml of bacteriophage conditions

production

were used: temperature:

the whole precul-

(Setric F-7-T Toulouse, medium.

30°C; constant

agitation. France)

For fermentation,

the

air flow: 60 l/h; agitation

rate: 300 rpm; initial percentage of dissolved oxygen: 90%. Bacteria in the log phase of growth were inoculated with bacteriophage at a multiplicity of infection (m.o.i.) ranging from 1 to 0.2. Bacterial lysis was followed by recording dissolved oxygen and optical

density.

Concentration and purification of crude lysate Phage particles were concentrated according to Yamamoto

et al. (1970) using 8%

polyethylene glycol 6000 (PEG) and 0.5 M NaCl at 4°C until complete precipitation. After centrifugation (2,000 X g, 15 min) the pellet was resuspended in phage buffer (Mannasse et al., 1972) and recentrifuged at 12,000 X g for 15 min. Then, phages were sedimented by high speed centrifugation (Beckman 60 Ti rotor; 78,000 X g, 1 h). The pellet was resuspended in 14 ml of phage buffer supplemented with 9.3 g of CsCl giving a refraction index of 1.379 at 25°C. After centrifugation (Beckman SW 50-l rotor; 100,000 X g, 15 h, 4”C), phage band was collected and dialysed against phage buffer. The amount (1959).

of phages recovered

was determined

by the method described

by Adams

RESULTS

Optical density and dissolved oxygen measure were used to follow phage production. As sensitive strain, Rhizobium meliloti M,,S was chosen to produce phage NM,. As shown in Fig. 1, at time zero, Rhizobium meliloti preculture was added into the fermenter vessel to obtain 2.108 bacteria/ml (OD = 0.2). The air flow rate which supplied 90% dissolved oxygen was employed to maintain bacterial growth. After 150 min of fermentation, the culture reached the exponential phase (4. IO9 bacteria/ml). Here, the oxygen consumption

involved

the decrease of dissolved

oxygen percentage.

A suitable phage suspension was then added (m.o.i. = 0.2). To allow bacteriophage adsorption, agitation and aeration were stopped during 20 min. This was accompanied by a decrease of dissolved oxygen which regained approximately its original level when agitation and aeration were re-established. About 100 min after the addition of phage, bacterial lysis was observed by decreasing of optical density. At OD = 0.05, complete lysis occurred and the mixture became almost clear. During this time, the percentage of dissolved oxygen reached its maximal value. Fermentation processes were characterized by (1) time elapsed between phage addition and the beginning of lysis; (2) duration of lysis. These parameters are the properties of each phage. As illustrated in Table 2, these two steps varied with respect

158

0.8

80

Fig. 1. Progrewon

with time of dissolved

indicates

the addition

TABLE

2

Time elapsed Phages

between

oxygen and optical density during phage NM, production

(arrow

of phages).

addition

of phage and starting

of the lysis and duration

Time between

addition

the beginning

of the lysis (min)

of phage and

of the lysis Duration (min)

NM,

100-120

240-270

NM, NM,

100-120

240-270

100-120

240-270

NM,

100-120

240-270

NM,

105-130

3 10-340

CM,

80-105

240-270

CM, CM,

90- 120 120-150

270-300

MM,

135-165

300-330

360-390

of the Iqsls

159

TABLE

3

Percentages

of phages

Phages

recovered

after PEG treatment

and CsCl gradient

Initial crude lysate titre

Phages

PFU/ml

PEG treatment

recovered*

after

centrifugation Phages

NM,

7.5 x 10’2

48

30

NM,

1.1 x 10’1

45

43

NM,

2.5 X 10’)

22

18.5

NM,

3.6 X 1O’j

36

31

NW

1.6 X 10’”

60

50

CM,

4.8 x 10”

16

CM,

1.3 x 10”

49

45

C&

1.2 x 10’3

90

84

MM,

2.3 X lOI

42

30

*Percent

recovered*

CsCl gradient

after

centrifugation

2.5

of initial crude lysate titre

to the phages examined. For example, these were respectively 100-120 min and 240-270 min for phage NM,. The latent period for most phage was found to extend from 90 to 120 min, but it was shorter for CM, and longer for MM,. The duration of lysis was about 4-6 h. The overall time required for phage production was about 8 h, including the bacterial growth. The lysates were then concentrated and purified. As shown in Table 3, the procedures resulted in a substantial loss of phages. With regard to the phages studied, PEG treatment was deleterious and reduced greatly the initial titre from 10 to 84%. In the same way, CsCl gradient

lowered

slightly

the percent

recovery.

DISCUSSION

The performance of a fermenter for aerobic process is governed by the efficiency of the apparatus in transferring oxygen from the air to the liquid medium and from this to the organisms. Therefore, a rapid growth without lag period of an aerobic bacterium such as Rhizobium mefiloti can be achieved using this procedure. In our case, bacterial growth reached up to the logarithmic phase after 2 h. Such growth is important for the efficacy of phage infective process. Therefore, large amounts of high titred bacteriophages were obtained in a short time. On the other hand, measurement of the dissolved oxygen enabled us to monitor bacterial growth and its lysis. This direct measure avoids contamination with atmospheric flora which could happen during sampling. Further, this method could be applied in non-homogeneous liquid medium when the OD measurements cannot be carried out. As shown in the results the latent period and duration of lysis change with different phages. Thus, these parameters were useful for monitoring the reproducibility of

160

phage production.

Indeed,

during

concentration

lysates, Rhizobium meliloti phages appeared to osmotic pressure of CsCl. In conclusion,

the procedure

described

stocks of phages which are necessary

and purification

to be sensitive

processes

to polyethylene

of crude glycol and

in this paper allowed us to obtain high titred

for biochemical

and genetic

studies.

ACKNOWLEDGEMENTS

We are grateful to H. Ah for his assistance during the preparation of this paper. The technical assistance of Andre Decq is also gratefully acknowledged. REFERENCES

Adams,

M.H.,

Bradley,

D.E.,

Corbin, Forrai,

1959, Bacteriophages 1967, Bacterial.

D., L. Barran

(Interscience

Publishers.

New York).

Rev. 31, 230.

and G. Ditta,

T., E. Vincze, Z. Banfalvi,

1983, Proc. Natl. Acad.

Sci. U.S.A.

G.B. Kiss, S.R. Gursmaran

80. 3005.

and A. Kondorosi,

1983, .I. Bactcrtol.

152.

635. Hershey,

A.D., G. Kalmanson

and J. Bronfenbrenner.

Kowalski,

M., 1967, Acta Microbial.

Kowalski,

M. and J. DenariP,

Krsmanovic-Simic, Mannasse.

G.B. and F.M. Ausubel,

Ruvkun,

G.B., V. Sundaresan

Sik, T. and L. Orosz, Swanstrom, Werquin, Yamamoto,

(London)

and F.M. Ausubel,

1971, Plant and Soil (special

M. and M.H. Adams, M., M.T. Ben Brahim K.R.. B.M. Alberts,

Set. Parts 276, Ser. D, 2745.

and E.G. Barnes,

1981, Nature

and S. Chatterjee,

46. 267.

Sci. Paris 275, Ser. D. 141.

1973, C.R. Acad.

R.R. Granados

Ruvkun,

1943, J. Immunol.

16, 7.

1972, C.R. Acad.

D. and M. Werquin,

R.J.. R.C. Staples,

Sik, T.. J. Horvath

Polon.

1972. Virology

47, 375.

289. 85.

1982. Cell 29, 551. volume),

57.

1980, Mol. Gen. Genet.

178, 511.

1951, Proc. Sot. Exp. Biol. Med. 78, 372.

and D. Krsmanovic-Simtc, R. Benzinger.

L. Lawhorne

1977. C.R. Acad. Sci. Paris 284, S&r. D. 1X51. and G. Treiber,

1970. Virology

40, 734.