Biochimie 70 (1988) 423-427 © Soci6t6 de Chimie biologique/Elsevier, Paris 423

Characterization of the bacteriophage B2 of Lactobacillus plantarum ATCC 8014

Ingolf F. NES*, Johanne BRENDEHAUG and Kurt O. von HUSBY

Norwegian Food Research Institute, PO Box 50, N-1432 Aas-NLH, Norway

(Received 15-7-1987, accepted after revision 27-10-1987)

Summary - Bacteriophage B2 ofLactobaciilus plantarum ATCC 8014, isolated in 1971, belonged to Bradley's group B. Electron microscopy revealed an isometric head (110 nm) and a long non-contrac- tile and flexible tail (500 nm) containing about 75 regularly aligned lateral striations. Burst size was 12-14 phages per infectious centre.

The latent period for phage development was 75 min and the rise period approximately 90 min. The phage particle contained 5 major proteins. The buoyant density of the phage in CsCl was mea- sured as 1.575 g/cm 3.

B2 genome was a linear double-stranded DNA molecule of 37 _+ 1% guanosine-cytosine. Its size was 73 kilobase pairs (kbp). Restriction analysis of the genome showed that 4 restriction enzymes (Xba I, Sac I, Bgl II and Sma I) gave single site cuts in the DNA, while Ava I and Sal I formed 2 and 5 cuts, respectively.

bacteriophage / lactobacillus

Introduction

The first phage active on lactobacilli was reported as early as 1934 [1]. Since then most studies of bacteriophages specific for the lacto- bacilli have been conducted on bacteriophages of the species involved with fermented milk products or lactic acid beverages. Only a few reports concern studies of bacteriophages spe- cific for L. plantarum strains. Recently one bac- teriophage has been described which belongs to Bradley's group A [2, 3], and an earlier report has identified another L. plantarum phage as a member of group D or group E of Bradley's classification of bacteriophages [4, 5].

In the course of a study on cell wall polymers some 16 years ago, the bacteriophage B2 was isolated from anaerobic sewage sludge as a lytic phage for the strain Lactobaciilus plantarum

A T C C 8014 I61 L nlantnrurn ATC'(" RfllA h~¢ . . . . . . t v . i - ~ - / ~ . . . . . ~ - ~ - - , = i a t ~ . . . . - - , ~ . . - v v , i t • i t a w . ~

long been used to assay various amino acids and vitamins [7]. In a previous study, it had been shown that L. plantarum ATCC 8014 was one of the most efficient meat starter cultures then available, but that phage B2 was able to disturb lactic acid fermentation in model expe- riments [8]. The present work exposes some of the physicochemical properties of phage B2.

Materials and methods

Growth of bacteria and phage propagation The bacteriophage B2 and its host L. plantarum ATCC 8014 were obtained from the American Type Culture Collection. The bacteria were grown in MRS broth [9] and the bacteriophages were propa- gated in the same medium without Tween 80. The

*Author to whom correspondence should be addressed.

424 LF. Nes et al.

nonionic detergent was left out of the medium in order to avoid foaming during handling of the lysed culture.

Bacteriophage numbers were determined as pla- que-forming units (pfu), as described earlier [8]. No difference in phage titre was observed in absence or in presence of CaCI2 in the media. The plaques appeared as small, clear zones o f = 0.5 - 1 mm dia- meter.

Large scale bacteriophage production was carried out as follows: 1,000 ml MRS prewarmed at 30 ° C were inoculated with a fresh overnight L. plantarum culture with an initia; OD o f = 0.05 at 600 nm (1 cm light pathway), measured in a Beckmann Model 25 spectrophotometer. The cells were grown to 0.3 - 0 . 40D at 30°C without shaking, and then infected with phage B2 at a multiplicity of infection (MOI) of 0.1 - 0.3. An increase in the absorbance (approxima- tely double) was observed within 2 h, which was followed by lysis of the bacteria observed by an absorbance decrease.

Three h after infect'ion, 3 ml chloroform were added to assure complete lysis of the cells. Cell debris and CHCI3 were removed by centrifugation. The bacteriophages were concentrated by adding polyethylene-glycol 6000 (10% w/v) and NaCI (0.5 M), as described earlier by Yamamoto et aL [10]. After storage on ice for at least 3 h the precipitate was collected by centrifugation. The pi'ecipitate was dissolved in buffer 1 (100 mM Tris-HCl, pH 8.0; 10 mM MgCI,). The bacteriophage was purified twice by CsCI isopycnic centrifugation in a Beckman Ti 50 fixed-angle rotor at 40,000 rpm for 45 h at 15°C. The B2 phage was stored in buffer 2 { 1 N / ' ~ m k A T . ; . L l P l _ t ] " "7 C . 1/'~ _ _ L 4 r L i _ J " ~ I x ~ 11LTv~7 l l l i V l l l l a - - l l q k . , l , l . l l l I . J , I U I l i l V l I Y I ~ , i 2 ) .

One-step growth curve and burst size One-step growth curves were performed and burst sizes were measured as described by Adams [11]. A culture of bacteria was infected with phage particles at an MOI of= 1, then samples were removed at dif- ferent times after infection and immediately assayed for phage.

Electron microscopy The electron micrographs of the phage particles were carried out with CsCl-purified concentrated phage stock by using standard techniques and stain- ing with 1% uranyl acetate, pH 4.

SDS-polyacrylamide gel electrophoresis To determine the size of the proteins of the phage particle, 12.5% sodium dodecyl sulphate (SDS)- polyacrylamide gel electrophoresis was utilized according to Laemmli and Favre [12]. Bio-Rad (Richmond, CA 94804, USA) standard proteins and 2-bacteriophage proteins were chosen as molecular weight standards. 2-proteins were obtained by dis-

rupting 2-$7 CI857ts with SDS prior to electrophor- esis. ,~-57 Ci857ts was grown as described earlier [13].

Isolation of DNA The phage B2 was extracted twice with water-satu- rated phenol followed by diethylether extraction. The DNA was dialyzed and stored in 10 mM Tris- HCI (pH 8.0), 0.1 mM EDTA.

Restriction enzyme analyses The DNA restriction enzymes were obtained from BRL (Gaithersburg, MD 20877, USA) and Pharma- cia (S-751 82 Uppsala, Sweden). DNA cleavage was carried out according to specifications of the suppli- ers. The DNA fragments were separated by standard agarose gel electrophoresis (0.5-0.7% agarose) at constant voltage (40-70 V) and visualized by UV- light (300 nm) after staining with ethidium bromide.

Chemical composition of DNA The guanosine-cytosine (GC) content of B2 DNA was carried out by DNA melting analysis according to Mandel and Marmur [14]. DNA isolated from Lactobacillus plantarum ATCC 8014 was used as a control for DNA melting analysis (its % GC value was confirmed to be 45 +l) . In addition, the base composition of B2 DNA was determined by formic acid hydrolysis [15] followed by HPLC analysis of the DNA hydrolysate on a Supelcosil LC 18 DB reversed-phase column (Supelco Inc., Bellefonte, PA 16823, USA). The column was connected to a Spectra Physics SP 8700 delivery system and the detector system was a Hewlett-Packard 1040 photo- U I U U ~ a l l a y UUtUt ; tur .

3'-end labelling of DNA The YOH ends of B2 DNA were labelled with termi- nal deoxynucleotidyl transferase and [a-32P]-ddATP using the Y-end labelling kit from Amersham (Little Chalfont, England HP7 9NA).

R e s u l t s

Morphology and protein composit ion

Figure 1 showns a micrograph of B2 phage par- ticles. T h e phage possesses an isometr ic head (1 l0 nm diameter ) and a relatively long and flexible tail of ~ 500 n m in length. T h e distal end of the tail con ta ined some s t ructura l ele- m e n t s of a base plate. However , the picture did not reveal any fine s t ruc ture . Along the tail, 75 regularly spaced t ransversal s tr iat ions were observed.

B2 particles had 5 major pro te in bands with calculated molecu la r weights of 37,000, 32,500,

Bacteriophage B2 of Lactobacillus plantarum A TCC 8014 425

-: (~ ~. iN N

S t a n d . m m o) proteins

l O O n m ~ ~ i l m m m

Fig. 1. Electron micrograph of B2 particles negatively stained with uranyl acetate.

92.s K -

s s . 2 K -

4 s K - -

31 K -

2 1 K -

1 4 K "

p r o t e i n s

- ST.S K

- s : K

- - 1 1 K

26,000, 25,000 and 21,000 respectively, as shown in Fig. 2. After CsCI isopycnic centrifu- gation, the buoyant density of the phage par- ticles was calculated to be 1.575 g/cm'.

Lytic characteristics

Typical results of a one-step growth experi- ment are shown in Fig. 3. It can be seen from this figure that the latent period is ~ 75 min, the rise period ~ 90 min, and the burst size 12- 14 phages per infectious centre.

Characteristics of the phage genome

The % GC of the phage DNA wz ~ determined to be 37 __. 1 by DNA melting a,:,alysis. The DNA consisted of the normal bases as judged by HPLC analysis of formic acid hydrolysates of the DNA (data not shown).

The size of the genome was determined by restriction enzyme analysis. Eco RI cleaved B2 DNA into 11 fragments; the genome size of B2 was given by the sum of the molecular sizes of these fragments, and was estimated to be 73 kbp. This size was consistent with the values which were calculated from various other res.- triction enzyme digests, and a restriction map of B2 was constructed (Fig. 4). Xba I, Sac I and Bgl II are single-site cutters, while Ava I and Sal I are 2- and 5-site cutters, respectively. Sma I gives a single cut in B2 DNA at ~ 18.5 kbp, very close to the Sal I site.

Fig. 2. SDS-polyacrylamide gel electrophoresis of B2 pro- teins. Standard proteins (lysozyme, soybean trypsin inhi- bitor, carbonic anhydras¢, ovalbumin, bovine serum albu- min and phosphorylas¢ B) and A-bacteriophage proteins (4 major bands) were used as molecular weight standards.

1 5 , . l i

~0

0 . i 4 - ,

o

r - . ~

I,L

0 i ..... , , 0 6 0 120 180

T IME ( r a i n )

Fig. 3. One-step growth curves of phage B2 propagated on L. plantarum ATCC 8014. The squares and triangles represent 2 independent experiments.

426 LF. Nes et al.

O"

A v a I q Xba I

4 S a l l , S m a l

4 Ava I

4 Sal I

• Sal I 9 "

- ( Sac I

q Sal I

o o -

f Bgl II S a i l

o

Fig. 4. Restriction map of B2 DNA.

The genome was labelled by [a-nP]-ddATP using terminal deoxynucleotidyl transferase. The terminally labelled genome was digested by Sai I enzyme. Out of 6 fragments, 2 were pre- ferentially labelled, showing that these 2 frag- ments terminated the linear genome. In addi- tion, agarose gel electrophoresis which is known to discriminate between various forms ofDNA (covalently closed circles, open circles, as well as linearized molecule) only showed one distinct band with B2 DNA. Both experi-

ments strongly suggest that B2-DNA is a linear molecule. Furthermore, the linear B2 genome is not circular permutated as seen in phage T4 of E. co/i. This conclusion is based upon the fact that only 2 restriction fragments were pre- ferentially labelled by the terminal deoxynuc- leotidyl transferase.

Discussion

Bacteriophage B2 is the first Lactobacillusplan- tarum phage whose properties have been inves- tigated in some detail. This phage has a large isometric head and a flexible non-contractile tail. This overall appearance classifies this phage as belonging to Bradley's group B [2]. This morphological type seems common among the phages of various lactobacilli.

From Lactobaci/lus caseL a closely related bacterium to L. plantarum, 3 virulent bacterio- phages termed PL 1 [16, 17], J 1 [18], and ~bFSV [19] also belong to group B. Both PL 1 and J 1 phages, which are closely related, have a genome of ~ 40 kbp while ~bFS-V genome was estimated to be 41.3 kbp.

Phage fri of L. plantarum, which was des- cribed earlier, belongs to Bradley's group A. It has a regular hexagonal head (90 nm in diamet- er) and a contractile tail (190 nm in length). Its lvtic cycle includes a latent period of 75 min~ l~ollowed by a rise period of 75 min [3].

Compared to the above-mentioned phages, B2 is one of the largest phages ever described among those of the so-called subgroup Strepto- bacterium of lactobacilli, with its 500-nm long tail and head diameter of 110 nm. Moreover, it has a very long linear genome (73 kbp) with no detectable cohesive ends, unlike PL1 and J1.

The burst size of PL-1 was estimated to be 200-600 phages per cell [17], while those of J1 and fri were both 200 phages [3, 17]. The burst size of ~bFSW-TI, a temperate phage almost indistinguishable from ~bFSV, was ~ 70 [20]. The rather low burst size (12-14 phages per cell) of B2 may reflect the large size of this phage.

The increased addition ofhomofermentative lactobacilli to various food and feed products as lactic starters or as dietary supplements will no doubt lead in the near future to growing con- cern with bacteriophage irffection of these cul- tures.

Bacteriophage B2 of Lactobaciilus piantarum A TCC 8014 427

Acknowledgments

The skillful assistance of Ms. Brit O. Pedersen and Ms. Birgitta Baardsen is greatly appreciated. The electron micrograph of the phage particles was gene- rously provided by Dr. T. Krekling at the Agricultu- ral University of Norway, As.

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