Physical mapping and partial genetic characterization of the Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539

Arch Virol (1999) 144: 1503–1512

Physical mapping and partial genetic characterization of the Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539 L. Auad1 , L. Räisänen2 , R. R. Raya1 , and T. Alatossava2 1

Centro de Referencia para Lactobacilos (CERELA), San Miguel de Tucumán, Argentine 2 Department of Biology, University of Oulu, Linnanmaa, Oulu, Finland Accepted April 20, 1999

Summary. A restriction map was constructed of the 37 kb genome of the temperate Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539. Restriction analysis and Southern hybridization experiments detected variable levels of homologous regions among the genomes of lb539 and the L. delbrueckii reference phages LL-H (virulent) and mv4 (temperate). The principal homology was observed at the regions encoding the structural proteins. These studies allowed us to construct a partial genetic map of phage lb539 for lysin, the main structural tail protein and the packaging region genes. Furthermore, a short 1.5 kb DNA fragment of the prolate-headed JCL1032 phage genome was observed to be highly homologous with the DNA of the isometric-headed lb539, mv4 and LL-H phages. The described distribution of the homologous regions between the genomes of the phages lb539, LL-H, mv4 and JCL1032 presented here supports the modular evolution theory of the bacteriophages.

Introduction Lactobacillus delbrueckii subsp. bulgaricus and subsp. lactis are two important lactic acid bacteria widely employed in the dairy industry as starter cultures in the manufacture of yogurt (subsp. bulgaricus) and cheeses (subsp. bulgaricus and lactis). One of the most critical problem in these processes is the contamination of the starters by bacteriophages that causes cellular lysis of the starter cells. As a consequence, the fermentation ceases and quality of the products may be lowered, causing significant economical losses. Temperate and virulent L. delbrueckii subsp. bulgaricus and lactis bacteriophages are highly related and have been classified in four groups (a to d) on the basis of immunoblotting test and DNA-DNA hybridization [14, 20]. Most of

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them belong to Siphoviridae family [14] and group B of Bradley’s classification [5] with isometric- (groups a, b and d) and prolate-head (group c). Group a is the largest and includes 5 temperate and 13 lytic phages of both subspecies. The best characterized is the virulent phage LL-H [2, 15]. Its genome has been completely sequenced and the integrase, structural proteins, lysin and early genes, as well as DNA packaging sequences, have been located [16]. The location of the attP site, pac site, and the genes encoding lysin, integrase and the mayor capsid protein of phage mv4 were also mapped and sequenced [8, 12, 24]. Group b is composed by virulent phages of L. bulgaricus and group c and d contain temperate bacteriophages of L. lactis. Groups a, c and d have similar host range, however they are different morphologically, serologically and molecularly. Previously, Forsman [9] described phage JCL1032, a new type of L. lactis phage that has intermediate characteristics between phages of groups a and b. It has prolate head (group c), a long cross-barred tail, and dsDNA of 45.8 kb with cos ends, which has few recognition sites for a variety of restriction enzymes like phage genomes of the group b. On the other hand, JCL1032 shares homology with the DNA of bacteriophages LL-H and mv4 belonging to the group a [1, 9]. Bacteriophage lb539 is a temperate phage isolated by induction with mitomycin C from L. bulgaricus CRL 539 [3]. Morphologically, it is a Bradley B phage [5] with an isometric head and a long noncontractile tail. The genome is a linear and double-stranded DNA molecule which contains pac sites. In previous dot-blot experiments, it was shown that phage lb539 belongs to L. delbrueckii group a: its genome hybridized with the genomes of phages mv4 and LL-H [3]. In this report, we have begun to characterize the genome of lb539 by restriction mapping of its 37 kb DNA, using the restriction enzymes EcoRI, EcoRV, SacII, SalI, PstI and XhoI, and by analyzing the homologous regions among L. delbrueckii bacteriophages lb539, mv4, LL-H, and JCL1032 genomes. From these results and from the SDS-PAGE analysis of phage lb539 structural proteins, a preliminary genetic map of lb539 has been constructed. Materials and methods Phages and bacteria The phages and bacteria used in this study are listed in Table 1. Bacteria were propagated at 37 ◦ C in MRS broth [7]. Agar was used at 1.5% to make solid media. Phages were propagated on L. delbrueckii subsp. lactis LKT in MRS containing CaCl2 (10 mM for lb539 and mv4 and 20 mM for LL-H and JCL1032).

Phage purification and DNA isolation Phage particles were purified either by ultracentrifugation (150 min, 100,000 × g) or by CsCl equilibrium density gradient centrifugation. Phage particles, resuspended en TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8) were then treated with 10% SDS and proteinase K (25 mg/ml). Phage DNA was extracted twice by phenol-chloroform-isoamyl alcohol and concentrated by ethanol precipitation.

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Table 1. Lactobacillus delbrueckii phages and strains used in this study

Phage LL-H mv4 lb539 JCL1032

Characteristics

Host range (subsp.) References bulgaricus lactis

Lytic, group aa Temperate, group aa Temperate, group aa NDb , group ca

− + + −

+ + + +

Strain L. delbrueckii subsp. lactis: LKT (CNRZ 700) ATCC 15808 (CNRZ 326) L. delbrueckii subsp. bulgaricus: CRL 539 Lysogenic strain of phage lb539 LT4 Lysogenic strain of phage mv4 CNRZ 1004LT4 LT4 cured of the prophage mv4

Alatossava et al. [1] Cluzel et al. [6] Auad et al. [3] Forsman [9]

Cluzel et al. [6] Cluzel et al. [6]

Auad et al. [3] Cluzel et al. [6] Lahbib-Mansais et al. [12]

a

According to the classification of Mata et al. [14] and Sechaud et al. [20] ND Not determined +/− indicate that cells are sensitive/resistant, respectively, to phage attack

b

Restriction analysis Single and double digestions of phage DNA were done. The digestions with restriction enzymes were performed according to the instructions of the manufacturer (Boehringer Manneheim GmbH, Mannheim, Germany). The fragments generated were separated on a 0.7% agarose by gel electrophoresis in Tris-acetate buffer as described by Sambrook et al. [19]. 1 kb DNA ladder (Gifco, Gaithersburg, U.S.A) was used as molecular DNA size standard. Hybridization analysis DNA fragments were transferred from agarose gels to nylon membranes (Hybond-N; Amersham) as described by Southern [22], and modified by Smith and Summers [21]. Probes were labelled with P32 with the Amersham’s Rediprime DNA labelling kit system. Membranes were hybridized overnight at 37 ◦ C in hybridization buffer containing the probe and formamide 30% (v/v), 5 × SSPE, 5 × Denhardt’s solution, 0.5% SDS and 100␮g/ml herring sperm DNA. After DNA hybridization, the strips were sequentially washed in 2 × SSPE, 0.1% SDS at room temperature for 2 min, and twice in SSPE 1 × 0.1% SDS at 50 ◦ C. These conditions demanded 74% homology, presuming a GC content of 50% in the genomes of the phages studied. To compare phage lb539 with phage LL-H we also used strong stringency conditions which demanded 82% homology. In this case, the temperature of hybridization was 65 ◦ C and the two last washes were done at 65 ◦ C instead of 50 ◦ C. Amplification and isolation of LL-H DNA fragments PCR technique was used to amplify LL-H DNA with the Gold AmpliTaq polymerase (Perkin Elmer). The primers used are listed in Table 2. The amplified products were extracted from agarose gels and purified using a QIAquick Gel Extraction Kit (QIAGEN).

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L. Auad et al. Table 2. Sequences of the primers used in PCR analysis to amplify LL-H DNA

Sequence and direction

LL-H genome nucleotide numbersa

Location

50 -TCTACTCTGGTGCATCC-30 −→ 50 -GACCTTGGCGGTCTTCC-30 ←− 50 -GGGACTTTCACCTGCCA-30 −→ 50 -GCTGGCGATCCGGATAT-30 ←− 50 -GGCCTGGCTTACGAA-30 −→ 50 -TCGCCAAAGTGGATCTG-30 ←− 50 -AGACGTCGCTGTTTCCA-30 −→ 50 -GCAAGCCGGCTCTTACC-30 ←−

23338–23354 23753–23737 9491–9507 10264–10248 33301–33315 34285–34269 34364–34380 697–681

lys lys ORF113B ORF148 ORF139 ORF205 ORF172 ORF172-ORF82

a

From Mikkonen et al. [16] SDS-polyacrylamide gel electrophoresis

SDS-denatured proteins of CsCl purified phage particles were separated in 12% acrylamide gel according to the method of Laemmli [11]. Protein molecular mass markers were used as references (SDS Molecular weight markers, Sigma). Proteins were detected by silver staining as described by Oakley et al. [17].

Results Restriction map of lb539 The restriction map of the bacteriophage lb539 was constructed with the enzymes: EcoRI, EcoRV, SacII, SalI, PstI and XhoI (Fig. 1a). The genome size of the phage lb539 was estimated to be 37 kb based on the sum of the sizes of the restriction fragments. The size of each fragment is also indicated in Fig. 1a. DNA homology The DNA-DNA homology between lb539 and the phages LL-H, mv4 and JCL1032 was analyzed by Southern hybridization. Total DNA from bacteriophage lb539 was used as probe against mv4, LL-H and JCL1032 DNA (Fig. 2). In all cases moderate stringency conditions were employed (minimal homology 74%). P32 labelled lb539 DNA hybridized with most mv4 genome, with the exception of a 3.4 kb SalI fragment close to the pac site of mv4 DNA. When the same probe was used against the digested LL-H DNA, all the DNA fragments hybridized, although the intensity of the signal was variable depending on the fragments involved in the hybridization. A region of 23.7 kb between the sites EcoRI (1.9) – XbaI (25.6) in the LL-H genome, showed the strongest signal (Fig. 2). However, under higher stringency conditions (minimal homology 82%), the homology observed was reduced, and signals were not longer detected in the region including part of the early gene region and of the genes for DNA packaging (from EcoRI-1.9 to EcoRI-29.0 in Fig. 2). P32 -labelled lb539 DNA against JCL1032 genome only hybridized with four HindIII fragments of 1.5, 2.3, 2.8 and 6.7 kb (Fig. 2). The highest signal was

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Fig. 1. a Restriction map of the bacteriophage lb539 constructed for enzymes EcoRI, SacII, SalI, PstI, EcoRV and XhoI. Fragments sizes are included with the exception of SalI-H (0.75 kb), SalI-G (0.85 kb) and PstI-H (0.6 kb); b Organization of the phage lb539 genome based on Southern hybridizations with probes of LL-H DNA derived PCR products. The locations of the homologous region to g17 (EcoRV 3.4 kb-Sacll 11 kb) and lysin (PstI 1.4 kb) genes in lb539 genome are indicated

registered in the HindIII fragment of 1.5 kb in size. In previous publications this fragment has also shown homology with the LL-H and mv4 genomes [1, 9]. To complement the DNA homology studies, Southern hybridization was also performed using total mv4, LL-H and JCL1032 DNA as probes against lb539 DNA. The results shown in Fig. 3 indicate the distribution of homologous regions in the genome of bacteriophage lb539. Because some DNA regions of the physical map of phage lb539 are not very dense in restriction digestion sites (Fig. 1a), some information of the homology distribution could be lost. However, it is evident that DNA homology along the genome of lb539 is distributed unequally as in a mosaic way. Partial genetic map of lb539 genome As Mata proposed [13], phage LL-H is a reference phage representative of group a. LL-H genome has been completely sequenced [15], so we designed specific primers to amplify genes from LL-H genome (Table 2) and used the PCR-products as probes against lb539 DNA. The amplified fragments from LL-H DNA were the lysin genes, the gene g17 which encodes LL-H main tail protein and the region before and covering DNA packaging genes. The position and nucleotide sequence of the primers are listed in Table 2. The PCR products were purified, labelled and probed against lb539

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total DNA digested with different endonucleases. The locations of the LL-Hhomologous genes on the lb539 genome are shown in Fig. 1b. Protein analysis The structural protein composition of purified lb539 phage particles was analyzed by SDS-PAGE. The protein profile observed was identical to the protein profiles of phages LL-H and mv4; two very intense bands of 34 kDa and 19 kDa could be detected corresponding to two major structural proteins (data not shown). This pattern seems to be a constant characteristic in phages belonging to group a [14, 23]. Discussion In previous works, it was showed that LL-H, mv4 and JCL1032 share DNA homology [1, 9]. In this study, these phages were compared with the bacteriophage lb539. Our results indicates that among lb539, mv4, LL-H, and JCL1032 exists a phylogenetic relationship. All of them (except JCL1032) belong to DNA homology group a [14] so they could come from a common ancestor. Although some of the genome characteristics (cos site and the paucity of recognition sites for some restriction enzymes) of the prolate-headed phage JCL1032 are more similar to group b phages [9, 10], phage JCL1032 has a DNA segment (1.5 kb HindIII fragment, see Fig. 2) which is homologous with the phage genomes studied, including lb539 DNA. The paucity of recognition sites for various endonucleases is a phenomenon previously described in lactic lactococcal phages [18]. Southern hybridization experiments showed that the strongest homology between lb539 and phages LL-H and mv4 is located in the region containing genes encoding phage structural proteins. These results are supported with the similar SDS-PAGE profiles of the main proteins of these phages [14, 23]. In JCL1032, a phage morphologically different, its protein composition is also different [9]. It has been proposed that the homology observed in the structural genes of phages belonging to group a would be caused by the presence of a common module for these genes while the regions without homology would be involved in lysogeny and replication functions [14]. The location of the g17, lysin and DNA packaging genes seems to be similar in both LL-H and lb539 phages (Fig. 1b). As in mv4, the lysin gene of lb539 has homology with the muramidase of phage LL-H. However, this homology is only partial since is not detectable at high stringency conditions (minimal homology 82%) (Fig. 2). In addition, we could not amplify the lysin gene of lb539 using the primers (Table 2) and the conditions described for LL-H [15] (data not shown). The mv4 and lb539 showed a cross-superinfection immunity [3] which suggests that the early gene regions of both bacteriophages are homologous. Our data are according to the modular evolution theory of bacteriophages [4]. Upon this theory, each virus is a favorable combination of modules which represent a biological function. The exchange of a given module for another with the same viral function occurs by recombination among a population of different

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phages related by their similar modular construction. This efficient mechanism offers a greater adaptation to new environmental conditions than evolution by linear descent. Acknowledgements This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) grant PIA 7175/96 and by Consejo de Ciencias y Técnicas (CIUNT; Universidad Nacional de Tucumán). We thank the Centre International for Mobility (CIMO), Finland (HA7 024) for generous support to L.A.

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