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- MATERIALS AND METHODS
Aims: To investigate the basic properties of six temperate and three virulent phages, active on Lactobacillus fermentum, on the basis of morphology, host ranges, protein composition and genome characterization.
Methods and Results: All phages belonged to the Siphoviridae family; two of them showed prolate heads. The host ranges of seven phages contained a common group of strains. SDS-PAGE protein profiles, restriction analysis of DNA and Southern blot hybridization revealed a high degree of homology between four temperate phages; partial homologies were also detected among virulent and temperate phages. Clustering derived from host range analysis was not related to the results of the DNA hybridizations.
Conclusions: The phages investigated have common characteristics with other known phages active on the genus Lactobacillus. Sensitivity to viral infection is apparently enhanced by the presence of a resident prophage.
Significance and Impact of the Study: These relationships contribute to the explanation for the origin of phage infection in food processes where Lact. fermentum is involved, such as sourdough fermentation.
- Top of page
- MATERIALS AND METHODS
There are few reports in the literature concerning Lact. fermentum phages. De Klerk et al. (1965), who first studied the fine structure of viral particles active on this species, described eight lytic phages belonging to the Myoviridae family and only one temperate phage ascribed to the Siphoviridae family, having an unsheathed tail 182 nm long with an icosahedral head 50 nm wide. Recently, Foschino et al. (1995) isolated two virulent Lact. fermentum phages from a sourdough sample for wheat bread production. The first, FE5-B1, presented the typical morphology of Myoviridae, with a sheathed contractile tail 170 nm long and an icosahedral head 83 nm wide; the second, Z63-B1, belonged to the Siphoviridae family, having a non-contractile tail 160 nm long and an isometric head 60 nm wide. Another virulent phage, Z63-B2 (Foschino et al. 1998), active on the same species but isolated from sourdoughs in a different bakery plant, was shown to be very similar to phage Z63-B1. All phages examined in the present study were ascribed to the Siphoviridae family; seven out of nine belonged to the B1 morphotype, while the other two phages revealed uncommon shapes and were attributed to the B3 morphotype according to Ackermann’s classification (Ackermann and Du Bow 1986). Phages of lactobacilli with prolate heads have rarely been isolated. The first, described by Séchaud et al. (1988), was the temperate phage 0235 harboured in Lact. delbrueckii subsp. lactis strain CNRZ 235; the second, active on the same species of bacterium, was the lytic phage JLC 1032 characterized by Forsman (1993). Another prolate-headed phage, named Φy8 and spontaneously released from Lact. acidophilus strain Y8, was isolated from fermented milk by Kiliçet al. (1996); the morphology of phages 064 and 0209 was similar to this one. All had unusual elongated flexible tails.
Data on host ranges showed two separated clusters containing both temperate and virulent phages. Viruses of the first group attacked a set of strains where, remarkably, six out of eight were lysogenic. Because of the effect of immunity, none of the temperate phages caused lysis of the respective harbouring strain. Phage 064 was unable to lyse strain CNRZ 209, which is lysogenic for phage 0209 and vice versa; the same observation applied to temperate phages 0BU130 and 0FE129. The second group consisted of the temperate phage FEM and the virulent phage BU77-B1, which both propagated only on four strains isolated in Italy from whey samples. They were completely inactive on strains that were sensitive to the viruses of the first cluster. Most of the cultures were insensitive to the attack of any phage; 14 out of 17 of these strains appeared to be non-lysogenic. Furthermore, analysis of the sensitivity patterns of the 31 strains revealed that cultures that proved to be sensitive to any phage could be found more frequently among lysogenic (six out of nine) than non-lysogenic strains (eight out of 22). Similar observations were made in the comparative study of Séchaud et al. (1992), carried out on 35 bacteriophages of Lact. helveticus; in this case, there were 77% sensitive strains among lysogenic cultures and 58% among non-lysogenic strains. In the same way, Fayard et al. (1993) reported that lysogenic strains of Streptococcus salivarius subsp. thermophilus were good, or sole, indicators for temperate phages which did not belong to the same immunity group of the harboured phage. Moreover, they observed that the passage of a native temperate phage in a lysogenic indicator strain expanded the host range and triggered a DNA rearrangement of the viral particle. Therefore, contrary to what has generally been described, these results suggest that sensitivity to viral infection is apparently enhanced by the presence of a resident prophage. However, phage resistance of strains is not necessarily affected by lysogeny, since the mechanism of immunity works only with closely-related phages (Davidson et al. 1990; Hill 1993). It is thus likely that the sensitivity pattern of a lysogenic strain is concerned more with its genetic properties and prophage than with the molecular characteristics of fibres or base plates of an outer infecting viral particle. In fact, grouping of phages obtained by host ranges is not related to the results of the DNA hybridization analysis. Viral particles which showed equal or narrow sensitive strain patterns, such as phages FEM and BU77-B1 or phages 017 and 0209, showed no evidence of genetic homology; on the other hand, the virulent phage Z63-B3, whose genome did not hybridize with any other, shared sensitive strains with six other phages.
Protein profiles obtained by SDS-PAGE of phages 064 and 0209 appeared identical to those of phages 0BU130 and 0FE129, while patterns of the remaining viral particles were all different. Numbers and values of molecular masses of the major proteins were similar to those reported in other studies concerning the characterization of phages active on lactobacilli (Mata et al. 1986; Séchaud et al. 1988; Davidson et al. 1990; Forsman 1993; Foschino et al. 1995).
Restriction analysis of genomes showed coincident patterns between DNAs of phages 064 and 0209, and between those of phages 0BU130 and 0FE129. All the other phages exhibited individual profiles of restriction. The calculated sizes of genomes agree with those reported in the literature for other bacteriophages of lactobacilli (Mata et al. 1986; Séchaud et al. 1988; Davidson et al. 1990; Forsman 1993; Foschino et al. 1995). Strict relationships between phages Z63-B2 and Z63-B1 have already been found at the physiological, structural and genetic level, confirming them as variants of one another (Foschino et al. 1995, 1998).
Southern hybridization experiments confirmed a high degree of homology between the genomes of temperate phages 064 and 0209, and those of temperate phages 0BU130 and 0FE129. Results of RAPD PCR with DNAs of strains CNRZ64 and CNRZ209 suggested that the respective phages, 064 and 0209, are identical viral particles harboured in two strains isolated from different specimens at different places and times. With regard to strains BU130 and FE129, the amplification patterns were coincident, so the two harboured phages, 0BU130 and 0FE129, are the same viral particle present in two isolates of a unique culture found in a sample of Grana cheese whey. DNAs from prolate-headed phages 064 and 0209 did not show any homology with genomic DNA of the other isometric-headed phages, which is in disagreement with Forsman (1993), who found few, but highly homologous, DNA regions between the genomes of these two morphotypes.
Despite the differences found in restriction enzyme digestion patterns, partial homologies were also detected among DNAs from virulent phages, Z63-B2 and BU77-B1, and temperate phages, 0BU130, 0FE129 and FEM. In a comparative study on Lact. casei phages reported by Forsman et al. (1993), similar results were obtained; lytic phages, ΦFSW and LC-Nu, and temperate phage PL-1 showed different structural proteins and DNA restriction profiles, but one-third of each viral genome proved to be highly homologous (> 85%) with those of the other phages. A significant relationship was found by Vasala et al. (1993) between the virulent phage, LL-H, and the temperate phage, mv4, both active on Lact. delbrueckii subsp. lactis. Although they were isolated in different places at different times, these phages revealed a high level of homology in the structure and organization of the gene cluster encoding structural proteins.
The demonstration of lysogeny is valuable at the applied level, as temperate phages are a potential source of virulent phages in food processing. Similarities in morphology, host range, structural proteins and DNA compositions between temperate and lytic phages reported in several studies (Mata et al. 1986; Séchaud et al. 1988; Davidson et al. 1990) support this thesis. Until now, phage infections in small bakeries and industries have not caused serious problems because most sourdough processes are still carried out in a batch system using mixed natural starters. These mixtures contain several strains, often belonging to different bacterial species and not all sensitive to a given phage strain (Ottogalli et al. 1996). The risk of a phage infection increases when a continuous system or selected culture is adopted. Information about the genetic characteristics of starter cultures and their interactions with bacteriophages can contribute to correct control procedures being applied during the fermentative process.