Characterization of the prolate-headed lactococcal bacteriophage OvML3: location of the lysin gene and its DNA homology with other prolate-headed phages

Abstract

4vML3 is a virulent prolate-headed bacteriophage that attacks a genetically well-studied strain family including Lactococcus Iactis subsp. Iactis strains 712, C2 and ML3. A restriction map was constructed for &ML3 using a wide variety of restriction endonucleases. The DNA was highly refractory to in uitro restriction; this is a common feature of lytic lactococcal phages. Genome size was estimated as 23 kb, which is similar to the sizes of other phages of the same morphological group. The presence of heat-dissociable DNA fragments was noted in gel electrophoresis of restriction enzyme digests. Cohesive ends were confirmed by pretreating the DNA with T4 ligase, which rendered the composite fragment insensitive to heat. The phage &ML3 genome thus consists of linear and non-permuted double-stranded DNA with complementary cohesive ends. The lysin gene from this phage acts as a specific lysis agent against the lactic streptococci. This gene has been cloned and sequenced previously. Further specific subcloning of EcoRV fragments of &ML3 DNA has located the lysin gene in the central region of the genome, orientated from right to left. This location was confirmed by hybridization of a lysin gene probe to @ML3 DNA. The lysin gene probe showed homology to a number of other prolate-headed phages including Pool. The lysin gene of PO01 was shown to be located in the central region of its genome. However, the isometric phage P107 did not hybridize, in spite of encoding its own lysin gene.

Purification and characterization of the lytic activity induced by the prolate-headed bacteriophage P001 in Lactococcus lactis

“…By analysing the restriction map of the PO01 genome we conclude that both N-termini are located respectively on the left hand side of the EcoRI-A fragment and the right hand side of the HpaII-A fragment spanning around 500 bp of the phage genome (Braun et al 1989). This region is approximately the same region, that was previously identified by hybridization with the m M L 3 lysin gene (Shearman et al 1991). Although only the oligonucleotide probe for the N-terminus of the small PO01 lysin hybridized with the m M L 3 lysin gene fragment used in this study, amino acid sequences with homologies to the N-termini of both PO01 enzymes could be found in the ovML3 and c2 lysins which are identical in these parts of the proteins ( Table 1).…”

“…1989;Powell et al 1989;Prevots et al 1990). Moreover, a lysin gene which codes for a lysozyme-like enzyme was identified by DNA hybridizations on the genome of several prolateheaded phages and localized in the central region of the POOl restriction map (Shearman et al 1991). Phage-induced lysins might be responsible for the so called 'nascent phage phenomenon', in which a multiple strain starter is lysed when infected with a phage specific for one of the strains.…”

Bacteriophage and bacteriophage resistance in lactic acid bacteria

“…The phage DNA is 23 kb in length, is composed of dsDNA, and is cohesiveended. The phage genome has been restriction mapped and, in common with other mapped bacteriophages, shows a paucity of restriction sites for in vitro digestion with commercial endonucleases [22]. The rcgion encoding the phage lysin was cloned in Agtl0 and selected on the basis of expression from recombinant a plaques [23].…”

Progress and potential in the biotechnology of lactic acid bacteria

“…Some analyses of DNA replication following bacteriophage infection have been reported [103][104][105] and amongst temperate bacteriophages a molecular analysis of the bacteriophage integration process involving classical attP/attB site specific integration has been described [106]. For some bacteriophage genomes cohesive ends have been defined and sequenced [107][108][109] whereas other examples of bacteriophages with circularly permuted structures and terminal redundancy have been defined [110][111][112]. Bacteriophage lysin genes from several bacteriophages have been cloned and sequenced [113][114][115][116] and Kim and Batt [117][118][119] have analysed genes for structural proteins of lactococcal bacteriophage F4-1.…”