Temperate phages TP901-1 and ϕLC3, belonging to the P335 species, apparently use different pathways for DNA injection inLactococcus lactissubsp.cremoris3107

Abstract

Five mutants of Lactococcus lactis subsp. cremoris 3107 resistant to phage TP901-1 were obtained after treatment with ethyl methanesulfonate. Two of the mutants were also resistant to phage phiLC3. The remaining three mutants were as sensitive as 3107. Mutants E46 and E100 did not adsorb the two phages. Mutants E119, E121 and E126 adsorbed phage phiLC3 as well as 3107 but phage TP901-1 with significantly reduced efficiency. All, except E46, could be lysogenized with phage TP901-BC1034, a derivative of TP901-1 harboring an erythromycin-resistance marker. However, the lysogenization frequency was 10(3)-10(4) fold higher for 3107 than for the mutants. Mitomycin C induction of lysogenized mutants 3107 indicated that phage propagation was not affected in these four mutants. Electron microscopy and analysis of total DNA of infected cells showed that DNA was liberated from the phage particle during infection of strain 3107 with TP901-1 and that intracellular phage DNA replication occurred. This was not the case for mutants E121 and E126. This strongly suggests that some step starting with triggering DNA release and ending with DNA injection is impaired during infection with TP901-1. As such impairment was not seen when infecting E119, E121 and E126 with phiLC3, we conclude that TP901-1 and phiLC3 either are differently triggered by their receptor or utilize different pathways of injection.

Differences in Lactococcal Cell Wall Polysaccharide Structure Are Major Determining Factors in Bacteriophage Sensitivity

“…The ISS1 insertion elements were shown to be located within a gene cluster that is presumed to be responsible for the biosynthesis of a CWPS. Interestingly, a similar sedimenting phenotype was observed in several (but not all) BIMs of L. lactis 3107, which had been isolated as being insensitive to phages TP901-1 and φLC3 (both of which belong to the P335 group) (25). Finally, L. lactis MG1363 mutants deficient in CWPS pellicle biosynthesis were shown to display a sedimenting phenotype and to be insensitive to the 936 group bacteriophage sk1 (20).…”

“…It has previously been shown that certain BIMs of L. lactis 3107, which had been generated by ethyl methanesulfonate exposure, display distinctly different infection profiles with respect to phages φLC3 and TP901-1 (25). Of five TP901-1-resistant BIMs generated, two were shown to be resistant to φLC3.…”

“…Spot assays and plaque assays were performed by the overlay method as previously described (42). Adsorption assays were performed as described previously (25). Briefly, cells at an optical density at 600 nm of 0.5 were harvested and resuspended in 0.02 volume of 10 mM MgSO 4 .…”

“…Supernatants were then assayed for free phages by standard plaque assays with an appropriate indicator strain. Frequency-of-lysogeny assays using erythromycin-tagged phage TP901 erm (a derivative of TP901-1, also designated TP901-BC1034) were performed as previously described (25). …”

Structural Aspects of the Interaction of Dairy Phages with Their Host Bacteria

“…Firstly, that the glycosyltransferase inactivation may prevent the transfer and decoration of the required pellicle monosaccharide component to the surface thus negating the primary contact between the phage RBP and its saccharide receptor; or secondly, if primary contact is established and a membrane component is required for the translocation of the DNA across the membrane, the membrane-associated protein inactivation prevents internalisation of the phage DNA. Chemical mutagenesis of the lactococcal host strain 3107, which is sensitive to the P335 phages LC3 and TP901–1, resulted in the isolation of five genetically and phenotypically distinct mutants resistant to one or both phages [37]. This analysis revealed that the injection pathways employed by these phages of the same species are distinct.…”