Host genetic requirements for DNA release of lactococcal phage TP901-1

Abstract

The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram-positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901-1 due to mutations that affect TP901-1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three-component glycosylation system (TGS) was shown to be required for TP901-1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope-associated component that triggers TP901-1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram-positive infecting phage.

FIGURE 1

Schematic representation of the genomic region containing csdG ₃₁₀₇ (L3107_1442), encoding a 891 AA product (A); and csdC ₃₁₀₇ (L3107_1875), encoding a 312 AA protein (B) in L. cremoris 3107. The mutations found in each strain resistant to TP901‐1 are indicated. E119 and E126 substitutions lead to the incorporation of a stop codon in csdG ₃₁₀₇ (expected products of 463 and 161 AA, respectively), whereas E121 insertion causes a translational frameshift that generates a pre‐mature stop codon in csdC ₃₁₀₇ (143 AA expected product).

FIGURE 2

Log₂ fold change in the amount of TP901‐1 DNA internalised in different strains compared with L. cremoris 3107, determined by qPCR. TP901‐1 ssb and rep genes (blue and orange bars, respectively) were detected and expressed relative to an internal control, the chromosomal host gene L3107_0192.

FIGURE 3

(A) SEC‐HPLC purification of rhamnan and PSP oligosaccharides extracted from cell walls of L. cremoris 3107 and TP901‐1‐resistant mutant E126. * indicates non‐saccharidic compounds. (B) Monosaccharide composition of purified rhamnan peaks relative to glucosamine (GlcNH₂ = 1) from cell walls of L. cremoris 3107 and its derivative E126 from a representative experiment. (C) Monosaccharide composition of purified PSP peaks relative to glucosamine (GlcNH₂ = 1) from cell walls of L. cremoris 3107 and its derivative E126 from a representative experiment.

FIGURE 4

Gas chromatography profiles corresponding to methylation analysis of LTA preparations of L. lactis IL1403 (as a control), L. cremoris 3107 and its mutant E126 following phenol extraction. Presence of terminal galactose shown with “t‐Gal” and absence of t‐Gal shown with a blue arrow. The figures were zoomed onto the region corresponding to hexoses.

FIGURE 5

Schematic representation of L. cremoris 3107 TGS required for TP901‐1 DNA release. CsdC₃₁₀₇, synthesises Und‐P‐Glc in the cytoplasmic side of the cell membrane using UDP‐Glc as a donor. The lipid intermediate, Und‐P‐Glc, is transferred across the membrane by a flippase, presumably encoded by L3107_0554. Finally, CsdG₃₁₀₇ transfers the Glc to a final acceptor, presumably a carbohydrate moiety, to form a saccharidic molecule that can trigger TP901‐1 DNA release. The stars indicate the pre‐mature stop codon locations within the TMHs in the corresponding products of mutants E119, E121 and E126. E121 renders a truncated CsdC₃₁₀₇ of 143 AA, while E119 and E126 produce a truncated CsdG₃₁₀₇ of 463 and 161 AA, respectively. Note that the long extracellular loop of CsdG₃₁₀₇ between TMHs 13th and 14th includes residues 466 to 859. Created with BioRender.com.