Ribitol-Containing Wall Teichoic Acid of Tetragenococcus halophilus Is Targeted by Bacteriophage phiWJ7 as a Binding Receptor

Abstract

Tetragenococcus halophilus, a halophilic lactic acid bacterium, is used in the fermentation process of soy sauce manufacturing. For many years, bacteriophage infections of T. halophilus have been a major industrial problem that causes fermentation failure. However, studies focusing on the mechanisms of tetragenococcal host-phage interactions are not sufficient. In this study, we generated two phage-insensitive derivatives from the parental strain T. halophilus WJ7, which is susceptible to the virulent phage phiWJ7. Whole-genome sequencing of the derivatives revealed that insertion sequences were transposed into a gene encoding poly(ribitol phosphate) polymerase (TarL) in both derivatives. TarL is responsible for the biosynthesis of ribitol-containing wall teichoic acid, and WJ7 was confirmed to contain ribitol in extracted wall teichoic acid, but the derivative was not. Cell walls of WJ7 irreversibly adsorbed phiWJ7, but those of the phage-insensitive derivatives did not. Additionally, 25 phiWJ7-insensitive derivatives were obtained, and they showed mutations not only in tarL but also in tarI and tarJ, which are responsible for the synthesis of CDP-ribitol. These results indicate that phiWJ7 targets the ribitol-containing wall teichoic acid of host cells as a binding receptor. IMPORTANCE Information about the mechanisms of host-phage interactions is required for the development of efficient strategies against bacteriophage infections. Here, we identified the ribitol-containing wall teichoic acid as a host receptor indispensable for bacteriophage infection. The complete genome sequence of tetragenococcal phage phiWJ7 belonging to the family Rountreeviridae is also provided here. This study could become the foundation for a better understanding of host-phage interactions of tetragenococci.

Keywords: Tetragenococcus halophilus; bacteriophage; host receptor; wall teichoic acid.

FIG 1

Biosynthetic pathways of Rbo- and Gro-WTA in S. aureus, adapted from previous reports (46–49). WTA synthesis is initiated with consecutive transfer of N-acetylglucosamine (GlcNAc) phosphate and N-acetylmannosamine (ManNAc) to the lipid carrier undecaprenylphosphate by TagO and TagA on the cytoplasmic side of the cell membrane. UDP-ManNAc is generated from UDP-GlcNAc by MnaA. In Gro-WTA synthesis, the primase TagB adds the first Gro-P to the disaccharide unit, and the polymerase TagF extends the Gro-P chain. The precursor CDP-glycerol is generated by TagD. In Rbo-WTA synthesis, the TagB reaction is followed by the addition of only one Gro-P unit by TarF. Subsequently, TarL polymerizes Rbo-P, and TarIJ is responsible for the generation of the precursor CDP-ribitol. Finally, the complete WTA polymers are translocated to the extracellular space by TagGH and are linked to the N-acetylmuramic acid residue of peptidoglycan by Lcp family proteins, primarily LcpA. The basic WTA polymers can be decorated with glycosyl residues and/or d-alanyl moieties; Rib-P, ribulose 5-phosphate; Rbo, ribitol; Gro, glycerol; P, phosphate; PP, diphosphate.

FIG 2

Characterization of phiWJ7. (A) Genetic map of the phiWJ7 genome. Arrows indicate the possible ORFs. Functional groups are categorized into patterns; striped, DNA replication and packaging; gray-shaded, structural proteins; dotted, bacterial lysis; blank, unknown. ITR indicates inverted terminal repeats and is marked by black triangles; ssDNA, single-stranded DNA. (B) Phylogeny of phiWJ7 and other Rountreeviridae phages based on the amino acid sequence relatedness of major capsid protein. The names of the six genera belonging to the family Rountreeviridae are denoted on the right side of each phage. Bacillus phage phi29 belongs to the family Salasmaviridae. (C) SEM image of WJ7 treated with phiWJ7. The black bar represents 500 nm.

FIG 3

Phage susceptibility of the host strains toward phiWJ7, phiWJ7_2, and phiWJ7_3. Each phage specimen was diluted and spotted on each host strain indicated on the left. The various dilutions of a phage were spotted on the same plate of a strain, although it is divided.

FIG 4

WTA synthesis genes of WJ7. (A) Schematic representation of tarIJL in T. halophilus WJ7 and S. aureus COL and the location of ISs transposed into tarL in WJ7R1 and WJ7R2. Percentage shows the amino acid identities between T. halophilus and S. aureus. The derivative names and IS names are indicated on the left and right of the arrows that represent the putative transposase, respectively. The locus tags of the WTA synthesis genes in COL and NBRC 12172 are designated in Table S4 in the supplemental material; aa, amino acids. (B) Chromosomal organization of the four loci harboring the genes involved in WTA synthesis in the NBRC 12172 and WJ7 genomes. Homologous ORFs with amino acid sequence identities above 90% are connected with gray dotted lines, except for transposases. The black arrows above each ORF indicate the position at which the primers were designed.

FIG 5

Extracted ion chromatogram of the benzoyl derivatives of Rbo (m/z 690.23), Gro (m/z 422.16), and D₅-Gro (1,1,2,3,3-D₅-glycerol; m/z 437.19) in the cell wall samples of WJ7 and WJ7R1. Extracted ion chromatograms of Gro and Rbo in the reference standard sample are also shown; Rt, retention time.

FIG 6

PhiWJ7 adsorption to the cell walls of WJ7 and the derivatives. Free phage titers were calculated as percentages of the control (without cell walls); Cont., control sample. Data are expressed as the mean with error bars representing ± standard deviation (SD; n = 3). Bars with asterisks are significantly different by Tukey’s multiple-comparison test (*, P < 0.05; **, P < 0.01). (A) Free phages represent total adsorption (the sum of phages reversibly and irreversibly adsorbed). (B) Free phages after 100-fold dilution of the samples represent irreversible adsorption.