# The cell surface-associated rhamnose-glucose polysaccharide represents the receptor of Streptococcus thermophilus bacteriophage P738

## TL;DR

This study identifies a specific sugar-based structure on Streptococcus thermophilus that allows a bacteriophage to infect it, helping to develop more robust bacterial cultures for dairy production.

## Contribution

The study identifies the rhamnose-glucose polysaccharide as the receptor for the P738 bacteriophage in Streptococcus thermophilus.

## Key findings

- Mutations in a glycosyltransferase gene within the rgp locus confer resistance to P738 phage infection.
- Loss of a terminal rhamnose residue in the RGP side chain prevents phage binding and infection.
- Fluorescent binding assays confirmed the role of the RGP side chain in phage adsorption.

## Abstract

Streptococcus thermophilus is a key lactic acid bacterial species extensively used in dairy fermentations, where bacteriophage (phage) infections pose a significant threat to production efficiency. While the cell surface receptors for four of the five currently known streptococcal phage genera have previously been identified, the specific host-encoded receptor for the P738 phage group has not been elucidated to date. In this study, P738 bacteriophage-insensitive mutants of S. thermophilus UCCSt50 were isolated, and whole-genome sequencing revealed mutations in a glycosyltransferase-encoding gene within the rgp locus, responsible for the biosynthesis of the cell wall-associated rhamnose-glucose polysaccharide (RGP). Compositional and methylation analysis demonstrated that loss of a terminal rhamnose residue from the RGP side chain underpinned the observed resistance to P738 binding and infection. Fluorescent binding assays validated the biological functionality of the predicted receptor binding protein of P738, confirming the critical role of the RGP side chain in phage adsorption. These findings reveal a strict requirement for a complete tetrasaccharide side chain structure for P738 binding, contrasting with the less stringent requirements observed for the Brussowvirus SW13. This work offers valuable insights to guide the development of phage-robust starter cultures through knowledge-based strain selection and rotation strategies, thereby supporting more sustainable and reliable dairy fermentation processes.

Streptococcus thermophilus is one of the most extensively applied members of the lactic acid bacteria, being extensively used as a bacterial starter culture in the production of fermented foods, including cheeses and yogurt. Bacteriophage infections present a significant threat to industrial dairy fermentations, which can lead to substandard production processes and product quality. Understanding phage-host interactions, which commence with the recognition and binding of a given bacteriophage to a particular host-encoded, cell surface-associated receptor, is essential for developing strategies to minimize phage-related risks in dairy fermentations. This study identifies the specific cell surface polysaccharide structure produced by Streptococcus thermophilus as the receptor required for infection by phage P738, bridging a key knowledge gap in phage-host interactions in this important industrial bacterial species. By elucidating how phage P738 recognizes and binds to its host, this work offers valuable information to aid strain selection and the development of phage-robust starter cultures.