Novel P335-like Phage Resistance Arises from Deletion within Putative Autolysin <i>yccB</i> in <i>Lactococcus lactis</i>

Lactococcus lactis and Lactococcus cremoris are broadly utilized as starter cultures for fermented dairy products and are inherently impacted by bacteriophage (phage) attacks in the industrial environment. Consequently, the generation of bacteriophage-insensitive mutants (BIMs) is a standard approach for addressing phage susceptibility in dairy starter strains. In this study, we characterized spontaneous BIMs of L. lactis DGCC12699 that gained resistance against homologous P335-like phages. Phage resistance was found to result from mutations in the YjdB domain of yccB, a putative autolysin gene. We further observed that alteration of a fused tail-associated lysin-receptor binding protein (Tal-RBP) in the phage restored infectivity on the yccB BIMs. Additional investigation found yccB homologs to be widespread in L. lactis and L. cremoris and that different yccB homologs are highly correlated with cell wall polysaccharide (CWPS) type/subtype. CWPS are known lactococcal phage receptors, and we found that truncation of a glycosyltransferase in the cwps operon also resulted in resistance to these P335-like phages. However, characterization of the CWPS mutant identified notable differences from the yccB mutants, suggesting the two resistance mechanisms are distinct. As phage resistance correlated with yccB mutation has not been previously described in L. lactis, this study offers insight into a novel gene involved in lactococcal phage sensitivity

Data_Sheet_2_Host-encoded, cell surface-associated exopolysaccharide required for adsorption and infection by lactococcal P335 phage subtypes.docx

Lactococcus lactis and Lactococcus cremoris compose commercial starter cultures widely used for industrial dairy fermentations. Some lactococcal strains may produce exopolysaccharides (EPS), which have technological applications, including texture production and phage resistance. Two distinct gene clusters associated with EPS production, designated 6073-like and 7127-like, were identified on plasmids in lactococcal strains. Infectivity of two subsets of P335 group phages, distinguished based on their single-component baseplate/receptor-binding protein nucleotide sequences, was correlated to the presence of a host-encoded 6073-like or 7127-like eps gene cluster. Furthermore, phages belonging to these subsets differentially adsorbed to lactococcal strains harboring the respective eps gene cluster. Loss of the respective EPS-encoding plasmid from a fully phage-sensitive strain resulted in loss of phage adsorption and resistance to the phage. Transmission electron microscopy (TEM) showed that the EPS produced by strains encoding the 6073-like or 7127-like eps gene clusters are cell-surface associated, which, coupled with phage plaquing and adsorption data, shows that specific capsular EPS are involved in host recognition by certain P335 phage subgroups. To our knowledge, this is the first description of the involvement of EPS produced via the Wzx/Wzy-dependent pathway in phage sensitivity of L. lactis or L. cremoris. This study also shows strains that do not appear to be phage-related based on plaque formation may still be related by phage adsorption and indicates that optimal formulation of phage-robust cultures should take into account the EPS type of individual strains.</p

Data_Sheet_1_Host-encoded, cell surface-associated exopolysaccharide required for adsorption and infection by lactococcal P335 phage subtypes.docx

Lactococcus lactis and Lactococcus cremoris compose commercial starter cultures widely used for industrial dairy fermentations. Some lactococcal strains may produce exopolysaccharides (EPS), which have technological applications, including texture production and phage resistance. Two distinct gene clusters associated with EPS production, designated 6073-like and 7127-like, were identified on plasmids in lactococcal strains. Infectivity of two subsets of P335 group phages, distinguished based on their single-component baseplate/receptor-binding protein nucleotide sequences, was correlated to the presence of a host-encoded 6073-like or 7127-like eps gene cluster. Furthermore, phages belonging to these subsets differentially adsorbed to lactococcal strains harboring the respective eps gene cluster. Loss of the respective EPS-encoding plasmid from a fully phage-sensitive strain resulted in loss of phage adsorption and resistance to the phage. Transmission electron microscopy (TEM) showed that the EPS produced by strains encoding the 6073-like or 7127-like eps gene clusters are cell-surface associated, which, coupled with phage plaquing and adsorption data, shows that specific capsular EPS are involved in host recognition by certain P335 phage subgroups. To our knowledge, this is the first description of the involvement of EPS produced via the Wzx/Wzy-dependent pathway in phage sensitivity of L. lactis or L. cremoris. This study also shows strains that do not appear to be phage-related based on plaque formation may still be related by phage adsorption and indicates that optimal formulation of phage-robust cultures should take into account the EPS type of individual strains.</p