Identification of Lactococcus lactis Genes Required for Bacteriophage Adsorption

Lactococcus lactis is widely used in starter cultures for cheese production. Bacteriophage contamination during the fermentation process is a major problem, causing lysis of the starter bacteria and consequently slow or failed fermentation of the milk. Bacteriophage infection requires specific recognition between the phage receptor-binding protein (RBP) and the host cell receptor. A better understanding of this recognition mechanism should increase the possibility of preventing phage infection. Bacterial receptors have been well studied in gram-negative bacteria, especially Escherichia coli. Phages attacking E. coli recognize either lipopolysaccharides or specific proteins of the outer membrane. For example, phage lambda initially interacts reversibly and then interacts irreversibly with the outer membrane protein LamB (32, 35), which facilitates the diffusion of maltose into the cell (10). A slightly more complex mechanism is utilized by phage T5, which initially binds reversibly to polymannose O antigens in lipopolysaccharides at the E. coli surface (13, 14) and then binds irreversibly to the ferrichrome transporter FhuA (5, 19). Finally, phage T4 binds reversibly with its long tail fibers to B-type lipopolysaccharides or to the outer membrane porin OmpC (15, 16, 28), whereupon the additional short tail fibers bind irreversibly to the lipopolysaccharide core region (27). For gram-positive bacteria the information on phage receptors is sparser. However, phages attacking L. lactis seem to bind initially to specific carbohydrate receptors exposed to the surface of the cell wall (29, 34, 40, 42). For many phages this binding step is reversible (29). Rhamnose, glucose, and galactose are often involved in this initial phage binding, as shown by the ability of these monosaccharides to competitively inhibit adsorption of several phages to cell surfaces (29, 40, 42). Binding to other carbohydrates, such as glucosamine and galactosamine, has been demonstrated for phage eb7 (20). For phages belonging to species c2, a secondary irreversible binding step requires binding to the membrane phage infection protein (PIP) (2, 12, 29, 41), while most phages of species 936, P335, and 949 do not use PIP as a secondary receptor (21); the only exception is 936-species phage kh (2, 29). Some of these phages may use other protein receptors in the membrane, as determined for 936-species phage sk1, which is able to bind to cell membranes deficient in PIP (2). The aim of this work was to identify genes in L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris Wg2 that are important for adsorption of the 936-species phages bIL170 and φ645, respectively. Phage bIL170 and its host, L. lactis IL1403, are an excellent model system because the complete genome sequences are known (4, 7). In L. lactis IL1403 a biochemical or biological role has been assigned to 64.2% of the genes (4), and genes assumed to be involved in biosynthesis of surface polysaccharides and the PIP gene have been identified (2, 4). In bIL170 the RBP gene has recently been identified to be orf20 (9). In contrast, the genome sequences of L. lactis Wg2 and phage φ645 are not available. φ645 was selected since it infects both L. lactis Wg2 and L. lactis IL1403, whereas bIL170 infects only L. lactis IL1403. Here we report that the genes identified, which are putatively involved in biosynthesis of cell wall polysaccharides in L. lactis IL1403 and L. lactis Wg2, are important for adsorption of phages bIL170 and φ645, respectively.