Identification of Lactococcus lactis Genes Required for Bacteriophage Adsorption

Abstract

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that 645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and 645 that showed no homology in the C-terminal part.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 ...

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