Phage-resistant mutants, isolated from cultures of Lactococcus lactis subsp. lactis C2 infected with phage c2, did not form plaques but bound phage normally. The mutants were sensitive to another phage, skl, although the number of plaques was reduced -56% and the plaques were four times smaller. Binding to phage skl was reduced about 10%. Another group of phage-resistant mutants, isolated from cultures infected with phage skl, bound normally to both phages c2 and skl but did not form plaques with either phage. Carbohydrate analyses by gas chromatography of the ceUl walls showed no significant differences in saccharide compositions between the wild-type and phage-resistant cells. However, a difference was observed in the interactions of the phage with the cytoplasmic membranes. Membranes from the wild-type cells, but not mutant cells, inactivated phage c2. Phage skl was not inactivated by membrane from either strain. Treatment of wild-type membranes with proteinase K eliminated the ability of the membrane to inactivate the phage, whereas treatment with mutanolysin had no effect. On the basis of this ability to inactivate the phage, a membrane protein was partially purified by gel filtration and ion-exchange chromatography. Under nondenaturing conditions, the phageinactivating protein has an apparent Mr of e350,000. The protein has an apparent subunit size of 32 kDa, which suggests that it normally exists as a multimer with 10 to 12 subunits or in association with other membrane components. It is proposed that this protein is required for phage c2 infection.In lactococci, carbohydrates of the exopolysaccharide may be commonly used as phage receptors (8). Previous studies from our laboratory have shown that the binding determinants of phages for Lactococcus lactis subsp. cremoris KH and subsp. lactis C2 include the rhamnose of the extracellular wall polysaccharides (16, 17). An exception to this was reported by Oram and Reiter (12), who found that membranes from L. lactis subsp. lactis ML3 inactivated phage m13. The phage-inactivating material appeared to be a protein, because trypsin digestion of the plasma membrane destroyed the phage-inactivating activity. In our previous study (16), treatment of the cell envelope (cytoplasmic membrane plus peptidoglycan and exopolysaccharides) from L. lactis subsp. lactis C2 with either mutanolysin or sodium dodecyl sulfate (SDS) alone did not completely eliminate the adsorption of some phages (for example, c2). This suggested that the adsorption of certain phages may require both a membrane component and a saccharide unit on the cell wall (peptidoglycan and exopolysaccharides).In this paper, we report a difference in the cytoplasmic membranes of phage c2-sensitive and -resistant strains of L. lactis subsp. lactis C2. This difference correlates with the presence of a protein and was used to purify and identify a protein with an apparent Mr of -350,000 and a subunit size of 32 kDa. We propose that this protein is required for phage c2 infection, apparently at a step subsequent t...

A receptor for bacteriophages of lactic acid bacteria, including Lactococcus lactis subsp. cremoris KH, was found on the cell wall and not on the cell membrane, as determined by a phage-binding assay of sodium dodecyl sulfate-and mutanolysin-treated cell walls. The cell wall carbohydrates of L. lactis subsp. cremoris KH were analyzed by gas chromatography and mass spectrometry and found to contain rhamnose, galactose, glucose and N-acetylglucosamine. Similar analysis of mutants that were reduced in the ability to bind phages kh, 643, c2, m13, and 1 indicated that galactose was essential for binding all phages. In addition, rhamnose was required for binding phages kh and m13. Inhibition studies of phage binding by using two different lectins with a specificity for galactose indicated that phage kh may not bind directly to galactose. Rather, galactose may be an essential structural component located in the vicinity of the receptor. Incubation of any of the five phages with rhamnose or of phage kh with purified cell walls inactivated the phages. Inactivation required divalent cations and was irreversible. Inactivation of phages was stereospecific for rhamnose, as neither L-(+)-nor D-(-)-fucose (the stereoisomers of rhamnose) inhibited the phage. Furthermore, phage infection of a culture was completely inhibited by the addition of rhamnose to the medium. Therefore, the receptor for phage kh appears to be a rhamnose component of the extracellular wall polysaccharide. * Corresponding author. t Oregon State University Agricultural Experiment Station communication no. 9022.

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