title: Subcellular location of phage infection protein (Pip) in Lactococcus lactis.
abstract: The amino acid sequence of the phage infection protein (Pip) of Lactococcus lactis predicts a multiple-membrane-spanning region, suggesting that Pip may be anchored to the plasma membrane. However, a near-consensus sortase recognition site and a cell wall anchoring motif may also be present near the carboxy terminus. If functional, this recognition site could lead to covalent linkage of Pip to the cell wall. Pip was detected in both plasma membranes and envelopes (plasma membrane plus peptidoglycan) isolated from the wild-type Pip strain LM2301. Pip was firmly attached to membrane and envelope preparations and was solubilized only by treatment with detergent. Three mutant Pip proteins were separately made in which the multiple-membrane-spanning region was deleted (Pip-Deltammsr), the sortase recognition site was converted to the consensus (Pip-H841G), or the sortase recognition site was deleted (Pip-Delta6). All three mutant Pip proteins co-purified with membranes and could not be solubilized except with detergent. When membranes containing Pip-Deltammsr were sonicated and re-isolated by sucrose density gradient centrifugation, Pip-Deltammsr remained associated with the membranes. Strains that expressed Pip-H841G or Pip-Delta6 formed plaques with near unit efficiency, whereas the strain that expressed Pip-Deltammsr did not form plaques of phage c2. Both membranes and cell-free culture supernatant from the strain expressing Pip-Deltammsr inactivated phage c2. These results suggest that Pip is an integral membrane protein that is not anchored to the cell wall and that the multiple-membrane-spanning region is required for productive phage infection but not phage inactivation.
authors.affiliation: Department of Microbiology, Oregon State University, OR 97331, USA.
authors.forename: Duane T
authors.initials: DT
authors.lastname: Mooney
authors.forename: Monica
authors.initials: M
authors.lastname: Jann
authors.forename: Bruce L
authors.initials: BL
authors.lastname: Geller
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D001435:Bacteriophages / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry; D013294:Lactococcus lactis / Q000737:chemistry* / Q000166:cytology / Q000821:virology; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D011817:Rabbits
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; C082934:phage infection protein, Lactococcus lactis