Graduate Thesis Or Dissertation
 

Functional and structural characterization of phage infection protein (Pip) in Lactococcus lactis

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/6108vf23p

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  • This thesis describes an analysis of the structure/function relationship of the phage infection protein (Pip) in Lactococcus lactis. Pip is a protein required for phage infection of L. lactis exclusively by phage c2 species. Pip, which shares no significant homology to previously studied proteins, contains 6 hydrophobic regions; one residing at the N-terminal region is a putative signal sequence, and the other five located in the C terminus, comprise the putative membrane-spanning domain (MSD). No other motifs or domain structures are identified in Pip. Facing outward from the cells lies a hydrophilic region including amino acids 100 to 700 of Pip (Pip 100-700). When purified, Pip 100-700 inactivated phage c2 in vitro. No other component was required for the inactivation. The inactivation of phage c2 was inhibited by adding Pip antiserum to the reaction, indicating that phage c2 inactivation is caused by Pip. The amino acid residues or regions required for inactivation of phage c2 were mapped by mutagenic analysis. A large deletion in Pip removes 451 amino acids from amino acid #193 to #644 of Pip resulting in full resistance to phage c2. An insertion mutation at amino acid #108 resulted in partial resistance to phage c2. These data suggest that the conformation of Pip is important for the binding. An island of charged amino acids from amino acid #227-231 (EIKEK) in Pip was site-directedly mutated into the neutral group AIAAA. When the plasmid containing these missense mutations was electroporated into JK1O1, the transformant remained sensitive to phage c2, indicating that the missense mutations were not critical for phage infection. The Pip gene was randomly mutated, expressed in a Pip negative background, and screened for phage resistance. Fully resistant, partially resistant and temperature sensitive mutants of Pip were identified. All of the mutants created from nonsense or frameshift mutations, resulted in truncations of Pip. Apparently, missense mutations in Pip do not cause phage resistance, at least under the conditions used for screening. All of the mutants have partial or total deletion of the MSD. While the partially resistant and totally resistant mutants have the MSD totally deleted, only parts of MSD in temperature sensitive mutants are deleted, suggesting that the MSD is required for phage infection, but not for inactivation. The lack of the MSD might cause mislocalization of Pip, resulting in partial or total resistance. The mechanism of resistance of the mutants was characterized by measuring absorbance of phage to cells expressing the mutated Pip. Phage c2 adsorbed to all of the mutants collected, suggesting that adsorption of phage c2 is not the cause of resistance. Pip expression was verified in all mutants selected by slot blot and Western blot. For partially or totally resistant mutants, Pip was found in both the whole cell extracts and the cell-free supernatants. The attached Pip enabled partially resistant and temperature-sensitive mutants to form plaques with phage c2. In addition to the Pip attached to the cells, Pip was also released into the growth medium in the mutants with a deleted MSD, shown by Western blots of cell-free supernatants of those mutants. Pip released in the cell-free supernatant was shown to inactivate phage c2, which was similar to the effect of purified Pip 100-700 on phage c2. In a previous report, a 35kDa protein was shown to inactivate c2 (Valyasevi, 1991). In this thesis, the 35kDa protein was further analyzed using a recently made antiserum to PiplOO-700. Western blot analysis of the 35kDa protein using Pip antiserum showed a signal at 94kDa, which was the full length Pip. There was no signal at 3 5kDa, suggesting that 3 5kDa protein is not the proteolytic product of Pip and the "purified" 35kDa sample was contaminated with Pip wild type. The contaminating Pip was probably the component that caused phage c2 inactivation.
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