Progress 12/01/00 to 11/30/03
Outputs
The deduced amino acid sequence of the phage infection protein (Pip) of Lactococcus lactis predicts a multiple membrane-spanning domain, suggesting that Pip may be anchored to the plasma membrane. However, a near-consensus sortase recognition site and cell wall anchoring motif may also be present near the carboxy end of Pip. 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 wild-type Pip strain LM 2301. Pip did not elute from membranes treated at pH 11 to remove peripheral membrane proteins, but was removed from envelopes treated with detergent. Three mutant Pip proteins were separately made in which the multiple membrane-spanning region was deleted (Pip-Dhy), the sortase recognition site was converted to the consensus (PipH841G), and the sortase recognition site (Pip-D6) was deleted. All three mutant Pip proteins
co-purified with membranes, and remained membrane-associated during pH 11 treatment. Pip-H841G and Pip-D6 were removed from envelopes by detergent treatment. Strains that expressed Pip-H841G or Pip-D6 plated phage c2 with near unit efficiency, whereas expression of Pip-Dhy did not lead to plaque formation. Viable cells, isolated membranes, and cell-free culture supernatant from a strain expressing Pip-Dhy inactivated phage c2. These results suggest the 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. Although, the region required for phage infection could not be pinpointed, the study provides more information about the function of Pip and might be helpful to find ways to study Pip further. The main points of the study are summarized as below: 1. Soluble region of Pip (Pip100-700), by itself, can inactivate phage in vitro. Pip100-700 may contain the actually binding
sites for phage c2. 2. Conformation of Pip may be important for phage infection. 3. Membrane-spanning domain is important for phage infection, but not for phage inactivation. 4. Missense mutations of Pip apparently do not change the phage-sensitivity of L. lactis strain. 5. For productive infection, binding of phage c2 to Pip might require multiple binding sites or multiple components. 6. The 35 kDa protein (Valyasevi, 1991) was not the proteolytic product of Pip. There is no apparent relationship between Pip and the 35 kDa protein, and there is no evidence that the 35kDa protein is involved in phage inactivation or infection.
Impacts
Milk is an important commodity in the U.S. From a nutritional standpoint, it is one of the most complete foods, supplying protein, lipids, carbohydrate, vitamins, and minerals. From an economic view, over 156 billion pounds of milk were produced in the US in 1997, with a total market value of nearly $21 billion. Cheese is the most important value-added product made from milk. In 1998, 56.4 % of the milk produced in the US was manufactured into cheese. Forty four percent of the cheese made in 1998 was American type cheeses (cheddars). In 1997, the wholesale market value of American type cheese in the US was $5.6 billion. Bacteriophage infection of cheese starter cultures is a serious and persistent problem that causes significant economic loss each year to the dairy industry. The pip strategy of phage resistance is the only strategy that has not selected mutant phages in the laboratory or factory. The pip, 2-step model of attachment is the most highly developed model
for investigating the initial steps of lactococcal phage infection. We have shown that a newfound knowledge of a phage receptor (pip) can lead to a novel and successful strategy of engineering phage resistance in L .lactis. Our research objectives have helped us understand the mechanism by which lactococcal phages infect their host. A better understanding of the mechanism of phage infection has enablee us to design better strategies of resistance to phages.
Publications
- Hang Ngo. 2003. Functional and Structural Characterization of Phage Infection Protein (Pip)in Lactococcus lactis. Master's Thesis.
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Progress 01/01/02 to 12/31/02
Outputs
We have completed objective 1 of the project, which is: To determine the sub-cellular location of Pip on the surfaces of L. lactis. Our results show that Pip is anchored to the plasma membrane and not to the cell wall. The significance of this is that it establishes the location of the bacteriophage receptor (Pip) to an area of the cell that is consistent with a role for Pip not only in the attachment of the phage to the cell surface, but also to the entry of the phage genome into the host cell. This helps us understand the mechanism of phage infection and suggests ways to prevent phage infection. We are nearing the completion of objective 2, which is: To identify the molecular region of Pip that anchors it to the cell surface. We have found that deleting or mutating the hexapeptide LPXTH has no effect on the sub-cellular location of Pip. Moreover, deleting the multiple membrane spanning region of Pip destabilizes the protein and lowers its capacity to function by
orders of magnitude. These results are consistent with the location of Pip in the plasma membrane and not in the cell wall.
Impacts
Cheese is the most important value-added product made from milk. In 1998, 56.4 % of the milk produced in the US was manufactured into cheese (USDA annual dairy report, 1999). Forty four percent of the cheese made in 1998 was American type cheeses (cheddars). In 1997, the wholesale market value of American type cheese in the US was $5.6 billion (USDA Dairy Market Statistics, 1997 Annual Summary). Bacteriophage infection of cheese starter cultures is a serious and persistent problem (Sanders, 1987; Klaenhammer and Fitzgerald, 1994;Forde and Fitzgerald, 1999) that causes significant economic loss each year to the dairy industry. The pip strategy of phage resistance is the only strategy that has not selected mutant phages in the laboratory or factory. The pip, 2-step model of attachment is the most highly developed model for investigating the initial steps of lactococcal phage infection. We have shown that a newfound knowledge of a phage receptor (pip) can lead to a novel
and successful strategy of engineering phage resistance in L. lactis. Our research objectives will help us understand the mechanism by which lactococcal phages infect their host. A better understanding of the mechanism of phage infection will enable us to design better strategies of resistance.
Publications
- D. T. Mooney and B. L. Geller. 2002. Analysis of the Subcellular Location of Phage Infection Protein (Pip) from Lactococcus lactis. Federation of European Microbiology Societies Seventh Symposium on Lactic Acid Bacteria: Genetics, Metabolism and Applications. Egmond aan Zee, The Netherlands. September 1-5. Book of Abstracts F24.
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