WO2007093849A2

WO2007093849A2 - P100 bacteriophage for control of listeria monocytogenes

Info

Publication number: WO2007093849A2
Application number: PCT/IB2006/004168
Authority: WO
Inventors: Martin J. Loessner
Original assignee: Micreos Food Safety BV
Current assignee: Micreos Food Safety BV
Priority date: 2005-09-16
Filing date: 2006-09-15
Publication date: 2007-08-23
Anticipated expiration: 2008-09-17
Also published as: WO2007093849A3

Abstract

Disclosed are compositions and methods for treating and preventing Listeriosis using recombinant bacteriophage (P100) and bacteriophage-derived endolysin (Ply100).

Description

PlOO BACTERIOPHAGE FOR CONTROL OF LISTERIA MONOCYTOGENES

I. CROSS-REFERENCE TO RELATED APPLICATIONS

1. This application claims benefit of U.S. Provisional Application No. 60/717,779, filed September 16, 2005, and of U.S. Provisional Application No. 60/783,747, filed March 17, 2006, which are hereby incorporated herein by reference in their entirety.

II. BACKGROUND

2. Listeria monocytogenes is a bacterial pathogen that contaminates many food products, the list of which includes but is not limited to soft cheeses, pates, ice cream, smoked and cured fish, frozen seafood, salads, and processed meats. When ingested, these bacteria can produce a disease termed listeriosis, characterized by a variety of symptoms and conditions, including diarrhea, abortion, and encephalitis. Collectively, in the industrialized nations, hundreds of deaths occur each year as a result of Listeria monocytogenes food contamination.

3. The food processing industry has not been sufficiently successful in eradicating Listeria monocytogenes bacteria from the environment of the processing plants. As a result, even foods that have been pasteurized at temperatures high enough to kill these bacteria nevertheless become contaminated, post-pasteurization. The bacteria gain access to the foodstuffs through one or more routes, including (i) from the raw materials (e.g. raw milk, and/or milk that has been pasteurized at low temperatures); (ii) from the processing machinery (in and on which the bacteria can grow as biofilms that are difficult to eradicate ); and (iii) from airborne bacteria present in the plant environment which can settle onto the surface of the foodstuffs during curing, packaging, and so on.

4. Despite the numerous methods used in the food industry to control and prevent L. monocytogenes contamination, the bacteria gain access to and persist in the environment of food processing plants. Moreover, they survive the very high concentrations of salt that are present in several food-making processes. The resulting contamination of the foodstuffs (including but not limited to cheeses, pates, cold cuts, hot dogs and other processed foods) leads to scores of deaths each year in developed nations, and also to product recalls whose retail worth each year, in the aggregate, is measured in the hundreds of millions of dollars.

5. The methods currently in use to control Listeria in the food industry include: (i) pasteurization of primary ingredients (e.g. milk) and heat treatment of the products, which is often unsuccessful because recontamination frequently occurs and many products cannot undergo a final (listeriocidal) heat treatment; (ii) application of physicochemical agents such as

I disinfectants, enzymes, antibiotics, etc., which experience has shown do not reduce the bacterial counts sufficiently; and (iii) attempts to break up biofilms mechanically, which leave sufficient residues of bacteria behind that the foodstuffs still become contaminated.

6. Additional methods must therefore be made available to the food processing industry in order to protect the health of consumers, and to reduce the exposure of numerous companies to the great cost and the loss of good will that result from such contaminations and recalls.

III. SUMMARY

7. Disclosed are compositions and methods related to pi 00, a phage which can be used in the treatment of listeria caused problems. IV. BRIEF DESCRIPTION OF THE DRAWINGS

8. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

9. Figure 1 shows the effect of phage PlOO on growth of L. monocytogenes on surface- ripened, soft cheese with a washed rind. All tested cheese were contaminated with L. monocytogenes on day 1 after cheese making. (A) PlOO was repeatedly applied to the cheese surface at different concentrations during all rind smearings until day 13. The data point for repeated low dose application on day 16 was not measured. (B) A single high dose of PlOO was added to the brine during first smearing of the cheese rind. The control cheeses received no phage. AU cheeses were packaged on day 16 after cheese making (indicated by a star).

10. Figure 2 shows the effect of phage PlOO on the growth of L. monocytogenes in cooked chicken fillet (vacuum, 7°C).

11. Figure 3 shows the evolution of the phage titer on the cooked ham during storage.

12. Figure 4 shows the effect of PlOO on the growth of the L. monocytogenes cocktail (LISl, LIS2 and LIS3) in the cooked ham (vacuum packaged and stored at 7°C).

13. Figure 5 shows the effect of PlOO on the evolution of the total aerobic psychrotrophic count in the cooked ham (vacuum packaged and stored at 7°C).

14. Figure 6 shows the effect of PlOO on the evolution of the number of lactic acid bacteria in the cooked ham (vacuum packaged and stored at 7°C). V. DETAILED DESCRIPTION

15. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

A. Definitions ^■ 16. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

17. Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of vames disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed the "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

18. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

19. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. 20. "Primers" are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.

21. "Probes" are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art. 22. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. 23. Disclosed are the components to be used to prepare the disclosed compositions as . . well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular vector is disclosed and discussed and a number of vector components including the promoters are discussed, each and every combination and permutation of promoters and other vector components and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods. B. Compositions

24. Provided herein are compositions and methods of treating and preventing Listeriosis. The compositions and methods can involve controlling Listeria monocytogenes contamination, such as for example, in a food product, on food processing equipment or on food storage containers. The compositions and methods can also involve treating an animal infected with Listeria monocytogenes. The compositions and methods can also involve delivering a nucleic acid to a Listeria monocytogenes bacterium. The compositions and methods can also involve detecting the presence of Listeria monocytogenes.

1. Listeriosis 25. Listeriosis is an infection resulting from the ingestion of foods contaminated by

Listeria monocytogenes, and is characterized by a variety of symptoms, from diarrhea to abortion and infections of the brain and central nervous system. Because of its high mortality rate of approximately 25-30% (Vasquez-Boland et al., 2001), the disease ranks among the most severe food-borne illnesses. It was estimated that approximately 2000 hospitalizations and 500 deaths occur annually in the United States alone, as a result of the consumption of foods contaminated with L. monocytogenes (Mead et al., 1999). Listeria does not belong to the normal flora of healthy animals or man, but is an environmental bacterium and usually contaminates foods during fermentation, processing, storage, or even packaging of foods. This includes most ready- to-eat products such as milk and cheeses (mostly soft cheese), cold cuts (different types of meats), hot dogs, smoked fish, seafoods, and various delicatessen items.

26. The currently available methods and procedures are insufficient to achieve full control of this organism, whether in the food itself as well or in the food production and processing equipment and related environments. Thus, there is a need for better methods to prevent contamination, and promising novel approaches should be considered and evaluated. Thus, provided herein, for example, are compositions comprising isolated, purified, or recombinant bacteriophages and bacteriophage-derived proteins, such as endolysins, for treating listeriosis.

2. Bacteriophages

27. Bacteriophages can be regarded as natural enemies of bacteria, and therefore are candidates to evaluate as agents for the control of foodborne bacterial pathogens, such as

Listeria. The attributes of phages include the following: (i) they are designed to kill live bacterial target cells, (ii) they generally do not cross species or genus boundaries, and will therefore not affect (a) desired bacteria in foods (e.g., starter cultures), (b) commensals in the gastrointestinal ^ tract, or (c) accompanying bacterial flora in the environment. Moreover, (iii) since phages are generally composed entirely of proteins and nucleic acids, their eventual breakdown products consist exclusively of amino acids and nucleic acids. Thus, they are not xenobiotics, and, unlike antibiotics and antiseptic agents, their introduction into and distribution within a given environment may be seen as a natural process. With respect to their potential application for the biocontrol of undesired pathogens in foods, feeds, and related environments, it should be considered that phages are the most abundant self-replicating units in our environment, and are present in significant numbers in water and foods of various origins, in particular fermented foods (reviewed by Sulakvelidze and Barrow, 2005). On fresh and processed meat and meat products, more than 108 viable phage per gram are often present (Kennedy and Bitton, 1987). It is a fact that phages are routinely consumed with food, in quite significant numbers. Moreover, phages are also normal commensals of humans and animals, and are especially abundant in the gastrointestinal tract (Furuse, 1987; Breitbart et al, 2003).

28. Because of their inherent specificity, phages harbor the potential for precise targeting of a bacterial contamination, without compromising the viability of other microorganisms' in the habitat. A number of recent reviews (Greer, 2005; Hudson et al., 2005; Sulakvelidze and Barrow, 2005; Withey et al., 2004) summarize the current status of using phage for the control of undesired bacteria in systems other than therapy of disease in humans and animals. The potential of phages for controlling foodborne pathogens is reflected in recent studies dealing with Salmonella (Goode et al., 2003;Leverentz et al., 2001; Whichard et al., 2003), Campylobacter (Atterbury et al., 2003; Goode et al., 2003), E. coli (HuV et al., 2005; Toro et al., 2005), and L. monocytogenes (Dykes and Moorhead, 2002; Leverentz et al., 2003, 2004). However, most of the phage-host systems are highly specific, which is a general limitation of using a limited number of characterized phages to attack an unknown diversity of a given target bacterium. Solutions to circumvent this problem can include (i) careful selection and pooling of different phages with different lysis ranges, and/or (ii) the use of single broad host range phages which are able to infect all (or a majority of) the targeted organisms. The latter solution permits a precise definition of the agent, and use of a single phage (rather than a pooled mixture) can be expected to facilitate the process of obtaining regulatory approval. 29. Almost all of the phages infecting organisms of the genus Listeria are temperate and feature a very narrow host range (Loessner and Rees, 2005). As disclosed herein, PlOO represents one of the few virulent phages for this genus, which is strictly lytic and therefore invariably lethal to a bacterial cell once an infection has been established., Moreover, PlOO features an unusually broad host range within the genus Listeria, similar to phage A511 (Loessner, 1991; Loessner and Busse, 1990; van der Mee-Marquet et al., 1997). As disclosed herein, more than 95% of approximately 250 different foodborne Listeria isolates belonging to serovar groups 1/2, 4 (L. monocytogenes), and 5 (L. ivanovii) were infected and killed by PlOO. (1) PlOO phage

30. Provided herein are compositions. These compositions can be used for treating and preventing Listeriosis, such as by controlling Listeria contamination in a food product, on food processing equipment or on food storage containers. The provided composition can comprise recombinant PlOO phage or recombinant PlOO polypeptides. Also disclosed are methods for decontaminating an object which has a particular pathogen, such as a Listeria bacterium, by incubating the object with or admninstering to the object, one or more of the compositions disclosed herein. As used herein, decontaminating means that the number of bacteria on an object have been reduced. It is not required that the bateria be completely eradicated or removed, although in certain embodiments this may occur. 31. It is understood that a strain of P 100 phage has been disclosed as a deposited phage in the ATCC as deposit designation no. PTA-4383. This PlOO phage is provided in United States Patent Application No. 10/516507 filed on December 1, 2004, which is herein incorporated by regeference in its entireity and at least for material relating to PlOO phage and their uses. Disclosed herein, is the sequence of the complete genome of this PlOO phage, as well as the open reading frames for the proteins produced by this phage. This information allows for the production of recombinant phage as well as recombinant versions of the proteins produced by this phage. These recombinant proteins can be used as disclosed herein, but can at least be used in conjunction with whole phage to reduce Listeria bacteria. Also disclosed are variants of the PlOO phage which are capable of being made and used based on the genetic information. Also disclosed herein are specific conditions for use of the PlOO phage as well as its variants.

32. Thus, provided is a composition comprising recombinant PlOO phage. In one aspect, the phage are do not include the PlOO phage designated as ATCC PTA-4383. In addition, also disclosed are embodiments of the proteins produced by PlOO which are recombinant. By "recombinant" is meant proteins which are produced within an environment which has been manipulated in some way by recombinant biotechnology. Thus, the herein provided recombinant PlOO phage can comprise a nucleic acid having mutations, substitutions, or deletions to the sequence set forth in SEQ JD NO:1 (PlOO genome).

η . 33, The folly annotated PlOO genome sequence (SEQ ID NO:1) has been deposited in GenBank under Accession No. DQ004855. The PlOO genome comprises 174 open reading frames (ORFs), shown in Table 3. Thus, the provided recombinant PlOO phage can be produced from a nucleic acid vector comprising all of these ORFs if at least one ORF comprises a mutation, substitution, and/or deletion to the sequence of the phage deposited in ATCC PTA- 4383. The provided recombinant PlOO phage can also be produced from a nucleic acid vector comprising less than all of these ORFs. The provided recombinant PlOO phage can also be produced from a vector comprising a mixture (chimera) of PlOO ORFs and ORFs from other phages (e.g. A511). It is understood that one of skill in the art using standard methods can routinely select ORFs for deletion, substitution or mutation for use in the herein provided methods. For example, substitution of the PlOO capsid protein encoding nucleic acids (gplO; nucleotides 5,472 to 6,497 of SEQ ID NO:1) with nucleic acids encoding other phage capsid proteins of a different phage family then P 100 can alter the specificity of the resulting phage.

(2) Other Bacteriophages 34. Also provided are other Listeria bacteriophages (see Table 1) that can be.used in combination with PlOO to control Listeria monocytogenes. Thus, provided is a composition comprising PlOO phage and one or more of A511, P35, Al 18, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971A, 02971 C, 907515, 12981, 11711 A, 00241, 13441, A500, A640. B021, PSA, B653, 90666, 90861, 910716, 93253, 52, 340, 312, 108, 10, 2425 A, 2425, or 3551 phage.

35. In one aspect, the recombinant PlOO phage that can be used herein can be defined based on homology to Pl 00. Thus, the herein disclosed recombinant PlOO phage can be 65%, 70%, 75%, 80%, 85%, 90%, 95% homologous to the PlOO phage as encoded by SEQ ED NO:1. In another aspect, the disclosed recombinant PlOO phage is not A511, P35, Al 18, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971 A, 02971C, 907515, 12981, 11711 A, 00241, 13441, A500, A640, B021, PSA, B653, 90666, 90861, 910716, 93253, 52, 340, 312, 108, 10, 2425 A, 2425, or 3551 phage.

Table 1. Listeria Phages.

Phage Host strain Serovar group of Host strain

A511 1001 polyvalent (virulent)

P35 1001 1/2 (virulent)

A118 1001 1/2

A502 1001 1/2

A006 1001 1/2

A005 1001 1/2

A620 1001 1/2

11355C 1764 1/2 Table 1. Listeria Phages.

Phage Host strain Serovar group of Host strain

00611 1764 1/2

43 1763 1/2

21 1997 1/2

2685 1764 1/2

4477 1764 1/2

01761 1764 1/2

12029 1763 1/2

717 1764 1/2

10993 1763 1/2

10072 1762 1/2

0297IA 1763 1/2

02971C 1764 1/2

907515 1764 1/2

12981 1764 1/2

11711 A 1764 1/2

00241 1764 1/2

13441 1764 1/2

A500 1042 4b

A640 1042 4b

B021 1042 4b

PSA 1042 4b

B653 3009 4b

90666 1761 4b

90861 1761 4b

910716 1761 4b

93253 1761 4b

52 1996 4b

340 1996 4b

312 1996 4b

108 1996 4b

107 1996 4b

2425A 11002 4b

2425 1999 •4b

3551 11001 4b

(3) PlOO polypeptides

36. Also provided herein is a composition comprising a recombinant phage PlOO polypeptide. For example, the provided polypeptide can be a large terminase, endolysin, portal protein, capsid protein, tail sheath protein, baseplate protein, tail protein, helicase, replicase, primase, exonuclease, dUTPase, ribonucleoside-diphosphate reductase alpha subunit, ribonucleoside-diphosphate reductase beta subunit, ribose-phosphage pyrophosphokinase, nicotinamid phosphoribosyl transferase, DNA polymerase, recombinase, sigma factor, alanyl- tRNA synthetase, ATPase, ligase, pyrphosphathydrolase, or repressor.

37. Also disclosed are the cells or mediums expressing the disclosed recombinant proteins. Thus, provided is a recombinant polypeptide produced by a cell, as well as the cell, comprising a nucleic acid having the sequence set forth in nucleotides 1,746 to 3,300 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 8,189 to 9,832 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 11,790 to 13,196 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 17,130 to 18,818 of SEQ ID NO: 1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 29,606 to 30,316 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 30,330 to 31,376 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 41,690 to 43,435 of SEQ ID NO: 1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 43,450 to 45,090 of SEQ ID NO: 1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 45,108 to 46,571 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 47,733 to 48,113 of SEQ ID NO: 1 , or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 50,227 to 51,288 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 51,335 to 51,982 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 53,678 to 55,027 of SEQ ID NO: 1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 55,238 to 56,269 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 56,445 to 57,476 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 60,267 to 61,181 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 61,192 to 62,985 of SEQ E) NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 66,415 to 66,729 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 66,812 to 67,648 of SEQ TD NO: 1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 67,983 to 70,091 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 72,029 to 73,273 of SEQ ID NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 73,712 to 74,350 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 80,225 to 80,707 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 83,439 to 84,455 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 117,468 to 116,527 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 120,547 to 120,344 of SEQ TD NO:1, or a fragment or conservative variant thereof. Also provided is a recombinant polypeptide produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 120,209 to 120,985 of SEQ E) NO:1, or a fragment or conservative variant thereof. 38. Also provided is a recombinant polypeptide encoded by a nucleic acid or nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having any of the sequences set forth in Table 3. For example, provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 1,746 to 3,300 of SEQ 3D NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ ID NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 8,189 to 9,832 of SEQ TD NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 11,790 to 13,196 of SEQ TD NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 17,130 to 18,818 of SEQ ID NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 29,606 to 30,316 of SEQ TD NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 30,330 to 31 ,376 of SEQ ID NO: 1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 41,690 to 43,435 of SEQ TD NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 43,450 to 45,090 of SEQ ID NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 45,108 to 46,571 of SEQ TD NO: 1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 47,733 to 48,113 of SEQ ID NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 50,227 to 51,288 of SEQ ID NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to anucleic acid having the sequence set forth in nucleotides 51,335 to 51,982 of SEQ ID NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 53,678 to 55,027 of SEQ ID NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 55,238 to 56,269 of SEQ ID NO : 1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 56,445 to 57,476 of SEQ ID NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 60,267 to 61 , 181 of SEQ ID NO : 1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 61,192 to 62,985 of SEQ ID NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 66,415 to 66,729 of SEQ ID NO: 1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 66,812 to 67,648 of SEQ ID NO: 1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 7,983 to 70,091 of SEQ ID NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to anucleic acid having the sequence set forth in nucleotides 72,029 to 73,273 of SEQ ID NO: 1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 73,712 to 74,350 of SEQ ID NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth

, _]3 in nucleotides 80,225 to 80,707 of SEQ ID NO: 1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 83,439 to 84,455 of SEQ ID NO: 1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 117,468 to 116,527 of SEQ ID NO: 1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 120,547 to 120,344 of SEQ ID NO: 1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions, or other conditions disclosed herein, to a nucleic acid having the sequence set forth in nucleotides 120,209 to 120,985 of SEQ ID NO: 1.

39. Also provided is a recombinant polypeptide or a nucleic acid having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid or nucleic acid disclosed herein, such as in Table 3. For example, provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 1,746 to 3,300 of SEQ ID NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions to a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ ID NO: 1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 8,189 to 9,832 of SEQ ID NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 11 ,790 to 13,196 of SEQ ID NO: 1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 17,130 to 18,818 of SEQ ID NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 29,606 to 30,316 of SEQ TD NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 30,330 to 31,376 of SEQ ID NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 41,690 to 43,435 of SEQ ID NO: 1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 43,450 to 45,090 of SEQ ID NO: 1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 45,108 to 46,571 of SEQ ID NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 47,733 to 48,113 of SEQ ID NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 50,227 to 51,288 of SEQ ID NO :1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 51 ,335 to 51 ,982 of SEQ E) NO : 1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 53,678 to 55,027 of SEQ E) NO :1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 55,238 to 56,269 of SEQ ID NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 56,445 to 57,476 of SEQ ID NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 60,267 to 61,181 of SEQ TD NO: 1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 61,192 to 62,985 of SEQ E⁾ NO: 1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 66,415 to 66,729 of SEQ ID NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 66,812 to 67,648 of SEQ E) NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 7,983 to 70,091 of SEQ E) NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 72,029 to 73,273 of SEQ E) NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 73,712 to 74,350 of SEQ TD NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 80,225 to 80,707 of SEQ E) NO: 1. Also provided is a recombinant polypeptide having 70%, 7,5%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 83,439 to 84,455 of SEQ E) NO:1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 117,468 to 116,527 of SEQ E) NO: 1. Also provided is a recombinant polypeptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 120,547 to 120,344 of SEQ E) NO:1. Also provided is a recombinant polypeptide encoded by a nucleic acid that can hybridize under stringent conditions to a nucleic acid having the sequence set forth in nucleotides 120,209 to 120,985 of SEQ E) NO:1.

(a) Endolysins

40. Phage-encoded lysins or endolysins are highly active enzymes which hydrolyze bacterial cell walls. These phage encoded cell wall lytic enzymes are synthesized late during virus multiplication and mediate the release of progeny virions. Endolysins can be used to lyse Listeria cells to recover nucleic acids or cellular protein for detection or differentiation. The endolysin can also be used to treat animals (including humans) which are infected with Listeria and to treat surfaces which may be contaminated with Listeria. Endolysin (ply) genes from Listeria phages (Al 18, A500 and A511) have been cloned and purified (Loessner, et al., Applied and Environmental Microbiology, August 1996, p. 3057-3060).

41. Provided herein is a recombinant PlOO endolysin polypeptide (Ply 100). The polypeptide can be isolated and purified from the herein disclosed recombinant PlOO phages. For example, PlylOO can be isolated by techniques known in the art including but not limited to lysis, chromatography, filtration, and centrifugation. The endolysin can be isolated from Listeria which have been incubated with Pl 00.

42. As provided herein, PlylOO can also be recombinantly expressed in a host bacteria (e.g. E. coli, L. lactis, S. aureus, and B. cereus) or other cell or other in vitro system as understood. Thus, the provided endolysin polypeptide can be produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ ID NO:1, or a fragment or conservative variant thereof. The provided endolysin polypeptide can also be produced by a cell comprising a nucleic acid that can hybridize under stringent conditions to a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ ID NO:1, or a fragment or conservative variant thereof.

43. The endolysin can be isolated from the host bacteria or the host bacteria containing the endolysin can be directly applied or administered without isolation of the endolysin. For example, a host bacteria which produces the endolysin can be administered to an animal or applied to a surface where the endolysin would be secreted into the food, onto the surface or into the animal's gut. The endolysin can then attack Listeria cells present in this environment.

44. One unit of endolysin activity is defined as the amount of endolysin necessary to decrease the optical density at 600 nm by 0.01/min, at pH 8.0 and 25⁰C. in a volume of 1 ml, when heat-killed, washed cells of Listeria monocytogenes are used as a substrate.

(b) PlOO Capsids 45. PlOO particles can be used for the delivery of a composition to ^Listeria cell. Thus, provided herein is a PlOO capsid polypeptide and phage particles produced therefrom. The PlOO capsid polypeptides can be recombinantly expressed in a host bacteria (e.g. E. coli, L. lactis, S. aureus, and B. cereus). Thus, the provided capsid polypeptide can be produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 11,790 to 13,196 of SEQ TD NO: 1, or a fragment or conservative variant thereof.

(4) Nucleic acids

46. Provided herein is an isolated nucleic acid encoding a bacteriophage Pl 00-specific polypeptide. Thus, provided is a nucleic acid comprising a fragment or conservative variant of the sequence set forth in SEQ ID NO: 1. Thus, provided is a nucleic acid comprising a sequence set forth in SEQID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. The provided nucleic acid can also comprise a nucleic acid that can hybridize under stringent conditions to a sequence set forth in SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO: 1. By "a sequence set forth in SEQ ID NO: 1 " is meant any portion or unique fragment of the PlOO genome such that the nucleic acid is at least 10, 20, 100 nucleotides in length.

47. For example, provided is a nucleic acid having the sequence set forth in nucleotides 1,746 to 3,300 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ ID NO: 1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 8,189 to 9,832 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1, . Also provided is a nucleic acid having the sequence set forth in nucleotides 11,790 to 13,196 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO: 1. Also provided is a nucleic acid having the sequence set forth in nucleotides 17,130 to 18,818 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 29,606 to 30,316 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 30,330 to 31,376 of SEQ ID NO:1, wherein thenucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 41,690 to 43,435 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 43,450 to 45,090 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 45,108 to 46,571 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 47,733 to 48,113 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 50,227 to 51,288 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO: 1. Also provided is a nucleic acid having the sequence set forth in nucleotides

51,335 to 51,982 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ TD NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 53,678 to 55,027 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 55,238 to 56,269 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 56,445 to 57,476 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 60,267 to 61,181 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 61,192 to 62,985 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ TD NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 66,415 to 66,729 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 66,812 to 67,648 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 7,983 to 70,091 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 72,029 to 73,273 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ ID NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 73,712 to 74,350 of SEQ E) NO:1, wherein the nucleic acid does not consist of SEQ TD NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 80,225 to 80,707 of SEQ TD NO:1, wherein the nucleic acid does not consist of SEQ E) NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 83,439 to 84,455 of SEQ TD NO: 1 , wherein the nucleic acid does not consist of SEQ TD NO: 1. Also provided is a nucleic acid having the sequence set forth in nucleotides 117,468 to 116,527 of SEQ TD NO: 1, wherein the nucleic acid does not consist of SEQ TD NO: 1. Also provided is a nucleic acid having the sequence set forth in nucleotides 120,547 to 120,344 of SEQ ID NO:1, wherein the nucleic acid does not consist of SEQ TD NO:1. Also provided is a nucleic acid having the sequence set forth in nucleotides 120,209 to 120,985 of SEQ TD NO: 1, wherein the nucleic acid does not consist of SEQ TD NO:1.

48. Also provided is a nucleic acid vector comprising a nucleic acid encoding a bacteriophage PlOO-specific polypeptide, wherein the nucleic acid does not consist of SEQ TD NO:1. Also provided is a cell comprising a nucleic acid encoding a bacteriophage P100-specific polypeptide, wherein the nucleic acid does not consist of SEQ TD NO: 1.

49. It is also understood that the disclosed compositions include combinations of the compositions. For example, disclosed are combinations of the specific PlOO proteins disclosed herein along with any A511, P35, A118, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971A, 02971C, 907515, 12981, 11711 A, 00241, 13441, A500, A640, B021, PSA, B653, 90666, 90861, 910716, 93253, 52, 340, 312, 108, 10, 2425A, 2425, or 3551 phage or A511, P35, Al 18, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971A, 02971C, 907515, 12981, 11711 A, 00241, 13441, A500, A640, B021, PSA, B653, 90666, 90861, 910716, 93253, 52, 340, 312, 108, 10, 2425A, 2425, or 3551 phage protein. 3. Use for controlling Listeria

50. Effective concentrations/ dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The concentrations/ dosages ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder are effected.

51. For example, the herein provided compositions can be used for controlling Listeria contamination in a food product, on food processing equipment or on food storage containers. The provided compositions can be applied on or into food products, and/or into various physical sites within the food processing plants, by a number of means including, but not limited to, admixing the compositions into the food products, spraying the compositions onto the foodstuffs, spraying the compositions onto the plant equipment, and/or directly applying the compositions to the plant equipment. Said applications significantly reduce the numbers of Listeria monocytogenes bacteria that would otherwise be present. The concentration of PlOO phage for administration on or into food products, and/or into various physical sites within the food processing plants is contemplated to be in the range of about 10³ to 10 ° pfu (plaque forming units) per mL, including about 10⁶ to 10⁹ pfu/ mL. The PlOO phage can be administered to the surface of the food products and/or physical sites within the food processing plants with a final surface concentration of about 10⁵ to 10⁸pfα/crn², including 10⁶ to 10⁷pfu/cm². The concentration of PlylOO for administration on or into food products, and/or into various physical sites within the food processing plants is contemplated to be in the range of about 2-2000 ng/ ml, such as about 20-200 ng/ ml. The compositions are administered until successful reduction or elimination of the Listeria monocytogenes is achieved or until the amount of Listeria monocytogenes is substantially reduced. 52. The herein provided compositions can also be used to treat animals, including humans, infected with Listeria monocytogenes. When administered to a subject, the dosages should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosages will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

53. Any suitable route of administration can be used to administer the phage including but not limited to: oral, aerosol or other device for delivery to the lungs, nasal spray, intravenous, intramuscular, intraperitoneal, intrathecal, vaginal, rectal, topical, lumbar puncture, intrathecal, and direct application to the brain and/or meninges. Excipients which can be used as a vehicle for the delivery of the phage, endolysin and/or host bacteria containing the endolysin will be apparent to those skilled in the art. For example, the compositions could be in lyophilized form and be dissolved just prior to administration by IV injection. The dosage of administration for PlOO phage is contemplated to be in the range of about 10³ to about 10¹³ pfu/per kg/per day, such as about 10¹² pfu/per kg/per day. The dosage of administration for the PlylOO is contemplated to be in the range of about 2-2000 ng/per g/per day, such as about 20-200 ng/per g/per day. The compositions are administered until successful elimination of the Listeria monocytogenes is achieved or until the amount of Listeria monocytogenes is substantially reduced. 54. Also provided is a method for detecting the presence of Listeria monocytogenes, comprising obtaining a sample suspected to contain Listeria monocytogenes, incubating said sample with recombinant PlOO phage or recombinant PlylOO, and detecting any change in said sample caused by PlOO, as an indication of the presence of Listeria monocytogenes. As an example, said change in said sample is due to lysis by PlOO or a detectable label or signal. As another example, the method can comprise recombinantly inserting a gene construct into the genome of PlOO before incubation with said sample, wherein expression of said gene construct results in a detectable signal in the presence of Listeria monocytogenes. The gene construct can, fore example, encode a bio-luminescent protein, such as one selected from the group consisting of luciferase and a fluorescent protein. Luciferase can be from bacteria or insects. The fluorescent protein can be green fluorescent protein or a variant thereof. The method can comprise immobilizing said Listeria monocytogenes on a solid support, such as a test strip, and detecting any change on said solid support. As an example, Listeria monocytogenes can be immobilized using ∑tnti-Listejia antibodies. Samples that can be tested using the provided method includes those obtained from patients suspected of being infected with Listeria monocytogenes or those obtained from a food product, food processing equipment or food storage¹ containers.

55. Also provided is the use of recombinant phage PlOO, or a nucleic acid or polypeptide derived therefrom, for the production of a pharmaceutical composition for the treatment of an animal infected with Listeria monocytogenes. Also provided is the use of a nucleic acid encoding a PlOO polypeptide for the production of a pharmaceutical composition for the treatment of an animal infected with Listeria monocytogenes. Also provided is the use of a recombinant PlOO polypeptide for the production of a pharmaceutical composition for the treatment of an animal infected with Listeria monocytogenes. 4. Combinations

56. Also provided is the use of the herein provided compositions in combination with other anti-Listerial agents known in the art. Examples of such anti-Listerial agents, which are preferentially combined with the provided compositions, include but are not limited to endolysins, surface disinfectants, antimicrobial agents, enzymes, surfactants, and other phage.

57. The provided compositions can be combined with listeriolysins which are enzymes which have been shown to selectively control Listeria in food and the environment. Thus, the herein provided compositions can be administered in combination with, for example, Plyl 18, Ply 500 orPly511 endolysins. 58. The provided compositions can be combined with known surface disinfectants such as (i) preservatives of various kinds, such as but not limited to benzoic acid and BHT; and (ii) various disinfectants with which the phages are compatible, such as but not limited to quaternary ammonium compounds.

59. The provided compositions can be combined with known antimicrobial agents (including antibiotics and chemotherapeutic agents) including but not limited to vancomycin, nisin, danofloxacin and neomycin.

60. The provided compositions can be combined with enzymes to aid in breaking up biofilms (e.g. biofilms found in food processing equipment). Such enzymes are known in the art and include but are not limited to polysaccharide depolymerase enzymes, and protease. 61. The provided compositions can be combined with known surfactants when used to treat food processing equipment. The surfactant helps to wet the surface so that the phage are properly distributed over the various surfaces, and to solubilize and remove dirt so that the Listeria are accessible to the phage. Suitable surfactants include but are not limited to Tween 80, 20 and 81 and Dobanols.

62. The provided compositions can be combined with phage specific for Listeria monocytogenes and/or phage specific for other bacteria known to contaminate food processing equipment and food products. Such bacteria include but are not limited to E. coli, and bacterial species from the genera Salmonella, Bacillus, Staphylococcus, Streptococcus, Clostridium, and Pseudomonas.

5. Administration

63. The herein provided phage can be applied in a liquid or a powdered form to food products and food processing equipment. If applied as a liquid, the phage are applied at a concentration of about 10³ to 10¹⁰ pfu/ mL, including about 10⁶ to 10⁹ pfu/ mL. If applied as a dry powder the phage are applied at a concentration of about 10³ to 10¹⁰ pfu/ mg, including about 10⁶ to 10⁹ pfu/ mg. The phage can be suspended in a suitable carrier prior to application or drying, including but not limited to protein solutions containing BSA, casein, whey protein, soy bean protein, etc and sugar based carriers containing sugars such as mannitol. The phage can be lyophilized or cryopreserved by vitrification and either suspended in a solution prior to application or applied directly as a dry powder.

64. Suitable amounts of phage for use in the present invention can be obtained by techniques known in the art, including but not limited to a batch technique where a culture of host bacteria is grown and then seeded with phage. After an amount of time suitable to allow maximal phage propagation and bacterial lysis, the culture is further lysed by physical or chemical means and the lysate spun down. The phage containing supernatant can be used as is or further purified using techniques such as ultrafiltration, chromatography and centrifugation.

65. The endolysin can be applied in a liquid or a powdered form to food products and food processing equipment. The endolysin is applied in a concentration between 2 to 2000 ng endolysin per ml or per gram of carrier, and preferably between 20 to 200 ng endolysin per ml or per gram of carrier.

66. Food products to which the herein provided compositions can be administered include, but are not limited to dairy products, meat products, fish products, unpasteurized food products, and salads. As used in the present application, the term "dairy product" is intended to include any food product made using milk or milk products, including but not limited to milk, yogurt, ice cream, cheese, butter, and cream. As used in the present application, the term "meat product" is intended to include any food product which contains animal tissue, including but not limited to beef, pork, and poultry. The term "ready to eat meat product" in intended to include any meat product which does not require cooking prior to consumption, including but not limited to pates, hot dogs, bologna, salami, and cold cuts. As used in the present application, the term "fish product" is intended to include any food product which contains tissue from an aquatic animal including but not limited to lobster, crab, fresh water and saltwater fish and other seafoods. As used in the present application, the term "unpasteurized food product" is intended to include any food product which is prepared using unpasteurized primary ingredients and which does not undergo a final (listeriocidal) heat treatment. As used in the present invention, the term "salad" is intended to include any food product which contains mixtures of vegetables or fruits, and particularly such mixtures as are presented for consumers to choose from in a display commonly referred to as a "salad bar". 6. Pharmaceutical carriers

67. The herein provided compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

68. The compositions maybe administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

69. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.

70. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog.

Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). hi general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either Tecycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses ana toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). 71. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

72. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

73. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

74. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

75. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

76. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

77. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders maybe desirable..

78. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

7. Use for identification of Listeria Phages

79. Also provided herein is a method of identifying, cloning, and isolating Listeria phages using the herein provided nucleic acid sequences. A detailed description of the protocol for homology-based PCR is found in Pytela R, et al. (Polymerase chain reaction cloning with degenerate primers: homology-based identification of adhesion molecules. Methods Enzymol 245:420, 1994). Briefly, primers are designed based upon conserved sequences found in multiple members of a protein family. Since sequence conservation is rarely perfect (especially at the nucleotide level), primers are said to be "degenerate," meaning that more than one nucleotide is permitted at certain positions. Primer mixtures are used to amplify DNA samples (cDNAs or genomic DNAs) thought to contain sequences encoding unknown members of the protein family. There are two major roles for this method: 1) clone novel members of known families and 2) clone homologues in different species.

80. Homology PCR can be used when target novel sequence must have at least two regions with sequences very similar to known proteins. (One primer is modeled after each of these two regions.) These regions may be small, perhaps as short as 12 nucleotides (4 amino acids). However, more reliable results are obtained with somewhat larger areas (18-24 nt, 6-8 aa). Ideally, regions should be separated by 150-500 nt. Avoid excessive codon degeneracy. Find an area where there is as much identity as possible between sequences. Avoid regions containing many leucine, serine, and arginine residues, since each can be encoded by six different codons. The ideal amino acids are tryptophan and methionine (one codon each). In addition, it may be useful to use deoxyinosine ("I" in the primer sequence) instead of mixing all four nucleotides at ^• completely degenerate positions. This can substantially reduce overall primer degeneracy and should reduce false priming. Make 3' ends of primers match well. If the 3' ends do not base pair with the target sequence, the polymerase will not extend from the primer. Adding restriction sites to 5' ends makes cloning easier. A few additional bases 5' to the site often improve digestion efficiency (see the appendix to the New England Biolabs Catalog for information about specific restriction enzymes). Other methods can be used to clone products made using primers without restriction sites (e.g., blunt-end or TA cloning).

81. Thus, the herein provided nucleic acids can be used to design degenerate primers for the cloning and sequencing of A511, P35, Al 18, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971A, 02971C, 907515, 12981, 11711 A, 00241, 13441, A500, A640, B021, PSA, B653, 90666, 90861, 910716, 93253, 52, 340, 312, 108, 10, 2425A, 2425, or 3551 phage. These degenerate primers can be used to clone the homologous genes for the indicated phage or other phage. 8. Sequence similarities

82. It is understood that as discussed herein the use of the terms homology and identity mean the same thing as similarity. Thus, for example, if the use of the word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences. Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not.

83. In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences. This identity of particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of genes and proteins herein disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.-

84. Another.way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

85. The same types, of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein. 86. For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above. For example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods. As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods. As yet another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages). 9. Selective Hybridization

87. The term hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene. Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide. The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.

88. Parameters for selective hybridization between two nucleic acid molecules are well known to those of skill in the art. For example, in some embodiments selective hybridization conditions can be defined as stringent hybridization conditions. For example, stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps. For example, the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5⁰C to 20°C below the Tm. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA- RNA hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is herein incorporated by reference for material at least related to hybridization of nucleic acids). A preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C. Stringency of hybridization and washing, if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for. Likewise, stringency of hybridization and washing, if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art. 89. Another way to define selective hybridization is by looking at the amount

(percentage) of one of the nucleic acids bound to the other nucleic acid. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid. Typically, the non-limiting primer is in for example, 10 or 100 or 1000 fold excess. This type of assay can he performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their k_<j, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their k_d. 90. Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended. Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.

91. Just as with homology, it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules. It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient. For example if 80% hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein. 92. It is understood that those of skill in the art understand that if a composition or method meets any one of these criteria for determining hybridization either collectively or singly it is a composition or method that is disclosed herein.

10. Nucleic acids 93. There are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example, Plyl 00, as well as any other proteins disclosed herein, as well as various functional nucleic acids. The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, that the expressed rrJRNA will typically be made up of A, C, G, and U. Likewise, it is understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantagous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment. a) Nucleotides and related molecules

94. A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. An non-limiting example of a nucleotide would be 3'-AMP (3'- adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).

95. A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties.

96. Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid. 97. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86, 6553-6556),

98. A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. . The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.

99. A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides. b) Sequences

100. There are a variety of sequences related to, for example, bacteriophages as well as any other protein disclosed herein that are disclosed on Genbank, and these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein. 101. A variety of sequences are provided herein and these and others can be found in

Genbank, at www.pubmed. gov. Those of skill in the art understand how to resolve sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences. Primers and/or probes can be designed for any sequence given the information disclosed herein and known in the art. c) Primers and probes

102. Disclosed are compositions including primers and probes, which are capable of interacting with the genes disclosed herein, hi certain embodiments the primers are used to support DNA amplification reactions. Typically the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred. In certain embodiments the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. Typically the disclosed primers hybridize with the nucleic acid or region of the nucleic acid or they hybridize with the complement of the nucleic acid or complement of a region of the nucleic acid. d) Functional Nucleic Acids

103. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting. For example, functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, and external guide sequences. The functional nucleic acid molecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.

104. Functional nucleic acid molecules can interact with any macromolecule, such as DNA₅ RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids can interact with the mRNA of PlOO or the genomic DNA of PlOO or they can interact with a polypeptide PlOO. Often functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.

105. Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (k_d)less than or equal to 10^"6, 10^"8, lO^"10, or 10^"12. A representative sample of methods and techniques which aid in the design and use of antisense molecules can be found in the following non-limiting list of United States patents: 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437.

106. Aptamers are molecules that interact with a target molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP (United States patent 5,631,146) and theophiline (United States patent 5,580,737), as well as large molecules, such as reverse transcriptase (United States patent 5,786,462) and thrombin (United States patent 5,543,293). Aptamers can bind very tightly with k_dS from the target molecule of less than 10^"12 M. It is preferred that the aptamers bind the target molecule with a k_d less than 10^"6, 10^"8, 10^"10, or 10^"12. Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule (United States patent 5,543,293). It is preferred that the aptamer have a k_d with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the k_d with a background binding molecule. It is preferred when doing the comparison for a polypeptide for example, that the background molecule be a different polypeptide. For example, when determining the specificity of P 100 aptamers, the background composition could be A511. Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,476,766, 5,503,978, 5,631,146, 5,731,424 , 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660 , 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698. 107. Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, (for example, but not limited to the following United States patents: 5,334,711, 5,436,330, 5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but not limited to the following United States patents: 5,631,115, 5,646,031, 5,683,902, 5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), and tetrahymena ribozymes (for example, but not limited to the following United States patents: 5,595,873 and 5,652,107). There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (for example, but not limited to the following United States patents: 5,580,967, 5,688,670, 5,807,718, and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in the following non-limiting list of United States patents: 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855, 5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756.

108. Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a k_d less than 10^" , 10^"8, 10^'10, or 10^"12. Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566, and 5,962,426.

109. External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNase P, which cleaves the I target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse

P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and Altaian, Science 5 238:407-409 (1990)).

110. Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukarotic cells. (Yuan et al., Proc. Natl. Acad. Sci. USA 89:8006- 8010 (1992); WO 93/22434 by Yale; WO 95/24489 by Yale; Yuan and Altman, EMBO J 14:159-168 (1995), and Carrara et al., Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)).

10 Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules be found in the following non-limiting list of United States patents: 5,168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162. 11. Nucleic Acid Delivery

111. In the methods described above which include the administration and uptake of 1.5 exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the disclosed nucleic acids can be in the form of naked DNA or RNA, or the nucleic acids can be in a vector for delivering the nucleic acids to the cells, whereby the antibody-encoding DNA fragment is under the transcriptional regulation of a promoter, as would be well understood by one of ordinary skill in the art. The vector can be a commercially available preparation, such as an 0 adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other 5 liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

112. As one example, vector delivery can be via a viral system, such as a retroviral 0 vector system which can package a recombinant retroviral genome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486, 1988; Miller et al., MoI. Cell. Biol. 6:2895, 1986). The recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding a broadly neutralizing antibody (or active fragment thereof). The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use ot retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994), adeno-associated viral (AAV) vectors (Goodman et al., Blood 84:1492-1500, 1994), lentiviral vectors (Naidini et al., Science 272:263-267, 1996), pseudotyped retroviral vectors (Agrawal et al., Exper. Hematol. 24:738-747, 1996). Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al., Blood 87:472-478, 1996). This disclosed compositions and methods can be used in conjunction with any of these or other commonly used gene transfer methods. 113. As one example, if the antibody-encoding nucleic acid is delivered to the cells of a subject in an adenovirus vector, the dosage for administration of adenovirus to humans can range from about 10⁷ to 10⁹ plaque forming units (pfu) per injection but can be as high as 10¹² pfu per injection (Crystal, Hum. Gene Ther. 8:985-1001, 1997; Alvarez and Curiel, Hum. Gene Ther. 8:597-613, 1997). A subject can receive a single injection, or, if additional injections are necessary, they can be repeated at six month intervals (or other appropriate time intervals, as determined by the skilled practitioner) for an indefinite period and/or until the efficacy of the treatment has been established.

114. Parenteral administration of the nucleic acid or vector, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. For additional discussion of suitable formulations and various routes of administration of therapeutic compounds, see, e.g., Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.

12. Expression systems

115. The nucleic acids that are delivered to cells typically contain expression controlling systems. For example, the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of KNA polymerase and transcription factors, and may contain upstream elements and response elements. a) Viral Promoters and Enhancers

116. Preferred promoters controlling transcription from vectors in mammalian host cells maybe obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as aHindin E restriction fragment (Greenway, PJ. et al., Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species also are useful herein.

117. Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M.L., et al., Mol. Cell Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., MoI. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

118. The promo tor and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways "to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs. 119. hi certain embodiments the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression of the region of the transcription unit to be transcribed, hi certain constructs the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR.

120. It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

121. Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contain a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct. b) Markers

122. The viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Preferred marker genes are the E. CoIi lacZ gene, which encodes β-galactosidase, and green fluorescent protein. 123. In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydro folate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are: CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.

124. The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1 : 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., MoI. Cell. Biol. 5: 410-413 (1985)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin. 13. Peptides a) Protein variants

125. As discussed herein there are numerous variants of the PlOO proteins that are herein contemplated. Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known hcquciiuc aie wen Known, for example Ml 3 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 2 and 3 and are referred to as conservative substitutions.

TABLE 2: Amino Acid Abbreviations

Amino Acid Abbreviations

Alanine Ala A allosoleucine AIIe

Arginine Arg R asparagine Asn N aspartic acid Asp D

Cysteine Cys C glutamic acid GIu E

Glutamine GIn Q

Glycine GIy G

Histidine His H

Isolelucine lie I

Leucine Leu L

Lysine Lys K phenylalanine Phe F proline Pro P pyroglutamic acid pGlu

Serine Ser S

Threonine Thr T

Tyrosine Tyr Y

Tryptophan Trp W

Valine VaI V

TABLE 3: Amino Acid Substitutions

Original Exemplary Conservative Substitutions -

Residue others are known in the art.

Ala Ser

Arg Lys; GIn

Asn GIn; His

Asp GIu

Cys Ser

GIn Asn, Lys

GIu Asp

GIy Pro TABLE 3: Amino Acid Substitutions

Original Exemplary Conservative Substitutions - Residue others are known in the art.

His Asn;Gm

He Leu; VaI

Leu He; VaI

Lys Arg; Gln

Met Leu; He

Phe Met; Leu; Tyr

Ser Thr

Thx Ser

Trp Tyr

Tyr Trp; Phe

VaI He; Leu

126. Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 3, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and/or glycosylation.

127. For example, the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.

128. Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also may be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.

129. Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post- translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl.

130. It is understood that one way to define the variants and derivatives of the disclosed proteins herein is through defining the variants and derivatives in terms of homology/identity to specific known sequences. For example, SEQ JD NO:1 sets forth the PlOO genome encoding PlOO proteins. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95% homology to the encoded proteins. Those of skill in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level. 131. Another way of calculating homology can be performed by published algorithms.

Optimal alignment of sequences for comparison maybe conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

132. The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. 133. It is understood that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations.

134. As this specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence.

135. It is understood that there are numerous amino acid and peptide analogs which can be incorporated into the disclosed compositions. For example, there are numerous D amino acids or amino acids which have a different functional substituent then the amino acids shown in Table 2 and Table 3. The opposite stereo isomers of naturally occurring peptides are. disclosed, as well as the stereo isomers of peptide analogs. These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way (Thorson et al., Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion in Biotechnology, 3:348-354 (1992); Ibba, Biotechnology & Genetic Engineering Reviews 13:197-216 (1995), Cahill et al., TIBS, 14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba and Hennecke, Bio/technology, 12:678-682 (1994) all of which are herein incorporated by reference at least for material related to amino acid analogs). 136. Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH₂NH-, -CH₂S-, -CH₂-CH₂ --, -CH=CH- (cis and trans), -COCH₂ --, - CH(OH)CH₂-, and -CHH₂SO- (These and others can be found in Spatola, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., ϊnt J Pept Prot Res 14:177-185 (1979) (-CH₂NH-, CH₂CH₂-); Spatola et al. Life Sci 38:1243-1249 (1986) (-CH H₂-S); Harm J. Chem. Soc Perkin Trans. 1307-314 (1982) (--CH-CH-, cis and trans); Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (-COCH₂-); Jennings-White et al. Tetrahedron Lett 23:2533 (1982) (-COCH₂-); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (-CH(OH)CH₂-); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (-C(OH)CH₂-); and Hruby Life Sci 31:189-199 (1982) (-CH₂-S-); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is -CH₂NH-. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g-aminobutyric acid, and the like.

137. Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.

138. D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L- lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. (Rizo and Gierasch Ann. Rev. Biochem. 61 :387 (1992), incorporated herein by reference).

14. Antibodies (1) Antibodies Generally

139. Provided herein are antibodies specific for the PlOO polypeptides disclosed herein. The term "antibodies" is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term "antibodies" are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with a PlOO polypeptide. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods. 140. The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chaiή(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. ScL USA, 81:6851-6855 (1984)).

141. The disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro, e.g., using the HIV Env-CD4-co-receptor complexes described herein.

142. The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains- of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.

143. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.

144. The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, MJ. Curr. Opin. Biotechnol. 3:348-354, 1992).

145. As used herein, the term "antibody" or "antibodies" can also refer to a human antibody and/or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

15. Computer readable mediums

146. It is understood that the disclosed nucleic acids and proteins can be represented as a sequence consisting of the nucleotides of amino acids. There are a variety of ways to display these sequences, for example the nucleotide guanosine can be represented by G or g. Likewise the amino acid valine can be represented by VaI or V. Those of skill in the art understand how to display and express any nucleic acid or protein sequence in any of the variety of ways that exist, each of which is considered herein disclosed. Specifically contemplated herein is the display of these sequences on computer readable mediums, such as, commercially available floppy disks, tapes, chips, hard drives, compact disks, and video disks, or other computer readable mediums. Also disclosed are the binary code representations of the disclosed sequences. Those of skill in the art understand what computer readable mediums. Thus, computer readable mediums on which the nucleic acids or protein sequences are recorded, stored, or saved. 147. Disclosed are computer readable mediums comprising the sequences and information regarding the sequences set forth herein. Also disclosed are computer readable mediums comprising the sequences and information regarding the sequences set forth herein. 16. Kits 148. Disclosed Jtierem are kits that are drawn to reagents that can be used in practicing the methods disclosed herein. The kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice, of the, disclosed methods. For example, the kits could include recombinant phage, as well as the buffers and enzymes required to use the phage as intended. C. Methods of making the compositions

149. The compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted. 1. Nucleic acid synthesis

150. For example, the nucleic acids, such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B). Synthetic methods useful for making oligonucleotides are also described by Ikuta et al., Ann. Rev. Biochem. 53 :323-356 (1984), (phosphotriester and phosphite- triester methods), and Narang et al., Methods EnzymoL, 65:610-620 (1980), (phosphotriester method). Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et al., Bioconjug. Chem. 5:3-7 (1994).

2. Peptide synthesis 151. One method of producing the disclosed proteins, such as SEQ TD NO:23, is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the disclosed proteins, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. (Grant GA (1992) Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis. Springer-Verlag Inc., NY (which is herein incorporated by reference at least for material related to peptide synthesis). Alternatively, the peptide or polypeptide is independently synthesized in vivo as described herein. Once isolated, these independent peptides or polypeptides may be linked to form a peptide or fragment thereof via similar peptide condensation reactions. 152. For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)). The first step is the chemoselective reaction of an unprotected synthetic peptide—thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J.Biol. Chem., 269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).

153. Alternatively, unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry TV. .Academic Press, New York, pp. 257-267 (1992)).

3. Process claims for making the compositions 154. Disclosed are processes for making the compositions as well as making the intermediates leading to the compositions. For example, disclosed are nucleic acids in SEQ DD O: 1. There are a variety of methods that can be used for making these compositions, such as synthetic chemical methods and standard molecular biology methods. It is understood that the methods of making these and the other disclosed compositions are specifically disclosed.

155. Disclosed are nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid comprising the sequence set forth in SEQ ID NO:1 and a sequence controlling the expression of the nucleic acid.

156. Also disclosed are nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence having 80% identity to a sequence set forth in SEQ ID NO:1, and a sequence controlling the expression of the nucleic acid. 157. Disclosed are nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence that hybridizes under stringent hybridization conditions to a sequence set forth SEQ ID NO: land a sequence controlling the expression of the nucleic acid.

158. Disclosed are cells produced by the process of transforming the cell with any of the disclosed nucleic acids. Disclosed are cells produced by the process of transforming the cell with any of the non-naturally occurring disclosed nucleic acids.

159. Disclosed are any of the disclosed peptides produced by the process of expressing any of the disclosed nucleic acids. Disclosed are any of the non-naturally occurring disclosed peptides produced by the process of expressing any of the disclosed nucleic acids. Disclosed are any of the disclosed peptides produced by the process of expressing any of the nόn-naturally disclosed nucleic acids.

160. Disclosed are recombinant phage produced by the process of transfecting a cell with the modified PlOO nucleic acid molecules disclosed herein.

D. Methods of using the compositions 1. Methods of using the compositions as research tools

161. The disclosed compositions can be used in a variety of ways as research tools. For example, the disclosed compositions, such as SEQ ID NO:1 can be used to study the interactions between bacteriophage and Listeria, by for example acting as inhibitors of binding.

162. The disclosed compositions can also be used diagnostic tools related to diseases, such as listeriosis.

163. The disclosed compositions can be used in any known method of screening assays, related to chip/micro arrays. The compositions can also be used in any known way of using me computer reaαaoie emDodiments of the disclosed compositions, for example, to study relatedness or to perform molecular modeling analysis related to the disclosed compositions.

E. Examples

164. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely, exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ⁰C or is at ambient temperature, and pressure is at or near atmospheric.

1. Example 1 a) Materials and methods

(1) Preparation, sequencing and bioinformatic analyses of the PlOO genome

165. Phage PlOO was first isolated from a sewage effluent sample taken irom a dairy plant in southern Germany. Liquid samples were centrifuged, filter-sterilized, and tested for presence of Listeria phages by spotting small drops on preformed lawns of a selection of different Listeria indicator strains as previously described (Loessner and Busse, 1990). One particular phage which formed large, clear plaques on most tested strains was isolated, purified, and designated as PlOO. A stock lysate of PlOO, containing approximately 3xlO⁹ pfu/ml (plaque forming units), was then prepared using L. monocytogenes WSLC 1001 as a host, and stored at 4°C.

166. Propagation of PlOO was performed using either L. monocytogenes WSLC 1001 or the non-pathogenic host L. innocua WSLC 2096 or WSLC 2321. Purification of virions by polyethylene-glycol precipitation and CsCl density gradient centrifugation, and extraction of the DNA molecules was performed as previously described (Loessner et al., 1994; Loessner and Scherer, 1995). The sequence of the PlOO double-stranded DNA genome was determined using a "shotgun" cloning strategy (Loessner et al., 2000; Zimmer et al., 2003), with some modifications. In brief, approximately lOμg purified DNA was disrupted into fragments of 0.5-5 kb size by mechanical shearing. Fragments of the desired size (1-2 kb) were inserted into a standard plasmid vector (pBluescript or pGEM), and cloned into E. coli XLl -Blue. Nucleotide sequencing of a total of approximately 700 inserts was performed using dye-labeled

, ςo oligonucleotide primers complementary to vector sequences flanking the inserts (forward and reverse), in an automated nucleotide sequencer (ABI 3700; Applied Biosystems). After approximately 50 contigs of various lengths could be assembled, gaps were closed by using phage DNA directly as template in the sequencing reaction, employing oligonucleotide primers complementary to the ends of the contigs (primer walking). Regions of low redundancy or showing sequence ambiguities were checked again by primer walking, or by sequencing a PCR amplification product designed to encompass the region of interest.

167. After the complete sequence was assembled, genome coordinates were defined: nucleotide position 1 (left end of the genome) was set directly upstream of the putative terminase subunit genes. The information encoded by the PlOO genome was then analyzed using Vector NTI software (version 8; InforMax), and the annotated genome and all predicted open reading frames (ORF), gene products (gp) and secondary structures were again confirmed by visual inspection. The basic prerequisites for an ORF were the presence of one of the three potential start codons ATG, TTG or GTG, a suitable ribosomal binding site (Loessner and Scherer, 1995; Loessner et al., 2000), and a length of at least 40 encoded amino acids. Nucleotide and amino acid sequence alignment searches (BlastN, BlastX, and BlastP) using the ORFs and deduced gene products, respectively, were performed with Vector NTIs integrated BLAST engine which used the non-redundant database (available through the NCBI web sites http://www.ncbi.nlm.nih.gov/). Searches for specific protein domains and conserved motifs with known function were performed using the PFAM tools (available online at http://pfam.wustl.edu/hmmsearch.shtml). Transmembrane domains were predicted by using the hidden Markov model (TMHMM) (available at http://www.cbs.dtu.dk/services/TMHMM/). Helix-turnhelix-Scans (HTH) were performed using SeqWeb Version 2.1.0 (GCG package) (accessed via the biocomputing services of the University of Zurich online at http://www.bio.unizh.ch/bioc/). Potential tRNA genes were identified using the bioinformatics tool provided by http://www.genetics.wustl.edu/eddy/tRNAscan-SE (Lowe and Eddy, 1997). Loops and hairpins were identified using HΣBIO software (Hitachi) and VectorNTI, and a preliminary graphical genetic map of PlOO was constructed using VectorNTI.

168. To screen all 174 gene products predicted to be encoded by the PlOO genome (Table 4) for possible similarities to currently known protein food allergens, another in silico analysis was performed based on local alignments to the amino acid sequences of the proteins contained in the Food Allergy Research and Resource Program (FARRP) allergen database available at http://www.allergenonline.com. Table 4. features ol t mcteπopnage rl 00 ORFs, gene products, homologies, and functional assignments.

ORF Start Stop GP GP Homologies other Function

(MW^a) (IP^a) phages^b gpi ¹ 52 438 ^■ 14.7 ^■ 8.82 — gp2 422 694 10.5 10.05 orfl l8 (LP65) gp3 700 1,116 15.3 4.36 orf34 (phage K) gp4 1,116 . 1,397 10.6 9.7 or135 (phage K);

1102phil-3; orfl l5

(LP65) gp5 1,747 3,300 59.1 5.83 orf35 (phage K); Large terminase

1102phil-3; orfl l5

(LP65); gp6 3,369 4,208 31.5 5.29 orf36 (phage K); gp7 4,213 4,410 7.7 5.32 — gp8 4,400 5,038 24.4 4.78 orfl (A511) (100%); orf36 (phage K) gp9 5,028 5,408 14.4 6.94 orf2 (A511) gplO 5,472 6,497 36.4 9.81 ply (ASH); and Endolysin (amidase) endolysins from other phages gpii 6,670 7,398 26.1 8.99 gpl2 7,500 7,820 12.2 9.82 gpl3 7,822 8,172 13.6 5.47 orf40 (phage K) gpl4 8,189 9,832 61.1 6.39 orf41 (phage K); Putative portal protein orfl 12 (LP65); other phages gpl5 9,931 10,725 29.7 5.1 orfl (A511), orf42

(phage K), orfl 11

(LP65) gplό 10,718 11,620 33.8 4.47 orf2 (A511); orf43

(phage K), gpl7 11,790 13,196 51.5 5.27 cps (A511 ); cps Maj or capsid protein

(Twort), cps (phage

K); orfl 09 (LP65) gpl8 13,278 13,613 12.9 8.95 — gpl9 13,620 14,501 33.2 4.99 orf3 (A511); orf45

(phage K); gp20 14,519 15,337 31.2 6.51 orf4 (A511); orf46

(phage K); orfl 07

(LP65) gp21 15,337 15,954 23.9 10.28 orf5 (A511); orf47

(phage K); orfl 06

(LP65) gp22 15,967 16,806 31.5 4.68 orf6 (A511); orf48

(phage K); orfl 05

(LP65) gp23 16,806 17,126 12.3 8.69 orf7 (A511) gp24 17,130 18,818 61.3 4.85 Tsh (A511 ), orf49 Tail sheath protein

(phage K); orfl 03

(LP65); Twort gp25 18,937 19,308 13.7 5.91 orf8 (A511); orf50

(phage K); orfl 02

(LP65) 1 able 4. features oi i Dacteπopnagc rlOO ORPs, gene products, homologies, and functional assignments.

ORF Start Stop GP GP Homologies other Function

(MW ^a) (IP^a) phages^b gp26 19,459 19,902 17.3 4.86 orf9 (A511); orf52

(phage K); orf 100

(LP65) gp27 19,970 20,557 23.1 4.14 orf54 (phage K); gp28 20,619 24,344 131 9.06 orf55 (phage K); orf

98 (LP65) gp29 24,393 26,780 88.4 5.15 orf56/57 (phage K); orf 134 (LP65) gp30 26,798 28,330 56.8 4.8 orf58 (phage K); orf97

(LP65) gp31 28,368 29,081 25.7 5.21 orf59 (phage K); orf 129/130 (LP65) gp32 29,086 29,619 20.2 5.07 orf60 phage K; orfl31

(LP65) gp33 29,606 30,316 26.3 4.74 orfδl (phage K); Putative baseplate orf 132 (LP65) protein gp34 30,330 31,376 39.2 5.02 orf62 (phage K); orf95 Tail protein

(LP65) gp35 31,412 35,341 146 4.84 orf63 (phage K); orf94

(LP65) gp36 35,458 35,979 19.1 5.91 orf64 (phage K); orf91

(LP65) gp37 35,996 39,451 128.2 5 orf65 (phage K); orf90

(LP65); other phages gp38 39,497 39,718 8.6 5.24 — gp39 39,922 41,013 39.2 7.01 gp20 (Al 18), (PBSX) gp40 41,045 41,455 15.2 4.5 — gp41 41,452 41,589 5.3 5.11 gpl7 (PSA) gp42 41,690 43,435 66.4 6.45 orf69 (phage K); Putative helicase orf 123 (LP65); other phages gp43 43,450 45,090 62.8 6.45 orf70 (phage K) putative replicase gp44 45,108 46,571 55.6 5.89 orf71 (phage K); orf76 Primase-helicase

(LP65); other phages gp45 46,586 47,638 39.8 4.92 — gp46 47,733 48,113 14.5 9.8 orf74 (phage K); orf70 Exonuclease

(LP65) gp47 48,184 49,617 53.8 5 • — gp48 49,637 50,227 22.9 7.02 orf75 (phage K) gp49 50,227 51,288 40.4 4.85 orf76 (phage K); orf68 Prirnase

(LP65) gp50 51,335 51,982 23.5 5.76 Proteins from several dUTPase phages and bacteria gp51 51,979 52,203 8.1 5 .28 gp52 52,200 52,523 12.2 4.48 gp53 ^' 52,516 52,938 16.1 5.33 orf77 (phage K) gp54 52,941 53,561 23.6 5.38 orf78 (phage K), gene2 (SPOl); (D 14),

(T5) Table 4. Features of bacteπopήaj ^e PlOO ORFs, gene products, homologies, and functional assignments.

ORF Start Stop GP GP Homologies other Function

(MW^a) (IP^a) phages'³ gp55 53,678 55,027 51.7 5.79 Ribonucleoside- diphosphate reductase alpha subunit gp56 55,238 56,269 38.8 5.62 Ribonucleoside- diphosphate reductase alpha subunit gp57 56,445 57,476 39.5 4.98 Ribonucleoside- diphosphate reductase beta subunit gp58 57,473 57,925 17.4 4.45 gp59 57,928 58,224 10.8 5.06 gp60 58,248 58,940 25.7 6.04 gp61 58,943 59,080 5 9.63 gp62 59,083 60,270 44.3 7.18 gp63 60,267 61,181 34.7 5.28 orfl l0 (phage K), Ribose-phosphate

(Felix 01) pyrophosphokinase gp64 61,192 62,985 67.9 5.31 orfll l (phage K) Nicotinamid phosphoribosyl' transferase gp65 63,081 65,540 95 7.31 orfl8 (phage K); orfl37 (LP65) gp66 65,634 66,422 30.8 9.47 orf84 (phage K); gp67 66,415 66,729 12 9.27 orf85 (phage K); DNA binding gp68 66,812 67,648 31.9 5.34 orf86/88/90 (phage K); DNA polymerase

SPOl; orf 59 (LP65) gp69 67,983 70,091 80.9 5.67 orf86/88/90 (phage K); DNA polymerase

SPOl; orf 59 (LP65) gp70 70,186 70,662 18.6 5.03 orf91 (phage K); gp71 70,700 71,959 46.7 4.92 orf22 phage (Twort); orf92 (phage K) gp72 72,029 73,273 46.1 7.75 orf93 (phage K), Recombinase recombinase A (LP65) gp73 73,335 73,712 14.5 8.95 — gp74 73,712 74,350 25.2 7.12 orf94 (phage K); many Potential sigma fact bacterial proteins gp75 74,409 74,570 6.1 3.93 — gp76 74,769 74,626 5.5 6.76 gp77 74,791 75,498 26.1 4.91 orf95 (phage K) gp78 75,606 75,992 15 4.93 — gp79 75,989 76,918 35.5 6.18 — gp80 76,977 78,248 47.5 7.87 orf98 (phage K); orf64

(LP6) gp81 78,268 78,651 13.9 9.6 — gp82 78,659 79,204 20.5 8.68 — gp83 79,259 79,456 7.1 8.21 — gp84 79,507 80,214 27 9.65 orflOl (phage K); orf45 (LP65) gpS5 80,225 80,707 18.6 10.35 orfl02 (phage K); Alanyl-tRNA synthetase gp86 80,767 81,651 33.2 5.31 — gp87 81,740 82,177 16.7 5.5 — Table 4. Features of bacteriophage PlOO ORFs, gene products, homologies, and functional assignments.

ORF Start Stop GP GP Homologies other Function (MW") (IP^a) phages'³ . gp88 82,183 82,629 17.4 4.48 — gp89 82,604 83,434 32 5.82 — gp90 83,439 84,455 38.1 5.26 orfl5(phageK) ATPase gp91 84,442 85,287 32.5 8.45 — gp92 85,349 85,816 17.8 5.08 gp93 85,849 86,421 21.4 9.43 gp94 86,418 86,996 21.7 9.89 gp95 86,989 87,321 12.6 9.81 gp96 ^' 87,607 88,335 27.5 5.32 orfl03(phageK), orf41 (LP65) gp97 88,350 88,817 17.9 4.38 orfl04(phageK) gp98 88,932 89,975 39.5 5.83 orfl05(phageK) gp99 90,023 90,592 21.2 5.29 — gplOO 90,595 91,128 19.7 7.85 — gplOl 91,143 91,901 29.3 9.36 — gplO2 91,914 92,204 11.6 9.4 — gplO3 92,914 92,633 10.8 8.31 — gplO4 93,942 94,724 30.4 5.52 — gplO5 94,891 95,100 8.1 3.95 — gplO6 95,213 95,476 10.4 10.1 •— gplO7 95,560 95,835 10.4 9.75 — gplO8 95,948 96,340 15 4.09 gplO9 97,353 97,607 9.1 6.16 — gpllO 97,604 97,864 9.6 4.45 — gplll 97,888 98,073 7.3 6.76 — gpll2 98,092 98,265 6.2 10.03 — gpll3 98,407 98,682 10.2 6.78 — gpll4 98,696 98,983 10.9 4.97 gpll5 99,137 99,409 10.3 4.37 gpH6 99,726 99,854 4.8 9.9 gpll7 100,157 100,561 15.2 4.79 Sensorprotein (phi13) gp37 (PSA) gpH8 100,564 100,782 8.2 5.6 gpll9 100,784 101,005 8.3 5.06 gpl20 101,012 101,248 9.2 4.58 gpl21 101,245 101,511 10.1 4.18 gpl22 101,504 101,995 18.4 5.07 gpl23 101,998 102,504 19.3 4.55 gpl24 102,515 103,699 46.6 6.77 gp52 (PSA) (EJ-I) gpl25 103,862 104,287 16.7 7.88 — gpl26 104,305 104,595 11.2 3.95 gp37 (PSA) gpl27 104,592 104,777 7.2 4.75 gpl28 104,777 105,124 13.3 5.35 — gpl29 105,156 105,416 10.1 4.08 —• gpl30 105,496 105,828 13 5.3 — gpl31 105,829 106,224 15.3 9.3 — gpl32 106,289 106,468 6.3 9.11 — gpl33 106,491 106,853 14.3 5.38 gpl34 106,853 107,209 13.8 5.91 — gpl35 108,026 107,253 30.3 5.59 gpl36 108,359 108,039 12.2 4.69 orf58(A118) Table 4. Features of bacteriophage .PlOO ( 3RPs, gene products, homologies, and functional assignments.

ORP Start Stop GP GP Homologies other Function

(MW^{£ l}) (iP^a) phages^b gpl37 108,660 108,352 11.8 6.82 — gpl38 109,183 108,674 20 9.67 — gpl39 109,396 109,205 7.3 8.22 — gpl40 109,686 109,402 10.5 4.17 — gpl41 110,157 109,876 10.9 8.8 — gpl42 110,441 110,217 8.8 8.23 — gpl43 110,984 110,442 21 6.99 — gpl44 111,208 110,981 9.2 4.24 — gpl45 112,464 111,211 48.2 5.89 • — gpl46 112,891 112,466 16.4 4.79 — gpl47 113,444 112,956 18.9 9.39 gpl48 114,082 113,450 23.9 9.53 gpl49 114,282 114,085 7.8 5.84 gpl50 114,784 114,272 19.2 8.35 gpl51 115,481 114,864 23.6 6.42 gpl52 115,696 115,478 8.1 8.97 gpl53 116,090 115,713 14.4 4.53 orfl (SPOl) gpl54 116,449 116,093 13.4 9.18 — gpl55 117,468 116,527 36.2 5.32 orf21 (phage K) Ligase gpl56 118,018 117,482 20.3 4.99 — gpl57 118,206 118,015 7.8 9.95 gpl58 118,710 118,207 19.3 9.05 orf4 (phage K) gpl59 118,981 118,712 10.4 9.22 gpl60 120,311 119,031 47.8 8.18 gplδl 120,547 120,344 7.8 9.23 — gpl62 120,971 120,540 16.8 5.54 Pyrophosphathydrolase gpl63 121,209 120,985 8.3 8.99 — gpl64 121,465 121,223 9.5 9.57 or06 (A118) Repressor gpl65 123,090 121,570 57.3 6.72 (KVP40) (Aehl)

(Felix 01) gpl66 124,019 123,801 8.2 9.62 — gpl67 125,497 125,090 16.5 10.06 — gpl68 125,720 125,523 7.7 6.94 — gpl69 128,127 127,855 10.2 5.8 gpl70 128,679 128,254 16.4 9.74 — gpl71 130,275 130,039 8.8 4.99 — gpl72 130,666 130,325 12.9 5.51 — gpl73 131,035 130,691 13.8 5.13 — gpl74 131,320 131,051 9.9 5.98 — tRNA-Met 123,714 123,784 — Anticodon CAT tRNA-Met tRNA-Pro 124,678 124,752 — Anticodon TGG tRNA-Pro tRNA-Arg 125,870 125,940 — Anticodon TCT tRNA-Arg tKNA-Gly 126,187 126,257 — Anticodon TCC tRNA-Gly tRNA-Asn 126,327 126,399 — Anticodon GTT tRNA-Asn tRNA-Ser 127,020 127,111 — Anticodon TGA tRNA-Ser tRNA-Phe 127,124 127,195 — Anticodon GAA tRNA-Phe tRNA-Lys 127,201 127,272 — Anticodon TTT tRNA-Lys tRNA-Tyτ 127,280 127,351 — Anticodon ATA tRNA-Tyr tRNA-Trp 127,398 127,469 — Anticodon CCA tRNA-Trp tRNA-Gln 127,473 127,544 — Anticodon TTG tRNA-Gln tRNA-Thr 127,563 127,634 — Anticodon TGT tRNA-Thr Table 4. Features of bacteriophage PlOO OKFs, gene products, homologies, and functional assignments.

ORF Start Stop GP GP Homologies other Function

(MW⁴) (IP^a) phages'³ tRNA-Tyr 127,717 127,798 — — Anticodon GTA tRNA-Tyr tRNA-Leu 128,160 128,242 — — Anticodon TAG tRNA-Leu tRNA-Asp 128,710 128,781 — — Anticodon GTC tRNA-Asp tRNA-Ile 128,886 128,957 — — Anticodon GAT tRNA-Ile tRNA-Ser 129,134 129,220 • — — Anticodon GCT tRNA-Ser tRNA-Cys 129,302 129,372 — . — Anticodon GCA tRNA-Cys a Predicted by computer analysis. b Only the most sigmWcant homologies are listed. Names of phages are in brackets; individual references are not listed. c Based upon homologies to other proteins.

(2) Repeated dose oral toxicity study in rats

169. A total of 10 healthy male and 10 healthy female Wistar albino rats (Ace Animals, Boyertown, USA) of about 8 weeks of age were used, with a pre-test body weight range of 202-231 g per male, and 193-214 gper female. Animals were randomly selected and assigned to two groups of five males and five females per group, and individually identified by ear tags. The rats were housed 1 per cage in stainless steel wire bottom cages, in a temperature controlled animal room, with a 12 h light/dark cycle. Fresh rodent chow diet was provided ad libitum, except for the fasting period of one day prior to sacrifice. Fresh water was available ad libitum. 170. As test material for the oral studies, purified and concentrated (5XlO¹ ' pfu/ml) phage PlOO particles suspended in phosphate-buffered saline pH 7.3 (PBS) was used. The slightly cloudy liquid was aliquoted in five tubes containing 12ml each, and stored at 4⁰C for the duration of the experiment. The phage suspension and control liquid (PBS) were orally administered once daily, over a five-day period, using a syringe and 16 gauge ball-tipped feeding needle. Animals in group 1 were dosed with 1.0ml of PlOO phage (5x10ⁿ phages), animals in group 2 (control group) received 1.0ml of PBS only.

171. Body weights were recorded pre-test and prior to termination. The animals were observed once daily for toxicity and pharmacological effects, and twice daily for morbidity and mortality. Food consumption was calculated at the end of the study. On day 8, all animals were anesthetized with ether, sacrificed, and exsanguinated.

172. AU animals were examined for gross pathology. The esophagus, stomach, duodenum, jejunum, ileum, cecum, and colon were preserved in 10% neutral buffered formalin. Histopathologic preparation (cross-sections and longitudinal sections) and microscopical analysis were performed according to standardized procedures. All results were evaluated based on the relationship between the dose levels and incidents or severity of responses (if any). Appropriate statistical evaluations were performed using Instat Statistics Version 2.0 software.

(3) Application of PlOO to control Listeria on a soft cheese model 173. To demonstrate the usefulness of PlOO for the control of L. monocytogenes on the surface of contaminated soft cheeses, several experiments were conducted. As a suitable test organism, L. monocytogenes strain LmC (serovar l/2c) was used, originally isolated from a dairy plant known to have a persistent Listeria contamination in the production equipment. The organism was cultivated on BHI agar (Oxoid, UK) at 30⁰C, and plates stored at 4⁰C. PlOO lysates were purified by tangential-flow ultrafiltration (30 kDa cut-off), and adjusted to approximately 1x10¹⁰ pfu/ml, in MOPS buffer (1OmM 3-(7V-morpholino) propanesulfonic acid, pH 7.3).

174. In preliminary experiments, an artificial cheese surface model (Ch-easy plates; NTZO) was employed to define the most suitable conditions for application of phage during ripening of cheese. Experimental modifications included (a) spiking the unripened cheese surface with Listeria cells at concentrations of 1 or 10 cfu/g of cheese, respectively, and (b) addition of phage PlOO at various intervals to the salt brine wash (15-20% NaCl, dissolved in water), resulting in different concentrations of phage on the cheese surface.

175. Based upon these optimization trials, PlOO was then used during production/ ripening of artificially contaminated surface ripened red-smear soft cheese (type "Munster"). The entire process was designed to simulate a commercial production process, and carried out in a fully equipped cheese-making pilot plant. Cheeses were made according to standard protocols, from pasteurized cow's milk, using a mesophilic starter culture and calf rennet. The acidified, clotted curd was cut, pressed in plastic cheese moulds, and treated in a brine bath (1.9M NaCl) for several hours (day 0). The unripened cheeses (45% fat in dry matter, weight approx. 180 g, single flat side surface approx. 65 cm²) were then surface-dried for approximately 20 h at controlled humidity. In all experimental setups, round flat cheese rinds (65 cm , corresponding to approximately 30- 40 g) were then removed with sterile knifes, and placed in large plastic petri dishes, rind-side up. The rinds were then smeared at days 1, 2, 3, 4, 6, 10, 13 with 210 μl of a smearing solution consisting of 1.9M NaCl and a mixed surface ripening flora {Brevibacterium linens (108 cfu/ml) and Debaryomyces hansenii (10⁸ cfu/ml) (the yeast was used on day 1 only)). To achieve even distribution of Listeria cells, they were added to the first washing solution (6x10³ cfu/ml), which resulted in a fairly consistent contamination density of approximately 2x10¹ cfii/cm². During ripening, cheeses were incubated at controlled temperature of 14°C and 98% relative humidity. On day 16, cheese were packaged in parchment composite paper, and stored at 6°C until the end of the experiment.

176. In a first set of experiments designed to evaluate the required concentration of PlOO, the phage was repeatedly applied to the cheese surface. Two different concentrations were used, a higher dose (3x10⁹ pfu/ml, resulting in phage titers on the cheese surface of approximately 6xlO⁷ pfu/ cm²), and a lower dose (1.5xlO⁸ pfu/ml, corresponding to approximately 2x10⁶ pfu/cm² on the surface). Phage was added to all washing/smearing solutions. In a second cheese-ripening experiment, only one single dose of phage was used (6xlO⁸ pfu/ml). To optimize the distribution of phage on the uneven cheese surface, 1.0ml of smearing solution was used per cheese surface, which resulted in a phage count of 6x10 pfu/cm². Control cheeses received Listeria cells but no phage.

177. For sampling, the cheese rinds (65 cm², corresponding to approximately 30-40 g) were homogenized with buffer (5OmM trisodium-citrate, pH 7.3; added to 250ml) using a Stomacher laboratory blender. The homogenate and decimal dilutions prepared thereof were . surface plated on Listeria selective Oxford agar plates (Oxoid), in triplicate. The plates were incubated at 37⁰C for 48 h, until typical Listeria colonies could be enumerated and viable counts calculated. The lower limit of detection was approximately 5 cfu/cm² of cheese.

178. To determine the possible development of resistance against PlOO, more than 30 of the Listeria colonies isolated from the Ch-easy plates during preliminary setups, and from cheeses treated with lower doses of PlOO were re-purified by repeated streaking on non-selective agar plates, and subsequently with Pl 00 in different lysis assays (liquid culture lysis assay and/or plaque formation in double-layer agar plates).

179. The titer of PlOO on the cheese surfaces was determined from the same homogenized samples. To avoid microbial contamination of the soft agar double layer plates, an antibiotic- resistant indicator host strain (L. ivanovii Sm¹) was used. Volumes of 0.1ml of decimal dilutions were mixed with 0.2ml of log-phase bacteria and 3.5ml BHI soft agar (0.4% agar), and poured onto the surface of a BHI plate (both media contained 300 μg streptomycin/ml). Following incubation for 16-24 h at 30⁰C, plaques could be counted. b) Results

(1) Sequencing and bioinformatics

180. The complete dsDNA genome sequence of P 100 of 131 ,384 bp was assembled from a highly redundant set of 1756 single sequence reads with an average length of 800 bp, yielding a total of 1,405,715 bp (corresponding to >10-fold average coverage). The fully annotated sequence has been deposited in GenBank, under Accession No. DQ004855 (SEQ DD NO:1).

181. A total of 174 open reading frames were identified, predicted to encode gene products (proteins) ranging from 5 kDa (gp61) to 146 kDa (gρ35) (Table 3). In addition, PlOO encodes a total of 18 tRNAs, located at the right end of the genome (nucleotide position 123,714-129,372). Solely on the basis of sequence similarities, putative functional assignments could be made to 25 of the predicted products, whereas the other proteins represent new entries in the database. 182. The bioinformatic analyses and annotations (in particular sequence alignments and motif searches) did not reveal any similarities of PlOO genes or gene products to any genes or proteins or other factors known or believed to play a direct or indirect role in the pathogenicity or virulence of X. monocytogenes (Vasquez-Boland et al., 2001), or of any other infectious, toxin-producing or otherwise harmful microorganism. 183. Pl 00 appears to be closely related to Listeria phage A511. They both -feature a broad (but nevertheless different) host range within the genus Listeria, and belong to the same morphotype family (Myoviridae; Zink and Loessner, 1992). The phenotypical observations correlate well with the now available genetic data, which revealed significant nucleotide sequence homologies ofPlOO to the A511 genome (Loessner and Scherer, 1995; Dorscht et al., submitted for publication). On an overall scale, PlOO also shared some sequence similarities with other known Myoviridae phages infecting Gram-positive bacteria of the low G+C cluster, such as Staphylococcus aureus phage K (O 'Flaherty et al., 2004) and Lactobacillus plantarum phage LP65 (Chibani-ChennouW et al., 2004a,b).

184. Alignments of the 174 predicted PlOO proteins with all proteins and polypeptides contained in the current food allergen database returned only one match: gp71, a 419 amino acid polypeptide encoded by orf71, which showed local similarity (e-value 8x10^"10) of short sequence stretches in its C-terminal portion to epitopes of wheat γ-gliadin. However, these similarities appear to be based upon specific local distribution of glutamine and proline residues in these proteins, and are not expected to cause immunological cross-reaction. (2) Repeated dose toxicology study in rats

185. Oral administration of a high dose of phage PlOO for five consecutive days, followed by a two day recovery period in male and female Wistar albino rats, revealed no in-life effects attributable to the material. No deaths were noted during the study. Body weight changes over the 8 day period were normal; an average increase of 48 g (males) and 24 g (females) was observed, with no differences between the test group and the control group. There were no significant (p60.05) differences in mean body weight or food consumption between the groups (data not shown). There were no abnormal physical signs or behavioral changes noted in any animal at any observation time point. There were no significant test-article related changes in any of the male or female rats given PlOO. Necropsy results (Table 5) were normal in all animals except one of the animals of the PlOO test group which showed a small red area in the mucosa at the junction of jejunum and ileum. Multiple thin sections from this area of the gastrointestinal tract were then examined, and all were within normal histological limits with no microscopic change to correlate with the gross observation.

186. It was concluded that the histomorphologic observations in the male and female rats of both groups of this study are typical of those which occur spontaneously in laboratory rats of this strain and age, and administration of PlOO phage had no effect on the type or incidence of these findings.

Tabled. Incidence of histomorphologic observations

Dose group PlOO Control

Sex M F M F

Number of animals/group 5 5 5 5

Stomach

# examined/normal 5/3 5/4 5/3 5/4

- dilatation, mucosal glands^a 2 1 2 1

Esophagus

# examined/normal 5/5 5/5 5/5 5/5

Duodenum

# examined/normal 5/5 5/5 5/5 5/5

Jejunum

# examined/normal 5/5 5/5 5/5 5/5

Cecum

# examined/normal 5/5 5/4 5/5 5/5

- inflammation, mucosa, chronic^a 1

Colon

# examined/normal 5/5 5/5 5/5 5/5

Ileojejunal junction^b

# examined/normal — 1/1 — — ^a Minimal degree. ^b Only the female rat which showed a slight red area was tested. (3) Efficacy of PlOO for control of L. monocytogenes on soft cheese

187. The results shown in Fig. 1 demonstrate the effect of PlOO on Z. monocytogenes contamination on a surface-ripened Munster-type soft cheese. The manufacturing process used was indistinguishable from that employed in commercial production of this type of cheese, including the specific parameters of inoculation with a standardized bacterial/ yeast ripening flora, ripening conditions (temperature and duration), washing of the rind, and time point of packaging.

188. The inhibitory effects of PlOO were clearly dose-dependent. In the first set of experiments (Fig. IA), a lower concentration of 1.5x10⁸ pfu/ml was repeatedly applied, which resulted in an approximately 2-3 log decrease of Listeria viable counts. Although this represents a massive reduction, it was not complete elimination. However, when a higher concentration of 3x10⁹ phages per ml smearing solution was used, complete eradication of viable X. monocytogenes was observed. This result was confirmed by selective enrichment and subsequent plating of cheese samples, which were negative for Listeria, In a subsequent experiment, only a single dose of phage was applied to the cheeses, shortly after contamination with Listeria cells. The larger volume of smearing liquid used here (1.0ml) permitted a better distribution of phage on the surface of the cheese. This approach also resulted in complete inhibition, i.e., Listeria viable counts were below the limit of detection at all times following application of PlOO. hi contrast, the untreated control cheeses supported growth of L. monocytogenes to titers of generally more than 10⁷ cfu/cm².

189. All of the Listeria clones re-isolated from Ch-easy plates and cheeses treated with lower concentrations of phage retained sensitivity to PlOO infection, i.e., we were unable to detect development of insensitivity or resistance against the phage among the surviving Listeria cells. It is also important to note that Phage Pl 00 did not noticeably affect the functioning of the natural flora and ripening process, i.e., there were no apparent changes of the PlOO treated product compared to the controls, in terms of general appearance or color.

190. Because it was a possibility that the virions could potentially be inactivated by the proteases secreted by the microbial ripening flora, we have monitored the stability of PlOO during the ripening process. However, repeated determination of phage titers recovered from the homogenized cheese surfaces before and after smearing indicated that it is sufficiently stable; no significant decrease or increase in phage titer was determined over a period of 6 days. 2. Example 2 a) Experimental setup 191. The test product was industrially prepared, cooked chicken fillet that was sliced into slices of approximately 17.5 g and approximately 1 dm² or 100 cm² and a thickness of approximately 1.5 mm. The product was treated in three different ways: (1) Blank: not inoculated product (2) LIS : product inoculated with a mixture of three Listeria monocytogenes (LISl, LIS2 and LIS3) strains at a realistic contamination level of 17 cfu/g (Table 6)

(3) . LIS + PHA: product inoculated with phages (at 7x10⁷ pfu/g or 1x10⁹ pfu/dm² or 1x10⁷ pfu/cm²) and a mixture of three Listeria monocytogenes

(LISl, LIS2 and LIS3) strains (at a level of 17 cfu/g)

192. From the appropriate dilution of the mixture of the three L.monocytogenes strains (5xlO⁸ cfu/g) and/or from the phage solution containing 2x 10¹⁰ /ml, 0.5 ml of inoculum was taken with a pipette, divided over and spread on the surface of ± 15O g of product (8 slices = 8 dm2 in total) with a spatula to reach the desired inoculation levels (phages: 7x10⁷ pfu/g or 1x10⁹ pfu/dm² or IxIO⁷ pfu/cm² and L.monocytogenes: 17 cfu/g).

193. After inoculation, the 150 g portions of product were vacuum packaged and stored at 7 ± 1°C in a ventilated refrigerator. Vacuum packaging was performed using a Multivac A300/42 (Hagenmϋller, Wolfertschwenden, Germany) gas packaging machine in a high barrier film (NX90, Euralpak, Wommelgem, Belgium) of 90 μm thickness with an oxygen transmission rate of 5.2 ml/m² 24h atm at 23⁰C and 85% of relative humidity.

194. At 4 different points in time the product was analysed on the presence of some microbial parameters:

(1) Total aerobic psychrotrophic count (2) Number of lactic acid bacteria

(3) Number of Listeria monocytogenes

195. Details of the investigated Listeria monocytogenes strains are listed below in Table 6. Table 6. Investigated Listeria monocytogenes strains

Code LFMFP'-code Species Origin/Type

LISl LFMFP 45 L. monocytogenes Isolated by LFMFP

LIS2 LFMFP 182 L. monocytogenes Scott A (serotype 4B)

LIS3 LFMFP 34 L. monocytogenes LMG 13305

¹ LFMFP = Laboratory of Food Microbiology and Food Preservation b) Results 196. The product was characterized by a pH of 6.25, a water activity of 0.9797 and a

NaCl-I evel of 3.11% (on the waterphase). Table 7 presents the results of the determination of the total aerobic psychrotrophic count, the number of lactic acid bacteria and the number of Z. monocytogenes, ha Figure 2, the effect of phage PlOO on the growth of L. monocytogenes in cooked chicken fillet (vacuum, 7°C) is presented. Table 7. Total aerobic psychrotrophic count, lactic acid bacteria and L. monocytogenes

0 3,2 3,2 3,2

7 8,6 8,3 8,3

14 8,5 8,4 8,5

21 8,8 8,7 8,5

Lactic acid bacteria (loglO cfu/g)

0 2,2 2,2 2,2

7 5,2 5,5 5,8

14 7,8 6,7 8,6

21 7,7 7,5 8,7

L. monocytogenes (loglO cfu/g)

0 l,0^a 1,0 1,0

7 1,0 2,5 2,0

14 1,0 4,6 1,8

21 I₁O 4,3 1,0 ^a Detection limit for the determination of the number of L. monocytogenes cells = 1 loglO cfu/g.

3. Example 3 a) Test product

197. The test product was 'Meesterlyck' cooked ham, an industrially prepared cured cooked ham that meets the criteria of Table 8. The product was produced by Brackenier NV (Oosterzele, Belgium) and after production, the cooked hams were sliced and vacuum packaged per 50Og in the same company. The shelf-life at 7°C was estimated at ± 2 weeks. Table 8. Criteria for 'Meesterlyck' cooked ham

Final product:

Chemical

Salt (NaCl): max. 2 %

Moisture/protein: max. 3,5

Total protein — non protein nitrogen — collagen 88,0 % van totaal eiwit

Sugars: max. 1,0 %

K-nitrate: max. 30 mg per kg

Na-nitrite: max. 30 mg per kg

Na-nitrite + K-nitrate: max. 50 mg per kg

Phosphate (P2O5)/protein: max. 2,2

Glutamic acid: max. 300 mg per kg

Citrate: max. 100 mg per kg

No non-meat proteins, starch or colourings

Bacteriological

Lactobacilli: max. 5.000 per gram

Enterobacteriaceae: max. 100 per gram

Salmonella and Listeria monocytogenes: absent in 25 gram

Escherichia coli: max. 10 per gram

Staphylococcen coagulase-positief: max. 100 per gram b) Experimental set-up

198. The test product was first characterized by determining the pH, water activity (aw), salt level, dry matter, lactate level and nitrite level.

199. The product was treated in four different ways: (1) Blank: not inoculated product

(2) LIS: product inoculated with a mixture of three Listeria monocytogenes (LISl, LIS2 and LIS3) strains (at a level of 10 cfu/g)

(3) LIS + PHA-I: product inoculated with phages (IxIO⁷ pfu/cm²) and a mixture of three Listeria monocytogenes (LISl, LIS2 andLIS3) strains (at a level of lO cfu/g)

(4) LIS +PHA-2: product inoculated with phages (5x10⁶ pfu/cm²) and a mixture of three Listeria monocytogenes (LISl, LIS2 and LIS3) strains (at a level of 10 cfu/g)

200. In the case of LIS: from the appropriate dilution of the mixture of the three L. monocytogenes strains (3^χ10⁸ cfu/g), 100 μ\ was divided over and spread on the surface of ± 150 g of product with a spatula to reach the desired inoculation level of L.monocytogenes (± 10 cfu/g).

201. In the case of LIS + PHA-I and LIS + PHA-2: more or less 30-60 seconds after inoculation with the L.monocytogenes mixture, the product was inoculated with the phage solution. From the appropriate dilution of the phage solution containing 2xl0¹⁰pfu/ml, 150 μl was divided over and spread on the surface of ± 150 g of product with a spatula to reach the desired inoculation levels (PHA-I: ± 1x10⁷ pfu/cm² and PHA-2: ± 5x10⁶ pfu/cm²).

202. After inoculation, the 15O g portions of product were vacuumpackaged and stored at 7 ± 1°C in a ventilated refrigerator up to THT+30%. Vacuum packaging was performed using a Multivac A300/42 (Hagenmiiller, Wolfertschwenden, Germany) gas packaging machine in a high barrier film (NX90, Euralpak, Wommelgem, Belgium) of 90 μm thickness with an oxygen transmission rate of 5.2 ml/m² 24h atm at 23⁰C and 85% of relative humidity.

203. The experiment was performed in triplicate. The different products were followed at 7°C and at regular time intervals the products were subjected to: ( 1 ) determination of some microbial parameters :

• Total aerobic psychrotrophic count

• Number of lactic acid bacteria

• Number of Listeria monocytogenes (2) phage number determination

204. Details of the investigated Listeria monocytogenes strains are listed below in Table 9. Table 9. Investigated Listeria monocytogenes strains

Code LFMFPl -code Species Origin/Type

LISl LFMFP 45 L. monocytogenes Isolated by LFMFP

LIS2 LFMFP 182 L. monocytogenes Scott A (serotype 4B)

LIS3 LFMFP 34 L. monocytogenes LMG 13305

1, LFMFP = Laboratory of Food Microbiology and Food Preservation c) Results (1) Product characterization

205. The chemical parameters that were analysed in order to characterize the cooked ham product are summarized in Table 10. Table 10. Chemical parameters characterizing the product pH aw NaCl (⁰Zo)¹ DM (%)² Nitrite (mg/kg) (D+L)-lactic acid (%) (as NaNO₂)

Duplicate 1 6.09 0.9826 1.7 29.9 <5 0.71

Duplicate 2 6.12 0.9827 1.7 27.0 6 062

^!, NaCl is determined according to the method of Mohr (titrimetric determination of chloride ions)

², DM = dry matter

(2) Phage titer determination

206. The results of the phage titer determinations of the cooked ham samples that were treated with the phage solution are summarised in Table 11. The evolution of the phage titers on the cooked ham during storage under vacuum at 7⁰C is presented in Figure 3.

Table 11. Phage titer (pfu/cm2) as a function of time of three replicate samples (Tl, T2 and T3) of the cooked ham and for both treatment levels (PHA-I and PHA-2)

PHA-2 (5e+6/cm²) PHA-I (le+7/cm²)

Time(d) Tl T2 T3 Tl T2 T3

0 3.95E+06 5.83E+06 2.54E+06 6.58E+05 1.13E+06 1.05E+06

3 5.45E+06 4.32E+06 6.39E+06 1.69E+06 5.55E+06 3.38E+06

6 3.85E+06 4.14E+06 2.44E+06 4.79E+06 6.11E+06 2.63E+06

10 4.89E+06 2.07E+06 2.16E+06 1.88E+06 1.32E+06 9.96E+05

207. The obtained phage titers on day 0 were more or less 0.5 log-cycle lower than the desired phage titers and this was the case for both of the two treatment levels (PHA-I and PHA- 2). On day 0 treatment level PHA-I was more or less 0.5 log-cycle higher than treatment level PHA-2 but this difference did not exist anymore on the other days of analysis. On day 3, 6 and 10, there was no significant difference anymore in the phage titers between the two different treatment levels. The results demonstrate the stability of phage PlOO on the cooked ham product since titers did not change significantly between day 0 and day 10 of the experiment. (3) Microbial analyses

208. The results from the microbial analyses of the three replicate samples of the four treatments are summarized in Tables 12, 13, 14 and 15.

Table 12. Total aerobic psychrotrophic count and lactic acid bacteria as a function of time of three replicate samples of the non-inoculated cooked ham

Time(d) Blank 1 Blank 2 Blank 3

Total aerobic psychrotrophic count (lo£ ϊlO cfu/g)

0 3.23 3.02 2.00

3 5.06 5.00 4.87

6 7.08 7.97 7.94

10 8.49 8.57 8.59

Lactic acid bacteria (loglO cfu/g)

0 1.85 2.83 1.00

3 4.79 4.90 4.52

6 6.63 7.81 7.79

10 7.13 6.85 7.00

Table 13. Total aerobic psychrotrophic count, lactic acid bacteria and number of L. monocytogenes as a function of time of three replicate samples of the Listeria-inoculated cooked ham

Time(d) LISl LIS2 LIS3

Total aerobic psychrotrophic count (loglO cfu/g)

0 3.23 3.02 2.00

3 6.45 6.48 6.09

6 8.19 7.73 8.00

10 8.16 8.34 8.32

Lactic acid bacteria (loglO cfu/g)

0 1.85 2.83 1.00

3 5.72 6.26 5.90

6 7.51 6.70 7.41

10 6.70 8.23 6.46

L. monocytogenes (log 10 cfu/g)

0 1.85 1.60 1.60

3 1.48 1.85 1.70

6 3.28 3.02 3.14

10 2.85 3.04 3.28

¹ Detection limit for the determination of the number of Z. monocytogenes cells = 1 loglO cfu/g.

Table 15. Total aerobic psychrotrophic count, lactic acid bacteria and number of L. monocytogenes as a function of time of three replicate samples of the Listeria-inoculated cooked ham that was treated with Listex at 5x10⁶ pfu/cm²

Time(d) (LIS + PHA-2)-! (LIS + PHA-2)-2 (LIS + PHA-2)-3

Total aerobic psychrotrophic count (logl 0 cfu/g)

0 3.23 3.02 2.00

3 5.34 5.40 4.86

6 7.63 8.03 8.03

10 8.98 8.93 8.93

Lactic acid bacteria (loglO cfu/g)

0 1.85 2.83 1.00

3 4.45 4.85 4.12

6 6.20 6.51 6.14

10 6.38 6.30 5.88

L. monocytogenes (loglO cfu/g)

0 1.00 1.00 1.00

3 1.00 1.30 1.00

6 2.02 1.54 2.00

10 1.70 2.11 1.48

Detection limit for the determination of the number of L. monocytogenes cells ■= 1 loglO cfu/g.

209. The evolution of the different microbial parameters as a function of time is presented in Figures 4, 5 and 6.

210. From the results of day 0 it can be seen that phage treatment (PHA-I and PHA-2) had an immediate effect of ± 0.5 loglO cfu/g on the number of L. monocytogenes on the cooked ham.

211. Treatment of the cooked ham with P 100 significantly inhibited the growth of L. monocytogenes compared to the cooked ham that was not treated with PlOO. On the cooked ham inoculated with the cocktail of L. monocytogenes and not treated with PlOO, the number of L. monocytogenes increased with 1.37 loglO cfu/g. On the cooked ham inoculated with the cocktail of L. monocytogenes and treated with PlOO at a level of 1x107 pfu/cm2 the number of L. monocytogenes increased with 0.40 loglO cfu/g. On the cooked ham inoculated with the cocktail of L. monocytogenes and treated with PlOO at a level of 5xlO⁶ pfu/cm2 the number of L. monocytogenes increased also with 0.76 loglO cfu/g.

212. Talcing into account the 95% confidence intervals no significant difference in antilisterial effect was observed between both treatment levels.

213. Treatment of the cooked ham with P 100 does not influence the presence of bacteria other than !,, monocytogenes since the evolution of the total aerobic psychrotrophic count and the number of lactic acid bacteria as a function of time does not differ between cooked ham samples treated with PlOO and cooked ham samples not treated with PlOO. F. References

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Claims

VI. CLAIMS

What is claimed is:

1. A composition for controlling Listeria contamination, comprising recombinant PlOO phage.

2. The composition of claim 1 , wherein the recombinant PlOO phage comprises a nucleic acid having mutations, substitutions, or deletions to the sequence set forth in SEQ ID NO:1.

3. The composition of claim 1 , further comprising A511 , P35, Al 18, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971A, 02971C, 907515, 12981, 11711 A, 00241, 13441, A500, A640, B021, PSA, B653, 90666, 90861, 910716, 93253, 52, 340, 312, 108, 10, 2425A, 2425, or 3551 phage.

4. The composition of claim 1, further comprising an agent selected from the group consisting of listeriolysin, a surface disinfectant, an antibiotic, a surfactant, an enzyme, and a phage specific for bacterial contaminants other than Listeria monocytogenes.

5. The composition of claim 1 , further comprising a pharmaceutically acceptable carrier.

6. A composition for controlling Listeria contamination, comprising Plyl 00 polypeptide.

7. The composition of claim 6, wherein the Plyl 00 polypeptide is produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ JD NO:1, or a fragment or conservative variant thereof.

8. The composition of claim 6, wherein the Plyl 00 polypeptide is produced by a cell comprising a nucleic acid that can hybridize under stringent conditions to a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ ID NO:1, or a fragment or conservative variant thereof.

9. The composition of claim 6, further comprising at least one variety of lytic phage from the Myoviridae family.

10. The composition of claim 6, wherein said lytic phage are selected from the group consisting of PlOO, A511, P35, Al 18, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971A, 02971C, 907515, 12981,

11711 A, 00241, 13441, A500, A640, B021, PSA, B653, 90666, 90861, 910716, 93253, 52, 340, 312, 108, 10, 2425 A, 2425, and 3551 phage.

11. The composition of claim 6, wherein said endolysin is recombinantly produced in a host bacteria.

12. A recombinant bacteriophage Plyl 00 polypeptide.

13. The polypeptide of claim 12, wherein the polypeptide is produced by a ceu comprising a. ' nucleic acid having the sequence set forth in nucleotides 1,746 to 3,300 of SEQ ID NO:1, nucleotides 5,472 to 6,497 of SEQ ID NO:1, nucleotides 8,189 to 9,832 of SEQ ID NO: 1, nucleotides 11,790 to 13,196 of SEQ ID NO: 1, nucleotides 17,130 to 18,818 of SEQ ID NO :1, nucleotides 29,606 to 30,316 of SEQ ID NO:1, nucleotides 30,330 to 31,376 of SEQ ID NO:1, nucleotides 41,690 to 43,435 of SEQ TD NO:1, nucleotides 43,450 to 45,090 of SEQ ID NO:1, nucleotides 45,108 to 46,571 of SEQ JD NO:1, nucleotides 47,733 to 48,113 of SEQ ID NO:1, nucleotides 50,227 to 51,288 of SEQ ID NO:1, nucleotides 51,335 to 51,982 of SEQ ID NO:1, nucleotides 53,678 to 55,027 of SEQ ID NO:1, nucleotides 55,238 to 56,269 of SEQ ID NO:1, nucleotides 56,445 to 57,476 of SEQ ID NO:1, nucleotides 60,267 to 61,181 of SEQ ED NO:1, nucleotides 61,192 to 62,985 of SEQ E) NO:1, nucleotides 66,415 to 66,729 of SEQ E⁾ NO:1, nucleotides 66,812 to 67,648 of SEQ E) NO:1, nucleotides 67,983 to 70,091 of SEQ E) NO:1, nucleotides 72,029 to 73,273 of SEQ E) NO:1, nucleotides 73,712 to 74,350 of SEQ E) NO:1, nucleotides 80,225 to 80,707 of SEQ E) NO:1, nucleotides 83,439 to 84,455 of SEQ E) NO:1, nucleotides 117,468 to 116,527 of SEQ E) NO:1, nucleotides 120,547 to 120,344 of SEQ E) NO:1, nucleotides 120,209 to 120,985 of SEQ E) NO:1, or a fragment or conservative variant thereof,

14. The polypeptide of claim 12, wherein the polypeptide is encoded by a nucleic acid that can hybridize under stringent conditions to a nucleic acid having the sequence set forth in nucleotides 1,746 to 3,300 of SEQ E) NO:1, nucleotides 5,472 to 6,497 of SEQ E) NO:1, nucleotides 8,189 to 9,832 of SEQ E) NO:1, nucleotides 11,790 to 13,196 of SEQ E) NO: 1, nucleotides 17,130 to 18,818 of SEQ E) NO: 1, nucleotides 29,606 to 30,316 of SEQ E) NO:1, nucleotides 30,330 to 31,376 of SEQ E) NO:1, nucleotides 41,690 to 43,435 of SEQ E) NO:1, nucleotides 43,450 to 45,090 of SEQ E) NO:1, nucleotides 45,108 to 46,571 of SEQ E) NO:1, nucleotides 47,733 to 48,113 ofSEQ E3 NO:l, nucleotides 50,227 to 51,288 of SEQ E) NO:1, nucleotides 51,335 to 51,982 of SEQ ID NO:1, nucleotides 53,678 to 55,027 of SEQ E) NO:1, nucleotides 55,238 to 56,269 of SEQ E) NO:1, nucleotides 56,445 to 57,476 of SEQ E) NO:1, nucleotides 60,267 to 61,181 of SEQ E⁾ NO:1, nucleotides 61,192 to 62,985 of SEQ E) NO:1, nucleotides 66,415 to 66,729 of SEQ E) NO:1, nucleotides 66,812 to 67,648 of SEQ E) NO:1, nucleotides 7,983 to 70,091 of SEQ ED NO:1, nucleotides 72,029 to 73,273 of SEQ E) NO:1, nucleotides 73,712 to 74,350 of SEQ E) NO:1, nucleotides 80,225 to 80,707 of SEQ TD NO:1, nucleotides 83,439 to 84,455 of SEQ TD NO:1, nucleotides 117,468 to 116,527 of SEQ TD NO:1, nucleotides 120,547 to 120,344 of SEQ ID NO:1, or nucleotides^' 120,209 to 120,985 of SEQ TD NO:1.

15. The polypeptide of claim 12, wherein the polypeptide has 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a polypeptide encoded by a nucleic acid having the sequence set forth in nucleotides 1,746 to 3,300 of SEQ E) NO:1, nucleotides 5,472 to 6,497 of SEQ E) NO:1, nucleotides 8,189 to 9,832 of SEQ 3D NO:1, nucleotides 11,790 to 13,196 of SEQ E) NO:1, nucleotides 17,130 to 18,818 of SEQ E) NO :1, nucleotides 29,606 to 30,316 of SEQ E) NO:1, nucleotides 30,330 to 31,376 of SEQ E⁾ NO:1, nucleotides 41,690 to 43,435 of SEQ E) NO:1, nucleotides 43,450 to 45,090 of SEQ E) NO:1, nucleotides 45,108 to 46,571 of SEQ E) NO:1, nucleotides 47,733 to 48,113 of SEQ E⁾ NO:1, nucleotides 50,227 to 51,288 of SEQ E) NO:1, nucleotides 51,335 to 51,982 of SEQ E) NO:1, nucleotides 53,678 to 55,027 of SEQ E) NO:1, nucleotides 55,238 to 56,269 of SEQ E) NO:1, nucleotides 56,445 to 57,476 of SEQ E⁾ NO:1, nucleotides 60,267 to 61,181 of SEQ E) NO:1, nucleotides 61,192 to 62,985 of.SEQ E) NO:1, nucleotides 66,415 to 66,729 of SEQ E) NO:1, nucleotides 66,812 to 67,648 of SEQ E) NO:1, nucleotides 7,983 to 70,091 of SEQ E) NO:1, nucleotides 72,029 to 73,273 of SEQ TD NO:1, nucleotides 73,712 to 74,350 of SEQ E) NO:1, nucleotides 80,225 to 80,707 of SEQ E) NO:1, nucleotides 83,439 to 84,455 of SEQ E) NO:1, nucleotides 117,468 to 116,527 of SEQ E) NO:1, nucleotides 120,547 to 120,344 of SEQ E) NO:1, or nucleotides 120,209 to 120,985 of SEQ E) NO:1.

16. An isolated nucleic acid encoding a bacteriophage PlOO-specific polypeptide, wherein the nucleic acid does not consist of SEQ TD NO:1.

17. The isolated nucleic acid of claim 16, wherein the nucleic acid has the sequence set forth in nucleotides 1,746 to 3,300 of SEQ TD NO:1, nucleotides 5,472 to 6,497 of SEQ TD NO: 1, nucleotides 8,189 to 9,832 of SEQ E) NO:1, nucleotides 11,790 to 13,196 of SEQ TD NO:1, nucleotides 17,130 to 18,818 of SEQ E) NO:1, nucleotides 29,606 to 30,316 of SEQ TD NO:1, nucleotides 30,330 to 31,376 of SEQ TD NO:1, nucleotides 41,690 to 43,435 of SEQ TD NO:1, nucleotides 43,450 to 45,090 of SEQ TD NO:1, nucleotides 45,108 to 46,571 of SEQ TD NO:1, nucleotides 47,733 to 48,113 of SEQ TD NO:1, nucleotides 50,227 to 51,288 of SEQ E) NO:1, nucleotides 51,335 to 51,982 of SEQ TD NO:1, nucleotides 53,678 to 55,027 of SEQ TD NO:1, nucleotides 55,238 to 56,269 of SEQ TD NO:1, nucleotides 56,445 to 57,476 of SEQ TD NO:1, nucleotides 60,267 to 61,181 of SEQ K) NO:1, nucleotides 61,192 to 62,985 of SEQ ID NO:1, nucleotides 66,415 to 66,729 of SEQ ID NO:1, nucleotides 66,812 to 67,648 of SEQ ID NO:1, nucleotides 67,983 to 70,091 of SEQ ID NO:1, nucleotides 72,029 to 73,273 of SEQ ID NO:1, nucleotides 73,712 to 74,350 of SEQ ID NO:1, nucleotides 80,225 to 80,707 of SEQ ID NO:1, nucleotides 83,439 to 84,455 of SEQ ID NO:1, nucleotides 117,468 to 116,527 of SEQ ID NO:1, nucleotides 120,547 to 120,344 of SEQ ID NO:1, nucleotides 120,209 to 120,985 of SEQ ID NO:1, or a fragment or conservative variant thereof. 18. The isolated nucleic acid of claim 16, wherein the nucleic acid can hybridize under stringent conditions to a nucleic acid having the sequence set forth in nucleotides 1 ,746 to 3,300 of SEQ ID NO:1, nucleotides 5,472 to 6,497 of SEQ ID NO:1, nucleotides 8,189 to 9,832 of SEQ ID NO:1, nucleotides 11,790 to 13,196 of SEQ ID NO:1, nucleotides 17,130 to 18,818 of SEQ ID NO:1, nucleotides 29,606 to 30,316 of SEQ ID NO:1, nucleotides 30,330 to 31,376 of SEQ ID NO:1, nucleotides 41,690 to 43,435 of SEQ ID NO:1, nucleotides 43,450 to 45,090 of SEQ ID NO:1, nucleotides 45,108 to 46,571 of SEQ ID NO:1, nucleotides 47,733 to 48,113 of SEQ ID NO:1, nucleotides 50,227 to 51,288 of SEQ ID NO:1, nucleotides 51,335 to 51,982 of SEQ ID NO:1, nucleotides 53,678 to 55,027 of SEQ ID NO:1, nucleotides 55,238 to 56,269 of SEQ ID NO:1, nucleotides 56,445 to 57,476 of SEQ ID NO:1, nucleotides 60,267 to 61,181 of SEQID NO:1, nucleotides 61,192 to 62,985 of SEQ ID NO:1, nucleotides 66,415 to 66,729 of SEQ ID NO:1, nucleotides 66,812 to 67,648 of SEQ ID NO:1, nucleotides 7,983 to 70,091 of SEQ ID NO:1, nucleotides 72,029 to 73,273 of SEQ ID NO:1, nucleotides 73,712 to 74,350 of SEQ ID NO:1, nucleotides 80,225 to 80,707 of SEQ ID NO:1, nucleotides 83,439 to 84,455 of SEQ H) NO:1, nucleotides 117,468 to 116,527 of SEQ ID NO:1, nucleotides 120,547 to 120,344 of SEQ ID NO:1, or nucleotides 120,209 to 120,985 of SEQ ID NO: 1.

19. A nucleic acid vector comprising the nucleic acid of claim 16.

20. A cell comprising the nucleic acid vector of claim 19.

21. A method for treating an animal infected with Listeria monocytogenes comprising administering an amount of recombinant phage PlOO suitable to reduce or eliminate said Listeria monocytogenes.

22. The method of claim 21, further comprising administering A511, P35, Al 18, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971A, 02971C, 907515, 12981, 11711 A, 00241, 13441, A500, A640, B021, PSA₅ B653, 90666, 90861, 910716, 93253, 52, 340, 312, 108, 10, 2425A, 2425, or 3551 phage.

23. A method for detecting the presence of Listeria monocytogenes, comprising obtaining a sample suspected to contain Listeria monocytogenes, incubating said sample with recombinant PlOO, and detecting any change in said sample caused by PlOO, as an indication of the presence of Listeria monocytogenes.

24. The method of claim 23, wherein said change in said sample is due to lysis by PlOO or a detectable label or signal.

25. The method of claim 23, further comprising recombinantly inserting a gene construct into the genome of PlOO before incubation with said sample, wherein^' expression of said gene construct results in a detectable signal in the presence of Listeria monocytogenes.

26. The method of claim 25 wherein said gene construct encodes a bio-luminescent protein. ^• 27. The method of claim 26, wherein said bioluminescent protein is selected from the group consisting of luciferase and a fluorescent protein.

28. The method of claim 27, wherein said luciferase is from bacteria or insects.

29. The method of claim 27, wherein said fluorescent protein is green fluorescent protein or a variant thereof.

30. The method of claim 23, further comprising immobilizing said Listeria monocytogenes on a solid support and detecting any change on said solid support.

31. The method of claim 30, wherein said Listeria monocytogenes are immobilized using ∑ωϊi-Listeria antibodies.

32. The method of claim 30, wherein said solid support is a test strip.

33. The method of claim 23, wherein said sample is obtained from a patient suspected of being infected with Listeria monocytogenes.

34. The method of claim 23, wherein said sample is obtained from a food product, food processing equipment or food storage containers.

35. A method for controlling Listeria contamination in a food product, on food processing equipment or on food storage containers, comprising applying recombinant PlylOO polypeptide to a food product, food processing equipment or food storage container in an amount sufficient to reduce the amount of Listeria.

36. The method of claim 35, wherein the recombinant PlylOO polypeptide is produced by a cell comprising a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ TD NO:l,^'or a fragment or conservative variant thereof. 5 /. ine metnoQ oi ciaiπi o / , wherein the recombinant Ply 100 polypeptide is proαuceα oy a cell comprising a nucleic acid that can hybridize under stringent conditions to a nucleic acid having the sequence set forth in nucleotides 5,472 to 6,497 of SEQ ID NO:1, or a fragment or conservative variant thereof.

38. The method of claim 35, further comprising applying at least one variety of lytic phage from the Myoviridae family to said food product, food processing equipment or food storage container.

39. The method of claim 38, wherein said lytic phage is selected from the group consisting of PlOO, A511, P35, Al 18, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971A, 02971C, 907515, 12981, 11711 A, 00241, 13441, A500, A640, B021, PSA, B653, 90666, 90861, 910716, 93253, 52, 340, 312, 108, 10, 2425 A, 2425, and 3551 phage.

40. The method of claim 35, further comprising applying endolysin from at least one other phage which infects Listeria or another bacterial genera.

41. The method of claim 40, wherein said other phage is A511, P35, A118, A502, A006, A005, A620, 11355C, 00611, 43, 21, 2685, 4477, 01761, 12029, 717, 10993, 10072, 02971A, 02971C, 907515, 12981, 11711 A, 00241, 13441, A500, A640, B021, PSA, B653, 90666, 90861, 910716, 93253, 52, 340, 312, 108, 10, 2425A, 2425, or 3551 phage.