Abstract:
The invention provides intracellular peptide toxins capable of killing bacterial and eukaryotic cells when present within the cell, while substantially lacking the ability to kill such cells when present externally. The invention also provides recombinant bacteriophage containing nucleic acid sequences encoding intracellular peptide toxins, and methods of using such bacteriophage to kill bacteria. Furthermore, the invention provides compositions, including pharmaceutical compositions, which can be used to kill bacteria or inhibit the growth of bacteria both in vitro and in vivo. Methods of treating a bacterial infection in a subject are also provided by the invention.

Description:
[0001]    This application is a continuation-in-part application of U.S. Ser. No. 10/025,598, which was filed Dec. 18, 2001, and which is incorporated here by reference in its entirety. 
     
    
     
       TECHNICAL FIELD  
         [0002]    This invention relates to compositions and methods for killing bacteria and other unwanted cells.  
         BACKGROUND  
         [0003]    Throughout recorded history virulent bacterial infections have been a bane to mankind. Until recently, it was assumed that drug antibiotics had largely eradicated virulent bacteria. It is now apparent, however, that bacteria have circumvented the effects of single-point targeted drug antibiotics. Consequently, there is a need to develop new anti-bacterial agents that can be used to supplement or replace conventional drug antibiotics.  
           [0004]    Like animal cells, bacterial cells are subject to infectious agents that are present in their environment. Viruses known as bacteriophage, or phage, specifically infect bacterial cells. Bacteriophage are the natural enemies of bacteria and, over the course of evolution, have developed proteins which enable them to infect a bacterial host cell, replicate their genetic material, usurp host metabolism, and ultimately kill their bacterial host cell.  
           [0005]    Research into the use of bacteriophage as therapeutic agents for treatment of bacterial infection began sometime in the late 19th century, predating the development of conventional drug antibiotics. By 1920, Edward Twort and Felix d&#39;Herelle, two noted pioneers in bacteriophage research, were isolating bacteriophage from several bacterial species and using them as anti-bacterial agents. During the early 1940&#39;s, however, antibiotics were introduced to the world as a broad range treatment for bacterial infections, and bacteriophage therapy research went into decline.  
           [0006]    Early clinical studies of phage therapy were plagued with poor experimental design, with few controls and little documentation, variable success due to the indiscriminate use of phage to treat a broad range of bacterial infections, and the use of procedures that introduced bacterial toxins into patients and loss of effectiveness of the isolated phage.  
           [0007]    The lack of knowledge and scientific expertise needed to understand bacteriophage and their interaction with bacteria also hindered efforts to improve phage therapy. For example, differences between the biological interaction of bacteriophage strains with their species-specific bacterial host in vitro as compared to in vivo have posed considerable difficulty. Although bacteriophage can be selected for their lytic virulence (immediately replicating and then inducing bacterial host cell lysis following infection) in vitro, such selection does not guarantee against the conversion of a seemingly lytic phage to a temperate phage (entering into a state of lysogeny via integration of the bacteriophage genome into the bacterial genome followed by a quiescent period during which lytic proteins are not expressed) in vivo. These conversions result in lysogenic bacteria that are resistant to further bacteriophage infection, thus reducing the effectiveness of phage therapy.  
           [0008]    Since the early 1940&#39;s drug antibiotics have become the choice for treating virulent bacterial infections. Several problems associated with this approach are now becoming evident. The misuse and overuse of drug antibiotics has contributed to the rise of antibiotic resistant bacterial strains. Moreover, since drug antibiotics are non-specific with respect to the types of bacteria that they effect, the bacterial flora that naturally occur within the body are killed along with the disease-causing bacterial pathogen. At least 200 identified bacterial species normally inhabit the human body, and many of these species synthesize and excrete vitamins vital for human health, promote the development of certain tissues, e.g., lymphatic tissue, e.g., Peyer&#39;s patches, and stimulate the production of cross-reactive “natural” antibodies that react with pathogenic bacteria. Moreover, natural bacterial flora greatly inhibit colonization by non-indigenous bacteria through normal niche colonization or by producing substances and bacteriocins that can inhibit and kill foreign bacteria. Conventional broad-spectrum antibiotics risk killing the non-pathogenic bacteria that are responsible for these beneficial effects.  
           [0009]    Bacterial drug resistance was evident at the onset of drug antibiotic therapy, and drug resistant virulent strains of both gram-negative bacteria (including pathogenic strains of  Escherchia coli ) and gram-positive bacteria (including pathogenic strains of Staphylococcus and Streptococcus) have become increasingly resistant to drug antibiotics. This increased resistance arises primarily from selection for virulent-resistance strains by the presence of drug antibiotics, resulting in the lateral transfer of resistance genes between different strains and species of bacteria. Epidemic outbreaks have been attributed to a single clone of a benign or virulent progenitor, as well as spontaneous multi-clonal populations within a community setting when drug antibiotic usage is increased. Although decreased usage of antibiotics may improve the odds of generating a population of virulent bacteria that are less resistance towards antibiotics, much contradictory evidence is beginning to surface. For example, a study in Finland found that the incidence of  Streptococcus pyogenes  resistance to macrolide decreased after macrolide treatment was reduced in favor of treatment with erythromycin. However, a follow-up study reported a subsequent 17% increase in  Streptococcus pyogenes  resistance to erythromycin. Another growing concern is the increasing number of multi-resistant bacteria. In 1968 approximately 12,500 people in Guatemala died from an epidemic of Shigella, caused by a bacterial strain that contained a plasmid encoding genes resistant to four different antibiotics (Davies (1996)  Nature  383:219). Population genetics studies of virulent bacteria causing disease outbreaks or increases in frequency and virulence have shown that the distinct clones responsible for the acute outbreaks are often characterized by unique combinations of virulence genes or alleles of those genes.  
           [0010]    Increasing drug antibiotic resistance has resulted in increased dosage levels and duration of antibiotic treatment. These practices are associated with hypersensitivity and serious side effects in a growing number of patients (see Cunha (2001)  Med Clin North Am  85:149; Kirjavainen and Gibson (1999)  Ann Med  31:288; Lee et al. (2000)  Arch Intern Med  160:2819; and Martinez et al. (1999)  Medicine  78:361). The increasing hypersensitivity and side effects are not being seriously addressed and have so far been clinically under-evaluated (Demoly et al. (2000)  Bull Acad Natl Med  184:761; and Gruchalla (2000)  Allergy Asthma Proc  21:39). As an example of one serious side effect that is becoming increasingly prevalent, especially in children, the use of antibiotics has been shown to be positively associated with the development of asthma and atopy. The mechanisms underlying these associations remain largely unknown (von Hertzen (2000)  Ann Med  32:397).  
           [0011]    Drug antibiotics and their effects are not isolated to individuals under the supervision of a doctor&#39;s care, but are a communal health issue. Molecular population studies have identified healthy humans that are VRE (vancomycin-resistant enterococci) carriers. An increase in VRE strains in healthy farm animals is associated with the increased use of the antibiotic avoparcin. There is currently a tentative link between the consumption of farm animals and VRE transference to people (Bates (1998)  J Hosp Infect  27:89). Data on antibiotic resistance profiles of several food born pathogens provides ample evidence that antibiotic resistance traits have entered the microflora of farm animals and the food supply produced from them (Teuber (1999)  Cell Mol Life Sci  56:755).  
         SUMMARY  
         [0012]    The present invention is based, at least in part, on the development of intracellular peptide toxins and peptide-like toxins. As the term “intracellular peptide toxins” implies, these compositions are toxic to a cell when internalized, but less toxic and preferably non-toxic when outside the cell. Such peptide toxins and peptide-like toxins are useful in the production of recombinant bacteriophage that effectively function as bacteriocides (i.e., toxin-phage bacteriocides) that can provide a viable alternative to conventional antibiotics. The peptide toxins of the invention can also be used to kill other types of unwanted cells, e.g., cancer cells, such as malignant and benign cancer cells.  
           [0013]    A toxin-phage bacteriocide (TPB) includes bacteriophage that have been genetically engineered to encode a peptide toxin that can be expressed within the bacterial host cell. Within the bacterial host cell, the peptide toxin is active and functions to kill the bacterial host cell. Importantly, the toxin-phage bacteriocide of the invention retains its activity as a bacteriophage, and is therefore capable of completing the lytic phase of its lifecycle. Completion of the lytic phase of its life-cycle results in both the production of additional toxin-phage bacteriocide and host cell lysis.  
           [0014]    The peptide toxins of the invention can be used to kill many types of unwanted cells by introducing the peptide toxin into the unwanted cells by genetic or other means, e.g., by receptor-specific uptake  
           [0015]    In one aspect, the invention features a method of producing a toxin-phage bacteriocide. The method includes: (a) identifying a bacteriophage that is capable of infecting a bacterial cell of interest; (b) preparing a recombinant bacteriophage genome by introducing a nucleic acid sequence that encodes an intracellular peptide toxin into the genome of the bacteriophage, wherein the nucleic acid sequence that encodes the peptide toxin is operatively linked to a promoter that is active within the bacterial cell of interest; and (c) allowing the formation of a toxin-phage bacteriocide particle that contains the recombinant bacteriophage genome.  
           [0016]    In preferred embodiments, the nucleic acid sequence that encodes an intracellular peptide toxin includes the nucleic acid of SEQ ID NO:1, which encodes the TPB peptide toxin A amino acid sequence (SEQ ID NO:2). In other embodiments, the nucleic acid sequence that encodes an intracellular peptide toxin encodes a peptide toxin other than the TPB peptide toxin A, e.g., a peptide toxin that is a variant of the amino acid sequence of TPB peptide toxin A, or a peptide toxin that functions analogously to the TPB peptide toxin A. In some embodiments, a variant of the TPB peptide toxin A includes at least one amino acid alteration, e.g., an insertion, deletion, or amino acid change. In preferred embodiments, the alteration is located at one or more of amino acids 16, 17, 18, 19, 20, 21, and 22 of SEQ ID NO:2. In other preferred embodiments, the alteration is a conservative amino acid substitution. The invention includes a peptide toxin that has 10, 9, 7, 6, 5, 4, 3, 2, or 1 amino acid alterations in the amino acid sequence of SEQ ID NO:2 yet retains the ability to kill a cell when present internally, but not externally. In certain embodiments the amino acid changes are amino acid alterations. The invention includes peptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 92%, 94%, 96%, or 98% identical to SEQ ID NO:2 yet retains that ability to kill a cell when present internally, but not externally. In still other preferred embodiments, the alteration does not change the net ionic charge of the resulting TPB peptide toxin variant, as compared to TPB peptide toxin A, under conditions of physiological pH. The following amino acid substitutions are among those considered conservative:  
                                                       For Amino Acid   Code   Replace with any of . . .                           Alanine   Ala   Gly, Cys, Ser           Arginine   Arg   Lys, His           Asparagine   Asn   Asp, Glu, Gln,           Aspartic Acid   Asp   Asn, Glu, Gln           Cysteine   Cys   Met, Thr, Ser           Glutamine   Gln   Asn, Glu, Asp           Glutamic Acid   Glu   Asp, Asn, Gln           Glycine   Gly   Ala           Histidine   His   Lys, Arg           Isoleucine   Ile   Val, Leu, Met           Leucine   Leu   Val, Ile, Met           Lysine   Lys   Arg, His           Methionine   Met   Ile, Leu, Val           Phenylalanine   Phe   Tyr, His, Trp           Proline   Pro           Serine   Ser   Thr, Cys, Ala           Threonine   Thr   Ser, Met, Val           Tryptophan   Trp   Phe, Tyr           Tyrosine   Tyr   Phe, His           Valine   Val   Leu, Ile, Met                      
 
           [0017]    To determine the percent identity of two amino acid sequences (for example, SEQ ID NO:2 and a variant thereof), the sequences are aligned for optimal comparison purposes. Gaps can be introduced in one or both of the sequences and non-identical sequences at either terminus can be disregarded (e.g., a sequence that contains SEQ ID NO:2 is a variant of SEQ ID NO:2, even if it has additional residues at either terminus, so long as it functions as an intracellular peptide toxin). While the variant may be longer than SEQ ID NO:2, it may also be shorter. For example, the variant can be 25-38 amino acids long. The amino acid residues at corresponding amino acid positions are compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position. The determination of percent identity between two amino acid sequences is accomplished using the BLAST® 2.0 program, which is available to the public. Sequence comparison is performed using an ungapped alignment and using the default parameters (Blossom 62 matrix, gap existence cost of 1, per residue gapped cost of 1, and a lambda ratio of 0.85). The mathematical algorithm used in BLAST programs is described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).  
           [0018]    The invention also features a nucleic acid molecule, e.g., an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide consisting essentially of SEQ ID NO:2 or a variant thereof. The invention also includes nucleic acid molecules encoding polypeptides consisting of SEQ ID NO:2 or a variant thereof.  
           [0019]    Preferably, the nucleic acid molecule encoding the TPB peptide toxin includes a bacterial promoter and other sequences required to direct transcription and translation of TPB peptide toxin in the bacterial cell being targeted. Those skilled in the art can readily obtain promoter sequences and other sequences required for expression.  
           [0020]    In preferred embodiments, homologous recombination is used to introduce the nucleic acid sequence that encodes the intracellular peptide toxin into the bacteriophage genome. In related embodiments, homologous recombination is carried out in vitro. In other related embodiments, homologous recombination is carried out in vivo. In other embodiments, the recombinant bacteriophage genome is packaged into bacteriophage particles in vitro or in vivo, thereby resulting in the production of toxin-phage bacteriocide particles.  
           [0021]    In a related aspect, the invention features compositions that include at least one toxin-phage bacteriocide. In preferred embodiments, the toxin phage bacteriocide includes a nucleic acid sequence encoding an intracellular peptide toxin. In particularly preferred embodiments, the toxin phage bacteriocide includes a nucleic acid sequence encoding the TPB peptide toxin A (SEQ ID NO:2). In other embodiments, the toxin phage bacteriocide includes a nucleic acid sequence encoding TPB peptide toxin A variants. The invention also includes compositions comprising two, three or more different toxin-phage bacteriocides and a pharmaceutically acceptable carrier.  
           [0022]    In preferred embodiments, the compositions include a single strain or multiple variant strains of toxin-phage bacteriocide that has been substantially purified away from the bacterial host cells used to produce or amplify the toxin-phage bacteriocide. In other preferred embodiments, the compositions include a toxin-phage bacteriocide that has been substantially purified away from the bacterial host cell medium in which the bacterial host cells were grown during the production or amplification of the toxin-phage bacteriocide. In other embodiments, the compositions include a toxin-phage bacteriocide that has been partially purified from the bacterial host cells and bacterial host cell medium used to produce or amplify the toxin-phage bacteriocide.  
           [0023]    In another aspect, the invention features a method of using a toxin-phage bacteriocide to kill a bacterial cell. The method involves contacting bacterial cells (e.g., bacterial cells that include one or more strains or species of bacteria) with a toxin-phage bacteriocide, such that at least one toxin-phage is able to bind to and infect at least one bacterial cell, and then allowing the toxin-phage that have infected bacterial cells to kill the bacterial cells. In preferred embodiments, the toxin-phage binds to and infects bacterial cells that are of a selected type. In other preferred embodiments, the toxin-phage does not bind to or infect bacterial cells that are not of the selected type. The contacting can occur within a patient, e.g., a human or animal patient, or in vitro. In vitro studies using the gram negative  Escheria coli  and gram positive  Bacillus subtilis  have found a 100% non-infectivity in the presence of a foreign toxin-phage.  
           [0024]    In some embodiments, an infected bacterial cell is killed as a result of the toxin-phage entering into the lytic phase of its life-cycle, such that the bacterial cell is killed by lysis. In other embodiments, the infected bacterial cell is killed as a result of the expression of the toxic peptide encoded by the nucleic acid molecule that was introduced into the genome of the toxin-phage. In still other embodiments, the bacterial cell is killed by a combination of the toxin-phage entering into the lytic phase of its life-cycle and the expression of the toxic peptide encoded by the nucleic acid molecule that was introduced into the bacterial cell by the toxin-phage. In other embodiments, a bacterial cell that is killed is either a gram-negative or a gram-positive bacterial cell.  
           [0025]    The invention includes a method of treating a bacterial infection, e.g., an antibiotic resistant bacterial infection.  
           [0026]    In another aspect, the invention features a pharmaceutical composition that includes at least one toxin-phage bacteriocide and at least one pharmaceutically acceptable carrier. In preferred embodiments, the pharmaceutical composition can be used in vivo, e.g., the pharmaceutical composition can be administered, e.g., by parenteral injection or orally, to a subject, to treat a bacterial infection present in the subject. In other embodiments, the pharmaceutical composition can be used topically to treat a bacterial infection present in or on a subject.  
           [0027]    In another aspect, the invention features a method of using a toxin-phage bacteriocide to treat a bacterial infection present in or on a subject. In some embodiments, the subject is a farm animal, e.g., a chicken, pig, goat, sheep, cow, or horse. In other embodiments the subject is a plant, e.g., an agricultural product or orchard tree. In other embodiments, the subject is a pet, e.g., a fish, bird, cat, or dog. In still other embodiments, the subject is a mammal, a primate, or a human. In preferred embodiments, the toxin-phage bactericide kills the bacteria that are the cause of the infection. In other embodiments, the toxin-phage bactericide slows or brings to a halt the spread of the bacterial infection. In preferred embodiments, the toxin-phage bactericide helps eliminate the bacterial infection. In other preferred embodiments, the toxin-phage bactericide does not kill the bacterial cells that are not the cause of the infection, e.g., bacterial cells that are normally present in the subject or are beneficial to the subject. In other embodiments, the infection constitutes a localized disease, e.g., a disease of the skin, nervous system, cardiovascular system, respiratory system, digestive system, and urinary and reproductive systems.  
           [0028]    In another aspect, the invention features a method of using a toxin-phage bacteriocide to prophylactically treat a potential bacterial infection in a subject. In some embodiments, the subject is a farm animal, e.g., a chicken, pig, goat, sheep, cow, or horse. In other embodiments the subject is a plant, e.g., an agricultural product or orchard tree. In other embodiments, the subject is a pet, e.g., a fish, bird, cat, or dog. In still other embodiments, the subject is a mammal, a primate, or a human. In preferred embodiments, the toxin-phage bactericide kills the bacteria that are the potential cause of infection. In other embodiments, the toxin-phage slows or brings to a halt the growth of the bacteria that are the potential cause of infection. In other preferred embodiments, the toxin-phage bactericide does not kill bacterial cells that are not the potential cause of infection, e.g., bacterial cells that are normally present in the subject or are beneficial to the subject. In other embodiments, the potential bacterial infection can result in acne, e.g., skin acne in a human. In other embodiments, the subject has an injury, e.g., a cut that breaks the outer dermal layer of the skin, an animal bite, a dermal burn, or a surgical wound or incision, or a surgically inserted device, e.g., a catheter, that is highly susceptible to bacterial infection. In still other embodiments, the potential bacterial infection can involve exposure to biological weapons, e.g., anthrax, plague, or tularemia.  
           [0029]    In another aspect, the invention features a method of treating an aqueous solution with a toxin-phage bacteriocide such that bacteria present in the solution are killed. In one embodiment, the resulting aqueous solution is partially sterilized and can subsequently be consumed by an animal, e.g., a farm animal, pet, mammal, primate, or human. Treatment of the aqueous solution will reduce the chance of bacterial infection resulting from consumption of the solution. In another embodiment, the aqueous solution is a solution that is subject to bacterial contamination, e.g., the water in a fish tank or wastewater, e.g., sewage. In other embodiments the invention features a method of treating a culture, e.g., an eukaryotic cell culture used for production of a recombinant protein with a toxin-phage bacteriocide.  
           [0030]    In another aspect, the invention features a method of treating a surface with one or more toxin-phage bacteriocides such that bacteria attached to the surface are killed or their growth is inhibited. In one embodiment, the surface is part of a device, e.g., a device that is used in medicine (e.g., surgical instruments, a stent, a catheter, an artificial organ, or an artificial heart valve), agriculture, industrial processes, or water and wastewater treatment. In another embodiment, the surface is covered with a biofilm. In other embodiments, the surface is treated regularly with a toxin-phage bacteriocide such that the formation of a biofilm is prevented or slowed.  
           [0031]    The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. 
       
    
    
     DESCRIPTION OF DRAWINGS  
       [0032]    [0032]FIG. 1 is a graph illustrating growth curves of  Escherichia coli  ( E. coli ) HB101 cells cultured in TB (Terrific Broth) and in the presence of a synthetically produced derivative of peptide toxin A. Different concentrations (0 (squares), 8.24 μM (circles), or 32.7 μM (triangles)) of the synthetic peptide were added to the growth media at To and cell growth was monitored for 24 hours (see Example 8). Growth of the cells over the first 6 hours at 37° C. is recorded in the graph. The X axis represents time (hours). The Y axis represents culture absorbance at 600 nm (OD 600 ).  
         [0033]    [0033]FIG. 2 is a graph illustrating growth curves of  Escherichia coli  ( E. coli ) HB101 cells as in FIG. 1. Growth of the cells over 24 hours at 37° C. is recorded in the graph.  
         [0034]    [0034]FIG. 3 is a graph illustrating growth curves of  Escherichia coli  ( E. coli ) HB101 cells cultured in LB broth and in the presence of a synthetically produced derivative of peptide toxin A or a variant of peptide toxin A (see Example 9). Different concentrations (0, 1 nM, 100 nM or 10 μM) of the synthetic peptides were added to the growth media at T 0  and cell growth at 37° C. was monitored for 72 hours.  
         [0035]    [0035]FIG. 4 is a graph illustrating growth curves of  Escherichia coli  ( E. coli ) HB101 cells cultured in Hanks Buffer and in the presence of a synthetically produced derivative of peptide toxin A or a variant of peptide toxin A (see Example 9). Different concentrations (0, 1 nM, 100 nM or 100 μM) of the synthetic peptides were added to the growth media at To and cell growth at 37° C. was monitored for 72 hours.  
         [0036]    [0036]FIG. 5 is a graph illustrating growth curves of the yeast  Pichia pastoris  cultured in YPD broth and in the presence of a synthetically produced derivative of peptide toxin A or a variant of peptide toxin A (see Example 9). Different concentrations (0, 1 nM, 100 nM or 10 μM) of the synthetic peptides were added to the growth media at T 0  and cell growth at 30° C. was monitored for 72 hours.  
         [0037]    [0037]FIG. 6 is a graph illustrating growth curves of the yeast  Pichia pastoris  cultured in Hanks Buffer and in the presence of a synthetically produced derivative of peptide toxin A or a variant of peptide toxin A (see Example 9). Different concentrations (0, 1 nM, 100 nM or 10 μM) of the synthetic peptides were added to the growth media at To and cell growth at 30° C. was monitored for 72 hours.  
         [0038]    [0038]FIG. 7 is a graph illustrating growth curves of untransformed BL21-DE3  E. coli  cells (circles) and BL21-DE3  E. coli  cells expressing TPB peptide toxin A C-terminally tagged with a HIS 12 peptide (squares) (see Example 1). Cell growth at was monitored for 7 hours.  
         [0039]    [0039]FIG. 8 is a graph illustrating growth curves of  E. coli  cells uninfected with phage (triangles), infected with wildtype lambda phage (squares), and infected with TPB phage (circles) (see Example 14). Cell growth at 37° C. was monitored for 400 minutes. 
     
    
     DETAILED DESCRIPTION  
       [0040]    The recombinant toxin-phage bacteriocide (TPB) of the invention is a genetically modified bacteriophage that has been modified to harbor a nucleotide sequence encoding a specialized intracellular peptide toxin. This peptide toxin, e.g., peptide toxin A (SEQ ID NO:2), is toxic to cells, e.g., bacterial cells when it is present inside the cell, but not when it is outside of a cell. The TPB allows efficient production of a peptide toxin within bacterial cells, thus killing the cells. The TPB of the invention are capable of killing a targeted species of bacteria during both lytic and lysogenic infection with a bacteriophage. This is in contrast to many therapeutic bacteriophage used previously, which can kill host bacteria only during the lytic phase. The TPB of the invention are species-specific. Therefore, significant numbers of commensal bacterial within the host will not become infected or killed by the TPB. Upon infection the TPB delivers its chromosomal DNA into the bacterial host cell. Lytic toxin-phage reproduction results in additional TPB that burst from the cell and infect additional bacterial host cells. Alternatively, depending on various environmental factors, some TPB infected bacterial cells enter lysogeny, incorporating the TPB chromosomal DNA into their own chromosomal DNA. Upon lysogenization of the bacterial cell, but not limited to this temporal event, the bacterial cell&#39;s transcriptional and translational apparatus produces the intracellular peptide toxin. The intracellular peptide toxin, when presented to a cell internally, kills the cell. Upon death of the cell, the intracellular peptide toxin is released into the extracellular environment. However, intracellular peptide toxins are not significantly toxic to cells when presented externally. For example, TPB peptide toxin A had no observable effect on cultures of  E. coli  or  Bacillus Subtilis  growing at 37° C. even when present at concentrations as high as 34.6 mM over a 25 hour period. Similarly, TPB peptide toxin A added to cultures of  Pichia pastoris  yeast cells had no observable effect. Finally, 10 μm TPB peptide toxin A had no observable effect on confluent mouse mammary carcinoma cells growing in EMT6 medium or Hanks Balanced Salt Solution over a 6 hour period.  
         [0041]    TPB can be designed to be specific for any selected strain of bacteria, thus desirable bacteria can be spared. Bacteriophage specific for a single bacterial host in nature have been found to remain within the host for as long as the bacterial host specific for that phage is present. Weber-Dabrowska, et al. (1987),  Arch Immunol Ther Exp  (Warsz) 35(5):563-8, tested for absorption of orally administered anti-staphylococcal and anti-pseudomomas phage in both urine and serum samples of patients with suppurative bacterial infections. No phage was present in any of the 56 patients prior to phage therapy. By day 10, 84% of the serum samples and 35% of urine samples contained phage, indicating bioavailability. The healthy control group exhibited a phage titer drop 100-fold between days 0-5. A comprehensive review of phage therapy (Alisky et al. (1998),  J of Infection  36:5) concluded that all studies with both human and animals showed no measurable antiphage antibodies generated.  
         [0042]    Without being bound by any particular theory, it appears that the TPB peptide toxin A, produced by a TPB of the invention, becomes introduced into internally available membranes of the cell. This has been observed to occur in both bacterial and yeast cells. In vitro studies using a lipid bilayer membrane model suggest that the toxin peptide permeabilizes membranes. Significantly, the TPB peptide toxin A does not appear to harm either bacterial cells or eukaryotic cells when applied externally, e.g., when introduced in a culture of growing cells.  
         [0043]    The peptide toxin A of the invention has also been found to be toxic to eukaryotic cell when presented internally. Thus, intracellular peptide toxins can be used to selectively target undesirable eukaryotic cells, e.g., cancer cells or virally infected cells, by selectively delivering the peptide toxins to the interior of the undesirable cells. Thus, the peptide toxins can be targeted to such cells in various ways, e.g., through receptor mediated targeting.  
         [0044]    This invention is further illustrated by the following examples that should not be construed as limiting.  
       EXAMPLE 1  
     Production of a Toxin Gene Master Stock  
       [0045]    A nucleic acid molecule encoding the TPB peptide toxin A can be prepared synthetically. The molecule has the sequence: ATG GAT TGG CTG AAA GCT CGG GTT GAA CAG GAA CTG CAG GCT CTG GAA GCA CGT GGT ACC GAT TCC AAC GCT GAG CTG CGG GCT ATG GAA GCT AAA CTT AAG GCT GAA ATC CAG AAG (SEQ ID NO:1). The nucleic acid molecule encodes a 39 amino acid peptide having the sequence: MDWLKARVEQELQALEARGTDSNAELRAMEAKLKAEIQK (SEQ ID NO:2).  
         [0046]    The TPB peptide toxin A encoding nucleic acid molecule (SEQ ID NO:1) was inserted into pET19b plasmid (Novagen, Inc.; Madison, Wis.). The expression vector BL21-Gold(DE3)pLysS (Stratagene, Inc.; La Jolla Calif.) was used for expression of the TPB peptide toxin A for in vitro studies. A TPB peptide toxin A encoding gene can be prepared by PCR amplifying a TPB peptide toxin A encoding nucleic acid molecule out of the pET19b plasmid, as discussed below, or by PCR amplification from a synthetically prepared nucleic acid molecule.  
         [0047]    The top strand 5′ oligonucleotide (SEQ ID NO:3) used for PCR amplification of the TPB peptide toxin A encoding gene included: an multiple cloning site (MCS), a promoter sequence that is functionally active in both gram-negative and gram-positive bacterial hosts, and a sequence homologous to the 5′ start region of the toxin gene sequence. It had the following sequence:  GCGTCCGGCGTAGAGGATCCAAGCTT TAATTTAAATTTTATTTGACAAAAATGGG CTCGTGTTGTACAAATGT ATGGATTGGCTGAAAGCTCGGGTTGAACAGG  (SEQ ID NO:3). The first underlined portion is the MCS sequence. Restriction endonucleases that are capable of cutting within this MCS sequence are shown in Table I.  
                                                         TABLE I                                   Enzyme   No.   Position   Sequence                                        AclWI   1   19   ggatc           AluI   1   23   ag/ct           AlwI   1   19   ggatc           BamHI   1   15   g/gatcc           BsiSI   1   5   c/cgg           Bsp143I   1   15   /gatc           BstI   1   15   g/gatcc           BstX2I   1   15   r/gatcy           BstYI   1   15   r/gatcy           CviJI   1   23   rg/cy           DpnI   1   17   ga/tc           DpnII   1   15   /gatc           HapII   1   5   c/cgg           HgaI   1   5   gacgc           HindIII   1   21   a/agctt           HpaII   1   5   c/cgg           Kzo9I   1   15   /gatc           MboI   1   15   /gatc           MflI   1   15   r/gatcy           MnlI   1   16   cctc           MseI   1   26   t/taa           MspI   1   5   c/cgg           NdeII   1   15   /gatc           NlaIV   1   17   ggn/ncc           PspN4I   1   17   ggn/ncc           Sau3AI   1   15   /gatc           Sse9I   1   27   /aatt           Tru1I   1   26   t/taa           Tru9I   1   26   t/taa           Tsp509I   1   27   /aatt           TspEI   1   27   /aatt           XhoII   1   15   r/gatcy                      
 
         [0048]    The central portion of the top strand 5′ oligonucleotide sequence (SEQ ID NO:3), which is not underlined, constitutes the VegI/II promoter sequence. The VegI/II promoter sequence has been shown by Pescheke et al. (1985),  J Mol Biol  186:547, to be active in both gram-negative and gram-positive bacterial cells. The second underlined portion of the top strand 5′ oligonucleotide sequence corresponds to the 5′ end of the TPB peptide toxin A gene sequence (SEQ ID NO:1). This sequence is capable of annealing to the bottom strand of the pET19b plasmid, e.g., in a PCR reaction.  
         [0049]    The bottom strand 3′ oligonucleotide (SEQ ID NO:4) used for PCR amplification of the TPB peptide toxin A encoding gene included a MCS site and a sequence complementary to the 3′ end of the toxin gene sequence. The terminator region present in the pET19b vector was not amplified so that the functional properties of the toxin peptide could be disrupted, rendering the gene product less toxic to the master stock host cell. The bottom strand 3′ oligonucleotide used for PCR amplification had the sequence:  CCATCGATGGCCGCTCGAG CTATTATTTCTGGATTTCAG (SEQ ID NO:4). The underlined portion of SEQ ID NO:4 constitutes the multiple cloning sites (MCS) sequence Restriction endonucleases that are capable of cutting within this MCS sequence are shown in Table II.  
                                                         TABLE II                                   Enzyme   #   Position   Sequence                                        AccBSI   1   16   gagcgg           AciI   1   14   ccgc           Ama87I   1   14   c/ycgrg           AvaI   1   14   c/ycgrg           BanIII   1   4   at/cgat           BcoI   1   14   c/ycgrg           Bsa29I   1   4   at/cgat           BscI   1   4   at/cgat           BseCI   1   4   at/cgat           BsoBI   1   14   c/ycgrg           BsoFI   1   11   gc/ngc           Bsp106I   1   4   at/cgat           BspDI   1   4   at/cgat           BspXI   1   4   at/cgat           BsrBI   1   6   gagcgg           BstD102I   1   16   gagcgg           Bsu15I   1   4   at/cgat           BsuRI   1   10   gg/cc           CfrI   1   8   y/ggccr           ClaI   1   4   at/cgat           CviJI   1   10   rg/cy           EaeI   1   8   y/ggccr           Eco88I   1   14   c/ycgrg           Fsp4HI   1   11   gc/ngc           HaeIII   1   10   gg/cc           ItaI   1   11   gc/ngc           PaeR7I   1   14   c/tcgag           PalI   1   10   gg/cc           Sfr274I   1   14   c/tcgag           TaqI   2   4, 15   t/cga           TthHB8I   2   4, 15   t/cga           XhoI   1   14   c/tcgag                      
 
         [0050]    The portion of the bottom strand 3′ oligonucleotide sequence that is not underlined is complementary to the 3′ end of the TPB peptide toxin A encoding gene sequence. This complementary sequence is capable of annealing to the top strand of the pET19b plasmid, e.g., in a PCR reaction.  
         [0051]    Following PCR amplification of the TPB peptide toxin A encoding gene using the top strand 5′ and the bottom strand 3′ oligonucleotides described above, the PCR product was gel purified (Qiagen, QIAquick Gel Extraction Kit, Cat.No.28704) and sequenced (by Research Genetics). Oligonucleotide primers used for sequencing included: GGCGTATCACGAGGCCC (SEQ ID NO:5); and GTGGCGCCGGTGATGCCGG (SEQ ID NO:6). SEQ ID NO:5 was used to sequence the PCR product from the 5′ direction, while SEQ ID NO:6 was used to sequence the PCR product from the 3′ direction.  
         [0052]    The purified PCR product was cut with the restriction endonucleases ClaI and BamHI and ligated into a pBR322 plasmid (ATCC 37017, 31344) that had been cut with the same enzymes. Insertion of the PCR product containing the TPB peptide toxin A gene PCR product into the pBR322 plasmid disrupted the tetR gene, negating tetracycline resistance. This disruption, in turn, allowed for a positive gene incorporation selection tool. Once a positive clone was identified, the region of the plasmid containing the TPB peptide toxin A gene PCR product was analyzed using restriction digests, and then sequenced.  
         [0053]    The resulting plasmid was transformed into competent HB101 (MAX Efficiency HB101 Competent Cells, Cat. No. 18296-012, Life Technologies), and a positive clone was chosen using ampicillin resistance as a selection criteria. A single colony clone was selected and cultured to exponential growth phase (LB, 37° C., 250 rpms), mixed with sterile glycerol (80:20 ratio) and stored in a −76° C. freezer.  
       EXAMPLE 2  
     Selection of Toxin Gene Integration Sites  
       [0054]    Both a gram-negative and a gram-positive bacterial species with their complimentary bacteriophage were chosen to illustrate the effectiveness of TPB peptide toxin A.  
         [0055]    [0055] Escherichia coli  (c600, ATCC Accession No. 23724) was chosen as an example of a gram-negative bacterial species that could be tested for the effects of a toxin-phage bacteriocide. There are many bacteriophage that are known to infect  E. coli,  one of which is lambda phage (ATCC Accession No. 23724-B2). The sequence of the lambda phage genome is described in Sanger et al. (1992)  J Mol Biol  162:729, the contents of which are incorporated herein by reference. The integration site for the TPB peptide toxin gene into the lambda phage genome was chosen to be between nucleotides 46,468 and 46,469. The nucleotide sequences of the regions immediately surrounding the chosen integration site are as follows: TTGCCCATATCGATGGGCAACTCATGCAATTATTGTGAG (SEQ ID NO:7); and CAATACACACGCGCTTCCAGCGGAGTATAAATGCCTAAAGTA (SEQ ID NO:8). SEQ ID NO:7 corresponds to the nucleotide sequence that is 5′ to the integration site, about nucleotides 46,430-46,468 of the lambda phage genome, while SEQ ID NO:8 corresponds to the nucleotide sequence that is 3′ to the integration site, about nucleotides 46,469-46,510 of the lambda phage genome.  
         [0056]    [0056] Bacillus subtilis  (BGSC #1L32, BGSC, Ohio State University, Columbus, Ohio) was chosen as an example of a gram-positive bacterial species that could be tested for the effects of a toxin-phage bacteriocide. There are many bacteriophage that are known to infect  B. subtilis,  one of which is phi-105 (BGSC #1A304(phi-105), BGSC, Ohio State University, Columbus, Ohio). The sequence of the phi-105 genome is available from the NCBI database on the Internet at ncbi.nim.nih.gov/entrez/query.fcgi. The integration site for the TPB peptide toxin gene into the phi-105 genome was chosen to be between nucleotides 38,448 and 38,449. The nucleotide sequences of the regions immediately surrounding the chosen integration site are as follows: GGGTAGTTGCATACCACTAAAGATGTTCAGGTGCACATG (SEQ ID NO:9); and AGCATTGGAGGAAAGGAACGCTTTAGGGGGAAGGGAAACC (SEQ ID NO:10). SEQ ID NO:9 corresponds to the nucleotide sequence that is 5′ to the integration site, about nucleotides 38,409-38,448 of the phi-105 genome, while SEQ ID NO:10 corresponds to the nucleotide sequence that is 3′ to the integration site, about nucleotides 38,449-38,488 of the phi-105 genome.  
       EXAMPLE 3  
     Introduction of a 3′ Terminator Sequence  
       [0057]    Before introducing the TPB peptide toxin A gene into the bacteriophage genomes, a terminator sequence can be added to the 3′ end of the toxin gene in order increase the stability of toxin gene RNA synthesized within the bacterial host cell. Addition of a terminator sequence to the 3′ end of the toxin gene can be accomplished by PCR, as it was in this example, as well as by other techniques known in the art, e.g., restriction fragment subcloning.  
         [0058]    The top strand 5′ oligonucleotide (SEQ ID NO:12) used to introduce the terminator sequence included the MCS sequences (SEQ ID NO:13) and a portion of the VegI/II promoter described in Example 1. The bottom strand 3′ oligonucleotide (SEQ ID NO:13) used to introduce the terminator sequence included a MCS sequence distinct from the MCS sequences described in Example 1, a 3′ terminator sequence, and a sequence complementary to the 3′ end of the TPB peptide toxin A gene.  
         [0059]    The top strand 5′ oligonucleotide used to add the terminator sequence to the TPB peptide toxin A gene had the sequence:  CGTCCGGCGTAGAGGATCCAAGCTT TAATTTAAATTTT (SEQ ID NO:11). The underlined portion of the top strand 5′ oligonucleotide sequence constitutes the MCS sequence. The multiple cloning sites sequence was introduced to allow versatility in manipulation of the PCR products and possible associated vectors. Restriction endonucleases that are capable of cutting within this MCS sequence are shown in Table I. The portion of the top strand 5′ oligonucleotide sequence that is not underlined corresponds to a portion of the VegI/II bacterial promoter added to the 5′ end of the TPB peptide toxin A gene produced in Example 1. The entire sequence of the top strand 5′ oligonucleotide sequence (SEQ ID NO:11) is capable of annealing to the TPB peptide toxin A gene construct produced in Example 1.  
         [0060]    The bottom strand 3′ oligonucleotide used to add the terminator sequence to the TPB peptide toxin A gene had the sequence:  
                                         (SEQ ID NO:12)                      CGGGAAGCTTGGATCCGCATAGC AAAACGGACATCACTCCGTTTCAATGG           AGGTGATGTCCGTTTT CCGCTCGAGCTATTATTTCTGGATTTCAGC .          
 
         [0061]    The first underlined portion of the bottom strand 3′ oligonucleotide sequence constitutes the MCS sequence. Restriction endonucleases that are capable of cutting within this MCS sequence are shown in Table III.  
                                                         TABLE III                                   Enzyme   #   Position   Sequence                                        AciI   1   18   ccgc           AclWI   1   15   ggatc           AluI   1   7   ag/ct           AlwI   1   15   ggatc           BamHI   1   11   g/gatcc           Bsp143I   1   11   /gatc           BstI   1   11   g/gatcc           BstX2I   1   11   r/gatcy           BstYI   1   11   r/gatcy           CviJI   1   7   rg/cy           DpnI   1   13   ga/tc           DpnII   1   11   /gatc           HindIII   1   5   a/agctt           Kzo9I   1   11   /gatc           MboI   1   11   /gatc           MflI   1   11   r/gatcy           NdeII   1   11   /gatc           NlaIV   1   13   ggn/ncc           PspN4I   1   13   ggn/ncc           Sau3AI   1   11   /gatc           XhoII   1   11   r/gatcy                      
 
         [0062]    The central portion of the bottom strand 3′ oligonucleotide sequence (SEQ ID NO:12), which is not underlined above, is the 3′ terminator sequence complement. The corresponding 3′ terminator sequence has been shown to form a stem-loop structure that is a positive retroregulator that stabilizes mRNAs in bacteria. This 3′ terminator sequence has been described in Wong and Chang (1986)  Proc Natl Acad Sci USA  83:3233. The second underlined portion of the bottom strand 3′ oligonucleotide sequence is the complement of the 3′ end of the TPB peptide toxin A gene sequence (SEQ ID NO:1). This sequence is capable of annealing to the bottom strand of the TPB peptide toxin A gene master stock plasmid produced in Example 1, e.g., in a PCR reaction.  
         [0063]    DNA isolated from the toxin gene bacterial stock produced in Example 1 was used as template for the PCR reaction involving the top strand 5′ and bottom strand 3′ oligonucleotides described above (SEQ ID NOS:11 and 12, respectively). Following PCR, the amplified TPB peptide toxin A gene containing the 3′ terminator sequence was gel purified (Qiagen, QIAquick Gel Extraction Kit, Cat.No.28704), analyzed by endonuclease restriction fragment analysis, and used in Example 4.  
       EXAMPLE 4  
     Recombinageneic Bacteriophage-Integrating Intracellular Peptide Toxin Genes  
       [0064]    Generation of toxin genes recombinagenic with a phage genome can be produced by introducing phage genomic sequences located 5′ and 3′ to a chosen integration site in the phage genome to the 5′ and 3′ ends, respectively, of a intracellular peptide toxin encoding gene.  
         [0065]    Generation of toxin genes recombinagenic with the lambda phage genome were produced by the addition of lambda phage genomic sequences located 5′(SEQ ID NO:7) and 3′ (SEQ ID NO:8) to the chosen integration site (see Example 2) to the 5′ and 3′ ends, respectively, of the TPB peptide toxin A gene produced in Example 3. A single round of PCR was used to make the additions. The primers used in the PCR reaction included a 5′ Lambda Oligonucleotide (SEQ ID NO:13), consisting of a MCS sequence, a 5′ homologous recombination sequence, a HindIII restriction site sequence, and a 5′ annealing sequence, and a 3′ Lambda Oligonucleotide (SEQ ID NO:14), consisting of a MCS sequence, a 3′ homologous recombination sequence, a second MCS sequence, and a 3′ annealing sequence.  
         [0066]    The 5′ Lambda Oligonucleotide had the sequence:  
                                         (SEQ ID NO:13)                      CCGGAATTCGCTAGCGGGCCCGAG TTGCCCATATCGATGGGCAACTCATG           CAATTATTGTGAG AAGCTTT AATTTAAATTTTATTTGACAAAAATGGG.          
 
         [0067]    The first underlined portion of the 5′ Lambda Oligonucleotide sequence constitutes MCS sequence. The MCS was introduced so that it would be easier to manipulate the PCR product for possible cloning into alternative vectors. Alternatively, the MCS region allows one to determine whether the toxin gene had integrated into the desired location in the lambda phage genome. Integration events that retain this MCS are not likely to have occurred in the desired location and can be discarded, whereas integration events that occurred via homologous recombination are likely to lack this MCS. Alternatively, the homologous recombinant sequence can be PCR amplified without the MCS and introduced into the phage genome. Restriction endonucleases that are capable of cutting within this MCS sequence are shown in Table IV.  
                                                         TABLE IV                                   Enzyme   #   Position   Sequence                                        AciI   1   17   ccgc           AcsI   1   4   r/aatty           Ama87I   1   19   c/ycgrg           ApaI   1   20   gggcc/c           ApoI   1   4   r/aatty           AspS9I   1   16   g/gncc           AsuI   1   16   g/gncc           AvaI   1   19   c/ycgrg           BanII   1   20   grgcy/c           BcoI   1   19   c/ycgrg           BfaI   1   11   c/tag           BmyI   1   20   gdgch/c           BsiSI   1   1   c/cgg           BsoBI   1   19   c/ycgrg           Bsp120I   1   16   g/ggccc           Bsp1286I   1   20   gdgch/c           BsuRI   1   18   gg/cc           Cac8I   1   12   gcn/ngc           Cfr13I   1   16   g/gncc           CviJI   1   18   rg/cy           Eco24I   1   20   grgcy/c           Eco88I   1   19   c/ycgrg           EcoRI   1   4   g/aattc           FauI   1   18   cccgc           FriOI   1   20   grgcy/c           HaeIII   1   18   gg/cc           HapII   1   1   c/cgg           HpaII   1   1   c/cgg           MaeI   1   11   c/tag           MspI   1   1   c/cgg           NheI   1   10   g/ctagc           NlaIV   1   18   ggn/ncc           PalI   1   18   gg/cc           PspN4I   1   18   ggn/ncc           PspOMI   1   16   g/ggccc           PstNHI   1   10   g/ctagc           Sau96I   1   16   g/gncc           SduI   1   20   gdgch/c           Sse9I   1   4   /aatt           Tsp509I   1   4   /aatt           TspEI   1   4   /aatt                      
 
         [0068]    The first portion of the 5′ Lambda Oligonucleotide (that is not underlined) constitutes the 5′ homologous recombination sequence, which was identified in Example 2 as the lambda phage sequence 5′ to the integration site. The second underlined portion of the 5′ Lambda Oligonucleotide constitutes a Hind III restriction site. Successful targeting of the toxin gene to the chosen site in the lambda phage genome will also result in the introduction of a new Hind III restriction site into the genome at the chosen site. Thus, restriction digest analysis of targeted lambda clones can help assess whether the targeting was successful and whether the toxin gene that has been introduced is intact, i.e., lacks deletions, rearrangements, etc. The second portion of the 5′ Lambda Oligonucleotide that is not underlined constitutes the 5′ annealing region, which is homologous to a portion of the VegI/II promoter sequence located at the 5′ end of the PCR product produced in Example 3. This sequence is designed to anneal to the PCR product of Example 3, thereby allowing PCR amplification of a toxin gene that contains lambda phage targeting sequences.  
         [0069]    The 3′ Lambda Oligonucleotide had the sequence:  CGCCCTAGGCGGCCGAGGACCC TACTTTAGGCATTTATACTCCGCTGGAAGCGC GTGTGTATT GGCATGCATCGATTAGT AAAACGGACATCACTCCG (SEQ ID NO:14). The first underlined portion of the 3′ Lambda Oligonucleotide sequence constitutes the first MCS sequence. The MCS was introduced so that it would be easier to manipulate the PCR product for possible cloning into alternative vectors. In addition, this multiple cloning sites sequence was introduced so that it would be easier to determine whether the toxin gene had integrated into the desired location in the lambda phage genome. Integration events that retain this MCS are not likely to have occurred in the desired location and can be discarded, whereas integration events that occurred via homologous recombination are likely to lack this MCS. Alternatively, the homologous recombinant sequence can be PCR amplified without the MCS and introduced into the phage genome. Restriction endonucleases that are capable of cutting within this MCS sequence are shown in Table V.  
                                                         TABLE V                                   Enzyme   #   Position   Sequence                                        AciI   1   12   ccgc           AspS9I   1   17   g/gncc           AsuI   1   17   g/gncc           AvaII   1   17   g/gwcc           AvrII   1   4   c/ctagg           BfaI   1   5   c/tag           BlnI   1   4   c/ctagg           Bme18I   1   17   g/gwcc           BsaJI   2   4, 13   c/cnngg           BsaOI   1   13   cgry/cg           BseDI   2   4, 13   c/cnngg           Bsh1285I   1   13   cgry/cg           BsiEI   1   13   cgry/cg           BsoFI   1   10   gc/ngc           BssT1I   1   4   c/cwwgg           BstMCI   1   13   cgry/cg           BstZI   1   10   c/ggccg           BsuRI   1   12   gg/cc           Cfr13I   1   17   g/gncc           CfrI   1   10   y/ggccr           CviJI   1   12   rg/cy           DraII   1   17   rg/gnccy           EaeI   1   10   y/ggccr           EagI   1   10   c/ggccg           EclXI   1   10   c/ggccg           Eco130I   1   4   c/cwwgg           Eco47I   1   17   g/gwcc           Eco52I   1   10   c/ggccg           EcoO109I   1   17   rg/gnccy           EcoT14I   1   4   c/cwwgg           ErhI   1   4   c/cwwgg           Fsp4HI   1   10   gc/ngc           HaeIII   1   12   gg/cc           HgiEI   1   17   g/gwcc           ItaI   1   10   gc/ngc           MaeI   1   5   c/tag           MnlI   1   18   cctc           NlaIV   1   19   ggn/ncc           PalI   1   12   gg/cc           PpuMI   1   17   rg/gwccy           Psp5II   1   17   rg/gwccy           PspN4I   1   19   ggn/ncc           Sau96I   1   17   g/gncc           SinI   1   17   g/gwcc           StyI   1   4   c/cwwgg           XmaIII   1   10   c/ggccg                      
 
         [0070]    The first portion of the 3′ Lambda Oligonucleotide that is not underlined constitutes the 3′ homologous recombination sequence, which was identified in Example 2 as the lambda phage sequence 3′ to the integration site. The second underlined portion of the 3′ Lambda Oligonucleotide constitutes a second MCS sequence. Successful targeting of the toxin gene to the chosen site in the lambda phage genome will also result in the introduction of the restriction sites present in this MCS into the genome at the chosen site. Thus, restriction digest analysis of targeted lambda clones can help assess whether the targeting was successful and whether the toxin gene that has been introduced is intact, i.e., lacks deletions, rearrangements, etc. Restriction endonucleases that are capable of cutting within this MCS sequence are shown in Table VI.  
                                                         TABLE VI                                   Enzyme   #   Position   Sequence                                        BanIII   1   9   at/cgat           BbuI   1   6   gcatg/c           Bsa29I   1   9   at/cgat           BscI   1   9   at/cgat           BseCI   1   9   at/cgat           Bsp106I   1   9   at/cgat           BspDI   1   9   at/cgat           BspXI   1   9   at/cgat           Bsu15I   1   9   at/cgat           Cac8I   1   4   gcn/ngc           ClaI   1   9   at/cgat           EcoT22I   1   8   atgca/t           Hsp92II   1   6   catg/           Mph1103I   1   8   atgca/t           NlaIII   1   6   catg/           NsiI   1   8   atgca/t           NspI   1   6   rcatg/y           PaeI   1   6   gcatg/c           Ppu10I   1   4   a/tgcat           SfaNI   1   10   gcatc           SphI   1   6   gcatg/c           TaqI   1   9   t/cga           TthHB8I   1   9   t/cga           Zsp2I   1   8   atgca/t                      
 
         [0071]    The second portion of the 3′ Lambda Oligonucleotide (SEQ ID NO:25) that is not underlined constitutes the 5′ annealing region (SEQ ID NO:28), which is complementary to a portion of the 3′ terminator sequence located at the 3′ end of the PCR product produced in Example 3. This sequence is designed to anneal to the PCR product of Example 3, thereby allowing PCR amplification of a TPB peptide toxin A gene that contains lambda phage targeting sequences.  
         [0072]    Generation of toxin genes recombinagenic with the phi-105 phage genome were produced by the addition of phi-105 phage genomic sequences located 5′ (SEQ ID NO:9) and 3′α(SEQ ID NO:10) to the chosen integration site (see Example 2) to the 5′ and 3′ ends, respectively, of the TPB peptide toxin A gene produced in Example 3. A single round of PCR was used to make the additions. The primers used in the PCR reaction included a 5′ Phi-105 Oligonucleotide (SEQ ID NO:15), consisting of a MCS sequence, a 5′ homologous recombination sequence, a HindIII restriction site sequence, and a 5′ annealing sequence, and a 3′ Phi-105 Oligonucleotide (SEQ ID NO:16), consisting of a MCS sequence, a 3′ homologous recombination sequence, a second MCS sequence, and a 3′ annealing sequence.  
         [0073]    The 5′ Phi-105 Oligonucleotide had the sequence:  
                                         (SEQ ID NO:15)                      CCGGAATTCGCTAGCGGGCCCGAG GGGTAGTTGCATACCACTAAAGATGT           TCAGGTGCACATG AAGCTT TAATTTAAATTTTATTTGACAAAAATGGG.          
 
         [0074]    The first underlined portion of the 5′ Phi-105 Oligonucleotide sequence constitutes the MCS sequence. The multiple cloning sites sequence was introduced so that it would be easier to manipulate the PCR product for possible cloning into alternative vectors. In addition, the MCS region allows one to determine whether the toxin gene had integrated into the desired location in the lambda phage genome. Integration events that retain this MCS are not likely to have occurred in the desired location and can be discarded, whereas integration events that occurred via homologous recombination are likely to lack this MCS. Alternatively, the homologous recombinant sequence can be PCR amplified without the MCS and introduced into the phage genome. Restriction endonucleases that are capable of cutting within this MCS sequence are shown in Table VII.  
                                                         TABLE VII                                   Enzyme   #   Position   Sequence                                        AciI   1   17   ccgc           AcsI   1   4   r/aatty           Ama87I   1   19   c/ycgrg           ApaI   1   20   gggcc/c           ApoI   1   4   r/aatty           AspS9I   1   16   g/gncc           AsuI   1   16   g/gncc           AvaI   1   19   c/ycgrg           BanII   1   20   grgcy/c           BcoI   1   19   c/ycgrg           BfaI   1   11   c/tag           BmyI   1   20   gdgch/c           BsiSI   1   1   c/cgg           BsoBI   1   19   c/ycgrg           Bsp120I   1   16   g/ggccc           Bsp1286I   1   20   gdgch/c           BsuRI   1   18   gg/cc           Cac8I   1   12   gcn/ngc           Cfr13I   1   16   g/gncc           CviJI   1   18   rg/cy           Eco24I   1   20   grgcy/c           Eco88I   1   19   c/ycgrg           EcoRI   1   4   g/aattc           FauI   1   18   cccgc           FriOI   1   20   grgcy/c           HaeIII   1   18   gg/cc           HapII   1   1   c/cgg           HpaII   1   1   c/cgg           MaeI   1   11   c/tag           MspI   1   1   c/cgg           NheI   1   10   g/ctagc           NlaIV   1   18   ggn/ncc           PalI   1   18   gg/cc           PspN4I   1   18   ggn/ncc           PspOMI   1   16   g/ggccc           PstNHI   1   10   g/ctagc           Sau96I   1   16   g/gncc           SduI   1   20   gdgch/c           Sse9I   1   4   /aatt           Tsp509I   1   4   /aatt           TspEI   1   4   /aatt                      
 
         [0075]    The first portion of the 5′ Phi-105 Oligonucleotide that is not underlined constitutes the 5′ homologous recombination sequence, which was identified in Example 2 as the phi-105 phage sequence 5′ to the integration site. The second underlined portion of the 5′ Phi-105 Oligonucleotide constitutes a Hind III restriction site. Successful targeting of the toxin gene to the chosen site in the phi-105 phage genome will also result in the introduction of a new Hind III restriction site into the genome at the chosen site. Thus, restriction digest analysis of targeted phi-105 clones can help assess whether the targeting was successful and whether the toxin gene that has been introduced is intact, i.e., lacks deletions, rearrangements, etc. The second portion of the 5′ Phi-105 Oligonucleotide that is not underlined constitutes the 5′ annealing region, which is homologous to a portion of the VegI/II promoter sequence located at the 5′ end of the PCR product produced in Example 3. This sequence is designed to anneal to the PCR product of Example 3, thereby allowing PCR amplification of a toxin gene that contains phi-105 phage targeting sequences.  
         [0076]    The 3′ Phi-105 Oligonucleotide had the following sequence:  
                                         (SEQ ID NO:16)                      CGCCCTAGGCGGCCGAGGACCC GGTTTCCCTTCCCCCTAAAGCGTTCCTT           TCCTCCAATGCT GGCATGCATCGATTAGT AAAACGGACATCACTCCG.          
 
         [0077]    The first underlined portion of the 3′ Phi-105 Oligonucleotide sequence constitutes the first MCS sequence. This multiple cloning site sequence was introduced so that it would be easier to manipulate the PCR product for possible cloning into alternative vectors. In addition, it would be easier to determine whether the toxin gene had integrated into the desired location in the lambda phage genome. Integration events that retain this MCS are not likely to have occurred in the desired location and can be discarded, whereas integration events that occurred via homologous recombination are likely to lack this MCS. Alternatively, the homologous recombinant sequence can be PCR amplified without the MCS and introduced into the phage genome. Restriction endonucleases that are capable of cutting within this MCS sequence are shown in Table VIII.  
                                                         TABLE VIII                                   Enzyme   #   Position   Sequence                                        AciI   1   12   ccgc           AspS9I   1   17   g/gncc           AsuI   1   17   g/gncc           AvaII   1   17   g/gwcc           AvrII   1   4   c/ctagg           BfaI   1   5   c/tag           BlnI   1   4   c/ctagg           Bme18I   1   17   g/gwcc           BsaJI   2   4, 13   c/cnngg           BsaOI   1   13   cgry/cg           BseDI   2   4, 13   c/cnngg           Bsh1285I   1   13   cgry/cg           BsiEI   1   13   cgry/cg           BsoFI   1   10   gc/ngc           BssT1I   1   4   c/cwwgg           BstMCI   1   13   cgry/cg           BstZI   1   10   c/ggccg           BsuRI   1   12   gg/cc           Cfr13I   1   17   g/gncc           CfrI   1   10   y/ggccr           CviJI   1   12   rg/cy           DraII   1   17   rg/gnccy           EaeI   1   10   y/ggccr           EagI   1   10   c/ggccg           EclXI   1   10   c/ggccg           Eco130I   1   4   c/cwwgg           Eco47I   1   17   g/gwcc           Eco52I   1   10   c/ggccg           EcoO109I   1   17   rg/gnccy           EcoT14   1   4   c/cwwgg           ErhI   1   4   c/cwwgg           Fsp4HI   1   10   gc/ngc           HaeIII   1   12   gg/cc           HgiEI   1   17   g/gwcc           ItaI   1   10   gc/ngc           MaeI   1   5   c/tag           MnlI   1   18   cctc           NlaIV   1   19   ggn/ncc           PalI   1   12   gg/cc           PpuMI   1   17   rg/gwccy           Psp5II   1   17   rg/gwccy           PspN4I   1   19   ggn/ncc           Sau96I   1   17   g/gncc           SinI   1   17   g/gwcc           StyI   1   4   c/cwwgg           XmaIII   1   10   c/ggccg                      
 
         [0078]    The first portion of the 3′ Phi-105 Oligonucleotide that is not underlined constitutes the 3′ homologous recombination sequence, which was identified in Example 2 as the phi-105 phage sequence 3′ to the integration site. The second underlined portion of the 3′ Phi-105 Oligonucleotide constitutes a second MCS sequence. Successful targeting of the toxin gene to the chosen site in the phi-105 phage genome will also result in the introduction of the restriction sites present in this MCS into the genome at the chosen site. Thus, restriction digest analysis of targeted phi-105 clones can help assess whether the targeting was successful and whether the toxin gene that has been introduced is intact, i.e., lacks deletions, rearrangements, etc. Restriction endonucleases that are capable of cutting within this MCS sequence are shown in Table IX.  
                                                         TABLE IX                                   Enzyme   #   Position   Sequence                                        BanIII   1   9   at/cgat           BbuI   1   6   gcatg/c           Bsa29I   1   9   at/cgat           BscI   1   9   at/cgat           BseCI   1   9   at/cgat           Bsp106I   1   9   at/cgat           BspDI   1   9   at/cgat           BspXI   1   9   at/cgat           Bsu15I   1   9   at/cgat           Cac8I   1   4   gcn/ngc           ClaI   1   9   at/cgat           EcoT22I   1   8   atgca/t           Hsp92II   1   6   catg/           Mph1103I   1   8   atgca/t           NlaIII   1   6   catg/           NsiI   1   8   atgca/t           NspI   1   6   rcatg/y           PaeI   1   6   gcatg/c           Ppu10I   1   4   a/tgcat           SfaNI   1   10   gcatc           SphI   1   6   gcatg/c           TaqI   1   9   t/cga           TthHB8I   1   9   t/cga           Zsp2I   1   8   atgca/t                      
 
         [0079]    The second portion of the 3′ Phi-105 Oligonucleotide that is not underlined constitutes the 5′ annealing region, which is complementary to a portion of the 3′ terminator sequence located at the 3′ end of the PCR product produced in Example 3. This sequence is designed to anneal to the PCR product of Example 3, thereby allowing PCR amplification of a TPB peptide toxin A gene that contains phi-105 phage targeting sequences.  
       EXAMPLE 5  
     In Vitro Homologous Recombination of an Intracellular Peptide Toxin Gene into a Bacteriophage Genome  
       [0080]    The homologous recombination event can be manipulated in vitro using isolated bacteriophage DNA added to a bacterial host cell supernatant seeded with the homologous recombination competent PCR product containing a intracellular toxin gene, using the basic protocols as described by Mackal, et al (1964),  PNAS  51:1172, the contents of which are incorporated herein by reference. Such a procedure was performed with the recombinagenic TPB peptide toxin gene PCR products produced in Example 4. After incubation at 37° C., the reaction mixtures are added to cell cultures of host cells and plated on the appropriate media by mixing in 2 mL of molten top agar poured onto a hardened bottom agar. Plates are incubated at 37° for the  E coli  C600 gram-negative bacteria, and 30° C. for the  B. subtilis  1L32 gram-positive bacteria. Plaques are screened for incorporation of the TPB peptide using Southern hybridization techniques. Plaques identified as positive are isolated and stocks are prepared from the single plaques. Chromosomal DNA isolated from these stocks is analyzed by restriction digestion, followed by sequencing.  
       EXAMPLE 6  
     In Vivo Homologous Recombination of an Intracellular Peptide Toxin Gene into a Bacteriophage Genome  
       [0081]    The homologous recombination event can be manipulated in vivo with competent bacterial cells lysogenic for the chosen bacteriophage. Competent  E coli  C600 gram-negative bacteria lysogenic for the wild-type lambda phage are prepared using the calcium chloride method, as described in  Molecular Cloning  (1989), 2 nd  Ed., Sambrook et al., Eds., Cold Spring Harbor Press, the contents of which are incorporated herein by reference. Competent  B. Subtilis  1L32 lysogenic for phi-105 phage are prepared using methods described by Errington &amp; Mandelstam (1983),  Journal of General Microbiology  129:2091, the contents of which are incorporated herein by reference. The recombinagenic PCR product containing the intracellular peptide toxin gene is added to the competent bacterial cells and heat shocked as described in Sambrook et al., supra. After a one hour incubation at 37° C., the reaction mixtures are added to cell cultures of host cells and plated on the appropriate media by mixing in 2 mL of molten top agar poured onto a hardened bottom agar. Plates are incubated at 37° for the  E coli  C600 gram-negative bacteria, and 30° C. for the  B. subtilis  1L32 gram-positive bacteria. Plaques are screened for incorporation of the TPB peptide using Southern hybridization techniques. Plaques identified as positive are isolated and stocks are prepared from the single plaques. Chromosomal DNA isolated from these stocks is analyzed by restriction digestion, followed by sequencing.  
       EXAMPLE 7  
     Use of a Toxin-Phage Bacteriocide to Kill Bacteria  
       [0082]    To test the effectiveness of TPB peptide toxin A, lambda phage (American Type Culture Collection (ATCC) Accession No. 23724-B2) was engineered to express TPB peptide toxin A. This modified phage killed 100% of  E. coli  (ATCC Accession No. 23724). No lysogenic colonies were observed.  
         [0083]    In addition, phi-105 phage (BGSC Accession No. 1A304 (phi105); Ohio State University, Columbus, Ohio) was engineered to express TPB peptide toxin A. This modified phage killed 100% of  B. subtilis  (BGSC Accession No. 1L32). No lysogenic clones were observed.  
       EXAMPLE 8  
     Analysis of the Effect of Extracellular Peptide Toxin A on Bacterial Growth  
       [0084]    To determine the extracellular toxicity of the toxin-phage bacteriocide (TPB) peptide toxin A, growth curves were generated for bacteria cultured in the presence of a synthetically produced derivative of the peptide. Because the toxin was not packaged in a phage particle, the bacterial cells presumably would not take it up. Two milliliters of TB (Terrific Broth) media was seeded with 50 μl of an  E. coli  HB101 cell culture grown overnight to saturation. The TPB peptide was added to the 2 mL culture at To. The growth of the bacterial cultures at 37° C. was assayed spectrophotometrically by monitoring the absorbance at 600 nm every hour for 24 hours (FIGS. 1 and 2). Two different concentrations of peptide (8.24 μM and 32.7 μM) were tested for an effect on cell growth.  
         [0085]    The growth curves illustrated in FIG. 1 revealed that the presence of peptide toxin A at concentrations as high as 32.7 μM did not alter cell growth, thus indicating that the peptide was non-toxic to the cells when present in the extracellular environment. Similar results were observed for the gram-positive bacterial strain  Bacillus subtilis  BGSC #1L32 (BGSC, Ohio State University, Columbus, Ohio).  
       EXAMPLE 9  
     Analysis of the Effect of an Extracellular Variant of Peptide Toxin A on Bacterial Growth  
       [0086]    A variant peptide toxin A was synthesized such that the amino acids Ala5 and Ala34 were deleted from the peptide. The extracellular toxicity of the peptide was tested by constructing growth curves of  E. coli  HB101 as described in Example 8. Cell growth was tested in two different types of culture media, LB Broth (FIG. 3) and Hanks Buffer (FIG. 4); and growth was monitored for 72 hours. Three different concentrations of peptides (1 nM, 100 nM, and 10 μM) were tested for an effect on cell growth. In a control experiment, peptide was not added to the culture.  
         [0087]    The growth curves in FIGS. 3 and 4 indicate that the cells cultured in the presence of the peptide toxin A variant grew at a rate equivalent to that of cells grown in the presence of the “wildtype” peptide or no peptide. The result was the same for cells cultured in LB Broth (FIG. 3) and cells grown in Hanks Buffer (FIG. 4), and was not affected by varying concentrations of peptide.  
         [0088]    The wildtype and variant peptides were also tested for an effect on the doubling time of the yeast strain  Pichia pastoris.  Cells were cultured at 30° C. in YPD Broth (FIG. 5) or Hanks Buffer (FIG. 6), and growth was monitored for 72 hours. The same three different peptide concentrations were tested as in the bacterial experiments described above.  
         [0089]    As was the result with bacterial growth, the growth curves in FIGS. 5 and 6 indicate that yeast cells cultured in the presence of the peptide toxin A variant grew at a rate equivalent to that of cells grown in the presence of the “wildtype” peptide or no peptide. The result was the same for cells cultured in YPD Broth and cells grown in Hanks Buffer, and was not affected by varying concentrations of peptide. Yeast cell growth was generally inhibited in Hanks Buffer (FIG. 6).  
         [0090]    These results indicated that minor changes in the toxin A peptide sequence could be engineered to have the same innocuous external effect on both bacterial and eukaryotic cell lines.  
       EXAMPLE 10  
     Analysis of Effect of Extracellular Peptide Toxin A on EMT6 Mammalian Cells in Culture  
       [0091]    Cultures of mammalian EMT6 cells were grown to confluency, and then various concentrations (0, 0.001 μM, 0.1 μM, and 10 μM) of the synthetic peptide toxin A mixed in Hanks Buffer solution were added to the culture media. The cultures were monitored by light microscopy at 40× magnification for 6.5 hours, over which period of time no changes were observed in cell morphology or confluency between untreated cultures, cultures treated with Hanks Buffer alone, and cultures treated with peptide toxin A in Hanks Buffer.  
         [0092]    In similar experiments, cultures of mammalian EMT6 cells were grown to confluency, and then various concentrations (0, 0.001 μM, 0.1 μM, and 10 μM) of the synthetic peptide toxin A mixed in Bovine serum solution were added to the culture media. The cultures were monitored by light microscopy at 40× magnification for 6.5 hours, over which period of time, no changes were observed in cell morphology or confluency between untreated cultures, cultures treated with Bovine serum alone, and cultures treated with peptide toxin A in Hanks Buffer.  
         [0093]    These results indicated that the peptide toxin A is non-toxic to mammalian cultured cells when present in the extracellular culture environment. This result was true regardless of whether the peptide was delivered as a solution in Hanks Buffer or as a solution in Bovine type serum.  
       EXAMPLE 11  
     Intracellular Bacterial Expression of Peptide Toxin A Fusion Proteins  
       [0094]    Attempts to express peptide toxin A alone in  E. coli  were unsuccessful. This is despite apparently successful attempts to clone a gene encoding the peptide alone into appropriate expression vectors; while cloning reactions appeared to be successful (as assessed by diagnostic PCR of ligation reactions), no positive clones have ever been achieved following transformation into competent host  E. coli  cells. To date, it has only been possible to express a chimeric form of the peptide, i.e., a HIS-tagged (from a pET19b vector; Novagen; Madison, Wis.) or a GST-(glutathione-S-transferase) tagged peptide. Therefore, peptide toxin A was cloned into a pET19b vector, and intracellular expression studies were performed using a peptide toxin A tagged at the C terminus with a HIS 12  peptide.  
         [0095]    The peptide toxin A-HIS fusion clone was transformed into BL21-DE3  E. coli  cells, and 60° C. freezer stocks were established for transformed and untransformed strains. To generate growth curves, transformed and untransformed cell cultures were seeded from the −60° C. stocks in 10 mL minimal media. After 2 hours shaking at 37° C., IPTG was added to both cultures to induce expression of the toxin A-HIS fusion gene in the transformed cells and to serve as a control for the untransformed cell culture. Cell growth was monitored spectrophotometrically at 600 nm for 7 hours. The resulting growth curves, shown in FIG. 7, indicated that the transformed BL21-DE3 cells did not grow significantly after expression of the toxin A-HIS fusion gene was induced at the 2 hour time point. Microscopic analysis revealed that 3 hours following induction of the toxin A fusion gene, cells clumped together, as compared to the control cultures. In addition, samples removed from the toxin-expressing cell culture were unable to re-propagate when placed in fresh media.  
         [0096]    These results indicated that the intracellular expression peptide toxin A fusion proteins inhibited bacterial cell growth.  
       EXAMPLE 12  
     Attempts to Generate anti-Peptide Toxin A Antibodies  
       [0097]    The production of antibodies specific against the peptide toxin A was attempted by Biosource International, Inc. (Camarillo, Calif.), employing a series of proprietary methods that included custom antigen design, immunization strategies and extensive affinity purification. Rabbits were used in attempts to produce the peptide toxin A antibodies, but all attempts failed. In addition, tetanus toxoid conjugates failed to generate high-affinity, high avidity, and high-specificity antibodies against peptide toxin A.  
         [0098]    The injected peptide toxin A did not appear to be harmful to the rabbits used in the attempts to inability to generate antibodies. This suggests that the toxin may not be harmful to mammals, generally, and particularly when the toxin is introduced into the body intravenously. Additional studies suggested that peptide toxin A is not harmful to fish. The peptide was added to standard fish food and fed to Medaka fish under controlled conditions. The fish remained healthy.  
       EXAMPLE 13  
     Methods used to Monitor the Successful Integration of the Toxin A Gene by Homologous Recombination  
       [0099]    A variety of methods were used to characterize the integration of the toxin phage gene into a bacteriophage chromosome.  
         [0100]    a) A Southern blotting technique was used to determine that the toxin A gene had integrated into the wildtype Phi 105 (Gram-positive phage) chromosome. DNA purified from wildtype Phi 105 and toxin-phage Phi 105 was analyzed on a 1% agarose gel alongside molecular weight controls. The gel was further analyzed using a Southern blotting method of analysis by chemiluminescent labeling. Positive signal was observed in the toxin-phage DNA lane but not in the wildtype phage DNA lane.  
         [0101]    b) A PCR-based technique employed oligonucleotides targeted to the 5′ and 3′ regions of the bateriophage chromosome insertion site. Amplification of a 450 bp product indicated insertion of the toxin A gene, while amplification of a 180 bp product indicated a wildtype bacteriophage DNA region. This approach not only verified insertion of the toxin A gene, but additionally verified that it was inserted into the appropriate target site.  
         [0102]    c) A plaque assay was a third method of testing for homologous recombination of the toxin A gene into the phage genome. By this method, completed homologous reaction mixes were added to an  E. coli  culture, mixed, and poured evenly onto an LB plate in top-agar. Different concentrations of the homologous reaction mix were mixed with the bacteria, resulting in slight to excessive bacterial lawn clearing. Areas that showed excessive clearing of the bacterial lawn were analyzed for positive toxin-phages. For the analysis, bacteriophage plaques were subcloned to generate single plaque colonies and tested for incorporation of the toxin A peptide gene. DNA was purified from the candidate toxin-phages and analyzed by a Southern blotting technique using chemiluminescence detection methods. DNA from wildtype lambda bacterial chromosomal DNA was analyzed as a negative control. This technique indicated the presence of toxin A sequence in DNA isolated from positive candidate toxin phage, but not in DNA isolated from wildtype lambda phage.  
       EXAMPLE 14  
     Effect of TPB Infection on Bacterial Growth  
       [0103]    Cultures of  E. coli  were grown overnight in LB broth at 37° C. and shaking at 250 rpm. Samples of the overnight cultures were removed and wildtype lambda phage or TPB-infected phage were added at an MOI (multiplicity of infection) of 1:1 (phage:bacteria). One control culture was not treated with phage. The samples were spectrophotometrically monitored at OD 600  every 30 minutes for 400 minutes while shaking at 250 rpm at 37° C.  
         [0104]    The growth curves, illustrated in FIG. 8, indicated that growth of bacteria infected with TPB phage was inhibited as compared to uninfected cells and cells infected with wildtype phage. Thus, TPB phage were more effective than wildtype lambda phage in decreasing  E. coli  population levels.  
       EXAMPLE 15  
     Identification of TPB Protein Expression Following Infection of Bacterial Cells  
       [0105]    Due to the inability to produce anti-toxin A peptide antibodies, an alternative and analogous experimental system was developed to determine if the incorporated toxin A peptide gene in the TPB could be expressed within the bacterial cell. In addition, due to the toxicity of the toxin A peptide, which results in the inability to directly and positively identify toxin A production, a fluorescent protein was utilized to facilitate an analogous identification and characterization of protein expression in infected bacterial cells. The toxin A gene was replaced with a gene encoding the Green Fluorescent Protein (GFP). The GFP gene was homologously recombined into a bacteriophage chromosome in exactly the same manner as the toxin A gene (supra). Gram-negative and gram-positive bacterial cells were infected with the resulting bacteriophage-GFP hybrids (called TPB-GFP) to test for GFP expression. Neither gram-negative nor gram-positive bacterial cells fluoresce under normal conditions.  
         [0106]    [0106] E. coli  (gram-negative) cells were infected with TPB-GFP. Cells were cultured in LB broth at 37° C., and visualized by light and fluorescence microscopy at 60× magnification.  
         [0107]    [0107] B. subtilis  (gram-positive) cells were infected with TPB-GFP. Cells were cultured in LB broth at 37° C., and visualized by light and fluorescent microscopy at 60× magnification.  
         [0108]    In a control experiment, wildtype  E. coli  were transformed with a plasmid expressing GFP. Cells were cultured in LB broth at 37° C., and visualized by light and fluorescent microscopy at 60× magnification.  
         [0109]    Fluorescent microscopy revealed that GFP was expressed in the gram-positive and gram-negative bacterial cells, and fluorescence was as bright or brighter than expression from a plasmid.  
         [0110]    These results indicated that when a protein-encoding gene (e.g., a GFP or toxin A gene) is incorporated into gram-negative or gram-positive bacteriophage, the gene can be expressed and a protein successfully synthesized in a phage-infected cell.  
         [0111]    A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.