# Patent

## Title
Compositions, methods and uses for a novel family of peptides

## Abstract

The present invention includes compositions and methods for the characterization and use of novel peptide from Brevibacillus sp., and peptides related thereto, including an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide that have two or more D-amino acids used as, e.g., an antimicrobial or even a feed additive.

## Background
This application claims benefit of U.S. Provisional application 60/540,569 filed Jan. 30, 2004.


TECHNICAL FIELD OF THE INVENTION
The present invention relates in general to the field of novel isolated and purified peptides, and more particularly, to the identification, characterization and use of a novel group of peptides from the newly discovered organism Brevibacillus texasporus. 
BACKGROUND OF THE INVENTION
This application claims priority to U.S. Provisional Patent Application Ser. No. 60/540,569, filed Jan. 30, 2004, relevant portions incorporated herein by reference. Without limiting the scope of the invention, its background is described in connection with antibiotics and feed additives.
Antibiotic overuse has led to widespread bacterial drug resistance. Novel antibiotics are needed to combat infections caused by bacterial resistant to conventional antibiotics. It is well known that microbes produce a huge variety of antibiotics to wage chemical warfare against competing microbes.
Many peptide antibiotics of microbial origin are synthesized by non-ribosomal peptide synthases (NRPS) and they contain unusual amino acids. NRPS enzymes usually have a co-linear modular architecture (Mootz, et al., 2002). The N-terminal to C-terminal order and specificities of the individual modules correspond to the sequential order and identities of the amino acid residues in the peptide product. Each NRPS module recognizes a specific amino acid and catalyzes stepwise condensation to form a growing peptide chain. The identity of the amino acid recognized by a particular module can be predicted by comparisons to other modules of known specificities (Challis, et al., 2000). Such strict correlation made it possible to identify genes encoding the NRPS enzymes for a number of microbial non-ribosomal peptides with known structures, as demonstrated by the identification of the mycobactin biosynthesis operon in the genome of Mycobacterium tuberculosis (Quadri, et al., 1998). Nevertheless, the art recognizes the continuing need to isolate, identify and characterize novel antimicrobial agents.
Examples of feed additives are widely known in the art. For example, U.S. Pat. No. 6,682,762 issued to Register, discloses one such Poultry and livestock feed additive. Briefly, this patent teaches a poultry and livestock feed additive composition containing 36 wt. % electrolytes, roughage and mineral oil to increase their dietary electrolyte balance. Addition of the electrolyte additive composition improves breeder hen performance as to egg production, body weight, and reduced mortality from heat stress. Broiler chickens on this diet result in increased processing yield, feed conversion and body weight. A method of preparing this dietary electrolyte feed for poultry and livestock is also described.
Yet another example of a feed additive is a taught by Nagai, et al., in U.S. Pat. No. 6,503,544, which teaches an animal feed additive that includes at least two components selected from the group consisting of the following three components (a), (b) and (c): (a) at least one herb selected from Pine Needle, Hawthorn Fruit, Bighead Atractylodes Rhizome, Milkvetch Root, Skullcap Root, Tangerine Fruit and Mint Siftings; (b) a live bacteria mixture composed of a yeast cell wall and a live bacteria preparation containing Lactobacillus acidophilus and/or Enterococcus faecium; and (c) an organic acid.
Feed additives may also include the byproducts of fermentation and other precesses, such as those taught by U.S. Pat. No. 5,863,574 issued to Julien for a feed additive for ruminant animals containing fungal and/or bacterial fermentation byproducts. The feed additive for ruminants, includes dried fungal and/or bacterial fermentation by products which provide glutamic acid fermentation solubles, dried corn fermentation solubles, or a mixture of dried glutamic acid fermentation solubles and dried corn fermentation solubles, wherein the dried solubles have been dried to a total moisture content of less than 30% by weight at a temperature not less than about 80° F. and not more than about 900° F.
SUMMARY OF THE INVENTION
In one embodiment, the present invention is a feed additive that includes an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids isolated from Brevibacillus sp. The carboxy-terminus —COOH group of the C-terminal Valine of the peptide may be reduced to —CH2OH, and may confer protease resistance to the peptide. The peptide feed additive may be stable at a pH of 1.0, at a pH 13.0, resistant to proteases or combinations thereof. Examples of the peptide may be selected from one or more of SEQ ID NOS: 1 to 20 (collectively called the BT peptides). It has been found that the peptide kills, gram positive bacteria, gram negative bacteria, fungi, protozoa or combinations thereof. The peptide may be isolated from Brevibacillus texasporus (ATCC PTA-5854) and may be added to feed at between about 0.5 and about 100 ppm. In one use, the peptide was added at between about 6 and about 12 ppm and demonstrated statistically significant growth stimulation.
The additive peptide may be added to a feed adapted for use by one or more of poultry, livestock, farm-raised fish, crabs, shrimp and fresh-water turtles. For example, the peptide may be included in a cereal-based animal feed, e.g., at least one cereal selected from barley, soya, wheat, triticale, rye and maize; and an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids isolated from Brevibacillus sp. The peptide-based feed additive may be include at between about 1 and 1000 ppm of an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide having two or more D-amino acids isolated from Brevibacillus sp. In fact, the present invention may be used with any of a large variety of feeds.
The present invention also includes an antimicrobial peptide that has two or more D-amino acids, carboxy-terminus reduced pH and heat stable isolated from Brevibacillus sp. For example, the present invention includes a biologically pure culture of microorganism Brevibacillus texasporus deposit No. ATCC PTA-5854) that produces an antimicrobial peptide that is carboxy-terminus reduced heat stable, amino terminus-methylated peptide and may include two or more D-amino acids. The feed additive may even be an isolated and purified microorganism of ATCC PTA-5854. The additive may be mixed with a feed for livestock selected from the group consisting of a milk replacer, a grower feed, a finisher feed, a pre-starter feed and a starter feed.
The present invention also includes a method for increasing body weight gain efficiency and feed efficiency in animals, by providing one or more of the BT peptides in an effective amount sufficient to increase growth in an animal feed. The animal feed is adapted for feeding livestock selected from the group consisting of, e.g., cattle, swine, chicken, horse, turkey, sheep, goat, farm-raised fish, crab, shrimp and turtle. Examples of feeds also include those for feeding birds selected from the group consisting of, e.g., chicken, turkey, duck, quail, Cornish hens, and pigeon. As such, the feed may be selected from the group consisting of, e.g., a cereal, soybean meal, isolated soybean protein, isolated soybean oil, isolated soybean fat, skimmed milk, fish meal, meat meal, bone meal, blood meal, blood plasma protein, whey, rice bran, wheat bran, a sweetener, a mineral, a vitamin, salt, and grass. Daily dose of the peptide ranges from about 0.01 to about 10 grams per kg body weight of the animal.
In yet another embodiment, the present invention is a broad spectrum antimicrobial compound for topical use comprising a peptide having two or more D-amino acids, carboxy-terminus reduced, pH and heat stable isolated from Brevibacillus sp. (Brevibacillus texasporus) For example, the peptide may have the sequence Me2Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-Vol (SEQ ID NO.: 1), wherein Vol is Valine alcohol, or any one of SED ID NOS.: 1-20.
Yet another embodiment is an isolated and purified nucleic acid having the sequence of BT operon (SEQ ID NO.: 21) that produces a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids. The isolated and purified nucleic acid that encode one or more polypeptide sequences for BT operon proteins (SEQ ID NOS.: 22 to 28) that include one or more enzymes used to make a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids. The invention also includes those isolated nucleic acids having at least 75% homology to SEQ ID NO.: 21. More specifically, the nucleic acid may encodes one or more polypeptide sequences for peptide synthesis operon proteins (SEQ ID NOS.: 22 to 28) that are enzymes used to make a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids. One or more BT operon polypeptides are expressed from SEQ ID NO.:21 and comprise one or more enzymes used to make a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids.
An isolated bacterial sample for use with the present invention may include an isolated bacterial strain of Brevibacillus texasporus E58. Another embodiment is an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide having two or more D-amino acids isolated from Brevibacillus sp that inhibits the growth of at least one bacterium selected from the group consisting of: Staphylococcus, Enterococcus, Pneumococcus, Bacilli, Methanococcus, Haemophilus, Archaeoglobus, Borrelia, Synedrocyptis, Mycobacteria, Pseudomonas and E. coli. A bacteria may be transformed with an isolated and purified nucleic acid having the sequence of BT operon (SEQ ID NO.: 21) that produces a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids. The protein expressed from the nucleic acid may include one or more BT operon proteins, or those related thereto. A vector may be modified or isolated that includes an isolated and purified nucleic acid having the sequence of BT operon (SEQ ID NO.: 21) that produces a heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids. One or more proteins may be expressed from the nucleic acid that encodes one or more BT operon proteins. The feed additive may also include an isolated and purified, heat stable, amino terminus-methylated carboxy-terminus reduced peptide that has greater than 75% sequence homology to SEQ ID NOS.: 1-20.
The present invention also relates to peptides, and non-ribosomal peptide synthases that synthesize these peptides containing unusual amino acids and other types of modifications. The invention also includes methods of producing and using the peptides alone or synergistically with conventional antibiotics in the treatment and prevention of various microbial infections and protozoal infections and disorders related to such infections; tumor cell proliferation, growth and spread; or as an immune modulating agents.



BRIEF DESCRIPTION OF THE DRAWINGS
For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:
FIG. 1A is a gel that shows the purification of peptide BT. Tricine gel separation of BT and the associated antibiotic activity. Chloroform extracted peptides were separated on a precast 16.5% Tricine gel (purchase from BioRad). One gel was stained with coomassie blue (left) to show peptide bands. Another gel (right) was overlaid with agar embedded with Bacillus cereus. Clear zones in the bacterial lawn correspond to the species that collapses into a single band with a molecular weight of  ̃1500. Molecular weight markers are as follows: triosephosphate isomerase 26.6 kD, myoglobin 17.0 kD, alpha-lactalbumin 14.4 kD, aprotinin 6.5 kD, insulin b chain, oxidized 3.5 kD, and bacitracin 1.4 kD.
FIG. 1B is a graph of mass spectrometry of chloroform-extracted BT Chloroform-extracted BT was ionized by addition of sodium chloride and then subjected to mass spectrometry analysis. Five ionized BT isomer groups (BT1555, BT1571, BT1583, BT1599 and BT1613) were detected and labeled.
FIG. 1C is a graph of mass spectrometry of purified BT1583. Fraction 33 of the C18 reverse phase HPLC was subjected to mass spectrometry analysis. Only protonated, sodium and potassium ionized BT1583 were detected;
FIG. 2A is a graph of BT1583 tandem mass spectrometry data. FIG. 2B is a partial BT1583 sequence structure deduced from amino acid composition and MS/MS experiments (Tables 1 and 2)(for complete motif and sequences see Tables 5 and 6, respectively);
FIGS. 3A to 3D are maps of the BT NRPS operon. FIG. 3A is a map of the construction of a supercontig from two contigs linked by a mate pair. Contig1 and contig 2 share a mate pair from a clone. The contigs are ordered and arranged to form a supercontig, which contains the sequences of contig 1 and contig 2, separated by an unsequenced gap region;
FIG. 3B is a map of the region sequenced in this work and the location of 9 ORFs found in the region. Six ORFs btA through btF encode the BT NRPS subunits (BtA, BtB, BtC, BtD, BtE and BtF);
FIG. 3C is a map of the domain organization of the BT NRPS subunits. The predicted amino acid substrate specificity of each module is marked in each A-domain;
FIG. 3D is a Phylogenetic tree of a multiple sequence alignment of all 13 binding pocket constituents as described in Table 3. The putative specificity was assigned using the partial BT1583 sequence. It is shown that those binding pockets of A-domains that supposedly activate the same or similar substrate cluster together;
FIGS. 3E-1 to 3E-12 are the nucleic acid sequence of the BT operon (SEQ ID NO.:21), the entire sequence is subdivided into 12 figures;
FIG. 3F is the amino acid sequence of BtA (SEQ ID NO.:22);
FIG. 3G is the amino acid sequence of BtB (SEQ ID NO.:23);
FIGS. 3H-1 and 3H-2 are the amino acid sequence of BtC (SEQ ID NO.:24), the entire sequence is divided into 2 figures;
FIG. 3I is the amino acid sequence of BtD (SEQ ID NO.:25);
FIG. 3J is the amino acid sequence of BtE (SEQ ID NO.:26);
FIG. 3K is the amino acid sequence of BtF (SEQ ID NO.:27);
FIG. 3L is the amino acid sequence of BtG (SEQ ID NO.:28);
FIGS. 4A to 4E are sequence alignment of conserved motifs and alignments of the adenylation, consensation, thilation, epimerization and reductase domains from the BT NRPS modules, respectively. Conserved motifs were identified according to (Marahiel, 1997). Consensus sequences were placed under each alignment. Residues agree with consensus were black shaded. All 12 C-domains were aligned together, with the * symbols indicate the start C domains that are known to be less conserved;
FIGS. 5A to 5E are ATP-PPi exchange assays for the relative substrate specificities of the purified A-domains of Modules 8, 5, 7 ,4 and 2, respectively, obtained from the ATP-PPi exchange assays were listed A) to D), respectively. The highest activity was defined as 100%. All 20 proteinogenic amino acids and L-Om were tested in each assay, and background was usually below 1%. Apparent Km of the A-domains toward specific amino acids were listed underneath; and
FIG. 6A is a summary of synthetic BT variants and FIG. 6B is a correlation between the BT variants and their properties for antibiotic activity and Pronase resistance as previously described.



DETAILED DESCRIPTION OF THE INVENTION
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.
As used herein, the term “domestic animal” describes, e.g., swine, cattle, horse, goat, sheep, deer, dog, cat and any of a number of useful rodents. The term “poultry” as used herein includes, e.g., chicken, hen, quail, turkey, guinea fowl and so forth. The term “nursery fishes” used here includes, e.g., carp, catfish, rainbow trout, ayu (sweet fish), eel, tilapia, conger, salmon, trout, red seabream, yellow tail, flounder, globefish, and so forth. A number of other animals are contemplated to also be useful, e.g., shrimp and prawn.
As used herein, the terms “additive” and “feed additive” are used to describe compositions from bacteria that may be used in conjunction with animal feed as feed additive resulting in an improvement of the health of livestock, poultry and fish, and a reduction of economic loss due to reduced or low weight and/or increasing the rate of growth (e.g., weight) of existing health animals. For example, the feed additive of the present invention may be used from bacterial isolates, partially or wholly degraded bacteria, isolated, isolated and purified from bacteria and/or synthesized synthetically in whole or in part. The additive or feed additive for the domestic animals, poultry and fishes may be of powder, grain or liquid form and will be used in accordance with the feeding condition and installations of the farm and the target animal.
Suitable animal feedstuffs include, e.g., green feed, silages, dried green feed, roots, tubers, fleshy fruits, grains and seeds, brewer's grains, pomace, brewer's yeast, distiller's spent grains, milling byproducts, byproducts of the production of sugar, starch and oil recovery and various food wastes. The feed additive of the present invention may be used alone or in conjunction with other well-known feed additives such as antioxidants or mixtures of various substances (mineral mixtures, vitamin mixtures) that can be added to such feeds for enhancement. Specific feeds may also adapted for certain animal species depending on age and stages of development.
Base feeds suitable for use in conjunction with the peptides of the present invention may be prepared as is well-known to the artisan skilled in the art of preparing feeds, e.g., they may use those as described in Kirk-Othmer, Encyclopedia of Chemical Technology, 4th Ed., vol. 10, pp. 288-300, Wiley, N.Y., 1993, relevant portions incorporated herein by reference. For example, the base feed may include one or more of the following ingredients: corn, sorghum, barley, wheat, soybean, peanut, canola, fish meal, milk products, fats and oils, vitamins and minerals.
The present inventor recognized that an NRPS operon may be a source of information that allows one to learn certain structural details of the peptide product. The identification of the BT NRPS operon results in critical refinements of the BT1583 peptide structure. Soil microorganisms were screened for strains that produce novel antibiotics. A Bacillus spp. E58 (ATCC PTA-5854) was isolated for its ability to produce an antibiotic BT against Staphylococcal aureus that cause life-threatening hospital-acquired infections in immunity-compromised patients The strain was named Brevibacillus texasporus based on its relatedness to Brevibacillus laterosporus. 
The modules of an NRPS are composed of smaller units or “domains” that each carries out a specific role in the recognition, activation, modification or joining of amino acid precursors to form the peptide product. One type of domain, the adenylation (A-) domain, is responsible for selectively recognizing and activating the amino acid that is to be incorporated by a particular module of the NRPS. Through analysis of the substrate-binding pocket of the A-domain of the PheA subunit of the Gramicidin S NRPS in combination with sequence comparison with other A-domains, it was possible to define ten residues that are the main determinants of the substrate specificity for an A-domain (Conti, et al., 1997; Stachelhaus, et al., 1999). The ten residues are considered an NRPS ‘codon’. The NRPS codon collection is still growing as new NRPS codons continue to be discovered. The present invention includes the isolated and purified nucleic acids and the proteins encoded thereby for a group of novel NRPS codons for Valine, Lysine, Ornithine and Tyrosine.
The amino acid activation step is ATP-dependent and involves the transient formation of an amino-acyl-adenylate. The activated amino acid is covalently attached to the peptide synthase through another type of domain, the thiolation (T-) domain that is generally located adjacent to the A-domain. The T-domain is post-translationally modified by the covalent attachment of a phosphopantetheinyl prosthetic arm to a conserved serine residue. The activated amino acid substrates are tethered onto the NRPS via a thioester bond to the phosphopantetheinyl prosthetic arm of the respective T-domains. Amino acids joined to successive units of the NRPS are subsequently covalently linked together by the formation of amide bonds catalyzed by another type of domain, the condensation (C-) domain. NRPS modules can also occasionally contain additional functional domains that carry out auxiliary reactions, the most common being epimerization of an amino acid substrate from the L- to the D-form. This reaction is catalyzed by a domain referred to as an epimerization (E-) domain that is generally located adjacent to the T-domain of a given NRPS module. Thus, a typical NRPS module has the following domain organization: C-A-T-(E).
Product assembly by NRPS involves three distinct phases, namely chain initiation, chain elongation, and chain termination (Keating & Walsh, 1999). Peptide chain initiation is carried out by specialized modules termed a “starter module” that comprises an A-domain and a T-domain. Elongation modules have, in addition, a C-domain that is located upstream of the A-domain. It has been experimentally demonstrated that such elongation domains cannot initiate peptide bond formation due to interference by the C-domain (Linne & Marahiel, 2000). All the growing peptide intermediates are covalently tethered to the NRPS during translocations as an elongating series of acyl-S-enzyme intermediates. To release the mature peptide product from the NRPS, the terminal acyl-S-enzyme bond must be broken. This process is the chain termination step and is usually catalyzed by a C-terminal thioesterase (TE) domain. Thioesterase-mediated release of the mature peptide from the NRPS enzyme involves the transient formation of an acyl-O-TE intermediate that is then hydrolyzed or hydrolyzed and concomitantly cyclized to release the mature peptide (Keating, et al., 2001). An alternative termination scheme involves reduction of the tethered C-terminal residue by a reductase (R-) domain that resides in the last NRPS module, resulting in release of a peptide with an alcoholic C-terminal residue (Gaitatzis, et al., 2001; Kessler, et al., 2004). Such reductase-mediated termination/C-terminal modification occurs in BT biosynthesis and contributes to super protease resistance of the BT peptides.
Identification and isolation of the NRPS operon was useful to the studies of a peptide antibiotic, however, identification of a specific NRPS operon remains a challenging task. Identification of an NRPS operon traditionally starts with identification of clones in a genomic BAC or cosmid library by hybridization with DNA probes from known NRPS genes or by gene fragments amplified by PCR of genomic DNA using degenerate primers. Because the amino acid sequences of NRPS domains are usually quite similar, such approaches can be successful, however, because probes or primers are often imperfect, some NRPS operons can be missed. Moreover, microbes often contain multiple NRPS operons, so that the probes or primers may reveal some NRPS operons but not the one sought. This often results in ill-fated efforts devoted towards an incorrect gene (Hopwood, 1997). A novel in silica approach was used as described herein to allow rapid and accurate identification of an NRPS operon.
Materials and Methods. Partial purification of BT. E58 B. texasporus cells were grown in one liter of LB in a 37° C. air shaker for three days. The culture was spun in a clinical centrifuge at 3000 rpm for 15 minutes. The supernatant was collected and 500 grams of ammonium sulfate was added and dissolved. The sample was spun in the clinical centrifuge at 3000 rpm for 15 minutes. The pellets were dissolved in 200 ml of distilled water. The solution was then boiled for 15 minutes and then cooled on ice. The sample was filtered with a 0.2 micron filter (Nalgen). The filtrate was mixed with 0.2 liter of chloroform at room temperature for 20 minutes with a stir bar. The mixture was separated into two phases through centrifugation in the clinical centrifuge at 3000 rpm for 15 minutes. The organic phase was collected and dried in a vacuum evaporator.
C18 reverse phase HPLC. The dried chloroform extract was dissolved in 2 ml of sterile distilled water. The solution was fractionated on a C18 reverse phase HPLC column in a gradient from 30% B to 55% Solution B (Solution B is 0.075% TFA in acetonitrile, Solution A is 0.1% TFA in water). Resultant fractions were dried and dissolved in sterile distilled water and analyzed for anti-S. aureus activity in a plate clear zone assay The peak fraction (Fraction 33) was subjected to amino acid composition, mass spectrometry, tandem mass spectrometry and chirality analyses.
Amino acid composition. Amino acid analysis was performed by the Protein Chemistry Laboratory at Texas A&M University in College Station, Tex. Samples were mixed with internal standards, dried in glass tubes in a vacuum concentrator and subjected to vapor phase hydrolysis by 6N HCl at 110° C. for 24 hours under argon atmosphere in the presence of phenol. The samples were subsequently reconstituted in borate buffer and transferred to a Hewlett Packard AminoQuant II system for automated derivatization and loading. The AminoQuant analyzes peptides and proteins by pre-column derivatization of hydrolyzed samples with o-phthalaldehyde (OPA) and 9-fluoromethyl-chloroformate (FMOC). The derivatized amino acids are separated by reverse phase HPLC and detected by UV absorbance with a diode array detector or by fluorescence using an in-line fluorescence detector.
Mass spectrometry and tandem mass spectrometry. Detection of D-form amino acid residues. The chiral analysis of amino acid residues in BT was performed by Commonwealth Biotechnologies, Inc. of Richmond, Va. BT was subjected to hydrolysis in 6N HCl in vacuum for 18 hours at 110° C. The amino acids were derivatized to FMOC amino acids and separated by HPLC chromatography. The elution profile of each amino acid was then determined on a chiral column. For both types of chromatography columns, peaks were identified by comparisons with appropriate standards.
Genomic DNA preparation. Log-phase E58 cells were harvested from an LB culture and lysed with Lysis Buffer [10 mM Tris (pH 8.0), 100 mM EDTA, 0.5% SDS]. RNase A was added to digest contaminating RNA. Genomic DNA was extracted with phenol/choloroform and then precipitated with ethanol. Dried DNA was resuspended in TE and an aliquot was run in 0.5% agarose gel for quality control.
Library construction and genome sequencing. The E58 genomic library construction, shot-gun sequencing and the assembly were performed by Agencourt Biosciences Corporation (Beverly, Mass.). Briefly, the whole genome library was constructed with an average insert length around 5 kb. 10,000 such clones were subject to automated DNA sequencing from both ends of the insert. 16,901 successfully sequenced reads were collected and assembled.
Nucleotide sequences and data analysis. All BLAST analyses against E58 genome were performed by use of WU BLAST software package (version 2.0) installed on a local computer (Gish, W. 1996-2003. http://blast.wustl.edu). Amino acid sequence homology searches were performed by use of the BLAST server at the National Center for Biotechnology Information (Bethesda, Md.) and nonredundant protein sequence database with default parameter values (Altschul, et al., 1990). Amino acid sequence alignments were performed by use of the CLUSTALW program (Thompson, et al., 1994) running at NPS@ web server at Institute of Biology and Chemistry of Proteins (Lyon, France).
The BT NRPS operon. The BT NRPS operon (Supercontig 3) contained 11 contigs, spanning a region of at least 46 kb. There were unsequenced regions, regions that were just sequenced once, and regions with bad sequencing quality. Also, carboxyl region of the thirteenth module was not covered by Supercontig 3. Three rounds of primer extension sequencing and one round of genome walking were performed to achieve the finishing of the NRPS operon. All original sequencing reads in Superontig 3 were extracted and reassembled using the SeqMan (Lasergene, DNASTAR Inc.). The default parameters were used for the reassembly. A higher stringency adopted by SeqMan caused the reassembled Supercotig 3 to break into 17 contigs with 16 unsequenced gaps. All contigs were further examined manually for single coverage and bad quality regions. Primers were designed to sequence into a gap as well as to obtain additional reads in the single coverage and low quality regions. New sequencing reads were joined with the original reads to create a new supercotig. The new supercotig was checked again for gaps, single coverage and low quality regions. After three rounds of such primer extension and reassembly, the putative BT NRPS operon was assembled into a single contig of 48,997 bp in length. To verify the assembled sequence, an EcoRI plus HindIII double digestion was performed with 20 clones that collectively spanned the whole region. Resultant digestion patterns were in perfect agreement with the restriction map predicted by the contig (data not shown). To sequence the downstream region of the contig, genome walking was successfully performed with E58 genomic DNA using GenomeWalker kit from Clontech. The effort resulted in a DNA sequence of 50,674 bp covering the putative BT NRPS operon.
Cloning, overexpression, and purification of His10-tagged BT A-domain proteins. DNA fragments encoding the A-domains of the BT NRPS Modules 8, 5, 7, 4 and 2 (Bt8A, Bt5A, Bt7A, Bt4A and Bt2A) were PCR-amplified and the PCR products were inserted into the His10-tag recombinant protein expression vector pET16b (Novagen). The A-domain borders were determined as defined by (Konz, et al., 1999). The expression constructs were transformed into the E.coli BL21 -AI strain (Invitrogen). Transformants were grown in L-broth at 37° C. to an A600 of 0.6 and then induced with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) plus 0.2% L-arabinose. The cells were allowed to grow for two additional hours at 30° C. before being harvested. Purification of the His10-tag recombinant proteins was achieved using the TALON metal affinity resins (BD Biosciences) under conditions recommended in the manual with modifications. Briefly, the E. coli cells were broken by sonication. Cell lysates were cleared by centrifugation at 25,000× g for one hour. His-tagged recombinant proteins were then incubated with the TALON resin, washed, and eluted with 500 mM imidazole. Eluted proteins were dialyzed against a buffer (50 mM HEPES, pH 8.0, 100 mM sodium chloride, 10 mM magnesium chloride, and 1 mM EDTA) and then analyzed with SDS PAGE plus Coomassie Blue staining. The recombinant proteins displayed apparent molecular weights compatible with calculated ones, and they appeared to be purified to homogeneity. Concentrations of the purified proteins were determined by using the calculated molar extinction coefficient for the A280.
ATP-PPi exchange assay. ATP-PPi exchange assays were performed to determine the substrate specificity of an A-domain. ATP-PPi exchanges were assayed as previously described (Stachelhaus, et al., 1998) with minor modifications. The assay mixture contained 50 mM HEPES (pH 8.0), 100 mM NaCl, 10 mM MgCl2, 2 mM ATP, 0.5 mM amino acid, 0.05 mM PPi, 0.15 μCi tetrasodium [32P]pyrophosphate. Exchange was initiated by addition of purified recombinant A-domain proteins to a total volume of 0.1 ml. The protein concentrations were 0.2 μM for Module 4 and Module 5 A-domains while 2 μM for Module 7 and Module 8 A-domains. After incubation at 37° C. for 15 min, the reaction was stopped by addition of 0.5 ml of Termination Mix (100 mM tetrasodium pyrophosphate, 3.5% HClO4, and 1.6% [w/v] activated charcoal). The charcoal was pelleted by centrifugation, washed first with 40 mM pyrophosphate plus 1.4% perchloric acid and then with water, and was re-suspended in 0.5 ml of water. The charcoal/water suspension was added to a scintillation vial containing 5.0 ml of scintillation fluid, and the bound radioactivity was determined by liquid scintillation counting. The apparent Km values were determined with substrate concentrations ranging from 0.1 to 10 mM.
MIC determination assays. Staphylococcal aureus was grown to mid-log phase in LB at 37° C., and diluted by 500-fold with fresh LB and dispensed into 96-well micro-titer plates. Different concentrations of peptides were added, and the micro-titer plates were incubated at 37° C. with shaking. A minimal inhibition concentration (MIC) was determined as the lowest peptide concentration that produced a clear well. All experiments were performed in triplicates, and highly consistent MICs were obtained.
Identification of the BT peptides. The bacterial strain E58 was isolated from soil in an effort to identify soil microorganisms that produce novel antibiotics against Staphylococcus aureus. E58 was found to be closely related to Brevibacillus laterosporus based on the 16S rDNA sequence homology (98.5% identity). E58 was named Brevibacillus texasporus and deposited to ATCC (catalog number PTA-5854). The antibiotic produced by E58 was named BT and its activity could be detected in the supernatant of a liquid E58 LB culture. Cell-free culture supernatant was, therefore, the starting material for BT purification. The antibiotic activity was precipitated by ammonium sulfate, which suggested that the antibiotic be a protein or peptide (data not shown). The activity was further extracted into chloroform, indicating that BT is made of small molecules. The antibiotic chloroform extract was evaporated in a vacuum evaporator, dissolved in water and then run on a SDS tricine gel. The two halves of a gel with identical lanes in each half were either stained for proteins/peptides or overlaid with agar embedded with BT-sensitive bacteria Bacillus cereus to test for antibiotic activity (FIG. 1A). Three species were visible after staining: the Bromophenol Blue dye originated from the gel loading buffer, an unknown peptide with a mass<1.4 kD and a third species with an antibiotic activity. This third species ran as a  ̃1.5 kD band at low concentrations (clearly visible on the original gel) and were later shown made up of a group of related peptides (see below). Their apparent masses increased with concentration suggesting that the peptides aggregate at higher concentrations. An antibiotic activity was seen associated with the peptides at higher concentrations, and we therefore concluded that the peptides likely conferred the BT antibiotic activity. The peptides were referred as the BT peptides. The BT peptides apparently were not toxic to B. cereus at the lower concentrations in this assay. Since the smallest detectable BT band ran at  ̃1.5 kD, therefore the BT peptides contained approximately 13 residues.
The chloroform-extracted BT was subject to a mass spectrometry assay. A group of peptides were detected in a range between 1550 and 1650 Daltons (FIG. 1B). The main species showed a molecular weight of 1583, and it was named BT1583. The other peptides were later shown to be isomers of BT1583 (Tables 5 and 6).
Partial BT Sequence Determination. The chloroform-extracted BT was purified further by C18 reverse phase HPLC (see Materials and Methods for details). BT1583 was purified to homogeneity in Fraction 33 of the C18 HPLC (FIG. 1C). An amino acid composition analysis of BT1583 (Fraction 33) showed BT1583 contained residues of Tyr, Lys, Leu, Ile, Val and Om. BT1583 was refractory to N-terminal sequencing and resistant to degradation by aminopeptidase M, suggesting that a non-standard N-terminal residue. BT1583 was also resistant to cleavage by carboxypeptidase Y, suggesting a non-standard C-terminal amino acid. Carboxyl-terminal sequencing was, therefore, not attempted.
Tandem mass spectrometry (MS/MS) was then chosen to sequence the BT1583 peptide. MS/MS data were obtained for BT1583 and they are shown in FIG. 2A and Table 2. The MS/MS data indicated that BT1583 contained 13 amino acid residues that correlated well with the amino acid composition. As expected, the masses of Residues 1 and 13 did not correspond to any standard amino acids. The last residue showed a mass of 103 daltons, which appeared to be compatible with a Valine having its C-terminus reduced from a carboxylic acid to an alcohol. The presence of a C-terminal alcoholic Valine was further confirmed by the presence of a reductase domain in the 13th Valine-specific module of the BT NRPS (see below). The identity of the N-terminal residue was more difficult to determine. Nonetheless, an N-terminal residue with a mass of 198 seemed to be compatible with the N,N-methylated form of Bmt {4-methyl-4-[(E)-2-butenyl]-4,N-methyl-Threonine} (Offenzeller, et al., 1996; Offenzeller, et al., 1993)
The presence of Ornithine in BT1583 indicated that BT1583 could not be synthesized by ribosomes. The presence of D-amino acids would strengthen this idea. We chose to assess the chiral properties of two of the most abundant residues in BT1583, Val and Leu. Chiral analyses revealed uniform L-Val residues but both L- and D-Leu residues at a ratio of 2:1.
The above biochemical and structural analyses were able to provide us with a partial BT1583 peptide sequence (Table 2 and FIG. 2B). The structures of the N- and C-terminal residues were not fully determined. Isoleucine and Leucine could not be distinguished. The position of the D-form Leu was not specified. Chiral properties of other residues in the peptide were not determined.
Shot-gun sequencing of the E58 genome. To better understand the structure and biosynthesis of the BT1583 peptide, we decided to identify the gene or operon that is responsible for the BT biosynthesis. The presence of non-proteinogenic Ornithine and D-form amino acids in the peptide led us to believe that BT1583 was synthesized by the NRPS in vivo (Marahiel, 1997). Most of the NRPS genes are co-linear reflecting a strict correlation between NRPS modules and the amino acid residues in the peptide product. If the BT NRPS operon is co-linear, it should encode 13 modules corresponding to the 13 amino acid residues in the BT1583 peptide. Assuming that on average, each module is encoded by an average 3.5 kb DNA fragment, a DNA fragment of 46 kb long would be necessary to accommodate the BT NRPS operon. As mentioned before, the traditional method to identify an NRPS operon involves probing a cosmid library with a generic probe. Since an imperfect generic probe may miss the target gene and there are usually multiple NRPS operons in a bacterial genome, such method frequently causes researchers to chase the wrong NRPS operon. To avoid such pitfall, we developed a genomic approach that provides an unbiased in silica overview of all NRPS operons in a genome to allow direct comparisons of the NRPS operons and therefore rational candidate operon selection. This novel approach resulted in rapid and accurate identification of the BT NRPS operon.
The E58 genome was estimated to be 5 Mb. An E58 genomic library was constructed with an average insert size of 5 kb. The whole genome was sequenced for a two-fold coverage. After sequence assembly, the E58 genome was represented by 1919 contigs with sizes ranging from 700 bp to 22.6 kb and 932 singlets. Such coverage would allow 99.995% of the genome to be represented by clones. Also, the average length of the gap between two neighboring contigs would be as small as 250 bp so that supercontigs could be constructed (see below). Moreover, supercontigs at such resolution would contain sufficient information to allow accurate in silica NRPS operon identification.
In silica identification of the BT NRPS operon. A three-step procedure was used to select the candidate BT NRPS operon. First, all contigs and singlets were searched for sequences encoding NRPS modules. Since E58 is related to B. subtilis, the putative peptide synthetase PPS1 from B. subtilis was chosen as the query sequence for BLAST analysis against a database containing all assembled E58 contigs. 128 contigs showed translated amino acid sequence similarities to PPS1, with P-values arranging from 0 to 1.
Second, supercontigs were constructed from the 128 contigs. Two sequencing reads from the ends of the same insert form a mate pair. A supercontig is a collection of contigs joined mate pairs that reside in different contigs. Identification of mate pairs allowed neighboring contigs to be ordered and orientated to form a supercontig (FIG. 3A). 31 supercontigs were successfully constructed to represent the whole E58 NRPS operon portfolio.
The candidate BT NRPS operon was selected from the E58 NRPS operon portfolio. The 31 supercontigs were examined for the possibility of harboring the BT NRPS operon, and Supercontig 3 (whose genetic features based on finished sequence are shown in FIG. 3B and 3C) was chosen as the candidate based on the following analyses.
Supercontig 3 potentially contained DNA sequence encoding 13 NRPS modules. Available information regarding the A-domian substrate specificities of Supercontig 3 showed compatibility with the partial BT1583 sequence. Complete sets of substrate specificity-conferring amino acid residues could be identified for eleven modules (except Modules 2 and 13 due to incomplete DNA sequence). Although not all specificity predictions could be made, good correlations were established between predicted NRPS amino acid substrates and the partial BT1583 sequence. Specifically, Module 4 was predicted to incorporate Ile, and Modules 9 and 12 were predicted to incorporate Leu (Table 3, see below for details). The partial BT1583 sequence had Leu or Ile at Positions 4, 9 and 12. Phylogenetic analysis of the substrate conferring amino acids of the eleven modules showed that modules expected to incorporate the same or highly similar amino acid did group together (FIG. 3D). For example, Modules 5, 6 and 8 that were all predicted to incorporate Val formed a cluster. Modules 7, 10 and 3 that were predicted to incorporate similar cationic amino acids (Lys and Orn respectively) formed another cluster.
The E-domain positions in the NRPS encoded by Supercontig 3 showed compatibility with the partial BT1583 peptide structure. Four E-domains were found in Modules 3, 7, 9 and 11 (FIG. 3C). Their positions were consistent with the aforementioned BT1583 chiral properties of all L-form Val residues and a 2:1 L- to D-form Leu residue ratio.
Supercontig 3 was therefore identified as the candidate locus for the BT NRPS operon. Primer extensions and genome walking were performed to obtain high quality sequence of the locus. The efforts resulted in a contig of 51,821 bp covering the putative BT NRPS operon (Genbank accession #), see FIG. 3F.
Putative BT NRPS subunits. Ten open reading frames (ORFs) were identified in the sequenced region through translation analysis and blast searches (Altschul, et al., 1997) (FIG. 3B). The middle six ORFs (named btA through btF) were predicted to encode six subunits of the BT NRPS (BtA through BtF), and their coordinates are listed in Table 4. Sequence analysis of the putative subunits confirmed the modular structure of a typical co-linear NRPS (FIG. 3C). The modules, each containing an A-domain and a T-domain, are linked by a C-domain. The loading module BtA has an A-domain followed by a T-domain. There are two noticeable overall features for the putative BT NRPS subunits. First, four out of six subunits exhibit a two-module structure. Second, all auxiliary E-domains are present at the end rather than in the middle of the putative NRPS subunits. Sequence alignments of conserved domains are shown in FIG. 4.
A reductase domain in Module 13. A domain of about 500 amino acids was identified at the C-terminus of BtF or Module 13. BLAST analysis showed that it has high similarity with several NADPH-dependent reductases from other NRPSs and polyketide synthetases. Its alignment with the reductase domains from MxcG of S. aurantiaca and Lys2 of S. cereviciae is shown in FIG. 4E. A similar reductase domain has also been identified in the Gramicidin A NRPS (Kessler, et al., 2004). All three reductases have been experimentally demonstrated to reduce their substrates to corresponding aldehydes in an NADPH-dependent reaction (Gaitatzis, et al., 2001; Kessler, et al., 2004; Sagisaka & Shimura, 1959). For myxochelin A and gramicidin A, the aldehydes are further reduced to alcohols. The exact mechanism for the second reduction step has not been identified. Either those reductase themselves or another proteins carry out the second reduction step, or the second reduction step is spontaneous. The MS/MS experiment suggested that the C-terminal residue of BT1583 might be the alcoholic form of Valine (FIG. 2B). The A-domain specificity prediction of the last putative BT NRPS module and the presence of a reductase domain in the module confirmed this proposal.
btG encodes an ABC transporter. btG is an ORF that is immediately downstream of btF, and it is transcribed in the same direction as are other bt ORFs. The initiation codon ATG is located 61 bp downstream of the btF stop codon. Translated amino acid sequence showed high similarity to members of the ATP-binding cassette (ABC) transporter super-family (data not shown). ABC transporter ORFs are found in typical NRPS operons. Their roles have been proposed to provide host with resistance to the peptide antibiotic product by pumping the peptide out of the cells. The exact role of the putative BtG ABC transporter needs to be established.
BT1583 peptide sequence refinement. The substrate specificity-conferring residues (Stachelhaus, et al., 1999) were extracted from all 13 A-domains and were compared to the collection of the amino acid-binding pocket constituents in the public NRPS codon database (raynam.chm.jhu.edu/ ̃nrps/index.html) (Challis, et al., 2000). Substrate specificity predictions were made based on the sequence alignments and they are listed in Table 3. The amino acid-binding pocket constituents of the first module showed a perfect match with an NRPS codon for Threonine/Dehydrothreonine, and it was predicted that Module 1 incorporates a Threonine derivative. N,N-methylated Bmt was proposed to be the N-terminal amino acid residue according to the MS/MS data (FIG. 2B and Table 2). Although the two proposals do not agree with each other 100%, both call for a Threonine derivative as the N-terminal amino acid residue.
As mentioned before three unambiguous specificity assignments could be made for Module 4 (Ile), Module 9 (Leu) and Module 12 (Leu) according to the NRPS codon database. These assignments were compatible with the partial BT1583 sequence and accordingly Positions 4, 9 and 12 of the BT1583 peptide were refined to Ile, Leu and Leu respectively. Since the only Ile of the BT1583 peptide had been assigned to Position 4, the remainder Leu was assigned to Position 2 of the BT1583 peptide. The A-domain specificity of Module 2 was therefore deduced to be Leu. These assignments in conjunction with the E-domain positional information allowed us to refine the BT1583 peptide sequence to (CH3)2-Bmt-Leu-dOrn-Ile-Val-Val-dLys-Val-dLeu-Lys-dTyr-Leu-Val-ol, wherein Val-ol is Valine alcohol.
Novel NRPS codons in BT biosynthesis. The amino acid-binding pocket constituents of Modules 5, 6 and 8 are identical. They differ with those of Module 13 by only one residue. No good matches were found for these sets of amino acid-binding pocket constituents in the NRPS codon database. However, they showed similarities to certain Ile, Leu or Val NRPS codons in the database. Since the partial BT1583 peptide sequence had Val residues at Positions 5, 6, 8 and 13, Modules 5, 6, 8 and 13 were deduced to incorporate Val. The amino acid-binding pocket constituents of Modules 5, 6, 8 and 13 represent potential novel NRPS codons for Val.
The amino acid-binding pocket constituents of Modules 7 and 10 are identical and they differ with those of Module 3 by only one residue. No match was found for these sets of amino acid-binding pocket constituents in the NRPS codon database. Since the partial BT1583 peptide sequence had Lys residues at Positions 7 and 10, the specificities of these modules were deduced to be Lys. Likewise the partial BT1583 peptide sequence had an Orn residue (which is highly similar to Lys in structure) at Position 3, and the specificity of Module 3 was therefore deduced to be Orn. The amino acid-binding pocket constituents of Modules 7 and 10 represent potentially the first NRPS codon for Lys, while those of Module 3 represent a potential novel NRPS codon for Orn.
The specificity prediction for Module 11 was quite ambiguous according the NRPS codon database. No good match was found for this set of amino acid-binding pocket constituents in the NRPS codon database. However, it showed similarities to certain Phe, Trp or Tyr NRPS codons in the database (data not shown). Since the partial BT1583 peptide sequence had Tyr residues at Position 11, the A-domain specificity of Module 11 was therefore deduced to be Tyr. The amino acid-binding pocket constituents of Module 11 represent a potential novel NRPS codon for Tyr.
Identity verification of the BT NRPS operon. Since the BT biosynthesis involves novel NRPS codons, experimental establishment of the novel codons (especially the novel Valine and Lysine codons) is critical to verifying the identity of the BT NRPS operon. In addition, since the placement of Ile at position 4 in BT1583 affects the placement of three Leu residues, the Module 4 codon also needed to be tested.
Since a purified recombinant A-domain of an NRPS module can selectively and efficiently activate the cognate amino acid substrate of the NRPS module in an ATP-PPi exchange assay (Konz, et al., 1999; Mootz & Marahiel, 1997), ATP-PPi exchange assays have been used to experimentally establish NRPS module specificities and novel NRPS codons. Recombinant A-domains of Modules 8, 5, 7 ,4 and 2 of the BT NRPS were produced and purified as described in Methods and Materials. Almost completely soluble recombinant A-domain proteins were obtained. A-domain specificities were determined in ATP-PPi exchange and aa Km assays (see Methods and Materials), and the results are shown in FIG. 5. All 20 proteinogenic amino acids and L-Orn were tested for each A-domain protein, and background noise in the experiments was usually below 1%.
The Module 8 A-domain protein was shown to activate L-Val (100%), with minor activation of L-Lys (10%) and L-Ile (4%). The apparent Km was determined to be 2.75 mM for L-Val. These results confirmed the novel Valine NRPS codon. Similarly, the Module 5 A-domain protein was found to activate L-Val (100%), L-Ile (23%), and L-Leu (17%). The apparent Km was determined to be 1.11 mM for L-Val and 2.78 mM for L-Ile, clearly showing that L-Val is the preferred substrate for Module 5.
L-Lys was the only amino acid that activated by the Module 7 A-domain protein. The apparent Km value was determined to be 1.12 mM. These results established the first Lys NRPS codon.
The Module 4 A-domain protein was shown to selectively activate L-Ile (100%), with minor activation of L-Val (9%) and L-Leu (7%). The apparent Km value for L-Ile was measured at 0.5 mM.
The Module 2 A-domain protein was found to be quite ambiguous. It activated L-Leu (98%) and L-Met (100%) with nearly equal efficiency, with significant activation of L-Val (67%) and minor activation of L-Ile (19%) and L-Phe (3.5%).
In general, all purified A-domain proteins were found to selectively activate predicted amino acid substrates in the ATP-PPi exchange assays. These results experimentally confirmed the identity of the BT NRPS operon.
Synthetic peptides. To further verify the BT peptide sequence as well as the identity of the BT NRPS operon, a synthetic peptide P81 (FIG. 6) was made (by Biomer Technology, Concord, Calif.) and tested for its properties. Since Bmt is not commercially available, we were not able to synthesize a peptide according to the refined BT1583 sequence and we used octanic acid-modified Threonine to synthesize the lipopeptide P81 to mimic BT1583. P81 showed full antibiotic activity and Pronase resistance as BT1583. These results lend support to the refined BT1583 peptide sequence and the identity of the BT NRPS operon.
To investigate the significance of the C-terminal alcoholic modification, an amide form of P81 (P59) was synthesized. P59 displayed antibiotic activity but no Pronase resistance. These results indicated that the C-terminal alcoholic modification plays a key role in conferring protease resistance to P81 and likely BT1583 as well.
Since the codon for the first BT NRPS module matches perfectly with known Thr NRPS codons, the possibility of an active BT isomer with an unmodified Thr at Position 1 needed to be investigated. An amide form of P81 (P58) was therefore synthesized, and P58 displayed poor antibiotic activity. This result confirmed that a Thr derivative (rather than unmodified) Thr needs to be at Position 1 to confer antibiotic activity.
The L- and D-form residues alternate in the middle of BT1583 with the exception of Position 5 (Val). Since the alternating chirality is a key structural feature for the peptide antibiotic Gramicidine A, we decided to investigate whether we missed the coding sequence of an E-domain for Module 5. A D/L alternating version of P59 (P80) was synthesized. P80 displayed no antibiotic activity. The above results confirmed not only the BT1583 peptide structure (with the exception of the N-terminal residue) but also the identity of the BT NRPS operon.
The BT1583 peptide structure, the BT NRPS operon and the BT NRPS allow us to propose a degenerate formula for isomers of BT1583 (Table 5). Based on the relative substrate selectivty of each module, the BT isomers likely to be produced by E58 in significant amounts were predicted and listed in Table 6. Most of the predicted BT isomers were experimentally verified by MS/MS (data not shown).
The structure and biosynthesis of the BT peptide was determined using an integrated approach of biochemistry, biophysics and genomics. Amino acid composition and tandem mass spectrometry experiments with purified BT1583 (the main BT isomer) produced a partial peptide structure. The presence of Ornithine and D-form residues in the partial structure indicated that the peptide was synthesized by a non-ribosomal peptide synthase in vivo. The BT NRPS operon was rapidly and accurately identified via a novel in silica gene hunting scheme. Sequence analysis of the BT NRPS operon revealed that it encodes a co-linear modular NRPS. The co-linear nature of the BT NRPS enabled us to use the BT operon genomic information and refine the BT1583 peptide sequence to Me2Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-Vol (SEQ ID NO.: 1), wherein Vol stands for Valine alcohol. Moreover, new NRPS codons for Valine, Lysine, Ornithine and Tyrosine were discovered and are reported here.
In silica NRPS operon identification. Traditional NRPS gene identification involves probing a genomic cosmid library with a generic probe. Such approach has the inherited shortcoming of causing researchers to chase the wrong gene in a genome with multiple NRPS operons. As shown herein, NRPS gene identification is improved for all NRPS operons in the genome when compared at the same time to find a best fit. Such comparison requires a draft genome. Fortunately, sequencing cost has decreased significantly to allow routine sequencing of microbial genomes. A two-fold coverage was sufficient for accurate NRPS operon identification. In actual BT NRPS operon selection, the following two sets of information are generated and compared to find the best candidate: the NRPS module clustering pattern of according to similarities of the substrate-binding pocket constituents; and positional information such as the positions of D-form residues. The module clustering technique is especially powerful in establishing the candidacy of an operon that involves novel NRPS codons (i.e., in the case of Modules 5, 6 and 8 of the BT NRPS operon). The in silica strategy is particularly useful for NRPS operon identification in strains (such as E58) that have a large number of NRPS operons in the genome.







TABLE 1







Amino acid composition of purified BT1583 peptide










Molar ratios




normalized to
Residues











Amino Acid
nmoles
Tyr
Ile
per peptide














Tyrosine
1.75
1.00
1.16
1


Valine
4.58
2.62
3.05
3


Isoleucine
1.50
0.86
1.00
1


Leucine
5.32
3.04
3.54
3


Lysine
3.57
2.04
2.38
2


Ornithine
1.2
0.69
0.80
1


Total # of derivatizable residues

10.25
11.93
11
















TABLE 2







Tandem mass spectrometry of BT1583













M/H+

Possible amino acid
M/H+ y-

Possible amino acid
Compiled (N to


b-series
ΔM
residue
series
ΔM
residue
C)
















198.1

(CH3)2-Bmt(?)



(CH3)2-Bmt(?)


311.16
113.06
L/I
1386.73
113.12
L/I
L/I


425.21
114.05
O
1273.61
114.04
O
O


538.28
113.07
L/I
1159.57
113.05
L/I
L/I


637.32
99.04
V
1046.52
198.08
V + V
V








V


864.42
227.10
V + K
848.44
128.07
K
K


963.46
99.04
V
720.37
99.04
V
V


1076.52
113.06
L/I
621.33


L/I


1204.58
128.06
K



K


1367.65
163.07
Y



Y


1480.81
113.16
L/I



L/I


1583.87
103.06
Valine alcohol



Valine alcohol
















TABLE 3







Predicted BT NRPS module substrate specificities and refinement of the BT1583


peptide structure. The residues were numbered according to the corresponding residues of PheA


(Conti, et al., 1997).













Predicted
Partial
Refined



PheA Numbering
Substrate
BT1583
BT1583




















Module
235
236
239
278
299
301
322
330
331
517
Specificity
Seq.
Seq.





1
D
F
W
N
I
G
M
V
H
K
Thr/Dht
(CH3)2-
(CH3)2-














Bmt*
Bmt


2
D
G
F
L
L
G
G
V
F
K
Ile/Leu
Leu/Ile
Leu**


3
D
S
G
P
S
G
A
V
D
K

Orn*
Orn


4
D
G
F
F
L
G
V
V
Y
K
Ile*
Leu/Ile
Ile


5
D
G
F
F
V
G
G
V
F
K
Ile/Leu/Val
Val*
Val


6
D
G
F
F
V
G
G
V
F
K
Ile/Leu/Val
Val*
Val


7
D
A
G
P
S
G
A
V
D
K

Lys*
Lys


8
D
G
F
F
V
G
G
V
F
K
Ile/Leu/Val
Val*
Val


9
D
A
W
F
L
G
N
V
V
K
Leu*
Leu/Ile
Leu


10
D
A
G
P
S
G
A
V
G
K

Lys*
Lys


11
D
A
A
A
V
V
G
V
A
K
Phe/Trp/Tyr
Tyr*
Tyr


12
D
A
W
F
L
G
N
V
W
K
Leu*
Leu/Ile
Leu


13
D
G
F
F
A
G
G
V
F
K
Ile/Leu/Val
Valine
Valine














alcohol*
alcohol





*The information was used for the BT1583 peptide sequence refinement.


**The Leu at this position was deduced from the fact that the only Ile had been assigned to Position 4.













TABLE 4







The BT NRPS operon (see FIGS. 3G–3L).












ORF

Gene product
















Start
End
Length
SEQ ID
length
MW
Homology



(nt)
(nt)
(bp)
NO.:
(amino acid)
(kD)
to


















btA
2,889
4,814
1,926
22
641
72.87
NRPS


btB
4,817
12,409
7,593
23
2,530
288.99
NRPS


btC
12,438
26,291
13,854
24
4,617
526.68
NRPS


btD
26,321
33,946
7,626
25
2,541
289.31
NRPS


btE
33,976
41,556
7,581
26
2,526
288.45
NRPS


btF
41,584
49,059
7,476
27
2,491
284.46
NRPS


btG
49,120
49,842
723
28
240
26.95
ABC transporter
















TABLE 5







A degenerate formula for the BT isomers



















1
2
3
4
5
6
7
8
9
10
11
12
13





Me2Bmt
L
dO
I
V
V
dK
V
dL
K
dY
L
Valine



M

V
I







alcohol



V


L



I



F





Numbers indicate the amino acid residue positions.













TABLE 6







Summary of BT isomers












Peptide sequences of the





Name
predicted products by the BT NRPS
MW
SEQ ID NO.:





BT1583
Me2Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-Vol
1583
 1



 


BT1601
Me2Bmt-M-dO-I-V-V-dK-V-dL-K-dY-L-Vol
1601
 2


 


BT1569V2
Me2Bmt-V-dO-I-V-V-dK-V-dL-K-dY-L-Vol
1569
 3


 


BT1583I2
Me2Bmt-I-dO-I-V-V-dK-V-dL-K-dY-L-Vol
1583
 4


 


BT1617
Me2Bmt-F-dO-I-V-V-dK-V-dL-K-dY-L-Vol
1617
 5


 


BT1597I5
Me2Bmt-L-dO-I-I-V-dK-V-dL-K-dY-L-Vol
1597
 6


 


BT1597L5
Me2Bmt-L-dO-I-L-V-dK-V-dL-K-dY-L-Vol
1597
 7


 


BT1615I5
Me2Bmt-M-dO-I-I-V-dK-V-dL-K-dY-L-Vol
1615
 8


 


BT1615L5
Me2Bmt-M-dO-I-L-V-dK-V-dL-K-dY-L-Vol
1615
 9


 


BT1583V2I5
Me2Bmt-V-dO-I-I-V-dK-V-dL-K-dY-L-Vol
1583
10


 


BT1583V2L5
Me2Bmt-V-dO-I-L-V-dK-V-dL-K-dY-L-Vol
1583
11


 


BT1597I2I5
Me2Bmt-I-dO-I-I-V-dK-V-dL-K-dY-L-Vol
1597
12


 


BT1597I2L5
Me2Bmt-I-dO-I-L-V-dK-V-dL-K-dY-L-Vol
1597
13


 


BT1631I5
Me2Bmt-F-dO-I-I-V-dK-V-dL-K-dY-L-Vol
1631
14


 


BT1631L5
Me2Bmt-F-dO-I-L-V-dK-V-dL-K-dY-L-Vol
1631
15


 


BT1569V4
Me2Bmt-L-dO-V-V-V-dK-V-dL-K-dY-L-Vol
1569
16


 


BT1587M2V4
Me2Bmt-M-dO-V-V-V-dK-V-dL-K-dY-L-Vol
1587
17


 


BT1555
Me2Bmt-V-dO-V-V-V-dK-V-dL-K-dY-L-Vol
1555
18


 


BT1569I2V4
Me2Bmt-I-dO-V-V-V-dK-V-dL-K-dY-L-Vol
1569
19


 


BT1603
Me2Bmt-F-dO-V-V-V-dK-V-dL-K-dY-L-Vol
1603
20









BT as a Feed Additive. (Semi-purified BT peptides rather than BT1583 were used in chicken growth promotion experiments.)
Based on the structure of the BT peptides and its biological and biochemical properties the present investigator produced sufficient material to test its use as a feed additive. In summary, the properties of the peptide used were as follows, 13 amino acid residues with numerous potential natural variants or isomers (>8) and derivatives (>30). Biologically, it was found that the BT peptides were a natural product produced by a Gram(+) bacterium. The BT peptide family is synthesized by a non-ribosome peptide synthase (NRPS), the cloning and characterization is disclosed herein. One such peptide, BT1583: Me2Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-Vol. (SEQ ID NO.: 1) (Vol stands for Valine alcohol) was selected for further studies because it is cationic and likely amphipathic, It contains unusual amino acid residues and/or includes multiple modifications.
BT1583 was also selected due to its high level of stability. The high stability observed for this peptide included one or more of the following characteristics: (1) no known enzymes can digest it; (2) it is not digested in the mouse or chicken GI track; (3) it can be autoclaved; (4) it survived the feed pelleting process; (5) it can stand extreme pHs (pH 1.0 and pH 13.0); and (6) the only known in vitro method to inactivate it is pH 1.0 plus 100° C. overnight.
In addition to the remarkable stability of BT1583, it demonstrated, in vitro, antibacterial against Gram-positive bacteria, e.g., for most Gram (+): MIC=1 microgram/ml. For Gram (−) the following were the antimicrobial activities observed: E. coli: MIC>20 microgram/ml; Pseudomonas and Salmonella: MIC>100 microgram/ml. BT1583 also shows antifungal, e.g., S. cerevisiae: MIC=50 microgram/ml. Anti-protozoal activity was also observed for BT1583 against, Tetrahymena: MIC=25 microgram/ml.
The E58 strain for producing BT1583 was selected because it was a fast growing and high peptide producer strain. Furthermore, in addition to fast peptide biosynthesis the strain is also grows in cheap media, e.g., with medium cost as low as 0.4 cents/L and a yield of, e.g., 0.5 g/L. Growth is generally carried out in an air shaker but may also be fermented. Furthermore, the peptide and the strain may be used without extensive adaptation of well-known procedures to an easy, one-step purification process.
The following tables and examples show the growth promotion capabilities and characteristics of the BT1583 peptide in Broiler Chicken, e.g., in a 21-day battery study.







TABLE 7







Summary of Growth Promoting Studies.












Feed conversion
Additional


Study
BT1583 concentration
improvement (point)
weight gain (%)














1-1
10
ppm
7
17


1-2
30
ppm
8
17


2-1
6
ppm
9
6.7


2-2
12
ppm
10
11


3-1
12
ppm
9
16


3-2
12
ppm with Coban
9
5.4





(vs Coban alone)


4-1
24
ppm with direct
13
7.1




coccidial challenge


4-2
48
ppm with direct
17
9.3




coccidial challenge









Briefly, the peptide was used in a semi-purified form to study the growth and feed conversion of 20-day old straight run broilers in batteries (Studies 2-1 and 2-2). Two amounts were tested against a feed control, peptide at 6 ppm and peptide at 12 ppm, 12 repetitions were carried out per treatment with 4 birds per pen. The diet used in the study was as follows.







TABLE 8







Basic Feed for Studies 2-1 and 2-2









PERCENT














INGREDIENTS (Mash Feed)




TAMU Corn
62.91



TAMU Dehulled Soybean Meal
30.67



DL Methionine
0.07



Blended A-V Fat
2.68



Limestone
1.45



Mono-Dicalcium Phosphate
1.58



Salt
0.33



TAMU Trace Minerals
0.05



TAMU Vitamins
0.25



NUTRIENT CONTENT (Calculated)



Metabolizable Energy (kcal/kg)
3100



Protein (%)
20.0



Lysine (%)
1.05



Methionine + Cystine (%)
0.72



Threonine (%)
0.75










BT1583 added in 200 grams of corn meal carrier
Table 9 shows the Statistics for a Dependent Variable: 20-day cumulative weight gain.
















Treatment
Mean
Std. Deviation
Number









Control
554.8236
38.13395
12



BT1583 @ 12 ppm
618.9340
46.79301
12



BT1583 @ 6 ppm
591.9750
47.93018
12



Total
588.5775
50.77136
36










Table 10 shows the Tests of Between-Subjects Effects
Dependent Variable: 20-day cumulative weight gain
















Type III Sum of






Source
Squares
df
Mean Square
F
Sig.




















Corrected
24868.642(a)
2
12434.321
6.279
.005


Model


Intercept
12471247.008
1
12471247.008
6297.459
.000


TRE
24868.642
2
12434.321

.005


Error
65351.939
33
1980.362
6.279


Total
12561467.588
36


Corrected
90220.580
35


Total





(a)R Squared = .276 (Adjusted R Squared = .232)






Table 11 shows the estimated marginal means for the study.
Dependent Variable: 20-day cumulative weight gain













95% Confidence Interval











Treatment
Mean
Std. Error
Lower Bound
Upper Bound





Control
554.824
12.846
528.687
580.960


BT1583 @
618.934
12.846
592.798
645.070


12 ppm


BT1583 @ 6 ppm
591.975
12.846
565.839
618.111









Table 12 shows the Post Hoc Tests for Homogeneous Subsets
Dependent Variable: 20-day cumulative weight gain Duncan














Subset













Treatment
N
1
2
















Control
12
554.8236




BT1583 @ 6 ppm
12

591.9750



BT1583 @ 12 ppm
12

618.9340



Sig.

1.000
.147







Means for groups in homogeneous subsets are displayed.



Based on Type III Sum of Squares



The error term is Mean Square(Error) = 1980.362.



a Uses Harmonic Mean Sample Size = 12.000.



b Alpha = .05.






Table 13 shows the Descriptive Statistics
Dependent Variable: 20-day cumulative feed conversion rate
















Treatment
Mean
Std. Deviation
N









Control
1.5922
.13721
12



BT1583 @ 12 ppm
1.4959
.10089
12



BT1583 @ 6 ppm
1.5065
.04795
12



Total
1.5315
.10841
36










Table 14 shows the Tests of Between-Subject Effects
Dependent Variable: 20-day cumulative feed conversion rate
















Type III Sum of






Source
Squares
df
Mean Square
F
Sig.




















Corrected
6.702E−02(a)
2
3.351E−02
3.212
.005


Model


Intercept
84.440
1
84.440
8092.585
.000


TRE
6.702E−02
2
3.351E−02
3.212
.053


Error
.344
33
1.043E−02
6.279


Total
84.851
36


Corrected Total
.411
35





(a)R Squared = .163 (Adjusted R Squared = .112)






Table 15 shows the Estimated Marginal Means
Dependent Variable: 20-day cumulative feed conversion rate













95% Confidence Interval











Treatment
Mean
Std. Error
Lower Bound
Upper Bound





Control
1.592
.029
1.532
1.652


BT1583 @
1.496
.029
1.436
1.556


12 ppm


BT1583 @ 6 ppm
1.506
.029
1.446
1.566









Table 16 shows the Post Hoc Tests for Homogeneous Subsets
Dependent Variable: 20-day cumulative feed conversion rate-Duncan














Subset













Treatment
N
1
2
















BT1583 @ 12 ppm
12
1.4959




BT1583 @ 6 ppm
12
1.5065



Control
12

1.5922



Sig.

.801
1.000







Means for groups in homogeneous subsets are displayed.



Based on Type III Sum of Squares



The error term is Mean Square (Error) = 1.043E−02.



a Uses Harmonic Mean Sample Size = 12.000.



b Alpha = .05.






To evaluate TAMUS BT1583 on growth and feed conversion of 3-wk old straight run broilers fed an industry type pelleted starter feed (in batteries, Studies 3-1 and 3-2). Briefly, the following six treatment regimens were examined: Control, Monensin at 90 ppm, BMD 50 at 50 ppm, BT1583 at 12 ppm, Monensin+BMD 50, Monensin+and BT1583 at 12 ppm. Eight (8) study repetitions per treatment were used, again with 4 birds per pen.







TABLE 17







Basic Feed for Studies 3-1 and 3-2.









PERCENT














INGREDIENTS (Pelleted Feed)




TAMU Corn
56.11



TAMU Dehulled Soybean Meal
35.90



DL Methionine
0.22



Blended A-V Fat
4.02



Limestone
1.43



Mono-Dicalcium Phosphate
1.55



Salt
0.46



TAMU Trace Minerals
0.05



TAMU Vitamins
0.25



NUTRIENT CONTENT (Calculated)



Metabolizable Energy (kcal/kg)
3100



Protein (%)
22.31



Lysine (%)
1.21



Methionine + Cystine (%)
0.92



Threonine (%)
0.84










BT1583 added via 200 grams of corn meal
Table 18 shows the Descriptive Statistics
Dependent Variable: 20-day cumulative weight gain
















Treatment
Mean
Std. Deviation
Number





















BT1583 @ 12 ppm
831.7396
40.47789
8



BMD @ 50 ppm
832.9688
30.12576
8



COB @ 90 ppm
792.8438
67.05913
8



COB + BT1583
835.7604
62.00447
8



COB + BMD
810.2188
74.64333
8



Control
719.7813
71.97.115
8



Total
803.8854
70.02414
48










Table 19 shows the Tests of Between-Subjects Effects
Dependent Variable: 20-day cumulative weight gain
















Type III Sum of






Source
Squares
df
Mean Square
F
Sig.




















Corrected
78986.007(a)
5
15797.201
4.380
.003


Model


Intercept
31019124.630
1
31019124.630
8600.903
.000


TRE
78986.007
5
15797.201
4.380
.003


Error
151472.835
42
3606.496


Total
31249583.472
48


Corrected
230458.842
47


Total





(a)R Squared = .343 (Adjusted R Squared = .264)






Table 20 shows the Estimated Marginal Means
Dependent Variable: 21-day cumulative weight gain













95% Confidence Interval











Treatment
Mean
Std. Error
Lower Bound
Upper Bound














BT1583
831.740
21.232
788.891
874.588


BMD
832.969
21.232
790.120
875.817


COB
792.844
21.232
749.995
835.692


COB + BT1583
835.760
21.232
792.912
878.609


COB + BMD
810.219
21.232
767.370
853.067


Control
719.781
21.232
676.933
762.630









Table 21 shows the Post Hoc Tests for Homogeneous Subsets
Dependent Variable: 21-day cumulative weight gain-Duncan














Subset













Treatment
N
1
2
















Control
8
719.7813




COB
8

792.8438



COB + BMD
8

810.2188



BT1583
8

831.7396



BMD
8

832.9688



COB + BT1583
8

835.7604



Sig.

1.000
.211







Means for groups in homogeneous subsets are displayed.



Based on Type III Sum of Squares



The error term is Mean Square(Error) = 3606.496.



a Uses Harmonic Mean Sample Size = 8.000.



b Alpha = .05.






Table 22 shows the Descriptive Statistics
Dependent Variable: 20-day cumulative feed conversion rate
















Treatment
Mean
Std. Deviation
N





















BT1583
1.3308
.03340
8



BMD
1.3397
.03132
8



COB
1.3712
.03023
8



COB + BT1583
1.2816
.02680
8



COB + BMD
1.3435
.02477
8



Control
1.4154
.03299
8



Total
1.3470
.04989
48










Table 23 shows the Tests of Between-Subjects Effects
Dependent Variable: 21-day cumulative feed conversion rate
















Type III Sum of






Source
Squares
df
Mean Square
F
Sig.




















Corrected
7.894E−02(a)
5
1.579E−02
17.442
.000


Model


Intercept
87.096
1
87.096
96218.356
.000


TRE
7.894E−02
5
1.579E−02
17.442
.000


Error
3.802E−02
42
9.052E−04


Total
87.213
48


Corrected
.117
47


Total





(a)R Squared = .675 (Adjusted R Squared = .636)






Table 24 shows the Estimated Marginal Means
Dependent Variable: 21-day cumulative feed conversion rate













95% Confidence Interval











Treatment
Mean
Std. Error
Lower Bound
Upper Bound





BT1583
1.331
.011
1.309
1.352


BMD
1.340
.011
1.318
1.361


COB
1.371
.011
1.350
1.393


COB + BT1583
1.282
.011
1.260
1.303


COB + BMD
1.344
.011
1.322
1.365


Control
1.415
.011
1.394
1.437









Table 25 shows the Dependent Variable:
20-day cumulative feed conversion rate-Duncan













Subset












Treatment
N
1
2
3
4















COB + BT1583
8
1.2816





BT1583
8

1.3308


BMD
8

1.3397
1.3397


COB + BMD
8

1.3435
1.3435


COB
8


1.3712


Control
8



1.4154


Sig.

1.000
.432
.053
1.000





Means for groups in homogeneous subsets are displayed.


Based on Type III Sum of Squares


The error term is Mean Square(Error) = 9.052E−04.


a Uses Harmonic Mean Sample Size = 8.000.


b Alpha = .05.






A more complete study to evaluate TAMUS BT1583 on growth and feed conversion of 42 day old straight run broilers in floor pens may be as follows: Treatments of six (6) groups, Control, Monensin at 90 ppm, BT1583 at 12 ppm, Monensin+BMD at 50 ppm, Monensin+BT1583 at 12 ppm and BMD at 50 ppm. 10 study repetitions per treatment were used to evaluate the effect of using the BT1583 peptide as a feed additive, this time with 40 birds per pen.
When used to promote growth in food-producing animals it was found that the BT1583 peptide provided about 10 points in feed conversion plus extra weight gains. One distinct advantage of the present invention is that no or very little absorption by the chicken GI track, thereby making it useful for widespread use. Furthermore, unlike conventional antibiotics, the present invention may target the bacterial membrane, and there is currently not a drug target that can be altered with one or two mutations to allow development of drug resistance. Furthermore, it was found that growth promotion via possible host immunity modulation is intrinsic to chicken and independent of drug resistance. Alternatively, but in no way limiting the present invention, the present invention may be used as an animal-use only antibiotic for bacterial infections. Also, to date, there has been no observed decrease in the growth promoting activity of the peptide.
A broiler floor pen trial to compare the performance of a new anti-microbial designated for this project as BT1583 alone and in combination with the widely used coccidiostat monensin (MON) to MON fed alone, MON in combination with the also widely used antimicrobial Bacitracin MD (BMD) and BMD alone.
The following levels of each treatment were evaluated:
1: Non-supplemented
2: Monensin (MON) 99 ppm
3: BT1583 12 ppm
4: BT1583 12 ppm+MON 99 ppm
5: MON 99 ppm+BMD 55 ppm
6: BMD 55 ppm
The study design included 10 pens per treatment and 40 birds per pen housed on day of hatch. Two basal corn-soy based diets of decreasing protein (approximately 23 to 20%) and increasing metabolizable energy (approximately 1400 to 1455 kcal/lb) were used from Day 0 to 21 (starter feed) and Days 22 to 42 (grower feed), respectively. Treatment premixes were measured and blended into diets at required levels. Between days 0 and 21 mortality was less than 1% with all birds growing optimally and of high health across all groups.
Beginning on study day 22, the study director modulated house temperature and air flow to mimic industry conditions conducive to outbreaks of colibacillosis within naive broiler flocks. This was done to stimulate a natural challenge for this study. Mortality climbed to a house average of approximately 10% by Day 42. A majority of these deaths occurred in groups not receiving BT1583 or MON. Lesions were consistent with those of colibacillosis (air sac, pneumonia, peri-hepatitis, peri-carditis and extreme morbidity). All mortality was documented (weight at death and post-mortem observations). All birds and feeds were weighed at 42 days. All remaining birds were euthanized on Day 42 by asphyxiation with the carcasses submitted for rendering.
All data were analyzed as described below and are displayed in Tables 26 through 30. The following variables were tested: Response Variables: Gain Per Bird, Feed Per Gain, Mortality (%), Adjusted Feed Per Gain. F test from One Way ANOVA with one blocking factor=location, at Day 42 using 0.05 level of significance.
All Response Variables: LSD T-test procedure for pair-wise comparisons with Type 1 error of means when ANOVA F ratio is significant, overall significance level of 0.05 used. Lines below means (see Table 30, below) indicate groups with insignificant differences in means.







TABLE 26







Weight gains (in lb) per bird










Treatment
Day 42 Gain/Bird














Non-supplemented
3.900d



Monensin (MON) 99 ppm
4.111bcd



BT1583 12 ppm
4.333ab



MON 99 ppm + BT1583 12 ppm
4.385a



MON 99 ppm + BMD 55 ppm
4.127c



BMD 55 ppm
3.971cd










Weight gains were heaviest for the 2 groups of broilers receiving BT1583 measured at 42 days with the MON+BT1583 significantly heavier (p<0.05) than that provided by the MON+BMD and MON groups.
Feed/Gain: Table 27 shows that MON+BT1583 fed broilers had the feed/gain values which were lower (p<0.05) than all other groups with the exception of the group receiving BT1583 alone.







TABLE 27







Feed/Gain










Treatment
Day 42 Feed/Gain














Non-supplemented
2.189c



Monensin (MON) 99 ppm
1.854b



BT1583 12 ppm
1.722ab



MON 99 ppm + BT1583 12 ppm
1.689a



MON 99 ppm + BMD 55 ppm
1.885bc



BMD 55 ppm
2.147c










Adjusted Feed/Gain: The total weight of mortality in each pen was added to the final live weight, that value reduced by subtracting the initial weight and then dividing that value into the Total feed consumed to calculate the Adjusted Feed/Gain.
Table 28 demonstrates the effects of the natural challenge on feed/gain values. Even with the adjustments for mortality, MON+BT1583 fed broilers had an adjusted feed/gain value which again was significantly lower (p<0.05) than all other groups with the exception of the group receiving BT1 583 alone.







TABLE 28







Feed/Gain Adjusted










Treatment
Day 42 Adjusted Feed/Gain














Non-supplemented
1.928c



Monensin (MON) 99 ppm
1.761b



BT1583 12 ppm
1.704ab



MON 99 ppm + BT1583 12 ppm
1.654a



MON 99 ppm + BMD 55 ppm
1.725b



BMD 55 ppm
1.838bc










Mortality: The majority of the deaths were caused by acute and chronic colibacillosis. Broilers receiving BT1583 or Monensin alone or in combination had lower mortality rates than the non-supplemented controls.







TABLE 29







Mortality by acute and chronic colibacillosis.










Treatment
Day 42 Mortality (%)














Non-supplemented
17.50c



Monensin (MON) 99 ppm
8.00a



BT1583
2.75a



MON 99 ppm + BT1583 12 ppm
3.50a



MON 99 ppm + BMD 55 ppm
7.75abc



BMD 55 ppm
18.25bc










Monensin is a polyether antibiotic that is approved and used as an anti-protozoal agent in the poultry industry. Slight efficacy by monensin and other polyether antibiotics against gram negative bacteria has been documented by many researchers and poultry industry personnel. BT1583 has also been stated to have efficacy against gram negative bacteria. Escherichia coli has been a major problem in the food industries both health wise and financially. Most products highly effective against this pathogen are too costly to use in broiler older than 21 days or have been pulled off the market due to similarities to human health products raising public health concerns. This study demonstrated that BT1583 is highly effective against colibacillosis in 3 to 4 week old naive broiler chickens raised under simulated commercial broiler conditions. The 20+ point weight gain advantages and 10+ point feed/gain advantages held by BT1583 over monensin and BMD fed alone and in combination observed on this trial is a strong indicator that this product may be an invaluable tool for the future of the poultry industry.







TABLE 30







P-Values Comparisons
















Day 42



Day 42
Day 42
Day 42
Day 42
Adjusted
Day 42


Wt/Gain
Indiv. Bird
Gain/Bird
Feed/Gain
Feed/Gain
Mortality


WtGn (lb)
Wt (lb)
(lb)
(FdWt/WTGn)
(Adj Feed/Gn)
(%)





Controls vs. MON
0.216456
0.153695
0.001923
0.023562
0.013284


Controls vs. BT1583
0.000028
0.000023
0.000134
0.002449
0.001433


Controls vs. MON + BT1583
0.000421
0.000333
0.000115
0.000147
0.006250


Controls vs. MON + BMD
0.004270
0.002816
0.096440
0.003283
0.179121


Controls vs. BMD
0.523702
0.546456
0.754966
0.231502
0.845776


MON vs. BT1583
0.177311
0.192494
0.159703
0.425156
0.172685


MON vs. MON + BT1583
0.018776
0.022738
0.041045
0.033717
0.234506


MON vs. MON + BMD
0.805698
0.905725
0.840264
0.275135
0.967501


MON vs. BMD
0.521589
0.443090
0.123047
0.453955
0.031824


BT1583 vs. MON + BT1583
0.645444
0.638704
0.472007
0.338775
0.663743


BT1583 vs. MON + BMD
0.014212
0.011862
0.254035
0.712535
0.336982


BT1583 vs. BMD
0.005943
0.005142
0.017789
0.091063
0.006670


MON + BT1583 vs. MON + BMD
0.006085
0.004962
0.145588
0.037877
0.386550


MON + BT1583 vs. BMD
0.006044
0.005385
0.013376
0.034621
0.003991


MON + BMD vs BMD
0.244875
0.214655
0.181891
0.205577
0.134645





(Note: Bold type and underlining indicate comparisons where p-value is less than 0.05)






It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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## Claims

1. A feed additive comprising: an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide with two or more D-amino acids isolated from Brevibacillus texasporus, comprising Me2Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-Vol (SEQ ID NO: 1), SEQ ID NO: 2-4 or SEQ ID NO: 5.

2. The additive of claim 1, wherein the Vol is valine alcohol produced by reducing —COOH group of the C-terminal Valine to —CH2OH.

3. The additive of claim 1, wherein the carboxy-terminus —COOH group of the C-terminal Valine is reduced to —CH2OH and confers protease resistance to the peptide.

4. The additive of claim 1, wherein the peptide is stable at a pH of 1.0, at a pH 13.0, resistant to proteases or combinations thereof.

5. The additive of claim 1, wherein the peptide is Me2Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-Vol (SEQ ID NO: 1).

6. The additive of claim 1, wherein the peptide kills, gram positive bacteria, gram negative bacteria, fungi, protozoa or combinations thereof.

7. The additive of claim 1, wherein the peptide is added at between about 0.5 and about 100 ppm.

8. The additive of claim 1, wherein the peptide is added at between about 6 and about 12 ppm.

9. The additive of claim 1, wherein the peptide is added to a feed adapted for use by one or more of poultry, livestock, farm-raised fish, crabs, shrimp and fresh-water turtles.

10. A cereal-based animal feed comprising: at least one cereal selected from barley, soya, wheat, triticale, rye and maize; and an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids isolated from Brevibacillus texasporus, wherein the peptide comprises the sequence Me2Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-Vol (SEQ ID NO: 1).

11. A peptide-based feed additive comprising: between about 1 and 1000 ppm of an isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids isolated from Brevibacillus texasporus, wherein the peptide comprises the sequence Me2Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-Vol (SEQ ID NO: 1).

12. An antimicrobial peptide comprising two or more D-amino acids, carboxy-terminus reduced pH and heat stable isolated from Brevibacillus texasporus, wherein the peptide comprises the sequence Me2Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-Vol (SEQ ID NO: 1).

13. A broad spectrum antimicrobial compound for topical use comprising a peptide comprising two or more D-amino acids, carboxy-terminus reduced, pH and heat stable isolated from Brevibacillus texasporus, wherein the peptide comprises the sequence Me2Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-Vol (SEQ ID NO: 1).

14. An isolated and purified, heat stable, amino terminus-methylated, carboxy-terminus reduced peptide comprising two or more D-amino acids isolated from Brevibacillus texasporus that inhibits the growth of at least one bacterium selected from the group consisting of: Staphylococcus, Enterococcus, Pneumococcus, Bacilli, Methanococcus, Haemophilus, Archaeoglobus, Borrelia, Synedrocyptis, Mycobacteria, Pseudomonas and E. coli., wherein the peptide comprises the sequence Me2Bmt-L-dO-I-V-V-dK-V-dL-K-dY-L-Vol (SEQ ID NO: 1).