Polynucleotides and polypeptides derived from lactobacillus rhamnosus hn001

Description of equivalent WO 2004031389 A1

POLYNUCLEOTIDES AND POLYPEPTIDES DERIVED FROM LACTOBACILLUS RHAMNOSUS HN001

Technical Field ofthe Invention

This invention relates to polynucleotides isolated from lactic acid bacteria, together with probes and primers specific to the polynucleotides; genetic constructs comprising the polynucleotides; biological materials, including plants, microorganisms and multicellular organisms, incorporating the polynucleotides; polypeptides expressed by the polynucleotides; and methods for using the polynucleotides and polypeptides.

Background ofthe Invention

The present invention relates to polynucleotides isolated from a specific strain of lactic acid bacteria, namely Lactobacillus rhamnosus HN001 (L. rhamnosus HN001). Lactic acid bacteria, and their enzymes, are the major determinants of flavor and fermentation characteristics in fermented dairy products, such as cheese and yogurt. Flavors are produced through the action of bacteria and their enzymes on proteins, carbohydrates and lipids.

Lactobacillus rhamnosus strain HN001 are heterofermentative bacteria that are Gram positive, non-motile, non-spore forming, catalase negative, facultative anaerobic rods exhibiting an optimal growth temperature of 37±1°C and an optimum pH of 6.0 - 6.5. Experimental studies have demonstrated that dietary supplementation with Lactobacillus rhamnosus strain HN001 induces a sustained enhancement in several aspects of both natural and acquired immunity (See PCT International Publication No. WO 99/10476). In addition, L. rhamnosus HN001, and certain other Gram-positive bacteria, can specifically and directly modulate human and animal health (See, for example, Tannock et al., Applied Environ. Microbiol. 66:2578-2588, 2000; Gill et al, Brit. J. Nutrition 83:167-176; Quan Shu et al, Food and Chem. Toxicol. 38:153-161, 2000; Quan Shu et al, Intl. J. Food Microbiol. 56:87-96, 2000; Quan Shu et al, Intl. Dairy J. 9:831-836, 1999; Prasad et al, Intl. Dairy J. 8:993-1002, 1998; Sanders and Huis in't Veld, Antonie van Leeuwenhoek 76:293-315, 1999; Salminen et al, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 211-253; Delcour et al, Antonie van Leeuwenhoek 76:159- 184, 1999; Blum et al, Antonie van Leeuwenhoek 76:199-205, 1999; Yasui et al, Antonie van Leeuwenhoek 76:383-389, 1999; Hirayama and Rafter, Antonie van Leeuwenhoek 76:391-394, 1999; Ouwehand, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 139-159; Isolauri et al, S 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 255-268; Lichtenstein and Goldin, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 269-277; El- Nezami and Ahokas, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 359-367; Nousianen et al, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 437-473; Meisel and Bockelmarm, Antonie van Leeuwenhoek 76:207- 215, 1999; Christensen et al, Antonie van Leeuwenhoek 76:217-246, 1999; Dunne et al, Antonie van Leeuwenhoek 76:279-292, 1999).

Beneficial health effects attributed to these bacteria include the following:

Increased resistance to enteric pathogens and anti-infection activity, including treatment of rotavirus infection and infantile diarrhea - due to increases in antibody production caused by an adjuvant effect; increased resistance to pathogen colonization; alteration of intestinal conditions, such as pH; and the presence of specific antibacterial substances, such as bacteriocins and organic acids.

Aid in lactose digestion - due to lactose degradation by bacterial lactase enzymes (such as beta-galactosidase) that act in the small intestine.

Anti-cancer (in particular anti-colon cancer) and anti-mutagenesis activities - due to anti- mutagenic activity; alteration of procancerous enzymatic activity of colonic microbes; reduction of the carcinogenic enzymes azoreductase, beta-glucuronidase and nitroreductase in the gut and/or faeces; stimulation of immune function; positive influence on bile salt concentration; and antioxidant effects.

Liver cancer reduction - due to aflatoxin detoxification and inhibition of mould growth.

Reduction of small bowel bacterial overgrowth - due to antibacterial activity; and decrease in toxic metabolite production from overgrowth flora. Immune system modulation and treatment of autoimmune disorders and allergies - due to enhancement of non-specific and antigen-specific defence against infection and tumors; enhanced mucosal immunity; . adjuvant effect in antigen-specific immune responses; and regulation of Thl/Th2 cells and production of cytokines.

Treatment of allergic responses to foods - due to prevention of antigen translocation into blood stream and modulation of allergenic factors in food.

Reduction of blood lipids and prevention of heart disease - due to assimilation of cholesterol by bacteria; hydrolysis of bile salts; and antioxidative effects.

Antihypertensive effect - bacterial protease or peptidase action on milk peptides produces antihypertensive peptides. Cell wall components act as ACE inhibitors

Prevention and treatment of urogenital infections - due to adhesion to urinary- and vaginal tract cells resulting in competitive exclusion; and production of antibacterial substances (acids, hydrogen peroxide and biosurfactants).

Treatment of inflammatory bowel disorder and irritable bowel syndrome - due to immuno-modulation; increased resistance to pathogen colonization; alteration of intestinal conditions such as pH; production of specific antibacterial substances such as bacteriocins, organic acids, hydrogen peroxide and biosurfactants; and competitive exclusion.

Modulation of infective endocarditis - due to fibronectin receptor-mediated platelet aggregation associated with Lactobacillus sepsis.

Prevention and treatment of Helicobacter pylori infection - due to competitive colonization and antibacterial effect.

Prevention and treatment of hepatic encephalopathy - due to inhibition and/or exclusion of urease-producing gut flora. Improved protein and carbohydrate utilization and conversion - due to production of beneficial products by bacterial action on proteins and carbohydrates.

Other beneficial health effects associated with L. rhamnosus include: improved nutrition; regulation of colonocyte proliferation and differentiation; improved lignan and isoflavone metabolism; reduced mucosal permeability; detoxification of carcinogens and other harmful compounds; relief of constipation and diarrhea; and vitamin synthesis, in particular folate.

Peptidases are enzymes that break the peptide bonds linking the amino group of one amino acid with the carboxy group (acid group) of an adjacent amino acid in a peptide chain. The bonds are broken in a hydrolytic reaction. There is a large family of peptidase enzymes that are defined by their specificity for the particular peptides bonds that they cleave (Barrett A J, Rawlings N D and Woessner J F (Eds.) 1998. Handbook of proteolytic enzymes. Academic Press, London, UK). The two main families are exopeptidases and endopeptidases. Exopeptidases cleave amino acids from the N- or C- terminus of a peptide chain, releasing free amino acids or short (di- and tri-) peptides. Different types of exopeptidases include:

• Aminopeptidases - release a free amino acid from the N-terminus of a peptide chain; • dipeptidyl-peptidases (also known as dipeptidyl-aminopeptidases) - release a dipeptide from the N-terminus of a peptide chain;

• tripeptidyl-peptidases (also known as tripeptidyl-aminopeptidases) - release a tripeptide from the N-terminus of a peptide chain);

• carboxypeptidases - release a free amino acid from the C-terminus of a peptide chain;

• peptidyl-dipeptidase - release a dipeptide from the C-terminus of a peptide chain;

• dipeptidases - release two free amino acids from a dipeptide; and

• tripeptidases - release a free amino acid and a dipeptide from a tripeptide.

Peptidases are important enzymes in the process of cheese ripening, and the development of cheese flavor. The hydrolysis of milk caseins in cheese results in textural changes and the development of cheese flavors. The raft of proteolytic enzymes that cause this hydrolysis come from the lactic acid bacteria that are bound up in the cheese - either starter cultures that grow up during the manufacture of the cheese, or adventitious and adjunct non- starter lactic acid bacteria that grow in the cheese as it ripens (Law and Haandrikman, Int. Dairy J. 7:1-11, 1997). Many other enzymes can also influence dairy product flavor, and functional and textural characteristics, as well as influencing the fermentation characteristics of the bacteria, such as speed of growth, acid production and survival (Urbach, Int. Dairy J. 5:877-890, 1995; Johnson and Somkuti, Biotech. Appl. Biochem. 13:196-204, 1991; El Soda and Pandian, J. Dairy Sci. 74:2317-2335, 1991; Fox et al, In Cheese: chemistry, physics and microbiology. Volume 1, General aspects, 2^nd edition, P Fox (ed) Chapman and Hall, London; Christensen et al, Antonie van Leeuwenhoek 76:217-246, 1999; Stingle et al, J. Bacteriol 20:6354-6360, 1999; Stingle et al, Mol. Microbiol. 32:1287-1295, 1999; Lemoine et al, Appl. Environ. Microbiol 63:1512-3518, 1997). Enzymes influencing specific characteristics and/or functions include the following:

Lysis of cells. These enzymes are mostly cell wall hydrolases, including amidases; muramidases; lysozymes, including N-acetyl muramidase; muramidase; N-acetyl- glucosaminidase; and N-acetylmuramoyl-L-alanine amidase. DEAD-box helicase proteins also influence autolysis.

■ Carbohydrate utilization. Lactose, citrate and diacetyl metabolism, and alcohol metabolism are particularly important. The enzymes involved include beta-galactosidase, lactate dehydrogenase, citrate lyase, citrate permease, 2,3 butanediol dehydrogenase (acetoin reductase), acetolactate decarboxylase, acetolactate synthase, pyruvate decarboxylase, pyruvate formate lyase, diacetyl synthase, diacetyl reductase, alcohol decarboxylase, lactate dehydrogenase, pyruvate dehydrogenase, and aldehyde dehydrogenase.

■ Lipid degradation, modification or synthesis. Enzymes involved include lipases, esterases, phospholipases, serine hydrolases, desaturases, and linoleate isomerase.

■ Polysaccharide synthesis. Polysaccharides are important not only for potential immune enhancement and adhesion activity but also for the texture of fermented dairy products. The enzymes involved are a series of glucosyl transferases, including beta-(l-3) glucosyl transferase, alpha-N acetylgalactosaminyl transferase, phosphogalactosyl transferase, alpha- glycosyl transferase, UDP-N-acetylglucosamine C4 epimerase and UDP-N-acetyl- glucosamine transferase.

■ Amino acid degradation. Enzymes include glutamate dehydrogenase, amino-transferases, amino acid decarboxylases, and enzymes involved in sulphur amino acid degradation including cystothione beta-lyase.

Sequencing of the genomes, or portions of the genomes, of numerous organisms, including humans, animals, microorganisms and various plant varieties, has been and is being carried out on a large scale. Polynucleotides identified using sequencing techniques may be partial or full-length genes, and may. contain open reading frames, or portions of open reading frames, that encode polypeptides. Putative polypeptides may be identified based on polynucleotide sequences and further characterized. The sequencing data relating to polynucleotides thus represents valuable and useful information.

Polynucleotides and polypeptides may be analyzed for varying degrees of novelty by comparing identified sequences to sequences published in various public domain databases, such as EMBL. Newly identified polynucleotides and corresponding polypeptides may also be compared to polynucleotides and polypeptides contained in public domain information to ascertain homology to known polynucleotides and polypeptides. In this way, the degree of similarity, identity or homology of polynucleotides and polypeptides having an unknown function may be determined relative to polynucleotides and polypeptides having known functions. Information relating to the sequences of isolated polynucleotides may be used in a variety of ways. Specified polynucleotides having a particular sequence may be isolated, or synthesized, for use in in vivo or in vitro experimentation as probes or primers. Alternatively, collections of sequences of isolated polynucleotides may be stored using magnetic or optical storage medium and analyzed or manipulated using computer hardware and software, as well as other types of tools. Summary ofthe Invention

The present invention provides isolated polynucleotides comprising a sequence selected from the group consisting of: (a) sequences identified in the attached Sequence Listing as SEQ ID NO: 1-121; (b) variants of those sequences; (c) extended sequences comprising the sequences set out in SEQ ID NOS: 1-121, and their variants; and (d) sequences comprising at least a specified number of contiguous residues of a sequence of SEQ ID NO: 1-121 -mers). Oligonucleotide probes and primers corresponding to the sequences set out in SEQ ID NO: 1- 121, and their variants are also provided. All of these polynucleotides and oligonucleotide probes and primers are collectively referred to herein, as "polynucleotides of the present invention."

The polynucleotide sequences identified as SEQ ID NO: 1-121 were derived from a microbial source, namely from fragmented genomic DNA of Lactobacillus rhamnosus, strain HNOOl, described in PCT International Publication No. WO 99/10476. Lactobacillus rhamnosus strain HNOOl are heterofermentative bacteria that are Gram positive, non-motile, non-spore forming, catalase negative, facultative anaerobic rods exhibiting an optimal growth temperature of 37+l°C and an optimum pH of 6.0 - 6.5. Experimental studies demonstrated that dietary supplementation with Lactobacillus rhamnosus strain HNOOl induced a sustained enhancement in several aspects of both natural and acquired immunity. A biologically pure culture of Lactobacillus rhamnosus strain HNOOl was deposited at the Australian Government Analytical Laboratories (AGAL), The New South Wales Regional Laboratory, 1 Suakin Street, Pymble, NSW 2073, Australia, as Deposit No. NM97/09514, dated 18 August 1997.

Certain ofthe polynucleotide sequences disclosed herein are "partial" sequences in that they do not represent a full-length gene encoding a full-length polypeptide. Such partial sequences may be extended by analyzing and sequencing various DNA libraries using primers and/or probes and well-known hybridization and/or PCR techniques. The partial sequences disclosed herein may thus be extended until an open reading frame encoding a polypeptide, a full-length polynucleotide and/or gene capable of expressing a polypeptide, or another useful portion of the genome is identified. Such extended sequences, including full-length polynucleotides and genes, are described as "corresponding to" a sequence identified as one of the sequences of SEQ ID NO: 1-121 or a variant thereof, or a portion of one ofthe sequences of SEQ ID NO: 1-121 or a variant thereof, when the extended polynucleotide comprises an identified sequence or its variant, or an identified contiguous portion (x-mer) of one of the sequences of SEQ ID NO: 1-121 or a variant thereof.

The polynucleotides identified as SEQ ID NO: 1-121 were isolated from Lactobacillus rhamnosus genomic DNA clones and represent sequences that are present in the cells from which the DNA was prepared. The sequence information may be used to identify and isolate, or synthesize, DNA molecules such as promoters, DNA-binding elements, open reading frames or full-length genes, that can then be used as expressible or otherwise functional DNA in transgenic organisms. Similarly, RNA sequences, reverse sequences, complementary sequences, antisense sequences and the like, corresponding to the polynucleotides of the present invention, may be routinely ascertained and obtained using the polynucleotides identified as SEQ ID NO: 1-121.

The present invention further provides isolated polypeptides encoded, or partially encoded, by the polynucleotides disclosed herein. In certain specific embodiments, the polypeptides of the present invention comprise a sequence selected from the group consisting of sequences identified as SEQ ID NO: 122-253, and variants thereof. Polypeptides encoded by the polynucleotides ofthe present invention may be expressed and used in various assays to determine their biological activity. Such polypeptides may be used to raise antibodies, to isolate corresponding interacting proteins or other compounds, and to quantitatively determine levels of interacting proteins or other compounds. Genetic constructs comprising the inventive polynucleotides are also provided, together with transgenic host cells comprising such constructs and transgenic organisms, such as microbes, comprising such cells.

The present invention also contemplates methods for modulating the polynucleotide and/or polypeptide content and composition of an organism, such methods involving stably incorporating into the genome ofthe organism a genetic construct comprising a polynucleotide of the present invention. In one embodiment, the target organism is a microbe, preferably a microbe used in fermentation, more preferably a microbe ofthe genus Lactobacillus, and most preferably Lactobacillus rhamnosus, or other closely microbial related species used in the dairy industry. In a related aspect, methods for producing a microbe having an altered genotype and/or phenotype is provided, such methods comprising transforming a microbial cell with a genetic construct of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to growth and multiplication. Organisms having an altered genotype or phenotype as a result of modulation of the level or content of a polynucleotide or polypeptide of the present invention compared to a wild-type organism, as well as components and progeny of such organisms, are contemplated by and encompassed within the present invention.

The isolated polynucleotides ofthe present invention may be usefully employed for the detection of lactic acid bacteria, preferably L. rhamnosus, in a sample material, using techniques well known in the art, such as polymerase chain reaction (PCR) and DNA hybridization, as detailed below.

The inventive polynucleotides and polypeptides may also be employed in methods for the selection and production of more effective probiotic bacteria; as "bioactive" (health- promoting) ingredients and health supplements for immune function enhancement; for reduction of blood lipids such as cholesterol; for production of bioactive material^' from genetically modified bacteria;^' as adjuvants; for wound healing; in vaccine development, particularly mucosal vaccines; as animal probiotics for improved animal health and productivity; in selection and production of genetically modified rumen microorganisms for improved animal nutrition and productivity, better flavor and improved milk composition; in methods for the selection and production of better natural food bacteria for improved flavor, faster flavor development, better fermentation characteristics, vitamin synthesis and improved textural characteristics; for the production of improved food bacteria through genetic modification; and for the identification of novel enzymes for the production of, for example, flavors or aroma concentrates.

The isolated polynucleotides of the present invention also have utility in genome mapping, in physical mapping, and in positional cloning of genes of more or less related microbes. Additionally, the polynucleotide sequences identified as SEQ ID NO: 1-121, and their variants, may be used to design oligonucleotide probes and primers. Such oligonucleotide probes and primers have sequences that are substantially complementary to the polynucleotide of interest over a certain portion ofthe polynucleotide. Oligonucleotide probes designed using the polynucleotides of the present invention may be used to detect the presence, and examine the expression patterns, of genes in any organism having sufficiently similar DNA and RNA sequences in their cells, using techniques that are well known in the art, such as slot blot DNA hybridization techniques. Oligonucleotide primers designed using the polynucleotides of the present invention may be used for polymerase chain reaction (PCR) amplifications. Oligonucleotide probes and primers designed using the polynucleotides ofthe present invention may also be used in connection with various microarray technologies, including the microarray technology of Affymetrix (Santa Clara, CA).

The polynucleotides of the present invention may also be used to tag or identify an organism or derived material or product therefrom. Such tagging may be accomplished, for example, by stably introducing a non-disruptive, non-functional, heterologous polynucleotide identifier into an organism, the polynucleotide comprising at least a portion of a polynucleotide ofthe present invention.

The polynucleotides of the present invention may also be used as promoters, gene regulators, origins of DNA replication, secretion signals, cell wall or membrane anchors for genetic tools (such as expression or integration vectors).

All references cited herein, including patent references and non-patent publications, are hereby incorporated by reference in their entireties.

Detailed Description The polynucleotides disclosed herein were isolated by high throughput sequencing of

DNA libraries from the lactic acid bacteria Lactobacillus rhamnosus as described below in Example 1. Cell wall, cell surface and secreted components of lactic acid bacteria are known to mediate immune modulation, cell adhesion and antibacterial activities, resulting in many beneficial effects including: resistance to enteric pathogens; modulation of cancer, including colon cancer; anti-mutagenesis effects; reduction of small bowel bacterial overgrowth; modulation of auto-immune disorders; reduction in allergic disorders; modulation of urogenital infections, inflammatory bowel disorder, irritable bowel syndrome, Helicobacter pylori infection and hepatic encephalopathy; reduction of infection with pathogens; regulation of colonocyte proliferation and differentiation; reduction of mucosal permeability; and relief of constipation and diarrhea. These cell components include, but are not limited to, peptidoglycans, teichoic acids, lipoteichoic acids, polysaccharides, adhesion proteins, secreted proteins, surface layer or S -layer proteins, collagen binding proteins and other cell surface proteins, and antibacterial substances such as bacteriocins and organic acids produced by these bacteria. Polynucleotides involved in the synthesis of these proteins and in the synthesis, modification, regulation, transport, synthesis and/or accumulation of precursor molecules for these proteins can be used to modulate the immune effects, antibacterial, cell adhesion and competitive exclusion effects ofthe bacteria or of components that might be produced by these bacteria. In order to function effectively as probiotic bacteria, L. rhamnosus HNOOl must survive environmental stress conditions in the gastrointestinal tract, as well as commercial and industrial processes. Modification of particular polynucleotides or regulatory processes has been shown to be effective against a number of stresses including oxidative stress, pH, osmotic stress, dehydration, carbon starvation, phosphate starvation, nitrogen starvation, amino acid starvation, heat or cold shock, and mutagenic stress. Polynucleotides involved in stress resistance often confer multistress resistance, i.e., when exposed to one stress, surviving cells are resistant to several non-related stresses. Bacterial genes and/or processes shown to be involved in multistress resistance include:

' Intracellular phosphate pools - inorganic phosphate starvation leads to the induction of pho regulon genes, and is linked to the bacterial stringent response. Gene knockouts involving phosphate receptor genes appear to lead to multistress resistance.

Intracellular guanosine pools - purine biosynthesis and scavenger pathways involve the production of phosphate-guanosine compounds that act as signal molecules in the bacterial stringent response. Gene knockouts involving purine scavenger pathway genes appear to confer multistress resistance.

Osmoregulatory molecules - small choline-based molecules, such as glycine-betaine, and sugars, such as trehalose, are protective against osmotic shock and are rapidly imported and/or synthesized in response to increasing osmolarity.

Acid resistance - lactobacilli naturally acidify their environment through the excretion of lactic acid, mainly through the cit operon genes responsible for citrate uptake and utilization.

Stress response genes - a number of genes appear to be induced or repressed by heat shock, cold shock, and increasing salt through the action of specific promoters.

The isolated polynucleotides of the present invention, and genetic constructs comprising such polynucleotides, may be employed to produce bacteria having desired phenotypes, including increased resistance to stress and improved fermentation properties. Many enzymes are known to influence dairy product flavor, functional and textural characteristics as well as general feπnentation characteristics such as speed of growth, acid production and survival. These enzymes include those involved in the metabolism of lipids, polysaccharides, amino acids and carbohydrates as well as those involved in the lysis of the bacterial cells.

The isolated polynucleotides and polypeptides of the present invention have demonstrated similarity to polynucleotides and/or polypeptides of known function. The identity and functions of the inventive polynucleotides based on such similarities are shown below in Table 1.

TABLE 1

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<img file="WO2004031389A1_D0008.tif" /><img file="WO2004031389A1_D0009.tif" /><img file="WO2004031389A1_D0010.tif" />

<img file="WO2004031389A1_D0011.tif" /><img file="WO2004031389A1_D0012.tif" /><img file="WO2004031389A1_D0013.tif" /><img file="WO2004031389A1_D0014.tif" />

<img file="WO2004031389A1_D0015.tif" /><img file="WO2004031389A1_D0016.tif" /><img file="WO2004031389A1_D0017.tif" />

<img file="WO2004031389A1_D0018.tif" /><img file="WO2004031389A1_D0019.tif" />

<img file="WO2004031389A1_D0020.tif" /> -39 162-164 Altered cell wall or cell surface Homologue of a bacterial hemolysin. characteristics, structures or functions. Hemolysin is a bacterial toxin gene that Modified adhesion to human or can be used to develop vaccines against animal cells or cell lines. pathogenic bacteria carrying the Production of desirable flavors. gene/protein. Transgenic microbes with Modified flavor, aroma and/or texture added, deleted or modified hemolysin attributes. protein gene can have a modified

Construction of genetic vectors for hemolytic activity in vitro (useful e.g. in controlled expression of RNA and/or bacteria-based assays) or in vivo (useful protein, fusion protein production, in enhancing/decreasing pathogenicity genetic modification, mutagenesis of bacteria, as desired (see e.g. J. Biol. amplification of genetic material or Chem. 267:10902-10909, 1992). for other genetic or protein Functional expression ofthe alpha- manipulations. hemolysin gene of Staphylococcus

Altered survival characteristics: aureus in intact E. coli and in cell survival of industrial processes, lysates. Deletion of five C-terminal growth or storage in product formats_* amino acids selectively impairs persistence in gut environment. hemolytic activity. The peptide itself can Altered metabolic properties. be used as a reagent e.g. in in vitro Altered probiotic attributes. assays of hemolytic activity. May be , Modified health properties (including invovled in scavenging iron from immunoregulatory, anticancer, gut environment, and therefore is involved health). in cell survival and metabolism, as well

Modified antibiotic resistance. as restricting growth of surrounding Improved antimicrobial properties. microbes. Improved fermentation properties or other industrially useful processes. Regulation of cell numbers and growth. , 41 165, 166 Altered cell wall or cell surface Homologue of penicillin-binding characteristics, structures or functions. proteins (PBPs). PBPs are enzymes Improved antimicrobial properties involved in the final stages of Modified adhesion to human or peptidoglycan biosynthesis. The C- animal cells or cell lines. terminal module binds penicillin and Production of desirable flavors. catalyzes peptidoglycan cross-linking. Modified flavor, aroma and/or texture The N-terminal domain can have attributes. transglycosylase activity (class A HMW

Construction of genetic vectors for PBPs). PBPs are the targets of beta- controlled expression of RNA and/or lactam antibiotics, which covalently protein, fusion protein production, bind to these proteins, inhibiting cell genetic modification, mutagenesis wall synthesis (Mariana et al, J. amplification of genetic material or Bacteriol. 182:1074-1079, 2000). for other genetic or protein Penicillin-binding protein gene can be manipulations. used for modifications in transgenic

Altered survival characteristics: bacteria, which can change their survival of industrial processes, susceptibility to penicillin (see e.g. <img file="WO2004031389A1_D0021.tif" /> controlled expression of RNA and/or strands) and a penicillin-sensitive protein, fusion protein production, transpeptidase c-terminal domain (cross- genetic modification, mutagenesis linking ofthe peptide subunits) amplification of genetic material or responsible for the final steps ofthe for other genetic or protein bacterial cell wall polymerization and manipulations. cross-linking, respectively (Zhao et al,

Altered survival characteristics: Protein Expr. Purif 16:331-339, 1999). survival of industrial processes, Penicillin-binding protein gene can be growth or storage in product formats, used for modifications in transgenic persistence in gut environment. bacteria, which can change their Altered metabolic properties. susceptibility to penicillin (see e.g. Altered probiotic attributes. . Smith and Klugman, Antimicrob. Modified health properties (including Agents Chemother. 42:1329-1333, immunoregulatory, anticancer, gut 1998). Alterations in PBP 1A essential- health). for high-level penicillin resistance in

Modified antibiotic resistance. Streptococcus pneumoniae. The DNA or Improved fermentation properties or protein can be used in various assays to other industrially useful processes. detect the presence ofthe gene or protein in various biological samples, where penicillin binding is of interest. 9, 234 Production of desirable flavors. Homologue of protein p60, encoded by

Modified flavor, aroma and/or texture the gene termed iap. p60 protein is a attributes. major extracellular product secreted by

Construction of genetic vectors for all isolates of Listeria monocytogenes. controlled expression of RNA and/or This protein has peptidoglycan protein, fusion protein production, hydrolase activity but also influences the genetic modification, mutagenesis uptake of L. monocytogenes by non- amplification of genetic material or phagocytic cells. Proteins related to p60 for other genetic or protein are found in all other Listeria species. It manipulations. has been shown that p60 protein is

Altered survival characteristics: among the strongest antigens in listeriae survival of industrial processes, for B- and T-cell responses. The protein growth or storage in product formats, p60 belongs to the E. coli nlpc / listeria persistence in gut environment. p60 family. This gene can be useful as a

Altered metabolic properties. probe to detect the presence ofthe

Altered probiotic attributes. gene/protein in various bacteria.

Modified adhesion to human or Deletion, addition and modification of animal cells or cell lines. the gene in transgenic bacteria can alter

Organisms or materials with improved|thι eir extracellular envelope structure, health properties (including thereby altering their growth and immunoregulatory, anticancer, gut pathogenicity characteristics. See e.g. health) Bubert et al, J. Bacteriol. 174:8166-

Altered resistance to antibiotics. 8171, 1992.

Improved antimicrobial properties.

170 Altered cell wall or cell surface Homologue of flotillin. Flotillins have characteristics, structures or functions, been found in mammalian, insect and Modified adhesion to human or bacterial cells and behave as resident <img file="WO2004031389A1_D0022.tif" /><img file="WO2004031389A1_D0023.tif" /><img file="WO2004031389A1_D0024.tif" />

<img file="WO2004031389A1_D0025.tif" /><img file="WO2004031389A1_D0026.tif" /><img file="WO2004031389A1_D0027.tif" />

<img file="WO2004031389A1_D0028.tif" /><img file="WO2004031389A1_D0029.tif" /><img file="WO2004031389A1_D0030.tif" /><img file="WO2004031389A1_D0031.tif" />

<img file="WO2004031389A1_D0032.tif" /><img file="WO2004031389A1_D0033.tif" /><img file="WO2004031389A1_D0034.tif" /><img file="WO2004031389A1_D0035.tif" /><img file="WO2004031389A1_D0036.tif" /> Construction of genetic vectors for autoregulated (Himpens et al, controlled expression of RNA and/or Microbiol. 146:3091-3098, 2000). protein, fusion protein production, Response regulators in bacteria are genetic modification, mutagenesis involved in the bacterium's ability to amplification of genetic material or monitor its surroundings and adapt to for other genetic or protein changes in its environment. Several of manipulations. these bacterial regulators are involved in

Altered survival characteristics: virulence and bacterial pathogenesis survival of industrial processes, within the host (see e.g. US Patent growth or storage in product formats, 5,910,572) The response regulators are persistence in gut environment. involved in production of virulence Regulation of metabolic processes. factors, motility, antibiotic resistance Altered probiotic attributes. and cell replication. Inhibitors of these Modified health properties (including proteins would be useful in preventing immunoregulatory, anticancer, gut bacterium from progressing to health, apoptosis). pathogenesis, thus useful in medical Modified antibiotic resistance. treatments against bacteria. May have Improved antimicrobial properties. utility as a controlled expression vector. Improved fermentation properties or other industrially useful processes.

Altered cell wall or cell surface Homologue of spinosyn biosynthesis. characteristics, structures or functions, Spinosyns are macrolides with a 21- Modified adhesion to human or carbon, tetracyclic lactone backbone to animal cells or cell lines. which the deoxysugars forosamine and Production of desirable flavors. tri-O-methylrhamnose are attached. Modified flavor, aroma and/or texture Macrolides interfere with the attributes. peptidlytransfer function ofthe

Construction of genetic vectors for ribosome. The macrolide antibiotics, controlled expression of RNA and/or which include erythromycin, protein, fusion protein production, azithromycin, and the streptogramin genetic modification, mutagenesis family among others, work by binding amplification of genetic material or the large ribosomal subunit. The for other genetic or protein molecular details ofthe binding site for manipulations. macrolides are not well understood. The

Altered survival characteristics: spinosyns, a novel family of insecticidal survival of industrial processes, macrocyclic lactones, are active on a growth or storage in product formats, wide variety of insect pests, especially persistence in gut environment. lepidopterans and dipterans (see Regulation of metabolic processes. WO9946387-A1: Biosynthetic genes for Altered probiotic attributes. spinosyn insecticide production ). This Modified health properties (including gene can be useful in a related immunoregulatory, anticancer, gut compound biosynthesis utilization for health, apoptosis). bioactive compounds. Modified antibiotic resistance. Improved antimicrobial properties. Improved fermentation properties or other industrially useful processes. 73 198 Altered cell wall or cell surface Homologue of EpsK protein. EpsK is characteristics, structures or functions. involved in exopolysaccharide Modified adhesion to human or biosynthesis. A broad variety of bacteria animal cells or cell lines. produce polysaccharides, which can Production of desirable texture. either be excreted into the environment Modified flavor, aroma and/or texture as exopolysaccharides (EPSs), form a attributes. capsule around the cell as capsular

Construction of genetic vectors for polysaccharides, or be attached to the controlled expression of RNA and/or cell membrane as the O antigens of protein, fusion protein production, lipopolysaccharides. The biosynthesis of genetic modification, mutagenesis polysaccharides that consist of repeating amplification of genetic material or units includes their assembly on a lipid for other genetic or protein carrier by sequential transfer of manipulations. monosaccharides from nucleotide sugars

Altered survival characteristics: by glycosyltransferases (GTFs) and the survival of industrial processes, subsequent polymerization and export of growth or storage in product formats, these repeating units. Secreted persistence in gut environment. exopolysaccharides contribute to the cell Altered metabolic properties. protection against environmental Altered probiotic attributes. influences, attachment to surfaces, Modified health properties (including nutrient gathering and to antigenicity. immunoregulatory, anticancer, gut Due to the variation of monosaccharide health, apoptosis). sequences, condensation linkages and Modified antibiotic resistance. non-carbohydrate decorations, an Improved antimicrobial properties. infinite array of structures can be Improved fermentation properties or provided by these sugar polymers. other industrially useful processes. Deletion, addition and modification of the gene in transgenic bacteria can alter their extracellular envelope structure, thereby altering their growth and pathogenicity characteristics.

74 199 Altered cell wall or cell surface Homologue of CpxA protein. CpxA is characteristics, structures or functions, involved in several diverse cellular Modified adhesion to human or processes, such as the functioning of animal cells or cell lines. acetohydroxyacid synthetase I, in the Production of desirable flavors. biosynthesis of isoleucine and valine, the Modified flavor, aroma and/or texture traJ protein activation activity for tra attributes. gene expression in F plasmid, and the

Construction of genetic vectors for synthesis, translocation, or stability of controlled expression of RNA and/or cell envelope proteins. It also activates protein, fusion protein production, CpxR by phosphorylation. The CpxA- genetic modification, mutagenesis CpxR two-component signal amplification of genetic material or transduction system regulates gene for other genetic or protein expression in adaptation to adverse manipulations. conditions. These include envelope

Altered survival characteristics: protein distress, heat shock, oxidative survival of industrial processes, stress, high pH, and entry into stationary <img file="WO2004031389A1_D0037.tif" />

<img file="WO2004031389A1_D0038.tif" /><img file="WO2004031389A1_D0039.tif" /><img file="WO2004031389A1_D0040.tif" /><img file="WO2004031389A1_D0041.tif" /><img file="WO2004031389A1_D0042.tif" /> animal cells or cell lines. also known as UDP-N-acetyl- Production of desirable flavors. muramoyl-L-alanyl-D-gamma-glutamyl- Modified flavor, aroma and/or texture L-lysyl-D-alanyl-D- attributes. alanine:undecaprenoid-alcohol-

Construction of genetic vectors for phosphate-phospho-N-acetylmuramoyl- controlled expression of RNA and/or pentapeptide-transferase. mraY protein, fusion protein production, catalyzes the formation of undecaprenyl- genetic modification, mutagenesis pyiOphosphoiyl-N-acetylmuramoyl- amplification of genetic material or pentapeptide from UDP-N- for other genetic or protein acetylmuramoyl-pentapeptide and manipulations. undecaprenyl-phosphate, the first step in

Altered survival characteristics: the lipid cycle reactions in biosynthesis survival of industrial processes, of bacterial cell wall peptidoglycans. growth or storage in product formats, Phospho-N-acetylmuramoyl- persistence in gut environment. pentapeptide-transferase is an integral Altered metabolic properties. membrane protein and belongs to the Altered probiotic attributes. glycosyltransferase family 4 mraY Modified health properties (including subfamily. immunoregulatory, anticancer, gut health).

Modified antibiotic resistance. Improved antimicrobial properties. Improved fermentation properties or other industrially useful processes.

Altered cell wall or cell surface Homologue of UPPS, encoding characteristics, structures or functions, undecaprenyl pyrophosphate synthetase Modified adhesion to human or (EC 2.5.1.31). UPPS catalyzes the Z- animal cells or cell lines. oligomerization of isopentenyl units Production of desirable flavors. with farnesyl pyrophosphate as a Modified flavor, aroma and/or texture priming substrate to give C50 and C55 attributes. prenyl pyrophosphates with Z,E mixed

Construction of genetic vectors for stereochemistry. Undecaprenyl controlled expression of RNA and/or pyrophosphate synthetase is required as protein, fusion protein production, a lipid carrier of glycosyl transfer in the genetic modification, mutagenesis biosynthesis of a variety of cell wall amplification of genetic material or polysaccharide components in bacteria. for other genetic or protein manipulations.

Altered survival characteristics: survival of industrial processes, growth or storage in product formats, persistence in gut environment. Altered metabolic properties. Altered probiotic attributes. Modified health properties (including immunoregulatory, anticancer, gut health). <img file="WO2004031389A1_D0043.tif" /><img file="WO2004031389A1_D0044.tif" /><img file="WO2004031389A1_D0045.tif" /><img file="WO2004031389A1_D0046.tif" />

<img file="WO2004031389A1_D0047.tif" /> Altered survival characteristics: of bacteria, families of CSPs, consisting survival of industrial processes, of three to nine members, have been growth or storage in product formats, described of which CspA in E. coli persistence in gut environment. (CspAΕ) and CspB in Bacillus subtilis Altered viability in response to stress (CspBB) are the best characterized. conditions. CspAΕ and CspBB are capable of

Altered metabolic properties or binding to single-stranded DNA and regulation of metabolic pathways. RNA, and based on these characteristics, Altered probiotic attributes. several functions for CSPs have been Improved fermentation properties or suggested, such as transcriptional other industrially useful processes. activators, RNA chaperones that facilitate the initiation of translation, and freeze-protective proteins. Recently it has been shown that CSPs might regulate the expression of cold- induced genes as antiterminators. Regulation of csp genes takes place at several levels, and for CspAΕ it was shown that cold- shock induction is achieved at the transcriptional level as well as at the level of mRNA and protein stability (Wouters et al, Appl. Environ. Microbiol. 67:5171-5178, 2001).

Production of desirable flavors. Homologue of fabF, beta-ketoacyl Modified flavor, aroma and/or texture synthase (acyl caπier protein). FabF is attributes. part ofthe fatty acid biosynthesis (fab)

Construction of genetic vectors for gene cluster (fabD-fabH-acpP-fabF) controlled expression of RNA and/or involved in fatty acid biosynthesis. FabF protein, fusion protein production, is the condensing enzyme thought to be genetic modification, mutagenesis responsible for elongation of fatty acids. amplification of genetic material or The fab genes are important in the for other genetic or protein production of butyric acid, with manipulations. important flavor and health impacts. It

Altered survival characteristics: also has antibiotic effects and may be survival of industrial processes, protective against colon cancer growth or storage in product formats, (Mortensen and Clausen, Scand. J. persistence in gut environment. Gastroenterol. Suppl. 216:132-148, Altered metabolic properties. 1996). Modified lipid, glycolipid or free fatty acid levels or functional properties. Modified production of short chain fatty acids.

Altered lipid metabolism. Altered probiotic attributes. Organisms or materials with improved health properties (including immunoregulatory, anticancer, gut <img file="WO2004031389A1_D0048.tif" /><img file="WO2004031389A1_D0049.tif" /> amplification of genetic material or reaction catalyzed by acetyl-CoA for other genetic or protein carboxylase proceeds via two half- manipulations. reactions. To carry out this two-step

Altered survival characteristics: reaction acetyl-CoA carboxylase survival of industrial processes, requires three distinct components: growth or storage in product formats, biotin carboxylase, biotin carboxyl persistence in gut environment. carrier protein, and carboxyltransferase. Altered metabolic properties. The biotin carboxylase component Modified lipid, glycolipid or free fatty catalyzesthe first half-reaction, which is acid levels or functional properties. an ATP-dependent carboxylation ofthe Modified production of short chain vitamin biotin. In vivo, biotin is fatty acids. covalently attached to the biotin

Altered lipid metabolism. carboxyl caπier protein designated as Altered probiotic attributes. BCCP. The second half-reaction, the Organisms or materials with improved transfer ofthe carboxyl group from health properties (including carboxybiotin to acetyl-CoA to make immunoregulatory, anticancer, gut malonyl-CoA, is catalyzed by the health) carboxyltransferase component. The chain length of newly synthesized fatty acids appears to depend on the concentration of malonyl-CoA (Janiyani et al, J. Biol. Chem. 276:29864-29870, 2001). Indications are that fatty acids in gram-positive bacteria act as signaling molecules that are important for cell differentiation (Marini et al, J. Bacteriol. 177:7003-7006, 1995).

Production of desirable flavors. Homologue of fabD, malonyl coenzyme Modified flavor, aroma and/or texture A-acyl carrier protein transacylase, attributes. which is part ofthe fatty acid

Construction of genetic vectors for biosynthesis (fab) gene cluster (fabD- controlled expression of RNA and/or fabH-acpP-fabF) involved in fatty acid protein, fusion protein production, biosynthesis. FabD overexpression leads genetic modification, mutagenesis to altered fatty acid composition in E. amplification of genetic material or coli, with increased amounts of cis- for other genetic or protein vaccenate incorporated into membrane manipulations. phospholipids. The fab genes are

Altered survival characteristics: important in the production of butyric survival of industrial processes, acid, with important flavor and health growth or storage in product formats, impacts. It also has antibiotic effects persistence in gut environment. and may be protective against colon Altered metabolic properties. cancer (Mortensen and Clausen, Scand. Modified lipid, glycolipid or free fatty [/. Gastroenterol. Suppl. 216:132-148, acid levels or functional properties. 1996). Modified production of short chain fatty acids. Altered lipid metabolism. <img file="WO2004031389A1_D0050.tif" /><img file="WO2004031389A1_D0051.tif" />

Isolated polynucleotides of the present invention include the polynucleotides identified herein as SEQ ID NO: 1-121; isolated polynucleotides comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-121; isolated polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NO: 1-121; isolated polynucleotides comprising a polynucleotide sequence that is complementary to any of the above polynucleotides; isolated polynucleotides comprising a polynucleotide sequence that is a reverse sequence or a reverse complement of any of the above polynucleotides; antisense sequences coπesponding to any of the above polynucleotides; and variants of any of the above polynucleotides, as that term is described in this specification.

The word "polynucleotide(s)," as used herein, means a single or double stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and coπesponding RNA molecules, including mRNA molecules, both sense and antisense strands of DNA and RNA molecules, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides. A polynucleotide ofthe present invention may be an entire gene, or any portion thereof. A gene is a DNA sequence which codes for a functional protein or RNA molecule. Operable antisense polynucleotides may comprise a fragment of the coπesponding polynucleotide, and the definition of "polynucleotide" therefore includes all operable antisense fragments. Antisense polynucleotides and techniques involving antisense polynucleotides are well known in the art and are described, for example, in Robinson-Benion, et al., "Antisense techniques," Methods in Enzymol 254(23): 363-375, 1995; and Kawasaki, et al.,Artific. Organs 20 (8): 836-848, 1996. The definitions of the terms "complement," "reverse complement," and "reverse sequence," as used herein, are best illustrated by the following examples. For the sequence 5' AGGACC 3', the complement, reverse complement, and reverse sequences are as follows: complement 3' TCCTGG 5' reverse complement 3' GGTCCT 5' reverse sequence 5' CCAGGA 3'

Preferably, sequences that are complements of a specifically recited polynucleotide sequence are complementary over the entire length ofthe specific polynucleotide sequence.

Identification of genomic DNA and heterologous species DNA can be accomplished by standard DNA DNA hybridization techniques, under appropriately stringent conditions, using all or part of a DNA sequence as a probe to screen an appropriate library. Alternatively, PCR techniques using oligonucleotide primers that are designed based on known DNA and protein sequences can be used to amplify and identify other identical or similar DNA sequences. Synthetic DNA corresponding to the identified sequences or variants thereof may be produced by conventional synthesis methods. All of the polynucleotides described herein are isolated and purified, as those terms are commonly used in the art.

The polynucleotides identified as SEQ ID NO: 1-121 contain open reading frames ("ORFs"), or partial open reading frames, encoding polypeptides. Additionally, polynucleotides identified as SEQ ID NO: 1-121 may contain non-coding sequences such as promoters and terminators that may be useful as control elements. Open reading frames encoding polypeptides may also be identified in extended or full-length sequences coπesponding to the sequences set out as SEQ ID NO: 1-121. Open reading frames may be identified using techniques that are well known in the art. These techniques include, for example, analysis for the location of known start and stop codons, most likely reading frame identification based on codon frequencies, similarity to known bacterial expressed genes, etc.

Tools and software suitable for ORF analysis include Gene Wise (The Sanger Center, Wellcome

Trust Genome Campus, Hinxton, Cambridge CB10 ISA, United Kingdom), Diogenes (Computational Biology Centers, University of Minnesota, Academic Health Center, UMHG

Box 43 Minneapolis MN 55455), and GRAIL (Informatics Group, Oak Ridge National

Laboratories, Oak Ridge, Tennessee, TN). Open reading frames and portions of open reading frames may be identified in the polynucleotides of the present invention. Once a partial open reading frame is identified, the polynucleotide may be extended in the area of the partial open reading frame using techniques that are well known in the art until the polynucleotide for the full open reading frame is identified. Thus, polynucleotides and open reading frames encoding polypeptides may be identified using the polynucleotides ofthe present invention. Once open reading frames are identified in the polynucleotides ofthe present invention, the open reading frames may be isolated and/or synthesized. Expressible genetic constructs comprising the open reading frames and suitable promoters, initiators, terminators, etc., which are well known in the art, may then be constructed. Such genetic constructs may be introduced into a host cell to express the polypeptide encoded by the open reading frame. Suitable host cells may include various prokaryotic and eukaryotic cells. In vitro expression of polypeptides is also possible, as well known in the art.

As used herein, the term "oligonucleotide" refers to a relatively short segment of a polynucleotide sequence, generally comprising between 6 and 60 nucleotides, and comprehends both probes for use in hybridization assays and primers for use in the amplification of DNA by polymerase chain reaction.

As used herein, the term "x-mer," with reference to a specific value of "x," refers to a polynucleotide comprising at least a specified number ("x") of contiguous residues of any of the polynucleotides identified as SEQ ID NO: 1-121. The value of x may be from about 20 to about 600, depending upon the specific sequence.

In another aspect, the present invention provides isolated polypeptides encoded, or partially encoded, by the above polynucleotides. In specific embodiments, such polypeptides comprise a sequence selected from the group consisting of SEQ ID NO: 122-253, and variants thereof. As used herein, the term "polypeptide" encompasses amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. The term "polypeptide encoded by a polynucleotide" as used herein, includes polypeptides encoded by a polynucleotide which comprises an isolated polynucleotide sequence or variant provided herein. Polypeptides of the present invention may be naturally purified products, or may be produced partially or wholly using recombinant techniques. Such polypeptides may be glycosylated with bacterial, fungal, mammalian or other eukaryotic carbohydrates or may be non-glycosylated.

Polypeptides of the present invention may be produced recombinantly by inserting a polynucleotide that encodes the polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a polypeptide encoding a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are Escherichia coli, Lactococcus lactis, Lactobacillus, insect, yeast or a mammalian cell line such as COS or CHO. The polynucleotide(s) expressed in this manner may encode naturally occuπing polypeptides, portions of naturally occurring polypeptides, or other variants thereof.

In a related aspect, polypeptides are provided that comprise at least a functional portion of a polypeptide having an amino acid sequence encoded by a polynucleotide of the present invention. As used herein, a "functional portion" of a polypeptide is that portion which contains the active site essential for affecting the function of the polypeptide, for example, the portion ofthe molecule that is capable of binding one or more reactants. The active site may be made up of separate portions present on one or more polypeptide chains and will generally exhibit high binding affinity.

Functional portions of a polypeptide may be identified by first preparing fragments of the polypeptide by either chemical or enzymatic digestion of the polypeptide, or by mutation analysis of the polynucleotide that encodes the polypeptide and subsequent expression of the resulting mutant polypeptides. The polypeptide fragments or mutant polypeptides are then tested to determine which portions retain biological activity, using, for example, the representative assays provided below.

Portions and other variants of the inventive polypeptides may be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques that are well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid- phase synthesis method, where amino acids are sequentially added to a growing amino acid chain (See Merrifield, J. Am. Chem. Soc. 85:2149-2154, 1963). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied Biosystems, Inc. (Foster City, CA), and may be operated according to the manufacturer's instructions. Variants of a native polypeptide may be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492, 1985). Sections of DNA sequences may also be removed using standard techniques to permit preparation of truncated polypeptides. In general, the polypeptides and polynucleotides disclosed herein are provided in an isolated, substantially pure, form. Preferably, the polypeptides and polynucleotides are at least about 80% pure; more preferably at least about 90% pure; and most preferably at least about 99% pure. As used herein, the term "variant" comprehends polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant polynucleotide and polypeptides sequences preferably exhibit at least 60%, more preferably at least 75%, more preferably yet at least 90%), and most preferably at least 95% identity to a sequence of the present invention. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100.

Polynucleotide and polypeptide sequences may be aligned, and the percentage of identical residues in a specified region may be determined against another polynucleotide or polypeptide, using computer algorithms that are publicly available. Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms. Polynucleotides may also be analyzed using the BLASTX algorithm, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. The percentage identity of polypeptide sequences may be examined using the BLASTP algorithm. The BLASTN, BLASTX and BLASTP programs are available on the NCBI anonymous FTP server and from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894, USA. The BLASTN algorithm Version 2.0.4 [Feb-24- 1998], Version 2.0.6 [Sept- 16- 1998] and Version 2.0.11 [Jan-20-2000], set to the parameters described below, is prefeπed for use in the determination of polynucleotide variants according to the present invention. The BLASTP algorithm, set to the parameters described below, is prefeπed for use in the determination of polypeptide variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, BLASTP and BLASTX, is described in the publication of Altschul, et al, Nucleic Acids Res. 25: 3389-3402, 1997.

The computer algorithm FASTA is available on the Internet and from the University of Virginia by contacting David Hudson, Vice Provost for Research, University of Virginia, P.O. Box 9025, Charlottesville, VA 22906-9025, USA. FASTA Version 2.0u4 [February 1996], set to the default parameters described in the documentation and distributed with the algorithm, may be used in the determination of variants according to the present invention. The use ofthe FASTA algorithm is described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444- 2448, 1988; and Pearson, Methods in Enzymol. 183: 63-98, 1990.

The following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity for polynucleotide sequences: Unix running command: blastall -p blastn -d embldb -e 10 -GO -E0 -r 1 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; - r Reward for a nucleotide match (BLASTN only) [Integer]; -v Number of one-line descriptions (V) [Integer]; -b Number of alignments to show (B) [Integer]; -i Query File [File In]; and -o BLAST report Output File [File Out] Optional.

The following running parameters are preferred for determination of alignments and similarities using BLASTP that contribute to the E values and percentage identity of polypeptide sequences: blastall -p blastp -d swissprotdb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; - E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line descriptions (v) [Integer]; -b Number of alignments to show (b) [Integer]; -I Query File [File In]; -o BLAST report Output File [File Out] Optional. The "hits" to one or more database sequences by a queried sequence produced by

BLASTN, FASTA, BLASTP or a similar algorithm, align and identify similar portions of sequences. The hits are aπanged in order ofthe degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length ofthe queried sequence. The BLASTN, FASTA, and BLASTP algorithms also produce "Expect" values for alignments. The Expect value (E) indicates the number of hits one can "expect" to see over a certain number of contiguous sequences by chance when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a database, such as the prefeπed EMBL database, indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size ofthe EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. By this criterion, the aligned and matched portions ofthe polynucleotide sequences then have a probability of 90% of being the same. For sequences. having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm.

According to one embodiment, "variant" polynucleotides and polypeptides, with reference to each of the polynucleotides and polypeptides of the present invention, preferably comprise sequences producing an E value of 0.01 or less when compared to the polynucleotide or polypeptide of the present invention. That is, a variant polynucleotide or polypeptide is any sequence that has at least a 99°/o probability of being the same as the polynucleotide or polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTN, FASTA, or BLASTP algorithms set at parameters described above. According to a prefeπed embodiment, a variant polynucleotide is a sequence having the same number or fewer nucleic acids than a polynucleotide of the present invention that has at least a 99% probability of being the same as the polynucleotide ofthe present invention, measured as having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at parameters described above. Similarly, according to a preferred embodiment, a variant polypeptide is a sequence having the same number or fewer amino acids than a polypeptide of the present invention that has at least a 99% probability of being the same as a polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTP algorithm set at the parameters described above. As noted above, the percentage identity is determined by aligning sequences using one of the BLASTN, FASTA, or BLASTP algorithms, set at the running parameters described above, and identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic or amino acids of the polynucleotide or polypeptide sequence of the present invention; and then multiplying by 100 to determine the percentage identity. For example, a polynucleotide of the present invention having 220 nucleic acids has a hit to a polynucleotide sequence in the EMBL database having 520 nucleic acids over a stretch of 23 nucleotides in the alignment produced by the BLASTN algorithm using the parameters described above. The 23 nucleotide hit includes 21 identical nucleotides, one gap and one different nucleotide. The percentage identity of the polynucleotide ofthe present invention to the hit in the EMBL library is thus 21/220 times 100, or 9.5%. The polynucleotide sequence in the EMBL database is thus not a variant of a polynucleotide ofthe present invention. In addition to having a specified percentage identity to an inventive polynucleotide or polypeptide sequence, variant polynucleotides and polypeptides preferably have additional structure and/or functional features in common with the inventive polynucleotide or polypeptide. Polypeptides having a specified degree of identity to a polypeptide of the present invention share a high degree of similarity in their primary structure and have substantially similar functional properties. In addition to sharing a high degree of similarity in their primary structure to polynucleotides of the present invention, polynucleotides having a specified degree of identity to, or capable of hybridizing to an inventive polynucleotide preferably have at least one of the following features: (i) they contain an open reading frame or partial open reading frame encoding a polypeptide having substantially the same functional properties as the polypeptide encoded by the inventive polynucleotide; or (ii) they contain identifiable domains in common.

Alternatively, variant polynucleotides of the present invention hybridize to the polynucleotide sequences recited in SEQ ID NO: 1-121, or complements, reverse sequences, or reverse complements of those sequences, under stringent conditions. As used herein, "stringent conditions" refers to prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65° C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.

The present invention also encompasses polynucleotides that differ from the disclosed sequences but that, as a consequence of the discrepancy of the genetic code, encode a polypeptide having similar enzymatic activity to a polypeptide encoded by a polynucleotide of the present invention. Thus, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NO: 1-121, or complements, reverse sequences, or reverse complements of those sequences as a result of conservative substitutions are encompassed within the present invention. Additionally, polynucleotides comprising sequences that differ from the inventive polynucleotide sequences or complements, reverse complements, or reverse sequences as a result of deletions and/or insertions totaling less than 10%) ofthe total sequence length are also contemplated by and encompassed within the present invention. Similarly, polypeptides comprising sequences that differ from the inventive polypeptide sequences as a result of amino acid substitutions, insertions, and/or deletions totaling less than 10% of the total sequence length are contemplated by and encompassed within the present invention, provided the variant polypeptide has similar activity to the inventive polypeptide. The polynucleotides of the present invention may be isolated from various libraries, or may be synthesized using techniques that are well known in the art. The polynucleotides may be synthesized, for example, using automated oligonucleotide synthesizers (e.g., Beckman Oligo 1000M DNA Synthesizer) to obtain polynucleotide segments of up to 50 or more nucleic acids. A plurality of such polynucleotide segments may then be ligated using standard DNA manipulation techniques that are well known in the art of molecular biology. One conventional and exemplary polynucleotide synthesis technique involves synthesis of a single stranded polynucleotide segment having, for example, 80 nucleic acids, and hybridizing that segment to a synthesized complementary 85 nucleic acid segment to produce a 5-nucleotide overhang. The next segment may then be synthesized in a similar fashion, with a 5-nucleotide overhang on the opposite strand. The "sticky" ends ensure proper ligation when the two portions are hybridized. In this way, a complete polynucleotide ofthe present invention may be synthesized entirely in vitro.

Certain ofthe polynucleotides identified as SEQ ED NO: 1-121 are generally refeπed to as "partial" sequences, in that they may not represent the full coding portion of a gene encoding a naturally occurring polypeptide. The partial polynucleotide sequences disclosed herein may be employed to obtain the coπesponding full-length genes for various species and organisms by, for example, screening DNA expression libraries using hybridization probes based on the polynucleotides of the present invention, or using PCR amplification with primers based upon the polynucleotides ofthe present invention. In this way one can, using methods well known in the art, extend a polynucleotide of the present invention upstream and downstream of the corresponding DNA, as well as identify the coπesponding mRNA and genomic DNA, including the promoter and enhancer regions, ofthe complete gene. The present invention thus comprehends isolated polynucleotides comprising a sequence identified in SEQ ID NO: 1-121, or a variant of one of the specified sequences, that encode a functional polypeptide, including full-length genes. Such extended polynucleotides may have a length of from about 50 to about 4,000 nucleic acids or base pairs, and preferably have a length of less than about 4,000 nucleic acids or base pairs, more preferably yet a length of less than about 3,000 nucleic acids or base pairs, more preferably yet a length of less than about 2,000 nucleic acids or base pairs. Under some circumstances, extended polynucleotides of the present invention may have a length of less than about 1,800 nucleic acids or base pairs, preferably less than about 1,600 nucleic acids or base pairs, more preferably less than about 1 ,400 nucleic acids or base pairs, more preferably yet less than about 1,200 nucleic acids or base pairs, and most preferably less than about 1,000 nucleic acids or base pairs.

Polynucleotides of the present invention comprehend polynucleotides comprising at least a specified number of contiguous residues ( -mers) of any of the polynucleotides identified as SEQ ID NO: 1-121 or their variants. According to preferred embodiments, the value of x is preferably at least 20, more preferably at least 40, more preferably yet at least 60, and most preferably at least 80. Thus, polynucleotides of the present invention include polynucleotides comprising a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer a 250-mer, a 300-mer, a 400-mer, a 500-mer or a 600-mer of a polynucleotide identified as SEQ ID NO: 1-121 or a variant of one of the polynucleotides identified as SEQ ID NO: 1-121.

Oligonucleotide probes and primers complementary to and/or coπesponding to SEQ ID NO: 1-121, and variants of those sequences, are also comprehended by the present invention. Such oligonucleotide probes and primers are substantially complementary to the polynucleotide of interest. An oligonucleotide probe or primer is described as "coπesponding to" a polynucleotide of the present invention, including one of the sequences set out as SEQ ID NO: 1-121 or a variant, if the oligonucleotide probe or primer, or its complement, is contained within one of the sequences set out as SEQ ID NO: 1-121 or a variant of one of the specified sequences. Two single stranded sequences are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared, with the appropriate nucleotide insertions and/or deletions, pair with at least 80%, preferably at least 90% to 95%, and more preferably at least 98% to 100%, of the nucleotides of the other strand. Alternatively, substantial complementarity exists when a first DNA strand will selectively hybridize to a second DNA strand under stringent hybridization conditions. Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, more usually less than about 500 mM and preferably less than about 200 mM. Hybridization temperatures can be as low as 5°C, but are generally greater than about 22°C, more preferably greater than about 30°C and most preferably greater than about 37°C. Longer DNA fragments may require higher hybridization temperatures for specific hybridization. Since the stringency of hybridization may be affected by other factors such as probe composition, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. DNA-DNA hybridization studies may performed using either genomic DNA or DNA derived by preparing cDNA from the RNA present in a sample to be tested.

In addition to DNA-DNA hybridization, DNA-RNA or RNA-RNA hybridization assays are also possible. In the first case, the mRNA from expressed genes would then be detected instead of genomic DNA or cDNA derived from mRNA of the sample. In the second case,

RNA probes could be used. Artificial analogs of DNA hybridizing specifically to target sequences could also be used.

In specific embodiments, the oligonucleotide probes and/or primers comprise at least about 6 contiguous residues, more preferably at least about 10 contiguous residues, and most preferably at least about 20 contiguous residues complementary to a polynucleotide sequence of the present invention. Probes and primers of the present invention may be from about 8 to 100 base pairs in length or, preferably from about 10 to 50 base pairs in length or, more preferably from about 15 to 40 base pairs in length. The primers and probes may be readily selected using procedures well known in the art, taking into account DNA-DNA hybridization stringencies, annealing and melting temperatures, potential for formation of loops and other factors, which are well known in the art. Tools and software suitable for designing probes, and especially for designing PCR primers, are available from Premier Biosoft International, 3786 Corina Way, Palo Alto, CA 94303-4504. Prefeπed techniques for designing PCR primers are also disclosed in Dieffenbach and Dyksler, PCR primer: a laboratory' manual, CSHL Press: Cold Spring Harbor, NY, 1995.

A plurality of oligonucleotide probes or primers corresponding to a polynucleotide of the present invention may be provided in a kit form. Such kits generally comprise multiple DNA or oligonucleotide probes, each probe being specific for a polynucleotide sequence. Kits of the present invention may comprise one or more probes or primers coixesponding to a polynucleotide of the present invention, including a polynucleotide sequence identified in SEQ ID NO: 1-121.

In one embodiment useful for high-throughput assays, the oligonucleotide probe kits of the present invention comprise multiple probes in an aπay format, wherein each probe is immobilized in a predefined, spatially addressable location on the surface of a solid substrate. Array foπnats which may be usefully employed in the present invention are disclosed, for example, in U.S. Patents No. 5,412,087, 5,545,531, and PCT Publication No. WO 95/00530, the disclosures of which are hereby incorporated by reference. Oligonucleotide probes for use in the present invention may be constructed synthetically prior to immobilization on an aιτay, using techniques well known in the art (See, for example, Gait, ed., Oligonucleotide synthesis a practical approach, IRL Press: Oxford, England, 1984). Automated equipment for the synthesis of oligonucleotides is available commercially from such companies as Perkin Elmer/ Applied Biosystems Division (Foster City, CA) and may be operated according to the manufacturer's instructions. Alternatively, the probes may be constructed directly on the surface of the aπay using techniques taught, for example, in PCT Publication No. WO 95/00530.

The solid substrate and the surface thereof preferably form a rigid support and are generally formed from the same material. Examples of materials from which the solid substrate may be constructed include polymers, plastics, resins, membranes, polysaccharides, silica or silica-based materials, carbon, metals and inorganic glasses. Synthetically prepared probes may be immobilized on the surface of the solid substrate using techniques well known in the art, such as those disclosed in U.S. Patent No. 5,412,087. In one such technique, compounds having protected functional groups, such as thiols protected with photochemically removable protecting groups, are attached to the surface of the substrate. Selected regions of the surface are then iπadiated with a light source, preferably a laser, to provide reactive thiol groups. This irradiation step is generally performed using a mask having apertures at predefined locations using photolithographic techniques well known in the art of semiconductors. The reactive thiol groups are then incubated with the oligonucleotide probe to be immobilized. The precise conditions for incubation, such as temperature, time and pH, depend on the specific probe and can be easily determined by one of skill in the art. The surface ofthe substrate is washed free of unbound probe and the iπadiation step is repeated using a second mask having a different pattern of apertures. The surface is subsequently incubated with a second, different, probe. Each oligonucleotide probe is typically immobilized in a discrete area of less than about 1 mm². Preferably each discrete area is less than about 10,000 mm², more preferably less than about 100 mm². In this manner, a multitude of oligonucleotide probes may be immobilized at predefined locations on the array.

The resulting aπay may be employed to screen for differences in organisms or samples or products containing genetic material as follows. Genomic or cDNA libraries are prepared using techniques well known in the art. The resulting target DNA is then labeled with a suitable marker, such as a radiolabel, chromophore, fluorophore or chemiluminescent agent, using protocols well known for those skilled in the art. A solution ofthe labeled target DNA is contacted with the surface ofthe aπay and incubated for a suitable period of time.

The surface of the aπay is then washed free of unbound target DNA and the probes to which the target DNA hybridized are determined by identifying those regions of the aπay to which the markers are attached. When the marker is a radiolabel, such as ³²P, autoradiography is employed as the detection method. In one embodiment, the marker is a fluorophore, such as fluorescein, and the location of bound target DNA is determined by means of fluorescence spectroscopy. Automated equipment for use in fluorescence scanning of oligonucleotide probe aπays is available from Affymetrix, Inc. (Santa Clara, CA) and may be operated according to the manufacturer's instructions. Such equipment may be employed to determine the intensity of fluorescence at each predefined location on the aπay, thereby providing a measure of the amount of target DNA bound at each location. Such an assay would be able to indicate not only the absence and presence of the marker probe in the target, but also the quantitative amount as well. The significance of such high-throughput screening system is apparent for applications such as microbial selection and quality control operations in which there is a need to identify large numbers of samples or products for unwanted materials, to identify microbes, or samples or products containing microbial material, for quarantine purposes, etc., or to ascertain the true origin of samples or products containing microbes. Screening for the presence or absence of polynucleotides of the present invention used as identifiers for tagging microbes and microbial products can be valuable for later detecting the genetic composition of food, fermentation and industrial microbes or microbes in human or animal digestive system after consumption of probiotics, etc.

In this manner, oligonucleotide probe kits of the present invention may be employed to examine the presence/absence (or relative amounts in case of mixtures) of polynucleotides in different samples or products containing different materials rapidly and in a cost-effective manner. Examples of microbial species which may be examined using the present invention, include lactic acid bacteria, such as Lactobacillus rhamnosus, and other microbial species.

Another aspect of the present invention involves collections of a plurality of polynucleotides of the present invention. A collection of a plurality of the 'polynucleotides of the present invention, particularly the polynucleotides identified as SEQ ID NO: 1-121, may be recorded and/or stored on a storage medium and subsequently accessed for purposes of analysis, comparison, etc. Suitable storage media include magnetic media such as magnetic diskettes, magnetic tapes, CD-ROM storage media, optical storage media, and the like. Suitable storage media and methods for recording and storing information, as well as accessing information such as polynucleotide sequences recorded on such media, are well known in the art. The polynucleotide information stored on the storage medium is preferably computer- readable and may be used for analysis and comparison ofthe polynucleotide information.

Another aspect of the present invention thus involves storage medium on which are recorded a collection ofthe polynucleotides ofthe present invention, particularly a collection of the polynucleotides identified as SEQ ID NO: 1-121. According to one embodiment, the storage medium includes a collection of at least 20, preferably at least 50, more preferably at least 100, and most preferably at least 200 of the polynucleotides of the present invention, preferably the polynucleotides identified as SEQ ID NO: 1-121, including variants of those polynucleotides.

Another aspect of the present invention involves a combination of polynucleotides, the combination containing at least 5, preferably at least 10, more preferably at least 20, and most preferably at least 50 different polynucleotides of the present invention, including polynucleotides selected from SEQ ID NO: 1-121, and variants of these polynucleotides.

In another aspect, the present invention provides genetic constructs comprising, in the 5 '-3' direction, a gene promoter sequence and an open reading frame coding for at least a functional portion of a polypeptide encoded by a polynucleotide of the present invention. In certain embodiments, the genetic constructs of the present invention also comprise a gene termination sequence. The open reading frame may be oriented in either a sense or antisense direction. Genetic constructs comprising a non-coding region of a gene coding for a polypeptide encoded by an inventive polynucleotide or a nucleotide sequence complementary to a non-coding region, together with a gene promoter sequence, are also provided. A terminator sequence may form part of this construct. Preferably, the gene promoter and termination sequences are functional in a host organism. More preferably, the gene promoter and termination sequences are common to those of the polynucleotide being introduced. The genetic construct may further include a marker for the identification of transformed cells.

Techniques for operatively linking the components of the genetic constructs are well known in the art and include the use of synthetic linkers containing one or more restriction endonuclease sites as described, for example, by Sambrook et al, in Molecular cloning: a laboratoiy manual Cold Spring Harbor Laboratories Press: Cold Spring Harbor, NY, 1989. The genetic constructs of the present invention may be linked to a vector having at least one replication system, for example, E. coli, whereby after each manipulation, the resulting construct can be cloned and sequenced and the coπectness ofthe manipulation determined.

Transgenic microbial cells comprising the genetic constructs of the present invention are also provided by the present invention, together with microbes comprising such transgenic cells, products and progeny of such microbes, and materials including such microbes. Techniques for stably incorporating genetic constructs into the genome of target microbes, such as Lactobacillus species, Lactococcus lactis or E. coli, are well known in the art of bacterial transformation and are exemplified by the transformation of E. coli for sequencing described in Example 1. Transgenic non-microbial cells comprising the genetic constructs of the present invention are also provided, together with organisms comprising such transgenic cells, and products and progeny of such organisms. Genetic constructs of the present invention may be stably incorporated into the genomes of non-microbial target organisms, such as fungi, using techniques well known in the art. In preferred embodiments, the genetic constructs of the present invention are erupted to transform microbes used in the production of food products, ingredients, processing aids, additives or supplements and for the production of microbial products for pharmaceutical uses, particularly for modulating immune system function and immunological effects, and in the production of chemoprotectants providing beneficial effects, probiotics and health supplements. The inventive genetic constructs may also be employed to transforai bacteria that are used to produce enzymes or substances such as polysaccharides, flavor compounds and bioactive substances, and to enhance resistance to industrial processes such as drying and to adverse stimuli in the human digestive system. The genes involved in antibiotic production, and phage uptake and resistance in Lactobacillus rhamnosus are considered to be especially useful. The target microbe to be used for transformation with one or more polynucleotides or genetic constructs of the present invention is preferably selected from the group consisting of bacterial genera Lactococcus, Lactobacillus, Streptococcus, Oenococcus, Lactosphaera, Trichococcus, Pediococcus and others potentially useful in various fermentation industries, and is most preferably selected from the group consisting of the following Lactobacillus species: Lactobacillus acetotolerans, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus ari∑onae, Lactobacillus aviarius, Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillus- brevis, Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus fructivorans, Lactobacillus gallinaram, Lactobacillus gasseri, Lactobacillus graminis, Lactobacillus hamsteri, Lactobacillus helveticus, Lactobacillus helveticus subsp. jugurti, Lactobacillus hetero, Lactobacillus hilgardii, Lactobacillus homohiochii, Lactobacillus japonicus, Lactobacillus johnsonii, Lactobacillus kefiri, Lactobacillus lactis, Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus oris, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus paracasei subsp. pseudoplantarum, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus ruminis, Lactobacillus sake, Lactobacillus salivarius, Lactobacillus salivarius subsp. salicinius, Lactobacillus salivarius subsp. salivarius, Lactobacillus sanfi'anciscensis, Lactobacillus sharpeae, Lactobacillus thermophilus, Lactobacillus vaginalis, Lactobacillus vermiforme, and Lactobacillus zeae.

In yet a further aspect, the present invention provides methods for modifying the concentration, composition and/or activity of a polypeptide in a host organism, such as a microbe, comprising stably incorporating a genetic construct of the present invention into the genome of the host organism by transforming the host organism with such a genetic construct. The genetic constructs ofthe present invention may be used to transform a variety of organisms including plants, such as monocotyledonous angiosperms (e.g., grasses, com, grains, oat, wheat and barley); dicotyledonous angiosperms (e.g., Arabidopsis, tobacco, legumes, alfalfa, oaks, eucalyptus, maple); gymnosperms, (e.g., Scots pine (Aronen, Finnish Forest Res. Papers, Vol. 595, 1996); white spmce (Ellis et al, Biotechnology 11:84-89, 1993); larch (Huang et al, In Vitjv Cell 27:201-207, 1991); and any kind of plant amenable to genetic engineering.

Thus, in yet another aspect, transgenic plant cells comprising the genetic constructs of the present invention are provided, together with plants comprising such transgenic cells, and fruits, seeds, products and progeny of such plants. Techniques for stably incorporating genetic constructs into the genome of target organisms, such as plants, are well known in the art and include Agrobacterium tumefaciens mediated introduction, elecfroporation, protoplast fusion, injection into reproductive organs, injection into immature embryos, high velocity projectile introduction and the like. The choice of technique will depend upon the target plant to be transformed. For example, dicotyledonous plants, and certain monocots and gymnosperms, may be transformed by Agrobacterium Ti plasmid technology, as described, for example by Bevan, Nucleic Acids Res. 12:8711-8721, 1984. Targets for the introduction of the genetic constructs include tissues, such as leaf tissue, disseminated cells, protoplasts, seeds, embiyos, meristematic regions, cotyledons, hypocotyls, and the like.

Once the cells are transformed, cells having the genetic construct incorporated in their genome are selected. Transgenic cells may then be cultured in an appropriate medium, using techniques well known in the art. In the case of protoplasts, the cell wall is allowed to refonn under appropriate osmotic conditions. In the case of seeds or embryos, an appropriate germination or callus initiation medium is employed. For explants, an appropriate regeneration medium is used. Regeneration of plants is well established for many species. For a review of regeneration of forest trees, see Dunstan et al, "Somatic embryogenesis in woody plants," in Thorpe, T.A., ed., In vitro embryogenesis of plants, (Current Plant Science and Biotechnology in Agriculture), 20(12):471-540, 1995. Specific protocols for the regeneration of spruce are discussed by Roberts et al. ("Somatic embryogenesis of Spruce," in Redenbaugh K, ed., Synseed: applications of synthetic seed to crop improvement, CRC Press: Ch.23:427-449, 1993). The resulting transformed plants may be reproduced sexually or asexually, using methods well known in the art, to give successive generations of transgenic plants and practically unlimited amounts of tagged plant-derived products.

The polynucleotides of the present invention may be further employed as non- disruptive tags for marking organisms, particularly microbes. Other organisms may, however, be tagged with the polynucleotides of the present invention, including commercially valuable plants, animals, fish, fungi and yeasts. Genetic constructs comprising polynucleotides of the present invention may be stably introduced into an organism as heterologous, non-functional, non-disruptive tags. It is then possible to identify the origin or source ofthe organism at a later date by determining the presence or absence of the tag(s) in a sample of material. Detection of the tag(s) may be accomplished using a variety of conventional techniques, and will generally involve the use of nucleic acid probes. Sensitivity in assaying the presence of probe can be usefully increased by using branched oligonucleotides, as described by Horn et al, Nucleic- Acids Res. 25(23):4842-4849, 1997, enabling detection of as few as 50 DNA molecules in the sample. Polynucleotides ofthe present invention may also be used to specifically suppress gene expression by methods that operate post-transcriptionally to block the synthesis of products of targeted genes, such as RNA interference (RNAi), and quelling. Briefly, traditional methods of gene suppression, employing anti-sense RNA or DNA, operate by binding to the reverse sequence of a gene of interest such that binding interferes with subsequent cellular processes and therefore blocks synthesis of the coπesponding protein. RNAi also operates on a post- translational level and is sequence specific, but suppresses gene expression far more efficiently. Exemplary methods for controlling or modifying gene expression using RNAi are provided in WO 99/49029 and WO 99/53050. In these methods, post-transcriptional gene silencing is brought about by a sequence-specific RNA degradation process which results in the rapid degradation of transcripts of sequence-related genes. Studies have shown that double-stranded RNA may act as a mediator of sequence-specific gene silencing (see, for example, Montgomery and Fire, Trends in Genetics, 14:255-258, 1998). Gene constructs that produce transcripts with self-complementary regions are particularly efficient at gene silencing. A unique feature of this post-transcriptional gene silencing pathway is that silencing is not limited to the cells where it is initiated. The gene-silencing effects may be disseminated to other parts of an organism and even transmitted through the germ line to several generations.

The polynucleotides of the present invention may thus be employed to generate gene silencing constructs and/or gene-specific self-complementary RNA sequences that can be delivered by conventional art-known methods to cells, such as microbial cells. Within genetic constructs, sense and antisense sequences can be placed in regions flanking an intron sequence in proper splicing orientation with donor and acceptor splicing sites, such that intron sequences are removed during processing of the transcript and sense and antisense sequences, as well as splice junction sequences, bind together to form double-stranded RNA. Alternatively, spacer sequences of various lengths may be employed to separate self-complementary regions of sequence in the construct. During processing of the gene construct transcript, intron sequences are spliced-out, allowing sense and anti-sense sequences, as well as splice junction sequences, to bind forming double-stranded RNA. Select ribonucleases then bind to and cleave the double-stranded RNA, thereby initiating the cascade of events leading to degradation of specific mRNA gene sequences, and silencing specific genes. Alternatively, rather than using a gene construct to express the self-complementary RNA sequences, the gene-specific double- stranded RNA segments are delivered to one or more targeted areas to be internalized into the cell cytoplasm to exert a gene silencing effect. The double-stranded RNA must have sufficient homology to the targeted gene to mediate RNAi and is preferably at least 25 nucleotides in length. Preferably, the double-stranded RNA corresponds specifically to a polynucleotide of the present invention. Gene silencing RNA sequences comprising the polynucleotides of the present invention are useful for creating genetically modified organisms with desired phenotypes as well as for characterizing genes (for example, in high-throughput screening of sequences), and studying their functions in intact organisms.

In another aspect, the present invention provides methods for using one or more of the inventive polypeptides or polynucleotides to treat disorders in a mammal, such as a human.

In this aspect, the polypeptide or polynucleotide is generally present within a composition, such as a pharmaceutical or immunogenic composition. Pharmaceutical compositions may comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable earner. Immunogenic compositions may comprise one or more of the above polypeptides and an immunostimulant, such as an adjuvant or a liposome, into which the polypeptide is incorporated.

Alternatively, a composition of the present invention may contain DNA encoding one or more polypeptides described herein, such that the polypeptide is generated in situ. In such compositions, the DNA may be present within any of a variety of delivery systems lαiown to those of ordinary skill in the art, including nucleic acid expression systems, and bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminator signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus Calmette-Guerin) that expresses an immunogenic portion ofthe polypeptide on its cell surface. In a prefeπed embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of a nonpathogenic, or defective, replication competent virus. Techniques for incorporating DNA into such expression systems are well known in the art. The DNA may also be "naked," as described, for example, in Ulmer et al, Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

While any suitable caπier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of caπier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the caπier preferably comprises water, saline, alcohol, a lipid, a wax or a buffer. For oral administration, any of the above earners or a solid caπier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as earners for the phaπnaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.

Any of a variety of adjuvants may be employed in the immunogenic compositions of the present invention to non-specifically enhance an immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bordetella pertussis or M. tuberculosis. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, MI), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and Quil A.

Routes and frequency of administration, as well as dosage, vary from individual to individual. In general, the inventive compositions may be administered by injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. In general, the amount of polypeptide present in a dose (or produced in situ by the DNA in a dose) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg per kg of host, and preferably from about 100 pg to about 1 μg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 ml to about 2 ml.

The following examples are offered by way of illustration and not by way of limitation.

Example 1

ISOLATION AND CHARACTERIZATION OF DNA SEQUENCES FROM

LACTOBACILLUS RHAMNOSUS STRAIN HNOOl

Lactobacillus rhamnosus strain HNOOl DNA libraries were constructed and screened as follows. DNA was prepared in large scale by cultivating the bacteria in 2 x 100 ml cultures with 100 ml MRS broth (Difco Laboratories, Detroit MI) and 1 ml Lactobacillus glycerol stock as inoculum, placed into 500 ml culture flasks and incubated at 37 °C for approx. 16 hours with shaking (220 rpm). The cultures were centrifuged at 3500 rpm for 10 min to pellet the cells. The supernatant was removed and the cell pellet resuspended in 40 ml fresh MRS broth and transfeπed to clean 500 ml culture flasks. Fresh MRS broth (60 ml) was added to bring the volume back to 100 ml and flasks were incubated for a further 2 hrs at 37°C with shaking (220 rpm). The cells were pelleted by centrifugation (3500 rpm for 10 min) and supernatant removed. Cell pellets were washed twice in 20 ml buffer A (50 mM NaCl, 30 mM Tris pH 8.0, 0.5 mM EDTA).

Cells were resuspended in 2.5 ml buffer B (25% sucrose (w/v), 50 mM Tris pH 8.0, 1 mM EDTA, 20 mg/ml lysozyme, 20 μg/ml mutanolysin) and incubated at 37 °C for 45 min. Equal volumes of EDTA (0.25 M) was added to each tube and allowed to incubate at room temperature for 5 min. 20% SDS (1 ml) solution was added, mixed and incubated at 65 °C for 90 min. 50 μl Proteinase K (Gibco BRL, Gaithersburg, MD) from a stock solution of 20 mg/ml was added and tubes incubated at 65 °C for 15 min.

DNA was extracted with equal volumes of phenol:chloroform:isoamylalcohol (25:24:1). Tubes were centrifuged at 3500 rpm for 40 min. The aqueous phase was removed to clean sterile Oak Ridge centrifuge tubes (30 ml). Crude DNA was precipitated with an equal volume of cold isopropanol and incubated at -20°C overnight.

After resuspension in 500 μl TE buffer, DNase-free RNase was added to a final concentraion of 100 μg/ml and incubated at 37°C for 30 min. The incubation was extended for a further 30 min after adding 100 μl Proteinase K from a stock solution of 20 mg/ml. DNA was precipitated with ethanol after a phenol:chloroform:isoamylalcohol (25:24:1) and a chloroform:isoamylalcohol (24:1) extraction and dissolved in 250 μl TE buffer.

DNA was digested with Sau3Al at a concentration of 0.004 U/μg in a total volume of

1480 μl, with 996 μl DNA, 138.75 μl 10X REACT 4 buffer and 252.75 μl H₂O. Following incubation for 1 hour at 37 °C, DNA was divided into two tubes. 31 μl 0.5 M EDTA was added to stop the digestion and 17 μl samples were taken for agarose gel analysis. Samples were put into 15 ml Falcon tubes and diluted to 3 ml for loading onto sucrose gradient tubes.

Sucrose gradient size fractionation was conducted as follows. 100 ml of 50% sucrose (w/v) was made in TEN buffer (1M NaCl, 20 mM Tris pH 8.0, 5 mM EDTA) and sterile filtered. Dilutions of 5, 10, 15, 20, 25, 30, 35 and 40% sucrose were prepared and overlaid carefully in Beckman Polyallomer tubes, and kept overnight at 4°C. TEN buffer (4 ml) was loaded onto the gradient, with 3 ml of DNA solution on top. The gradients were centrifuged at 26K for 18 hours at 4°C in a Centricon T-2060 centrifuge using a Kontron TST 28-38 rotor. After deceleration without braking (approx. 1 hour), the gradients were removed and fractions collected using an auto Densi-Flow (Haake-Buchler Instruments). Agarose gel was used to analyze the fractions. The best two pairs of fractions were pooled and diluted to contain less than 10% sucrose. TEN buffer (4 ml) was added and DNA precipitated with 2 volumes of 100% ice cold ethanol and an overnight incubation at -20°C. DNA pellets were resuspended in 300 μl TE buffer and re-precipitated for approx. 6 hours at -20°C after adding 1/10 volume 3 M NaOAC pH 5.2 and 2 volumes of ethanol. DNA was pelleted at top speed in a microcentrifuge for 15 min, washed -with 70% ethanol and pelleted again, dried and resuspended in 10 μl TE buffer.

DNA was ligated into dephosphoiylated -S wHI-digested pBluescript SK II⁺ and dephosphoiylated -5αn.HI-digested lambda ZAP Express using standard protocols. Packaging of the DNA was done using Gigapack III Gold packaging extract (Stratagene, La Jolla, CA) following the manufacturer's protocols. Packaged libraries were stored at 4 °C.

Mass excision from the primary packaged phage library was done using XL 1 -Blue MRF' cells and ExAssist Helper Phage (Stratagene). The excised phagemids were diluted with NZY broth (Gibco BRL, Gaithersburg, MD) and plated out onto LB-kanamycin agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) and isopropylthio-beta- galactoside (IPTG). After incubation, single colonies were picked for PCR size determination before the most suitable libraries were selected for sequencing.

Of the colonies picked for DNA minipreps and subsequent sequencing, the large majority contained an insert suitable for sequencing. Positive colonies were cultured in LB broth with kanamycin or ampicillin depending on the vector used, and DNA was purified by means of rapid alkaline lysis minipreps (solutions: Qiagen, Venlo, The Netherlands; clearing plates, Millipore, Bedford, MA). Agarose gels at 1% were used to screen sequencing templates for chromosomal contamination and concentration. Dye terminator sequencing reactions were prepared using a Biomek 2000 robot (Beckman Coulter, Inc., Fullerton, CA) and Hydra 96 (Robbins Scientific, Sunnyvale, CA) for liquid handling. DNA amplification was done in a 9700 PCR machine (Perkin Elmer/Applied Biosystems, Foster City, CA) according to the manufacturer's protocol.

The sequence of the genomic DNA fragments were determined using a Perkin Elmer/Applied Biosystems Division Prism 377 sequencer. The DNA clones were sequenced from the 5' and/or 3' end, and are identified as SEQ ID NO: 1-121 disclosed herein.

This example not only shows how the sequences were obtained, but also that a bacterium (E. coli) can be stably transformed with any desired DNA fragment of the present invention for permanent marking for stable inheritance.

The determined DNA sequences were compared to and aligned with known sequences in the public databases. Specifically, the polynucleotides identified in SEQ ID NO: 1-121 were compared to polynucleotides in the EMBL database as of August 12, 2002, using BLASTN algorithm Version 2.0.11 [Jan-20-2000], set to the following running parameters: Unix running command: blastall -p blastn -d embldb -e 10 -G 0 -E 0 -r 1 -v 30 -b 30 -i queryseq -o results. Multiple alignments of redundant sequences were used to build up reliable consensus sequences. The polypeptides identified in SEQ ID NO: 122-253 were compared to polypeptides in the SwissPROT-TrEMBL database as of August 12, 2002, using BLASTP algorithm Version 2.0.11 [Jan-20-2000], set to the following running parameters: Unix running command: blastall -p blastp -d swissprottrembledb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results.

BLASTN Polynucleotide Analysis

The sequences of SEQ ID NO: 1-18, 20-50, 52-62, 64-69, 71-83, 85-93 and 95-122^' were determined to have less than 50% identity, deteπnined as described above, to sequences in the EMBL database using the computer algorithm BLASTN, as described above. The sequence of SEQ ID NO: 94 was determined to have less than 75% identity, detemiined as described above, to sequences in the EMBL database using the computer algorithm BLASTN, as described above. Finally, the sequence of SEQ ID NO: 19 was determined to have less than 98% identity, deteπnined as described above, to sequences in the EMBL database using the computer algorithm BLASTN, as described above.

BLASTP Amino Acid Analysis

The amino acid sequences of SEQ ID NO: 124, 133, 134, 137-139, 141, 148, 150-156, 159, 162, 164-168, 170-172, 174, 175, 178, 184, 187, 188, 190, 194, 195, 198-200, 202, 203, 205-208, 212-214, 216, 221-224, 227, 229, 234, 235, 237, 240, 242-245, 249 and 252 were determined to have less than 50% identity, determined as described above, to sequences in the SWISSPROT-TrEMBL database using the BLASTP computer algoritlim as described above. The amino acid sequences of SEQ ID NO: 123, 125-129, 131, 144, 149, 158, 160, 161, 163, 169, 173, 176, 179-181, 183, 185, 186, 191-193, 197, 201, 209, 211, 215, 217, 218, 225, 226, 228, 230-233, 238, 239, 247, 248, 250, 251, 253, 254 and 256 were deteπnined to have less than 75%) identity, deteπnined as described above, to sequences in the SWISSPROT-TrEMBL database using the computer algorithm BLASTP, as described above. The amino acid sequences of SEQ ED NO: 132, 135, 142, 145-147, 157, 182, 204, 219, 241, 246 and 255 were determined to have less than 90% identity, deteπnined as described above, to sequences in the SWISSPROT-TrEMBL database using the computer algorithm BLASTP, as described above. The amino acid sequences of SEQ ID NOS: 140 and 236 were determined to have less than 98%) identity, determined as described above, to sequences in the SWISSPROT-TrEMBL database using the computer algoritlim BLASTP, as described above.

BLASTX Polynucleotide Analysis

The cDNA sequences of SEQ ID NO: 1-10, 12-18, 20-30, 32-42, 44-50, 52, 53, 55, 58, 59, 61, 62, 64, 66-69, 71-77, 79-83, 85-88, 90, 92, 95-105, 107-109, 111-114, 116-119, 121 and 122 were determined to have less than 50% identity, determined as described above, to sequences in the SWISSPROT-TrEMBL database using the computer algoritlim BLASTX, as described above. The cDNA sequences of SEQ ID NO: 11, 19, 43, 54, 57, 60, 65, 70, 78, 89, 91, 93, 106, 110, 115 and 120 were determined to have less than 75% identity, deteπnined as described above, to sequences in the SWISSPROT-TrEMBL database using BLASTX, as described above. The cDNA sequences of SEQ ID NO: 31, 51, 56 and 63 were determined to have less than 90% identity, determined as described above, to sequences in the SWISSPROT- TrEMBL database using BLASTN, as described above. The cDNA sequence of SEQ ED NO: 94 was determined to have less than 98% identity, detemiined as described above, to sequences in the SWISSPROT-TrEMBL database using BLASTX, as described above.

Based on similarity to known sequences, the isolated polynucleotides of the present invention identified as SEQ ID NO: 1-121 were putatively identified as encoding polypeptides having similarity to the polypeptides shown above in Table 1. The amino acid sequences encoded by the DNA sequences of SEQ ID NO: 1-121 are provided in SEQ ID NO: 122-253, respectively.

Several of the sequences provided in SEQ ID NO: 1-121 were found to be full-length and to contain open reading frames (ORFs). These full-length sequences, the location of ORFs (by nucleotide position) contained within these sequences, and the coπesponding amino acid sequences are provided in Table 2 below.

TABLE 2

<img file="WO2004031389A1_D0052.tif" /><img file="WO2004031389A1_D0053.tif" /><img file="WO2004031389A1_D0054.tif" />

SEQ ID NO: 106, 107, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 and 119 are full-length sequences of SEQ ID NO: 5, 12, 16, 44, 65, 71, 72, 78, 79, 81, 83, 103 and 21, respectively, with SEQ ID NO: 108 being a full-length sequence of SEQ ID NO: 15 and 42. SEQ ID NO: 253 is the full-length sequence of SEQ ED NO: 99.

SEQ ID NO: 1-253 are set out in the attached Sequence Listing. The codes for nucleotide sequences used in the attached Sequence Listing, including the symbol "n," conform to WIPO Standard ST.25 (1998), Appendix 2, Table 1. All references cited herein, including patent references and non-patent publications, are hereby incorporated by reference in their entireties.

While in the foregoing specification this invention has been described in relation to certain prefeπed embodiments, and many details have been set forth for puiposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles ofthe invention.