AU2002216499A1

AU2002216499A1 - Lactobacillus rhamnosus polynucleotides, polypeptides and methods for using them

Info

Publication number: AU2002216499A1
Application number: AU2002216499A
Authority: AU
Inventors: James Dekker; Matthew Glenn; Ilkka J Havukkala; Mark William Lubbers
Original assignee: Genesis Research and Development Corp Ltd; ViaLactia Biosciences NZ Ltd
Current assignee: Genesis Research and Development Corp Ltd; ViaLactia Biosciences NZ Ltd
Priority date: 2000-11-28
Filing date: 2001-11-28
Publication date: 2002-06-11
Also published as: US6476209B1; NZ534397A; WO2002044383A1; NZ526078A

Description

LACTOBACILLUS RHAMNOSUS POLYNUCLEOTIDES, POLYPEPTIDES AND METHODS FOR USING THEM

Technical Field of the Invention

This invention relates to polynucleotides isolated from lactic acid bacteria, namely Lactobacillus rhamnosus, including partial and extended sequences, as well as to probes and primers specific to the polynucleotides; DNA 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 of the Invention

The present invention relates to polynucleotides isolated from a specific strain of lactic acid bacteria, namely Lactobacillus rhamnosus HNOOl (L. rhamnosus HNOOl). 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 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±1°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 {See PCT International Publication No. WO 99/10476). In addition, L. rhamnosus HNOOl, 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. 629-367; 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 Bockelmann, 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 and 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 utilisation and conversion - due to production of beneficial products by bacterial action on proteins and carbohydrates.

Other beneficial health effects associated with L. rhamnosus and its components 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 tripeptides). Different types of exopeptidases include:

• Aminopeptidases - release a free amino acid from the N-terminus of a peptide chain;

• dipeptidyl-peptidase (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.

Endopeptidases hydrolyze peptide bonds internally within a peptide and are classified on the basis of their mode of catalysis: serine-endopeptidases - depend on serine (or threonine) as the nucleophile in the catalytic reaction; cysteine-endopeptidases - depend on the sulphydryl group of cysteine as the nucleophile in the catalytic reaction; aspartic-endopeptidases - contain aspartate residues that act as ligands for an activated water molecule which acts as the nucleophile in the catalytic reaction; and metallo-endopeptidases - contain one or more divalent metal ions that activate the water molecule that acts as the nucleophile in the catalytic reaction.

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 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. 74:2317-2362, 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. 20:6624- 6360, 1999; Stingle et al, Mol. Microbiol. 32:1287-1295, 1999; Lemoine et al, Appl.

Environ. Microbiol. 63:1512-6218, 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- acetylglucosaminidase; 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 decaboxylase, 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 are important 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-acetylglucosamine transferase.

^■ Amino acid degradation. Enzymes include glutamate dehydrogenase, aminotransferases, 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 putative 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 of the 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 NOS: 1-59; (b) variants of those sequences; (c) extended sequences comprising the sequences set out in SEQ ID NOS: 1-59, and their variants; and (d) sequences comprising at least a specified number of contiguous residues of a sequence of SEQ ID NOS: 1-59 (r-mers). Oligonucleotide probes and primers corresponding to the sequences set out in SEQ ID NOS: 1-59 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 NOS: 1-59 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±1°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 AustraUan 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. The polynucleotides identified as SEQ ID NOS: 1-59 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 then can 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 NOS: 1-59. Many of the polynucleotide sequences disclosed herein are "full-length" sequences, in that they represent a full-length gene encoding a full-length polypeptide and confirm an open reading frame. Some of the polynucleotides are partial length, in that they do not represent a full-length gene. Such partial length sequences may be extended as described herein to obtain the full-length sequences. 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 NOS: 1-59.

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: 62-120, and variants thereof. Polypeptides encoded by the polynucleotides of the 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. The polypeptides of the present invention may also be used as nutritional additives and as additives in dairy processing and fermentation processing. Several polypeptides of the present invention also have human and animal health related benefits.

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. Desired attributes may be produced in food, such as dairy (including milk protein hydroly sates and cheese), in living organisms and in non-food systems, by directed activity or directed suppression of activity of the appropriate enzyme. Directed activity of an enzyme may be achieved by introducing an appropriate polynucleotide or polypeptide to a bacterial strain (including strain HNOOl, or starter cultures), or by administering an enzyme preparation.

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 of the organism a genetic construct comprising a polynucleotide of the present invention. . Such modulation may involve up regulating or down regulating expression from one or more polynucleotides of the present invention. Up regulation may be accomplished, for example, by providing multiple gene copies, modulating expression by modifying regulatory elements or the like. Similarly, down regulation may be accomplished using known antisense and gene silencing techniques. In one embodiment, the target organism is a microbe, preferably a microbe used in fermentation, more preferably a microbe of the 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 of the 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 NOS: 1-59, and their variants, may be used to design oligonucleotide probes and primers. Oligonucleotide probes and primers have sequences that are substantially complementary to the polynucleotide of interest over a certain portion of the 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 PCR amplifications. Oligonucleotide probes and primers designed using the polynucleotides of the 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 incorporate regulatory elements such as promoters, gene regulators, origins of DNA replication, secretion signals, cell wall or membrane anchors for genetic tools (such as expression or integration vectors).

The polynucleotide sequences, encoded polypeptides and genetic constructs of this invention are useful for improving the properties of microbes that are used in the manufacture of milk-derived products, such as cheeses, yogurt, fermented milk products, sour milks, and buttermilk. Microbial metabolism during fermentation, which results in the breakdown of proteins, lipids and lactose in milk, influences the speed of ripening, the texture and consistency of fermented milk products, and the development of flavors and aromas during ripening. Undesirable flavors in milk products are produced, for example, by the food of milk-producing animals, microbial action, and enzymatic activity during fermentation, and require removal. The present invention provides polynucleotides and polypeptides and methods for their use in modifying the flavor, aroma, texture and health-related benefits of milk-derived products. Methods are described for modulating the polynucleotide content or composition of microbes used in the dairy industry by transforming the microbes with one or more polynucleotides sequences of Lactobacillus rhamnosus strain HNOOl. The inventive polynucleotides also include sequences encoding polypeptides that increase the survivability of microbes during industrial fermentation processes, wherein exposure to osmotic, temperature and other stresses can lead to reduced microbial viability, impaired metabolic activity and suboptimal fermentation conditions. While the present invention is described with particular reference to milk-derived products, it will be recognized that microbes such as Lactobacillus, which are used in the dairy industry, are also used in the production of other foods and beverages (e.g., fermented vegetables, beer, wines, juices, sourdough breads). It is expected that the polynucleotides described herein and their methods of use can be used for the processing of these foods and beverages as well.

This invention also provides transgenic microbial populations comprising expressible polynucleotide sequences of Lactobacillus rhamnosus strain HNOOl which health-related benefits. For example, the polypeptides encoded by the inventive sequences include enzymes that detoxify carcinogens, degrade allergenic proteins and lactose, and produce bioactive peptides and biogenic amines. Microbes transformed with these polynucleotide sequences can be taken internally as a probiotic composition or alternatively, the microbes or their encoded polypeptides can be added to products to provide health-related benefits. Nonpathogenic bacteria, preferably lactic-acid producing species of Bacillus, Lactobacillus, Sporolactobacillus or Bifidobacterium, that are able to colonize the gastrointestinal tract, preferably the gastrointestinal tract of a mammal, are useful for preventing or reducing pathogen colonization of the gastrointestinal mucosa, and for replacing normal flora that are depleted, for example, by drug therapy. The polynucleotide sequences of this invention can be used to transform microbes for use in a therapeutic composition that is effective for treating or preventing a gastrointestinal condition or disorder caused by the presence of pathogenic microbes in the gastrointestinal tract or by the absence of normal intestinal microbes in the intestinal tract. Such probiotic compositions can be administered alone or in combination with another pharmaceutical agent, depending on the condition that is to be treated. All references cited herein, including patent references and non-patent publications, are hereby incorporated by reference in their entireties.

Brief Description of the Drawings Fig. 1 shows the nucleotide sequence containing L. rhamnosus strain HNOOl fibronectin binding protein AB2 showing ATG initiation and translation stop codons (boxed).

Fig. 2 shows the amino acid sequence of HNOOl fibronectin binding protein AB2, with the subcloned fibronectin binding domain boxed. Fig. 3 demonstrates specific blocking of fibronectin binding to HNOOl cells by the fibronectin binding protein domain of AB2.

Detailed Description

The polynucleotides disclosed herein were isolated by high throughput sequencing of DNA libraries from the lactic acid bacteria Lactobacillus rhamnosus as described 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 of the 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 have 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 fermentation 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 putative identity and functions of the inventive polynucleotides based on such similarities are shown below in Table 1.

TABLE 1

Isolated polynucleotides of the present invention include the polynucleotides identified herein as SEQ ID NOS: 1-59; isolated polynucleotides comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 1-59; isolated polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NOS: 1-59; 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 corresponding 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 corresponding 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 of the 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 corresponding 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' 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 NOS: 1-59 may contain open reading frames ("ORFs"), or partial open reading frames, encoding polypeptides. Additionally, polynucleotides identified as SEQ ID NOS: 1-59 may contain non-coding sequences such as promoters and terminators that may be useful as control elements. Additionally, open reading frames encoding polypeptides may be identified in extended or full-length sequences corresponding to the sequences set out as SEQ ID NOS: 62-120. 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. Suitable tools and software for ORF analysis are available, for example, on the

Internet at the NCBI's site. Additional tools and software suitable for ORF analysis, include

GeneWise (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 of the present invention.

Once open reading frames are identified in the polynucleotides of the 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 ("JC") of contiguous residues of any of the polynucleotides identified as SEQ ID NOS: 1-59. 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, including the polypeptides of SEQ ID NOS: 62-120. 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 occurring 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 of the 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 Merrifϊeld solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain (See Merrifield, /. 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 mutagensis (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 disclosed herein are prepared in an isolated, substantially pure form. Preferably, the polypeptides 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 sequences preferably exhibit at least 40%, more preferably at least 60%, more preferably yet at least 75%, and most preferably at least 90% identity to a sequence of the present invention. Variant polypeptide sequences preferably exhibit at least 50%, 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 are available 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 preferred for use in the determination of polynucleotide variants according to the present invention. The BLASTP algorithm, set to the parameters described below, is preferred 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 at NCBI's website at URL http://www.ncbi.nlm.nih.gov/BLAST/newblast.html and 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 of the 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 arranged in order of the 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 of the 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 preferred 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 of the 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 of the 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% 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 preferred 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 of the 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 of the 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 of the 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 NOS: 1-59, 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 as a polypeptide encoded by a polynucleotide of the present invention. Thus, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NOS: 1-59, 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% of the 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 of the present invention may be synthesized entirely in vitro.

Certain of the polynucleotides identified as SEQ ID NOS: 1-59 are generally referred to as "partial" sequences, in that they do 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 corresponding 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 of the 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 corresponding mRNA and genomic DNA, including the promoter and enhancer regions, of the complete gene. The present invention thus comprehends isolated polynucleotides comprising a sequence identified in SEQ ID NOS: 1-59, 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 pahs. 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 and polypeptides of the present invention comprehend polynucleotides and polypeptides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides and polypeptides identified as SEQ ID NOS: 1-59 and 62-120, 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, or a 300-mer, 400-mer, 500-mer or 600-mer of a polynucleotide or polypeptide identified as SEQ ID NOS: 1-59 and 62-120 or a variant thereof.

Oligonucleotide probes and primers complementary to and/or corresponding to SEQ ID NOS: 1-59, 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 "corresponding to" a polynucleotide of the present invention, including one of the sequences set out as SEQ ID NOS: 1-59 or a variant, if the oligonucleotide probe or primer, or its complement, is contained within one of the sequences set out as SEQ ID NOS: 1-59 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. In addition, 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 suitable for designing PCR primers, are available on the

Internet, for example, at the Horizon Press site. In addition, a software program suitable for designing probes, and especially for designing PCR primers, is available from Premier

Biosoft International, 3786 Corina Way, Palo Alto, CA 94303-4504. Preferred 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 corresponding to a polynucleotide of the present invention, including a polynucleotide sequence identified in SEQ ID NOS: 1-59. In one embodiment useful for high-throughput assays, the oligonucleotide probe kits of the present invention comprise multiple probes in an array format, wherein each probe is immobilized in a predefined, spatially addressable location on the surface of a solid substrate. Array formats 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 incoφorated by reference.

Oligonucleotide probes for use in the present invention may be constructed synthetically prior to immobilization on an array, 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 array 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 irradiated 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 of the substrate is washed free of unbound probe and the irradiation 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 array 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 of the labeled target DNA is contacted with the surface of the array and incubated for a suitable period of time.

The surface of the array 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 array 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 arrays 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 array, 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 and/or polypeptides of the present invention. A collection of a plurality of the polynucleotides and/or polypeptides of the present invention, particularly the polynucleotides and polypeptides identified as SEQ ID NOS: 1-59 and 62-120, 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 of the polynucleotide information.

Another aspect of the present invention thus involves storage medium on which are recorded a collection of the polynucleotides and/or polypeptides of the present invention, particularly a collection of the polynucleotides and/or polypeptides identified as SEQ ID NOS: 1-59 and 62-120. 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 NOS: 1-59, 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 and/or polypeptides of the present invention, including polynucleotides and polypeptides selected from SEQ ID NOS: 1-59 and

62-120, and variants of these polynucleotides and polypeptides.

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 the above polynucleotides 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 laboratory 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 correctness of the 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 in Example 1. Transgenic, non-inicrobial, 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 employed 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 transform 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 selected, most preferably, from the group consisting of Lactobacillus species in the following list: Lactobacillus acetotolerans, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus arizonae, 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 gallinarum, 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 sanfranciscensis, Lactobacillus sharpeae, Lactobacillus thermophilus,

Lactobacillus vaginalis, Lactobacillus vermiforme, 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 of the present invention may be used to transform a variety of organisms. Organisms which may be transformed with the inventive constructs include plants, such as monocotyledonous angiosperms (e.g., grasses, corn, 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 spruce (Ellis et al, Biotechnology 11:84-89, 1993); and larch (Huang, et al, In Vitro 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, electroporation, 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, embryos, 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 reform 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. Polynucleotides of the present invention may also be used to specifically suppress gene expression by methods such as RNA interference (RNAi), which may also include cosuppression and quelling. This and other techniques of gene suppression are well known in the art. A review of this technique is found in Science 288:1370-1372, 2000. Traditional methods of gene suppression, employing antisense RNA or DNA, operate by binding to the reverse sequence of a gene of interest such that binding interferes with subsequent cellular processes and thereby blocks synthesis of the corresponding protein. RNAi also operates on a post-transcriptional level and is sequence specific, but suppresses gene expression far more efficiently

Studies have demonstrated that one or more ribonucleases specifically bind to and cleave double-stranded RNA into short fragments. The ribonuclease(s) remains associated with these fragments, which in turn specifically bind to complementary mRNA, i.e. specifically bind to the transcribed mRNA strand for the gene of interest. The mRNA for the gene is also degraded by the ribonuclease(s) into short fragments, thereby obviating translation and expression of the gene. Additionally, an RNA polymerase may act to facilitate the synthesis of numerous copies of the short fragments, which exponentially increases the efficiency of the system. A unique feature of this gene suppression 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.

Specifically, polynucleotides of the present invention are useful for generating gene constructs for silencing specific genes. Polynucleotides of the present invention may be used to generate genetic constructs that encode a single self-complementary RNA sequence specific for one or more genes of interest. Genetic constructs and/or gene-specific self- complementary RNA sequences may be delivered by any conventional method known in the art. Within genetic constructs, sense and antisense sequences flank an intron sequence arranged in proper splicing orientation making use of donor and acceptor splicing sites. Alternative methods may employ spacer sequences of various lengths rather than discrete intron sequences to create an operable and efficient construct. During post-transcriptional processing of the gene construct product, intron sequences are spliced-out, allowing sense and antisense sequences, as well as splice junction sequences, to bind forming double- stranded RNA. Select ribonucleases 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. Using this cellular pathway of gene suppression, gene function may be studied and high-throughput screening of sequences may be employed to discover sequences affecting gene expression. Additionally, genetically modified microbes and higher order organisms may be generated.

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 6200 rpm for 10 min to pellet the cells. The supernatant was removed and the cell pellet resuspended in 40 ml fresh MRS broth and transferred 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 (6200 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 6200 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 Sα«3AI 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 H₂0. 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, 62 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 analyse 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 dephosphorylated SαmHI-digested pBluescript SK II⁺ and dephosphorylated ifømHI-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 XLl-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.

To extend the sequences of the inserts from these clones, primers were designed from the determined nucleotide sequences so that the primer sequences are located approximately 100 bp downstream of the 5' end and 100 bp upstream of the 3' end of the determined nucleotide sequence. Selection of primers were done with the Gap4 Genome Assembly

Program (Bonfield et al, Nucleic Acids Res. 24:4992-4999, 1995 using the following parameters: No. of bases ahead: 40; No. of bases back: 40; Minimum melting temperature:

55°C; maximum melting temperature: 60°C; minimum length: 17 bp; maximum length: 20 bp; minimum GC-content: 40%; maximum GC-content: 60%. Sequencing of clones was done as described above. The determined nucleotide sequences are identified as SEQ ID NOS: 1-59 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: 1-59 were compared to polynucleotides in the EMBL database as of the end of July 2000, 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. Based on similarity to known sequences, the isolated polynucleotides of the present invention identified as SEQ ID NOS: 1-59 were 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: 1-59 are provided in SEQ ID NO: 62-120, respectively.

Several of the sequences provided in SEQ ID NO: 1-59 were found to be full-length and to contain open reading frames (ORFs). Specifically, SEQ ID NOS: 1; 2; 4-9; 11; 17; 18; 22; 30; 32; 38; 40; 41; 50; 51; 55; 57 and 59 were found to be full-length. The location of ORFs (by nucleotide position) contained within SEQ ID NOS: 1-62, and the corresponding amino acid sequences are provided in Table 2 below.

TABLE 2

Example 2 Isolation and Characterisation of Fibronectin Binding Protein from L. rhamnosus HNOOl The full-length polynucleotide sequence of fibronectin binding protein from L. rhamnosus strain HNOOl, given in SEQ ID NO: 59 is shown in Fig. 1 with ATG initiation and translation stop codons (boxed). Based on amino acid sequence similarity between the Streptococcus pyogenes fibronectin bind protein FBP54 and the HNOOl protein AB2, nucleotides encoding the fibronectin binding domain from AB2 was amplified by PCR and cloned into the pGEX-6P-3 expression vector. The primer sequences used are given in SEQ ID NOS: 60 and 61 and were tagged with EcoRl and Bamϋl restriction endonuclease recognition sequences, respectively, to facilitate cloning. Cloned AB2 fibronectin binding domain sequence was transformed into E. coli DH5 cells according to standard laboratory methods. The polypeptide sequence of fibronectin binding protein AB2 is given in SEQ ID NO: 120 and shown in Fig. 2, showing the fibronectin binding domain (boxed). The AB2 fibronectin binding domain was expressed as a fusion protein with glutathione S-transferase (GST), and purified using Glutathione Sepharose 4B resin (Pharmacia Biotech) according to the manufacturer's instructions. An aliquot of the purified AB2 fibronectin binding domain- GST fusion protein was checked by SDS-PAGE analysis. To analyze the fibronectin binding activity of the cloned AB2 fragment, a competition assay was performed such that the ability of unlabeled AB2 fibronectin binding domain-GST fusion protein to block the binding of iodine- 125 labeled fibronectin to HNOOl was compared to GST and bovine serum albumin (BSA).

A commercial preparation of fibronectin (Sigma) was radio-iodinated 0.1 mCi iodine-

125 (Amersham Pharmacia) using IODO-BEADS iodination reagent (Pierce) and separated from unincorporated iodine- 125 and excess sodium iodide- 125 using a PD-10 desalting column (Amersham-Pharmacia) according to the manufacturer's instructions except that the elution was performed in phosphate buffered saline in twelve 500 μl aliquots. Radioactivity in eluted fractions was quantitated on a Bioscan Quick Count QC-4000/XER Benchtop

Radioisotope Counter (Bioscan, Inc.) and fractions containing the first peak of radioactivity (corresponding to labeled fibronectin) were pooled and diluted to 1.5 ng/μl.

A 100 μl aliquot of an overnight culture of HNOOl cells was inoculated into 10 ml MRS broth and incubated at 37 °C for 18 hrs, after which a series of 100 μl aliquots were taken for assay. To each aliquot of cells, 30 ng of iodine- 125-labeled fibronectin and 30 μg of unlabeled protein in 250 μl phosphate buffered saline pH 7.4 (PBS). After 1 hr incubation at room temperature, 1.5 ml of ice-cold PBS with 0.1% Tween 20 (Sigma) was added, cultures centrifuged to pellet cells and supernatants removed. Radioactive levels in the pellets were measured as above. Results were compared to blank control without HNOOl cells to measure background protein binding. Results are the means triplicate samples and presented as the percent radioactivity (dpm) of the bacterial pellet compared to the radioactivity of the added iodine- 125-labeled fibronectin ± twice the standard error (Table 1, Figure 3).

Table 3. Proportion of radioactively-labeled fibronectin bound to HNOOl cells after co- incubation with unlabeled AB2 fibronectin binding domain-GST-fusion protein, BSA, GST only, and background fibronectin binding (no cell control).

The results in Fig. 3 indicate that while in the presence of 30 μg BSA 4.7% of the total fibronectin added at the start of the assay remained bound to HNOOl cells after washing, this level dropped to 2.3% in the presence of unlabeled AB2 fibronectin binding domain-GST fusion protein. This was significantly lower than fibronectin binding in the presence of

GST protein alone and approached the level of background fibronectin binding seen in the absence of HNOOl cells. Thus, the HNOOl AB2 protein contains a domain capable of specifically blocking fibronectin binding to HNOOl cells indicating that the AB2 gene encodes a fibronectin binding protein.

Fibronectin binding proteins are expressed by a number of bacteria and been shown to be essential for bacterial adhesion to human epithelial cell surfaces (Holmes et al, Mol.

Microbiol. 41: 1395-1408, 2001; Peacock et al, Microbiology 145:3477-3486, 1999), and may also bind fibrinogen (Kushiro et al, J. Mol. Microbiol. Biotechnol. 3:563-571, 2001). Applications for HNOOl fibronectin binding protein AB2 include:

• enhanced adhesion to intestinal surface and cell lines;

• enhanced survival in intestinal environment;

• altered metabolic characteristics; • altered adhesion characteristics

• altered flavor or aroma characteristics;

• enhanced probiotic effects;

• human immune system interaction

• reagents to block or modify adherence of bacteria to mucosal surfaces; and • development of vaccine carriers

These attributes may be produced in food, such as dairy, systems (including milk protein hydrolysates and cheese) by directed activity of the enzyme, either in a bacterial strain (including strain HNOOl, or starter cultures) or as an enzyme preparation.

SEQ ID NOS: 1-120 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.

While in the foregoing specification this invention has been described in relation to certain preferred embodiments, and many details have been set forth for purposes 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 of the invention.

Claims

We claim:

1. An isolated polynucleotide comprising a nucleotide sequence present in Lactobacillus rhamnosus strain HNOOl that encodes a polypeptide having at least one of the following activities: enzyme activity; anti-infection activity; lactose digestion modulating activity; immune system modulating activity; amino acid, lipid, vitamin or carbohydrate metabolic activity; flavor, texture or aroma modulating activity; multistress resistance and survival activity; antigenic activity; adhesion activity; and regulatory activity.

2. An isolated polynucleotide of claim 1, comprising a nucleotide sequence selected from the group consisting of: (1) the sequences recited in SEQ ID NOS: 1-59; and (2) sequences comprising a nucleotide sequence producing an Expectation ("E") value of 0.01 or less when compared to a sequence of (1) above using the BLASTN algorithm version 2.04 set to the default parameters described in the specification, above.

3. An isolated polynucleotide of claim 1 comprising a nucleotide sequence having at least 75% identical nucleotides to a compare sequence selected from the nucleotide sequences recited in SEQ ID NOS: 1-59, the percentage identical nucleotides being determined by aligning the sequence and the compare sequences using the BLASTN algorithm version 2.04 set at default parameters, identifying the number of identical nucleotides over aligned portions of the sequence and the compare sequences, dividing the number of identical nucleotides by the total number of nucleic acids of the compare sequence, and multiplying by 100 to determine the percentage identical nucleotides.

4. An isolated polynucleotide of claim 1 comprising a nucleotide sequence that hybridizes to a polynucleotide comprising a sequence recited in SEQ ID NOS: 1-59 under stringent hybridization conditions;

5. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (1) complements of the sequences recited in SEQ ID NOS: 1-59; (2) reverse complements of the sequences recited in SEQ ID NOS: 1-59; and (3) reverse sequences of the sequences recited in SEQ ID NOS: 1-59.

6. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (1) sequences comprising a nucleotide sequence that is a 200-mer of a sequence of claim 2; (2) sequences comprising a nucleotide sequence that is a 100-mer of a sequence of claim 2; and (3) sequences comprising a nucleotide sequence that is a 40-mer of a sequence recited in claim 2.

7. An isolated polynucleotide of claim 1 comprising a nucleotide sequence that differs from a nucleotide sequence recited in SEQ ID NOS: 1-59 as a result of conservative substitutions.

8. An isolated polynucleotide of claim 1 comprising a nucleotide sequence that differs from a nucleotide sequence recited in SEQ ID NOS: 1-59 as a result of deletions and/or insertions and/or substitutions totaling less than 10% of the total sequence length.

9. An isolated polynucleotide of claim 1 comprising a nucleotide sequence that differs from a nucleotide sequence recited in SEQ ID NOS: 1-59 as a result of substitutions, insertions, and/or deletions totaling less than 15% of the total sequence length.

10. An isolated polypeptide encoded by an isolated polynucleotide of any of claims 1-9.

11. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of : (1) the sequences recited in SEQ ID NOS: 62-120; (2) sequences producing an producing an Expectation ("E") value of 0.01 or less when compared to a sequence recited in (1) above using the BLASTP algorithm version 2.0.11 set to default parameters; (3) sequences comprising an amino acid sequence having at least 75% identical amino acid residues with a compare sequence selected from the amino acid sequences recited in (1) and (2) above, the percentage identical amino acids being determined by aligning the sequence and the compare sequences using the BLASTP algorithm version 2.0.11 set at default parameters, identifying the number of identical amino acids over aligned portions of the sequence and the compare sequences, dividing the number of identical amino acids by the total number of amino acids of the compare sequence, and multiplying by 100 to determine the percentage identical amino acids; (4) sequences differing by codon alterations that reflect the degeneracy of the genetic code; and (5) functionally similar sequences differing only by conservative amino acid substitutions.

12. A fusion protein comprising at least one polypeptide according to claim 11.

13. A kit comprising a plurality of oligonucleotide probes or primers comprising at least 10 contiguous residues complementary to 10 contiguous residues of a nucleotide sequence recited in claim 2.

14. A genetic construct comprising a polynucleotide of claim 2.

15. The genetic construct of claim 14, wherein said polynucleotide encodes a polypeptide that modifies the flavor, aroma, texture and health-related benefits of milk-derived products selected from the group consisting of: histidinol-phosphate aminotransferase, tyrosine aminotransferase, cysteine desulfurase, lipase, 0-acetylserine sulfhydrylase, surface protein, Group B streptococcal oligopeptidase, Pz-peptidase, dipeptidase, acylaminoacyl-peptidase, carboxylesterase, glycerophosphodiester phosphodiesterase, bifunctional alcohol dehydrogenase and acetaldehyde dehydrogenase, short-chain alcohol dehydrogenase, branched chain amino acid transport system II carrier protein, malolactic enzyme, pyruvate dehydrogenase, El (lipoamide) alpha subunit, formate C-acetyltransferase, 6-phosphogluconate dehydrogenase, 5- methyltetrahydropteroyltriglutamate homocysteine methyltransferase, S- methylmethionine permease, 6-phospho-beta-galactosidase, gamma-glutamyl phosphate reductase, dihydrofolate reductase, lactate dehydrogenase, lysine, aminopeptidase pepS, fibronectin binding protein AB2, prolinase (pepR), Xaa-Pro dipeptidase and human bile salt export pump.

16. The genetic construct of claim 14, wherein said polynucleotide encodes a polypeptide that increases the survivability of a microbe used in the manufacture of dairy products and probiotic supplements, wherein said polypeptide is selected from the group consisting of: transmembrane adhesion protein (SEQ ID NO: 1), major cell adherence molecule of Campylobacter jejuni and Campylobacter coli, collagen/mucin binding protein (SEQ ID NO: 8), ATP-dependent ClpC proteinase regulatory protein, O-sialoglycoprotein endopeptidase, human bile salt export pump, adhesin (SEQ ID NO: 28), bifunctional HPr Kinase/P- Ser-HPr phosphatase, malolactic enzyme, magnesium transporter, dTDP-4-keto-E rhamnose reductase, glucose inhibited division protein, glucose- 1 -phosphate thymidylyl transferase, phosphate starvation-induced protein, GTP binding protein, gamma-glutamyl phosphate reductase, Pav A, aminopeptidase pepS, heat-inducible transcription repressor protein, phosphoribosylaminoimidazolecarboxyamide formyltransferase/imp cyclohydrolase, hexulose-6-phosphate isomerase and fibronectin binding protein AB2.

17. A transgenic cell comprising a genetic construct according to any of claims 14-16.

18. A genetic construct comprising, in the 5 '-3' direction:

(a) a gene promoter sequence;

(b) a polynucleotide sequence comprising at least one of the following: (1) a polynucleotide coding for at least a functional portion of a polypeptide encoded by a nucleotide sequence described in claim 2; and (2) a polynucleotide comprising a non-coding region of a gene coding for an polypeptide encoded by a nucleotide sequence selected from the group consisting of sequences recited in claim 2; and

(c) a gene termination sequence.

19. The genetic construct of claim 14-16 or 18 wherein the polynucleotide is in a sense orientation.

20. The genetic construct of claim 14-16 or 18 wherein the polynucleotide is in an antisense orientation.

21. The genetic construct of claim 18, wherein the gene promoter sequence and gene termination sequences are functional in a prokaryote or eucaryote.

22. A method for modulating the polynucleotide content or composition of an organism comprising transforming the organism with a genetic construct of claim 14-16 or 18.

23. A method for improving the properties of microbes used in the manufacture of milk-derived products and probiotic supplements, which comprises modulating the polynucleotide content or composition of said microbes by transforming said microbes with one or more polynucleotide sequences selected from the group consisting of:

(a) Lactobacillus rhamnosus strain HNOOl sequences encoding polypeptides that modify the flavor, aroma, texture and health-related benefits of milk-derived products; and

(b) Lactobacillus rhamnosus strain HNOOl sequences encoding polypeptides that increase the survivability of said microbes in dairy product manufacturing processes.

24. The method of claim 23, wherein said polypeptides are selected from the group consisting of: histidinol-phosphate aminotransferase, tyrosine aminotransferase, cysteine desulfurase, lipase, 0-acetylserine sulfhydrylase, surface protein, Group B streptococcal oligopeptidase, Pz-peptidase, dipeptidase, acylaminoacyl-peptidase, carboxylesterase, glycerophosphodiester phosphodiesterase, bifunctional alcohol dehydrogenase and acetaldehyde dehydrogenase, short-chain alcohol dehydrogenase, branched chain amino acid transport system II carrier protein, malolactic enzyme, pyruvate dehydrogenase, El (lipoamide) alpha subunit, formate C-acetyltransferase, 6-phosphogluconate dehydrogenase, 5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase, S-methylmethionine permease, 6-phospho-beta- galactosidase, gamma-glutamyl phosphate reductase, dihydrofolate reductase, lactate dehydrogenase, lysin, aminopeptidase pepS, fibronectin binding protein AB2, prolinase (pepR), Xaa-Pro dipeptidase, human bile salt export pump, transmembrane adhesion protein (SEQ ID NO: 1), major cell adherence molecule of Campylobacter jejuni and Campylobacter coli, collagen/mucin binding protein (SEQ ID NO: 8), ATP-dependent ClpC proteinase regulatory protein, O-sialoglycoprotein endopeptidase, adhesin (SEQ ID NO: 28), bifunctional HPr Kinase/P-Ser-HPr phosphatase, malolactic enzyme, magnesium transporter, dTDP-4-keto- . rhamnose reductase, glucose inhibited division protein, glucose- 1 -phosphate thymidylyl transferase, phosphate starvation-induced protein, GTP binding protein, Pav A, heat- inducible transcription repressor protein, hosphoribosylaminoimidazolecarboxyamide formyltransferase/imp cyclohydrolase, and hexulose-6-phosphate isomerase.

25. A method for modifying the flavor, aroma, texture and/or nutritional and health 'benefits of milk-derived products, which comprises adding one or more polypeptides to the milk being processed, wherein said polypeptides are selected from the group consisting of Lactobacillus rhamnosus strain HNOOl histidinol-phosphate aminotransferase, tyrosine aminotransferase, cysteine desulfurase, lipase, O- acetylserine sulfhydrylase, surface protein, Group B streptococcal oligopeptidase, Pz- peptidase, dipeptidase, acylaminoacyl-peptidase, carboxylesterase, glycerophosphodiester phosphodiesterase, bifunctional alcohol dehydrogenase and acetaldehyde dehydrogenase, short-chain alcohol dehydrogenase, branched chain amino acid transport system II carrier protein, malolactic enzyme, pyruvate dehydrogenase, El (lipoamide) alpha subunit, formate C-acetyltransferase, 6- phosphogluconate dehydrogenase, 5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase, S-methylmethionine permease, 6-phospho-beta- galactosidase, gamma-glutamyl phosphate reductase, dihydrofolate reductase, lactate dehydrogenase, lysine, aminopeptidase pepS, fibronectin binding protein AB2, prolinase (pepR), Xaa-Pro dipeptidase and human bile salt export pump.

26. A method of identifying an organism or reproductive material or an extract therefrom as having a specific origin, the method comprising detecting in the genetic complement of the organism, material or extract the presence or absence of a polynucleotide identifier representative of said origin, the polynucleotide identifier comprising a sequence recited in SEQ ID NOS: 1-59.

27. The method of claim 26 wherein the organism is a bacterial cell or a yeast cell.

28. The method of claim 26 wherein the presence or absence of the polynucleotide identifier is detected by isolating DNA from the organism or material and contacting the isolated DNA with at least one oligonucleotide probe specific for the polynucleotide identifier.

29. The method of claim 26 wherein the isolated DNA is contacted with a plurality of oligonucleotide probes in an array format.