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Polynucleotides, materials incorporating them, and methods for using them Number:7,125,698 from the United States Patent and Trademark Office (PTO) owispatent

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Title: Polynucleotides, materials incorporating them, and methods for using them

Abstract: Novel polynucleotides isolated from Lactobacillus rhamnosus, as well as oligonucleotide probes and primers, genetic constructs comprising the polynucleotides, biological materials, including plants, microorganisms and multicellular organisms incorporating the polynucleotides, polypeptides expressed by the polynucleotides, and methods for using the polynucleotides and polypeptides are disclosed.

Patent Number: 7,125,698 Issued on 10/24/2006 to Glenn,   et al.


Inventors: Glenn; Matthew (Whenuapai, Auckland, NZ), Havukkala; Ilkka J. (Remucra, Auckland, NZ), Lubbers; Mark (Palmerston North, NZ), Dekker; James (Palmerston North, NZ)
Appl. No.: 10/264,213
Filed: October 3, 2002


Related U.S. Patent Documents

Application NumberFiling DatePatent NumberIssue Date
09971536Oct., 2001
09634238Aug., 20006544772
60152032Sep., 1999
60152031Sep., 1999
60147853Aug., 1999
60147852Aug., 1999

Current U.S. Class: 435/193 ; 426/534; 435/183; 435/194; 530/350
Current International Class: C12N 9/10 (20060101)
Field of Search: 435/193,183,194 426/534 530/350


References Cited [Referenced By]

U.S. Patent Documents
6379663 April 2002 Gill et al.
6476209 November 2002 Glenn et al.
6544772 April 2003 Glenn et al.
Foreign Patent Documents
WO 01/77335 Oct., 2001 WO
WO 02/44383 Aug., 2002 WO

Other References

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NCBI Database, Accession No. ZP.sub.--00046718, submitted Sep. 30, 2004. cited by other .
Stentz, Regis, et al., "Molecular Cloning and Analysis of the ptsHI Operon in Lactobacillus sake", Applied and Environmental Microbiology, vol. 63, No. 6, pp. 2111-2116 (1997). cited by other .
Luesink, Evert J., et al., "Molecular Characterization of the Lactococcus lactis ptsHI Operon and Analysis of the Regulatory Role of HPr", Journal of Bacteriology, vol. 181, No. 3, pp. 764-771 (1999). cited by other .
Groisillier, Agnes, et al., "Comparison of partial malolactic enzyme gene sequences for phylogenetic analysis of some lactic acid bacteria species and relationships with the malic enzyme", International Journal of Systematic Bacteriology, vol. 49, pp. 1417-1428 (1999). cited by other .
GenBank Accession No. AF098777, submitted Jun. 2, 1999. cited by other .
Kunji, Edmund R.S., et al., "The proteolytic systems of lactic acid bacteria", Antonie van Leeuwenhoek, vol. 70, pp. 187-221 (1996). cited by other .
Branny, Pavel, et al., "An operon encoding three glycolytic enzymes in Laciobacillus delbrueckii subsp. bulgaricus: glyceraldehyde-3-phosphate dehydrogenase, phoshoglycerate kinase and triosephosphate isomerase", Microbiology, vol. 144, pp. 905-914 (1998). cited by other .
GenBank Accession No. AJ000339, submitted May 8, 1998. cited by other .
Hidalgo, Elena, et al., "Molecular Cloning and DNA Sequencing of the Escherichia coli K-12 ald Gene Encoding Aldehyde Dehydrogenase", Journal of Bacteriology, vol. 173, No. 19, pp. 6118-6123 (1991). cited by other .
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Primary Examiner: Graser; Jennifer E.
Attorney, Agent or Firm: Sleath; Janet Speckman Law Group PLLC

Parent Case Text



CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent application Ser. No. 09/971,536, filed Oct. 2, 2001, which is a continuation-in-part of U.S. patent application Ser. No. 09/634,238, filed Aug. 8, 2000 now U.S. Pat. No. 6,544,772, which claims priority to U.S. Provisional Patent Application 60/147,853, filed Aug. 9, 1999, U.S. Provisional Patent Application 60/147,852, filed Aug. 9, 1999, U.S. Provisional Patent Application 60/152,032, filed Sep. 1, 1999, and U.S. Provisional Patent Application 60/152,031, filed Sep. 1, 1999.
Claims



We claim:

1. An isolated polypeptide comprising SEQ ID NO: 218.

2. An isolated polynucleotide comprising an amino acid sequence selected from the group consisting of: (a) sequences having at least 75% identity to SEQ ID NO: 218; (b) sequences having at least 90% identity to SEQ ID NO: 218; and (c) sequences having at least 95% identity to SEQ ID NO: 218, wherein the polypeptide has undecaprenyl-phosphate glycosyl-1-phosphate transferase activity.

3. A fusion protein comprising at least one polypeptide according to claim 1.

4. An isolated polypeptide encoded by a polynucleotide of SEQ ID NO: 93.

5. A composition comprising a polypeptide according to claim 1 and at least one component selected from the group consisting of: physiologically acceptable carriers and immunostimulants.

6. A method for modifying at least one property of a product, food, food additive, nutritional supplement or probiotic supplement, wherein the product, food, food additive, nutritional supplement or probiotic supplement is prepared from milk and the property is selected from the group consisting of: flavor; aroma; texture; nutritional benefits; immune system modulating properties; and health benefits, the method comprising adding a polypeptide of claim 1 to the milk.

7. A food product comprising an isolated polypeptide of claim 1.

8. The food product of claim 7, wherein the food product is derived from milk.

9. A fusion protein comprising at least one polypeptide according to claim 2.

10. A composition comprising a polypeptide according to claim 2 and at least one component selected from the group consisting of: physiologically acceptable carriers and immunostimulants.

11. A food product comprising an isolated polypeptide of claim 2.

12. The food product of claim 11, wherein the food product is derived from milk.
Description



TECHNICAL FIELD OF THE INVENTION

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

REFERENCE TO SEQUENCE LISTING SUBMITTED ON COMPACT DISC

This application incorporates by reference in its entirety the Sequence Listing that is provided in duplicate on compact discs that accompany the application. Each CD contains the following file: 1043c3 SEQLIST.txt, having a date of creation of Oct. 3, 2002 and a file size of 659 KB.

BACKGROUND OF THE INVENTION

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

Lactobacillus rhamnosus strain HN001 are heterofermentative bacteria that are Gram positive, non-motile, non-spore forming, catalase negative, facultative anaerobic rods exhibiting an optimal growth temperature of 37.+-.1.degree. C. and an optimum pH of 6.0 6.5. Experimental studies demonstrated that dietary supplementation with Lactobacillus rhamnosus strain HN001 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 HN001, and certain other Gram-positive bacteria can specifically and directly modulate human and animal health (See, for example, Tannock et al., Applied Environ. Microbiol. 66:2578 2588, 2000; Gill et al., Brit. J. Nutrition 83:167 176; Quan Shu et al., Food and Chem. Toxicol. 38:153 161, 2000; Quan Shu et al., Intl. J. Food Microbiol. 56:87 96, 2000; Quan Shu et al., Intl. Dairy J. 9:831 836, 1999; Prasad et al., Intl. Dairy J. 8:993 1002, 1998; Sanders and Huis in't Veld, Antonie van Leeuwenhoek 76:293 315, 1999; Salminen et al., 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 211 253; Delcour et al., Antonie van Leeuwenhoek 76:159 184, 1999; Blum et al., Antonie van Leeuwenhoek 76:199 205, 1999; Yasui et al., Antonie van Leeuwenhoek 76:383 389, 1999; Hirayama and Rafter, Antonie van Leeuwenhoek 76:391 394, 1999; Ouwehand, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 139 159; Isolauri et al., S 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 255 268; Lichtenstein and Goldin, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 269 277; El-Nezami and Ahokas, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 359 367; Nousianen et al., 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 437 473; Meisel and 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 Th1/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 include: improved nutrition; regulation of colonocyte proliferation and differentiation; improved lignan and isoflavone metabolism; reduced mucosal permeability; detoxification of carcinogens and other harmful compounds; relief of constipation and diarrhea; and vitamin synthesis, in particular folate.

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

Exopeptidases cleave amino acids from the N- or C-terminus of a peptide chain, releasing free amino acids or short (di- and tri-) peptides. Different types of exopeptidases include: Aminopeptidases--release a free amino acid from the N-terminus of a peptide chain; dipeptidyl-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.

Peptidases are important enzymes in the process of cheese ripening and the development of cheese flavor. The hydrolysis of milk caseins in cheese results in textural changes and the development of cheese flavors. The raft of proteolytic enzymes that cause this hydrolysis come from the lactic acid bacteria that are bound up in the cheese--either starter cultures that grow up during the manufacture of the cheese, or adventitious and adjunct non-starter lactic acid bacteria that grow in the cheese as it ripens (Law and Haandrikman, Int. Dairy J. 7:1 11, 1997).

Many other enzymes can also influence dairy product flavor, and functional and textural characteristics, as well as influencing the fermentation characteristics of the bacteria, such as speed of growth, acid production and survival (Urbach, Int. Dairy J. 5:877 890, 1995; Johnson and Somkuti, Biotech. Appl. Biochem. 13:196 204, 1991; El Soda and Pandian, J. Dairy Sci. 74:2317 2335, 1991; Fox et al., In Cheese: chemistry, physics and microbiology. Volume 1, General aspects, 2.sup.nd edition, P Fox (ed) Chapman and Hall, London; Christensen et al., Antonie van Leeuwenhoek 76:217 246, 1999; Stingle et al., J. Bacteriol. 20:6354 6360, 1999; Stingle et al., Mol. Microbiol. 32:1287 1295, 1999; Lemoine et al., Appl. Environ. Microbiol. 63:1512 3518, 1997). Enzymes influencing specific characteristics and/or functions include the following: Lysis of cells. These enzymes are mostly cell wall hydrolases, including amidases; muramidases; lysozymes, including N-acetyl muramidase; muramidase; N-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 decarboxylase, acetolactate synthase, pyruvate decarboxylase, pyruvate formate lyase, diacetyl synthase, diacetyl reductase, alcohol decarboxylase, lactate dehydrogenase, pyruvate dehydrogenase, and aldehyde dehydrogenase. Lipid degradation, modification or synthesis. Enzymes involved include lipases, esterases, phospholipases, serine hydrolases, desaturases, and linoleate isomerase. Polysaccharide synthesis. Polysaccharides are important not only for potential immune enhancement and adhesion activity but are important for the texture of fermented dairy products. The enzymes involved are a series of glucosyl transferases, including beta-(1-3) 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 b e 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: 1 121; (b) variants of those sequences; (c) extended sequences comprising the sequences set out in SEQ ID NOS: 1 121, and their variants; and (d) sequences comprising at least a specified number of contiguous residues of a sequence of SEQ ID NOS: 1 121 (x-mers). Oligonucleotide probes and primers corresponding to the sequences set out in SEQ ID NOS: 1 121, and their variants are also provided. All of these polynucleotides and oligonucleotide probes and primers are collectively referred to herein, as "polynucleotides of the present invention."

The polynucleotide sequences identified as SEQ ID NOS: 1 121 were derived from a microbial source, namely from fragmented genomic DNA of Lactobacillus rhamnosus, strain HN001, described in PCT International Publication No. WO 99/10476. Lactobacillus rhamnosus strain HN001 are heterofermentative bacteria that are Gram positive, non-motile, non-spore forming, catalase negative, facultative anaerobic rods exhibiting an optimal growth temperature of 37.+-.1.degree. C. and an optimum pH of 6.0 6.5. Experimental studies demonstrated that dietary supplementation with Lactobacillus rhamnosus strain HN001 induced a sustained enhancement in several aspects of both natural and acquired immunity. A biologically pure culture of Lactobacillus rhamnosus strain HN001 was deposited at the Australian Government Analytical Laboratories (AGAL), The New South Wales Regional Laboratory, 1 Suakin Street, Pymble, NSW 2073, Australia, as Deposit No. NM97/09514, dated 18 Aug. 1997.

Certain of the polynucleotide sequences disclosed herein are "partial" sequences in that they do not represent a full-length gene encoding a full-length polypeptide. Such partial sequences may be extended by analyzing and sequencing various DNA libraries using primers and/or probes and well-known hybridization and/or PCR techniques. The partial sequences disclosed herein may thus be extended until an open reading frame encoding a polypeptide, a full-length polynucleotide and/or gene capable of expressing a polypeptide, or another useful portion of the genome is identified. Such extended sequences, including full-length polynucleotides and genes, are described as "corresponding to" a sequence identified as one of the sequences of SEQ ID NOS: 1 121 or a variant thereof, or a portion of one of the sequences of SEQ ID NOS: 1 121 or a variant thereof, when the extended polynucleotide comprises an identified sequence or its variant, or an identified contiguous portion (x-mer) of one of the sequences of SEQ ID NOS: 1 121 or a variant thereof.

The polynucleotides identified as SEQ ID NOS: 1 121 were isolated from Lactobacillus rhamnosus genomic DNA clones and represent sequences that are present in the cells from which the DNA was prepared. The sequence information may be used to identify and isolate, or synthesize, DNA molecules such as promoters, DNA-binding elements, open reading frames or full-length genes, that 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: 1 121.

The present invention further provides isolated polypeptides encoded, or partially encoded, by the polynucleotides disclosed herein. In certain specific embodiments, the polypeptides of the present invention comprise a sequence selected from the group consisting of sequences identified as SEQ ID NO: 122 253, and variants thereof. Polypeptides encoded by the polynucleotides 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.

Genetic constructs comprising the inventive polynucleotides are also provided, together with transgenic host cells comprising such constructs and transgenic organisms, such as microbes, comprising such cells.

The present invention also contemplates methods for modulating the polynucleotide and/or polypeptide content and composition of an organism, such methods involving stably incorporating into the genome of the organism a genetic construct comprising a polynucleotide of the present invention. In one embodiment, the target organism is a microbe, preferably a microbe used in fermentation, more preferably a microbe 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: 1 121, and their variants, may be used to design oligonucleotide probes and primers. Such oligonucleotide probes and primers have sequences that are substantially complementary to the polynucleotide of interest over a certain portion 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 polymerase chain reaction (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, Calif.).

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

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

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

DETAILED DESCRIPTION

The polynucleotides disclosed herein were isolated by high throughput sequencing of DNA libraries from the lactic acid bacteria Lactobacillus rhamnosus as described 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 HN001 must survive environmental stress conditions in the gastrointestinal tract, as well as commercial and industrial processes. Modification of particular polynucleotides or regulatory processes has been shown to be effective against a number of stresses including oxidative stress, pH, osmotic stress, dehydration, carbon starvation, phosphate starvation, nitrogen starvation, amino acid starvation, heat or cold shock and mutagenic stress. Polynucleotides involved in stress resistance often confer multistress resistance, i.e., when exposed to one stress, surviving cells are resistant to several non-related stresses. Bacterial genes and/or processes shown to be involved in multistress resistance include:

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

Intracellular guanosine pools--purine biosynthesis and scavenger pathways involve the production of phosphate-guanosine compounds that act as signal molecules in the bacterial stringent response. Gene knockouts involving purine scavenger pathway genes appear to confer multistress resistance. Osmoregulatory molecules--small choline-based molecules, such as glycine-betaine, and sugars, such as trehalose, are protective against osmotic shock and are rapidly imported and/or synthesized in response to increasing osmolarity. Acid resistance--lactobacilli naturally acidify their environment through t he 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 identity and functions of the inventive polynucleotides based on such similarities are shown below in Table 1.

TABLE-US-00001 TABLE 1 SEQ ID SEQ ID NO: NO: DNA PROT Category Description 1 122 Construction of genetic vectors for Homologue of purL, encoding a controlled expression of RNA and/or phosphoribosylformylglycinamidine protein, fusion protein production, (FGAM) synthetase (EC 6.3.5.3). PurL genetic modification, mutagenesis catalyzes the fourth step in the amplification of genetic material or biosynthesis of purines. It is involved for other genetic or protein in resistance environmental stress manipulations. conditions and the stringent response Production of desirable flavors. through the control of intracellular Modified flavor, aroma and/or texture phosphate levels. Purines also play attributes. essential roles in many other cellular Altered survival characteristics: functions, including DNA replication, survival of industrial processes, transcription, intra-and extra-cellular growth or storage in product formats, signaling, energy metabolism, and as persistence in gut environment. coenzymes for many biochemical Altered viability in response to stress reactions. conditions. Altered metabolic properties or regulation of metabolic pathways. Altered probiotic attributes. 2 123 Construction of genetic vectors for Homologue of 5'-Phosphoribosyl-5- controlled expression of RNA and/or aminoimidazole (AIR) carboxylase protein, fusion protein production, (EC 4.1.1.21). AIR carboxylase is genetic modification, mutagenesis responsible for CO.sub.2 fixation during amplification of genetic material or purine biosynthesis. It catalyzes the for other genetic or protein carboxylation of AIR to 5'- manipulations. phosphoribosyl-5-aminoimidazole-4- Production of desirable flavors. carboxylic acid, in the de novo Modified flavor, aroma and/or texture biosynthesis of purine nucleotides. attributes. AIR carboxylase is composed of two Altered survival characteristics: nonidentical subunits, the catalytic survival of industrial processes, subunit is encoded by the purE gene, growth or storage in product formats, while the CO.sub.2-binding subunit is persistence in gut environment. encoded by the purK gene. These two Altered viability in response to stress genes form an operon in which the conditions. termination codon of the purE gene Altered metabolic properties or overlapped the initiation codon of the regulation of metabolic pathways. purK gene. The purEK operon is Altered probiotic attributes. regulated by the purR gene product, and a purR regulatory-protein-binding site related to the sequences found in other pur loci was identified in the purEK operon control region. It is involved in resistance environmental stress conditions and the stringent response through the control of intracellular phosphate levels. Purines also play essential roles in many other cellular functions, including DNA replication, transcription, intra- and extra-cellular signaling, energy metabolism, and as coenzymes for many biochemical reactions. 3 124 Construction of genetic vectors for Homologue of amino acid antiporters controlled expression of RNA and/or gadC, Xasa and acsA. Amino acid protein, fusion protein production, antiporters are integral membrane genetic modification, mutagenesis proteins involved in the transport of amplification of genetic material or amino acids into the cell and in for other genetic or protein extreme acid resistance. GadC is manipulations. homologous to putative glutamate- Production of desirable flavors. gamma-aminobutyrate antiporters of Modified flavor, aroma and/or texture Escherichia coli and Shigella flexneri attributes. and contains 12 putative membrane- Altered survival characteristics: spanning domains. It belongs to the survival of industrial processes, amino acid-polyamine-organocation growth or storage in product formats, (APC) superfamily, and the Xasa persistence in gut environment. family of transporters. It is involved in Altered viability in response to stress glutamate-dependent acid resistance conditions. and in antiport of glutamate and Altered amino acid metabolism. glutamate-gamma-aminobutyrate Altered metabolic properties or (GABA). The chloride-dependent regulation of metabolic pathways. expression is activated by gadR. GadC Altered probiotic attributes. is involved in tolerance to environmental stress conditions such as high salt and low pH. 4 125, 126 Construction of genetic vectors for Homologue of the B-subunit of controlled expression of RNA and/or phosphate-specific transporter (PstB). protein, fusion protein production, PstB is an ATP binding cassette genetic modification, mutagenesis (ABC) protein. Phosphate-specific amplification of genetic material or transporters (Pst) in bacteria are for other genetic or protein involved in phosphate transport. Pst is manipulations. a multisubunit system and belongs to Production of desirable flavors. the ABC superfamily of transporters. Modified flavor, aroma and/or texture (TC# 3.A.1.7.1) (Novak et al., J attributes. Bacteriol. 181: 1126 1133, 1999). Altered survival characteristics: Utility as a controlled expression survival of industrial processes, vector and in the control of growth or storage in product formats, intracellular phosphate levels persistence in gut environment. important for resistance to Altered phosphate metabolism. environmental stress conditions and Altered viability in response to stress induction of the stringent response. conditions. Altered metabolic properties or regulation of metabolic pathways. Altered probiotic attributes. 5, 106 127, 230 Construction of genetic vectors for Homologue of PstA/PstC, which are controlled expression of RNA and/or the two hydrophobic subunits of a protein, fusion protein production, phosphate-specific transporter (PstB), genetic modification, mutagenesis an ATP binding cassette (ABC) amplification of genetic material or protein. Phosphate specific transporter for other genetic or protein (Pst) in bacteria is involved in manipulations. phosphate transport. Pst is a Production of desirable flavors. multisubunit system and belongs to the Modified flavor, aroma and/or texture ABC superfamily of transporters. (TC# attributes. 3.A.1.7.1) (Novak et al., J. Bacteriol. Altered survival characteristics: 181: 1126 1133, 1999). Utility as a survival of industrial processes, controlled expression vector and in the growth or storage in product formats, control of intracellular phosphate persistence in gut environment. levels important for resistance to Altered phosphate metabolism. environmental stress conditions and Altered viability in response to stress induction of the stringent response. conditions. Altered metabolic properties or regulation of metabolic pathways. Altered probiotic attributes. 6 10 128, 130 133 Construction of genetic vectors for Homologue of a response regulator controlled expression of RNA and/or belonging to the family of 2- protein, fusion protein production, component signal transduction proteins genetic modification, mutagenesis phosphorylated by a specific sensor amplification of genetic material or kinase (phoR). PhoR for other genetic or protein activates/represses Pho regulon gene manipulations. transcription in response to phosphate Production of desirable flavors. starvation. The gene is involved in cell Modified flavor, aroma and/or texture cycle control, polysaccharide synthesis attributes. and intestinal adhesion, also Altered survival characteristics: multistress resistance. It is part of a survival of industrial processes, phosphate (PHO) regulon which is growth or storage in product formats, regulated by extracellular phosphate persistence in gut environment. and consists of 20 phosphate-regulated Altered phosphate metabolism. promotors, 10 regulatory genes and 2 Altered viability in response to stress phosphate transport systems. Under conditions. conditions of phosphate limitation, the Altered metabolic properties or response regulator PhoB is regulation of metabolic pathways. phosphorylated by the histidine kinase Altered probiotic attributes. PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon (Novak et al., J. Bacteriol. 181: 1126 1133, 1999). Utility as a controlled expression vector and in the control of intracellular phosphate levels important for resistance to environmental stress conditions and induction of the stringent response. 6 129 Construction of genetic vectors for Homologue of the response regulator controlled expression of RNA and/or PnpR. PnpR is part of a two- protein, fusion protein production, component regulatory system, PnpR- genetic modification, mutagenesis PnpS, and a downstream ABC amplification of genetic material or transporter, similar to the Pst system in for other genetic or protein E. coli, including a gene encoding a manipulations. PhoU protein. The E. coli Pst system Production of desirable flavors. belongs to the superfamily of ABC Modified flavor, aroma and/or texture transporters. It is part of a phosphate attributes. (PHO) regulon which is regulated by Altered survival characteristics: extracellular phosphate and consists of survival of industrial processes, 20 phosphate-regulated promotors, 10 growth or storage in product formats, regulatory genes and 2 phosphate persistence in gut environment. transport systems. Under conditions of Altered phosphate metabolism. phosphate limitation, the response Altered viability in response to stress regulator PhoB is phosphorylated by conditions. the histidine kinase PhoR and binds to Regulation of metabolic pathways. promoters that share a consensus PHO Altered metabolic properties or box. Under conditions of phosphate regulation of metabolic pathways. excess, PhoR, Pst, and PhoU Altered probiotic attributes. downregulate the PHO regulon (Novak et al., J. Bacteriol. 181: 1126 1133, 1999). Utility in immune modulation, gut adhesion, cell wall synthesis and polysaccharide production, survival, controlled expression vector. 11 134 Construction of genetic vectors for Homologue of the histidine kinase controlled expression of RNA and/or PhoR, which is involved in the E. coli protein, fusion protein production, Pst system. PhoR is part of a genetic modification, mutagenesis phosphate (PHO) regulon and amplification of genetic material or phosphorylates under conditions of for other genetic or protein phosphate limitation the response manipulations. regulator PhoB. Under conditions of Production of desirable flavors. phosphate excess, PhoR, Pst, and Modified flavor, aroma and/or texture PhoU down regulate the PHO regulon attributes. (Novak et al., J. Bacteriol. 181: 1126 1133, Altered survival characteristics: 1999) which consists of 20 survival of industrial processes, phosphate-regulated promoters, 10 growth or storage in product formats, regulatory genes and 2 phosphate persistence in gut environment. transport systems. Utility in immune Altered phosphate metabolism. modulation, gut adhesion, cell wall Altered viability in response to stress synthesis and polysaccharide conditions. production, survival, controlled Regulation of metabolic pathways. expression vector. Altered metabolic properties or regulation of metabolic pathways. Altered probiotic attributes. 12, 107 135, 231 Construction of genetic vectors for Homologue of PnpS (which in turn is a controlled expression of RNA and/or homologue of PhoR), which is part of protein, fusion protein production, a two-component regulatory system, genetic modification, mutagenesis PnpR-PnpS, and a downstream ATP- amplification of genetic material or binding cassette (ABC) transporter, for other genetic or protein similar to the Pst system in E. coli manipulations. including a gene encoding a PhoU Production of desirable flavors. protein. The E. coli Pst system belongs

Modified flavor, aroma and/or texture to the superfamily of ABC attributes. transporters. It is part of a phosphate Altered survival characteristics: (PHO) regulon which is regulated by survival of industrial processes, extracellular phosphate and consists of growth or storage in product formats, 20 phosphate-regulated promoters, 10 persistence in gut environment. regulatory genes and 2 phosphate Altered phosphate metabolism. transport systems. Under conditions of Altered viability in response to stress phosphate limitation, the response conditions. regulator PhoB is phosphorylated by Regulation of metabolic pathways. the histidine kinase PhoR and binds to Altered metabolic properties or promoters that share a consensus PHO regulation of metabolic pathways. box. Under conditions of phosphate Altered probiotic attributes. excess, PhoR, Pst, and PhoU downregulate the PHO regulon. PnpS (Novak et al., J. Bacteriol. 181: 1126 1133, 1999). Utility in immune modulation, gut adhesion, cell wall synthesis and polysaccharide production, survival, controlled expression vector. 13, 14 136, 137 Altered cell wall or cell surface Homologue of penicillin-binding characteristics, structures or protein 1B (Pbp1b) or murein functions. polymerase. Penicillin-binding Improved antimicrobial properties proteins (PBPs), targets of beta-lactam Modified adhesion to human or antibiotics, are membrane-bound animal cells or cell lines. enzymes essential for the biosynthesis Production of desirable flavors. of the bacterial cell wall. PBPs possess Modified flavor, aroma and/or texture a penicillin-insensitive attributes. transglycosylase N-terminal domain Construction of genetic vectors for (formation of linear glycan strands) controlled expression of RNA and/or and a penicillin-sensitive protein, fusion protein production, transpeptidase C-terminal domain genetic modification, mutagenesis (cross-linking of the peptide subunits) amplification of genetic material or responsible for the final steps of the for other genetic or protein bacterial cell wall polymerization and manipulations. cross-linking, respectively (Zhao et al., Altered survival characteristics: Protein Expr. Purif. 16: 331 339, survival of industrial processes, 1999). Utility in immune modulation, growth or storage in product formats, gut adhesion, cell wall synthesis and persistence in gut environment. polysaccharide production. Altered metabolic properties. Altered probiotic attributes. Modified health properties (including immunoregulatory, anticancer, gut health). Modified antibiotic resistance. Improved fermentation properties or other industrially useful processes. 15, 42, 138, 167, Altered cell wall or cell surface Homologue of penicillin-binding 108 232 characteristics, structures or protein 5 (Pbp5) also known as functions. muramoylpentapeptide Improved antimicrobial properties carboxypeptidase (EC 3.4.17.8, Modified adhesion to human or formerly EC 3.4.12.6). Pbp5 is a animal cells or cell lines. bacterial enzyme that requires a Production of desirable flavors. divalent cation for activity. Does not Modified flavor, aroma and/or texture cleave the C-terminal D-alanine from attributes. the reaction product, UDP-N-acetyl- Construction of genetic vectors for muramoyl-L-alanyl-D-g-glutamyl-6- controlled expression of RNA and/or carboxy-L-lysyl-D-alanine. protein, fusion protein production, Competitively inhibited by penicillins genetic modification, mutagenesis and cephalosporins. Penicillin-binding amplification of genetic material or proteins (PBPs), targets of beta-lactam for other genetic or protein antibiotics, are membrane-bound manipulations. enzymes essential for the biosynthesis Altered survival characteristics: of the bacterial cell wall. (Sifaoui et survival of industrial processes, al., Antimicrob. Agents Chemother. growth or storage in product formats, 45: 2594 2597, 2001). Utility in persistence in gut environment. immune modulation, gut adhesion, cell Altered metabolic properties. wall synthesis and polysaccharide Altered probiotic attributes. production. Modified health properties (including immunoregulatory, anticancer, gut health). Modified antibiotic resistance. Improved fermentation properties or other industrially useful processes. 16, 109 139, 233 Altered cell wall or cell surface Homologue of the hydrophobic characteristics, structures or transmembrane protein psaC. PsaC is a functions. member of the of ABC superfamily, Modified adhesion to human or involved in the transport of nutrients, animal cells or cell lines. translocation of signal molecules and Production of desirable flavors. chemotaxis (Janulczyk et al., Mol. Modified flavor, aroma and/or texture Microbiol. 34: 596 606, 1999). May be attributes. employed in immune modulation, gut Construction of genetic vectors for adhesion, cell wall synthesis, survival, controlled expression of RNA and/or and polysaccharide production. protein, fusion protein production, genetic modification, mutagenesis amplification of genetic material or for other genetic or protein manipulations. Altered survival characteristics: survival of industrial processes, growth or storage in product formats, persistence in gut environment. Altered metabolic properties. Altered probiotic attributes. Modified health properties (including immunoregulatory, anticancer, gut health). Modified antibiotic resistance. Improved antimicrobial properties. 17 140 Production of desirable flavors. Homologue of plnH. PlnH is the Modified flavor, aroma and/or texture accessory factor for ABC transporter attributes. plnG with strong similarities to the Construction of genetic vectors for proposed transport proteins of several controlled expression of RNA and/or other bacteriocins and to proteins protein, fusion protein production, implicated in the signal-sequence- genetic modification, mutagenesis independent export of E. coli amplification of genetic material or hemolysin (Huhne et al., Microbiol. for other genetic or protein 142: 1437 1448, 1996). LcnD is an manipulations. accessory protein of Lactococcus lactis Altered survival characteristics: with similarities to other proteins survival of industrial processes, involved in the secretion of various growth or storage in product formats, polypeptides. They operate in persistence in gut environment. conjunction with a protein from the Altered metabolic properties. family of ABC1 transporters. The Altered probiotic attributes. accessory proteins of Gram-negative Modified health properties (including bacteria are proposed to form a family immunoregulatory, anticancer gut of so-called membrane fusion proteins. health). It is hypothesized that they connect the Modified antibiotic resistance. inner and the outer membranes to Improved antimicrobial properties. facilitate the passage of substrates. CvaA, a member of the membrane fusion protein family, involved in the secretion of colicin V, has been shown to interact with both a cytoplasmic membrane protein (the ABC transporter) and a protein present in the outer membrane (Franke et al., J. Biol. Chem. 274: 8484 8490, 1999). May be employed as an antibacterial for control of infection and food preservation. 18, 121 141, 252 Homologue of Glyceraldehyde 3- phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) is a tetrameric NAD- binding enzyme common to both the glycolytic and gluconeogenic pathways that catalyzes reversibly the oxidative phosphorylation of D- glyceraldehyde 3-phosphate (G3P) to form 1,3-diphosphoglycerate (1,3- dPG) in the presence of NAD and inorganic phosphate. This enzyme is useful in manipulating alcohol dehydrogenation in vitro or in vivo, e.g. in fermentation processes or with transgenic bacteria with deleted, added or modified alcohol dehydrogenase gene. It can also be useful as a controlled expression vector. 19 142 Removal of undesirable flavor Homologue of the histidine-containing characteristics. protein ptsH, also known as the Production of desirable flavors. phosphocarrier protein HPr. Hpr is a Modified flavor, aroma, or texture component of the attributes. phosphoenolpyruvate-dependent sugar Construction of genetic vectors for phosphotransferase system (PTS), a controlled expression of RNA and/or major carbohydrate active-transport protein, fusion protein production, system. The phosphoryl group from genetic modification, mutagenesis phosphoenolpyruvate (PEP) is amplification of genetic material or transferred to the phosphoryl carrier for other genetic or protein protein HPr by enzyme I. Phospho- manipulations. HPr then transfers it to the permease Altered survival characteristics: (enzymes II/III). HPr is common to all survival of industrial processes, PTS and belongs to the HPr family. growth or storage in product formats, The HPr family consists of bacterial persistence in gut environment. proteins, all of which function as Modified carbohydrate levels or phosphoryl transfer proteins. They are functional properties. energy-coupling constituents of the Altered metabolic properties. phosphotransferase system (PTS) Modified carbohydrate metabolism. (TC # 4.A.1 4.A.6). which catalyzes sugar Altered probiotic attributes. uptake via a group translocation Improved fermentation properties or mechanism. The E. coli genome other industrially useful processes. encodes five HPr paralogues that Organisms or materials with function in PTS-related regulatory improved health properties (including capacities. May be employed in immunoregulatory, anticancer, gut survival and carbohydrate metabolism health, lactose tolerance) and as a controlled expression vector. 20 143 Removal of undesirable flavor Homologue of gamma (acyl carrier characteristics. protein) subunit of citrate lyase. Citrate Production of desirable flavors. lyase (EC 4.1.3.6.) is part of the citrate Modified flavor, aroma, or texture metabolism pathway and catalyzes the attributes. cleavage of citrate to oxaloacetate and Construction of genetic vectors for acetate and is composed of three controlled expression of RNA and/or subunits (alpha, beta, and gamma). protein, fusion protein production, Lactobacillae contribute through the genetic modification, mutagenesis citrate metabolism actively to the amplification of genetic material or flavor development of fermented dairy for other genetic or protein products (e.g., Dutch cheeses). It is manipulations. also involved in citrate metabolism Altered survival characteristics: pathway that results in lactic acid survival of industrial processes, production and acid tolerance (Magni growth or storage in product formats, et al., J. Bacteriol. 181: 1451 1457, persistence in gut environment. 1999) and may be employed in Modified carbohydrate levels or survival and carbohydrate metabolism. functional properties. Altered metabolic properties. Modified citrate metabolism. Altered probiotic attributes. Improved fermentation properties or other industrially useful processes. Organisms or materials with improved health properties (including immunoregulatory, anticancer, gut health, lactose tolerance) 20 144 Removal of undesirable flavor Homologue of beta (citryl-S-ACP characteristics. lyase) subunit of citrate lyase. Citrate Production of desirable flavors. lyase (EC 4.1.3.6.) is part of the citrate Modified flavor, aroma, or texture metabolism and catalyzes the cleavage attributes. of citrate to oxaloacetate and acetate Construction of genetic vectors for and is composed of three subunits controlled expression of RNA and/or (alpha, beta, and gamma). protein, fusion protein production, Lactobacillae contribute through

the genetic modification, mutagenesis citrate metabolism actively to the amplification of genetic material or flavor development of fermented dairy for other genetic or protein products (e.g., Dutch cheeses). It is manipulations. also involved in citrate metabolism Altered survival characteristics: pathway that results in lactic acid survival of industrial processes, production and acid tolerance (Magni growth or storage in product formats, et al., J. Bacteriol. 181: 1451 1457, persistence in gut environment. 1999) and may be employed in Modified carbohydrate levels or survival and carbohydrate metabolism. functional properties. Altered metabolic properties. Modified citrate metabolism. Altered probiotic attributes. Improved fermentation properties or other industrially useful processes. Organisms or materials with improved health properties (including immunoregulatory, anticancer, gut health, lactose tolerance). 20 145 Removal of undesirable flavor Homologue of alpha subunit characteristics. (citrate: acetyl-ACP transferase) of Production of desirable flavors. citrate lyase. Citrate lyase (EC 4.1.3.6.) Modified flavor, aroma, or texture is part of the citrate metabolism and attributes. catalyzes the cleavage of citrate to Construction of genetic vectors for oxaloacetate and acetate and is controlled expression of RNA and/or composed of three subunits (alpha, protein, fusion protein production, beta, and gamma). Lactobacillae genetic modification, mutagenesis contribute through the citrate amplification of genetic material or metabolism actively to the flavor for other genetic or protein development of fermented dairy manipulations. products (e.g., Dutch cheeses). It is Altered survival characteristics: also involved in citrate metabolism survival of industrial processes, pathway that results in lactic acid growth or storage in product formats, production and acid tolerance (Magni persistence in gut environment. et al., J. Bacteriol. 181: 1451 1457, Modified carbohydrate levels or 1999) and may be employed in functional properties. survival and carbohydrate metabolism. Altered metabolic properties. Modified citrate metabolism. Altered probiotic attributes. Improved fermentation properties or other industrially useful processes. Organisms or materials with improved health properties (including immunoregulatory, anticancer, gut health, lactose tolerance). 21, 119 146, 243 Removal of undesirable flavor Homologue of malic enzyme (EC characteristics. 1.1.1.39). Malic enzyme catalyzes L- Production of desirable flavors. malate oxidative decarboxylation and Modified flavor, aroma, or texture pyruvate reductive carboxylation and a attributes. malate transport protein (similar to Construction of genetic vectors for citP) involved in membrane potential controlled expression of RNA and/or generation via malate/lactate exchange. protein, fusion protein production, Because lactobacilli appear not to have genetic modification, mutagenesis a functioning Krebs cycle, this enzyme amplification of genetic material or may be involved in carbohydrate for other genetic or protein metabolism, amino acid biosynthesis manipulations. or L-malate utilization pathways. Altered survival characteristics: survival of industrial processes, growth or storage in product formats, persistence in gut environment. Modified carbohydrate levels or functional properties. Altered metabolic properties. Modified carbohydrate metabolism. Altered probiotic attributes. Improved fermentation properties or other industrially useful processes. Organisms or materials with improved health properties (including immunoregulatory, anticancer, gut health, lactose tolerance). 22 147 Construction of genetic vectors for Homologue of otsA,


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Linkgrinder is a free service that searches the Internet and indexes all files found so that you may search quickly and easily for shared files. These files are created and made available individually by users whose identity we are not aware of and who we have no control over. In essence we function like a search engine tool; these files ARE NOT STORED OR SERVED BY OUR NETWORK. We are not responsible for any materials obtained by using our service. We do not monitor any of the contents of these files. These files may contain viruses, illegal materials, materials inappropriate for minors, offensive files and the like. BY USING OUR SERVICE, YOU ASSUME FULL RESPONSIBILITY FOR DOWNLOADING THESE MATERIALS AND WILL INDEMNIFY US FOR ANY DAMAGES THAT MAY BE INCURRED.

For More Specific Information VIEW OUR TERMS OF SERVICE.

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