Polynucleotide encoding a Lactobacillus gasseri...

Details Polynucleotide encoding a Lactobacillus gasseri... Polynucleotide encoding a Lactobacillus gasseri...

2001-05-21

2003-12-16

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C536S023200, C536S023700, C435S320100, C435S201000, C435S419000, C435S325000, C435S254110, C435S252900, C435S455000, C435S468000, C435S471000

Reexamination Certificate

active

06664097

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns &bgr;-glucuronidase (GUS) proteins, DNA encoding the same, and methods of use thereof.
BACKGROUND OF THE INVENTION
&bgr;-Glucuronidase protein (GUS) and the gene encoding this protein (gusA) are widely used as reporter genes and proteins in molecular biology. Bacterial &bgr;-glucuronidase activity has been considered for many years to be almost unique to
Escherichia coli
and closely related Enterobacteriaceae (Wilson et al. (1992) The
Escherichia coli
gus operon: induction and expression of the gus operon in
E. coli
and the occurrence and use of GUS in other bacteria. In. S. R. Gallagher (ed.), GUS Protocols: using the GUS gene as a reporter of gene expression. Academic Press, San Diego, Calif.). However, evidence has slowly been accumulating to indicate that &bgr;-glucuronidase activity can also be found in a limited number of other bacteria, particularly gram-positive inhabitants of the GI tract (Akao (2000)
Biol. Pharm. Bull.
23:149-154; Akao (2000)
Biol. Pharm. Bull.
22:80-82; Hawkesworth et al. (1971)
J. Med. Microbiol.
4:451-459; McBain and Macfarlane (1998)
J. Med. Microbiol.
47:407-415). The gusA gene can also be found in Shigella species but activity is absent in many of the common, agriculturally-important bacterial species, such as Rhizobium, Agrobacterium, and Pseudomonas (GUS Protocols, 7-17 (S. Gallagher Ed. 1992)).
Lactobacillus gasseri
ADH is a human intestinal isolate that was identified by its ability to adhere to intestinal epithelial cells (Kleeman and Klaenhammer (1982)
J. Dairy Sci.
65:2063-2069).
L. gasseri
is one of a number of indigenous lactobacilli that are commonly associated with the microflora of a healthy human GI tract (Molin et al. (1993)
J. Appl. Bacteriol.
74:314-323; Song et al. (2000)
FEMS Microbiol. Lett.
187:167-173). A number of these lactobacilli are currently under investigation to determine the mechanistic basis of a variety of proposed probiotic activities (Klaenhammer (1998)
Int. Dairy J.
8:497-506). It remains an important objective to characterize the physiological and enzymatic activities of this group of organisms and ultimately to identify the genetic factors responsible for those activities. Studies with various Lactobacillus species, including
L. gasseri
, have consistently shown their ability to reduce the amount of fecal &bgr;-glucuronidase activity and lower the occurrence of cancer indicators present in the GI tract (de Roos and Katan (2000)
Am. J. Clin. Nutr.
71:405-411; Jin et al. (2000)
Poult. Sci
79:886-891; Ling et al. (1992)
Ann. Nutr. Metab.
36:162-166; McConnell and Tannock (1993)
J. Appl. Bacteriol.
74:649-651; Pedrosa et al. (1995)
Am. J Clin. Nutr.
61:353-359). The mechanisms by which lactobacilli lower the amount of &bgr;-glucuronidase activity in the gut remain unknown but may be the reflection of a variety of activities including, but not limited to, the exclusion or antagonism of typically &bgr;-glucuronidase-positive enterobacteria. Because lactobacilli colonize the proximal region of the small intestine, it is reasonable to expect them to be frequently exposed to &bgr;-D-glucuronides excreted via bile into the GI tract. Indeed, their frequent exposure to bile is reflected in the common occurrence of conjugated bile acid hydrolysis among different species (Christiaens et al. (1992)
Appl. Environ. Microbiol.
58:3792-3798; Elkins and Savage (1998)
J. Bacteriol.
180:4344-4349). Lactobacilli themselves have not traditionally been associated with &bgr;-glucuronidase activity, however, and there have been, to date, only two reports of &bgr;-glucuronidase-like activity in lactobacilli (McConnell and Tannock (1993)
J. Appl. Bacteriol.
74:649-651; Pham et al (2000)
Appl. Environ. Microbiol.
66:2302-2310). It has been unclear, however, whether this &bgr;-glucuronidase activity was the result of a true &bgr;-glucuronidase enzyme or reflected the activity of some other enzyme.
A disadvantage of currently available GUS proteins is that they have limited activity in acidic pH environments. Since acidic pH environments characterize a variety of industrial fermentation processes in which current GUS proteins cannot be be effectively used, it would be extremely useful to have new GUS proteins that operate at an acidic pH.
SUMMARY OF THE INVENTION
Accordingly, the invention provides isolated polynucleotides encoding the protein beta-glucuronidase (GUS), and which are preferably operable at a pH of less than 7 (e.g., are operable at a pH of 4 or 5). The polynucleotide sequence may be selected from the group consisting of:
(a) DNA having the nucleotide sequence given herein as SEQ ID NO:1 (which encodes the protein having the amino acid sequence given herein as SEQ ID NO:2);
(b) polynucleotides (e.g., cDNAs) that hybridize to DNA of (a) above (e.g., under stringent conditions) and which encode the protein &bgr;-glucuronidase (GUS); and
(c) polynucleotides that differ from the DNA of (a) or (b) above due to the degeneracy of the genetic code, and which encode the protein encoded by a DNA of (a) or (b) above.
The present invention further provides vector (e.g., an expression vector) containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence is contained within a host cell.
The invention further provides a protein or fragment thereof encoded by a polynucleotide as given above (e.g., the protein provided herein as SEQ ID NO: 2). Such proteins may be isolated and/or purified in accordance with known techniques.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding GUS under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
The invention also provides an antibody (e.g., a polyclonal antibody, a monoclonal antibody) which specifically binds to a protein as given above.


REFERENCES:
Akao, Taiko,Purification and Characterization of Glycyrrhetic Acid Mono-glucuronide &agr;-D-Glucuronidase in Eubacterium sp. GLH, Biol. Pharm. Bull., vol. 22, No. 1, pp. 80-82 (1999).
Akao, Taiko,Competition in the Metabolism of Glycyrrhizin with Glycyrrhetic Acid Mono-Glucuronide by Mixed Eubacterium sp. GLH and Ruminococcus sp. PO1-3, Biol. Pharm. Bull., vol. 23, No. 2, pp. 149-154 (2000).
De Roos, Nicole M., et al.,Effects of probiotic bacteria on diarrhea, lipid metabolism, and carcinogenesis: a review of papers published between 1988 and 1998, Am. J. Clin. Nutr., vol. 71, pp. 405-411 (2000).
Jin, L.Z., et al.,Digestive and Bacterial Enzyme Activities in Broilers Fed Diets Supplemented with Lactobacillus Cultures, Poultry Science, vol. 79, No. 6, pp. 886-891 (2000).
Klaenhammer, Todd R.,Functional Activities of Lactobacillus Probiotics: Genetic Mandate, Int. Dairy Journal, vol. 8, pp. 497-505 (1998).
Kleeman, E.G., et al.,Adherence of Lactobacillus Species to Human Fetal Intestinal Cells, J. Dairy Sci., vol. 65, No. 11, pp. 2063-2069 (1982).
McBain, A. J., et al.,Ecological and physiological studies on large intestinal bacteria in relation to production of hydrolytic and reductive enzymes involved in formation of genotoxic metabolites, J. Med. Microbiol., vol. 47, pp. 407-416 (1998).
McConnell, M.A., et al.,A note on lactobacilli and &agr;-glucuronidase activity in the intestinal contents of mice, Journal of Applied Bacteriology, vol. 74, pp. 649-651 (1993).
Pham, P.L., et al.,Production of Exopolysaccharide by Lactobacillus rhamnosus R and Analysis of Its Enzymatic Degradation during Prolonged Fermentation, Applied and Environmental Microbiology, vol. 66, No. 6, pp. 2302-2310 (Jun. 2000).
Wilson, Kate J., et al.,The Escherichia coli gus Operon: Induction and Expression of the gus Operon in E. coli and the Occurrence and Use of GUS in

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