Acid-inducible promoters for gene expression

Details Acid-inducible promoters for gene expression Acid-inducible promoters for gene expression

2000-08-11

2001-06-05

LeGuyader, John L. (Department: 1635)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S252300, C435S252900, C435S320100, C435S471000, C536S023100, C536S024100

Reexamination Certificate

active

06242194

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns promoter elements in general, along with DNA constructs comprising such promoters operably associated with a DNA in a recombinant DNA molecule, host cells containing the same, and methods of using the same.
BACKGROUND OF THE INVENTION
The gastrointestinal tract is the most densely colonized region of the human body (Savage,
Ann. Rev, Microbiol.
31, 107 (1977); Tannock, Normal microflora (Chapman and Hall, London 1995)) and the accumulated evidence indicates that this collection of microbes has a powerful influence on the host in which it resides. Comparisons between germ free and conventional animals have shown that many biochemical, physiological and immunological functions are influenced by the presence of the diverse and metabolically active bacterial community residing in the gastrointestinal tract (Marteau and Rambaud,
FEMS Microbiol. Rev.
12, 207 (1993); Norin et al.,
Appl. Environ. Microbiol.
74, 1850 (1991); Tannock, supra). Lactobacilli are important residents of the microflora (Ahrne et al.,
J. Appl. Microbiol.
85, 88 (1998); Kimura et al.,
Appl. Environ. Microbial.
63, 3394 (1997)), and have been the subject of intense and growing interest because of their possible role in the maintenance of gastrointestinal health (Bengmark,
Gut
42, 2 (1998)). Of immense importance to lactobacilli functioning in this role is the ability to endure in the harsh conditions of the gastrointestinal tract, where the gastric pH frequently falls below 2.0 in healthy individuals (McLauchlan et al.,
Gut
30, 573 (1998)).
Changes in extracellular pH have been shown to influence the expression of a variety of genes from many different bacteria (reviewed in Olson,
Mol. Microbiol.
8, 5 (1993)). In the presence of a low external pH (<3.5),
L. acidophilus
is able to maintain cytoplasmic pH at values close to neutral (Kashket,
FEMS Microbiol. Rev.
46, 233 (1987)). However, the mechanisms by which
L. acidophilus
responds and adapts to extremely acidic conditions remain poorly defined, For several organisms that inhabit the gastrointestinal tract, the F
1
F
0
-ATPase is an important element in the response and tolerance to low pH. In the fermentative bacterium,
Enterococcus
(En.)
hirae,
maintenance of cytoplasmic pH has been shown to occur via amplification of the proton translocating ATPase (Kobayashi et al.,
J. Bacteriol.
158, 1157 (1984); Kobayashi et al.,
J. Biol. Chem.
261, 627 (1986)). Similarly, a short exposure of
Salmonella typhimurium
to pH 6.0 induces the synthesis of the F
1
F
0
-ATPase (Foster and Hall,
J. Bacteriol.
173, 5129 (1990); Foster and Hall,
J. Bacteriol.
172, 771 (1990)). Nanen and Hutkins (
J. Dairy Sci.
74, 747 (1991)) have demonstrated that the specific activity of H
+
-ATPases from several lactic acid bacteria increases as the extracellular pH moves from neutral to 5.0. Alternatively, changes in environmental pH appear to have little influence on the expression of the atp operon, whose genes code for the various subunits of the H
+
-ATPase, in
Escherichia coli
(Kasimoglu et al.,
J. Bacteriol.
178, 5563 (1996)). Likewise, expression of the atp operon of
Bacillus subtilis
appears to be constitutive (Santana et al.,
J. Bacteriol.
176, 6802 (1994)).
The identification of conditionally expressed genes provides a wealth of insight into the physiological consequences of and responses to a given stimulus. In the case of
L. acidophilus,
a significant challenge remains in understanding the intestinal roles and activities of this organism. An important element in this regard is the determination of which characteristics are important for the survival and success of this organism in the gastrointestinal tract. While differential display (Liang and Pardee,
Science
257, 967 (1992); Welsh et al.,
Nucleic Acids Res.
20, 4965 (1992)) has been used extensively to identify conditionally expressed genes in eukaryotes, the application of this methodology in prokaryotes has not been explored to a comparatively significant extent (Abu Kwaik and Pederson,
Mol. Microbiol.
21, 543 (1996); Fislage,
Electrophoresis
19, 613 (1998); Fislage et al.,
Nucleic Acids Res.
25, 1830 (1997); Wong and McClelland,
Proc. Natl. Acad. Sci. USA
91, 639 (1994); Zhang and Normark,
Science
273, 1234 (1996)). Practical problems with the method are presented by: the relatively large proportion of structural RNA species in the total RNA; the low level of polyadenylation of mRNA (Sarkar,
Ann. Rev. Biochem.
66, 173 (1997)), which prohibits the use of 3′ dT anchored primers; and the structural instability and short half life of low abundance mRNA species of prokaryotes as compared to eukaryotes (Higgins et al.,
Curr. Opin. Genet. Dev.
2, 739 (1992)).
SUMMARY OF THE INVENTION
A first aspect of the present invention is an isolated polynucleotide encoding an acid-inducible, or acid-responsive, promoter element. The promoter element may be:
(a) DNA having the sequence:
CGGTACTAAG TAAACACCTT TTCACAAAAA ATATTTACTC TAATGCGCTT TCATTTTACA CAAAGAAGAT ATTTGGTGTT AAGATGATTT ACGTGTTCGA GTTTTATTCA ACACGAGAAG GGAGGTCACG AAGTA (SEQ ID NO: 1), comprising the F
1
F
0
-ATPase promoter of
Lactobacillus acidophilus;
or
(b) DNA that hybridizes to DNA of (a) above (e.g., under stringent conditions) and encodes an acid-inducible promoter.
Preferably, the promoter is a proton (H
+
) translocating ATPase promoter, and preferably the promoter is an F-type ATPase promoter.
The acid responsive promoter of the invention has applications in a number of scenarios. The promoter may be used for the expression of gene products during the normal course of fermentation by cells such as bacterial cells, particularly lactic acid bacteria, in dairy, meat, vegetable, cereal, and other bioconversions. The promoter may be used for the induction of gene products upon exposure of lactic acid bacteria to acid environments, including suspension into acidified foods or entry into the gastrointestinal tract or other body cavities as a probiotic bacteria.
The promoter may be used for the acid-responsive expression of enzymes, vaccines, proteins, peptides, etc. from lactic acid bacteria intended to serve as delivery or production systems. Specific examples include: Acid-induced expression of lactase expression from Lactobacillus species in the stomach or gastrointestinal tract (GIT) to facilitate lactose digestion; Acid-induced expression of vaccines from Lactobacillus or Lactococcal species in the appropriate body cavity in order to promote immunological responses and oral-induced mucosal immunity; acid-induced expression of holins/lysins to promote bacteria cell lysis or permeability and, thereby, release any of the above compounds into food, bioreactors, or the GIT of man or animals.
Accordingly, a second aspect of the invention is a recombinant DNA molecule comprising a promoter operably associated with a DNA of interest, wherein said promoter is an acid inducible promoter as described above. The DNA of interest may encode a protein or peptide, the production of which is upregulated upon induction, may encode an antisense oligonucleotide that causes the downregulation of a different gene upon induction of the acid inducible promoter, may encode a ribozyme, etc.
A third aspect of the invention is a vector (e.g., a plasmid) comprising a recombinant DNA molecule as described above.
Further aspects of the invention include a method of transforming a cell comprising providing a vector as described above and then transforming said cell with said vector, along with recombinant DNA molecules as described above. Where the DNA of interest is to be transcribed within the cell, the cell may be one in which the promoter is operable (that is, induced by acid pH as described herein, or simply constitutively active). The DNA of interest may be from a different organism than the host cell (that is, a heterologous DNA of interest), or may be from the same organism as is the host cell, although in a recombinant DNA molecule (in which case the reco

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