Antisense RNA expression strategies effective against...

Details Antisense RNA expression strategies effective against... Antisense RNA expression strategies effective against...

2000-08-21

2004-02-03

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

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

Bacteria or actinomycetales; media therefor

C435S006120, C435S320100, C536S023100, C536S024100, C536S024500

Reexamination Certificate

active

06686192

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to bacteriophage resistant recombinant bacteria and their use in fermentation.
BACKGROUND OF THE INVENTION
The dairy industry has harnessed certain members of the heterogeneous group of lactic acid bacteria, including the thermophilic bacterium
Streptococcus thermophilus,
as starter cultures or culture adjuncts to drive the lactate fermentations in the manufacture of a variety of fermented products. This organism grows best at the high end of the mesophilic range, about 42-25° C., thus it survives and produces acid at temperatures higher than can be tolerated by the mesophilic lactic acid bacteria. This characteristic makes
S. thermophilus
useful in the fermentation of dairy products, such as yogurt and Swiss and Italian cheeses, that are ordinarily manufactured or incubated at elevated temperatures.
Pasteurized milk, the primary substrate for fermented products, and in some instances the starter cultures themselves, have been be shown to be a natural reservoir for virulent bacteriophages capable of infecting and, inevitably, lysing the starter culture(s) during product manufacture (Bruttin et al,
Appl. Environ. Microbiol.
63:3144-3150 (1997); Moineau et al.,
J Dairy Sci.
79:2104-2111 (1996)). Depending upon the severity and temporal progression of the lytic infection, the concomitant loss of fermentative capacity associated with starter culture lysis can significantly retard or halt batch fermentations; thereby inflicting upon the dairy industry significant losses of time and production capital each year. With the advent of biotechnology, the dairy industry is seeking to identify novel phage defense strategies capable of extending the utility of industrial starter cultures.
Expression of antisense RNA silences gene expression in vivo through the formation of a double stranded target mRNA::antisense RNA duplexes (Inouye, M.
Gene
72:25-34 (1988)). Duplex formation is believed to interfere with protein translation by (i) masking the ribosome binding site (RBS), which prevents ribosome loading, and/or (ii) destabilizing the mRNA by targeting it for RNase-mediated degradation (Inouye, M.
Gene
72:25-34 (1988)). In theory, the best candidates for effective silencing by means of antisense RNA strategies will be genes that are (i) essential for bacteriophage maturation, (ii) transiently expressed and/or coded for by unstable mRNA species, (iii) expressed at low levels and/or expressed early, (iv) inefficiently translated, and (v) coded for by mRNA species that form secondary structures that are conducive to recognition of the antisense RNA molecule.
SUMMARY OF THE INVENTION
A first aspect of the present invention is an antisense oligonucleotide and a nucleic acid encoding an antisense oligonucleotide that binds to a bacterial cell bacteriophage RNA and inhibits the replication of that bacteriophage in a host bacterial cell (e.g.,
Streptococcus thermophilius
). For example, the RNA targeted by the antisense oligonucleotide may be mRNA encoding a bacteriophage protein, such as a phage helicase or a phage primase. The present invention may be carried out with any bacteriophage, including cos-type and pac-type bacteriophage. The antisense oligonucleotide is preferably at least 8 nucleotides in length. Examples include antisense oligonucleotides that comprise a continuous fragment at least 8 nucleotides in length, in antisense orientation, of the sequences given herein as SEQ ID NO: 1 or SEQ ID NO: 2.
A second aspect of the present invention is a construct comprising a promoter that is operably associated with the oligonucleotide described above. The promoter regulates transcription of the oligonucleotide in the antisense orientation, such that a sufficient amount of antisense RNA is transcribed to block translation of the phage-encoded replication machinery. Preferably the promoter regulates transcription of the antisense RNA constitutively.
A third aspect of the present invention comprises a bacterial cell, preferably
S. thermophilus,
which harbors a recombinant DNA vector containing an oligonucleotide as which encodes an antisense oligonucleotide as described above.
A fourth aspect of the present invention involves a recombinant nucleic acid vector comprising a bacteriophage origin of replication (ori) operatively associated with a nucleic acid sequence that blocks translation of phage-encoded replication machinery. When a bacterial cell, harboring the nucleic acid vector, is infected with phage, the nucleic acid vector exponentially replicates to increase the dose of antisense RNA.
These aspects are more completely described hereinbelow. In addition, other aspects of the present invention not explicitly set forth herein will become apparent to those skilled in the art.


REFERENCES:
patent: 5538864 (1996-07-01), Hill et al.
Sturino, Joseph M., et al.,Expressioni of Antisense RNA Targeted against Streptococcus thermophilus Bacteriophages, Applied and Environmental Microbiology,vol. 68, No. 2, pp. 588-596 (Feb. 2002).
Excerpt, Ph.D. Thesis of Shirley Walker, pp. 50-51 (1999).
Sturino, Joseph M., et al.,Construction of Effective Antisense Phage Defense Strategies for Lactic Acid Bacteria, Molecular Genetics of Bacteria&Phages(2000).
Sturino, Joseph M., et al.,Targeting Phage DNA Replication Functions with Antisense RNA, Molecular Genetics of Bacteria&Phages(2000).
Walker, Shirley A., et al,Molecular Characterization of a Phage-Inducible Middle Promoter and Its Transcriptional Activator from the Lactococcal Bacteriophage &phgr;31, Journal of Bacteriology,vol. 180, No. 4, pp. 921-931 (Feb. 1998).
International Search Report, International Application No. PCT/US01/26033 dated Feb. 21, 2002.
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Bruttin, Anne, et al.,Molecular Ecology of Streptococcus thermophilus Bacteriophage Infections in a Cheese Factory, Applied and Environmental Microbiology,vol. 63, No. 8, pp. 3144-3150 (1997).
Foley, Sophie, et al.,A Short Noncoding Viral DNA Element Showing Characteristics of a Replication Origin Confers Bacteriophage Resistance to Streptococcus thermophilus, Virology,vol. 250, pp. 1-11 (1998).
Stanley, Elizabeth, et al.,Identification of four loci isolated from two Streptococcus thermophilus phage genomes responsible for mediating bacteriophage resistance, FEMS Microbiology Letters,vol. 182, pp. 271-277 (2000).
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Schleifer, Karl Heinz, et al.,Revival of the Species Streptococcus thermophilus(ex Orla-Jensen, 1999)nom. rev., System Appl. Microbiol.,vol. 14, pp. 386-388 (1991).
Le Marrec, Claire, et al.,Two Groups of Bacteriophages Infecting Streptococcus thermophilus Can Be Distinguished on the Basis of Mode of Packaging and Genetic Determinants for Major Structural Proteins, Applied and Environmental Microbiology,vol. 63, No. 8, pp. 3246-3253 (Aug. 1997).
Bull, J.J., et al.,Viral escape from antisense RNA, Molecular Microbiology,vol. 28, No. 4, pp. 835-846 (1998).
Coleman, Jack, et al.,A novel immune system against bacteriophage infection using complementary RNA(micRNA),Nature,vol. 315, pp. 601-603 (Jun. 1985).
Hill, C., et al.,Future Prospects for Culture Improvement, Dairy Starter Cultures,pp. 249-255.
Walker, Shirley A., et al.,An Explosive Antisense RNA Strategy for Inhibition of a Lactococcal Bacteriophage, Applicated and Environmental Microbiology,vol. 66, No. 1, pp. 310-319 (Jan. 2000).
Zirnstein, Gerald, et al.,Streptococcus thermophilus, Streptococcus,pp. 2127-2133.

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