Microorganisms and assays for the identification of antibiotics

Details Microorganisms and assays for the identification of antibiotics Microorganisms and assays for the identification of antibiotics

2001-03-20

2004-12-14

Housel, James (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C424S009200, C514S001000, C530S300000, C530S350000, C435S007100

Reexamination Certificate

active

06830898

ABSTRACT:

BACKGROUND OF THE INVENTION
Antimicrobial or antibiotic treatment is a well-accepted therapy for fighting microbial infections that takes advantage of the existence of biological processes that are unique to bacteria or fungi, that can be safely inhibited to the detriment of the bacteria, without producing undesired or harmful side effects in the individual receiving such therapy. However, due at least in part to the continual evolution of microbial resistance to the available classes of antibiotics, and in part to the recent slowdown in the introduction of novel antimicrobials to market, there exists a need for the development of screening assays that target previously unexploited biochemical systems in microbes. In particular, there exists the need for the identification of new bacterial targets for use in drug discover programs designed to identify agents having potential use as anti-infective agents with novel modes of actions.
SUMMARY OF THE INVENTION
The present invention is based at least in part, on the identification of a novel target for use in screening assays designed to identify antimicrobial agents. In particular, the present invention is based on the identification and characterization of a previously unidentified microbial pantothenate kinase gene, coax. The coaX gene was first identified in
B. subtilis
where it is one of two genes encoding functional pantothenate kinase. Initially the present inventors identified and cloned the
B. subtilis
coaA gene (previously termed yqjS) that encodes a pantothenate kinase homologous to the CoaA enzyme previously characterized in
E. coli
. A second gene (previously termed yacB) has also been identified and cloned by the present inventors that is not homologous to any previously described pantothenate kinase. This latter pantothenate kinase-encoding gene has been renamed coaX. The coax gene could be deleted from
B. subtilis
strains with an intact coaA gene, but it could not be deleted from a strain containing a deletion in the coaA gene, indicating that the coaX gene is not essential in
B. subtilis
strains with a wild-type coaA gene. Homologs of the coaX gene can be found in a number of bacterial species, including but not limited to
Aquifex aeolicus, Bacillus anthracis, Bacillus halodurans, Bacillus stearothermophilus, Caulobacter crescentus, Chlorobium tepidum, Clostridium acetobutylicum, Dehalococcoides ethenogenes, Deinococcus radiodurans, Desulfovibrio vulgaris, Geobacter sulfurreducens, Pseudomonas putida, Rhodobacter capsulatus, Thiobacillus ferrooxidans, Streptomyces coelicolor, Synechocystis
sp.,
Thermotoga maritima, Bordetella pertussis, Borrelia burgdorferi, Campylobacter jejuni, Clostridium difficile, Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae, Porphyromonas gingivalis, Pseudomonas aeruginosa, Pseudomonas syringae
pv tomato,
Treponema pallidum, Xylella fastidiosa
and
Mycobacterium tuberculosis
. More importantly, however, this novel pantothenate kinase gene has been found to be the sole essential pantothenate kinase in troublesome pathogens including, but not limited to,
Bordetella pertussis, Borrelia burgdorferi, Campylobacter jejuni, Helicobacter pylori, Neisseria meningitidis, Pseudomonas aeruginosa, Treponema pallidum
and
Xylella fastidiosa
. Accordingly, the coaX gene represents an attractive target for screening for new antibacterial compounds to combat these pathogenic microorganisms, particularly microorganisms in which coaX is the sole pantothenate kinase-encoding gene.
Accordingly, the present invention features isolated CoaX proteins, in particular, proteins encoded by the coax gene in bacteria. The invention also features isolated nucleic acid molecules and/or genes, e.g., bacterial nucleic acid molecules and/or genes, in particular, isolated bacterial coaX nucleic acid molecules and/or genes. Also featured are vectors that contain isolated coaX nucleic acid molecules and/or genes as well as mutant coaX nucleic acid molecules and/or genes. Also featured are recombinant microorganisms (e.g., microorganisms belonging to the genus
Escherchia
or
Bacillus
, for example,
E. coli
or
B. subtilis
) containing isolated coaX nucleic acid molecules and/or genes or mutant coaX nucleic acid molecules and/or genes of the present invention. In particular, the invention features recombinant microorganisms that produce the CoaX proteins of the present invention, e.g., pantohthenate kinase proteins encodes by the coaX nucleic acid molecules and/or genes of the present invention.
Also featured are methods for identifying CoaX modulators utilizing, for example, isolated CoaX proteins of the present invention or recombinant microorganisms expressing the CoaX proteins of the present invention.
Other features and advantages of the invention will be apparent from the following detailed description and claims.


REFERENCES:
patent: 2002/0160456 (2002-10-01), Kleanthous et al.
patent: 2002/0164588 (2002-11-01), Eisenberg et al.
patent: WO 01/21772 A2 (2001-03-01), None
patent: WO 01/49721 (2001-07-01), None
DeShazer et al., Journal of Bacteriology, vol. 177(13), pp. 3801-3807 (Jul. 1995).*
Printout of SEQ ID NO: 74 from U.S. patent application Publication 2002/0160456.*
Printouts of search reports for U.S. application-09-813-453A-xx.rapb.*
Scott et al., Nature Genetics, 21:440-443 (1999).*
Skolnick et al., Trends in Biotechnology, 18(1): 34-39 (2000).*
Peter Bork, Genome Research, 10: 398-400 (2000).*
U.S. patent application Ser. No. 09/667569, Yocum, et al., filed Sep. 2000.
Calder et al. “Cloning and characterization of a eukaryotic pantothenate kinase gene (panK) fromAsperigillus nidulans.” (1999)J. Biol. Chem. 274:2014-2020.
DeShazer et al. “Identification of aBordetella pertussisregulatory factor required for transcription of the pertussis toxin operon inEscherichia coli.” J. Bacteriol. (Jul. 1995) 177(13):3801-7.
Dunn et al. “Isolation of temperature-sensitive pantothenate kinase mutants ofSalmonella typhimuriumand mapping of thecoaAgene.” (1979)J. Bacteriol. 140:805-808.
Flamm et al. “The nucleotide sequence of theEscherichia coli rtsgene.” (1988) Gene (Amst.) 74:555-558.
GenBank™ Accession No. AAB64970; Ydr531wp; CAI: 0.14 [Saccharomyces cerevisiae].
Rock et al. “Pantothenate kinase regulation of the intracellular concentration of coenzyme A.” (2000)J. Biol. Chem. 275:1377-1383.
Song et al. “Cloning, sequencing, and expression of the pantothenate kinase (coaA) gene ofEscherichia coli.” (1992)J. Bacteriol. 174:6411-6417.
Trias et al. “Innovative approaches to novel antibacterial drug discovery.”Curr. Opin. Biotechnol. 1997 Dec;8(6):757-62.
Vallari et al. “Isolation and characterization of temperature-sensitive pantothenate kinase (coaA) mutants ofEscherichia coli.” (1987)J. Bacteriol. 169:5795-5800.
Wood et al. “The Bvg accessory factor (Baf) enhances pertussis toxin expression inEscherichia coliand is essential forBordetella pertussisviability.”FEMS Microbiol. Lett. (Dec. 1, 2000) 193(1):25-30.
Wood et al. “The effect of the Bvg accessory factor (Baf) on the expression of pertussis toxin inBordetella pertussisandEscherichia coliand on the viability ofB. pertussis.” Abstracts of the General Meeting of the American Society for Microbiology May 1997; Abstr. B-349.
EMBL Acc. No: U12020 forBordetella pertussisBvg accessory factor (baf) gene, complete cds.

Affiliated with

Also associated with

Rating

Microorganisms and assays for the identification of antibiotics does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Microorganisms and assays for the identification of antibiotics, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Microorganisms and assays for the identification of antibiotics will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3307727