Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1997-05-05
2002-08-06
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S004000, C435S193000, C435S194000
Reexamination Certificate
active
06428971
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of cell biology, more specifically the teichoic acid pathway. Genes and proteins related to this pathway include: Teichoic Acid Polymerase (or TAP), and CDP-Glycerol:Poly(glycerophosphate) Glycerophosphotransferase.
INFORMATION DISCLOSURE
A. L. Honeyman, G. C. Stewart, “Identification of the protein encoded by rodC, a cell division gene from
Bacillus subtilis” Mol. Microbiol.
(1988) 2:735-741.
A. L. Honeyman, G. C. Stewart, “The nucleotide sequence of the rodC operon of
Bacillus subtilis. Mol. Microbiol.
(1989) 3:1257-1268.
C. Mauel, M. Young, P. Margot, D. Karamata “The essential nature of teichoic acids in
Bacillus subtilis
as revealed by insertional mutagenesis”
Mol. Gen. Genet.
(1991) 215:388-394.
C. Mauel, M. Young, D. Karamata, “Genes concerned with synthesis of poly(glycerol phosphate), the essential teichoic acid in
Bacillus subtilis
strain 168, are organized in two divergent transcription units”
J. Gen. Microbiol.
(1991) 137:929-941.
Y. S. Park, T. D. Sweitzer, J. E. Kison, C. Kent. “Expression, purification, and characterization of CTP:Glycerol-3-phosphate cytidyltransferase from
Bacillus subtilis.” J. Biol. Chem.
(1993) 268:16648-16654.
BACKGROUND OF THE INVENTION
The spread of antibiotic resistance in gram positive pathogenic bacteria is a serious problem which is only beginning to be registered in the clinic. The incidence of drug resistance is increasing—especially in
Staphylococcus aureus, Streptococcus pneumonia,
and the enterococci. Methicillin resistant
S. aureus
(MRSA), penicillin resistant
S. pneumoniae,
and vancomycin resistant enterococci, pose a serious threat to compromised patients. Vancomycin is the only antibiotic effective against MRSA. See, C. T. Walsh, “Vancomycin resistance: decoding the molecular logic”
Science
(1993) 261:308-309; I. R. Friedland, “Therapy of penicillin- and cephalosporin-resistant pneumococcal infections”
Trends Clinic Pract.
(1993) 25:451-455 and S. Dutka-Malen, and P. Courvalin, “Update on glycopeptide resistance in enterococci”
Antimicrob News
(1990) 7:81-88.
The cell wall teichoic acid pathway is found in the majority of gram positive bacteria, and studies with
Bacillus subtilis
have revealed that it is essential to cell viability. See, C. Mauel, M. Young, P. Margot, D. Karamata, “The essential nature of teichoic acids in
Bacillus subtilis
as revealed by insertional mutagenesis”
Mol Gen Genet
(1991) 215:388-394. The essential nature of cell wall teichoic acid may be due to the covalent attachment that it forms with peptidoglycan.
Cell wall teichoic acid, like peptidoglycan, is synthesized at the outer surface of the cell membrane using a nucleotide precursor (CDPglycerol) as the building block. Teichoic acid is a polymer of polyglycerolphosphate that is covalently attached to the peptidoglycan of gram positive bacteria. The enzyme CDP-Glycerol: Poly(glycerophosphate) glycerophosphotransferase catalyzes the polymerization of glycerolphosphate monomers from CDP-glycerol into a chain of polyglycerolphosphate linked via 1,3-phosphodiester bonds. Lipoteichoic acid is a related polymer of polyglycerolphosphate which is anchored to the cell membrane but is not attached to peptidoglycan.
There is an obvious clinical need for new antimicrobial agents which inhibit novel targets. In order to screen for unique inhibitors, essential metabolic pathways of gram positive pathogens, such as the cell wall teichoic acid pathway must be identified and their respective enzymes studied, cloned and made into useful assays and screens in order to identify novel antimicrobial agents.
SUMMARY OF THE INVENTION
This invention discloses a method of measuring and assaying the activity of the TAP enzyme. This invention also demonstrates how a common commercially available material may be used as a substrate for an important biological reaction that has previously had no substrate available for evaluating this reaction. This invention teaches the researcher and clinician that lipoteichoic can be used as a substrate to elucidate the presence and even the activity of the TAP enzyme. An embodiment of this invention is the application of this teaching to create an assay that enables one to monitor the activity of the TAP enzyme.
This invention also discloses, for the first time, the sequence of an active TAP enzyme and the nucleic acid sequence of the DNA that codes for this sequence.
This invention includes: the entire DNA sequence shown in FIG.
3
and Sequence Listing I.D. no. 1, and the DNA from residues 4 to 2274, first to last restriction site, and the DNA residues 24 to 2264. The coding DNA sequence shown in
FIG. 3
, alternatively named, “the rodC gene.” The DNA sequences corresponding to the sequence in
FIG. 3
where the residue at position 1872 is thymine in place of cytosine.
A bacterial DNA sequence that is capable of hybridizing to the DNA sequence of
FIG. 3
, under standard stringent conditions, to about 70 or more including, 75, 80, 85, 90, 95 or greater percent homology and having the ability to catalyze the reaction of CDP-glycerol plus H
2
O into teichoic or lipoteichoic acid.
The DNA sequence from
Staphylococcus aureus
that codes for the protein or protein sequence fragment from
Staphylococcus aureus
having at least 70% homology to related fragments described by FIG.
3
and
FIG. 4
, and that yield fragments of 7.0 kb, 5 kb, and 4.2 kb after EcoRI digest, or that yield fragments of 4.5, 3.3, 2.8 kb, after HindIII digest.
In addition to the DNA sequence, this invention describes various mutants, including: A collection of randomly mutated rodC genes. A selection of one or more randomly mutated rodC genes. A collection of bacteria having randomly mutated rodC genes. A selection of one or more bacteria having a random mutation selected from the collection of bacteria. The mutated bacteria selected from a mutant form of
B. subtilis
or
S. aureus.
Various proteins and peptide fragments from the expressed DNA are also described. The entire protein sequence shown in FIG.
3
and
FIG. 4
, Sequence I.D. NO. 2, the protein sequence from residues 1-746, and the protein sequence shown in FIG.
3
and
FIG. 4
where valine is the amino acid at position 616 in place of alanine. Also described are the protein sequence fragment from
Staphylococcus aureus
having at least 70% homology to related fragments described by FIG.
3
and
FIG. 4
, that yield fragments of 7.0 kb, 5 kb, and 4.2 kb after EcoRI digest; and the protein sequence fragment from
Staphylococcus aureus
having at least 70 % homology to related fragments described by FIG.
3
and
FIG. 4
that yield fragments of 4.5, 3.3, 2.8 kb, after HindIII digest. The protein disclosed in the Southern Blot shown in
FIG. 2
is described as well.
In addition to the DNA and proteins disclosed herein, this invention comprises various intermediates, intermediate vectors, plasmids and transformed or mutated cell lines. This invention comprises the DNA of the sequence disclosed in
FIG. 3
incorporated into a vector selected from a cloning vector, a shuttle vector or an expression vector, any of these vectors may be plasmid vectors. The cloning vector or plasmid can be selected from any widely available or commercially available plasmids. The plasmid can be any suitable pUC type or pBR type of plasmid, such as pUC18, or pUC19, or any other suitable plasmid such as pBR322. The vector may be a typical shuttle, vector The shuttle vector may be a plasmid such as, pMK4, or pYL112&Dgr;119. An expression vector may also he used, the expression vector is a plasmid with a very strong promoter, such as the following very strong promoters: pTrc99A, pDR540, or pET-21(+). In this nomenclature pTRC99A would be the name of the plasmid. Each plasmid used for expression of proteins has a unique promoter as follows: pTRC99A (trc promoter), pDR540 (tac promoter), pET-21(+) (T7 promoter).
Examples of plasmids would be a plasmid named pRODCAP18 comprising the cloned rodC gene, placed into the cloning vector, pUC18, th
Morin Sara E.
Shinabarger Dean L.
Swaney Steven M.
Meuting Raasch & Gebhardt, P.A.
Pharmacia & Upjohn Company
Slobodyansky Elizabeth
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