Staphylococcus strains comprising an inducible gene encoding...

Details Staphylococcus strains comprising an inducible gene encoding... Staphylococcus strains comprising an inducible gene encoding...

2000-02-10

2002-09-17

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

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

Bacteria or actinomycetales; media therefor

C435S471000, C435S477000, C435S481000, C536S023100, C536S024100

Reexamination Certificate

active

06451582

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to genetically engineered organisms useful for screening pharmaceutical compounds having antimicrobial capability and, more particularly, to recombinant organisms in which the expression of an RNA polymerase specificity factor can be regulated. The present invention is more specifically directed to recombinant bacterial strains in which the expression of RNA polymerase &sgr; subunit can be regulated by the addition of an exogenous effector molecule. In particular, the present invention relates to a new recombinant staphylococcal bacterial strain having an engineered inducer-responsive gene for regulating expression of RNA polymerase a subunit and methods for utilizing the same in high throughput screening to detect antimicrobial compounds pharmaceutically useful against staphylococcal bacteria.
BACKGROUND OF THE INVENTION
Numerous pathogenic organisms, such as
Staphylococcus aureus
(“
S. aureus
”), are responsible for infectious disease and health-related problems in humans and other animals throughout the United States and the world. As treatments are developed for combating a particular organism, such as treatments incorporating newly developed antibiotics and chemical compounds effective at eliminating existing strains of a particular organism, newer strains of such organisms emerge which are resistant to the existing treatments. Accordingly, there remains a constant need for the development of new ways for pharmaceutically combating pathogenic organisms.
Methods for combating an organism by interfering with genetic processes essential to survival and growth of the organism are becoming of increasing interest. In particular, researchers are directing their attention to chemical compounds which interfere with genetic transcription processes required for growth or continued existence of pathogenic organisms.
The expression of genetic information in an organism ultimately occurs via proteins, and particularly by enzymatic proteins which catalyze metabolic reactions. The flow of genetic information from DNA to protein occurs generally in two steps, termed “transcription” and “translation”. Transcription is the first step in the flow of genetic information, whereby DNA-encoded genetic information is copied into RNA. The further conversion of RNA into protein occurs by the process of translation. A “gene” is broadly a region of DNA which encodes one protein, and the transcription-translation process of forming that protein is termed “expression” of the gene.
In particular, transcription involves the synthesis of an RNA chain representing one strand of a DNA duplex. Importantly, RNA synthesis is catalyzed by an enzyme known as RNA polymerase. Transcription begins when RNA polymerase binds to a special region at the beginning of a DNA gene known as a promoter site.
RNA polymerase is generally comprised of two components, a core enzyme and a specificity factor. The specificity factor is particularly concerned with recognition and binding of the enzyme to the promoter region of a particular gene or set of genes on a DNA template. While the core enzyme component of RNA polymerase has the ability to synthesize RNA on a DNA template, it cannot initiate transcription at the promoter site without an associated specificity factor. The function of the specificity factor, thus, is to insure that RNA polymerase binds in a stable manner to DNA only at appropriate promoter sites. Consequently, the specificity factor directs binding of RNA polymerase at cognate promoter sequences, thereby initiating expression of only those selected genes incorporating the cognate promoter sequences.
Accordingly, the expression of a particular gene or set of genes can be controlled by regulating production of a corresponding specificity factor. In particular, the expression of a particular gene or set of genes can be inhibited by blocking the associated specificity factor, thereby preventing the binding of RNA polymerase to the gene promoter sequences. As a result, an attractive target for the treatment of a pathogenic organism would be the discovery of chemical agents which block an RNA polymerase specificity factor required for the expression of a gene or set of genes essential for continued existence of the organism.
One particular pathogenic organism of concern is the bacterium
S. aureus
, which is an opportunistic human pathogen and is the primary cause of nosocomial bacterial infections in the United States.
S. aureus
is associated with a number of life threatening systemic illnesses, such as bacteremia/sepsis, toxic shock syndrome and toxic epidermal necrolysis, as well as common bacterial infections of the skin. The recent emergence of methicillin-resistant and vancomycin-resistant strains of
S. aureus
has focused renewed attention on the need for development of new classes of antibiotics to combat such bacterial strains. A promising way of pharmaceutically combating bacterial strains, including
S. aureus
and other staphylococcal strains, is to interfere with genetic transcription processes relating to growth of the bacteria.
As in other eubacteria, the RNA polymerase of
S. aureus
is composed of two components, the core enzyme and a specificity factor. The core enzyme has a subunit composition of (&agr;
2
&bgr;&bgr;′. The &bgr; and &bgr;′ subunits together make up the catalytic center of the enzyme while the &agr; subunit is required for assembly of the core enzyme, as well as other functions in promoter recognition.
The specificity factor in
S. aureus
is one of several &sgr; subunits or factors. The principle &sgr; factor, encoded and expressed in
S. aureus
by the chromosomal gene plaC, is required for the expression of essential housekeeping genes required for bacterial growth. As a result, this &sgr; factor is an attractive target for the discovery of chemical agents which possess antibacterial properties by binding with or blocking the &sgr; factor function, since the blocking of or interfering with the &sgr; factor function can prevent expression of these housekeeping genes and retard or prevent growth of the bacteria.
U.S. Pat. Nos. 5,585,277 and 5,679,582 to Bowie et al. disclose methods for screening chemical compounds for potential pharmaceutical or antimicrobial effectiveness. In particular, these patents teach methods for identifying possible therapeutic ligands which bind to target proteins. The methods of these patents may be useful in affinity-based assays for the initial identification of chemical compounds as in vitro inhibitors of an RNA polymerase and more specifically an RNA polymerase specificity factor, such as the primary &sgr; subunit in staphylococcal bacteria. However, there remains a need in the art for screening methods, and recombinant organisms useful therein, which monitor the in vivo effects of chemical compounds on the growth and/or survival of a target organism. Specifically, there remains a need in the art for recombinant organisms, and production methods therefor, in which the production of an RNA polymerase specificity factor which encodes a gene essential for growth or continued existence of the organism can be specifically controlled for use in screening processes. In particular, there remains a need in the art for a staphylococcal bacterial strain in which the production of RNA polymerase &sgr; subunit can be specifically controlled for use in screening methods. Moreover, there is a need in the art for specific methods and compositions that allow the identification of antimicrobial agents which interact with and/or modify the function of the RNA polymerase &sgr; subunit thereby inhibiting bacterial growth.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide an organism in which the production of an RNA polymerase specificity factor can be controlled.
It is a further object of the present invention to provide a recombinant organism having a regulatable gene which encodes an RNA polymerase specificity factor required for expression of a gene

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