Staphylococcal GTPase obg nucleotide sequence encoding...

Details Staphylococcal GTPase obg nucleotide sequence encoding... Staphylococcal GTPase obg nucleotide sequence encoding...

2001-02-23

2004-03-16

Graser, Jennifer E. (Department: 1645)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069300, C435S320100, C435S325000, C435S362000, C435S365000, C435S367000, C435S252300, C435S252330, C435S069700, C536S023700

Reexamination Certificate

active

06706495

ABSTRACT:

FIELD OF THE INVENTION
The present invention generally relates to nucleotide sequences that encode proteins which are essential for bacterial growth. More particularly, the present invention is directed to a GTPase obg gene encoding a GTP-binding protein in Staphylococcal bacterial strains. Specifically, the present invention is directed to a Staphylococcus aureus (“
S. aureus
”) obg gene that is capable of expression in a host cell to produce enzymatically functional
S. aureus
GTP-binding protein. Additionally, the present invention pertains to recombinant expression vectors incorporating the GTPase obg gene of the present invention. The present invention is further directed to Staphylococcal GTP-binding protein, methods for producing GTP-binding protein, and methods for using GTP-binding protein as a novel thereapeutic target in affinity-based pharmacological screening procedures for the discovery of antibiotics active against
S. aureus
and other Staphylococcal bacteria.
BACKGROUND OF THE INVENTION
Numerous pathogenic organisms 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 continual need for the development of new ways for pharmaceutically combating pathogenic organisms.
One particular organism of concern is the bacterium
S. aureus
, which is an opportunistic human pathogen both in the community and in hospitals, and is the primary cause of nosocomial bacterial infections in the United States.
S. aureus
has a highly invasive nature and 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. Once the organism enters the bloodstream, patients are at risk of developing serious diseases such as endocarditis, osteomyelitis, and septic shock.
Despite the development and use of newer antimicrobial agents to combat
S. aureus
infections, the morbidity and mortality from serious
S. aureus
infections remain high. One reason is that
S. aureus
is adept in developing resistance to multiple antibiotics. The recent emergence of methicillin-resistant and vancomycin-resistant strains of
S. aureus
in Japan, and subsequently in the United States, has further highlighted the importance of finding alternative approaches to the prevention and treatment of Staphylococcal infections, and has focused renewed attention on the need for development of new classes of antibiotics to combat such bacterial strains. Despite the imminent crisis in
S. aureus
antibiotic resistance, the identification of novel targets for the development of novel antimicrobial agents remains elusive.
One promising way of pharmaceutically combating bacterial strains, including
S. aureus
and other Staphylococcal strains, is to interfere with genetic processes relating to growth and/or viability of the bacteria. Methods for combating organisms 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 that interfere with such processes.
A potential target for use with screening processes to identify chemical compounds that are useful in combating pathogenic organisms is a GTPase superfamily of GTP(guanosinetriphosphate)-binding proteins that includes G-proteins, elongation factors in
E. coli
, mammalian Ras, and procaryotic proteins such as Era, FtsZ, and Fth, etc. These GTPase regulatory molecules are classified as belonging to the GTPase superfamily due to a common ability to bind guanine nucleotides and hydrolyse GTP. March, “Membrane-Associated GTPases in Bacteria”, Molec. Microbiol., Vol. 6, pp. 1253-57, 1992.
GTP-binding proteins are important signaling molecules in bacteria as well as in eukaryotic cells. GTP-binding proteins have been recognized for many years as components of signal transduction pathways in eukaryotes. Only recently, however, has it been discovered that prokaryotes contain GTP-binding proteins that are essential for growth and/or viability of the organism. The involvement of these bacterial proteins in signal transduction in prokaryotes, however, is still not entirely clear.
One member of this superfamily of GTP-binding proteins which is of particular interest is the protein expressed by the obg gene (short for spo
OB
-associated
G
TP-binding protein). The obg gene specifically encodes a GTP-binding protein which is essential for bacterial growth and which is structurally conserved across an extraordinarily wide range of bacterial species. Obg was initially identified as a gene dowstream of the stage 0 sporulation gene spoOB in Bacillus subtilis in 1989. Trach et al., “
The Bacillus subtilis spoOB Stage
0
Sporulation Operon Encodes An Essential GTP
-
Binding Protein
” J. Bacteriol., Vol. 171, pp. 1362-71, 1989. Transcription analysis of this operon revealed that spoOB and obg are cotranscribed.
Various observations have been made about the Obg protein in certain organisms. Obg in
Bacillus subtilis
has been shown to bind GTP by the cross-linking method. Trach et al., supra.
Bacillus subtilis
Obg has also been characterized by its enzymatic activity with respect to GTP hydrolysis. Welsh et al., “Biochemical Characterization of the Essential GTP-Binding Protein Obg of
Bacillus subtilis
”, J. Bacteriol., Vol. 176, pp. 7161-68, 1994. It has also been demonstrated that Obg plays a crucial role in sporulation induction in
Bacillus subtilis
and
Streptomyces griseus
. Kok et al., “Effects on
Bacillus subtilis
of a Conditional Lethal Mutation in the Essential GTP-Binding Protein Obg”, J. Bacteriol., Vol. 176, pp. 7155-60, 1994; Okamoto et al., “Molecular Cloning and Characterization of the obg Gene of
Streptomyces griseus
in Relation to the Onset of Morphological Differentiation”, J. Bacteriol., Vol. 179, pp. 170-79, 1997.
Very little is known, however, about the physiological function of Obg. Obg homologs have recently been discovered in a diverse range of organisms ranging from bacteria to archaea to humans, and the evolutionary conservation between distantly related species suggests that this family of GTP-binding proteins has a fundamental, but unknown, cellular function. It has been proposed that, by monitoring the intracellular GTP pool size, Obg is involved in sensing changes in the nutritional environment leading ultimately to morphological differentiation. Okamoto et al., supra.
Obg is a unique GTPase in that it possesses an extended N-terminal glycine-rich domain not found in eukaryotic or archaeal homologs. An isolated
Bacillus subtilus
temperature-sensitive obg mutant was found to carry two closely linked missense mutations in the N-terminal domain, suggesting that this portion of obg is essential for cellular function. Kok et al., supra.
Very little is known about the essential functions of Obg, however. To date, Obg has been validated to be essential for growth in both Gram-negative bacteria (
E. coli, Caulobacter crescentus
) and Gram-positive bacteria (
Bacillus subtilis
). Maddock et al., “Identification of an Essential
Caulobacter crescentus
Gene Encoding a Member of the Obg Family of GTP-Binding Proteins”, J. Bacteriol., Vol. 179, pp. 6426-31, 1997; Arigoni et al., “A Genome-Based Approach for the Identification of Essential Bacterial Genes”, Nature Biotechnology, Vol. 16, pp. 851-56, 1998; Trach et al., supra. In addition, depletion of Obg has been shown to cause cessation of
Bacillus subtilis
cell growth. Vidwans et al., “Possible Role for the Essential GTP-Binding Protein Obg in Regulating the Initiation of Sporulation in
Bacillus subtilis
”, J. Bacter

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