Title of Invention	"A METHOD OF SCREENING ANTI-TUBERCULAR COMPOUND".
Abstract	The present invention discloses a use of Mycobacterium smegmatis as a surrogate host to study hypoxia responsive promoters of Mycobacterium tuberculosis and more particularly the invention is directed to a rapid method of screening anti-tubercular compound.

Full Text

Field of the present invention The present invention relates to a use of Mycobacterium smegmatis as a surrogate host to study hypoxia-responsive promoters of Mycobacterium tuberculosis. Also, it relates to identification of candidate antimycobacterial compounds and their effect on promoter activity and bacterial viability, under both hypoxic and aerobic conditions. Background of the present invention Tuberculosis continues to be the leading cause of death in the world due to infection with a single microbial agent. About one in every three individuals worldwide is infected with M. tuberculosis but the vast majority of these individuals do not show any symptoms of the disease and act as latent carriers. These persistent or latent bacilli act as reservoirs of the disease and help in its transmission to non-infected individuals. The current anti-tubercular drugs target actively replicating bacilli and therefore persistent bacteria are refractory to the action of conventional antitubercular drugs. The identification of novel gene targets is a focus of active research worldwide with the objective of targeting the persistent bacteria. The screening methods currently in use employ aerobic cultures of M. tuberculosis and hence are unlikely to identify compounds active against persistent bacteria. The additional disadvantage of M tuberculosis - based assays is that it is a slow growing pathogen and requires specialized laboratories and dedicated equipment. Evidence from many sources suggest that non-replicative persistence of M. tuberculosis is triggered by hypoxic or anaerobic conditions and / or nutrient limitation that is likely to prevail within granulomas (Wayne and Sohaskey, 2001). Thus hypoxia is one of the important signals regulating M. tuberculosis gene expression. In a recent study it was found that the expression of >100 genes is modulated when M. tuberculosis is subjected to defined hypoxic conditions (Sherman et al., 2001). Among the genes that are upregulated is a two-component system devR-devS. Since two-component systems are known to play a key role in adaptation of bacteria to different environmental stimuli, therefore the upregulation of this system under hypoxia suggested that this system was essential for adaptation of M. tuberculosis to hypoxia. The genes encoding the DevR-DevS two-component system (Rv3133c-Rv3132c) were first identified and partially characterized in M. tuberculosis in the inventor's laboratory (Dasgupta et al., 2000). The enhanced expression of devR-devS two-component system of M tuberculosis during hypoxia is most likely the result of enhanced transcription from its promoter(s). Earlier studies have shown that Rv3134c, a gene that is located upstream of devR is co-transcribed with devR and devS (Dasgupta et al., 2000). This . indicated that the common promoter responsible for transcription of all three genes is located upstream of Rv3134c. Since M. tuberculosis genes are often transcribed by multiple promoters hence additional promoter elements could be present upstream of devR gene also. DevR was recently shown to play a crucial role in hypoxia viability and in the hypoxia response (Boon et al, 2001; Park et al., 2003). DevR-DevS/Rv2027c were recently established as a bonafide two-component system of M. tuberculosis (Saini et al. 2004 a,b; Roberts et al. 2004). Selected compounds were demonstrated to inhibit DevS/Rv2027c phosphorylation in vitro (Tyagi and Saini, US and PCT patents applied). M. tuberculosis being a slow growing pathogen is relatively difficult to handle and hence M. smegmatis, a fast growing non-pathogenic species of the same genus is often used to study those physiological aspects of M tuberculosis that are common to both species. The physiological behaviour of M smegmatis under hypoxic conditions is very much similar to that of M tuberculosis (Dick et al., 1998). Also, the highly conserved homologues of many of the M. tuberculosis hypoxia-responsive genes such as devR-devS are present in M. smegmatis (Tyagi and Sharma, 2002, Zahrt and Deretic, 2001) and show upregulation under hypoxic conditions (Mayuri et al., 2002). Noting the similarities in gene expression during hypoxia response and the conservation of transcription machinery (Bashyam et al. 1996) in the two species it seemed appropriate to use M. smegmatis as a surrogate host to study the activity of M. tuberculosis devR-devS promoter. To assess the activity of M. tuberculosis devR promoter(s) in M smegmatis the Rv3134c- and devR- upstream regions were therefore cloned into a reporter plasmid vector and electroporated into M. smegmatis. The M. smegmatis cultures containing these constructs were then assessed for promoter activity under both hypoxic as well as aerobic conditions. The M. smegmatis model can be used to study hypoxia-responsive gene activity and evaluate the efficacy of candidate compounds in blocking hypoxia-modulated gene expression. Reporter assays: The use of reporter technology offers tremendous advantages in estimating the regulatory potential of a DNA fragment (even when nothing else is known about it) by linking it to genes whose products are stable and easily assayable. Many different reporter systems have been developed to study gene expression in mycobacteria including P-galactosidase (lacZ), Chloramphenicol Acetyltransferase (cat) , Alkaline phosphatase (phoA), Catechol 2,3-Dioxygenase (xylE), luciferase or Lux and GFP (Tyagi et al, 2000). Among these the lacZ encoded P-galactosidase reporter system has been used extensively because it is simple to detect and to assay. Resazurin-based assays: Resazurin (Alamar Blue) oxidation-reduction dye is a general indicator of cell growth and / or viability; the blue, non-fluorescent oxidized form becomes pink and fluorescent upon reduction (Collins and Franzblau, 1997). Growth can therefore be measured with a fluorimeter or spectrophotometer or determined by a visual colour change. Resazurin reduction is being evaluated as a sensitive, rapid, inexpensive alternative to the current methods for drug susceptibility determination (Collins and Franzblau, 1997; Franzblau et al. 1998; Bastian et al. 2001; Palomino et al. 2002; Martin et al. 2003). This rapid method has potential for application in high throughput format for screening novel antimycobacterial compounds (Collins and Franzblau, 1997). Objects of the present invention The main object of the present invention is to develop a simple and fast process for evaluating promoter activity of M. tuberculosis promoters in hypoxic conditions using M.smegmatis as a surrogate host. Another main object of the present invention is to develop a method of identifying anti-tubercular molecules. Yet another object of the present invention is to use M. smegmatis as a model for studying hypoxia- responsive promoters of M tuberculosis. Yet another object of the present invention is to show that the promoter(s) of the devR-devS locus of M tuberculosis are active and upregulated under hypoxic conditions in M. smegmatis also. Still another object of the present invention is to analyze the effect of different compounds on its activity (or that of any other hypoxia-responsive promoter) in a whole cell assay system. Still another object of the present invention is to demonstrate the utility of the M smegmatis whole cell assay system to evaluate the different compounds on hypoxic gene expression and bacterial viability. Still another object of the present invention is to study suitability of M. smegmatis for these assays that (a) screen for compounds that interfere with the hypoxia- responsiveness of the devR-devS promoter and (b) identify inhibitors of mycobacterial viability under hypoxic and aerobic conditions. Still another object of the present invention is to demonstrate that inhibitors of in vitro phosphorylation of histidine kinases do not inhibit activity in a non specific manner by inducing aggregation of the histidine protein kinases DevS or Rv2027c. Brief description of the accompanying drawings Fig.l. Map of pDK16 reporter plasmid. pDK16 vector containing the promoterless lacZ reporter gene and the kanamycin resistance cassette. The att-int region which enables it to undergo homologous recombination with the mycobacterial genome is shown. The unique restriction sites upstream of lacZ reporter gene which can be used for cloning are indicated. Due to presence of colEl origin of replication pDK16 can also be propagated in E. coli. A transcriptional terminator, TER 4, derived from fd phage is present downstream of lacZ to prevent read though of transcription into downstream regions. Fig. 2. Schematic representation of the devR-devS locus in M. tuberculosis. Solid black arrows represent the regions of M. tuberculosis genome used for promoter studies. A 701-bp fragment (nt 3500655 to 3501355, Cole et al., 1998) was PCR-amplified from M. tuberculosis DNA using R7 and R9c primers and cloned into pDK16 to generate pDP3. Similarly, a 1.45-kb fragment (nt 3499904 to 3501355, ref. Cole et al., 1998) was amplified using primers R7 and R3 and cloned to generate pDP2 Fig. 3. Growth curve of M smegmatis. A typical growth curve of M smegmatis culture transformed with pDK16 is shown. Viability (cfu ml-1) and A590 of aerobic and hypoxia adapted cultures are shown. 'f' and 'd' indicate fading and decolourisation of methylene blue dye at ~24h and ~48h respectively. Fig. 4. p-galactosidase activity of M smegmatis cultures carrying pDP2 and pDP3 grown under aerobic and hypoxic conditions. Activities are the average of at least 3 independent experiments and are represented after subtraction of activities obtained with vector control. T and 'd' refer to fading and decolorization of the redox indicator dye, methylene blue. Fig. 5. RT-PCR performed with RNA isolated from M. smegmatis cultures carrying pDP2 construct. R8 and R9c primers map in the Rv3135- Rv3134c intergenic region of M. tuberculosis. Lane 1, aerobic culture; lane 2, hypoxic culture; +, DNA positive control. Fig. 6. ß-galactosidase activity of M smegmatis cultures carrying pDP2w and pDP3w grown under aerobic and hypoxic conditions. Activities are the average of at least 3 independent experiments and are represented after subtraction of activities obtained with vector control, 'f and 'd' refer to fading and decolorization of the redox indicator dye, methylene blue. Fig. 7. RT-PCR performed with RNA isolated from M. smegmatis cultures carrying pDP2w construct. R8 and R9c primers map in the Rv3135- Rv3134c intergenic region of M. tuberculosis. Lane 1, aerobic culture; lane 2, hypoxic culture; +, DNA positive control The sequences mapping upstream of devR and Rv3134c were likely to contain promoter(s) driving transcription of these genes. The upstream regions of devR and Rv3134c were therefore PCR-amplified and cloned into the integration-proficient reporter plasmid vector pDK16 (Fig. 1, Jain et al, 1997, Tyagi, A.K. and Kaushal, D., unpublished results), to generate pDP3 and pDP2 (Fig. 2). Fig. 8. Effect of BPB on promoter activity and viability of M. smegmatis carrying the Rv3135-2 promoter construct under hypoxic and aerobic conditions of growth. % promoter activities under hypoxia (panel A) and aerobic conditions (panel D) are represented after subtraction of activities obtained with vector control. Viability status of M. smegmatis assessed as % reduction in CFU/ml under hypoxia (panel B) and aerobic conditions (panel E); and as % reduction in RFU under hypoxia (panel C) and aerobic condition (panel F) are presented. RFU refers to relative fluorescence units obtained after reduction of the redox dye resazurin. Fading and decolorisation of the redox dye methylene blue was observed at ~24h and ~48h respectively. Fig. 9. Effect of 2-MBI on promoter activity and viability of M. smegmatis carrying the Rv3135-2 promoter construct under hypoxic and aerobic conditions of growth. % promoter activities under hypoxia (panel A) and aerobic conditions (panel D) are represented after subtraction of activities obtained with vector control. Viability status of M. smegmatis assesse'd as % reduction in CFU/ml under hypoxia (panel B) and aerobic conditions (panel E); and as % reduction in RFU under hypoxia (panel C) and aerobic condition (panel F) are presented. RFU refers to relative fluorescence units obtained after reduction of the redox dye resazurin. Fading and decolorisation of the redox dye methylene blue was observed at ~24h and ~48h respectively. Fig. 10. BPB concentration - dependent inhibition of autophosphorylation of DevSioi and effect of BPB on protein aggregation. A. Autoradiogram of SDS-PAGE profile of DevSaoi autophosphorylation reaction products. B. Silver stained SDS-PAGE profile of cross- linked fractions which show that the addition of BPB does not cause aggregation of the sensor kinase. C. Silver stained SDS-PAGE profile of non cross-linked fractions. Fig. 11. 2-MBI concentration - dependent inhibition of autophosphorylation of DevSaoi and effect of 2-MBI on protein aggregation. A. Autoradiogram of SDS-PAGE profile of DevS2oi autophosphorylation reaction products. B. Silver stained SDS-PAGE profile of cross- linked fractions which show that the addition of 2-MBI does not cause aggregation of the sensor kinase. Fig. 12. Silver stained SDS-PAGE profile of cross- linked fractions which show that the addition of BPB does not cause aggregation of the Rv2027194. Fig. 13. Silver stained SDS-PAGE profile of cross- linked fractions which show that the addition of 2-MBI does not cause aggregation of the Rv2027194. Statement of Invention Accordingly, the present invention relates to a method of screening anti-tubercular compound, said method comprising the steps: a. constructing plasmid such as herein described by cloning Rv3134c and devR upstream regions from DNA of Mycobacterium tuberculosis under transcriptional control of divergent promoters P Rv3134-devR and PRVSUS optionally with a reporter gene, b. electroporating the plasmid into an electro competent Mycobacterium smegmatis, c. culturing the Mycobacterium smegmatis under hypoxic and/or aerobic conditions such as herein described and in presence and/or absence of test compound, and d. determining anti-tubercular activity of test compound in terms of down regulation or up-regulation of Rv3J34c and devR promoter activity as herein described, wherein down-regulation is indicative of the test compound being an anti-tubercular compound. Detailed description of the present invention Accordingly, the present invention relates to a simple and fast process for evaluating promoter activity of 'persistent' M. tuberculosis in hypoxic conditions using M. smegmatis as a surrogate host, said process comprising steps of: a. cloning Rv3134c- and devR- upstream regions from DNA of M. tuberculosis in an integration-proficient reporter plasmid vector to generate constructs, b. electroporating the constructs into electrocompetent M. smegmatis, c. culturing the electroporated M. smegmatis under hypoxic condition, and d. evaluating up-regulated promoter activity using a reporter gene. In an embodiment of the .present invention, the divergent reporter plasmid vector is pDK16. In another embodiment of the present invention, the promoters express genes from a group comprising Rv3134c, devR and devS from the complementary DNA strand, and Rv3135 from the direct DNA strand. In yet another embodiment of the present invention, the divergent promoters are P*V.J/.? dtevR and PRv3135- In still another embodiment of the present invention, the constructs are pDP3, pDP2, pDP3w, and pDP2w. In still another embodiment of the present invention, the reporter gene is selected from a group comprising p-galactosidase (lacZ), Chloramphenicol Acetyltransferase (cat), alkaline phosphatase (phoA), Catechol 2,3- dioxygenase (xylE), luciferase [lux} and GFP. In still another embodiment of the present invention, the reporter gene is P-galactosidase (lacZ). In still another embodiment of the present invention, the promoters are upregulated under hypoxic conditions. In still another embodiment of the present invention, said process helps determine the efficacy of candidate compounds in blocking hypoxia-modulated gene expression. In still another embodiment of the present invention, culturing in Dubos medium with about 0.2% Tween-80, about 0.05% glycerol (DTG medium) supplemented with about 10% ADC and kanamycin at about 20 µg.g/ml. In still another embodiment of the present invention, a method of identifying anti-tubercular molecules, said method comprising steps of: • cloning Rv3134c- and devR- upstream regions from DNA of M tuberculosis in an integration-proficient reporter plasmid vector to generate constructs, • electroporating the constructs into electrocompetent M. smegmatis, • culturing electroporated M smegmatis under hypoxic condition, • evaluating M. smegmatis viability in both presence and absence of anti- tubercular test compound, using resazurin reduction test, and • identifying anti-tubercular compounds. In still another embodiment of the present invention, anti-tubercular molecules are selected from a group comprising bromophenol blue (BPB) and 2-mercaptobenzimidazole (2-MBI). The slow growth and pathogenicity of M. tuberculosis often poses a problem for scientists who want to study various physiological aspects of this organism. This has prompted the use of M smegmatis as a surrogate host for studying those physiological aspects of M. tuberculosis that are common to both species. The physiological behaviour of M. smegmatis cultures is notably similar to that of M. tuberculosis under hypoxic conditions. Moreover the devR-devS and Rv3134c genes of M smegmatis too are upregulated under hypoxic conditions just as their counterparts in M. tuberculosis. The similarity in sequences and hypoxic response of these genes suggest that the molecular mechanism underlying the dormancy response is conserved in the two species. This invention describes cloning of the M. tuberculosis Rv3134c-devR-devS and its divergent promoter (Rv3135-2) into an integration-proficient, reporter plasmid pDK16, its introduction into the rapid growing non pathogenic mycobacterium M smegmatis, culture under aerobic and hypoxic conditions and measurment of (3-galactosidase activities in M. smegmatis cultures carrying these constructs. Activities were low under aerobic conditions but were upregulated many folds under hypoxia. At least two M. tuberculosis promoters mapping in the Rv3134c-devR-devS locus are faithfully regulated in M. smegmatis. The use of resazurin, a redox dye, in monitoring M smegmatis viability has also been demonstrated. Two compounds bromophenol blue (BPB) and 2-mercaptobenzimidazole (2-MBI) shown earlier to be inhibitors of DevR-DevS two-component system in vitro phosphorylation assays were assessed in M smegmatis for their effect on the hypoxia-responsive Rv3135-2 promoter and on culture viability under aerobic and hypoxic conditions. The induction in promoter activity under hypoxic conditions and bacterial viability (assessed by cfu and resazurin reduction) were inhibited in the presence of the compounds. The magnitude of resazurin reduction mirrored the viability as determined by the classical cfu assay. Therefore M. smegmatis is a suitable culture system for evaluating the efficacy of candidate compounds in blocking hypoxia-modulated gene expression and interfering with mycobacterial viability. Cross-linking experiments with purified proteins in the presence of BPB and 2-MBI show the effect of the inhibitors on autophosphorylation to be specific and not due to compound-mediated aggregation of the proteins. The slow growth and pathogenicity of M. tuberculosis often poses a problem for scientists who want to study various physiological aspects of this organism. This has prompted the use of M smegmatis as a surrogate host for studying those physiological aspects of M. tuberculosis that are common to both species. The physiological behaviour of M smegmatis cultures is notably similar to that of M. tuberculosis under hypoxic conditions. Moreover the devR-devS and Rv3134c genes of M smegmatis too are upregulated under hypoxic conditions just as their counterparts in M. tuberculosis. The similarity in sequences and hypoxic response of these genes suggest that the molecular mechanism underlying the dormancy response is conserved in the two species. This invention describes cloning of the M. tuberculosis Rv3134c-devR-devS promoter into an integration-proficient, reporter plasmid pDK16, it introduction into the rapid growing non pathogenic mycobacterium M smegmatis, culture under aerobic and hypoxic conditions, measurement of p-galactosidase activities of M smegmatis cultures carrying these constructs. Activities were low under aerobic conditions but were upregulated many folds under hypoxia. The promoter-fusion experiment described here establish that the reported hypoxia-dependent upregulation of Rv3J34c, devR and devS genes is due to induction of at least two M. tuberculosis promoters that are recognized and regulated in M. smegmatis and hence M smegmatis can serve as a suitable host for studying hypoxia-responsive promoters of M. tuberculosis. It has promise for evaluating the efficacy of candidate compounds in blocking hypoxia-modulated gene expression. In an embodiment of the present invention, wherein a simple and fast process for evaluating promoter activity of persistent M. tuberculosis in hypoxic conditions and also mycobacterial viability using M. smegmatis as a surrogate host, said process comprising steps of: o cloning Rv3134c- and devR- upstream regions from DNA of M.tuberculosis in an integration-proficient reporter plasmid vector to generate constructs, o electroporating the constructs into electrocompetent M. smegmatis, o culturing the electroporated M. smegmatis under hypoxic condition, and o evaluating promoter activity using a reporter gene. o evaluating mycobacterial viability using classical colony counts. o developing the resazurin reduction test to monitor M. smegmatis viability and activity of antimycobacterial compounds especially under hypoxic conditions o demonstrating the inhibitory action of compounds bromophenol blue and 2-mercaptobenzimidazole on reporter gene activity and viability In another embodiment of the present invention, wherein the reporter plasmid vector is pDK16. In yet another embodiment of the present invention, wherein the promoters express genes from a group comprising Rv3134c, devR and devS from the complementary DNA strand, and Rv3135 from the direct DNA strand. In still another embodiment of the present invention, wherein the constructs are pDP3, pDP2, pDP3w, and pDP2w. In still another embodiment of the present invention, wherein the reporter gene is selected from a group comprising ß-galactosidase (lacZ), Chloramphenicol Acetyltransferase (cat), alkaline phosphatase (phoA), Catechol 2,3- dioxygenase (xylE), luciferase (lux) and GFP. In still another embodiment of the present invention, wherein the reporter gene is p- galactosidase (lacZ). In still another embodiment of the present invention, wherein the promoters are upregulated under hypoxic conditions. In still another embodiment of the present invention, wherein said process helps determine the efficacy of candidate compounds in blocking hypoxia-modulated gene expression. In still another embodiment of the present invention, wherein PRV3134c-devR and PRV3135 are divergent promoters, both being induced under hypoxic conditions. Their induction leads to increased synthesis of p-galactosidase. Therefore this divergent promoter set can be used to simultaneously overexpress two proteins under hypoxic conditions. This can be achieved by placing their coding regions along with suitable translation recognition sequences downstream of the two divergent promoters. Moreover, this promoter set is an example. Such an approach is likely to succeed with other hypoxia- responsive promoters placed divergently in mycobacterial genomes including that of M tuberculosis. In still another embodiment of the present invention, wherein the promoters described herein are examples. Any hypoxia-responsive promoter of mycobacteria including M. tuberculosis can be utilized in the M. smegmatis system for basic studies and drug screening using whole-cell assays. In still another embodiment of the present invention, wherein the lacZ is reporter gene in this example. Any other reporter can be utilized, e.g. GFP. (A). Cloning of putative promoter regions into reporter plasmid vector: All routine recombinant DNA work was performed as described (Sambrook and Russell, 2001). M. tuberculosis H37Rv DNA was prepared by boiling cells in the presence of 0.1% Triton X-100 and the supernatant was used as a source of template DNA in PCR. (B). Electroporation of M. smegmatis: Preparation of electrocompetent cells: 100 ml of M. smegmatis (LR222) culture was grown in Middlebrook 7H9 media supplemented with ADC to an A590 of- 0.6. Cells were checked for purity by Ziehl-Neelsen staining and harvested at 5000 rpm at RT for 15 mins. The pellet was washed twice with 50 ml of 10% glycerol and twice with 25 ml of 10 % glycerol. Finally the pellet was resuspended in 1 ml of 10 % glycerol. 50-100 µ1 aliquots were subjected to slow freezing at-70°C. Electroporation: An aliquot of electrocompetent cells (~ 50 µl) was thawed at room temperature and 0.5 to 1 µg of DNA was added to the cells and gently mixed. The tubes were incubated at room temperature for 5 mins and then transferred to a 2 mm gap cuvette. The mix was pulsed at 2.5 kV /cm and a resistance of 125 Ω using a BTX electroporator (Electrocell manipulator 600). After pulsing, cells were recovered in Middlebrook 7H9 + 10 % ADC at 37°C overnight followed by plating on Middlebrook 7H10 agar plates containing Kanamycin (20 µg /ml) and cycloheximide (50 µg /ml). Plates were incubated at 37 °C for 3-4 days. (C). M. smegmatis culture conditions: Mycobacterial glycerol stocks were maintained at -70°C. Primary cultures were obtained by inoculating 1 % (v/ v) glycerol stock of the desired culture in Dubos medium supplemented with 10 % Albumin Dextrose complex (ADC) containing Kanamycin (20 )µg/ml) and Cycloheximide (50 µg/ml) and incubating it under shaking conditions for ~ 48 hours till A590 of 0.8 - 1.0 (log phase) was reached. The growth medium used to obtain primary cultures in the antimycobacterial,susceptibility studies, was Dubos medium with 0.2% Tween-80, 0.05% glycerol (DTG medium) supplemented with 10% ADC and kanamycin (20 µg/ml). The culture was fully aerated as head to air space ratio (HSR) of 4 was maintained. All primary cultures were checked to be free of contamination by Ziehl Neilsen (acid fast) staining. The primary culture was then vigorously vortexed with 5-7 sterile glass beads (3-4 mm diameter) in order to break the clumps, left undisturbed for 10 mins to allow the clumps to settle down and the homogeneous culture suspension at the top was used to inoculate a secondary culture for (i) assessment of lacZ promoter activity in aerobically grown cells (0 day or baseline) and (iii) determination of cell viability (cfu plating and resazurin reduction assay). (D). Establishment of a model for dormancy induced by hypoxia in M. smegmatis: A modified version of the methodology described by Dick et al (1998) was followed. Briefly, the A590 of the supernatant from the bead vortexed primary culture was measured and the primary culture was used to inoculate Dubos medium supplemented with 10 % ADC, and antibiotics to obtain a starting A590 of- 0.025. The cultures were mixed vigorously and 10 ml aliquots of the cultures were dispensed into 15 ml Corning polystyrene tubes, maintaining an HSR of 0.5. The caps were tightly screwed and sealed with Parafilm. The tubes were maintained at 37°C under static conditions of growth to give rise to a self-generated oxygen depletion gradient. Two control cultures containing the redox- sensitive dye, methylene blue (final concentration of 1.5 µg/ml) were used to monitor the depletion of oxygen in the sealed tubes. The cultures under static conditions of growth were used as follows: at various time points, (up to day 15) cultures in duplicates (2 tubes/ culture for each time point) were vigorously mixed, vortexed with ~ 5 sterile glass beads (3-4 mm diameter) for 2-4 min, allowed to stand for 5 min and then used for viability (cfu) plating and assessment of P galactosidase activity. (E). Aerobic growth control cultures: In order to compare the results obtained under hypoxic conditions with those under aerobic / shaking conditions, the supernatant from the bead-vortexed primary culture was inoculated into 100 ml of Dubos medium supplemented with 10 % ADC, kanamycin (20 (µg/ml) and cycloheximide (50 (µg/ml) in 500 ml conical flasks (HSR = 4) to obtain a starting A590 of ~ 0.025. The secondary culture was subjected to shaking at 190 rpm at 37°C and at various time points aliquots were used for assessment of promoter activity using P- galactosidase assays and cfu plating. (F). Establishment of growth curve: The growth characteristics of the secondary culture under static, hypoxic conditions and under shaking, aerobic conditions were assessed by: • A590 measurement of 1 ml or 500 µl aliquots (1: 3 or 1: 6 dilution respectively) of the aerobically growing culture and 1 ml of the supernatant of the bead vortexed static cultures. • Viability (cfu/ml) determination by plating serial dilutions of 900 µl aliquots of the culture (104 - 108 for aerobic cultures and 104 - 108 dilutions for static cultures) on Dubos agar plates containing 10 % ADC, kanamycin (20 µg/ml), cycloheximide (50 µg/ml). Alternatively, 450(4µl aliquots of the serially diluted cultures were plated on LB-Glycerol (0.05%) agar plates containing kanamycin (20 (4,µg/ml), during antimycobacterial susceptibility assays. The plates were incubated at 37°C for 3 days and the colonies were counted thereafter. (G). p-galactosidase assay: The activity of the M. tuberculosis devR promoter was measured in Miller units using the P-galactosidase assay. Cells containing pDP2 and pDP3 constructs of M. tuberculosis (Fig. 2) were used for studies along with a control culture where the cells carried only the pDK16 vector control. An aliquot of the culture was first used for determination of A590. Culture aliquots (3 ml) for the P-galactosidase assay were pelleted at 5000 rpm for 5 min. The pellets were resuspended in 3 ml of Z buffer containing P mercaptoethanol. To this 4 drops of chloroform and 2 drops of 0.1 % SDS were added. The suspension was vortexed for 30 sec and incubated at 30°C for 5 mins allowing lysis to take place. Then 400 µ1 of the substrate ONPG (4 mg/ml in Z buffer) was added to each tube. The suspension was mixed well and incubated in dark at 30 °C till yellow colour developed (usually ~ 25 mins). The debris were then removed by pelleting at 5000 rpm for 5 mins. The reaction was stopped by adding to the supernatant 1 ml of 1M sodium carbonate and A420 was measured. P galactosidase activity (Miller units) = 1000xA42o reaction time x culture volume x ASOO Reaction time was generally ~30 mins (starting after addition of ONPG till stopping by addition of 1 M sodium carbonate). (H). RT-PCR: Semi-quantitative RT-PCR was performed using RNA isolated from aerated and hypoxic cultures (day 2) of M. smegmatis. RNA input in the reaction was first normalized by RT-PCR targeting 23 S rDNA. M. tuberculosis-specific primers R8 and R9c targeting the intergenic region, were then used to amplify a 263 bp fragment from M. smegmatis cultures carrying pDP2 or pDP2w. (I). Demonstrate the use of reporter assay vis-a vis viability assay for simple and rapid screening of candidate compounds blocking hypoxia- modulated gene upregulation: The effect of candidate compounds, BPB and 2-MBI, on Rv3135-2 promoter activity and cell viability was studied under both hypoxia and aerobic conditions in a set-up essentially similar to that described above under (D) and (E) respectively. Serial twofold dilutions of the compound from its stock (in DMSO) were made in the growth medium prior to addition of M smegmatis culture. Culture: During the hypoxia set up with the candidate compounds, 5 ml aliquots of DTG medium supplemented with 10% ADC was dispensed in 15 ml Corning polystyrene tubes excepting one tube which contained 10 ml medium for preparing the first dilution of the series. The requisite amount of BPB from 0.5 M stock and 2-MBI from 0.33 M stock were added to the first tube of each series so as to obtain a final 2X concentration of the compounds, 12 mM BPB and 10 mM 2-MBI respectively. Subsequently, serial two-fold dilutions of the compounds were made by transferring 5 ml medium to the next tube down the series and finally 5 ml was discarded from the last tube. Thereafter, primary cultures of M. smegmatis carrying either the Rv3135-2 promoter construct or the pDK16 vector control were diluted to an optical density of 0.05 in fresh DTG medium supplemented with 10% ADC and kanamycin (40 µg/ml). 5 ml of this secondary culture was added to each tube of dilution series and mixed well so as to obtain a final volume of 10 ml , maintaining an HSR of 0.5 and starting cell density of A590 ~ 0.025. The caps were tightly screwed and sealed with parafilm. Two control sets, for each construct, without any compound were also set up in a similar way and served as no drug/compound control (essential to note the effect of the compound on promoter as well as cell viability). Aerobic set up was also perfomed in the similar manner (as described for hypoxia), in 15 ml Falcon polystyrene tubes, preparing twofold dilution of the compounds in 2 ml DTG medium supplemented with 10% ADC and adding 2 ml secondary culture to obtain a final culture volume of 4 ml (HSR ~ 4). Cells were grown with shaking from an initial cell density of A590 ~0.025. Processing: At various time points, cultures (aerobic and hypoxic) were vigorously mixed, vortexed with ~ 5 sterile glass beads (3-4 mm diameter) for 2-4 minutes, allowed to stand for 5 minutes, requisite volume of culture (3 ml for p-galactosidase assay, 50 µl for CPU plating and 1 ml for resazurin reduction test and A590 measurement) was spun down at 5000 rpm (Rota 4R-V/FM, Plastocrafts, India) at 4°C for 20 minutes and given a single wash with an equal amount of DTG medium and finally resuspended in DTG in volume original to the culture processed. This was done to remove any trace amounts of the colored compound present in the medium which otherwise might interfere with the reporter (P galactosidase activity) and viability assays (CPU and RFU) performed thereafter. The wash step is unnecessary when analyzing colorless compounds. (J). Utility of resazurin reduction in monitoring the viability of M smegmatis construct in the presence of aerobic and hypoxic cultures. 200 (0,1 culture aliquots were dispensed into 96-well microtitre plate with black base. 30 [4,1 of resazurin (0.02% in water) was added to each sample well in dark, and incubated at 37°C upto 2 hrs. After every 30 minutes of incubation, the wells were read in spectrofluorometer (spectraMAX GEMINLYS) at an excitation wavelength 530 nm and emission of 590 nm thereby measuring the amount of fluorescence (RFU or relative fluorescence units) obtained by reduced state of resazurin and estimating cell viability or inhibition of viability. Assessing candidate compounds BPB and 2-MBI: The viability of M. smegmatis Rv3135-2 promoter construct and pDK16 vector control was determined by a novel resazurin (Alamar Blue) reduction assay using the formula: (Formula Removed) Test well contained various concentrations of the candidate compound (BPB or 2-MBI) and control well was the culture grown in absence of the compound. Maximum resazurin reduction was obtained after 1 hour of incubation in the presence of resazurin and did not alter much after prolonged incubation with the dye. The aforementioned invention of the instant application is further substantiated with the help of the examples. However, these examples do not construe to limit the scope of the invention. Example No. 1. The devR-devS two-component system of M tuberculosis is upregulated under hypoxic conditions. The upregulation is most probably a result of increased transcription from the promoters. Earlier studies had shown that Rv3134c-devR-devS are co-transcribed (Dasgupta et al., 2000). The sequences mapping upstream of devR and Rv3134c were likely to contain promoter(s) driving transcription of these genes. The upstream regions of genes Rv3134c and devR were therefore cloned into the vector pDK16 (Fig. 1) resulting in pDP3 and pDP2 (Fig. 2). Both constructs supported a low p-galactosidase activity that was consistently higher than that of the vector control in aerated cultures. Subsequently the activity of putative PRV3134c andPRV3134C-devR promoters (in pDP3 and pDP2, respectively) was assessed in cultures grown under hypoxic conditions. Oxygen depletion was monitored via the fading and decolourization of methylene blue which occurred by 24 hours and 48 hours, respectively. Under these conditions, the various cultures grew in an exponential manner to a cell density of ~ 2 x 10 cells ml" and then entered a plateau phase by day 6 or 7, which extended till the experiment was terminated on day 15. In contrast, under aerobic conditions, cells grew exponentially to an initial cell density of 3 x 108 to 1 x io9 cells ml-1 and shifted thereafter to stationary phase (Fig. 3). Example No. 2 In cultures carrying pDP3 (PRV3134c promoter construct), a 3.3-fold induction in lacZ activity was noted just 24 hours after the initiation of static culture. A decline in activity was noted thereafter till day 4; lacZ activity was then stably maintained at 1.5- fold of basal level till the end of the experiment. In cultures carrying pDP2 (PRV3134C-devR promoter construct), a 5.7-fold induction in lacZ activity was noted at 24 hours, which was followed by a steady decline in activity till day 4; and thereafter lacZ activity was maintained at 2.2-fold of basal activity till the end of the experiment (Fig.4). Comparison of the ß-galactosidase activities on day 1 of hypoxic cultures carrying pDP2 versus pDP3 pointed to the dual promoter (PRV3134c-devR) being ~ 2.6-fold more active than the PRV3134c promoter. RT-PCR assays were also performed to confirm the hypoxia specific upregulation of devR promoter(s). An ~ 3-fold increase in transcript levels from this region was noted in hypoxic cultures as compared to aerated cultures of M. smegmatis carrying pDP2 plasmid (Fig. 5). This indicated that this locus was driven by at least two hypoxia-responsive promoters; one mapping upstream of Rv3134c and the other upstream of devR. Both these promoters were recognized in M. smegmatis and were upregulated under hypoxic conditions. Example No. 3 The Rv3135 gene is situated upstream of Rv3134c and is transcribed from the sense strand. The Rv3134c- and devR- upstream regions were also cloned into pDK16 in the opposite orientation (resulting in pDP3w and pDP2w) to assess the activity of P/M/JJ promoter, responsible for transcription of Rv3135. M. smegmatis cultures carrying these constructs too exhibited weak activity under aerobic conditions. A 3.6-fold and 3.3-fold induction in activities was noted with the pDP2w and pDP3w constructs, respectively at 24 hours (Fig. 6). This was followed by a reduction in activity and subsequent maintenance of activity at 1.5- and 1.2-fold of basal activity, respectively until day 15. The activity of the aerated control cultures of M. smegmatis remained unchanged over the same period. RT-PCR assay of this region using RNA from M smegmatis cultures carrying pDP2w plasmid too showed an ~3 fold induction under hypoxia as compared to aerated cultures (Fig. 7). In summary, both the PRV3134c-devR-devs as well as the divergent promoter PRV3135 were active in M. smegmatis and were modulated under hypoxic conditions. Example No. 4. A rapid and fast assay to screen candidate compounds blocking hypoxia-modulated gene expression was demonstrated by studying the effect of BPB and 2-MBI on Rv3135-2 promoter activity in M.smegmatis under hypoxia and aerobic conditions of growth. The effect of BPB concentrations (ranging from 0.375 mM to 6 mM) on the promoter activity was very prominent, though different, under both hypoxic and aerobic conditions. Unlike aerobic conditions where lower concentrations of BPB (ranging between 0.375-1.5 mM) did not have any affect on promoter activity, these concentrations of BPB moderately reduced Rv3135-2 promoter activity under hypoxia (Fig.8. A and D panels). Also, higher concentrations of BPB severely affected the promoter activity resulting in complete loss of activity. This was accompanied by a loss in viability also, under both hypoxic and aerobic conditions (Fig.8. B, C, E and F panels). Thus, BPB at lower concentrations, seems to show an inhibitory effect on the Rv3135-2 promoter activity in hypoxic but not in aerobic cultures (Fig.8. A and D panels). In contrast, 2-MBI showed a similar pattern of dosage-dependent inhibiton of promoter activity and cell viability, under both hypoxic and aerobic conditions (Fig.9.). The promoter activity was inversely proportional to the concentration of 2-MBI under both hypoxic and aerobic conditions of growth. Thus the effect of the candidate compounds on the promoter activity mirrors their effect on viability. Hence, use of promoter assay to study the effect of candidate compounds and rapidly screen compounds blocking hypoxia-modulated gene expression is justified. It is noted that these compounds have distinct effects on promoter activity and cell viability under hypoxic and aerobic culture conditions. In the case of BPB and 2- MBI, the effect of the compounds on promoter activity is mirrored in their effect on cell viability. However, these assays should enable the identification of inhibitory compounds that exercise their effect either on promoter activity or cell viability or both. Example No.5. Screening of antibacterial and antimycobacterial compounds is generally performed by conventional bacteriological techniques. Recently the use of resazurin/Alamar Blue in a microtitre plate format has been described (Collins and Franzblau, 1997). The assay has also found application in determining MICs and susceptibility status of clinical isolates of M. tuberculosis (Yajko et al, 1995, Franzblau et al, 1998, Palomino et al., 2002, Martin et al, 2003). These assays are time consuming owing to the slow growth rate of M. tuberculosis. Here we demonstrate the use of M. smegmatis to monitor cell viability under hypoxic and aerobic conditions of growth. This assay is also compatible with testing the effect of inhibitory compounds on cell viability. This is demonstrated with the use of inhibitory compounds BPB and 2-MBI (Fig. 8. and Fig. 9, panels C and F). A decline in the fluorescence of resazurin with increasing concentration of BPB was observed under hypoxia reaching complete inhibition at 6 mM concentration. A similar decline was also observed in viability assessed by the conventional plating assay (Fig.8. C and B panels). Low concentrations of BPB partially affected the viability of M. smegmatis under aerobic conditions. 2-MBI affected the viability of aerobic cultures in a dose dependent manner as measured by both reduction in CFU as well as RFU. Hence resazurin reduction as measured by reduction in RFU can be used as a simple and rapid means to determine cell viability and studying the affect of candidate compound on the viability of M. smegmatis under both hypoxia as well as aerobic conditions. Resazurin reduction assay holds another advantage over conventional plating technique for viability determination where the results are available within 2 hours of incubation with dye for the former in contrast to prolonged incubation of 72 hours for the latter. Example 6: Inhibition of DevS201 and Rv2027194 in an in vitro autophosphorylation assay is genuine and specific by BPB and 2-MBI and not due to compound-mediated protein aggregation. BPB and 2-MBI have been shown to inhibit autophosphorylation of DevS201 and Rv2027194 previously (Tyagi and Saini, 2003). Recent studies (Stephenson et al., 2000; Foster et al, 2004) concluded that certain inhibitors of histidine kinases exert their action through aggregation of the enzyme. To check whether the inhibiting effect of BPB and 2-MBI was due to non-specific aggregation of protein, cross-linking experiments with glutaraldehyde were performed on these proteins incubated with the inhibitors. To elaborate, DevS2oi/Rv2027i94 (15 µM) was incubated with the above-mentioned compounds (in a concentration course ranging from 5 mM to 10 uM. 25 mM stocks of the inhibitors were prepared in DMSO) in the phosphorylation reaction buffer (50 mM Tris. HC1, pH 8.0, 50 mM KC1 and 25 mM MgCl2) for 30 minutes at 25 C in a 20 µL reaction. The autophosphorylation reaction was initiated by the addition of 1 µCi of [y32P] ATP and 50 µM of cold ATP. The reaction contents were mixed and incubated for an additional 60 minutes at 25 C for phosphorylation. Subsequently, the samples were split into two equal fractions. For one fraction the reaction was terminated immediately. The other fraction was cross-linked for 30 minutes at room temperature by the addition of glutaraldehyde to a final concentration of 0.3% in the reaction. The autophosphorylation and cross-linking reactions were terminated by addition of SDS-PAGE sample buffer containing glycine to a final concentration of 400 mM. The samples were then analyzed on 12.5 % SDS-PAGE. Finally the gels were silver-stained and higher oligomeric forms of protein were visualized. In such a reaction the compounds showed inhibition of autophosphorylation as reported earlier (Fig. 10 and 11). The cross-linked and silver stained gels showed dimeric and other higher oligomeric forms of the protein. In the non cross-linked gels the dimeric and higher oligomeric forms were absent. In the absence of inhibitors, cross-linked DevS2oi and Rv2027i94 resolved as both monomer and dimer forms (Fig. 10-13). As expected, the autophosphorylation activity of DevS2o1 and Rv2027i94 decreased with the increasing concentration of BPB and 2-MBI. However increasing concentrations of the inhibitors did not have any effect on the oligomeric state of DevS2oi/Rv2027194 as determined by silver-stained gels. These experiments validated that the effect of the inhibitors on autophosphorylation was genuine and not due to the aggregation of the proteins in the presence of the inhibitor. Main advantages of the present invention M. tuberculosis being a slow growing, pathogen is relatively difficult to handle and hence M. smegmatis, a fast growing, non-pathogenic species of the same genus is often used to study those physiological aspects of M. tuberculosis that are common to both species. The physiological behaviour of M smegmatis under hypoxic conditions is very much similar to that of M. tuberculosis. Also, the highly conserved homologues of many of the M. tuberculosis hypoxia-responsive genes such as devR-devS are present in M. smegmatis and show upregulation under hypoxic conditions. Further promoter(s) of three M. tuberculosis genes (Rv3134c, devR and Rv3135) that are hypoxia-responsive were shown to be induced during hypoxia in M. smegmatis. These features suggest that M. smegmatis may serve as a suitable host for studying many features of M tuberculosis hypoxia response. Further promoter assays in M. smegmatis should prove useful for screening for novel compounds that can interfere with the hypoxia response. Such compounds are likely to prove useful in the development of novel antirubercular drugs that hamper adaptation of mycobacteria to hypoxia and perhaps a persistent granulomatous lifestyle which is the hall mark of tuberculosis. References: 1. Ahmed SA, Gogal RJVI and Walsh JE. (1994). A new rapid and simple non- radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H] thymidine incorporation assay. J Immunol Methods 170:211- 224. 2. Bashyam MD, Kaushal D, Dasgupta, SK and Tyagi AK. (1996). A study of the mycobacterial transcriptional apparatus: identification of novel features in promoter elements. J Bacteriol 178, 4847 - 4853. 3. Bastian I, Rigouts L, Palomino JC and Portaels F (2001). Kanamycin susceptibility testing by Mycobacterium tuberculosis using mycobacterium growth indicator tube and a colorimetric method. Antimicrob agents chemother 45(6), 1931-1936. 4. Boon C, Li R, Qi R and Dick T (2001). Proteins of Mycobacterium bovis BCG induced in Wayne dormancy model. J Bacteriol 182, 2672 - 2676. 5. Cole ST, Brosch R, Parkhill J and 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence Nature 393, 537-544. 6. Collins LA and Franzblau SG (1997). Microplate Alamar Blue assay versus Bactec 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob agents chemother 41(5), 1004-1009. 7. Dasgupta N, Kapur V, Singh KK, Das TK, Sachdeva S, Jyotisri K and Tyagi JS (2000). Characterization of a two-component system, devR-devS, of Mycobacterium tuberculosis. Tuber Lung Dis 803,141-159. 8. Dick T, Lee BH and Oei BM (1998). Oxygen depletion induced dormancy in Mycobacterium smegmatis. FEMS Microbiol Lett 163,159 - 164. 9. Foster JE, Sheng Q, McClain JR, Bures M, Nicas TI, Henry K, Winkler, ME and Gilmour R (2004). Kinetic and mechanistic analyses of new classes of inhibitors of two-component signal transduction systems using a coupled assay containing HpkA-DrrA from Thermotoga maritima. Microbiol 150, 885-896. 10. Franzblau SG, Witzig SR, Mclaughlin C, Torres P, Madico G, Hernandez A, Degnan MT, Cook MB, Quenzer VK, Fergusan RM and Oilman RH (1998). Rapid low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J Clin Microbiol 36(2), 362-366. 11. Jain S, Kaushal D, Dasgupta SK and Tyagi AK (1997). Construction of shuttle vectors for genetic manipulation and molecular analysis of Mycobacteria. Gene 190,37-44. 12. Martin A, Camacho M, Portaels F and Palomino JC (2003). Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second-line drugs: rapid, simple, and inexpensive method. Antimicrob agents chemother 47(11), 3616-3619. 13. Mayuri, Bagchi G, Das TK and Tyagi JS (2002). Molecular analysis of the dormancy response in M. smegmatis: expression analysis of genes encoding the DevR-DevS two-component system, Rv3134c and chaperone a-crystallin homologues. FEMS Microbiol Lett 211, 231 - 237. 14. Palomino JC, Martin A, Camacho M, Guerra H, Swings J and Portaels F (2002). Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob agents chemother 46(8), 2720-2722. 15. Park HD, Guinn, KM, Harrell MI, Liao R, Voskuil MI, Tompa M, Schoolnik GK and Sherman DR (2003). Rv3133c/dos# is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol Microbiol 48, 833 - 843. 16. Roberts DM, Liao RP, Wisedchaisri G, Hoi WG and Sherman DR (2004). Two sensor kinases contribute to the hypoxic response of Mycobacterium tuberculosis. J Biol Chem 279:23082-7. 17. Saini DK, Malhotra V and Tyagi JS (2004). Cross talk between DevS sensor kinase homologue, Rv2027c, and DevR response regulator of Mycobacterium tuberculosis.FEBS Lett 565:75-80. 18. Saini DK, Malhotra V, Dey D, Pant N, Das TK and Tyagi JS (2004). DevR- DevS is a bonafide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR. Microbiology 150:865-875. 19. Sambrook J and Russell DW (2001) Molecular cloning; a laboratory manual Third Ed., Cold Spring Harbour Press, New York. 20. Sherman DR, Voskuil M, Schnappinger D, Liao R, Harrell M and Schoolnik GK (2001). Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding a-crystallin. Proc Natl Acad Sci U.S.A. 98, 7534 - 7539. 21. Stephenson K, Yamaguchi Y and Hoch JA (2000). The mechanism of action of inhibitors of bacterial two-component signal transduction systems. J Biol Chem 275, 38900-38904. 22. Tyagi AK, Dasgupta SK and Jain S (2000). In Molecular Genetics of Mycobacteria (Hatfull G.F. & Jacobs, W.R. Jr. Ed.) ASM Press, Washington DC, 131-147. 23. Tyagi JS and Saini DK (2003). A screening method for developing drugs against pathogenic microbes having two-component system. US Non- Provisional application and PCT application no. PCT/IB03/04555 claiming priority from US Provisional application no. 60/418,337 of October 16, 2002. 24. Tyagi JS and Sharma D (2002). Mycobacterium smegmatis and tuberculosis. Trends Microbiol 10, 68 - 69. 25. Wayne LG and Sohaskey CD (2001). Nonreplicating persistence of Mycobacterium tuberculosis. Annu Rev Microbiol 55, 139-163. 26. Yajko DM, Madej JJ, Lancaster MV, Sanders CA, Cawthon VL, Gee B, Babst A and Hadley WK (1995). Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis. J Clin Microbiol 33(9), 2324-2327. 27. Zahrt TC and Deretic V (2001). Mycobacterium tuberculosis signal transduction system required for persistent infections. Proc Natl Acad Sci U.S.A. 98, 12706 -12711. Wo Claim: 1. A method of screening anti-tubercular compound, said method comprising the steps: a. constructing plasmid such as herein described by cloning Rv3I34c and devR upstream regions from DMA of Mycobacfer/um tuberculosis under transcriptional control of divergent promoters P Rv3134-devR and PRV 3135 optionally with a reporter gene, b. electroporating the plasmid into an electro competent Mycobacfer/um smegmaf/s, c. culturing the Mycobacterium smegmatis under hypoxic and/or aerobic conditions such as herein described and in presence and/or absence of test compound, and d. determining anti-tubercular activity of test compound in terms of down regulation or up-regulation of Rv3734c and devR promoter activity as herein described, wherein down-regulation is indicative of the test compound being an anti-tubercular compound. 2. A method as claimed in claim 1, wherein the plasmids are selected from pDP3, pDP2, pDP3w and pDP2w. 3. A method as claimed in claim 1 , wherein the reporter gene is selected from a group comprising ß-galactosidase (lacZ), Chloramphenicol Acetyltransferase (cat), alkaline phosphatase (phoA), catechol 2,3-dioxygenase (xylE), luciferase (lux) and GFP. 4. A method as claimed in claim 3, wherein the reporter gene is ß-galactosidase (lacZ). 5. A method of screening anti-tubercular compound substantially as herein described with reference to the drawings and foregoing examples.

Documents:

0981-del-2003-abstract.pdf

0981-del-2003-claims.pdf

0981-del-2003-correspondence-others.pdf

0981-del-2003-correspondence-po.pdf

0981-del-2003-description (complete).pdf

0981-del-2003-description (provisional).pdf

0981-del-2003-drawings.pdf

0981-del-2003-form-1.pdf

0981-del-2003-form-18.pdf

0981-del-2003-form-2.pdf

0981-del-2003-form-26.pdf

0981-del-2003-form-3.pdf

0981-del-2003-form-5.pdf