tuberculosis H37Rv

and VPCI591. (A)- Diagrammatic representation [not to scale] of the mce1 operon. Arrows indicate the position of primers. The hatched box depicts IGPr region. (B)- Fold difference in transcript level in VPCI591 over that of M.tuberculosis H37Rv for Rv0167, Rv0170 and Rv0174 in log phase (dotted) and stationary phase (hatched). The fold difference observed is the average of three independent experiments. Error bars represent the standard deviation. Effect of the regulatory sequence of IGPr on heterologous promoter To examine if the negative regulatory site, -100 to +1 region of IGPr functions independent of the associated promoter activity, we cloned it downstream of a heterologous promoter in pSdps1, driving the expression of β-galactosidase [23]. pSdps1 has 1 kb upstream region Screening Library concentration of the gene MSMEG_6467 BGB324 cell line from M.smegmatis. The promoter in pSdps1 is inducible under CHIR98014 clinical trial glucose starvation; at 0.02% glucose in Middlebrook 7H9 liquid medium in stationary phase [23]. By inclusion of +1 to -100 from IGPr of H37Rv (pDPrBRv) the promoter activity decreased by 35% relative to the control plasmid pSdps1 (895 versus 1358 units, Figure 7). When +1 to -100 from VPCI591 was cloned downstream to dps promoter (pDPrB591), the repression was reversed and the promoter

activity was enhanced by 25% over that of pSdps1 (1709 versus 1358 units). This shows that negative regulation by IGPr functions in the context of a heterologous promoter also. Figure 7 Regulation of heterologous promoter by IGPr. dps promoter activity under induced conditions in different constructs in terms of β-gal activity units expressed as nmol ONPG converted to o-nitrophenol per min per milligram of protein. The transformants were grown in Middlebrook 7H9 medium supplemented with 0.02% glucose (Induced). Each experiment was carried out in triplicates and S.D is indicated

as error bars. Discussion The mce1 operon is different from other three mce operons in having Rv0166, a fatty acyl CoA synthetase that catalyzes the initial step in lipid degradation [4, 24]. On the other hand, mce4 operon is known to be a part of the regulon involved in cholesterol metabolism, however it seems to be just one of the many possible lipid oxyclozanide substrates. Furthermore, it is speculated that mce1 operon may not have a role in cholesterol import as the loss of Mce1 transporter system does not appear to affect the residual uptake of cholesterol in mce4- deficient strain [25]. The presence of 200 base pairs of non-coding sequence between Rv0166 and Rv0167 is yet another feature peculiar to mce1 operon among the other four operons present in M.tuberculosis. In most other operons and also the other genes within mce1 operon, the intergenic distance is not more than one or two codons and often the translation initiation site of one gene is within the coding sequence of the adjacent gene [12].