Figure 3 Growth of the mycobacterial strains in low and high nitrogen broth culture. A. OD600 of wild type M. bovis was inoculated to an initial optical density of 0.006 – 0.008 in 7H9 medium containing (●) low nitrogen (3.8 mM ammonium sulphate) and (▲) high nitrogen (60 mM ammonium sulphate). B. OD600 of wild type M. smegmatis and MSFP in low and high nitrogen broth culture. Wild type M. smegmatis, low nitrogen (■), high nitrogen (□); MSFP, low nitrogen (●), high nitrogen (○). Data is mean ± SD of values obtained from three independent cultures. LN, low nitrogen; HN, high nitrogen. Relative quantification of glnA1 transcript of recombinant M. smegmatis strains Semi-quantitative RT-PCR assays

were performed with RNA obtained from different strains grown in Selleckchem LXH254 Ralimetinib purchase low and high nitrogen condition. M. smegmatis strain (MSFP and MSP1) showed up-regulation of glnA1 transcript in low nitrogen as compared to high nitrogen condition. The glnA1 transcript of M. bovis was also higher in low nitrogen than in high nitrogen condition, while MSP2 had no effect on glnA1 mRNA level in different nitrogen

conditions (Figure 4A, panel i and iii). Figure 4 Analysis of glnA1 transcription in mycobacterial strains in low and high nitrogen condition. A. For semi-quantitative reverse transcriptase PCR analysis, mycobacterial strains were grown in low and high nitrogen condition. glnA1 transcripts in (i) low nitrogen and (iii) high nitrogen condition. sigA loading control of respective test samples in low nitrogen (ii) and (iv) high nitrogen condition. (v) Genomic DNA contamination PCR analysis by sigA amplification without reverse transcriptase of respective test samples grown in low and high nitrogen condition. Lane M, marker; lane PC, positive control. B. For real-time (qRT-PCR) analysis, the expression profiles of glnA1 gene in low nitrogen (black bars) and high nitrogen (grey bars) conditions were compared with respect to their corresponding M. smegmatis wild-type strain in low nitrogen. Data shown are linear fold change normalized to sigA expression level. The transcripts were quantified by a SYBR Green-based real-time

PCR assay as described under “Materials and Methods.” The experiments were repeated three times, and data from one of the representative experiments are presented. LN, low nitrogen; HN, high nitrogen; LC, loading control. Non-specific serine/threonine protein kinase Real time PCR was performed further to study glnA1 expression quantitatively in low and high nitrogen conditions for MSFP, MSP1, MSP2, wild type M. smegmatis and M. bovis strains. The glnA1 expression levels in wild type M. smegmatis in low nitrogen condition was taken as the reference point in order to PLX3397 purchase calculate the fold change in recombinant strains. The data obtained from real time PCR was normalized to sigA expression levels, as an internal control. It was observed that in case of nitrogen starvation, the expression of glnA1 gene in MSFP and MSP1 strains was highly up-regulated.