Thin sections (100 nm) were obtained using Leica Ultracut (Leica, Germany) and collected on Nickel grids (200 mesh; Electron Microscopy Sciences). For localization, monoclonal anti-PLG antibody (1:100) (Sigma) was used. The grids were washed and subsequently treated with gold (10 nm) conjugated – anti mouse IgG. Mice pre-immune
serum was used as a negative control. The immunolabeled sections Autophagy Compound Library chemical structure were stained with uranyl acetate and viewed using a Jeol 2100 F transmission electron microscope (Jeol Analytic Instruments) at an acceleration voltage of 120 KV. Biofilm formation Biofilm formation was observed by growing static cultures of mycobacteria without shaking in 7H9 medium without Tween 80 at 37°C. Biofilm formation was assayed by crystal violet staining method developed by Reicht et al.[19, 20]. Briefly, 200 μl of stationary phase cultures (A600 normalized to 1) were added to 7H9 medium in polystyrene culture plates for biofilm formation and in culture tubes for pellicle formation. After incubation of static culture of M. smegmatis strains for 2 days and M. bovis for 2–3 weeks, biofilm was quantified by removing the medium carefully and staining with 1% crystal violet for 45 min. PCI-34051 research buy The wells were washed three times with water and air-dried. The dye was solubilized with 80% ethanol and A550 was measured. Results Generation
of glnA1 promoter Selleckchem Crenolanib variants Figure 2 shows a schematic representation of the deletion variants of the promoter. M. bovis contains two native promoters P1 and P2 within 320 bp upstream of glnA1 gene
(start codon designated as +1). 124 bp upstream of glnA1 start codon was taken as P1 promoter. Further, from 320 bp upstream sequence, 31 bp (-46 to -76) was deleted from Branched chain aminotransferase the native promoter and taken as P2 promoter. The native, P1 and P2 promoter with glnA1 gene were used for further characterization in response to nitrogen limitation and excess. Figure 2 Schematic representation of glnA1 promoter. glnA1 gene with two promoters P1 and P2. +1 represents glnA1 translational start site. The red arrow represents the transcriptional start site. The black arrow represents the position of primers used to make deletion variants of the glnA1 promoter. Growth characteristics M. bovis strain was grown in low and high nitrogen medium and growth profile was studied by measuring optical density at 600 nm. No significant difference was observed in the growth of M. bovis when cultured in low nitrogen medium as compared to growth in high nitrogen medium (Figure 3A). This indicated that M. bovis was able to acquire nitrogen from other sources in the medium (L-glutamic acid, ferric ammonium citrate and ammonium sulphate). Same was the case when growth of wild type M. smegmatis and MSFP was studied in low and high nitrogen conditions (Figure 3B).