It binds to upstream sequence Dabrafenib of glnA1 and activates transcription during nitrogen starvation (Figure 1). Furthermore, in high nitrogen conditions to evade the depletion of cellular glutamate levels due to conversion of all glutamate to glutamine the GS enzyme is modified post translationally [12]. In case of the nitrogen sufficiency, GlnE protein acts as a negative regulator and it adenylylates the GS enzyme at a conserved tyrosine residue at 406 position [13]. Hence, the adenylylated form of GS becomes inactive (Figure 1). Figure 1 Pictorial representation depicting role
of glutamine synthetase in nitrogen metabolism and PLG synthesis. In low nitrogen conditions GlnR acts as a positive regulator and activates transcription of glnA1 gene. In high nitrogen conditions GlnE acts as a negative regulator and adenylylated GS protein, which thus becomes inactive. GS, glutamine synthetase; ↑↑↑, up-regulation. In this study, we investigated the behaviour of glnA1 gene of M. bovis both at the mRNA and protein levels in response to nitrogen availability. The present study emphasizes on the effect of nitrogen concentration selleck compound on expression levels of glnA1 gene from the two different promoters when present independently or together. We have also studied the effect of nitrogen concentration on PLG layer synthesis in the cell wall of mycobacteria. Methods Bacterial strains
and growth conditions The bacterial strains and plasmids used
in this study are listed in Table 1. M. bovis and M. smegmatis strains were routinely cultured in 7H9 broth (Difco) supplemented with 10% (v/v) albumin, dextrose and catalase (ADC), 0.2% (v/v) glycerol and 0.05% (v/v) Tween 80, at 37°C with shaking at 150 rpm. Escherichia coli DH5α (Novagen) was used for cloning experiments. E. coli DH5α was grown in Luria-Bertani medium. Kanamycin was used at concentration of 25 μg/ml for mycobacteria and 50 μg/ml for E. coli strains. Table 1 Plasmids and strains used in this study Plasmids Relevant characteristics Source/Reference pGEM-T Nintedanib (BIBF 1120) Easy amp R ori pUC (Cloning vector) Promega pMV261 kan R (Mycobacterial shuttle non-integrative vector) Stover et al., 1991 [14] pDS1 pGEM-T Easy containing glnA1 coding sequence with native promoter This work pDS2 pMV261 containing glnA1 coding sequence with native promoter This work pDS3 pGEM-T Easy containing glnA1 coding sequence with P1 promoter This work pDS4 pMV261 containing glnA1 coding sequence with P1 promoter This work pDS5 pMV261 containing glnA1 coding sequence with P2 promoter This work Strains Relevant characteristics Source/Reference DH5α supE44 ΔlacU(Φ80lacZΔM15) hsdR17 rec1 endA1 gyrA96 thi-1 relA1 Novagen M. bovis AN5 Wild Type ATCC M. smegmatis mc2 Wild Type ATCC MSFP M. smegmatis containing pDS2 This work MSP1 M. smegmatis containing pDS4 This work MSP2 M.