, 2008). Two additional genes, contained Ibrutinib ic50 in an

operon with amtB, have also been proposed to be under GlnR control: glnK (msmeg_2426; PII-type protein) and glnD (msmeg_2427; an adenylyl-transferase enzyme) (Amon et al., 2008). Therefore, glnA1, amtB and amt1 were selected for analysis in this study. Four other genes were also chosen for investigation owing to their proposed role in nitrogen metabolism in Mycobacteria (Amon et al., 2009). These were amtA (msmeg_4635), an ammonium transporter; nirB (msmeg_0427), a nitrite reductase enzyme; gltD (msmeg_3226), a glutamate synthase enzyme involved in glutamate synthesis during nitrogen limitation; and glnE (msmeg_4293), a bifunctional adenylyl-transferase thought to modulate GS enzymatic activity. Expression of glnR (msmeg_5784) itself was also included. Wild-type and mutant strains were grown in nitrogen-limiting or nitrogen-excess conditions for 13 h. Expression values for each gene analysed were compared with the housekeeping gene sigA (msmeg_2758) whose expression did not alter in the conditions tested (data not shown). Genes previously shown to be under GlnR control, glnA1, amtB and amt1, were all highly up-regulated in the wild type during nitrogen limitation when compared with their expression in nitrogen-excess conditions (Fig. 2). glnA1 expression was induced approximately 13-fold, amtB 153-fold and amt1 219-fold (Table 3). However, there was no induction of these genes in either GlnR mutant

grown under nitrogen limitation Carnitine palmitoyltransferase II (Fig. 2 and Table 3). To account for the fact that the GlnR mutants deplete the external ammonium at a slower rate than the wild type (Fig. 1) and see more may not be stressed at 13 h, we repeated the qRT-PCR using samples taken at 19 h, when external nitrogen was no longer detectable. However, there was also no induction of gene expression in either mutant at this later time point (data not shown). Transcriptional control of other genes proposed to be involved in mycobacterial nitrogen metabolism, not shown previously to be under GlnR control, was subsequently investigated. Figure 3 shows that amtA, gltD and nirB were up-regulated in the wild-type strain in response

to nitrogen limitation at 13 h, compared with nitrogen excess, while glnE expression was down-regulated. During nitrogen limitation, amtA was induced approximately 337-fold, nirB 103-fold and gltD 8-fold; glnE was down-regulated 3-fold. Again, no significant change in the expression levels of these genes was observed in the GlnR mutants at either 13 h (Fig. 3 and Table 3) or 19 h (data not shown). To exclude the possibility that the GlnR_D48A mutation inhibited glnR expression, leading to the observed null phenotype of this strain, transcriptomic analysis of glnR was performed. No significant change in glnR expression was observed under nitrogen-limiting conditions for either the wild type or GlnR_D48A mutant (Table 3 and Fig. 2), confirming previous observations that M.