Aspartate D48 is essential for the GlnR-mediated transcriptional response to nitrogen limitation in Mycobacterium smegmatis.

Nitrogen is an essential element required for bacterial growth and consequently bacteria must adapt to situations of nitrogen limitation for survival. The transcriptional response to nitrogen limitation in Mycobacterium smegmatis is thought to be regulated by GlnR, although, to date, only five nitrogen metabolism genes have been shown to be under its direct control. GlnR belongs to the OmpR family of two-component response regulators that are typically activated by phosphorylation of a conserved aspartate residue. The M. smegmatis GlnR protein contains the highly conserved aspartate residue (D48) corresponding to the phosphorylation sites identified in other OmpR family regulators. In this study, we replaced GlnR D48 with alanine and constructed a GlnR deletion mutant. Under nitrogen-limiting conditions, both the GlnR_D48A and GlnR deletion mutants exhibited reduced growth rates compared with wild type. Transcriptional analysis showed both mutants failed to up-regulate the expression of GlnR-controlled genes under nitrogen-limiting conditions. We therefore demonstrate that the GlnR aspartate (D48) residue is essential for its function as a nitrogen-stress transcriptional response regulator in M. smegmatis.