A single regulator NrtR controls bacterial NAD⁺ homeostasis via its acetylation.

Abstract

Nicotinamide adenine dinucleotide (NAD⁺) is an indispensable cofactor in all domains of life, and its homeostasis must be regulated tightly. Here we report that a Nudix-related transcriptional factor, designated MsNrtR (MSMEG_3198), controls the <i>de novo</i> pathway of NAD⁺biosynthesis in <i>M. smegmatis</i>, a non-tuberculosis <i>Mycobacterium</i>. The integrated evidence <i>in vitro</i> and <i>in vivo</i> confirms that MsNrtR is an auto-repressor, which negatively controls the <i>de novo</i> NAD⁺biosynthetic pathway. Binding of MsNrtR cognate DNA is finely mapped, and can be disrupted by an ADP-ribose intermediate. Unexpectedly, we discover that the acetylation of MsNrtR at Lysine 134 participates in the homeostasis of intra-cellular NAD⁺ level in <i>M. smegmatis</i>. Furthermore, we demonstrate that NrtR acetylation proceeds via the non-enzymatic acetyl-phosphate (AcP) route rather than by the enzymatic Pat/CobB pathway. In addition, the acetylation also occurs on the paralogs of NrtR in the Gram-positive bacterium <i>Streptococcus</i> and the Gram-negative bacterium <i>Vibrio</i>, suggesting that these proteins have a common mechanism of post-translational modification in the context of NAD⁺ homeostasis. Together, these findings provide a first paradigm for the recruitment of acetylated NrtR to regulate bacterial central NAD⁺ metabolism.