Mutations affecting nitrogen regulation.

Functions of the mutated genes are summarized, and the location of all mutations are shown on the operon below the schematic. Mutations are shown if they appear at >10% frequency in more than one flask in an ALE lineage, and ALE numbers are in bold if the mutation appears in the endpoint clone. The mutations indicated with a dashed arrow occured in the ΔhisD strain and a solid arrow if they occured in ΔhisD strain’s partner MSE strain. (A) Mutations were acquired within the open reading frame of both genes comprising the nitrogen sensing two-component regulatory system. Shown in the schematic is the regulatory cascade in which nitrogen concentration is sensed (via GlnK or GlnB) by GlnL. In response to low nitrogen availability GlnL is autophosphorylated resulting in a subsequent transfer of the phosphorus group to GlnG. Phosphorylated GlnG upregulates general functions associated with nitrogen starvation, including increasing GlnK expression [77]. (B) Further, mutations were observed in the ORF of GlnK, one of two nitrogen metabolism regulators, sharing most functions with GlnB. Both genes become uridylylated in response to high concentrations of 2-oxoglutarate and ATP and low concentrations of L-glutamine, which is an indication of nitrogen limitation. GlnK-UMP can activate GLNS deadenylation, thus increasing its activity. Unlike GlnB, when GlnK is in a deuridylylated state (indicative of high nitrogen availability) it can be sequestered by the AmtB ammonium transporter reducing the transporter’s activity [75].