An extragenic fix for a broken enzyme

In this work, we show how exploration of genomic sequence space yields an unexpected evolutionary 'fix' that allows the cell to bypass the phenotypic defects associated with a dysfunctional enzyme. We identified a transposon (Tn) insertion in the gene (argA) encoding N-acetylglutamate synthase (NAGS) that leads to arginine auxotrophy in Pseudomonas aeruginosa. This Tn insertion generates a truncated version of the ArgA protein (ArgA_188-432) that retains NAGS activity but, unlike the wild-type protein, is unrestrained by arginine feedback inhibition. However, the Tn::argA mutant readily re-acquired a prototrophic phenotype through the acquisition of spontaneous secondary mutations outside of the argA locus. Whole-genome sequencing and complementation analyses of one such 'bypass mutant' allowed us to trace this epistatic effect to an Ala128→Val substitution in AmbA, a LysE-type transmembrane transporter. Using a variety of genetic approaches, we show that the ambA ^Ala128Val mutation gives rise to a change/expansion of function in the encoded gene product and that this mutation is directly or indirectly responsible for bypassing the auxotrophy associated with the original Tn::argA mutation. Further analysis of the Tn::argA ambA ^Ala128Val mutant revealed that, compared with the 'wild-type' progenitor, the mutant exhibited large-scale changes in its proteome and metabolome consistent with elevated intracellular arginine and enhanced polyamine degradation. We conclude that the ambA ^Ala128Val mutation overcomes the phenotypic impact of the dysregulated ArgA_188-432 enzyme, possibly by enabling efflux of toxic polyamines (which we also show can competitively inhibit NAGS).