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-acetyl glutamate synthase that leads to arginine auxotrophy in Pseudomonas aeruginosa. This Tn insertion generates a truncated version of the ArgA protein (ArgA188-432) that retains N-acetyl glutamate synthase 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 ambAAla128Val 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 ambAAla128Val 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 ambAAla128Val mutation overcomes the phenotypic impact of the dysregulated ArgA188-432 enzyme, possibly by enabling efflux of toxic polyamines (which we also show, can competitively inhibit N-acetyl glutamate synthase).