Mohan, Veena, Labrini, Edoardo, Aksoy, Cemile Selin, Behrends, Volker ORCID: https://orcid.org/0000-0003-4855-5497, Evangelista, Laila C., Spring, David R. and Welch, Martin (2026) An extragenic fix for a broken enzyme. Microbiology, 172 (10).

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Abstract

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 (ArgA188-432) 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 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 NAGS).

Abbreviations

AAK, amino acid kinase; AMB, l-2-amino-4-methoxy-trans-3-butenoic acid; CF, cystic fibrosis; CoF, change-of-function; DTNB, 5′,5-dithiobis(2-nitrobenzoic acid); EoF, expansion-of-function; FA, formic acid; FDR, False Discovery Rate; GABA, γ-aminobutyrate; gDNA, genomic DNA; GNAT, Gcn5-related N-acetyltransferase; LC, liquid chromatography; NAG, N-acetyl l-glutamate; NAGS, N-acetylglutamate synthase; Ni-NTA, Nickel-Nitrilotriacetic Acid; OAT, ornithine acetyltransferase; PA, Pseudomonas aeruginosa; PCA, principal component(s) analysis; TMHMM, Transmembrane helices; Hidden Markov Model; TMT, tandem mass tagging; Tn, transposon; WGS, whole-genome sequencing.

Item Type:	Article
Identifier:	10.1099/mic.0.001760
Keywords:	arginine metabolism, auxotrophy, enzyme regulation, evolution, proteomics, Pseudomonas aeruginosa
Subjects:	Natural sciences > Cell and molecular biology
Date Deposited:	16 Oct 2026
Dates:	Date Publication status 5 October 2026 Published
School, department or research centre:	School of Medicine and Biosciences
Keywords:	arginine metabolism, auxotrophy, enzyme regulation, evolution, proteomics, Pseudomonas aeruginosa
URI:	https://repository.uwl.ac.uk/id/eprint/15550

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