The Cysteine Desulfurase IscS Is a Significant Target of 2-Aminoacrylate Damage in Pseudomonas aeruginosa.

ABSTRACT

Pseudomonas aeruginosa encodes eight members of the Rid protein superfamily. PA5339, a member of the RidA subfamily, is required for full growth and motility of P. aeruginosa. Our understanding of RidA integration into the metabolic network of P. aeruginosa is at an early stage, with analyses largely guided by the well-established RidA paradigm in Salmonella enterica. A P. aeruginosa strain lacking RidA has a growth and motility defect in a minimal glucose medium, both of which are exacerbated by exogenous serine. All described ridA mutant phenotypes are rescued by supplementation with isoleucine, indicating the primary generator of the reactive metabolite 2-aminoacrylate (2AA) in ridA mutants is a threonine/serine dehydratase. However, the critical (i.e., phenotype determining) targets of 2AA leading to growth and motility defects in P. aeruginosa remained undefined. This study was initiated to probe the effects of 2AA stress on the metabolic network of P. aeruginosa by defining the target(s) of 2AA that contribute to physiological defects of a ridA mutant. Suppressor mutations that restored growth to a P. aeruginosa ridA mutant were isolated, including an allele of iscS (encoding cysteine desulfurase). Damage to IscS was identified as a significant cause of growth defects of P. aeruginosa during enamine stress. A suppressing allele encoded an IscS variant that was less sensitive to damage by 2AA, resulting in a novel mechanism of phenotypic suppression of a ridA mutant. IMPORTANCE 2-aminoacrylate (2AA) is a reactive metabolite formed as an intermediate in various enzymatic reactions. In the absence of RidA, this metabolite can persist in vivo where it attacks and inactivates specific PLP-dependent enzymes, causing metabolic defects and organism-specific phenotypes. This work identifies the cysteine desulfurase IscS as the critical target of 2AA in Pseudomonas aeruginosa. A single substitution in IscS decreased sensitivity to 2AA and suppressed growth phenotypes of a ridA mutant. Here, we provide the first report of suppression of a ridA mutant phenotype by altering the sensitivity of a target enzyme to 2AA.