Perturbation of the MetJ regulon impacts the consequences of 2-aminoacrylate stress in Salmonella enterica

Abstract

In the absence of the broadly conserved deaminase RidA (Reactive intermediate deaminase A), Salmonella enterica and other organisms accumulate the reactive enamine species 2-aminoacrylate (2AA). Free 2AA, generated from serine by the serine/threonine dehydratase IlvA, reacts with and covalently inactivates a subset of pyridoxal 5'-phosphate-dependent enzymes. The metabolic stress caused by 2AA generates growth defects in S. enterica, including (i) when l-alanine is used as a nitrogen source, (ii) when pyruvate is used as a carbon source or (iii) in the presence of exogenous serine. Although the enzymatic targets of 2AA are consistent between growth conditions, the consequences of 2AA-dependent damage differ depending on the distribution of metabolic flux required in different conditions. Analysing the suppressors of a ridA mutant has furthered our understanding of the RidA stress paradigm and, more generally, how a metabolic network responds to perturbation. Many such suppressors modulate the metabolic network to eliminate 2AA production by IlvA. Here, we describe that eliminating the MetJ transcriptional repressor allows a ridA mutant to grow in the presence of 2AA stress in each of the three conditions. The mechanisms by which a ΔmetJ suppresses a ridA mutant are nuanced and medium-dependent, emphasizing that consequences of 2AA stress differ based on environmental and metabolic context.

Keywords: 2-aminoacrylate stress; MetJ; MetR; RidA.

Fig. 1.. The RidA paradigm and relevant targets of 2AA stress in S. enterica. The PLP-dependent serine/threonine dehydratase IlvA (EC 4.3.1.19) generates 2AA, which tautomerizes to 2-iminopropionate (2IP). 2AA can be deaminated by water, or the RidA deaminase, to form pyruvate. In the absence of RidA, 2AA persists in the cell long enough to damage PLP-dependent enzymes. Depending on the unique structure of an organism’s metabolic network and the nutrient composition of its surroundings, damage to certain key enzymes may generate a growth defect. In S. enterica, relevant targets of 2AA include, but are not limited to, (i) IlvE (EC 2.6.1.27), an aminotransferase required for the last step of isoleucine biosynthesis; (ii) GlyA (serine hydroxymethyl transferase, EC 2.1.2.1), the predominant source of glycine and one-carbon units; and (iii) DadX (EC 5.1.1.1), the catabolic alanine racemase required for l-alanine utilization. Damage to these enzymes has been associated with growth defects on minimal media with pyruvate provided as the sole carbon source, exogenous serine or l-alanine provided as the sole nitrogen source, respectively. Abbreviations: 2KMV, 2-keto-3-methylvalerate; THF, tetrahydrofolate; 5,10-mTHF, 5,10-methylene-tetrahydrofolate; DadA, d-amino acid dehydrogenase (EC 1.4.5.1).

Fig. 2.. Deletion of metJ improves the growth of an alr ridA mutant. Strains with the indicated genotypes [alr (squares; DM14178), alr ridA (circles; DM17781) and alr ridA metJ (triangles; DM17788)] were grown on (a) minimal glucose medium with l-alanine (5 mM) as the sole nitrogen source and (b) minimal pyruvate medium. Control growth is shown as alr ridA in the presence of isoleucine in each medium. Growth was monitored by following OD at 650 nm over 25–35 h as indicated. Data shown are averages from three biological replicates with standard error bars.

Fig. 3.. Deletion of metJ improves the growth of an alr ridA mutant with exogenous serine. Strains with the indicated genotypes [alr (circles; DM14178), alr ridA (squares; DM17781) and alr ridA metJ (triangles; DM17788)] were grown on minimal glucose medium with serine (5 mM). The alr ridA mutant was also grown with serine and methionine (open squares). Growth was monitored by OD (650 nm) over 24 h. Data shown are averages from three biological replicates with standard error bars.

Fig. 4.. Stimulation of growth by homocysteine is MetR-dependent. All strains had an alr mutation and carried additional lesions as indicated. ridA (diamonds; DM17781), ridA metJ (solid squares/circles/triangles; DM17984) and ridA metJ metR (open circles/triangles; DM18542) were grown on minimal glucose medium with serine (5 mM) and additional supplements as indicated: Met (methionine, 0.3 mM) and Homocys (homocysteine, 0.2 mM). Data shown are averages from three biological replicates with standard error bars.