Tetrahydrofolate levels influence 2-aminoacrylate stress in Salmonella enterica

Abstract

In Salmonella enterica, the absence of the RidA deaminase results in the accumulation of the reactive enamine 2-aminoacrylate (2AA). The resulting 2AA stress impacts metabolism and prevents growth in some conditions by inactivating a specific target pyridoxal 5'-phosphate (PLP)-dependent enzyme(s). The detrimental effects of 2AA stress can be overcome by changing the sensitivity of a critical target enzyme or modifying flux in one or more nodes in the metabolic network. The catabolic L-alanine racemase DadX is a target of 2AA, which explains the inability of an alr ridA strain to use L-alanine as the sole nitrogen source. Spontaneous mutations that suppressed the growth defect of the alr ridA strain were identified as lesions in folE, which encodes GTP cyclohydrolase and catalyzes the first step of tetrahydrofolate (THF) synthesis. The data here show that THF limitation resulting from a folE lesion, or inhibition of dihydrofolate reductase (FolA) by trimethoprim, decreases the 2AA generated from endogenous serine. The data are consistent with an increased level of threonine, resulting from low folate levels, decreasing 2AA stress.IMPORTANCERidA is an enamine deaminase that has been characterized as preventing the 2-aminoacrylate (2AA) stress. In the absence of RidA, 2AA accumulates and damages various cellular enzymes. Much of the work describing the 2AA stress system has depended on the exogenous addition of serine to increase the production of the enamine stressor. The work herein focuses on understanding the effect of 2AA stress generated from endogenous serine pools. As such, this work describes the consequences of a subtle level of stress that nonetheless compromises growth in at least two conditions. Describing mechanisms that alter the physiological consequences of 2AA stress increases our understanding of endogenous metabolic stress and how the robustness of the metabolic network allows perturbations to be modulated.

Keywords: 2-aminoacrylate stress; RidA; alanine racemase; folate biosynthesis; metabolic integration.

Fig 1

RidA is required for full use of L-alanine as a nitrogen source. The left panel shows the growth of strains DM9404 (wild type), DM14178 (alr), DM17594 (alr ridA), and DM13760 (dadX) when L-alanine is provided as the sole source of nitrogen. The inset on the right schematically shows the pathway for L-alanine utilization. L-alanine is converted to D-alanine by one of two PLP-dependent racemases (DadX, Alr). DadX is the primary catabolic alanine racemase and is required for the use of L-alanine as a nitrogen source. D-alanine is deaminated to pyruvate by the FAD-dependent oxidoreductase DadA (EC 1.4.99), releasing NH₃ that is used as a source of nitrogen. All data are an average of three biological replicates with standard error bars.

Fig 2

folE alleles increase the utilization of L-alanine as a nitrogen source. Growth of the parent and three suppressor mutants is shown in the NCN glucose medium with L-alanine as N source (A), and minimal glucose no-carbon E (NCE) medium (NH4⁺ as N source) (B). Strains were the parental alr ridA (DM17527, open triangles), alr ridA folE183 (DM17578, open squares), alr ridA folE181 (DM17576, closed circles), and alr ridA fol182 (DM17577, closed squares). Data are an average of three biological replicates with standard error bars.

Fig 3

Suppressing allele folE181 is recessive. Two single colonies from each relevant strain were patched onto NB, and the replica was printed to the medium indicated. Growth was observed after 18- and 41-h incubation. Strains were A (alr); B (alr ridA); C (alr ridA folE181); D (alr ridA folE181/pBAD24-folE); and E (alr ridA folE181/VOC). All media was supplemented with 0.2% L-arabinose to induce expression of the plasmid-encoded folE.

Fig 4

Biosynthetic pathway for tetrahydrofolate. A simplified schematic of the folate biosynthetic pathway that results in the formation of tetrahydrofolate is shown. FolE catalyzes the first committed step in the pathway and FolA the last. DHNP can be taken up and enter the pathway as indicated. Trimethoprim can be taken up and inhibits the FolA enzyme. Abbreviations: DHNP, D-erythro-7,8-dihydroneopterin; 7,8-DHF, 7,8-dihydrofolate; THF, tetrahydrofolate; GTP, guanosine triphosphate.

Fig 5

Inhibition of folate biosynthesis allows utilization of L-alanine. Strains DM17594 (alr ridA; circles) and DM17593 (alr ridA folE181; squares) were grown in NCN medium with L-alanine as nitrogen source with (open symbols) or without (closed symbols) added TMP (0.24 µg/mL). Data are an average of three biological replicates with standard error bars.

Fig 6

folE alleles restore growth of an alr ridA mutant on pyruvate medium. Strains DM17594 (alr ridA; circles), DM17577 (alr ridA folE182; squares), and DM17578 (alr ridA folE183; triangles) were grown in a minimal medium with pyruvate as a carbon source. Additions Ile (filled circles), and TMP (half-filled circles) were added to DM17594. Data are an average of three biological replicates with standard error bars.

Fig 7

Reduced folate synthesis impacts thiamine synthesis. Soft agar was seeded with DM17577 (alr ridA folE) and overlaid on four minimal NCE glucose plates containing different nutrients; (A) no addition, (B) isoleucine, (C) thiamine, and (D) histidine. Two microliters containing 158 nmoles of adenine were spotted at The position indicated by the white X. The plates were incubated at 37C overnight, and growth was detected as turbidity-present over plates except in the zone of killing around adenine on plates A and B.