Endogenous Synthesis of 2-Aminoacrylate Contributes to Cysteine Sensitivity in Salmonella enterica

Ernst, Dustin C.; Lambrecht, Jennifer A.; Schomer, Rebecca A.; Downs, Diana M.

doi:10.1128/jb.01960-14

Cited by 36 publications

(45 citation statements)

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“…IlvE activity was measured in crude lysates as previously described (8). Cultures were grown at 37°C with aeration to stationary phase in 2 ml of NB.…”

Section: Methodsmentioning

confidence: 99%

“…Previous analysis of S. enterica showed that PLP-dependent serine/threonine dehydratases (IlvA/TdcB; EC 4.3.1.19), cysteine desulfhydrase (CdsH; EC 4.4.1.1), and diaminopropionate ammonia-lyase (DpaL; EC 4.3.1.15) generated free 2AA from exogenous serine, cysteine, and diaminopropionate, respectively, leading to complete growth inhibition in the absence of RidA (7,8; D. C. Ernst, M. E. Anderson, and D. M. Downs, submitted for publication). In the absence of exogenous 2AA precursors, basal IlvA activity produced sufficient 2AA from endogenous serine to cause a minor growth defect in ridA strains grown in minimal glucose medium (9).…”

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confidence: 99%

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2-Aminoacrylate Stress Induces a Context-Dependent Glycine Requirement in ridA Strains of Salmonella enterica

Ernst

Downs

2016

J Bacteriol

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The reactive enamine 2-aminoacrylate (2AA) is a metabolic stressor capable of damaging cellular components. Members of the broadly conserved Rid (RidA/YER057c/UK114) protein family mitigate 2AA stress in vivo by facilitating enamine and/or imine hydrolysis. Previous work showed that 2AA accumulation in ridA strains of Salmonella enterica led to the inactivation of multiple target enzymes, including serine hydroxymethyltransferase (GlyA). However, the specific cause of a ridA strain's inability to grow during periods of 2AA stress had yet to be determined. Work presented here shows that glycine supplementation suppressed all 2AA-dependent ridA strain growth defects described to date. Depending on the metabolic context, glycine appeared to suppress ridA strain growth defects by eliciting a GcvB small RNA-dependent regulatory response or by serving as a precursor to one-carbon units produced by the glycine cleavage complex (GCV). In either case, the data suggest that GlyA is the most physiologically sensitive target of 2AA inactivation in S. enterica. The universally conserved nature of GlyA among free-living organisms highlights the importance of RidA in mitigating 2AA stress. IMPORTANCEThe RidA stress response prevents 2-aminoacrylate (2AA) damage from occurring in prokaryotes and eukaryotes alike. 2AA inactivation of serine hydroxymethyltransferase (GlyA) from Salmonella enterica restricts glycine and one-carbon production, ultimately reducing fitness of the organism. The cooccurrence of genes encoding 2AA production enzymes and serine hydroxymethyltransferase (SHMT) in many genomes may in part underlie the evolutionary selection for Rid proteins to maintain appropriate glycine and one-carbon metabolism throughout life. R id (RidA/YER057c/UK114) proteins are found throughout each kingdom of life, indicating that these proteins play fundamental roles in cellular processes. Bioinformatics analyses identified eight subgroups within the Rid protein family (1). The archetypal RidA subfamily is broadly distributed and includes the best-studied RidA enzyme from Salmonella enterica, while the remaining seven Rid subgroups are primarily restricted to bacteria. In S. enterica, RidA is known to preempt metabolic damage caused by the unbound reactive enamine 2-aminoacrylate (2AA) (2). In the absence of RidA, 2AA persisted in solution long enough to inactivate multiple target enzymes in vivo and in vitro (2-4). RidA proteins isolated from organisms spanning the tree of life displayed similar enamine and imine deaminase activities (5, 6), suggesting that RidA enzymes have a conserved role in preventing 2AA stress.2AA stress arises as a consequence of some pyridoxal 5=-phosphate (PLP)-dependent enzyme activities. Previous analysis of S. enterica showed that PLP-dependent serine/threonine dehydratases (IlvA/TdcB; EC 4.3.1.19), cysteine desulfhydrase (CdsH; EC 4.4.1.1), and diaminopropionate ammonia-lyase (DpaL; EC 4.3.1.15) generated free 2AA from exogenous serine, cysteine, and diaminopropionate, respectively, leading ...

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“…IlvE activity was measured in crude lysates as previously described (8). Cultures were grown at 37°C with aeration to stationary phase in 2 ml of NB.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

2-Aminoacrylate Stress Induces a Context-Dependent Glycine Requirement in ridA Strains of Salmonella enterica

Ernst

Downs

2016

J Bacteriol

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“…CdsH was provided by D. C. Ernst and was purified as previously described (37). A typical reaction mixture for monitoring the release of pyruvate at A 230 included Tris buffer (50 mM, pH 8.6), pyridoxal-5-phosphate (PLP) (25 M), purified CdsH (0.005 to 5 M), and cysteine, cystine, or S-sulfocysteine (1 mM).…”

Section: Methodsmentioning

confidence: 99%

The Cysteine Desulfhydrase CdsH Is Conditionally Required for Sulfur Mobilization to the Thiamine Thiazole in Salmonella enterica

2014

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Thiamine pyrophosphate is a required coenzyme that contains a mechanistically important sulfur atom. In Salmonella enterica, sulfur is trafficked to both thiamine biosynthesis and 4-thiouridine biosynthesis by the enzyme ThiI using persulfide (R-S-S-H) chemistry. It was previously reported that a thiI mutant strain could grow independent of exogenous thiamine in the presence of cysteine, suggesting there was a second mechanism for sulfur mobilization. Data reported here show that oxidation products of cysteine rescue the growth of a thiI mutant strain by a mechanism that requires the transporter YdjN and the cysteine desulfhydrase CdsH. The data are consistent with a model in which sulfide produced by CdsH reacts with cystine (Cys-S-S-Cys), S-sulfocysteine (Cys-S-SO 3 ؊ ), or another disulfide to form a small-molecule persulfide (R-S-S-H). We suggest that this persulfide replaced ThiI by donating sulfur to the thiamine sulfur carrier protein ThiS. This model describes a potential mechanism used for sulfur trafficking in organisms that lack ThiI but are capable of thiamine biosynthesis.

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“…Whole-genome sequencing was used to identify the causative suppressor mutations in S. enterica DM13631 and DM13718 (ptsI611). The protocol used for preparation of genomic DNA has been described previously (36).…”

Section: Methodsmentioning

confidence: 99%

Induction of the Sugar-Phosphate Stress Response Allows Saccharomyces cerevisiae 2-Methyl-4-Amino-5-Hydroxymethylpyrimidine Phosphate Synthase To Function in Salmonella enterica

2015

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Thiamine pyrophosphate is a required cofactor for all forms of life. The pyrimidine moiety of thiamine, 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P), is synthesized by different mechanisms in bacteria and plants compared to fungi. In this study, Salmonella enterica was used as a host to probe requirements for activity of the yeast HMP-P synthase, Thi5p. Thi5p synthesizes HMP-P from histidine and pyridoxal-5-phosphate and was reported to use a backbone histidine as the substrate, which would mean that it was a single-turnover enzyme. Heterologous expression of Thi5p did not complement an S. enterica HMP-P auxotroph during growth with glucose as the sole carbon source. Genetic analyses described here showed that Thi5p was activated in S. enterica by alleles of sgrR that induced the sugar-phosphate stress response. Deletion of ptsG (encodes enzyme IICB [EIICB] of the phosphotransferase system [PTS]) also allowed function of Thi5p and required sgrR but not sgrS. This result suggested that the role of sgrS in activation of Thi5p was to decrease PtsG activity. In total, the data herein supported the hypothesis that one mechanism to activate Thi5p in S. enterica grown on minimal medium containing glucose (minimal glucose medium) required decreased PtsG activity and an unidentified gene regulated by SgrR. IMPORTANCEThis work describes a metabolic link between the sugar-phosphate stress response and the yeast thiamine biosynthetic enzyme Thi5p when heterologously expressed in Salmonella enterica during growth on minimal glucose medium. Suppressor analysis (i) identified a mutant class of the regulator SgrR that activate sugar-phosphate stress response constitutively and (ii) determined that Thi5p is conditionally active in S. enterica. These results emphasized the power of genetic systems in model organisms to uncover enzyme function and underlying metabolic network structure.T hiamine pyrophosphate (TPP) is a cofactor for many central metabolic enzymes and is required at low levels by all organisms. Humans require dietary intake of thiamine, which is biosynthesized by a variety of plants, bacteria, and fungi. TPP is composed of two independently synthesized moieties, 5-(2-hydroxyethyl)-4-methylthiazole phosphate (THZ-P) and 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P). In bacteria and plants, the first steps of the HMP-P biosynthesis pathway are shared with purine biosynthesis (Fig. 1) (1-3). In these organisms, the radical S-adenosylmethionine (SAM) enzyme ThiC catalyzes an intramolecular rearrangement of the purine intermediate 5-aminoimidazole ribotide (AIR) to the pyrimidine HMP-P (4, 5).In contrast, fungi do not contain a ThiC homolog; under aerobic conditions, HMP-P is synthesized by the Thi5p enzyme family. In vivo labeling in yeast implicated histidine and pyridoxine as precursors to HMP-P biosynthesis under aerobic conditions ( , and other species have between zero and five copies of this gene (9). Genetic analysis in S. cerevisiae demonstrated that the four enzymes are f...

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Endogenous Synthesis of 2-Aminoacrylate Contributes to Cysteine Sensitivity in Salmonella enterica

Cited by 36 publications

References 50 publications

2-Aminoacrylate Stress Induces a Context-Dependent Glycine Requirement in ridA Strains of Salmonella enterica

2-Aminoacrylate Stress Induces a Context-Dependent Glycine Requirement in ridA Strains of Salmonella enterica

The Cysteine Desulfhydrase CdsH Is Conditionally Required for Sulfur Mobilization to the Thiamine Thiazole in Salmonella enterica

Induction of the Sugar-Phosphate Stress Response Allows Saccharomyces cerevisiae 2-Methyl-4-Amino-5-Hydroxymethylpyrimidine Phosphate Synthase To Function in Salmonella enterica

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