Interpreting the role of antioxidants in vivo: A cautionary tale.

Bacteria have a remarkable ability to sense environmental stresses and to respond to these stressors by adapting their metabolism and physiology. In recent publications, investigators have suggested that multiple stresses that cause cell death share the mechanistic feature of stimulating the formation of reactive oxygen species (ROS). A central piece of evidence cited in these claims is the ability of exogenous antioxidant compounds to mitigate stress-related cell death. The validity of attributing a positive effect of exogenous antioxidants to ROS-mediated stress is challenged by an important study by Korshunov and Imlay in this issue of Molecular Microbiology. This study reports biochemical data that convincingly show that some commonly used antioxidants quench oxidants orders of magnitude too slowly to have a significant effect on the concentration of ROS in the cell. Under conditions where antioxidants minimize cell death, they also slow growth. Significantly, slowing cell growth by other means has the same restorative effect as adding an antioxidant. Based on the solid biochemical and genetic data, Korshunov and Imlay make the case for discarding the use of antioxidants to diagnose conditions that generate increased internal ROS production.

Tetrahydrofolate levels influence 2-aminoacrylate stress in Salmonella enterica.

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.

Functional characterization of the dbu locus for D-branched-chain amino acid catabolism in Pseudomonas putida.

Pseudomonas putida is a metabolically robust soil bacterium that employs a diverse set of pathways to utilize a wide range of nutrients. The versatility of this microorganism contributes to both its environmental ubiquity and its rising popularity as a bioengineering chassis. In P. putida, the newly named dbu locus encodes a transcriptional regulator (DbuR), D-amino acid oxidase (DbuA), Rid2 protein (DbuB), and a putative transporter (DbuC). Current annotation implicates this locus in the utilization of D-arginine. However, data obtained in this study showed that genes in the dbu locus are not required for D-arginine utilization, but, rather, this locus is involved in the catabolism of multiple D-branched-chain amino acids (D-BCAA). The oxidase DbuA was required for catabolism of each D-BCAA and D-phenylalanine, while the requirements for DbuC and DbuB were less stringent. The functional characterization of the dbu locus contributes to our understanding of the metabolic network of P. putida and proposes divergence in function between proteins annotated as D-arginine oxidases across the Pseudomonas genus.IMPORTANCEPseudomonas putida is a non-pathogenic bacterium that is broadly utilized as a host for bioengineering and bioremediation efforts. The popularity of P. putida as a chassis for such efforts is attributable to its physiological versatility and ability to metabolize a wide variety of compounds. Pathways for L-amino acid metabolism in this microbe have been rather well studied, primarily because of their relevance to efforts in foundational physiology research, as well as the commercial production of economically pertinent compounds. However, comparatively little is known about the metabolism of D-amino acids despite evidence showing the ability of P. putida to metabolize these enantiomers. In this work, we characterize the D-BCAA catabolic pathway of P. putida and its integration with the essential L-BCAA biosynthetic pathway. This work expands our understanding of the metabolic network of Pseudomonas putida, which has potential applications in efforts to model and engineer the metabolic network of this organism.

Reactive Enamines and Imines In Vivo: Lessons from the RidA Paradigm.

Metabolic networks are webs of integrated reactions organized to maximize growth and replication while minimizing the detrimental impact that reactive metabolites can have on fitness. Enamines and imines, such as 2-aminoacrylate (2AA), are reactive metabolites produced as short-lived intermediates in a number of enzymatic processes. Left unchecked, the inherent reactivity of enamines and imines may perturb the metabolic network. Genetic and biochemical studies have outlined a role for the broadly conserved reactive intermediate deaminase (Rid) (YjgF/YER057c/UK114) protein family, in particular RidA, in catalyzing the hydrolysis of enamines and imines to their ketone product. Herein, we discuss new findings regarding the biological significance of enamine and imine production and outline the importance of RidA in controlling the accumulation of reactive metabolites.

2-Aminoacrylate stress damages diverse PLP-dependent enzymes in vivo.

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for a class of enzymes that catalyze diverse reactions in central metabolism. The catalytic mechanism of some PLP-dependent enzymes involves the generation of reactive enamine intermediates like 2-aminoacrylate (2AA). 2AA can covalently modify PLP in the active site of some PLP-dependent enzymes and subsequently inactivate the enzyme through the formation of a PLP-pyruvate adduct. In the absence of the enamine/imine deaminase RidA, Salmonella enterica experiences 2AA-mediated metabolic stress. Surprisingly, PLP-dependent enzymes that generate endogenous 2AA appear to be immune to its attack, while other PLP-dependent enzymes accumulate damage in the presence of 2AA stress; however, structural determinants of 2AA sensitivity are unclear. In this study, we refined a molecular method to query proteins from diverse systems for their sensitivity to 2AA in vivo. This method was then used to examine active site residues of Alr, a 2AA-sensitive PLP-dependent enzyme, that affect its sensitivity to 2AA in vivo. Unexpectedly, our data also showed that a low level of 2AA stress can persist even in the presence of a functional RidA. In summary, this study expands our understanding of 2AA metabolism and takes an initial step toward characterizing the structural determinants influencing enzyme susceptibility to damage by free 2AA.

Genetic Analysis Using Vitamin B6 Antagonist 4-Deoxypyridoxine Uncovers a Connection between Pyridoxal 5'-Phosphate and Coenzyme A Metabolism in Salmonella enterica.

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for organisms in all three domains of life. Despite the central role of PLP, many aspects of vitamin B6 metabolism, including its integration with other biological pathways, are not fully understood. In this study, we examined the metabolic perturbations caused by the vitamin B6 antagonist 4-deoxypyridoxine (dPN) in a ptsJ mutant of Salmonella enterica serovar Typhimurium LT2. Our data suggest that PdxK (pyridoxal/pyridoxine/pyridoxamine kinase [EC 2.7.1.35]) phosphorylates dPN to 4-deoxypyridoxine 5'-phosphate (dPNP), which in turn can compromise the de novo biosynthesis of PLP. The data are consistent with the hypothesis that accumulated dPNP inhibits GlyA (serine hydroxymethyltransferase [EC 2.1.2.1]) and/or GcvP (glycine decarboxylase [EC 1.4.4.2]), two PLP-dependent enzymes involved in the generation of one-carbon units. Our data suggest that this inhibition leads to reduced flux to coenzyme A (CoA) precursors and subsequently decreased synthesis of CoA and thiamine. This study uncovers a link between vitamin B6 metabolism and the biosynthesis of CoA and thiamine, highlighting the integration of biochemical pathways in microbes. IMPORTANCE PLP is a ubiquitous cofactor required by enzymes in diverse metabolic networks. The data presented here expand our understanding of the toxic effects of dPN, a vitamin B6 antagonist that is often used to mimic vitamin B6 deficiency and to study PLP-dependent enzyme kinetics. In addition to de novo PLP biosynthesis, we define a metabolic connection between vitamin B6 metabolism and synthesis of thiamine and CoA. This work provides a foundation for the use of dPN to study vitamin B6 metabolism in other organisms.

Pyridoxal and α-Ketoglutarate Independently Improve Function of Saccharomyces cerevisiae Thi5 in the Metabolic Network of Salmonella enterica.

Microbial metabolism is often considered modular, but metabolic engineering studies have shown that transferring pathways, or modules, between organisms is not always straightforward. The Thi5-dependent pathway(s) for synthesis of the pyrimidine moiety of thiamine from Saccharomyces cerevisiae and Legionella pneumophila functioned differently when incorporated into the metabolic network of Salmonella enterica. Function of Thi5 from Saccharomyces cerevisiae (ScThi5) required modification of the underlying metabolic network, while LpThi5 functioned with the native network. Here we probe the metabolic requirements for heterologous function of ScThi5 and report strong genetic and physiological evidence for a connection between alpha-ketoglutarate (αKG) levels and ScThi5 function. The connection was built with two classes of genetic suppressors linked to metabolic flux or metabolite pool changes. Further, direct modulation of nitrogen assimilation through nutritional or genetic modification implicated αKG levels in Thi5 function. Exogenous pyridoxal similarly improved ScThi5 function in S. enterica. Finally, directly increasing αKG and PLP with supplementation improved function of both ScThi5 and relevant variants of Thi5 from Legionella pneumophila (LpThi5). The data herein suggest structural differences between ScThi5 and LpThi5 impact their level of function in vivo and implicate αKG in supporting function of the Thi5 pathway when placed in the heterologous metabolic network of S. enterica. IMPORTANCE Thiamine biosynthesis is a model metabolic node that has been used to extend our understanding of metabolic network structure and individual enzyme function. The requirements for in vivo function of the Thi5-dependent pathway found in Legionella and yeast are poorly characterized. Here we suggest that αKG modulates function of the Thi5 pathway in S. enterica and provide evidence that structural variation between ScThi5 and LpThi5 contributes to their functional differences in a Salmonella enterica host.

Mechanism of Pyridoxine 5'-Phosphate Accumulation in Pyridoxal 5'-Phosphate-Binding Protein Deficiency.

The pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) plays an important role in vitamin B6 homeostasis. Loss of this protein in organisms such as Escherichia coli and humans disrupts the vitamin B6 pool and induces intracellular accumulation of pyridoxine 5'-phosphate (PNP), which is normally undetectable in wild-type cells. This accumulated PNP could affect diverse metabolic systems through the inhibition of some PLP-dependent enzymes. In this study, we investigated the as-yet-unclear mechanism of intracellular accumulation of PNP due to the loss of PLPBP protein encoded by yggS in E. coli. Genetic studies using several PLPBP-deficient strains of E. coli lacking a known enzyme(s) in the de novo or salvage pathways of vitamin B6, including pyridoxine (amine) 5'-phosphate oxidase (PNPO), PNP synthase, pyridoxal kinase, and pyridoxal reductase, demonstrated that neither the flux from the de novo pathway nor the salvage pathway solely contributed to the PNP accumulation caused by the PLPBP mutation. Studies of the strains lacking both PLPBP and PNPO suggested that PNP shares the same pool with PMP, and showed that PNP levels are impacted by PMP levels and vice versa. Here, we show that disruption of PLPBP perturbs PMP homeostasis, which may result in PNP accumulation in the PLPBP-deficient strains. IMPORTANCE A PLP-binding protein (PLPBP) from the conserved COG0325 family has recently been recognized as a key player in vitamin B6 homeostasis in various organisms. Loss of PLPBP disrupts vitamin B6 homeostasis and perturbs diverse metabolisms, including amino acid and α-keto acid metabolism. Accumulation of PNP is a characteristic phenotype of PLPBP deficiency and is suggested to be a potential cause of the pleiotropic effects, but the mechanism of this accumulation has been poorly understood. In this study, we show that fluxes for PNP synthesis/metabolism are not responsible for the accumulation of PNP. Our results indicate that PLPBP is involved in the homeostasis of pyridoxamine 5'-phosphate, and that its disruption may lead to the accumulation of PNP in PLPBP deficiency.

The Rid family member RutC of Escherichia coli is a 3-aminoacrylate deaminase.

The Rid protein family (PF14588, IPR006175) is divided into nine subfamilies, of which only the RidA subfamily has been characterized biochemically. RutC, the founding member of one subfamily, is encoded in the pyrimidine utilization (rut) operon that encodes a pathway that allows Escherichia coli to use uracil as a sole nitrogen source. Results reported herein demonstrate that RutC has 3-aminoacrylate deaminase activity and facilitates one of the reactions previously presumed to occur spontaneously in vivo. RutC was active with several enamine-imine substrates, showing similarities and differences in substrate specificity with the canonical member of the Rid superfamily, Salmonella enterica RidA. Under standard laboratory conditions, a Rut pathway lacking RutC generates sufficient nitrogen from uracil for growth of E. coli. These results support a revised model of the Rut pathway and provide evidence that Rid proteins may modulate metabolic fitness, rather than catalyzing essential functions.

Loss of YggS (COG0325) impacts aspartate metabolism in Salmonella enterica.

YggS is a pyridoxal 5'-phosphate (PLP)-binding protein of the conserved COG0325 family. Despite a connection with vitamin B6 homeostasis in many species, neither a precise biochemical activity nor the molecular mechanism of how YggS contributes to cellular function has been described. In a transposon mutagenesis screen, we found that insertions in aspC (encoding a PLP-dependent aspartate aminotransferase, EC 2.6.1.1) in a Salmonella enterica strain lacking yggS caused a synthetic growth defect, which could be rescued by the addition of exogenous aspartate. Characterization of spontaneous suppressors which improved the growth of the yggS aspC double mutant suggested that this synthetic aspartate limitation was dependent on TyrB, a PLP-dependent aromatic amino acid aminotransferase (EC 2.6.1.57). Genetic and biochemical data were consistent with the hypothesis that TyrB activity was inhibited by accumulated pyridoxine 5'-phosphate and α-keto acids caused by a yggS mutation. This study provides data consistent with a working model implicating YggS in modulating concentrations of B6 vitamers via transamination.

Functional characterization of the HMP-P synthase of Legionella pneumophila (Lpg1565).

The production of the pyrimidine moiety in thiamine synthesis, 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P), has been described to proceed through the Thi5-dependent pathway in Saccharomyces cerevisiae and other yeast. Previous work found that ScThi5 functioned poorly in a heterologous context. Here we report a bacterial ortholog to the yeast HMP-P synthase (Thi5) was necessary for HMP synthesis in Legionella pneumophila. Unlike ScThi5, LpThi5 functioned in vivo in Salmonella enterica under multiple growth conditions. The protein LpThi5 is a dimer that binds pyridoxal-5'-phosphate (PLP), apparently without a solvent-exposed Schiff base. A small percentage of LpThi5 protein co-purifies with a bound molecule that can be converted to HMP. Analysis of variant proteins both in vivo and in vitro confirmed that residues in sequence motifs conserved across bacterial and eukaryotic orthologs modulate the function of LpThi5. IMPORTANCE: Thiamine is an essential vitamin for the vast majority of organisms. There are multiple strategies to synthesize and salvage this vitamin. The predominant pathway for synthesis of the pyrimidine moiety of thiamine involves the Fe-S cluster protein ThiC. An alternative pathway utilizes Thi5, a novel enzyme that uses PLP as a substrate. The Thi5-dependent pathway is poorly characterized in yeast and has not been characterized in Bacteria. Here we demonstrate that a Thi5-dependent pathway is necessary for thiamine biosynthesis in Legionella pneumophila and provide biochemical data to extend knowledge of the Thi5 enzyme, the corresponding biosynthetic pathway, and the role of metabolic network architecture in optimizing its function.

Balancing cost and benefit: How E. coli cleverly averts disulfide stress caused by cystine.

Building a robust, stable network must include strategies to minimize perturbations caused by environmental stress, while optimizing cellular fitness. The introduction of oxygen into the Earth's atmosphere brought challenges for the microbes that had evolved enzyme machinery and metabolic network stability in the anoxic world. Unable to generate new enzyme paradigms and metabolic networks de novo, organisms have evolved strategies to neutralize the impact of oxygen that can be added to and integrated into the existing metabolic framework. This issue of Molecular Microbiology includes a paper by Korshunov et al. in which the authors describe an elegant strategy that Escherichia coli has evolved to minimize metabolic stress that results from the acquisition and use of cystine, the oxidized form of cysteine, as a source of cellular sulfur. This study highlights how a strategy involving both cost and benefit can result in a functional, but energy intensive mechanism for this bacterium to thrive in an oxic world.

Inhibition of glycine cleavage system by pyridoxine 5'-phosphate causes synthetic lethality in glyA yggS and serA yggS in Escherichia coli.

The YggS/Ybl036c/PLPBP family includes conserved pyridoxal 5'-phosphate (PLP)-binding proteins that play a critical role in the homeostasis of vitamin B6 and amino acids. Disruption of members of this family causes pleiotropic effects in many organisms by unknown mechanisms. In Escherichia coli, conditional lethality of the yggS and glyA (encoding serine hydroxymethyltransferase) has been described, but the mechanism of lethality was not determined. Strains lacking yggS and serA (3-phosphoglycerate dehydrogenase) were conditionally lethality in the M9-glucose medium supplemented with Gly. Analyses of vitamin B6 pools found the high-levels of pyridoxine 5'-phosphate (PNP) in the two yggS mutants. Growth defects of the double mutants could be eliminated by overexpressing PNP/PMP oxidase (PdxH) to decrease the PNP levels. Further, a serA pdxH strain, which accumulates PNP in the presence of yggS, exhibited similar phenotype to serA yggS mutant. Together these data suggested the inhibition of the glycine cleavage (GCV) system caused the synthetic lethality. Biochemical assays confirmed that PNP disrupts the GCV system by competing with PLP in GcvP protein. Our data are consistent with a model in which PNP-dependent inhibition of the GCV system causes the conditional lethality observed in the glyA yggS or serA yggS mutants.

Absence of MMF1 disrupts heme biosynthesis by targeting Hem1pin Saccharomyces cerevisiae.

The RidA subfamily of the Rid (YjgF/YER057c/UK114) superfamily of proteins is broadly distributed and found in all domains of life. RidA proteins are enamine/imine deaminases. In the organisms that have been investigated, lack of RidA results in accumulation of the reactive enamine species 2-aminoacrylate (2AA) and/or its derivative imine 2-iminopropanoate (2IP). The accumulated enamine/imine species can damage specific pyridoxal phosphate (PLP)-dependent target enzymes. The metabolic imbalance resulting from the damaged enzymes is organism specific and based on metabolic network configuration. Saccharomyces cerevisiae encodes two RidA homologs, one localized to the cytosol and one to the mitochondria. The mitochondrial RidA homolog, Mmf1p, prevents enamine/imine stress and is important for normal growth and maintenance of mitochondrial DNA. Here, we show that Mmf1p is necessary for optimal heme biosynthesis. Biochemical and/or genetic data herein support a model in which accumulation of 2AA and or 2IP, in the absence of Mmf1p, inactivates Hem1p, a mitochondrially located PLP-dependent enzyme required for heme biosynthesis.

An Unexpected Role for the Periplasmic Phosphatase PhoN in the Salvage of B6 Vitamers in Salmonella enterica.

Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B6, essential for cellular function in all domains of life. In many organisms, such as Salmonella enterica serovar Typhimurium and Escherichia coli, this cofactor can be synthesized de novo or salvaged from B6 vitamers in the environment. Unexpectedly, S. enterica strains blocked in PLP biosynthesis were able to use exogenous PLP and pyridoxine 5'-phosphate (PNP) as the source of this required cofactor, while E. coli strains of the same genotype could not. Transposon mutagenesis found that phoN was essential for the salvage of PLP and PNP under the conditions tested. phoN encodes a class A nonspecific acid phosphatase (EC 3.1.3.2) that is transcriptionally regulated by the PhoPQ two-component system. The periplasmic location of PhoN was essential for PLP and PNP salvage, and in vitro assays confirmed PhoN has phosphatase activity with PLP and PNP as substrates. The data suggest that PhoN dephosphorylates B6 vitamers, after which they enter the cytoplasm and are phosphorylated by kinases of the canonical PLP salvage pathway. The connection of phoN with PhoPQ and the broad specificity of the gene product suggest S. enterica is exploiting a moonlighting activity of PhoN for PLP salvage.IMPORTANCE Nutrient salvage is a strategy used by species across domains of life to conserve energy. Many organisms are unable to synthesize all required metabolites de novo and must rely exclusively on salvage. Others supplement de novo synthesis with the ability to salvage. This study identified an unexpected mechanism present in S. enterica that allows salvage of phosphorylated B6 vitamers. In vivo and in vitro data herein determined that the periplasmic phosphatase PhoN can facilitate the salvage of PLP and PNP. We suggest a mechanistic working model of PhoN-dependent utilization of PLP and PNP and discuss the general role of promiscuous phosphatases and kinases in organismal fitness.

Pyridoxal Reductase, PdxI, Is Critical for Salvage of Pyridoxal in Escherichia coli.

Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B6 and an essential cofactor in all organisms. In Escherichia coli, PLP is synthesized via the deoxyxylulose 5-phosphate (DXP)-dependent pathway that includes seven enzymatic steps and generates pyridoxine 5'-phosphate as an intermediate. Additionally, E. coli is able to salvage pyridoxal, pyridoxine, and pyridoxamine B6 vitamers to produce PLP using kinases PdxK/PdxY and pyridox(am)ine phosphate oxidase (PdxH). We found that E. coli strains blocked in PLP synthesis prior to the formation of pyridoxine 5'-phosphate (PNP) required significantly less exogenous pyridoxal (PL) than strains lacking pdxH and identified the conversion of PL to pyridoxine (PN) during cultivation to be the cause. Our data showed that PdxI, shown to have PL reductase activity in vitro, was required for the efficient salvage of PL in E. coli The pdxI+E. coli strains converted exogenous PL to PN during growth, while pdxI mutants did not. In total, the data herein demonstrated that PdxI is a critical enzyme in the salvage of PL by E. coliIMPORTANCE The biosynthetic pathway of pyridoxal 5'-phosphate (PLP) has extensively been studied in Escherichia coli, yet limited information is available about the vitamin B6 salvage pathway. We show that the protein PdxI (YdbC) is the primary pyridoxal (PL) reductase in E. coli and is involved in the salvage of PL. The orthologs of PdxI occur in a wide range of bacteria and plants, suggesting that PL reductase in the B6 salvage pathway is more widely distributed than previously expected.

The Role of YggS in Vitamin B6 Homeostasis in Salmonella enterica Is Informed by Heterologous Expression of Yeast SNZ3.

YggS (COG0325) is a pyridoxal 5'-phosphate (PLP)-binding protein proposed to be involved in homeostasis of B6 vitamers. In Salmonella enterica, lack of yggS resulted in phenotypes that were distinct and others that were similar to those of a yggS mutant of Escherichia coli Like other organisms, yggS mutants of S. enterica accumulate endogenous pyridoxine 5'-phosphate (PNP). Data herein show that strains lacking YggS accumulated ∼10-fold more PLP in growth medium than a parental strain. The deoxyxylulose 5-phosphate-dependent biosynthetic pathway for PLP and the PNP/pyridoxamine 5'-phosphate (PMP) oxidase credited with interconverting B6 vitamers were replaced with a single PLP synthase from Saccharomyces cerevisiae The impact of a yggS deletion on the intracellular and extracellular levels of B6 vitamers in this restructured strain supported a role for PdxH in PLP homeostasis and led to a general model for YggS function in PLP-PMP cycling. Our findings uncovered broader consequences of a yggS mutation than previously reported and suggest that the accumulation of PNP is not a direct effect of lacking YggS but rather a downstream consequence.IMPORTANCE Pyridoxal 5'-phosphate (PLP) is an essential cofactor for enzymes in all domains of life. Perturbations in PLP or B6 vitamer content can be detrimental, notably causing B6-dependent epilepsy in humans. YggS homologs are broadly conserved and have been implicated in altered levels of B6 vitamers in multiple organisms. The biochemical activity of YggS, expected to be conserved across domains, is not yet known. Herein, a simplified heterologous pathway minimized metabolic variables and allowed the dissection of this system to generate new metabolic knowledge that will be relevant to understanding YggS.

Putative Horizontally Acquired Genes, Highly Transcribed during Yersinia pestis Flea Infection, Are Induced by Hyperosmotic Stress and Function in Aromatic Amino Acid Metabolism.

While alternating between insects and mammals during its life cycle, Yersinia pestis, the flea-transmitted bacterium that causes plague, regulates its gene expression appropriately to adapt to these two physiologically disparate host environments. In fleas competent to transmit Y. pestis, low-GC-content genes y3555, y3551, and y3550 are highly transcribed, suggesting that these genes have a highly prioritized role in flea infection. Here, we demonstrate that y3555, y3551, and y3550 are transcribed as part of a single polycistronic mRNA comprising the y3555, y3554, y3553, y355x, y3551, and y3550 genes. Additionally, y355x-y3551-y3550 compose another operon, while y3550 can be also transcribed as a monocistronic mRNA. The expression of these genes is induced by hyperosmotic salinity stress, which serves as an explicit environmental stimulus that initiates transcriptional activity from the predicted y3550 promoter. Y3555 has homology to pyridoxal 5'-phosphate (PLP)-dependent aromatic aminotransferases, while Y3550 and Y3551 are homologous to the Rid protein superfamily (YjgF/YER057c/UK114) members that forestall damage caused by reactive intermediates formed during PLP-dependent enzymatic activity. We demonstrate that y3551 specifically encodes an archetypal RidA protein with 2-aminoacrylate deaminase activity but Y3550 lacks Rid deaminase function. Heterologous expression of y3555 generates a critical aspartate requirement in a Salmonella entericaaspC mutant, while its in vitro expression, and specifically its heterologous coexpression with y3550, enhances the growth rate of an Escherichia coli ΔaspC ΔtyrB mutant in a defined minimal amino acid-supplemented medium. Our data suggest that the y3555, y3551, and y3550 genes operate cooperatively to optimize aromatic amino acid metabolism and are induced under conditions of hyperosmotic salinity stress.IMPORTANCE Distinct gene repertoires are expressed during Y. pestis infection of its flea and mammalian hosts. The functions of many of these genes remain predicted or unknown, necessitating their characterization, as this may provide a better understanding of Y. pestis specialized biological adaptations to the discrete environments of its two hosts. This study provides functional context to adjacently clustered horizontally acquired genes predominantly expressed in the flea host by deciphering their fundamental processes with regard to (i) transcriptional organization, (ii) transcription activation signals, and (iii) biochemical function. Our data support a role for these genes in osmoadaptation and aromatic amino acid metabolism, highlighting these as preferential processes by which Y. pestis gene expression is modulated during flea infection.

Analyses of variants of the Ser/Thr dehydratase IlvA provide insight into 2-aminoacrylate metabolism in Salmonella enterica.

RidA is a conserved and broadly distributed protein that has enamine deaminase activity. In a variety of organisms tested thus far, lack of RidA results in the accumulation of the reactive metabolite 2-aminoacrylate (2AA), an obligate intermediate in the catalytic mechanism of several pyridoxal 5'-phosphate (PLP)-dependent enzymes. This study reports the characterization of variants of the biosynthetic serine/threonine dehydratase (EC 4.3.1.19; IlvA), which is a significant generator of 2AA in the bacteria Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa and the yeast Saccharomyces cerevisiae Two previously identified mutations, ilvA3210 and ilvA3211, suppressed the phenotypic growth consequences of 2AA accumulation in S. enterica Characterization of the respective protein variants suggested that they affect 2AA metabolism in vivo by two different catalytic mechanisms, both leading to an overall reduction in serine dehydratase activity. To emphasize the physiological relevance of the in vitro enzyme characterization, we sought to explain in vivo phenotypes using these data. A simple mathematical model describing the impact these catalytic deficiencies had on 2AA production was generally supported by our data. However, caveats arose when kinetic parameters, determined in vitro, were used to predict formation of the isoleucine precursor 2-ketobutyrate and model in vivo (growth) behaviors. Altogether, our data support the need for a holistic approach, including in vivo and in vitro analyses, to generate data used in understanding and modeling metabolism.

Conserved Pyridoxal 5'-Phosphate-Binding Protein YggS Impacts Amino Acid Metabolism through Pyridoxine 5'-Phosphate in Escherichia coli.

Escherichia coli YggS (COG0325) is a member of the highly conserved pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) family. Recent studies suggested a role for this protein family in the homeostasis of vitamin B6 and amino acids. The deletion or mutation of a member of this protein family causes pleiotropic effects in many organisms and is causative of vitamin B6-dependent epilepsy in humans. To date, little has been known about the mechanism by which lack of YggS results in these diverse phenotypes. In this study, we determined that the pyridoxine (PN) sensitivity observed in yggS-deficient E. coli was caused by the pyridoxine 5'-phosphate (PNP)-dependent overproduction of Val, which is toxic to E. coli The data suggest that the yggS mutation impacts Val accumulation by perturbing the biosynthetic of Thr from homoserine (Hse). Exogenous Hse inhibited the growth of the yggS mutant, caused further accumulation of PNP, and increased the levels of some intermediates in the Thr-Ile-Val metabolic pathways. Blocking the Thr biosynthetic pathway or decreasing the intracellular PNP levels abolished the perturbations of amino acid metabolism caused by the exogenous PN and Hse. Our data showed that a high concentration of intracellular PNP is the root cause of at least some of the pleiotropic phenotypes described for a yggS mutant of E. coliIMPORTANCE Recent studies showed that deletion or mutation of members of the YggS protein family causes pleiotropic effects in many organisms. Little is known about the causes, mechanisms, and consequences of these diverse phenotypes. It was previously shown that yggS mutations in E. coli result in the accumulation of PNP and some metabolites in the Ile/Val biosynthetic pathway. This work revealed that some exogenous stresses increase the aberrant accumulation of PNP in the yggS mutant. In addition, the current report provides evidence indicating that some, but not all, of the phenotypes of the yggS mutant in E. coli are due to the elevated PNP level. These results will contribute to continuing efforts to determine the molecular functions of the members of the YggS protein family.