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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. [ABSTRACT FROM AUTHOR]

BackgroundIt is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5'-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.ResultsPhylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5'-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.ConclusionsLike the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5'-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

Metabolism is an integrated network of biochemical pathways that assemble to generate the robust, responsive physiologies of microorganisms. Despite decades of fundamental studies on metabolic processes and pathways, our understanding of the nuance and complexity of metabolism remains incomplete. The ability to predict and model metabolic network structure, and its influence on cellular fitness, is complicated by the persistence of genes of unknown function, even in the best-studied model organisms. This review describes the definition and continuing study of the Rid superfamily of proteins. These studies are presented with a perspective that illustrates how metabolic complexity can complicate the assignment of function to uncharacterized genes. The Rid superfamily of proteins has been divided into eight subfamilies, including the well-studied RidA subfamily. Aside from the RidA proteins, which are present in all domains of life and prevent metabolic stress, most members of the Rid superfamily have no demonstrated physiological role. Recent progress on functional assignment supports the hypothesis that, overall, proteins in the Rid superfamily modulate metabolic processes to ensure optimal organismal fitness. [ABSTRACT FROM AUTHOR]

Phosphoribosylamine (PRA) is the first intermediate in the common purine/thiamine biosynthetic pathway and is primarily synthesized by the product of the purF gene, glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14). Past genetic and biochemical studies have shown that multiple mechanisms for the synthesis of PRA independent of PurF are present in Salmonella enterica. Here, we describe mutant alleles of the essential prsA gene, which encodes PRPP synthetase (E.C. 2.7.6.1), that allow PurF-independent thiamine synthesis. The mutant alleles resulted in reduced PrsA activity in extracts, caused nutritional requirements indicative of PRPP limitation and allowed non-enzymic formation of PRA due to a build-up of ribose 5-phosphate (R5P). These results emphasize the balance that must be reached between pathways competing for the same substrate to maintain robustness of the metabolic network. DOI 10.1099/mic.0.033050-0

In Salmonella enterica, tricarballylate (Tcb) catabolism requires function of TcuB, a membrane-bound protein that contains [4Fe-4S] clusters and heme. TcuB transfers electrons from reduced flavin adenine dinucleotide in the Tcb dehydrogenase (TcuA) to electron acceptors in the membrane. We recently showed that functions needed to assemble [Fe-S] clusters (i.e., the iscRSUA-hscBA-fdx operon) compensate for the lack of ApbC during growth of an apbC strain on Tcb. ApbC had been linked to [Fe-S] cluster metabolism, and we showed that an apbC strain had decreased TcuB activity. Here we report findings that expand our understanding of the regulation of expression of the iscRSUA genes in Salmonella enterica. We investigated why low levels of glucose or other saccharides restored growth of an apbC strain on Tcb. Here we report the following findings. (i) A [less than or equal to] 1 mM concentration of glucose, fructose, ribose, or glycerol restores growth of an apbC strain on Tcb. (ii) The saccharide effect results in increased levels of TcuB activity. (iii) The saccharide effect depends on the global regulatory protein Cra. (iv) Putative Cra binding sites are present in the regulatory region of the iscRSUA operon. (v) Cra protein binds to all three sites in the iscRSUA promoter region in a concentrationdependent fashion. To our knowledge, this is the first report of the involvement of Cra in [Fe-S] cluster assembly.

Iron-sulfur clusters may have been the earliest catalytic cofactors on earth, and most modern organisms use them extensively. Although members of the Archaea produce numerous iron-sulfur proteins, the major cluster assembly proteins found in the Bacteria and Eukarya are not universally conserved in archaea. Free-living archaea do have homologs of the bacterial apbC and eukaryotic NBP35 genes that encode iron-sulfur cluster carrier proteins. This study exploits the genetic system of Salmonella enterica to examine the in vivo functionality of apbC/NBP35 homologs from three archaea: Methanococcus maripaludis, Methanocaldococcus jannaschii, and Sulfolobus solfataricus. All three archaeal homologs could correct the tricarballylate growth defect of an S. enterica apbC mutant. Additional genetic studies showed that the conserved Walker box serine and the Cys-X-X-Cys motif of the M. maripaludis MMP0704 protein were both required for function in vivo but that the amino-terminal ferredoxin domain was not. MMP0704 protein and an MMP0704 variant protein missing the N-terminal ferredoxin domain were purified, and the Fe-S clusters were chemically reconstituted. Both proteins bound equimolar concentrations of Fe and S and had UV-visible spectra similar to those of known [4Fe-4S] cluster-containing proteins. This family of dimeric iron-sulfur carrier proteins evolved before the archaeal and eukaryal lineages diverged, representing an ancient mode of cluster assembly.

The response of a cell to integrated stresses was investigated using environmental and/or genetic perturbations that disrupted labile iron homeostasis and increased oxidative stress. The effects of the perturbations were monitored as nutritional requirements, and were traced to specific enzymic targets. A yggX gshA cyaY mutant strain required exogenous thiamine and methionine for growth. The thiamine requirement, which had previously been linked to the Fe-S cluster proteins ThiH and ThiC, was responsive to oxidative stress and was not directly affected by manipulation of the iron pool. The methionine requirement was associated with the activity of sulfite reductase, an enzyme that appeared responsive to disruption of labile iron homeostasis. The results are incorporated in a model to suggest how the activity of iron-containing enzymes not directly sensitive to oxygen can be decreased by oxidation of the labile iron pool.

Strains of Salmonella enterica lacking YggX and the cellular reductant glutathione exhibit defects similar to those resulting from iron deficiency and oxidative stress. Mutant strains are sensitive to hydrogen peroxide and superoxide, deregulate the expression of the Fur-regulated gene entB, and fail to grow on succinate medium. Suppression of some yggX gshA mutant phenotypes by the cell-permeable iron chelator deferoxamine allowed the conclusion that increased levels of cellular Fenton chemistry played a role in the growth defects. The data presented are consistent with a scenario in which glutathione acts as a physiological chelator of the labile iron pool and in which YggX acts upstream of the labile iron pool by preventing superoxide toxicity.

Mutants of Salmonella enterica lacking apbC have nutritional and biochemical properties indicative of defects in [Fe-S] cluster metabolism. Here we show that apbC is required for S. enterica to use tricarballylate as a carbon and energy source. Tricarballylate catabolism requires three gene products, TcuA, TcuB, and TcuC. Of relevance to this work is the TcuB protein, which has two [4Fe-4S] clusters required for function, making it a logical target for the apbC effect. TcuB activity was 100-fold lower in an apbC mutant than in the isogenic [apbC.sup.+] strain. Genetic data show that derepression of the iscRSUA-hscAB-fdx-orf3 operon or overexpression of iscU from a plasmid compensates for the lack of ApbC during growth on tricarballylate. The studies described herein provide evidence that the scaffold protein IscU has a functional overlap with ApbC and that ApbC function is involved in the synthesis of active TcuB.

The YjgF/YER057c/UK114 family of proteins is conserved across the three domains of life, yet no biochemical function has been clearly defined for any member of this family. In Salmonella enterica, a deletion of yjgF results in a requirement for isoleucine when the mutant strain is grown in glucose-serine or pyruvate medium. Feedback inhibition of IlvA is required for the curative effect of isoleucine on glucose-serine medium. On pyruvate medium, yjgF mutants are unable to synthesize enough isoleucine for growth. From this study, we conclude that the isoleucine requirement of a yjgF mutant on pyruvate is a consequence of the decreased transaminase B (IlvE) activity that has previously been characterized in these mutants.

Cobalt is essential for growth of Salmonella enterica and other organisms, yet this metal can be toxic when present in excess. Wild-type Salmonella exhibits several metabolic defects when grown in the presence of cobalt, some of which generate visible growth consequences. Work herein identifies sulfur assimilation, iron homeostasis, and Fe-S cluster metabolism as targets for cobalt toxicity. In each ease it is proposed that cobalt exerts its effect by one of two mechanisms: direct competition with iron or indirectly through a mechanism that involves the status of reduced thiols in the cell. Cobalt toxicity results in decreased siroheme production, increased expression of the Fur regulon, and decreased activity of Fe-S cluster proteins. The consequences of reduced sulfite reduetase activity in particular are exacerbated by the need for glutathione in cobalt resistance. Significantly, independent metabolic perturbations could be detected at cobalt concentrations below those required to generate a detectable growth defect.

In Salmonella enterica, the biosynthetic pathways for the generation of purines and the essential cofactor thiamine pyrophosphate branch after sharing five enzymatic steps. Phosphoribosyl amine (PRA) is the first intermediate in the common portion of the pathway and is generated from phosphoribosylpyrophosphate and glutamine by the PurF enzyme (phosphoribosylpyrophosphate amidotransferase). A null mutation in yjgF allows PurF-independent PRA formation by an unknown mechanism. The tryptophan biosynthetic enzyme complex anthranilate synthase-phosphoribosyitransferase, composed of the TrpD and TrpE proteins, was shown to be essential for PRA formation in strains lacking both yjgF and purF. The activity generating PRA in a yjgF mutant background has features that distinguish it from the TrpDE-mediated PRA formation shown previously for this enzyme in strains with an active copy of yjgF. The data presented here are consistent with a model in which the absence of YjgF uncovers a new catalytic activity of TrpDE.

Several cellular pathways have been identified which affect the efficiency of thiamine biosynthesis in Salmonella enterica. Mutants defective in iron-sulfur (Fe-S) cluster metabolism are less efficient at synthesis of the pyrimidine moiety of thiamine. These mutants are compromised for the conversion of aminoimidazole ribotide (AIR) to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P), not the synthesis of AIR. The gene product ThiC contains potential ligands for an Fe-S cluster that are required for function in vivo. The conversion of AIR to HMP-P is sensitive to oxidative stress, and variants of ThiC have been identified that have increased sensitivity to oxidative growth conditions. The data are consistent with ThiC or an as-yet-unidentified protein involved in HMP-P synthesis containing an Fe-S cluster required for its physiological function.

A mutant gyrA allele resulting in an A271E substitution in the DNA gyrase protein generated astrain unable to grow on the [C.sub.4]-dicarboxylates succinate, malate, and fumarate. Bacteria harboring gyrA751 displayed decreased negative supercoiling in cells. Expression of the dctA gene, which encodes the [C.sub.4]-dicarboxylate transporter, was reduced in a gyrA751 mutant, providing the first evidence that dctA expression is supercoiling sensitive and uncovering a simple metabolic screen for lesions in gyrase that reduce negative supercoiling.

A combination of in silico, in vivo and in vitro approaches is used to achieve a full understanding of microbial metabolism. Microbial metabolism is best appreciated when a complex system that is more than the sum of its parts is considered along with an in vivo, phenotypically driven method, which has provided the means to describe the role of uncharacterized open reading frames (ORFs) in the context of metabolism.

An allele of rpoD (rpoD1181) that results in increased synthesis of the pyrimidine moiety of thiamine in Salmonella enterica was identified. The S508Y substitution caused by rpoD1181 is analogous to the S506F derivative of the Escherichia coli protein. The properties of this E. coli mutant protein have been well characterized in vitro. Identification of a metabolic phenotype caused by the rpoD1181 allele of S. enterica allows past in vitro results to be incorporated in continuing efforts to understand cellular processes that are integrated with the thiamine biosynthetic pathway.

The YjgF/YER057c/UK114 family is a highly conserved class of proteins that is represented in the three domains of life. Thus far, a biochemical function demonstrated for these proteins in vivo or in vitro has yet to be defined. In several organisms, strains lacking a YjgF homolog have a defect in branched-chain amino acid biosynthesis. This study probes the connection between yjgF and isoleucine biosynthesis in Salmonella enterica. In strains lacking yjgF the specific activity of transaminase B, catalyzing the last step in the synthesis of isoleucine, was reduced. In the absence of yjgF, transaminase B activity could be restored by inhibiting threonine deaminase, the first enzymatic step in isoleucine biosynthesis. Strains lacking yjgF showed an increased sensitivity to sulfometruron methyl, a potent inhibitor of acetolactate synthase. Based on work described here and structural reports in the literature, we suggest a working model in which YjgF has a role in protecting the cell from toxic effects of imbalanced ketoacid pools.

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. [ABSTRACT FROM AUTHOR]

In bacteria, the biosynthetic pathway for the hydroxymethyl pyrimidine moiety of thiamine shares metabolic intermediates with purine biosynthesis. The two pathways branch after the compound aminoimidazole ribotide. Past work has shown that the first common metabolite, phosphoribosyl amine (PRA), can be generated in the absence of the first enzyme in purine biosynthesis, PurF. PufF-independent PRA synthesis is dependent on both strain background and growth conditions. Standard genetic approaches have not identified a gene product singly responsible for PufF-independent PRA formation. This result has led to the hypothesis that multiple enzymes contribute to PRA synthesis, possibly as the result of side products from their dedicated reaction. A mutation that was able to restore PRA synthesis in a purF gnd mutant strain was identified and found to map in the gene coding for the TrpD subunit of the anthranilate synthase (AS)-phosphoribosyl transferase (PRT) complex. Genetic analyses indicated that wild-type AS-PRT was able to generate PRA in vivo and that the P362L mutant of TrpD facilitated this synthesis. In vitro activity assays showed that the mutant AS was able to generate PRA from ammonia and phosphoribosyl pyrophosphate. This work identifies a new reaction catalyzed by AS-PRT and considers it in the context of cellular thiamine synthesis and metabolic flexibility.

Research has been conducted on purine biosynthetic gene mutations. The effect of these mutations on unanticipated phenotypes resulting from the purine, histidine and thiamine pyrophosphate biosynthetic pathways has been investigated, and the results are reported.

Biochemical analysis reveals that the mutant aminoimidazole ribonucleotide synthase protein is defective in ATP binding and this defect can be corrected by high levels of formyl-glycinamidine ribonucleotide (FGAM) in Salmonella enterica. Data indicate that that FGAM compensates for the decreased affinity for ATP by the mutant enzyme.