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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.

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 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.

Research has been conducted on the Salmonella enterica serovar Typhimurium's isc gene cluster. The study of the metabolic consequences of two insertions in this cluster is presented.

Research has been conducted on the PurF-independent thiamine synthesis in Salmonella entrica serovar Typhimurium. The results of the mutation isolation in a number of loci are dicussed.

A study concerning the structural characterization of the ApbE lipoprotein is examined. Experiments reveal that it is exposed to the periplasmic space.

Research was conducted to examine the proposed synthesis that the pyrimidine moiety of thiamine can be characterized independently of the first enzyme of the de novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in Salmonella typhimurium. Isotope labeling experiments were carried out with two compounds, (2-(super 13)C)glycine and (super 13C)formate. Results demonstrate that PurF-independent TPP synthesis did not function at the same capacity as that involving the PurF enzyme.

Research was conducted to examine the regulation of thiamine pyrophosphate synthesis in Salmonella typhimurium. Results revealed the existence of new gene products, apbE single mutants and apbE purfF double mutants, that require thiamine. The apbE locus was identified as the product used in thiamine synthesis. It has been observed to produce a 36-kDa membrane-associated lipoprotein.

A research study was conducted to determine the importance of rseC, a gene in the rpoE cluster of the Salmonella thyphimurium (S typhimurium). Some of the methods used were the insertion of rseB, genetic analysis, descriptive analysis and complementation analysis. Results revealed that rseC, the fourth gene in the rpoE cluster, plays a distinctive role in the thiamine synthesis in S thyphimurium independent from rpoE .

Thiamine pyrophosphate (TPP) in Salmonella typhimurium is a necessary cofactor for several enzymes in the cell. The identification and characterization of a 2-gene operon at 46 Cs on the s. typhimurium chromosome involved in thiamine biosynthesis is described. It shows that thiM is an operon with thiD and is transcriptionally regulated by TTA, suggesting a biosynthetic role in TPP synthesis.

The initial physical and genetic characterization of a thiI, a new locus that is necessary for 4-methyl-5-(beta-hydroxyethyl)-thiazole synthesis, is presented. This new gene is found to be a 1,449-bp open reading frame situated at minute 9.65 on the S. typhimurium chromosome. Research results also reveal that it encodes a 55-kDa 483-amino-acid protein. ThiI is not transcriptionally regulated by thiamine pyrophosphate.

Derivatives of purF mutants from Salmonella typhimurium that no longer needed panthotenate for thiamine-independent growth were isolated. Analysis of these mutants indicated that they were defective in succinate dehydrogenase (Sdh), an enzyme of the tricarboxylic acid cycle. Phenotypic studies showed that a defect in Sdh decreased the amount of thiamine needed by S. typhimurium. This reduced requirement correlated with levels of succinyl-coenzyme A.

Thiamine is thought to be synthesized by two alternative pathways, one involving the first four enzymes of the purine pathway and a second that can function independently of the purine pathway. Insertion mutations in purG and purI prevent thiamine synthesis through the alternative pyrimidine biosynthetic (APB) pathway under aerobic but not anaerobic growth conditions. In contrast, point mutations in purG and purI caused one of three distinct phenotypes: [Pur.sup.-] [Apb.sup.-], [Pur.sup.-] [Apb.sup.+], or [Pur.sup.+] [Apb.sup.-]. Analysis of these three mutant classes demonstrated two genetically separable functions for PurG and PurI in thiamine synthesis. In addition to their known enzymatic role in de novo purine synthesis, we propose that PurG and PurI play a novel, possibly nonenzymatic role in the APB pathway. Suppression analysis of Put Apb mutants identified two new genetic loci involved in the APB pathway, apbB and apbD. We show here that mutations in apbB and apbD cause distinct, allele-specific suppression of the thiamine requirement of purG and purI mutants. Our results suggest that PurG and PurI and one or more components of the APB pathway may function as a complex needed for aerobic function of the APB pathway.

The first five steps in de novo purine biosynthesis are involved in the formation of the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine. We show here that the first enzyme in de novo purine biosynthesis, PurF, is required for thiamine synthesis during aerobic growth on some but not other carbon sources. We show that PurF-independent thiamine synthesis depends on the recently described alternative pyrimidine biosynthetic (APB) pathway. Null mutations in zwf (encoding glucose-6-P dehydogenase), gnd (encoding gluconate-6-P dehydrogenase), purE (encoding aminoimidazole ribo-nucleotide carboxylase), and purR (encoding a regulator of gene expression) were found to affect the function of the APB pathway. A model is presented to account for the involvement of these gene products in thiamine biosynthesis via the APB pathway. Results presented herein demonstrate that function of the APB pathway can be prevented either by blocking intermediate formation or by diverting intermediate (s) from the pathway. Strong genetic evidence supports the conclusion that aminoimidazole ribotide (AIR) is an intermediate in the APB pathway.

Exogenous thiamine or thiamine phosphate forms thiamine pyrophosphate (TPP) that suppresses the transcription of the Salmonella typhimurium thi operon present at 90 min of the bacterial genome. The thi operon at 10 min is unaffected by TPP. Mutants unable to produce thiamine require either the thiamine thiazole moiety or thiamine for their growth. Most of the mutants have mutations in the 90 min area. These mutants require the thiazole moiety for growth and contain no thiamine phosphate kinase that is encoded by the thiL gene.

The ability of exogenous pantothenate to act as replacement for the needed thiamine by purF mutants of Salmonella typhimurium is negated by blocks in the oxidative pentose phosphate pathway, gnd which encodes gluconate 6-phosphate dehydrogenase or zwf which encodes glucose 6-phosphate dehydroenase. The defects resulting from the mutations can be remedied by adding more ribose 5-P which indicates that ribose 5-P is important in replacing thiamine by pantothenate.

Characterization of apbA, the new genetic locus identified in thiamine biosynthesis in Salmonella typhimurium, shows that a functional APB pathway is essential for thiamine synthesis. The phenotype of apbA mutants reveals that the pur genetic background controls the functioning of this pathway under aerobic or anaerobic growth conditions. Cloning and sequencing of the apbA gene shows that it contains a polypeptide, which is a predictor of the NAD/flavin adenine dinucleotide-binding pocket in the protein.

Evidence is presented for a novel synthesis pathway of the pyrimidine moiety of thiamine in Salmonella typhimurium. The pathway, designated as alternative pyrimidine biosynthesis (APB), bypasses the de novo synthesis of pyrimidine and is expressed during anoxic growth conditions. APB utilizes pantothenate as a precursor in pyrimidine biosynthesis. Another pyrimidine biosynthetic pathway, the purF function, which is responsible for growth in the presence of pantothenate, was shown to be affected by temperature and pantothenate levels.

EPR spectroscopic studies are conducted to understand the interaction of AdoMet with active ThiC from Salmonella enterica, which generates a free radical on the [alpha]-carbon of an amino acid residue. The AdoMet-dependent generation of free radical provides evidence for the complicated rearrangement catalyzed by ThiC, which employ free radical chemistry for radical SAM enzymes.

The purification and characterization of the ThiC protein from Salmonella enterica is reported. The results obtained provide insight into the activity of ThiC, which carries an oxygen labile [Fe-S] cluster critical for generation of 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP).

The first nutritional and biochemical activity of mutants of Salmonella enterica ApbC in the context of iron-sulfur ([Fe-S]) cluster metabolism is described. The result data shed light on the ability of ApbC to coordinate an [4Fe-4S] cluster across its dimer interface and transfer the cluster to an apoprotein acting as an [Fe-S] cluster scaffold protein, a function discovered for its eukaryotic homologues.

Research has been conducted on Salmonella enterica mutant strain which uses exogenous aminoimidazole riboside as a thiamine source. The characterization of this strain has been carried out, and the details are reported.

Research has been conducted on Fe-S cluster metabolism defect in Salmonella enterica serovar Typhimurium. Results demonstrate that this defect is affected by biochemical and nutritional properties associated with apbC or apbE mutant strains.

The oxidative pentose phosphate pathway is needed for function of the alternative pyrimidine biosynthetic pathway, a pathway which permits thiamine synthesis in the absence of the PurF enzyme in Salmonella typhimurium. Mutants that do not need the function of the oxidative pentose phosphate pathway for thiamine synthesis were isolated and characterized. Further phenotypic studies of these mutants showed that they were also affected by the presence of serine in the medium, indicating a partial defect in isoleucine biosynthesis.