- Research article
- Open Access
Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family
© Niehaus et al.; licensee BioMed Central. 2015
- Received: 3 November 2014
- Accepted: 27 April 2015
- Published: 15 May 2015
It 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.
Phylogenetic 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.
Like 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.
- Metabolite repair
- damage pre-emption
- reactive enamine/imine
- carbamoyl phosphate
- FAD-dependent amine oxidase
- comparative genomics
Many metabolites are prone to spontaneous and enzymatic side-reactions that form damaged compounds – a phenomenon known as metabolite damage [1-3]. Damaged metabolites are often unusable and may become toxic if they accumulate, so they must be dealt with . Two common solutions are (i) to repair the damaged metabolite by reconverting it to its original form , or (ii) to pre-empt damage by converting a potentially harmful compound into a benign one before damage occurs . Cells may also simply excrete damage products [6,7].
Certain members of the diverse and widely distributed YjgF/YER057c/UK114 family were recently shown to pre-empt metabolite damage by deaminating reactive intermediates of the branched-chain amino acid (BCAA) biosynthesis pathway, and were accordingly named RidA (reactive intermediate deaminase A). Specifically, the isoleucine biosynthesis enzyme threonine dehydratase produces enamines that tautomerize to imines (both of which are very reactive), and RidA proteins catalyze hydrolysis of the imines to 2-oxoacids (which are relatively stable) [8,9]. In bacteria and plants, the serine-derived enamine 2-aminoacrylate (2AA) can attack the pyridoxal 5'-phosphate (PLP) cofactor of branched-chain aminotransferase, which inactivates this enzyme and perturbs BCAA synthesis; RidA proteins forestall this damage by hydrolyzing the corresponding imine 2-iminopropanoate (2IP) [9,10]. 2AA could in principle attack any PLP-enzyme, and has been shown to inactivate alanine racemase , serine hydroxymethyltransferase , and aspartate aminotransferase , so that the protective effects of RidA likely extend beyond BCAA biosynthesis.
A few other disparate activities have been assigned to YjgF/YER057c/UK114 family members. These include in vitro molecular chaperone activity for Drosophila melanogaster DUK114 , endoribonuclease activity for rat L-PSP , and specialist chorismatase  and 2-aminomuconate deaminase [17,18] activities in bacterial aromatic metabolism.
There is good reason to think that YjgF/YER057c/UK114 family proteins have other – and widespread – roles in metabolic processes. The family occurs in nearly every organism in all domains of life, and some species encode multiple members. Salmonella enterica, for instance, has three YjgF/YER057c/UK114 family genes, and Streptomyces coelicolor has eleven. Although YjgF/YER057c/UK114 proteins or domains are uniformly small (~130 amino acids), their sequences are diverse and some members share <8% sequence identity [19,20]. The phylogenetic distribution pattern and sequence diversity strongly imply multiple roles, and several have been suggested in pyrimidine degradation [21,22], mitochondrial maintenance [23,24], and metabolic regulation .
To explore additional functional roles, we made phylogenetic and comparative genomic analyses of the YjgF/YER057c/UK114 family. This work enabled prediction of two novel roles for RidA proteins, for which experimental support was obtained by biochemical, genetic, and metabolomic approaches.
Phylogenetic analysis and nomenclature of the YjgF/YER057c/UK114 family
Rid subfamily nomenclature
Annotation in SEED database
Annotation in NCBI CDD
RidA/YER057c/UK114 superfamily protein
RidA/YER057c/UK114 superfamily, group 1
RidA/YER057c/UK114 superfamily, group 2, YoaB-like protein
RidA/YER057c/UK114 superfamily, group 3
RidA/YER057c/UK114 superfamily, group 4
RidA/YER057c/UK114 superfamily, group 5
RidA/YER057c/UK114 superfamily, group 6
RidA/YER057c/UK114 superfamily, group 7, YjgH-like protein
Crystal structures and mutagenesis studies have identified functionally important residues in the RidA subfamily. The highly conserved Arg107 forms salt bridges with the carboxylate oxygens of benzoate in human hp14.5  and the corresponding Arg105 hydrogen bonds with the carboxylate oxygens of serine, threonine, and 2-oxobutanoate in Escherichia coli TdcF (paralogous to S. enterica RidA)  (Figure 1B). Changing this arginine to alanine almost completely abolished imine-hydrolyzing activity in plant and S. enterica RidA proteins [8,9]. It has been suggested that conserved Tyr17 and Glu120 residues of E. coli TdcF also play a role in substrate binding and positioning of a water molecule used for imine hydrolysis, but replacing the corresponding residues with alanine had little effect on the activity of S. enterica RidA .
We analyzed residue conservation for all the Rid family proteins listed in the NCBI Conserved Domain Database, paying particular attention to the predicted active site residues (Figure 1C). The Rid1, Rid2, and Rid3 subfamilies retain the conserved arginine and glutamate residues found in RidA. The Rid4, Rid5, Rid6, and Rid7 subfamilies have the glutamate residue but the arginine residue is replaced by tryptophan in Rid4 and Rid7, and is variable in Rid5 and Rid6. The tyrosine is conserved, or conservatively replaced by phenylalanine, in all subfamilies except Rid3 where it is replaced by isoleucine and Rid2 where it is variable. The presence or absence of the critical arginine residue thus separates Rid subfamilies into two groups: (i) subfamilies RidA and Rid1-Rid3 that have this residue and are predicted to hydrolyze imines, and (ii) subfamilies Rid4-Rid7 that lack it and presumably serve other functions. Finally, it should be noted that a small minority (~10%) of RidA proteins have a serine residue instead of arginine, suggesting there may be functional diversity even within the RidA subfamily (Figure 1C).
Comparative genomic analysis of the Rid family
We used comparative genomic analysis to explore the possibility that Rid family proteins have functions besides hydrolysis of threonine dehydratase-derived imines, as suggested by their sequence diversity and phylogenetic distribution. The analysis was made on a representative set of 981 prokaryotic genomes (Additional file 1) (see Methods for details) using the SEED database and its tools, which enable ready detection of gene clustering patterns . Gene clustering in prokaryotes strongly implies a functional relationship, particularly when clusters occur in more than one configuration and in diverse organisms . Full results of the analysis are available at the SEED database (see Methods).
To home in on functional associations, we mapped the frequency with which Rid genes cluster with individual arginine and pyrimidine metabolism genes. This analysis showed that clustering centered on genes for carbamoyl phosphate-related enzymes, none of which have a PLP cofactor or are known to form enamines or imines (Figure 5B). This association suggests a novel role for Rid proteins in carbamoyl phosphate metabolism.
RidA hydrolyzes reactive imines produced by amino acid oxidases
To assess the ability of Rid family proteins to hydrolyze imines produced by FAD-dependent amine oxidases we used an assay based on the ability of imines to react rapidly with semicarbazide to produce semicarbazones, which absorb in the UV range [9,39]. The competition between semicarbazone formation and Rid-mediated hydrolysis of the enzymatically produced imines can thus be monitored spectrophotometrically. We chose to assay the three Rid proteins of S. enterica because S. enterica RidA is well studied and so can serve as a benchmark for imine-hydrolyzing activity, and because S. enterica Rid2 (YoaB) has the catalytically important arginine residue and S. enterica Rid7 (STM1549) does not.
We then tested the ability of the S. enterica proteins to hydrolyze other amino acid-derived imines. First, the rate of semicarbazone formation was determined in assays containing LOX and various amino acid substrates without Rid proteins (Additional file 2: Table S1). Amino acids with polar and charged side groups proved to be very poor substrates, and thus non-charged methionine, phenylalanine, and glutamine were chosen to test further. The amount of LOX in the assay was adjusted so that the rate of semicarbazone formation was the same for each amino acid substrate, thus allowing direct comparison of imine-hydrolyzing capabilities. At 10 μM, Rid7 did not significantly reduce semicarbazone formation for any amino acid tested, indicating that it cannot hydrolyze any of the amino acid-derived imines (Figure 6C). RidA hydrolyzed the methionine-derived imine nearly as effectively as the leucine-derived imine, reducing the rate of semicarbazone formation by 91% compared to 93%, and was only slightly less effective (86% reduction in rate of semicarbazone formation) in hydrolyzing the glutamine-derived imine (Figure 6C). Rid2 hydrolyzed the glutamine- and leucine-derived imines at similar rates (44% and 46% reduction in semicarbazone), and hydrolysed the methionine-derived imine more slowly (23% reduction in semicarbazone). RidA and Rid2 also hydrolyzed the phenylalanine-derived imine, although relatively slowly, reducing semicarbazone formation by 35% and 12%, respectively. These results show that RidA and to a lesser extent Rid2, but not Rid7, can hydrolyze several imino acids and that RidA and Rid2 differ in substrate preference.
Genetic evidence supports a RidA-carbamoyl phosphate connection
To explore the functional connection between Rid proteins and carbamoyl phosphate predicted by comparative genomics, we engineered S. enterica to accumulate carbamoyl phosphate, and ablated genes encoding each of the three Rid family proteins. S. enterica is particularly appropriate for this experiment because its RidA gene clusters with the arginine metabolic genes encoding ornithine carbamoyltransferase and Arg deiminase, the regulatory gene argR, the pyrimidine metabolic gene encoding Asp carbamoyltransferase, and carbamate kinase, suggesting that S. enterica RidA plays a role in carbamoyl phosphate metabolism. The arginine biosynthesis pathway was blocked by disrupting the gene encoding ornithine carbamoyltransferase (ArgI), and an expression plasmid containing the gene encoding the CarB subunit of carbamoyl phosphate synthetase (CPS) was introduced. This subunit is sufficient to produce carbamoyl phosphate provided that the medium contains ammonium . The plasmid used (pCA24N-carB) was isopropyl β-D-thiogalactopyranoside (IPTG)-inducible . We reasoned that an IPTG-inducible growth defect in strains lacking a Rid family member would confirm a role for this Rid protein in carbamoyl phosphate metabolism.
Metabolomic analysis indicates division of labor among S. enterica Rid proteins
The more general metabolic impact of ablating Rid family members was surveyed using a gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) metabolomics platform. We compared single ridA and triple ridA Rid2 Rid7 knockout strains with wild-type S. enterica. In total, 277 compounds were detected in all three strains, of which 144 were positively identified. We then calculated the relative -fold change for each compound between the mutant strains and wild-type. About 22% of compounds in the single RidA knockout had significant (P ≤ 0.05) -fold changes (60 of 277 total compounds; 37 of 143 identified compounds), while ~31% of compounds in the triple knockout showed significant -fold changes (87 of 277 total compounds; 52 of 143 identified compounds) (Figure 8B). Besides confirming that Rid2 and Rid7 cannot replace RidA, these data suggest that Rid2 and Rid7 have roles of their own, i.e. that there is division of labor among S. enterica Rid proteins. There was much overlap in the profiles of the two knockout strains; 78% of the compounds that changed significantly in the single knockout also did so in the triple knockout (Figure 8B). Furthermore, the changes in overlapping compounds were always in the same direction (increase or decrease), and were generally of similar magnitude (Figure 8C).
The compounds showing significant -fold changes came from diverse sectors of metabolism. One sector was sulfur metabolism; cysteine showed the largest change of any identified compound – almost 14-fold in the triple knockout (Figure 8C). A second sector was BCAA metabolism; isoleucine and leucine changed in both knockouts, as did other BCAA pathway metabolites (Figure 8C). Several compounds involved in arginine, proline, and pyrimidine metabolism changed, possibly due to a lesion in carbamoyl phosphate metabolism (Figure 8C). Purine-related metabolites were also affected, as were various sugars and sugar derivatives (Figure 8C).
Separating the Rid family into subfamilies, and analyzing patterns of residue conservation and chromosomal clustering in these subfamilies, led to one finding that was broadly predictable from previous work, and two that were not. The broadly predictable finding was that the largest and most widely distributed subfamily, RidA, most probably pre-empts damage by hydrolyzing 2IP (the tautomer of 2AA) that comes from other PLP-dependent enzymes besides the known source threonine dehydratase. 2AA is formed in the normal catalytic or side-reactions of L-cysteine desulfurase  and D-cysteine desulfhydrase , cystathionine beta-lyase , and tryptophanase , with each of which RidA clusters (Figure 3).
The first novel finding was that the RidA subfamily, the Rid2 subfamily, and probably the Rid1 and Rid3 subfamilies, which all retain the arginine residue shown to be essential for 2IP hydrolysis [8,9], can hydrolyze imines produced by FAD-dependent amine oxidases. This finding extends the known imine-hydrolyzing function of Rid proteins to a wider range of imines that are formed by enzymes in pathways very different to those involving the imine-forming PLP enzymes. The imine compounds formed by amine oxidases could potentially react with, and hence damage, various cellular components [39,42]. Thus, the physiological implications are that enamine/imine damage to metabolism is not restricted to that from 2AA/2IP and that, collectively, Rid family proteins with the conserved arginine residue could act as wide-spectrum enamine/imine damage-pre-emption enzymes.
The second novel finding was that the RidA subfamily, and possibly other Rid subfamilies, are closely connected to carbamoyl phosphate metabolism. Under physiological conditions, carbamoyl phosphate breaks down spontaneously and rapidly (half-life ~5 min at 37°C) to phosphate and isocyanate, which tautomerizes to cyanate . Isocyanate is a potent damage agent because it reacts readily with amino, thiol, carboxyl, and other groups, and so carbamoylates amino acids, proteins and other molecules . Because isocyanate and carbamoyl groups have some similarity to imines, and RidA proteins have imine hydrolase activity, it is reasonable to infer that this subfamily can pre-empt carbamoylation damage in an analogous way to its action in pre-empting imine damage. In this connection, it may be noted that a simple possibility – that RidA proteins hydrolyze carbamoyl phosphate or isocyanate to release ammonia – was not supported by pilot experiments with S. enterica RidA (Additional file 2: Figure S3). Another possibility is that Rid proteins protect a carbamoyl phosphate-related enzyme or metabolite from damage caused by 2AA or another enamine/imine-containing compound.
The Rid protein family comprises a large, widely distributed – and probably archetypal – subfamily (RidA) and seven smaller subfamilies (Rid1 through Rid7) found mainly in bacteria. The RidA through Rid3 subfamilies share a catalytically critical arginine residue and apparently serve to hydrolyze the reactive imines generated by PLP-dependent enzymes or FAD-dependent amine oxidases, thereby pre-empting the damage these imines would otherwise cause. The RidA subfamily most probably has an additional damage-pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined.
The biochemical activities and physiological functions of the Rid4 through Rid7 subfamilies remain completely unknown. Thus far it is clear that these four subfamilies are most likely not imine hydrolases since they lack the arginine residue on which imine hydrolase activity depends, and because no imine hydrolase activity was detected for S. enterica Rid7. And although the Rid1 through Rid3 subfamilies are predicted – and in the case of S. enterica Rid2, demonstrated – to have imine hydrolase activity in vitro, nothing is known about the in vivo substrates for these three subfamilies.
In sum, the Rid family as a whole still has only a few known functions, and additional functions surely remain to be discovered. It is nonetheless clear that RidA proteins are iconic examples of the emerging principle of metabolite damage pre-emption ; the same seems likely to prove true of proteins from other Rid subfamilies.
Sequences were from GenBank or the SEED database . Sequences were analyzed with the NCBI CDD database and CDTree tool. To analyze residue conservation, protein alignments were taken from NCBI CDD, trimmed with Galaxy tools (https://usegalaxy.org/root), and used to create graphics with WebLogo (http://weblogo.berkeley.edu/) [45,46]. Crystal structure analysis and graphics were made with PyMol (http://www.pymol.org/). Comparative genomic analyses were made with SEED . Figure 2 was created using iTOL tools [47,48].
A set of 981 representative genomes was chosen as follows. The algorithm for computing molecular operational taxonomic units (MOTUs) based on DNA barcode data [49,50] was used to group ~12,600 prokaryotic genomes available in the SEED database in January 2014 into ~1000 such groups. One representative genome from each MOTU was then selected based on the amount of published data (when available) and the level of research interest for different microorganisms within the MOTU. The resulting set of 927 eubacterial and 54 archaeal genomes is a convenient set that accurately represents the diversity of sequenced prokaryotes, and is not skewed by the overabundance of genomes for medically or industrially important organisms (e.g. E. coli). The Rid family was analysed in the SEED database within the functional and genomic contexts provided by SEED subsystem ‘RidA family in 981 representative prokaryotes’ available at http://pubseed.theseed.org//SubsysEditor.cgi?page=ShowSubsystem&subsystem=RidA_family_in_981_representative_prokaryotes.
The small size and sequence variability of Rid family proteins preclude accurate detection and annotation of this family in public genome databases. We therefore performed an exhaustive global search for members of this family in all prokaryotic genomes in the SEED database (~8,150 at the time of this work). To do so, 24 diverse representative Rid family DNA sequences were selected (Additional File 3) and used as queries for a global BLAST search of prokaryotic genomes with relaxed search parameters (E-value of e-4) to ensure detection of all relevant targets, including RidA domains in fusions with other proteins. A list of raw BLAST hits was generated (179,483 total) and mapped to the corresponding open reading frames in each of the SEED genomes. The resultant preliminary list of potential RidA-related ORFs (26,879 total) was analyzed and filtered by cross-mapping with the NCBI Conserved Domain Database collection using custom software tools (available upon request) with the goals (i) to filter out false positives (erroneously detected ORFs unrelated to the Rid family); (ii) to classify the Rid family into coherent subgroups ; (iii) to detect fusion events of Rid domains with other protein families; and (iv) to assign accurate consistent annotations to all identified bona fide members of the Rid family in the SEED database (20,658 ORFs total). Based on this analysis, each of the identified Rid family proteins (stand-alone or fused) was assigned to one of eight subfamilies according to the NCBI Conserved Domain Database classification (see cd00448: http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=100004) and uniformly annotated with one of the Role names from the SEED controlled vocabulary as shown in Table 1.
Chemicals and enzymes were from Sigma Aldrich (St. Louis, MO).
cDNAs and expression constructs
For expression of C-terminal His-tagged proteins in E. coli, sequences encoding predicted proteins without their stop codons were PCR-amplified (RidA, primers catgccatggctatgagcaaaactattgcgacgg and tccgctcgaggcgacgaacagcgatcgcttcaatc; STM1549, primers catgccatggctatgacgcaxacgtatcgcggtttttcc and tccgctcgagggggattcgggcaataacctttatttcg; YoaB, primers catgccatggctatgtctatcgtgcgtattgatxgc and tccgctcgagtaccgccgcgacaatcttaatctc) from genomic DNA of S. enterica subsp. enterica serovar Typhimurium str. LT2. Amplicons were digested with NcoI and XhoI and ligated into the matching sites of pET28b. All constructs were sequence-verified. The ASKA plasmid pCA24N-carB was used for CarB overexpression . This plasmid features an IPTG-inducible promoter that controls expression of carB, and confers chloramphenicol resistance.
argI833::tn10 ridA3::MudJ pCA24N-carB (-gfp)
argI833::tn10 pCA24N-carB (-gfp)
argI833::tn10 STM1549-26::kan pCA24N-carB (-gfp)
argI833::tn10 ΔyoaB626 pCA24N-carB (-gfp)
ridA3::tn10 ΔyoaB624::cat STM1549-26::kan
Protein expression and isolation
Proteins were expressed and purified as previously described . Briefly, E. coli strain BL21 (DE3) RIPL harboring each expression construct was grown in 200 mL of LB medium with 50 mg/L kanamycin at 37°C until OD at 600 nm reached 0.8. Cultures were then cooled to 22°C and isopropyl-3-D-thiogalactoside and ethanol were added to final concentrations of 0.5 mM and 4% v/v, respectively. Cultures were incubated for a further 20 h at 22°C and cells were collected and stored at -80°C. Proteins were purified from bacterial lysates with Ni-NTA superflow resin (Qiagen, Valencia, CA) columns according to the manufacturer’s protocol. Proteins were passed through PD-10 columns (GE Healthcare, Cleveland, OH) equilibrated with 5 mM triethanolamine-HCl, pH 7.6, 10% (v/v) glycerol, then concentrated to 80-120 mg/mL with Amicon Ultra-4 3,000 NMWL centrifugal filters (Millipore, Billerica, MA). Aliquots (5 μL) were frozen in liquid nitrogen and stored at -80°C.
To assess the ability of Rid proteins to hydrolyze the imine derived from leucine, asays (100 μL) contained 50 mM potassium pyrophosphate, pH 8.7, 10 mM semicarbazide-HCl (neutralized), 1 μg (3.5 units) bovine liver catalase, 0.5 μg (3 units) Crotalus adamanteus LOX, and the indicated amount of S. enterica RidA, Rid2, or Rid7. Reactions were started by adding L-leucine (final concentration 5 mM), and absorbance at 248 nm was monitored at 22°C. To assess activity against imines derived from other amino acids, assays were as above except that the amount of LOX was adjusted so that the rate of semicarbazone formation was constant (leucine, 1 μg; methionine, 1.25 μg; phenylalanine, 2 μg; glutamine, 5 μg).
Growth experiments with S. enterica
Cultures were grown at 37°C with shaking in nutrient broth (Difco) containing 20 μg/mL chloramphenicol until stationary phase and then diluted 1:100 in 5 mL of the same medium in 18 x 150 mm borosilicate culture tubes. When present, IPTG was added to a final concentration of 0.1 mM. Growth was monitored by optical density at 650 nm, and analyzed using GraphPad Prism Software (version 6.0).
Bacterial cultures were grown overnight in M9 minimal medium plus 0.2% glucose and used to inoculate 2 mL of fresh medium to an optical density of 0.05 at 600 nm. Cultures were grown at 37°C with shaking for 5-6 h until optical density reached 1.7 ± 0.1 at 600 nm, then an equivalent of 1 mL culture at an optical density of 2.0 was collected in 1.5-mL Eppendorf tubes, centrifuged at 16,000 × g for 15 s; the pellet was frozen in liquid nitrogen. The harvesting procedure was completed in <30 s. Samples were stored at -80°C. Either six (LC-MS) or 12 (GC-MS) independent cultures for each strain were analyzed. Extraction for LC-MS was conducted by adding 1.1 mL cold, degassed methanol: water (3:1) to each sample and vortexing for 20 sec prior to sonicating for 5 min. Samples were vortexed for another 20 sec then kept at -20°C for 30 min, followed by centrifugation at 14,000 × g for 5 min. The resulting supernatant was transferred to 1.5 mL Eppendorf tubes and dried with a LabConco Centrivap Concentrator (LabConco Corporation, Kansas City, MO) overnight. The dried material was dissolved in 100 μL of 5 mM ammonium acetate, 0.2% (v/v) acetic acid and analyzed by LC-MS. Chromatography was performed on an Agilent 1290 Infinity LC System (Agilent Technologies, Santa Clara, CA) using a Waters Acquity 1.7 μm BEH HILIC 150 x 2.1 mm HPLC column as previously described . Extraction for GC-MS analysis was conducted by adding 1 mL of cold, degassed acetonitrile:isopropanol:water (3:3:2) and vortexing for 10 sec. Samples were placed on shaker at 4°C for 4 min then centrifuged at 14000 × g for 2 min. Aliquots (450 μL) were transferred to 1.5 mL Eppendorf tubes and dried as above. As previously described , the dried material was derivatized with methoxyamine hydrochloride in pyridine followed by N-tert-butylmethylsilyl-N-methyltrifluoroacetamide and then analyzed by GC-MS. Raw data are available at metabolomicsworkbench.org.
This research was supported by the U.S. National Science Foundation award MCB-1153413. The work was further supported by GM95837 from the NIH to DMD. The authors are grateful for GC-TOF-MS data generated by the West Coast Metabolomics Center, Megan Showalter, Mimi Swe and Carol Tran, partly funded by grant NIH U24 DK097154. We thank Jennifer Lambrecht for constructing the original yoaB::cat and STM1549::kan isolates which were reconstructed for this study.
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