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Research Article
18 March 2020

A Pathway for Degradation of Uracil to Acetyl Coenzyme A in Bacillus megaterium

ABSTRACT

Bacteria utilize diverse biochemical pathways for the degradation of the pyrimidine ring. The function of the pathways studied to date has been the release of nitrogen for assimilation. The most widespread of these pathways is the reductive pyrimidine catabolic pathway, which converts uracil into ammonia, carbon dioxide, and β-alanine. Here, we report the characterization of a β-alanine:pyruvate aminotransferase (PydD2) and an NAD+-dependent malonic semialdehyde dehydrogenase (MSDH) from a reductive pyrimidine catabolism gene cluster in Bacillus megaterium. Together, these enzymes convert β-alanine into acetyl coenzyme A (acetyl-CoA), a key intermediate in carbon and energy metabolism. We demonstrate the growth of B. megaterium in defined medium with uracil as its sole carbon and energy source. Homologs of PydD2 and MSDH are found in association with reductive pyrimidine pathway genes in many Gram-positive bacteria in the order Bacillales. Our study provides a basis for further investigations of the utilization of pyrimidines as a carbon and energy source by bacteria.
IMPORTANCE Pyrimidine has wide occurrence in natural environments, where bacteria use it as a nitrogen and carbon source for growth. Detailed biochemical pathways have been investigated with focus mainly on nitrogen assimilation in the past decades. Here, we report the discovery and characterization of two important enzymes, PydD2 and MSDH, which constitute an extension for the reductive pyrimidine catabolic pathway. These two enzymes, prevalent in Bacillales based on our bioinformatics studies, allow stepwise conversion of β-alanine, a previous “end product” of the reductive pyrimidine degradation pathway, to acetyl-CoA as carbon and energy source.

INTRODUCTION

Many bacteria possess biochemical pathways for the degradation of the pyrimidine ring, enabling them to utilize pyrimidines as a nitrogen source for growth (1). Three pyrimidine degradation pathways have been reported in bacteria, known as the reductive (Pyd) (2, 3), oxidative (4, 5), and pyrimidine utilization (RUT) (3) pathways. Of the three, the Pyd pathway is the most widespread in bacteria and has been studied in Escherichia coli B (6). In the Pyd pathway, uracil is first reduced to dihydrouracil by dihydropyrimidine dehydrogenase (PydA), followed by sequential hydrolysis by dihydropyrimidinase (PydB) and ureidopropionase (PydC) (2, 7). One of the pyrimidine nitrogen atoms is released as ammonia, and one of the carbon atoms is released as carbon dioxide. The rest of the atoms are trapped in β-alanine.
Recently, we reported an extended Pyd pathway in Lysinibacillus massiliensis, involving a β-alanine:2-oxoglutarate aminotransferase (PydD1) and an NAD(P)H-dependent malonic semialdehyde reductase (PydE) (8). PydD1 generates glutamate, an intermediate in nitrogen metabolism, thus enabling the assimilation of the second pyrimidine nitrogen atom. PydE converts the toxic intermediate malonic semialdehyde into 3-hydroxypropionate (3-HP), which is excreted into the medium (8). In addition to being incorporated into bacterial nitrogen metabolism, β-alanine could also in principle be incorporated into bacterial carbon metabolism, allowing pyrimidines to be used as a carbon source for growth. However, studies to date have focused almost exclusively on the utilization of pyrimidines as a nitrogen source. To our knowledge, the utilization of pyrimidines as a carbon source by bacteria has only been reported in Hydrogenomonas facilis, although the enzymes involved have not been identified (9).
A pathway for the utilization of β-alanine as a carbon source has been reported in 12-aminododecanate-degrading Pseudomonas sp. strain AAC (10). In this bacterium, β-alanine is converted to malonic semialdehyde by a highly promiscuous ω-aminotransferase that is also involved in the degradation of 12-aminododecanate. Malonic semialdehyde is then oxidized to acetyl coenzyme A (acetyl-CoA) by an NAD+-dependent malonic semialdehyde dehydrogenase. We hypothesized that the incorporation of similar enzymes into the Pyd pathway might allow pyrimidines to serve as a carbon source for growth of some bacteria and thus carried out a systematic bioinformatics investigation of variants of the Pyd pathway in bacteria.
Here, we report the biochemical characterization of a β-alanine:pyruvate aminotransferase (PydD2) and an NAD+-dependent malonic semialdehyde dehydrogenase (MSDH), associated with a Pyd gene cluster in Bacillus megaterium (Fig. 1A). These enzymes extend the Pyd pathway by converting β-alanine into acetyl-CoA, a key intermediate in carbon and energy metabolism (Fig. 1B). The physiological relevance of this extended Pyd pathway was supported by the growth of B. megaterium with uracil as the sole carbon and energy source. We describe a bioinformatics study exploring the prevalence of variants of the Pyd pathway in bacteria in the phylum Firmicutes and find that, while the 3-HP-producing pathway is more prevalent in fermenting bacteria in the order Clostridiales, the acetyl-CoA-producing pathway is more prevalent in the metabolically diverse bacteria in the order Bacillales (Fig. 1).
FIG 1
FIG 1 Variants of the extended reductive pyrimidine catabolism (Pyd) pathway. (A) Representative extended Pyd pathway gene clusters in Firmicutes bacteria. In addition to PydABC, many Bacillales bacteria contain a β-alanine:pyruvate aminotransferase (PydD2) and malonic semialdehyde dehydrogenase (MSDH), while many Clostridiales bacteria contain a β-alanine:2-oxoglutarate aminotransferase (PydD1) and malonic semialdehyde reductase (PydE). (B) Comparison of the two extended reductive pyrimidine degradation pathways, involving either PydD1 and PydE as previously reported (8) or PydD2 and MSDH as described in this study.

RESULTS

Recombinant production of PydD2 and MSDH.

B. megaterium PydD2 and MSDH were recombinantly produced in E. coli and purified to near homogeneity (see Fig. S1 in the supplemental material). The UV-visible (UV-Vis) spectrum of the purified recombinant PydD2 contains characteristic absorbance at 330 nm and 410 nm, which correlate to the enolimine and ketoenamine forms of the PLP (pyridoxal phosphate) cofactor, respectively (see Fig. S2 in the supplemental material).
PydD2 shares 42% sequence identity with the previously reported taurine:pyruvate aminotransferase (Tpa) from Bilophila wadsworthia, which catalyzes the transamination of taurine, a sulfonate analog of β-alanine (11). MSDH shares 56% sequence identity with a previously reported malonic and methylmalonic semialdehyde dehydrogenase (IolA) from Bacillus subtilis, putatively involved in myo-inositol catabolism (12).

β-Alanine:pyruvate aminotransferase activity of PydD2.

The kinetics of PydD2-catalyzed transamination was measured using a coupled spectrophotometric assay with the L. massiliensis PydE, which catalyzes the NADPH-dependent reduction of malonic semialdehyde (Fig. 2A and B), assayed at the optimal pH of 9.0 (see Fig. S3 in the supplemental material). Steady state kinetic parameters for PydD2 were determined (Fig. 2C and D) and are summarized in Table 1. Under the same reaction conditions, activity was not detected with 2-oxoglutarate replacing pyruvate as the amine acceptor (Fig. 2B).
FIG 2
FIG 2 PydE-coupled activity assays for PydD2. Detection of malonic semialdehyde generated by PydD2-catalyzed β-alanine:pyruvate transamination (A) and β-alanine:2-oxoglutarate transamination (B). Steady-state kinetics of PydD2 were determined by varying the concentration of β-alanine (C) or pyruvate (D), while fixing the concentration of the other substrate at 25 mM. Assays monitor NADPH consumption accompanying malonic semialdehyde reduction by PydE.
TABLE 1
TABLE 1 Kinetic parameters of PydD2
SubstrateApparent kcat (s−1)Apparent Km (mM)Apparent kcat/Km (M−1 s−1)
β-Alanine1.5 ± 0.113.7 ± 2.6109 ± 22
Pyruvate1.1 ± 0.115.7 ± 2.370 ± 12
PydD2 catalyzed the formation of l-α-alanine from β-alanine and pyruvate, as detected by an assay with alanine dehydrogenase (ALD) (Fig. 3). The PydD2 reaction products were analyzed by derivatization with 2,4-dinitrophenylhydrazine (DNPH) followed by liquid chromatography-mass spectrometry (LC-MS). The DNPH (compound 1) and acetaldehyde-DNPH (compound 5) standards eluted at 11.5 and 19.5 min (Fig. 4A), and their identities were confirmed by electrospray ionization-MS (ESI-MS) (m/z) (Fig. 4B). Pyruvate-DNPH isomers (compounds 3 and 4), eluting at 13.9 and 18.0 min, were prominent in the assay mixture without PydD2 but were diminished in the complete assay, accompanied by the appearance of a peak eluting at 13.3 min, corresponding to malonic semialdehyde-DNPH (compound 2) (Fig. 4A). The negative ion mass spectrum of this peak suggested decarboxylation accompanying the ionization process (Fig. 4B), as previously observed for the product of L. massiliensis PydD1 (8). A peak corresponding to acetaldehyde-DNPH was also present in this assay, suggesting spontaneous decarboxylation of malonic semialdehyde during the reaction or workup (Fig. 4).
FIG 3
FIG 3 Detection of l-α-alanine as a PydD2 reaction product. (A) Scheme for spectrophotometric assay involving l-α-alanine dehydrogenase (ALD). (B) Assays monitoring NADH formation accompanying l-α-alanine oxidation by ALD.
FIG 4
FIG 4 LC-MS analyses of product formation in PydD2-catalyzed transamination. (A) Elution profiles of the products of PydD2-catalyzed β-alanine:pyruvate transamination, monitoring the absorbance at 360 nm. The complete assay contains PydD2 and both substrates (β-alanine and pyruvate), controls omitting the enzyme or both substrates, DNPH, and DNPH-acetaldehyde standards are included. The break in the vertical axis of the plot is indicated in dotted lines. (B) ESI(−) m/z spectra of the DNPH derivative peaks in panel A with compounds 1 to 5 displayed in order. Retention time is indicated in each spectrum.

Malonic semialdehyde dehydrogenase activity of MSDH.

MSDH catalyzed the CoA-dependent oxidation of malonic semialdehyde, as determined using a spectrophotometric assay monitoring NADH formation (Fig. 5A). The first-order rate constant (kobs) of MSDH was 1.1 ± 0.1 s−1. The MSDH reaction products were analyzed by derivatization by LC-MS. The acetyl-CoA product coeluted with the standard at 6.7 min on the extracted ion chromatogram (Fig. 5B), and its identity was confirmed by ESI-MS (m/z) (Fig. 5C).
FIG 5
FIG 5 MSDH catalyzes the oxidation and decarboxylation of malonic semialdehyde, forming acetyl-CoA. (A) Assays monitoring the formation of NADH accompanying the malonic semialdehyde oxidation. (B) LC-MS extracted ion chromatographs monitoring the formation of acetyl-CoA. A commercial acetyl-CoA standard and negative controls omitting NAD+, CoA-SH, PydD2, or MSDH are included. (C) ESI(+) m/z spectrum of the acetyl-CoA peak in panel B.

Pyrimidines as the sole carbon source support growth of Bacillus megaterium.

Next, we investigated the physiological relevance of the extended Pyd gene cluster in the utilization of pyrimidines as a carbon and energy source. B. megaterium could grow using defined medium with uracil or β-alanine as the sole carbon source (Fig. 6). The final cell density was similar to cells grown using glucose as the carbon source. No growth was observed in the negative control where the carbon source was omitted.
FIG 6
FIG 6 Uracil or β-alanine as the sole carbon source supports the growth of Bacillus megaterium. The sole carbon source used in each of the culture media is labeled.

Bacteria in the orders Bacillales and Clostridiales contain distinct Pyd gene cluster variants.

A sequence similarity network (SSN) of 1,071 PydA homologs from bacteria in the phylum Firmicutes was constructed (see Fig. S4 in the supplemental material). We focused our analysis on the two largest clusters, within which many of the PydA homologs are associated with PydB and PydC, suggesting involvement in the Pyd pathway. Cluster 1 (324 sequences) contains mostly sequences from bacteria in the order Clostridiales (238 sequences) and a number of sequences from Lactobacillales (46 sequences), while cluster 2 (443 members) contains almost exclusively sequences from Bacillales (429 sequences) (Fig. 7A). The gene clusters in Clostridiales bacteria are typified by that of Clostridium chromiireducens, and those in Bacillales bacteria are typified by that of B. megaterium (Fig. 1B).
FIG 7
FIG 7 The SSN of PydA homologs in Firmicutes bacteria. Only two clusters are shown, which contain PydA homologs associated with other Pyd pathway enzymes. The full SSN is presented in Fig. S4 in the supplemental material. (A) Cluster 1 is dominated by Clostridiales bacteria (red) with some Lactobacillales (yellow). Cluster 2 is dominated by Bacillales (blue). (B) Sequences containing a PydB homolog belonging to the clusters UniRef50_D4MUM2 or UniRef50_Q45515 within their genome neighborhood (blue-green). (C) Sequences containing a putative PydD homolog belonging to the aminotransferase family (PFam family PF00202) within their genome neighborhood (dark blue). (D) Sequences containing a putative PydE homolog belonging to the metal-dependent alcohol dehydrogenase family (PF00465, dark red) or a putative MSDH homolog belonging to the aldehyde dehydrogenase family (PF00171, purple) directly adjacent to PydD.
The genome neighborhood of many of the 546 of the PydA homologs in these two clusters contain a PydB homolog closely related to either C. chromiireducens or B. megaterium PydB (belonging to the UniRef50_D4MUM2 or UniRef50_Q45515 clusters, respectively). Each member of a UniRef50 cluster shares ≥50% sequence identity and ≥80% overlap with the seed sequence of the cluster (13) (Fig. 7B; see also Data Set S1 in the supplemental material), suggesting involvement in the Pyd pathway.
The genome neighborhood of 437 of the PydA homologs in these two clusters contain a putative PydD homolog belonging to the aminotransferase family (PFam family PF00202) (Fig. 7C). Of these PydD homologs, 83 occur immediately adjacent to a putative PydE homolog belonging to the metal-dependent alcohol dehydrogenase family (PF00465), and 226 occur immediately adjacent to a putative MSDH homolog belonging to the aldehyde dehydrogenase family (PF00171) (Fig. 7D). The PydDE combination is more common in Clostridiales bacteria, while the PydD-MSDH combination is more common in Bacillales bacteria.

DISCUSSION

The biochemical characterization of the β-alanine:pyruvate aminotransferase PydD2 and the malonic semialdehyde dehydrogenase MSDH, associated with the PydABC gene cluster in B. megaterium, provides a pathway for the conversion of β-alanine derived from the Pyd pathway into acetyl-CoA, for incorporation into carbon and energy metabolism. The growth of B. megaterium in defined medium with uracil as its sole carbon and energy source is consistent with the suggested physiological function of this pathway.
Unlike the previously characterized L. massiliensis PydD1, which exhibits a preference for 2-oxoglutarate as the amine acceptor, B. megaterium PydD2 is specific for pyruvate as the amine acceptor. The use of pyruvate as the amine acceptor may be favored in the carbon metabolism pathway because it generates l-α-alanine, enabling the regeneration of pyruvate by alanine dehydrogenase, with the elimination of ammonia. We observed that several of the Bacillales Pyd2-containing gene clusters also contain a putative alanine dehydrogenase (PF01262) (see Fig. S5 in the supplemental material). A similar trend in amine acceptor preference was observed for aminotransferases involved in the bacterial degradation of 2-aminoethylsulfonate (taurine), which is structurally similar to β-alanine and degraded using similar enzymes. The utilization of taurine as a nitrogen source by Chromohalobacter salexigens (14), and hypothetically by Bifidobacterium kashiwanohense (15), involves a taurine:2-oxoglutarate aminotransferase, while utilization of taurine as a carbon and energy source by Bilophila wadsworthia (11) and Ruegeria pomeroyi (16) involves a taurine:pyruvate aminotransferase. Growth of B. wadsworthia on taurine is accompanied by the induction of alanine dehydrogenase (17), providing evidence for its role in this pathway.
Of the PydA homologs analyzed in Firmicutes bacteria, nearly all of those in Bacillales and half of those in Clostridiales bacteria contained a PydB homolog in their genome neighborhood (Fig. 7B), suggesting their involvement in the Pyd pathway. Among these PydA sequences, many are associated with a putative β-alanine aminotransferase (Fig. 1 and 7C) and either a metal-dependent alcohol dehydrogenase or a CoA-dependent aldehyde dehydrogenase, suggesting alternative pathways for β-alanine degradation. Strikingly, the former pathway is present almost exclusively in Clostridiales bacteria, while the latter is present almost exclusively in Bacillales bacteria. While Clostridiales bacteria are fermenting bacteria, Bacillales include metabolically diverse bacteria that carry out aerobic and anaerobic respiration. We hypothesize that this enables them to utilize the acetyl-CoA as a substrate for energy generation through the oxidative citric acid cycle, explaining the differential occurrence of variants of the Pyd pathway.

MATERIALS AND METHODS

General.

Bacillus megaterium (DSM 32) was purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). Lysogeny broth (LB) medium (Difco) was purchased from Becton Dickinson (MD, USA). The rich growth medium for Bacillus megaterium was prepared by dissolving 5 g peptone (AoBox, Beijing, China) and 3 g meat extract (AoBox, Beijing, China) in 1 liter distilled water and adjusting the pH to 7.0. For agar plates of this rich medium, 1.5% agar was added. To prepare the defined medium for the growth of Bacillus megaterium, 9 g Na2HPO4·12H2O, 1.5 g KH2PO4, 10 mg MnSO4·H2O, 0.2 g MgSO4·7H2O, 0.2 g NH4Cl, 10 mg CaCl2·2H2O, 5 mg Na2·EDTA·2H2O, 1.5 mg CoCl2·6H2O, 1 mg ZnCl2, 0.1 mg H3BO3, 0.2 mg CuCl2·2H2O, 0.1 mg Na2MoO4·2H2O, 0.2 mg NiSO4·6H2O, 1 mg FeSO4·7H2O, 0.2 mg Na2SeO3, 0.4 mg Na2WO4·2H2O, 20 μg biotin, 20 μg folic acid, 100 μg pyridoxine-HCl, 50 μg thiamine-HCl, 50 μg riboflavin, 50 μg nicotinic acid, 50 μg d-Ca-pantothenate, 1 μg vitamin B12, 50 μg p-aminobenzoic acid, and 50 μg lipoic acid were dissolved in 1 liter distilled water. Twenty millimolar glucose, 40 mM β-alanine, or 30 mM uracil as the sole carbon source was added. The medium was sterilized by filtration through a sterile 0.22-μm filter.
Acetonitrile was purchased from Concord Technology (Tianjin, China). Formic acid was purchased from Merck (NJ, USA). All protein purification chromatographic experiments were performed on an ÄKTA pure or ÄKTA prime plus fast protein liquid chromatography (FPLC) machine equipped with appropriate columns (GE Healthcare, USA). Protein concentrations were calculated from the absorbance at 280 nm measured using NanoDrop One (Thermo Fisher Scientific). NAD(P)H-coupled activity assays were carried out by monitoring the absorbance at 340 nm using a plate reader (Tecan M200).

Cloning, expression, and purification of PydD2 and MSDH.

Colony PCRs were performed on B. megaterium colonies to amplify the pydD2 (UniProt accession number A0A0B6ASC9) and MSDH (mmsA) (UniProt accession number A0A0B6AI58) genes, using the primer pairs 1F/1R and 2F/2R (Table 2), respectively. The amplified DNA fragments were inserted by Gibson assembly at the SspI site of a modified pET-28a(+) vector (HT), containing a His6 tag followed by a tobacco etch virus (TEV) protease cleavage site. The resulting plasmids HT-PydD2 and HT-MSDH were confirmed by sequencing.
TABLE 2
TABLE 2 Oligonucleotides used for cloning
OligonucleotideTargetSequence (5′ to 3′)Application
1FPydD25'-CCTGTACTTCCAATCCAATATGGTACAGGTCAGTCAAA-3'Construct HT-PydD2
1RPydD25'-TCCGTTATCCACTTCCAATCTATTTGATGCTGTCAAGC-3'Construct HT-PydD2
2FMSDH5'-TGAAAACCTGTACTTCCAATCCAATATGTCAGTTGTGAATAATGATACGC-3'Construct HT-MSDH
2RMSDH5'-ATCCGTTATCCACTTCCAATTTAAAAGTCAAAGCGTGATGTA-3'Construct HT-MSDH
PydD2 and MSDH were recombinantly produced and purified by Talon Co2+-affinity chromatography, following a previously described protocol for L. massiliensis PydD1 (8). The purified PydD2 and MSDH were quantified using their calculated extinction coefficients (ε280 = 60,740 M−1 cm−1) and (ε280 = 36,900 M−1 cm−1), respectively. The purity of both proteins was analyzed by SDS-PAGE.

Activity assay for PydD2.

The β-alanine:pyruvate aminotransferase activity of PydD2 was measured using a coupled spectrophotometric assay in the presence of excess L. massiliensis PydE (8). In a typical assay, a 200-μl reaction mixture containing 0.1 to 0.4 μM PydD2, 1.2 μM L. massiliensis PydE, 10 μM PLP (pyridoxal phosphate), 25 mM pyruvate or 2-oxoglutarate, 25 mM β-alanine, and 0.4 mM NADPH in 100 mM Tris-HCl, pH 9.0, was prepared, and the decrease in absorbance at 340 nm was monitored over 1.5 min. The optimal pH of PydD2 was determined by carrying out the assay in different buffers {morpholineethanesulfonic acid [MES], pH 6.0; HEPES, pH 7.0, 7.5, and 8.0; Tricine, pH 8.5; Tris-HCl, pH 9.0; Na2CO3-NaHCO3, pH 9.5; 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid [CAPSO], pH 10.0}.
To obtain the steady state kinetic parameters, the concentration of one substrate (β-alanine or pyruvate) was varied in the presence of excess (25 mM) of the cosubstrate. A total of 0.4 μM PydD2 was used for the transamination from β-alanine to pyruvate in the presence of 1.2 μM PydE and 0.4 mM NADPH. The decrease in absorbance at 340 nm was monitored over 1.5 min.

Detection of alanine as a PydD2 reaction product.

To carry out the transamination reaction, a 200-μl reaction mixture containing 100 mM Tris-HCl, pH 9.0, 20 μM PLP, 5 mM pyruvate, 5 mM β-alanine, and 1 μM PydD2 was incubated at 35°C in a water bath for 30 min. The reaction was then heat inactivated in a boiling water bath for 2 min. To detect the amino acid product, 100 μl of the reaction mixture was mixed with a 100-μl solution containing 10 mM NAD+ and 72 mU of Bacillus subtilis alanine dehydrogenase (ALD) in the same reaction buffer. The absorbance at 340 nm was monitored over 50 s.

LC-MS detection of malonic semialdehyde as a PydD2 reaction product.

A 200-μl reaction mixture containing 100 mM Tris-HCl, pH 9.0, 10 μM PLP, 25 mM pyruvate, 25 mM β-alanine, and 4 μM PydD2 was incubated at room temperature (RT) for 30 min. Negative controls include assays omitting either the enzyme or both substrates (β-alanine and pyruvate). The product was derivatized with 2,4-dinitrophenylhydrazine (DNPH) (J&K) and analyzed by LC-MS as previously described (8).

MSDH activity assay.

Malonic semialdehyde, the substrate of MSDH, is chemically unstable and was generated in situ with excess PydD2, β-alanine, and pyruvate. A 200-μl reaction mixture containing 100 mM phosphate buffer, pH 8.2, 0.5 μM MSDH, 1 mM coenzyme A (CoASH), 1 mM NAD+, 7.5 μM PydD2, 150 mM β-alanine, and 150 mM pyruvate was prepared, and the increase in absorbance at 340 nm was monitored over 1.5 min.

LC-MS detection of acetyl-CoA as an MSDH reaction product.

A 200-μl reaction mixture containing 100 mM Tris-HCl, pH 9.0, 10 μM PLP, 25 mM β-alanine, 25 mM pyruvate, 1 mM CoA, 1 mM NAD+, 4 μM PydD2, and 12 μM MSDH was incubated at RT for 30 min. MSDH, NAD+, CoA, or PydD2 was omitted in the negative controls. Protein was removed by extraction with an equal volume phenol/chloroform/isopentanol (25:24:1). Following centrifugation, the aqueous layer was filtered through a 0.22-μm nylon membrane filter prior to LC-MS analysis.
LC-MS analysis was performed on an Agilent 6420 triple quadrupole LC-MS instrument (Agilent Technologies). The drying gas temperature was maintained at 350°C with a flow rate of 12 liters min−1 and a nebulizer pressure of 25 lb/in2. LC-MS analysis was carried out on an Agilent Zorbax SB-C18 column (4.6 by 250 mm; product number 880975-902). A linear gradient of acetonitrile (4 to 35% in H2O) containing 0.1% formic acid and a flow rate of 1.0 ml/min for 15 min was used to elute.

Bacterial culture using pyrimidine as the sole carbon source.

Bacillus megaterium was grown in a 50-ml centrifuge tube containing 20 ml liquid rich medium in a shaker incubator at 220 rpm and 30°C overnight. A total of 100 μl of this starter culture was transferred into 20 ml of defined medium containing different carbon sources (20 mM glucose, 40 mM β-alanine, or 30 mM uracil). A negative control omitting the carbon source was included. The optical density at 600 nm (OD600) of each culture was then recorded every 2 to 3 h.

Bioinformatics.

A list of 1,071 PydA candidates containing both dihydroorotate dehydrogenase (InterPro domain number IPR005720) (18) and 4Fe4S ferredoxin (InterPro domain number IPR017896) domains, from bacteria in the phylum Firmicutes, was compiled from the UniProt database (19). A sequence similarity network (SSN) was constructed using the web-based enzyme function initiative enzyme similarity tool (EFI-EST) (20) and visualized using Cytoscape v3.5 (21). The E value threshold was adjusted to 10−140 (greater than ∼60% sequence identity is required to draw an edge). The sequence length was restricted to >350 amino acids to exclude partial sequences. The genome neighborhoods of the PydA candidates within a 10-open reading frame window were examined using the web-based enzyme function initiative genome neighborhood tool (22).

ACKNOWLEDGMENTS

We thank the instrument analytical center of the School of Pharmaceutical Science and Technology at Tianjin University for providing the LC-MS analysis and Zhi Li, Xinghua Jin, and Xiangyang Zhang for the helpful discussion.
This work was supported by the National Key R&D Program of China (no. 2019YFA0905700), the National Science Foundation of China (no. 31870049) (Y.Z.), and the Agency for Science, Research, and Technology of Singapore Visiting Investigator program grant no. 1535j00137 (H.Z.).
The authors declare no conflict of interest.
D.Z., J.Y., Y.Z., and D.L. designed and carried out biochemical experiments with PydD2 and MSDH. Y.W. designed and carried out experiments with bioinformatics and was involved in conceptualizing the project and writing the manuscript. E.L.A., H.Z., and Y.Z. were involved in conceptualizing the project, getting grants for the project, overall supervision of the project, and writing the manuscript.

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Information & Contributors

Information

Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 86Number 718 March 2020
eLocator: e02837-19
Editor: Rebecca E. Parales, University of California, Davis
PubMed: 31953335

History

Received: 5 December 2019
Accepted: 10 January 2020
Published online: 18 March 2020

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Keywords

  1. β-alanine
  2. acetyl-CoA
  3. aminotransferase
  4. carbon source
  5. malonic semialdehyde dehydrogenase
  6. pyridoxal phosphate
  7. pyrimidine
  8. pyruvate
  9. reductive pathway
  10. sequence similarity network

Contributors

Authors

Di Zhu
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China
Yifeng Wei
Institute of Chemical and Engineering Sciences, Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore
Jinyu Yin
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China
Dazhi Liu
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China
Ee Lui Ang
Institute of Chemical and Engineering Sciences, Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore
Institute of Chemical and Engineering Sciences, Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China

Editor

Rebecca E. Parales
Editor
University of California, Davis

Notes

Address correspondence to Huimin Zhao, [email protected], or Yan Zhang, [email protected].
Di Zhu and Yifeng Wei contributed equally to this work. Author order was determined in order of increasing seniority.

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