Modular Isotopomer Synthesis of γ‑Hydroxybutyric Acid for a Quantitative Analysis of Metabolic Fates

Herein we report a study combining metabolomics and mass isotopomer analysis used for investigation of the biochemical fate of γ-hydroxybutyric acid (GHB). Using various ¹³C incorporation labeling patterns into GHB, we have discovered that GHB is catabolized by previously unknown processes that include (i) direct β-oxidation to acetyl-CoA and glycolate, (ii) α-oxidation to 3-hydroxypropionyl-CoA and formate, and (iii) cleavage of C-4 to yield 3-hydroxypropionate and CO₂. We further utilized the unique attributes of our labeling patterns and the resultant isotopomers to quantitate relative flux down the identified pathways

Using Isotopic Tools to Dissect and Quantitate Parallel Metabolic Pathways

Using Isotopic Tools to Dissect and Quantitate Parallel Metabolic Pathway

Table_1_Isolation of endophytic fungi from Cotoneaster multiflorus and screening of drought-tolerant fungi and evaluation of their growth-promoting effects.docx

In the context of climate change and human factors, the drought problem is a particularly serious one, and environmental pollution caused by the abuse of chemical fertilizers and pesticides is increasingly serious. Endophytic fungi can be used as a protection option, which is ecologically friendly, to alleviate abiotic stresses on plants, promote plant growth, and promote the sustainable development of agriculture and forestry. Therefore, it is of great significance to screen and isolate endophytic fungi that are beneficial to crops from plants in special habitats. In this study, endophytic fungi were isolated from Cotoneaster multiflorus, and drought-tolerant endophytic fungi were screened by simulating drought stress with different concentrations of PEG-6000, and the growth-promoting effects of these drought-tolerant strains were evaluated. A total of 113 strains of endophytic fungi were isolated and purified from different tissues of C. multiflorus. After simulated drought stress, 25 endophytic fungi showed strong drought tolerance. After ITS sequence identification, they belonged to 7 genera and 12 species, including Aspergillus, Fusarium, Colletotrichum, Penicillium, Diaporthe, Geotrichum, and Metarhizium. According to the identification and drought stress results, 12 strains of endophytic fungi with better drought tolerance were selected to study their abilities of dissolving inorganic phosphorus and potassium feldspar powder and producing indole-3-acetic acid (IAA). It was found that the amount of dissolved phosphorus in 7 strains of endophytic fungi was significantly higher than that of CK, and the content of soluble phosphorus was 101.98–414.51 μg. ml−1; 6 endophytic fungi had significantly higher potassium solubilization than CK, and the content of water-soluble potassium ranged from 19.17 to 30.94 mg·l−1; 6 strains have the ability to produce IAA, and the yield of IAA ranged between 0.04 and 0.42 mg. ml−1. This study for the first time identified the existence of endophytic fungi with drought tolerance and growth-promoting function in C. multiflorus, which could provide new direction for plant drought tolerance and growth promotion fungi strain resources. It also provides a theoretical basis for the subsequent application of endophytic fungi of C. multiflorus in agricultural and forestry production to improve plant tolerance.</p

Metabolomics and Mass Isotopomer Analysis as a Strategy for Pathway Discovery: Pyrrolyl and Cyclopentenyl Derivatives of the Pro-Drug of Abuse, Levulinate

We recently reported that levulinate (4-ketopentanoate) is converted in the liver to 4-hydroxypentanoate, a drug of abuse, and that the formation of 4-hydroxypentanoate is stimulated by ethanol oxidation. We also identified 3 parallel β-oxidation pathways by which levulinate and 4-hydroxypentanoate are catabolized to propionyl-CoA and acetyl-CoA. We now report that levulinate forms three seven-carbon cyclical CoA esters by processes starting with the elongation of levulinyl-CoA by acetyl-CoA to 3,6-diketoheptanoyl-CoA. The latter γ-diketo CoA ester undergoes two parallel cyclization processes. One process yields a mixture of tautomers, i.e., cyclopentenyl- and cyclopentadienyl-acyl-CoAs. The second cyclization process yields a methyl-pyrrolyl-acetyl-CoA containing a nitrogen atom derived from the ε-nitrogen of lysine but without carbons from lysine. The cyclic CoA esters were identified in rat livers perfused with levulinate and in livers and brains from rats gavaged with calcium levulinate ± ethanol. Lastly, 3,6-diketoheptanoyl-CoA, like 2,5-diketohexane, pyrrolates free lysine and, presumably, lysine residues from proteins. This may represent a new pathway for protein pyrrolation. The cyclic CoA esters and related pyrrolation processes may play a role in the toxic effects of 4-hydroxypentanoate

Chemo-genomic characterization of antifolate resistance determinants in <i>M</i>. <i>smegmatis</i>.

(A) Simplified enzymatic conversions of folate derivatives in de novo biosynthesis and the one-carbon metabolic network in bacteria. Abbreviations: H4PteGlun, tetrahydrofolate (green) serves as carrier for one-carbon groups. AICART, aminoimidazolecarboxamide ribonucleotide transferase; DHFS, dihydrofolate synthase; DHFR, dihydrofolate reductase; DHPS, dihydropteroate synthase; FTD, 10-formyltetrahydrofolate dehydrogenase; FTS, 10-formyltetrahydrofolate synthetase; Gly, glycine; GTP, guanosine triphosphate; H2PteGlun, dihydrofolate; Hcy, homocysteine; Met, methionine; MS, methionine synthase; MTCH, methylenetetrahydrofolate cyclohydrolase; MTD, methylenetetrahydrofolate dehydrogenase; MTHFR, methylenetetrahydrofolate reductase; MTHFS, 5,10-methenyltetrahydrofolate synthetase; pABA, para-aminobenzoic acid; PGT, phosphoribosyl glycinamide transferase; Pte, pteroate; PteGlu1, folic acid; Ser, serine; SHMT, serine hydroxymethyltransferase; TS, thymidylate synthase. Two different types of TS have been described: ThyA and ThyX. While most organisms contain either ThyA or ThyX, some organisms including M. tuberculosis have both. Reactions directly involved in the methylfolate trap (MS) and thymineless death (TS) are highlighted in yellow and red, respectively. (B) Genome distributions of antifolate resistance determinants in M. smegmatis. Laboratory assigned catalog numbers (n = 1–50, S1 Table) were plotted against their corresponding locus tags (msmeg_No.). (C) A typical SULFA susceptibility and chemical complementation assay of M. smegmatis strains. A pool of antifolate sensitive mutants was replicated onto NE plates, in top-down order: (i) control, (ii) SCP, (iii) SCP plus PteGlu1, (iv) SCP plus 5-CHO-H4PteGlu1, (v) SCP plus 5-CH3-H4PteGlu1, and (vi) SCP plus pABA. SCP was used at 10.5 μg/ml while supplements were used at 0.3 mM final concentration. Colonies marked with “C” were from the parental strain mc2155, which was used as a control. Colonies marked with asterisks were from the “white” mutants.</p

Genetic and chemical induction of the methylfolate trap during <i>Salmonella</i> infection of macrophages.

(A) Survival of Salmonella strains in macrophages treated with SULFAs. Macrophages J774A.1 were infected for 1 h followed by 18 h chase, during which cells were untreated or treated with 1 mg/ml SMZ. Colony forming units (c.f.u.) were determined by serial dilution and plating method. (B) Cellular uptake and conversion of exogenous B12 in mammalian cells requires transcobalamin (TC) and CblC proteins, respectively. Antivitamin B12 molecules such as EtPhCbl inhibit transcobalamin and CblC, thereby restricting B12 bioavailability to intracellular bacteria. (C) Depletion of CblC expression, detected by Western Blot using a specific antibody (top), caused B12 starvation (middle) and increased SULFA sensitivity (bottom) of intracellular Salmonella. siRNA transfected THP-1 macrophages were infected with S. typhimurium cells expressing β-galactosidase from a B12 starvation-responsive promoter for 1 h, followed by 18 h chase, during which the infected macrophages were treated without or with 1 mg/ml SMZ. B12 starvation was estimated by determining β-galactosidase activity while Salmonella survival measured by c.f.u. counting. (D) Chemical restriction of B12 sensitizes intracellular S. typhimurium to SULFA treatment. Macrophages J774A.1 were infected with S. typhimurium cells harboring a B12 molecular probe for 1 h followed by 18 h chase, during which cells were untreated or treated with 1 mg/ml SMZ or/and 50 nM EtPhCbl. B12 starvation was estimated through measuring enzymatic activity (top) and expression of β-galactosidase by Western Blot (middle). Salmonella survival from the corresponding macrophages was measured through c.f.u. counting (bottom). Error bars represent standard deviations from biological triplicates. ns, no significant difference compared to control groups.</p

Metabolic dynamics of the methylfolate trap in <i>Salmonella typhimurium</i> SULFA resistance.

(A) Wide-spectrum SULFA susceptibility of S. typhimurium metH(+) and metH(-) analyzed by 10X serial dilution. Cultures were diluted starting with OD1 and 5 μl cell suspensions were spotted onto LB agar in the absence (control) or presence of different SULFAs, used at the indicated concentrations. These SULFA drugs are classified into all four subgroups, in left-right order: short-acting (blue), intermediate-acting (yellow), long-acting (green), and ultra-long-acting (pink), respectively. Growth was recorded after 48 h at 37°C. (B) Viability of S. typhimurium metH(+) (red) and metH(-) (blue) on LB agar 24 h post-SMZ addition (125 μg/ml). Colony forming units (c.f.u.) were determined and normalized to c.f.u. values of the inoculation input (0 h). The y-axis represents c.f.u. fold-change on a log10 scale of SMZ-treated (+SMZ, hatched bars) and control non-treated samples (-SMZ, empty bars). Error bars represent standard deviations from biological triplicates. **** pC) SULFA susceptibility of S. typhimurium strains in liquid LB medium. Cultures of metH(+) (red) and metH(-) (blue) growing at OD1 was added with 2.5 mg/ml SMZ (arrow). Growth was monitored by measuring OD600. (D) Dynamics of the folate pool in S. typhimurium metH(+) (red) and metH(-) (blue) strains. At selected time points following SULFA treatment, cells were collected and folate extracted and analyzed by LC-MS/MS. Bars represent the combined levels of all 5-CH3-H4PteGlun species (top), all non-methylated folate species (middle), and total folate (bottom) following SMZ addition. s, significant difference between metH(-) and corresponding metH(+) samples; ns, no significant difference. (E) Dynamics of 41 metabolites in metH(+) (upper) and metH(-) (lower) strains. Metabolites are shown with their fold change over time (0–8 hours post SMZ addition). At selected time points following SMZ treatment, cells were collected and metabolites extracted and analyzed by LC-MS/MS. Signal intensity was normalized to OD600nm at each time point. Relative levels are expressed as the log ratio of the normalized signal intensity of SMZ-treated cells at each time point to the normalized signal intensity of the no drug control sample at t = 0 (n = 3). The data shown in all figures represents the mean of biological repeats (n ≥ 3) with standard deviations. In the experiments demonstrated in Fig 5C–5E, SMZ was added at 2.5 mg/ml when cultures reached OD1.</p

Methylfolate trap in <i>Mycobacterium smegmatis</i>.

<p>(<b>A</b>) A model depicting the chemical conversions and factors involved in the methylfolate trap-mediated SULFA sensitivity. The CH₃- group in 5-CH₃-H₄PteGlu_n is first transferred to the B₁₂ cofactor, which further transfers it to homocysteine (Hcy) to make methionine (Met). The MetH reaction thereby recycles 5-CH₃-H₄PteGlu_n back to free H₄PteGlu_n which continues the flow of the one-carbon network. (<b>B</b>) Chemical complementation of <i>M</i>. <i>smegmatis</i> “white” mutants mapped to <i>metH</i> or <i>cobIJ</i>. The strains exhibited increased SULFA susceptibility and impaired 5-CH₃-H₄PteGlu₁ utilization. Approximately 5x10³ cells were spotted onto NE medium added with 10.5 μg/ml SCP with or without exogenous supplements. Unlike wild type and other mutants, these mutants were unable to use 5-CH₃-H₄PteGlu₁ to antagonize SCP. Exogenous B₁₂ restored 5-CH₃-H₄PteGlu₁ utilization and SCP resistance to <i>cobIJ</i> but not <i>metH</i> mutants. (<b>C</b>) Effect of <i>metH</i> and <i>cobIJ</i> on the folate pool in <i>M</i>. <i>smegmatis</i>. Growing cultures of <i>M</i>. <i>smegmatis</i> strains were treated with 285 μg/ml SCP for 30 min followed by folate extraction and LC-MS/MS analysis. Data shows the combined levels of all 5-CH₃-H₄PteGlu_n species (top), all non-methyl folate species (middle), and the total folate (bottom). Bars represent means of biological triplicates with standard deviations. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference between the indicated strains. (<b>D</b>) Targeted mutagenesis confirms the roles of <i>metH</i> and <i>cobIJ</i> in methylfolate trap-induced SULFA sensitivity and 5-CH₃-H₄PteGlu₁ utilization in <i>M</i>. <i>smegmatis</i>. Paper discs were embedded with 0.5 mg SCP and placed at the center of the medium surface, seeded with bacterial strains. Exogenous B₁₂ and 5-CH₃-H₄PteGlu_n were used at 0.3 and 1 mM, respectively. Genetic complementation was achieved by <i>in trans</i> expression of <i>metH</i> or <i>cobIJ</i>. B₁₂ alone restored wild type SULFA resistance level to <i>Ms</i>Δ<i>cobIJ</i>, whereas the combination of 5-CH₃-H₄PteGlu₁ and B₁₂ completely abolished SULFA resistance to all strains but <i>Ms</i>Δ<i>metH</i>.</p

Methylfolate trap in <i>Mycobacterium tuberculosis</i>.

<p>(<b>A</b>) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B₁₂ and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD₆₀₀ of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10⁴ cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B₁₂ alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. (<b>B</b>) H37Rv-derived strains were grown to OD₆₀₀ of 1 and 5 μl cultures were spotted onto 7H10-OADC or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B₁₂, and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10⁻⁶ dilution plates provide a visual comparison between strains in viability (top). (<b>C</b>) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE (<a href="http://prosite.expasy.org/" target="_blank">http://prosite.expasy.org</a>). Domains are labeled as the cofactors to which they bind. (<b>D</b>) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B₁₂ and methionine was analyzed using the MTT method. Cultures growing at an OD₆₀₀ of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10⁴ cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B₁₂ (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. (<b>E</b>) Survival of H37Rv (Red), its derived <i>metH</i> mutant (RvΔ<i>metH</i>, Blue) and the complemented strain (RvΔ<i>metH</i>/<i>metH</i>, Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p<0.01; ns, no significant difference compared to corresponding H37Rv sample. The data presented is the representative of four independent experiments. (<b>F</b>) Survival of H37Rv (Red), CDC1551 (Blue), and the CDC1551 strain <i>in trans</i> expressing the intact <i>metH</i> gene from H37Rv (CDC1551/<i>metH</i>, Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p<0.01; ns, no significant difference compared to H37Rv.</p

Susceptibility of bacterial strains to antifolates.

<p>Susceptibility of bacterial strains to antifolates.</p