Metabolomics responses and tolerance of Pseudomonas aeruginosa under acoustic vibration stress.

Sound has been shown to impact microbial behaviors. However, our understanding of the chemical and molecular mechanisms underlying these microbial responses to acoustic vibration is limited. In this study, we used untargeted metabolomics analysis to investigate the effects of 100-Hz acoustic vibration on the intra- and extracellular hydrophobic metabolites of P. aeruginosa PAO1. Our findings revealed increased levels of fatty acids and their derivatives, quinolones, and N-acylethanolamines upon sound exposure, while rhamnolipids (RLs) showed decreased levels. Further quantitative real-time polymerase chain reaction experiments showed slight downregulation of the rhlA gene (1.3-fold) and upregulation of fabY (1.5-fold), fadE (1.7-fold), and pqsA (1.4-fold) genes, which are associated with RL, fatty acid, and quinolone biosynthesis. However, no alterations in the genes related to the rpoS regulators or quorum-sensing networks were observed. Supplementing sodium oleate to P. aeruginosa cultures to simulate the effects of sound resulted in increased tolerance of P. aeruginosa in the presence of sound at 48 h, suggesting a potential novel response-tolerance correlation. In contrast, adding RL, which went against the response direction, did not affect its growth. Overall, these findings provide potential implications for the control and manipulation of virulence and bacterial characteristics for medical and industrial applications.

Lowering of lysophosphatidylcholines in ovariectomized rats by Curcuma comosa.

Decline of ovarian function in menopausal women increases metabolic disease risk. Curcuma comosa extract and its major compound, (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (DPHD), improved estrogen-deficient ovariectomized (OVX) rat metabolic disturbances. However, information on their effects on metabolites is limited. Here, we investigated the impacts of C. comosa ethanol extract and DPHD on 12-week-old OVX rat metabolic disturbances, emphasizing the less hydrophobic metabolites. Metabolomics analysis of OVX rat serum showed a marked increase compared to sham-operated rat (SHAM) in levels of lysophosphatidylcholines (lysoPCs), particularly lysoPC (18:0) and lysoPC (16:0), and of arachidonic acid (AA), metabolites associated with inflammation. OVX rat elevated lysoPCs and AA levels reverted to SHAM levels following treatments with C. comosa ethanol extract and DPHD. Overall, our studies demonstrate the effect of C. comosa extract in ameliorating the metabolic disturbances caused by ovariectomy, and the elevated levels of bioactive lipid metabolites, lysoPCs and AA, may serve as potential biomarkers of menopausal metabolic disturbances.

Aspergillus niger upregulated glycerolipid metabolism and ethanol utilization pathway under ethanol stress.

The knowledge of how Aspergillus niger responds to ethanol can lead to the design of strains with enhanced ethanol tolerance to be utilized in numerous industrial bioprocesses. However, the current understanding about the response mechanisms of A. niger toward ethanol stress remains quite limited. Here, we first applied a cell growth assay to test the ethanol tolerance of A. niger strain ES4, which was isolated from the wall near a chimney of an ethanol tank of a petroleum company, and found that it was capable of growing in 5% (v/v) ethanol to 30% of the ethanol-free control level. Subsequently, the metabolic responses of this strain toward ethanol were investigated using untargeted metabolomics, which revealed the elevated levels of triacylglycerol (TAG) in the extracellular components, and of diacylglycerol, TAG, and hydroxy-TAG in the intracellular components. Lastly, stable isotope labeling mass spectrometry with ethanol-d6 showed altered isotopic patterns of molecular ions of lipids in the ethanol-d6 samples, compared with the nonlabeled ethanol controls, suggesting the ability of A. niger ES4 to utilize ethanol as a carbon source. Together, the studies revealed the upregulation of glycerolipid metabolism and ethanol utilization pathway as novel response mechanisms of A. niger ES4 toward ethanol stress, thereby underlining the utility of untargeted metabolomics and the overall approaches as tools for elucidating new biological insights.

The effects of disruption in membrane lipid biosynthetic genes on 1-butanol tolerance of Bacillus subtilis.

Microbes with enhanced 1-butanol tolerance have the potentials to be utilized in various biotechnological processes. To achieve the rational design of such strains, we previously conducted an untargeted metabolomics analysis of Bacillus subtilis under 1-butanol stress and uncovered a novel type of microbial responses as the alterations in the glycerolipid and phospholipid composition. However, the current knowledge about the relevance of these changes on 1-butanol tolerance remains quite limited. Here, we constructed the B. subtilis mutants with disruption in the pssA, ugtP (U), mprF (M), yfnI, and yfnI/mprF genes in the membrane lipid biosynthetic pathways. The 1-butanol tolerance test indicated markedly increased and decreased 1-butanol resistance in M and U compared to the wild-type strain, respectively, and slight effects in other strains under high stress level. Further examination of the lipid contents of these strains in the presence of 1-butanol by liquid chromatography-mass spectrometry demonstrated an elevated ratio of neutral and anionic to cationic lipids in direct relation with an improved 1-butanol tolerance. Last, cell morphological studies showed the shortening of only the U cells, compared to the wild-type. All strains including U were capable of elongating by 14-24% under 1-butanol stress. Together, the studies indicated the involvement of membrane lipid biosynthetic genes, which regulated glycerolipid and phospholipid composition, on 1-butanol tolerance and allowed for the procurement of M with enhanced 1-butanol tolerance trait, highlighting the usefulness of the overall approaches on discovery of novel biological insights and engineering of microorganisms with desired resistance characteristics.

Serum lipidomics analysis of ovariectomized rats under Curcuma comosa treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcuma comosa Roxb. (C. comosa) or Wan Chak Motluk, Zingiberaceae family, has been used in Thai traditional medicine for the treatment of gynecological problems and inflammation. AIM OF THE STUDY: This study aimed to investigate the therapeutic potential of C. comosa by determining the changes in the lipid profiles in the ovariectomized rats, as a model of estrogen-deficiency-induced hyperlipidemia, after treatment with different components of C. comosa using an untargeted lipidomics approach. MATERIALS AND METHODS: Lipids were extracted from the serum of adult female rats subjected to a sham operation (SHAM; control), ovariectomy (OVX), or OVX with 12-week daily doses of estrogen (17ß-estradiol; E2), (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (DPHD; a phytoestrogen from C. comosa), powdered C. comosa rhizomes or its crude ethanol extract. They were then analyzed by liquid chromatography-mass spectrometry, characterized, and subjected to the orthogonal projections to latent structures discriminant analysis statistical model to identify tentative biomarkers. RESULTS: Levels of five classes of lipids (ceramide, ceramide-1-phosphate, sphingomyelin, 1-O-alkenyl-lysophosphatidylethanolamine and lysophosphatidylethanolamine) were elevated in the OVX rats compared to those in the SHAM rats, while the monoacylglycerols and triacylglycerols were decreased. The E2 treatment only reversed the levels of ceramides, whereas treatments with DPHD, C. comosa extract or powder returned the levels of all upregulated lipids back to those in the SHAM control rats. CONCLUSIONS: The findings suggest the potential beneficial effects of C. comosa on preventing the increased ceramide levels in OVX rats, a possible cause of metabolic disturbance under estrogen deficiency. Overall, the results demonstrated the power of untargeted lipidomics in discovering disease-relevant biomarkers, as well as evaluating the effectiveness of treatment by C. comosa components (DPHD, extract or powder) as utilized in Thai traditional medicine, and also providing scientific support for its folklore use.

Comparative protein profiles of Butea superba tubers under seasonal changes.

Seasonal changes are major factors affecting environmental conditions which induce multiple stresses in plants, leading to changes in protein relative abundance in the complex cellular plant metabolic pathways. Proteomics was applied to study variations in proteome composition of Butea. superba tubers during winter, summer and rainy season throughout the year using two-dimensional polyacrylamide gel electrophoresis coupled with a nanoflow liquid chromatography coupled to electrospray ionization quadrupole-time-of-flight tandem mass spectrometry. A total of 191 protein spots were identified and also classified into 12 functional groups. The majority of these were mainly involved in carbohydrate and energy metabolism (30.37 %) and defense and stress (18.32 %). The results exhibited the highest numbers of identified proteins in winter-harvested samples. Forty-five differential proteins were found in different seasons, involving important metabolic pathways. Further analysis indicated that changes in the protein levels were due mainly to temperature stress during summer and to water stress during winter, which affected cellular structure, photosynthesis, signal transduction and homeostasis, amino-acid biosynthesis, protein destination and storage, protein biosynthesis and stimulated defense and stress mechanisms involving glycolytic enzymes and relative oxygen species catabolizing enzymes. The proteins with differential relative abundances might induce an altered physiological status within plant tubers for survival. The work provided new insights into the better understanding of the molecular basis of plant proteomes and stress tolerance mechanisms, especially during seasonal changes. The finding suggested proteins that might potentially be used as protein markers in differing seasons in other plants and aid in selecting B. superba tubers with the most suitable medicinal properties in the future.

Untargeted metabolomics analysis revealed changes in the composition of glycerolipids and phospholipids in Bacillus subtilis under 1-butanol stress.

1-Butanol has been utilized widely in industry and can be produced or transformed by microbes. However, current knowledge about the mechanisms of 1-butanol tolerance in bacteria remains quite limited. Here, we applied untargeted metabolomics to study Bacillus subtilis cells under 1-butanol stress and identified 55 and 37 ions with significantly increased and decreased levels, respectively. Using accurate mass determination, tandem mass spectra, and synthetic standards, 86 % of these ions were characterized. The levels of phosphatidylethanolamine, diglucosyldiacylglycerol, and phosphatidylserine were found to be upregulated upon 1-butanol treatment, whereas those of diacylglycerol and lysyl phosphatidylglycerol were downregulated. Most lipids contained 15:0/15:0, 16:0/15:0, and 17:0/15:0 acyl chains, and all were mapped to membrane lipid biosynthetic pathways. Subsequent two-stage quantitative real-time reverse transcriptase PCR analyses of genes in the two principal membrane lipid biosynthesis pathways revealed elevated levels of ywiE transcripts in the presence of 1-butanol and reduced expression levels of cdsA, pgsA, mprF, clsA, and yfnI transcripts. Thus, the gene transcript levels showed agreement with the metabolomics data. Lastly, the cell morphology was investigated by scanning electron microscopy, which indicated that cells became almost twofold longer after 1.4 % (v/v) 1-butanol stress for 12 h. Overall, the studies uncovered changes in the composition of glycerolipids and phospholipids in B. subtilis under 1-butanol stress, emphasizing the power of untargeted metabolomics in the discovery of new biological insights.

Pueraria mirifica leaves, an alternative potential isoflavonoid source.

We investigated the major leaf isoflavonoid contents of Pueraria mirifica from three different cultivars (PM-III, PM-IV, and PM-V) using reverse RP-HPLC analysis. The proportions and net levels of puerarin, daidzin, genistin, and daidzein in P. mirifica leaves were found to depend on the plant cultivar and to correlate with cultivation temperature and rainfall amount. The crude leaf-extracts were tested using the Yeast Estrogen Screen (YES) assay with both human estrogen receptors (hERα and hERß). Their estrogenic activity was higher when determined by the YES system containing hERß than that with hERα and was also higher when the Δsnq2 than the wildtype yeast was employed. The results open the possibility of selecting and cultivating certain P. mirifica cultivars at a farm scale to produce a sufficient supply of leaf material to act as a starting source for the commercial scale extraction of these major isoflavonoids.

Superoxide dismutase isozyme detection using two-dimensional gel electrophoresis zymograms.

Superoxide dismutases (SODs) are ubiquitous antioxidant enzymes involved in cell protection from reactive oxygen species. Their antioxidant activities make them of interest to applied biotechnology industries and are usually sourced from plants. SODs are also involved in stress signaling responses in plants, and can be used as indicators of these responses. In this article, a suitable method for the separation of different SOD isoforms using two-dimensional-gel electrophoresis (2D-GE) zymograms is reported. The method was developed with a SOD standard from bovine erythrocytes and later applied to extracts from Stemona tuberosa. The first (non-denaturing isoelectric focusing) and second (denaturing sodium dodecylsulphate-polyacrylamide gel electrophoresis) dimensions of duplicate 2D-GE gels were stained with either Coomassie brilliant blue G-250 for total protein visualization, or SOD activity (zymogram) using riboflavin/nitroblue tetrazolium. For confirmation, putative SOD activity positive spots were subject to trypsin digestion and nano-liquid chromatography tandem mass spectrometry, followed by searching the MASCOT database for potential identification. The method could separate different SOD isoforms from a plant extract and at least partially maintain or allow renaturation to the native forms of the enzyme. Peptide sequencing of the 2D-GE suggested that the SODs were resolved correctly, identifying the control CuZn-SOD from bovine erythrocytes. The two SODs from S. tuberosa tubers were found to be likely homologous of a CuZn-SOD. SOD detection and isoform separation by 2D-GE zymograms was efficient and reliable. The method is likely applicable to SOD detection from plants or other organisms. Moreover, a similar approach could be developed for detection of other important enzymes in the future.

Identification of Pseudomonas aeruginosa phenazines that kill Caenorhabditis elegans.

Pathogenic microbes employ a variety of methods to overcome host defenses, including the production and dispersal of molecules that are toxic to their hosts. Pseudomonas aeruginosa, a Gram-negative bacterium, is a pathogen of a diverse variety of hosts including mammals and the nematode Caenorhabditis elegans. In this study, we identify three small molecules in the phenazine class that are produced by P. aeruginosa strain PA14 that are toxic to C. elegans. We demonstrate that 1-hydroxyphenazine, phenazine-1-carboxylic acid, and pyocyanin are capable of killing nematodes in a matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of phenazine-1-carboxylic acid and pyocyanin are pH-dependent at non-overlapping pH ranges. We found that acidification of the growth medium by PA14 activates the toxicity of phenazine-1-carboxylic acid, which is the primary toxic agent towards C. elegans in our assay. Pyocyanin is not toxic under acidic conditions and 1-hydroxyphenazine is produced at concentrations too low to kill C. elegans. These results suggest a role for phenazine-1-carboxylic acid in mammalian pathogenesis because PA14 mutants deficient in phenazine production have been shown to be defective in pathogenesis in mice. More generally, these data demonstrate how diversity within a class of metabolites could affect bacterial toxicity in different environmental niches.

Discovery of a protein-metabolite interaction between unsaturated fatty acids and the nuclear receptor Nur77 using a metabolomics approach.

Neuron-derived clone 77 (Nur77) is an orphan nuclear receptor with currently no known natural ligands. Here we applied a metabolomics platform for detecting protein-metabolite interactions (PMIs) to identify lipids that bind to Nur77. Using this approach, we discovered that the Nur77 ligand-binding domain (Nur77LBD) enriches unsaturated fatty acids (UFAs) in tissue lipid mixtures. The interaction of Nur77 with arachidonic acid and docosahexaenoic acid was subsequently characterized using a number of biophysical and biochemical assays. Together these data indicate that UFAs bind to Nur77LBD to cause changes in the conformation and oligomerization of the receptor. UFAs are the only endogenous lipids reported to bind to Nur77, which highlights the use of metabolomics in the discovery of novel PMIs.

Untargeted metabolomics.

Along with genes and proteins, metabolites play important roles in sustaining life. Their functions include "primary" functions in metabolism and energy storage, as well as "secondary" functions in cell-to-cell signaling, metal acquisition, and virulence. There remains much to be learned about the in vivo roles of metabolites. Approaches that accelerate measurement of metabolite levels directly from cells and tissues should increase our understanding of the diverse roles of metabolites and potentially lead to discovery of novel metabolites and metabolic pathways. Metabolomics is an important comparative tool to study global metabolite levels in samples under various conditions. In this unit, the steps needed to perform a mass spectrometry (MS)--based untargeted metabolomics experiment using bacterial supernatants are detailed. In contrast to a targeted metabolomics experiment, which measures ions from known metabolites, an untargeted metabolomics experiment registers all ions within a certain mass range, including ions belonging to structurally novel metabolites. The protocols in this unit describe the conditions necessary for analyzing hydrophobic metabolites and provide an example of how to structurally characterize a novel metabolite.

Exploring disease through metabolomics.

Metabolomics approaches provide an analysis of changing metabolite levels in biological samples. In the past decade, technical advances have spurred the application of metabolomics in a variety of diverse research areas spanning basic, biomedical, and clinical sciences. In particular, improvements in instrumentation, data analysis software, and the development of metabolite databases have accelerated the measurement and identification of metabolites. Metabolomics approaches have been applied to a number of important problems, which include the discovery of biomarkers as well as mechanistic studies aimed at discovering metabolites or metabolic pathways that regulate cellular and physiological processes. By providing access to a portion of biomolecular space not covered by other profiling approaches (e.g., proteomics and genomics), metabolomics offers unique insights into small molecule regulation and signaling in biology. In the following review, we look at the integration of metabolomics approaches in different areas of basic and biomedical research, and try to point out the areas in which these approaches have enriched our understanding of cellular and physiological biology, especially within the context of pathways linked to disease.

Regulation of alkyl-dihydrothiazole-carboxylates (ATCs) by iron and the pyochelin gene cluster in Pseudomonas aeruginosa.

Using the pyochelin (pch) gene cluster as an example, we demonstrate the utility of untargeted metabolomics in the discovery and characterization of secondary metabolites regulated by biosynthetic gene clusters. Comparison of the extracellular metabolomes of pch gene cluster mutants to the wild-type Pseudomonas aeruginosa (strain PA 14) identified 198 ions regulated by the pch genes. In addition to known metabolites, we characterized the structure of a pair of novel metabolites regulated by the pch gene cluster as 2-alkyl-4,5-dihydrothiazole-4-carboxylates (ATCs), using a combination of mass spectrometry, chemical synthesis, and stable isotope labeling. Subsequent assays revealed that ATCs bind iron and are regulated by iron levels in the media in a similar fashion as other metabolites associated with the pch gene cluster. Further genetic complementation and overexpression analyses of the pch genes revealed ATC production to be dependent on the pchE gene in the pch gene cluster. Overall, these studies highlight the ability of untargeted metabolomics to reveal regulatory connections between gene clusters and secondary metabolites, including novel metabolites.