Functional groups matter: metabolomics analysis of Escherichia coli exposed to trans-cinnamic acid and its derivatives unveils common and unique targets.

Phenolic acids are derivatives of benzoic and cinnamic acids, which possess important biological activities at certain concentrations. Trans-cinnamic acid (t-CA) and its derivatives, such as p-coumaric acid (p-CA) and ferulic acid (FA) have been shown to have antibacterial activity against various Gram-positive and -negative bacteria. However, there is limited information available concerning the antibacterial mode of action of these phenolic acids. In this study, we aimed to ascertain metabolic alterations associated with exposure to t-CA, p-CA, and FA in Escherichia coli BW25113 using a nuclear magnetic resonance (NMR)-based metabolomics approach. The results showed that t-CA, p-CA, and FA treatments led to significant changes (p < 0.05) in the concentration of 42, 55, and 74% of the identified metabolites in E. coli, respectively. Partial least-squares discriminant analysis (PLS-DA) revealed a clear separation between control and phenolic acid groups with regard to metabolic response. Moreover, it was found that FA and p-CA treatment groups were clustered closely together but separated from the t-CA treatment group. Arginine, putrescine, cadaverine, galactose, and sucrose had the greatest impact on group differentiation. Quantitative pathway analysis demonstrated that arginine and proline, pyrimidine, glutathione, and galactose metabolisms, as well as aminoacyl-tRNA and arginine biosyntheses, were markedly affected by all phenolic acids. Finally, the H2O2 content of E. coli cells was significantly increased in response to t-CA and p-CA whereas all phenolic acids caused a dramatic increase in the number of apurinic/apyrimidinic sites. Overall, this study suggests that the metabolic response of E. coli cells to t-CA is relatively different from that to p-CA and FA. However, all phenolic acids had a certain impact on oxidative/antioxidant status, genomic stability, arginine-related pathways, and nucleic acid metabolism.

NMR metabolomics analysis of Escherichia coli cells treated with Turkish propolis water extract reveals nucleic acid metabolism as the major target.

AIMS: Propolis is a resinous bee product containing several hundred biologically active compounds. Although the antibacterial activity of propolis has been demonstrated in many in vitro studies, less is known about its mode of action. In this study, we aimed to shed some light on the antibacterial mechanism of action of propolis against Escherichia coli BW25113 using a nuclear magnetic resonance (NMR) based metabolomics approach. METHODS: E. coli BW25113 cells were subjected to different sub-lethal concentrations (0, 2, 4, and 6 mg/mL) of Turkish propolis water extract (PWE). The 500-MHz 1H NMR spectroscopy was then employed to ascertain the metabolic profiles of E. coli extracts. RESULTS: A total of 52 metabolites were identified from the NMR spectra, belonging to 17 main classes, such as amino acids and peptides, purines, and fatty acids. Twelve out of these 52 metabolites displayed remarkable changes at all tested PWE concentrations when compared to control conditions (P < .05). Levels of 28 more metabolites were significantly altered in at least one of the three PWE treatments. The results of partial least squares discriminant analysis showed that there was a clear separation between control and propolis-treated cells and that putrescine, adenine, adenosine, guanosine, glucose, N6-acetyllysine, and acetamide had the highest effect on group differentiation. Finally, quantitative pathway analysis revealed that purine metabolism was significantly affected by PWE treatments. CONCLUSIONS: Our results suggest that PWE inhibits the growth of E. coli BW25113 by affecting nucleic acid metabolism to a great extent. To the best of our knowledge, this is the first study to evaluate the global metabolic response of a bacterium to propolis.

Identification of interaction partners using protein aggregation and NMR spectroscopy.

The interaction among proteins is one of the most fundamental methods of information transfer in the living system. Many methods have been developed in order to identify the interaction pairs or groups either in vivo or in vitro. The in vitro pulldown/coprecipitation assay directly observes the protein that binds to the target. This method involves electrophoresis, which is a technique of a low resolution as well as a low throughput. As a better alternative, we wish to propose a new method that is based on the NMR spectroscopy. This method utilizes the aggregation of the target protein and the concomitant signal disappearance of the interacting partner. The aggregation is accomplished by the elastin-like polypeptide, which is fused to the target. If a protein binds to this supramolecular complex, its NMR signal then becomes too broadened in order to be observed, which is the basic phenomenon of the NMR spectroscopy. Thus, the protein that loses its signal is the one that binds to the target. A compound that interferes with these types of bindings among the proteins can be identified by observing the reappearance of the protein signals with the simultaneous disappearance of the signals of the compound. This technique will be applied in order to find an interaction pair in the information transfer pathway as well as a compound that disrupts it. This proposed method should be able to work with a mixture of proteins and provide a higher resolution in order to find the binding partner in a higher throughput fashion.

Development of an Autoinducible Plasmid for Recombinant Protein Production.

BACKGROUND: The production of recombinant proteins in E. coli involves such factors as host strains, expression vectors, culture media, and induction methods. The typical procedure to produce heterologous proteins consists of the following: (1) insertion of the target gene into a suitable vector to construct an overexpression plasmid, (2) transformation of a strain specialized for protein production with the constructed plasmid DNA, (3) growth of the host in a suitable medium and induction of the protein production at a right moment, and (4) further growth to get the maximum yield. There are hurdles involved in each of these steps, and researchers have developed many materials or methods, which often require special recipes or procedures. OBJECTIVE: To eliminate the special requirements for recombinant protein production by using readily available materials. Also to save time and effort in the routine protein production work. METHODS: We started with a vector capable of producing a target protein fused to the C-terminus of the maltose-binding protein (MBP). The mCherry (red fluorescent protein) gene was fused to MBP. It acted as a reporter in the initial screening procedure. The original lethal gene (barnase) was replaced with sacB. We chose 3 stationary phase promoters and made hybrids of them by mixing halves from each one. The T5 promoter was replaced with these stationary phase promoters or their hybrids. The best plasmid was selected by the color intensity of the cell pellet. MBP and GST genes were inserted in the place of sacB, and their production yields were compared with the original plasmid in the conventional way of expression. RESULTS: We constructed an expression plasmid with an autoinducible promoter working in a host that was not specially designed for protein production and in a TB medium that did not contain any secret ingredient, nor was it difficult to prepare unlike Studier's defined medium. This plasmid also contains a color indicator that turns red when protein production is successful. We tested our system with the maltose-binding protein (MBP) and the glutathione S-transferase (GST), and showed that both proteins were produced to a level comparable to what the commercial medium and/or the specialized strain yielded. CONCLUSION: We developed a plasmid equipped with an autoinducible promoter, a hybrid of the two promoters which were activated at the stationary phase. This plasmid does not need a special E. coli strain nor a sophisticated nor an expensive medium. It produces an intense red (or pink) color, which can be used as an indicator of a successful production of the target protein and as a predictive measure of the amount of the produced target protein. We speculate that this plasmid will have its greatest advantage when growing cells at low temperatures, which would inevitably take a long time.

A simple and sensitive detection of the binding ligands by using the receptor aggregation and NMR spectroscopy: a test case of the maltose binding protein.

Protein-ligand interaction is one of the highlights of molecular recognition. The most popular application of this type of interaction is drug development which requires a high throughput screening of a ligand that binds to the target protein. Our goal was to find a binding ligand with a simple detection, and once this type of ligand was found, other methods could then be used to measure the detailed kinetic or thermodynamic parameters. We started with the idea that the ligand NMR signal would disappear if it was bound to the non-tumbling mass. In order to create the non-tumbling mass, we tried the aggregates of a target protein, which was fused to the elastin-like polypeptide. We chose the maltose binding proteinas a test case, and we tried it with several sugars, which included maltose, glucose, sucrose, lactose, galactose, maltotriose, and ß-cyclodextrin. The maltose signal in the H-1 NMR spectrum disappeared completely as hoped around the protein to ligand ratio of 1:3 at 298 K where the proteins aggregated. The protein signals also disappeared upon aggregation except for the fast-moving part, which resulted in a cleaner background than the monomeric form. Since we only needed to look for a disappearing signal amongst those from the mixture, it should be useful in high throughput screening. Other types of sugars except for the maltotriose and ß-cyclodextrin, which are siblings of the maltose, did not seem to bind at all. We believe that our system would be especially more effective when dealing with a smaller target protein, so both the protein and the bound ligand would lose their signals only when the aggregates formed. We hope that our proposed method would contribute to accelerating the development of the potent drug candidates by simultaneously identifying several binders directly from a mixture.

Relationship between recombinant protein expression and host metabolome as determined by two-dimensional NMR spectroscopy.

Escherichia coli has been the most widely used host to produce large amounts of heterologous proteins. However, given an input plasmid DNA, E. coli may produce soluble protein, produce only inclusion bodies, or yield little or no protein at all. Many efforts have been made to surmount these problems, but most of them have involved time-consuming and labor-intensive trial-and-error. We hypothesized that different metabolomic fingerprints might be associated with different protein production outcomes. If so, then it might be possible to change the expression pattern by manipulating the metabolite environment. As a first step in testing this hypothesis, we probed a subset of the intracellular metabolites by partially labeling it with 13C-glucose. We tested 71 genes and identified 17 metabolites by employing the two-dimensional NMR spectroscopy. The statistical analysis showed that there existed the metabolite compositions favoring protein production. We hope that this work would help devise a systematic and predictive approach to the recombinant protein production.

Application of two-dimensional NMR spectroscopy to metabotyping laboratory Escherichia coli strains.

NMR Spectroscopy has been established as a major tool for identification and quantification of metabolites in a living system. Since the metabolomics era began, one-dimensional NMR spectroscopy has been intensively employed due to its simplicity and quickness. However, it has suffered from an inevitable overlap of signals, thus leading to inaccuracy in identification and quantification of metabolites. Two-dimensional (2D) NMR has emerged as a viable alternative because it can offer higher accuracy in a reasonable amount of time. We employed (1) H,(13) C-HSQC to profile metabolites of six different laboratory E. coli strains. We identified 18 metabolites and observed clustering of six strains according to their metabolites. We compared the metabolites among the strains, and found that a) the strains specialized for protein production were segregated; b) XL1-Blue separated itself from others by accumulating amino acids such as alanine, aspartate, glutamate, methionine, proline, and lysine; c) the strains specialized for cloning purpose were spread out from one another; and d) the strains originating from B strain were characterized by succinate accumulation. This work shows that 2D-NMR can be applied to identify a strain from metabolite analysis, offering a possible alternative to genetic analysis to identify E. coli strains.

Amphiphilic thiol functional linker mediated sustainable anti-biofouling ultrafiltration nanocomposite comprising a silver nanoparticles and poly(vinylidene fluoride) membrane.

We develop sustainable anti-biofouling ultrafiltration membrane nanocomposites by covalently immobilizing silver nanoparticles (AgNPs) onto poly(vinylidene fluoride) (PVDF) membrane mediated by a thiol-end functional amphiphilic block copolymer linker. Field emission scanning electron microscopy (FE-SEM) and energy-dispersive X-ray spectroscopy (EDXS) measurements reveal that the AgNPs are highly bound and dispersed to the PVDF membrane due to the strong affinity of the AgNPs with the thiol-modified block copolymeric linkers, which have been anchored to the PVDF membrane. The membrane performs well under water permeability and particle rejection measurements, despite the high deposition of AgNPs on the surface of membrane. The Ag-PVDF membrane nanocomposite significantly inhibits the growth of bacteria on the membrane surface, resulting in enhanced anti-biofouling property. Importantly, the AgNPs are not released from the membrane surface due to the robust covalent bond between the AgNPs and the thiolated PVDF membrane. The stability of the membrane nanocomposite ensures a sustainable anti-biofouling activity of the membrane.

Dosage Effects of Salt and pH Stresses on Saccharomyces cerevisiae as Monitored via Metabolites by Using Two Dimensional NMR Spectroscopy.

Saccharomyces cerevisiae, which is a common species of yeast, is by far the most extensively studied model of a eukaryote because although it is one of the simplest eukaryotes, its basic cellular processes resemble those of higher organisms. In addition, yeast is a commercially valuable organism for ethanol production. Since the yeast data can be extrapolated to the important aspects of higher organisms, many researchers have studied yeast metabolism under various conditions. In this report, we analyzed and compared metabolites of Saccharomyces cerevisiae under salt and pH stresses of various strengths by using two-dimensional NMR spectroscopy. A total of 31 metabolites were identified for most of the samples. The levels of many identified metabolites showed gradual or drastic increases or decreases depending on the severity of the stresses involved. The statistical analysis produced a holistic outline: pH stresses were clustered together, but salt stresses were spread out depending on the severity. This work could provide a link between the metabolite profiles and mRNA or protein profiles under representative and well studied stress conditions.

Recombinant production, isotope labeling and purification of ENOD40B: a plant peptide hormone.

The plant peptide hormone ENOD40B was produced in a protein production strain of Escherichia coli harboring an induction controller plasmid (Rosetta(DE3)pLysS) as a His6-tagged ubiquitin fusion protein. The fusion protein product was denatured and refolded as part of the isolation procedure and purified by immobilized metal ion chromatography. The peptide hormone was released from its fusion partner by adding yeast ubiquitin hydrolase (YUH) and subsequently purified by reversed phase chromatography. The purity of the resulting peptide fragment was assayed by MALDITOF mass spectrometry and NMR spectroscopy. The final yields of the target peptide were 7.0 mg per liter of LB medium and 3.4 mg per liter of minimal medium.

Stress adaptation of Saccharomyces cerevisiae as monitored via metabolites using two-dimensional NMR spectroscopy.

Many studies on yeast metabolism are focused on its response to specific stress conditions because the results can be extended to the human medical issues. Most of those works have been accomplished through functional genomics studies. However, these changes may not show a linear correlation with protein or metabolite levels. For many organisms including yeast, the number of metabolites is far fewer than that of genes or gene products. Thus, metabolic profiling can provide a simpler yet efficient snapshot of the system's physiology. Metabolites of Saccharomyces cerevisiae under various stresses were analyzed and compared with those under the normal, unstressed growth conditions by two-dimensional NMR spectroscopy. At least 31 metabolites were identified for most of the samples. The levels of many identified metabolites showed significant increase or decrease depending on the nature of the stress. The statistical analysis produced a holistic view: different stresses were clustered and isolated from one another with the exception of high pH, heat, and oxidative stresses. This work could provide a link between the metabolite profiles and mRNA or protein profiles under representative and well-studied stress conditions.

The sigma-1 receptor protects against cellular oxidative stress and activates antioxidant response elements.

Sigma-1 receptors are associated with Alzheimer's disease, major depressive disorders, and schizophrenia. These receptors show progrowth/antiapoptotic properties via their chaperoning functions to counteract ER (endoplasmic reticulum) stress, to block neurodegeneration, and to regulate neuritogenesis. The sigma-1 receptor knock out mouse offered an opportunity to assess possible mechanisms by which the sigma-1 receptor modulates cellular oxidative stress. Nuclear magnetic resonance (NMR) metabolomic screening of the WT (wild type) and sigma-1 KO (knockout) livers was performed to investigate major changes in metabolites that are linked to oxidative stress. Significant changes in protein levels were also identified by two-dimensional (2D) gel electrophoresis and mass spectrometry. Increased levels of the antioxidant protein peroxiredoxin 6 (Prdx6), and the ER chaperone BiP (GRP78) compared to WT littermates were detected. Oxidative stress was measured in WT and sigma-1 KO mouse liver homogenates, in primary hepatocytes and in lung homogenates. Furthermore, sigma-1 receptor mediated activation of the antioxidant response element (ARE) to upregulate NAD(P)H quinone oxidoreductase 1 (NQO1) and superoxide dismutase 1 (SOD1) mRNA expression in COS cells was shown by RT PCR. These novel functions of the sigma-1 receptor were sensitive to well-known sigma ligands via their antagonist/agonist properties.

Tracing Metabolite Footsteps of Escherichia coli Along the Time Course of Recombinant Protein Expression by Two-Dimensional NMR Spectroscopy.

The recombinant expression of proteins has been the method of choice to meet the demands from proteomics and structural genomics studies. Despite its successful production of many heterologous proteins, Escherichia coli failed to produce many other proteins in their native forms. This may be related to the fact that the stresses resulting from the overproduction interfere with cellular processes. To better understand the physiological change during the overproduction phase, we profiled the metabolites along the time course of the recombinant protein expression. We identified 32 metabolites collected from different time points in the protein production phase. The stress induced by protein production can be characterized by (A) the increased usage of aspartic acid, choline, glycerol, and N-acetyllysine; and (B) the accumulation of adenosine, alanine, oxidized glutathione, glycine, N-acetylputrescine, and uracil. We envision that this work can be used to create a strategy for the production of usable proteins in large quantities.

Recombinant Expression, Isotope Labeling and Purification of the Vitamin D Receptor Binding Peptide.

The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquitin molecule in Rosetta(DE3)pLysS, a protein production strain of Escherichia coli harboring an induction controller plasmid. The fusion protein was bound to the immobilized metal ions, and the denaturation and renaturation of the fusion protein were performed as a part of the purification procedure. After the elution of the fusion protein, the peptide hormone was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The purity of the resulting peptide fragment was checked by MALDI-TOF mass and NMR spectroscopy. The final yields of the target peptide were around 5 and 2 mg per liter of LB and minimal media, respectively. The recombinant expression and purification of this peptide will enable structural and functional studies using multidimensional NMR spectroscopy and X-ray crystallography.

Discovery of novel human phenylethanolamine N-methyltransferase (hPNMT) inhibitors using 3D pharmacophore-based in silico, biophysical screening and enzymatic activity assays.

With the aid of receptor-oriented pharmacophore-based in silico screening, we established three pharmacophore maps explaining the binding model of hPNMT and a known inhibitor, SK&F 29661 (Martin et al., 2001). The compound library was searched using these maps. Nineteen selected candidate inhibitors of hPNMT were screened using STD-NMR and fluorescence experiments. An enzymatic activity assay based on HPLC was additionally performed. Consequently, three potential hPNMT inhibitors were identified, specifically, 4-oxo-1,4-dihydroquinoline-3,7-dicarboxylic acid, 4-(benzo[d][1,3]dioxol-5-ylamino)-4-oxobutanoic acid, and 1,4-diaminonaphthalene-2,6-disulfonic acid. These novel inhibitors were retrieved using Map II comprising one hydrogen bond acceptor, one hydrogen bond donor, one lipophilic feature, and shape constraints, including a hydrogen bond between Lys57 of hPNMT and a hydrogen bond donor of the inhibitor, and stacked hydrophobic interactions between the side-chain of Phe182 and an aromatic region of the inhibitor. Water-mediated interactions between Asn267 and Asn39 of hPNMT and the amide or amine group of three potent inhibitors were additional important features for hPNMT activity. The binding model presented here may be applied to identify inhibitors with higher potency. Moreover, our novel compounds are valuable candidates for further lead optimization of PNMT inhibitors.

Stationary phase expression, purification, and characterization of XorKI, a restriction endonuclease from Xanthomonas oryzae pv. oryzae.

An endonuclease from Xanthomonas oryzae pathovar oryzae KACC 10331, XorKI, was heterologously produced in Escherichia coli by applying the stationary state induction method. The yield was 5.4 mg of XorKI per liter of LB medium. XorKI existed in multiple oligomeric forms as evidenced by gel filtration chromatography. The specific activity of purified XorKI was 323000 units per mg.

Recombinant expression, purification, and characterization of XorKII: a restriction endonuclease from Xanthomonas oryzae pv. oryzae.

An endonuclease from Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, XorKII, was recombinantly produced in Escherichia coli by applying the stationary state induction method, which was necessary to prevent the unwanted lysis of E. coli cells. XorKII was purified by immobilized metal affinity chromatography on an FPLC system. The yield was 3.5mg of XorKII per liter of LB medium. The purified recombinant XorKII showed that it recognized and cleaved to the same site as PstI. It behaved as a dimer as evidenced by the size exclusion chromatography. The specific activity of the purified XorKII was determined to be 31,300 U/mg. The enzyme activity was monitored by cleaving lambda DNA or YEp24 plasmid as substrates. The enzyme was the most active at 10mM Tris-HCl pH 7.0, 10 mM MgCl(2), 1mM dithiothreitol at 37 degrees C. XorKII was easily inactivated by heating at 65 degrees C for 5 min, but retained most of the original activity after incubation at 37 degrees C for 24h.

Recombinant expression and purification of functional XorII, a restriction endonuclease from Xanthomonas oryzae pv. oryzae.

An endonuclease from Xanthomonas oryzae pathovar oryzae KACC 10331, XorII, was recombinantly produced in Escherichia coli using a T7 system. XorII was purified using a combination of ion exchange and immobilized metal affinity chromatography (IMAC). An optimized washing protocol was carried out on an IMAC in order to obtain a high purity product. The final amount of purified XorII was approximately 2.5 mg/L of LB medium. The purified recombinant XorII was functional and showed the same cleavage pattern as PvuI. The enzyme activity tested the highest at 25 degrees in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM dithiothreitol at a pH of 7.9.

Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin.

An antimicrobial peptide, piscidin, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The peptide was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The expression and purification process of piscidin encountered several problems such as the lysis of the bacterial cell upon induction of the peptide production, the unwanted cleavage of the fusion protein inside the bacterial cell, and high tendency to aggregate in the aqueous environment. Such problems were alleviated by employing ubiquitin as a fusion partner for piscidin, growing the cells at a lower temperature, and changing the order of the purification steps. The yields of the fusion protein and the peptide were around 15 and 1.5 mg per liter of LB or minimal medium, respectively. The recombinant expression and purification of piscidin will enable its structural and dynamic studies using multidimensional NMR spectroscopy.