Microbial Metabolism Studies Research Articles

Genome-Scale Metabolic Models in Fungal Pathogens: Past, Present, and Future.

Fungi are diverse organisms with various characteristics and functions. Some play a role in recycling essential elements, such as nitrogen and carbon, while others are utilized in the food and drink production industry. Some others are known to cause diseases in various organisms, including humans. Fungal pathogens cause superficial, subcutaneous, and systemic infections. Consequently, many scientists have focused on studying the factors contributing to the development of human diseases. Therefore, multiple approaches have been assessed to examine the biology of these intriguing organisms. The genome-scale metabolic models (GEMs) have demonstrated many advantages to microbial metabolism studies and the ability to propose novel therapeutic alternatives. Despite significant advancements, much remains to be elucidated regarding the use of this tool for investigating fungal metabolism. This review aims to compile the data provided by the published GEMs of human fungal pathogens. It gives specific examples of the most significant contributions made by these models, examines the advantages and difficulties associated with using such models, and explores the novel approaches suggested to enhance and refine their development.

Gut microbial metabolites of dietary polyphenols and their potential role in human health and diseases.

Polyphenols contribute as one of the largest groups of compounds among all the phytochemicals. Common sources of dietary polyphenols are vegetables, fruits, berries, cereals, whole grains, etc. Owing to their original form, they are difficult to get absorbed. Dietary polyphenols after undergoing gut microbial metabolism form bioaccessible and effective metabolites. Polyphenols and derived metabolites are all together a diversified group of compounds exhibiting pharmacological activities against cardiovascular, cancer, oxidative stress, inflammatory, and bacterial diseases. The formed metabolites are sometimes even more bioavailable and efficacious than the parent polyphenols. Studies on gut microbial metabolism of dietary polyphenols have introduced new approach for the use of polyphenol-rich food in the form of supplementary diet. This review provides insights on various aspects including classification of polyphenols, gut microbiota-mediated metabolism of polyphenols, chemistry of polyphenol metabolism, and pharmacological actions of gut microbial metabolites of polyphenols. It also suggests the use of polyphenols from marine source for the microbial metabolism studies. Till date, gut microbial metabolism of polyphenols from terrestrial sources is extensively studied as compared to marine polyphenols. Marine ecosystem is a profound but partially explored source of phytoconstituents. Among them, edible seaweeds contain high concentration of polyphenols, especially phlorotannins. Hence, microbial metabolism studies of seaweeds can unravel the pharmacological potential of marine polyphenol-derived metabolites.

Metabolic turnover of cysteine-related thiol compounds at environmentally relevant concentrations by Geobacter sulfurreducens.

Low-molecular-mass (LMM) thiol compounds are known to be important for many biological processes in various organisms but LMM thiols are understudied in anaerobic bacteria. In this work, we examined the production and turnover of nanomolar concentrations of LMM thiols with a chemical structure related to cysteine by the model iron-reducing bacterium Geobacter sulfurreducens. Our results show that G. sulfurreducens tightly controls the production, excretion and intracellular concentration of thiols depending on cellular growth state and external conditions. The production and cellular export of endogenous cysteine was coupled to the extracellular supply of Fe(II), suggesting that cysteine excretion may play a role in cellular trafficking to iron proteins. Addition of excess exogenous cysteine resulted in a rapid and extensive conversion of cysteine to penicillamine by the cells. Experiments with added isotopically labeled cysteine confirmed that penicillamine was formed by a dimethylation of the C-3 atom of cysteine and not via indirect metabolic responses to cysteine exposure. This is the first report of de novo metabolic synthesis of this compound. Penicillamine formation increased with external exposure to cysteine but the compound did not accumulate intracellularly, which may suggest that it is part of G. sulfurreducens' metabolic strategy to maintain cysteine homeostasis. Our findings highlight and expand on processes mediating homeostasis of cysteine-like LMM thiols in strict anaerobic bacteria. The formation of penicillamine is particularly noteworthy and this compound warrants more attention in microbial metabolism studies.

Complete metabolic study by dibutyl phthalate degrading Pseudomonas sp. DNB-S1.

The primary purpose of this study was to systematically explore the complete metabolic pathway and tolerance mechanism of strain DNB-S1 to dibutyl phthalate (DBP), and the effect of DBP on energy metabolism of DNB-S1. Here, DNB-S1, a strain of Pseudomonas sp. that was highly effective in degrading DBP, was identified, and differentially expressed metabolites and metabolic networks of DBP were studied. The results showed that the differentially expressed metabolites were mainly aromatic compounds and lipid compounds, with only a few toxic intermediate metabolites. It speculated that phthalic acid, salicylic acid, 3-hydroxybenzoate acid, 3-Carboxy-cis, cis-muconate, fumarypyravate were intermediate metabolites of DBP. Their up-regulation indicated that there were two metabolic pathways in the degradation of DBP (protocatechuate pathway and gentisate pathway), which had been verified by peak changes at 290nm, 320nm, 330nm, and 375nm in the enzymatic method. Also, aspartate, GSH, and other metabolites were up-regulation, indicating that DNB-S1 had a high tolerance to DBP and maintained cell homeostasis, which was also one of the essential reasons to ensure the efficient degradation of DBP. Altogether, this study firstly proposed two pathways to degrade DBP and comprehensively explored the effect of DBP on the metabolic function of DNB-S1, which enriched the study of microbial metabolism of organic pollutants, and which provided a basis for the application of metabolomics.

Optimization for microbial incorporation and efficiency of photodynamic therapy using variation on curcumin formulation.

A mixture of curcuminoids: curcumin, desmethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are named natural curcumin. It is a lipophilic photosensitizer (PS) highly soluble in an organic solvent such as dimethyl sulfoxide (DMSO). Curcumin is a PS used for microbial inactivation using photodynamic action. However, this solvent has high cytotoxicity and is unavailable in formulations for clinical use. This study aimed to investigate the interactions of curcuminoids syrup with Streptococcus sp., a gram-positive coccus and one of the major pharyngeal pathogens, responsible for diseases such as pharyngitis. Bacteria were incubated with curcuminoids (natural curcumin, synthetic, DMC, BDMC) at 37 °C in formulations: 1) syrup (water + sucrose) 2) solution alcohol + DMSO. Was centrifuged, and the supernatant collected for absorbance analysis. The results obtained correlating the absorbance with the supernatant to the absorbance of the default concentration. A study of microbial metabolism by growth curve was carried out to justify the result. The incorporation of curcumin in syrup is superior to alcohol/DMSO solution by microorganisms. Curcumin incorporation by S. mutans, S. pyogenes, isolated bacteria was 24, 26, 27 % in syrup and 10, 13, 5 % in alcohol/DMSO, respectively. Also, the presence of carbohydrate in a solution can activate the bacterial metabolism, getting better uptake results and photodynamic inactivation to natural curcumin and DMC. Such finds care optimizes the use of curcumin without complications generated by the solvent.

Real-Time Monitoring of Microbial Fermentation End-Products in Biofuel Production with Titrimetric Off-Gas Analysis (TOGA)

Abstract. Bioprocesses can be utilized for the sustainable production of biofuels and other value-added co-products. However, most bioprocesses targeting production of low value, high-volume products cannot yet compete with conventional technologies and require further development. On-line monitoring can help expedite bioprocess development by allowing dynamic study of microbial metabolism, optimal process control, and product quality assurance. Titrimetric off-gas analysis (TOGA) is an on-line sensor system that combines a membrane inlet mass spectrometer (MIMS) with on-line titration techniques to allow dynamic study of biological processes, including fermentation. In this study, TOGA was applied to fermentation of 2 g L -1 cellobiose with Clostridium thermocellum. Ethanol, CO 2 , H 2 , and total hydrogen ion production (HP) were quantified in real time. Concurrent off-line analysis was performed for verification using high-performance liquid chromatography and gas chromatography. Errors in the on-line data with respect to off-line analyses were found to be 4.8% ±3.7%, 7.1% ±3.5%, and 10.7% ±9.0% for H 2 , CO 2 , and ethanol, respectively. The discrepancies between the on-line and off-line analyses were not statistically significant with 95% confidence intervals. The titration system effectively maintained the process pH at 7.2 ±0.02 and also measured the hydrogen ion production (HP), which could be 88.4% accounted for in the production of CO 2 and organic acids, as determined by off-line analyses. The instrument was shown to be a viable on-line sensor that can monitor multiple physiologically relevant variables and contribute insight to the development and control of fermentation with C. thermocellum. The methodology can easily be extended to other fermentation processes.

Microbial metabolites of 8-prenylnaringenin, an estrogenic prenylflavanone

Microbial metabolism studies of the phyto-estrogen (+/-)-8-prenylnaringenin (8-PN) (1) has led to the isolation of three pairs of metabolites (2-4). The structures of these compounds were identified as 5,4'-dihydroxy-7,8-[2-(1-hydroxy-1-methylethyl)-2,3-dihydrofurano]flavanones (2), 8-prenylnaringenin 7-O-beta-D-glucopyranosides (3), and 8-prenylnaringenin 7-O-beta-D-(6'''-O-alpha-hydroxypropionyl)-glucopyranosides (4) on the basis of the spectroscopic analysis.

Community dynamics of anaerobic bacteria in deep petroleum reservoirs

The nature, activity and metabolism of microbes that inhabit the deep subsurface environment are a matter of ongoing debate. The analysis of oil samples from three different basins in South America, central Europe and the Middle East indicates the presence of intact phospholipids and suggests that indigenous bacteria inhabit petroleum reservoirs in sediment depths of up to 2,000 m. The nature, activity and metabolism of microbes that inhabit the deep subsurface environment are a matter of ongoing debate1,2,3,4,5,6,7. Primarily limited by temperature8, little is known about secondary factors that restrict or enhance microbial activity9,10 or about the extent of a habitable environment deep below the surface. In particular, the degraders of chemically inert organic substrates remain elusive9. Petroleum reservoirs can be regarded as natural bioreactors and are ideally suited for the study of microbial metabolism in the deep subsurface. Here we analyse series of oil samples that were biodegraded to different degrees. We find fatty acids after hydrolysis of purified crude oil fractions, indicating the presence of intact phospholipids and suggesting that indigenous bacteria inhabit petroleum reservoirs in sediment depths of up to 2,000 m. A major change in bacterial community structure occurs after the removal of n-alkanes, indicating that more than one consortium is responsible for petroleum degradation11. Our results suggest that further study of petroleum fluids will help understand bacterial metabolism and diversity in this habitat of the deep subsurface.

The Application of19F Nuclear Magnetic Resonance to Investigate Microbial Biotransformations of Organofluorine Compounds

Fluorinated organic compounds, although rare in nature, are significant environmental contaminants owing to the numerous applications for which this class of compounds is employed. It is important that biodegradation of these compounds can be readily assessed in order to provide information on their fate in the environment. Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy has emerged as a very useful technique to readily determine the catabolism of fluorinated aromatic compounds by microorganisms, either in whole cell or cell-free systems. The principal advantage of this technique is that fluorinated compounds can be observed directly in the culture supernatant or enzyme assay, without purification or derivatization. In this review an account of the application of 19F NMR in the study of microbial metabolism of organofluorine compounds is presented.

Metabolism of quercetin and rutin by the pig caecal microflora prepared by freeze-preservation

Several studies confirm a protection of cardiovascular diseases and certain forms of cancer by dietary flavonoid intake. The bioavailability of flavonoids is influenced by the metabolism of the microflora in the intestine. Using a new in vitro model system the deglycosylation of the flavonol rutin and the degradation of its aglycone quercetin were investigated by using fresh pig caecal inocula in comparison to inocula prepared before by freeze-preservation between 6 wk and 5 months. The incubation experiments led to the same pattern of phenolic degradation products in comparable amounts in both preparations using HPLC-DAD and GC-flame ionization detection (GC-FID) or GC-MS detection within 24-48 h of incubation. With the preservation of the microbial vitality and the metabolic efficiency by freeze-preparation over several months the experimental design of microbial metabolism studies will be independent in time and locality.

Microbial metabolism of the environmental estrogen bisphenol A.

Preliminary microbial metabolism studies of bisphenol A (BPA) (1) on twenty six microorganisms have shown that Aspergillus fumigatus is capable of metabolizing BPA. Scale-up fermentation of 1 with A. fumigatus gave a metabolite (2) and its structure was established as bisphenol A-O-beta-D-glucopyranoside (BPAG) based on spectroscopic analyses.

In vivo NMR system for evaluating oxygen-dependent metabolic status in microbial culture

To optimize an appropriate microbial culture in a fermentor, precise control of the medium's dissolved oxygen tension (DOT) is crucial. In particular, to study the effect of DOT on cellular metabolic status by using in vivo nuclear magnetic resonance (NMR) measurements, the set-up of the experiment must be optimized to maintain DOT in the culture. In the conventional method, DOT is monitored by a sensor inside a fermentor and is controlled by changing the agitation rate. Here, we report a novel and accurate system that minimizes time lag by an automated aeration flow control device, allowing an NMR spectrometer to monitor representative metabolites in real-time. To fulfill these two objects, the fermentor was composed of a fermentation vessel and two outer tubes, through which the medium was circulated by rotary pumps. One tube monitored DOT in via a sensor, and at the same time the other tube monitored metabolites via an NMR spectrometer. In this study, we used this system to analyze the responses of Escherichia coli cells under various oxygen conditions. The results validated the use of this system in the study of microbial metabolism.

Microbial and mammalian metabolism studies of the semisynthetic antimalarial, anhydrodihydroartemisinin.

Microbial metabolism studies of the semisynthetic antimalarial anhydrodihydroartemisinin (1), have shown that it is metabolized by a number of microorganisms. Large scale fermentation with Streptomyces lavendulae L-105 and Rhizopogon species (ATCC 36060) have resulted in the isolation of four microbial metabolites. These metabolites have been identified as a 14-carbon rearranged product (2), 9 beta-hydroxyanhydrodihydroartemisinin (3), 11-epi-deoxydihydroartemisinin (4), and 3 alpha-hydroxydeoxyanhydrodihydroartemisinin (5). Microbial metabolites were completely characterized by spectral methods, including 1H-NMR and 13C-NMR spectroscopy. The structure and stereochemistry of metabolite 2 were unequivocally established by X-ray crystallographic analysis. Thermospray mass spectroscopy/high-performance liquid chromatographic analyses of plasma from rats used in mammalian metabolism studies of 1 have shown microbial metabolite 3 to be the major mammalian metabolite. In vitro antimalarial testing has shown metabolite 3 to possess antimalarial activity.

Microbial production of a crisnatol metabolite.

Microbial metabolism studies of crisnatol (1), a new DNA intercalator, has resulted in the isolation and characterization of a major metabolite identified as the C-1 hydroxylated crisnatol (2). The structure of the metabolite was established by comparison of its spectral data to that of crisnatol. Complete 13C-NMR assignments for crisnatol and its C-1 hydroxylated metabolite were also made.

Structure elucidation and thermospray high-performance liquid chromatography/mass spectroscopy (HPLC/MS) of the microbial and mammalian metabolites of the antimalarial arteether.

Microbial metabolism studies of the antimalarial drug arteether (1) have shown that arteether is metabolized to six new metabolites in addition to those previously reported (3). Large-scale fermentations with Cunninghamella elegans (ATCC 9245) and Streptomyces lavendulae (L-105) have resulted in the characterization of these metabolites primarily by two-dimensional nuclear magnetic resonance (2D-NMR) methods as 9 beta-hydroxyarteether (2), a ring rearrangement metabolite (3), 3 alpha-hydroxy-11-epi-deoxydihydroartemisinin (4), 9 alpha-hydroxyarteether (5), 2 alpha-hydroxyarteether (6), and 14-hydroxyarteether (7). Thermospray mass spectroscopy/high-performance liquid chromatographic analyses have shown that four of these metabolites (2, 5, 6, 7) are also present in rat liver microsome preparations.

Microbial metabolism studies of the antimalarial drug arteether.

Microbial metabolism studies of the antimalarial drug arteether (1) have shown that arteether is metabolized by a number of microorganisms. Large-scale fermentation with Aspergillus niger (ATCC 10549) and Nocardia corallina (ATCC 19070) have resulted in the isolation of four microbial metabolites which have been characterized using two-dimensional nuclear magnetic resonance (2D-NMR) techniques. These metabolites have been identified as "AEM1" (2), 3 alpha-hydroxydeoxyarteether (3), 3 alpha-hydroxydeoxydihydroartemisinin (4), and deoxydihydroartemisinin (5).

Flowthrough Reactor Flasks for Study of Microbial Metabolism in Sediments

Flowthrough reactor flasks are described that allow continuous low-level nutrient input to mixed anoxic sediments without dilution of the sediment. The flasks were tested by simulating sulfate inputs into sediments collected from a freshwater eutrophic lake. After an initial 2-day adaptation within the reactor system, rates of methane production and sulfate consumption were constant for the duration of a 12-day incubation. A sulfate input rate of 0.15 mmol liter of sediment day resulted in an equivalent rate of sulfate removal, which was unaffected by inputs of acetate (1.0 mmol liter of sediment day). The rate of methane production in control reactors, 0.18 mmol liter of sediment day, was doubled by the addition of acetate, whereas sulfate consumption was only stimulated by additions of high concentrations of sulfate plus acetate (1.5 and 1.0 mmol liter of sediment day, respectively). The reactor system appears to be effective in maintaining the balance between sulfate reduction and methane production in freshwater sediments and is potentially useful for study of the response of sediment populations to varying inputs of naturally occurring substrates, selected inhibitors, or xenobiotic compounds.

Novel sulfur-containing microbial metabolite of primaquine.

Microbial metabolism studies of the antimalarial drug primaquine, using Streptomyces roseochromogenus (ATCC 13400) have produced an N-acetylated metabolite and a methylene-linked dimeric product, both of which have been previously reported, and a novel sulfur-containing microbial metabolite. The structure of the metabolite as a sulfur-linked dimer was proposed on the basis of spectral and chemical data. The molecular formula C34H44N6O4S was established from field-desorption mass spectroscopy and analytical data. The 1H- and 13C-nuclear magnetic resonance spectral data firmly established that the novel metabolite was a symmetrically substituted dimer of primaquine N-acetate with a sulfur atom linking the two units at C-5. The metabolite has been shown to be a mixture of stereoisomers which can equilibrate in solution. This observation was confirmed by microbial synthesis of the metabolite from optically active primaquine.

Fungal metabolism of 4-methylprimaquine.

4-Methylprimaquine (4-MPQ), an 8-aminoquinoline antimalarial drug, was subjected to microbial metabolism studies in an effort to isolate, identify, and predict the probable mammalian metabolite(s). Preliminary screening with a number of strains of Aspergillus indicated that several species were capable of metabolizing 4-MPQ to one major metabolite based on thin-layer chromatographic analysis. Preparative scale conversion of 4-MPQ using whole-cell suspensions of Aspergillus ochraceus afforded one major metabolite (4-MPQ-I). Based on spectroscopic and chemical evidence, the structure of the metabolite is proposed as 6-methoxy-8-(3-carboxyl-l-methylpropylamino)-lepidine.

Analysis of drugs by microcalorimetry: Isothermal power-conduction calorimetry and thermometric titrimetry

Microcalorimetric analysis has been the subject of a few review's in recent years, but these reviews have mainly dealt with the wide-ranging capabilities of calorimetric assay. This review, however, discusses the experimental basis and practical exploitation of the method in the particularly important area of pharmaceuticals. This field of analysis embraces both conventional chemical assays and bioassays which involve living microbial species. The review highlights the design of calorimetric instruments appropriate for study of microbial metabolism and interaction with drug substances. For comprehensiveness, both microcalorimetric and thermometric assay systems are discussed and critically assessed.