Metabolism of nitropolycyclic aromatic hydrocarbons by human intestinal microflora

Anaerobic bacterial suspensions from human and rat feces and intestinal contents, and pure cultures of anaerobic bacteria metabolized 1-nitropyrene and 6-nitrobenzo[a]pyrene to 1-aminopyrene and 6-aminobenzo[a]pyrene, respectively. The metabolites were isolated by reversed-phase high performance liquid chromatography and identified by comparison of their chromatographic and mass spectral properties with those of authentic compounds. The results suggest that anaerobic intestinal bacteria could play a significant role in the metabolism of potentially carcinogenic nitropolycyclic aromatic hydrocarbons.     

Microbiological diagnosis of suppurative-inflammatory processes of the E. N. T. organs and the maxillo-mandibulo-facial region under suspicion of anaerobic non-clostridial infection

The etiological role of non-sporulating anaerobic bacteria as causative agents of suppurative-inflammatory diseases (SID) of the maxillo-mandibulo-facial region and the E. N. T. organs as studied. Express diagnosis of anaerobic infection was carried out by means of gas-liquid chromatography. The species-specific composition of the microflora of the suppurative focus was investigated. It was established that only obligate anaerobes in monoculture or in association were isolated from patients with so-called "sterile" inoculations when strictly anaerobic bacteriological technique was used. The use of anaerobic bacteriological technique of investigation enlarged the spectrum of the microflora isolated from the suppurative focus from 5 to 26 species. Results of chromatographic and bacteriological examinations were compared; the main causes of obtaining false-positive and false-negative results of chromatography were analysed. Statistical processing using factor analysis has shown that the information power of chromatographic examination of the metabolites of anaerobic bacteria is higher in comparison with the main clinical-laboratory indices, but statistical processing using cluster analysis and correlation analysis has revealed that an index like metabolic activity reflects the degree of real participation of anaerobic microflora in the development of the pathological process, and can be used in the clinic for the evaluation of the degree of severity of the course of the SID and of the effectiveness of treatment.     

High resolution deuterium NMR studies of bacterial metabolism

High resolution deuterium NMR spectra were obtained from suspensions of five bacterial strains: Escherichia coli, Clostridium perfringens, Klebsiella pneumoniae, Proteus mirabilis, and Staphylococcus aureus. Deuterium-labeled D-glucose at C-1, C-2, and C-6 was used to monitor dynamically anaerobic metabolism. The flux of glucose through the various bacterial metabolic pathways could be determined by following the disappearance of glucose and the appearance of the major end products in the 2H NMR spectrum. The presence of both labeled and unlabeled metabolites could be detected using 1H NMR spectroscopy since the proton resonances in the labeled species are shifted upfield due to an isotopic chemical shift effect. The 1H-1H scalar coupling observed in both the 2H and 1H NMR spectra was used to assign definitively the resonances of labeled species. An increase in the intensity of natural abundance deuterium signal of water can be used to monitor pathways in which a deuteron is lost from the labeled metabolite. The steps in which label loss can occur are outlined, and the influence these processes have on the ability of 2H NMR spectroscopy to monitor metabolism are assessed.     

An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry

A method for the global analysis of yeast intracellular metabolites, based on electrospray mass spectrometry (ES-MS), has been developed. This has involved the optimization of methods for quenching metabolism in Saccharomyces cerevisiae and extracting the metabolites for analysis by positive-ion electrospray mass spectrometry. The influence of cultivation conditions, sampling, quenching and extraction conditions, concentration step, and storage have all been studied and adapted to allow direct infusion of samples into the mass spectrometer and the acquisition of metabolic profiles with simultaneous detection of more than 25 intracellular metabolites. The method, which can be applied to other micro-organisms and biological systems, may be used for comparative analysis and screening of metabolite profiles of yeast strains and mutants under controlled conditions in order to elucidate gene function via metabolomics. Examples of the application of this analytical strategy to specific yeast strains and single-ORF yeast deletion mutants generated through the EUROFAN programme are presented.     

Autoregulatory factors of secondary metabolism and morphogenesis in actinomycetes

The Gram-positive bacterial genus Streptomyces possesses interesting biological aspects, such as the ability to produce a wide variety of secondary metabolites and a mycelial form of growth that culminates in sporulation. A close relationship of secondary metabolism and cell differentiation has been well recognized; secondary metabolism might be a physiological expression of cell differentiation. A variety of diffusible low-molecular-weight chemical substances have been found to function in general as regulatory factors, like "hormones" in eukaryotes, for secondary metabolism and cell differentiation. Among these factors, A-factor has been most extensively studied. This review summarizes recent research on the chemical structures, functions, biosyntheses, and mode of action of these regulatory factors.     

Quantification of metabolic flux in plant secondary metabolism by a biogenetic organizational approach

Metabolic engineering represents a promising approach to enhance the yield of valuable natural products from plants. A method to quantify flux through metabolite measurements is necessary for the analysis of native and modified pathways. Rather than focusing only on the accumulation of the final products, analyzing a wide range of secondary metabolites has significant advantages. We propose a model that organizes the flux analysis by grouping metabolites of similar biosynthetic origin. To this end, we have quantified temporal profiles of metabolites from several branches of the indole alkaloid pathway in Catharanthus roseus hairy root cultures. By analyzing these data, we are able to examine the distribution of flux around key branchpoints. Furthermore, this analysis provides crucial information such as an estimate of total flux to secondary metabolism.     

Comparative profiling analysis of central metabolites in Euglena gracilis under various cultivation conditions

Comparative metabolic profiling analysis was performed to investigate light- and aeration-dependent regulation of central metabolism in Euglena gracilis. The metabolic profiles of E. gracilis were significantly altered in response to changes in aeration conditions. While many glycolytic intermediates and amino acids accumulated in aerobically grown E. gracilis, a significant reduction in these metabolites was observed for cells under anaerobic conditions, which resulted in elevated production of wax ester.     

Determination of human fibroblasts metabolism in vitro by gas chromatography-mass spectrometry of cell-excreted metabolites

In order to develop an approach to the study of cell metabolism in vitro, we undertook the determination of metabolites excreted by human skin diploid fibroblasts into culture medium using high-resolution gas chromatography in combination with mass spectrometry. Chromatographic and mass spectrometric characteristics of 29 metabolites have been obtained, and 11 of the metabolites have been identified. The excreted metabolites reflect the activity of certain metabolic processes in fibroblasts in vitro. A comparison of chromatographic and mass spectrometric characteristics of the cell metabolites and of those excreted from the body in urine showed most of the metabolites excreted by fibroblasts to be different from the urine metabolites. The possibility of secondary conversion of cell metabolites in the organism and the specificity of metabolism in cells of different tissues are discussed.     

Metabolism of dietary genotoxins by the human colonic microflora; the fecapentaenes and heterocyclic amines

The microflora of the human colon is a complex ecosystem of anaerobic bacteria which have the capability of enzymatically transforming a variety of dietary (or biliary) compounds to genotoxic metabolites. In the past, most investigators studying the interplay between diet and colonic flora and its role in the etiology of cancers focused on the reductive and glycosidic potential of the bacterial enzymes--many of which reverse the oxidative and conjugative reactions performed by the liver. Recent work in our laboratory has focused on the metabolism of two relatively new classes of genotoxins, the fecapentaenes and the heterocyclic amines (pyrolysis carcinogens). The fecapentaenes (conjugated ether lipids) are produced in the colon by Bacteroides spp. from polyunsaturated ether phospholipids (plasmalogens) whose natural origin and function are unknown. The fecapentaenes are potent direct-acting genotoxins that are detected in the feces of most individuals on normal western diets. The heterocyclic amines, which originate from fried or broiled proteinaceous foods, normally require activation by the liver before being potent mutagens or carcinogens. However, the "IQ" subclass (e.g. IQ and MeIQ) can be activated in the colon by Eubacterium and Clostridium species to a 7-hydroxy form which is directly mutagenic in Salmonella. Although there is no direct evidence that the fecapentaenes or the 7-hydroxy "IQ" compounds influence risk for colon cancer, the potency and prevalence of these bacterial metabolites is cause for concern.     

A comparison of the metabolism of 25-hydroxyvitamine D3 by chick renal tubules, homegenates and mitochondria

The metabolism of 25-hydroxycholecalciferol by isolated chick renal tubules has been investigated by the use of a new chromatographic method employing silica-treated paper as the supporting phase. This method has been found to be superior to Sephadex LH20 column chromatography in resolving the metabolites of 25-hydroxycholecalciferol, particularly in separating 1,25-dyhydroxycholecalciferol from other metabolites. With this method several heretofore undescribed metabolites of 25-hydroxycholecalciferol have been recognized and partially characterized.     

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.     

Gas chromatographic metabolic profiling: a sensitive tool for functional microbial ecology

Microbial metabolomics, which consists of a non-targeted analysis of the metabolites released from ('exometabolome') or existing in ('endometabolome') a cell has mostly been used to study the metabolism of particular microbes. Metabolomes also represent a picture of microbial activity and we suggest that the exometabolome may also contain pertinent information for studying microbial interaction networks. Gas chromatography coupled to mass spectrometry is the most commonly used technique in metabolomics studies. It allows a wide range of metabolites to be detected but requires the derivatisation of compounds prior to detection. This type of non-targeted analysis can introduce biases to the detection and quantification of the different metabolites, particularly at the extraction and derivatisation steps. The aims of this study, therefore, were to quantify the sources of variability and to test the sensitivity of the GC metabolic profiling approach to small environmental changes such as shifts in temperature. The temperature sensitivity of metabolic profiles was compared with that of catabolic profiles obtained using Biolog microplates. Analytical variability was compared with biological variability by incubating bacterial strains isolated from soil with fructose at 20 degrees C and by replicating each step of the protocol (incubation, extraction and derivatisation). For both the endo- and the exometabolome, more than 70% of the total variability was of biological origin and principal components analysis clearly separated the strains along the first ordination axis. The endometabolome distinguished bacterial strains at the species level only, whereas separation was evident at the species and group level with the exometabolome. Temperature had a significant but differential effect on the metabolite production of the bacterial strains whilst their catabolic profiles remained relatively unaffected. The exometabolome was more sensitive to temperature shifts than the endometabolome, suggesting that this pool may be of interest for studies in environmental functional ecology.     

Gas chromatographic metabolic profiling: A sensitive tool for functional microbial ecology

Microbial metabolomics, which consists of a non-targeted analysis of the metabolites released from (‘exometabolome’) or existing in (‘endometabolome’) a cell has mostly been used to study the metabolism of particular microbes. Metabolomes also represent a picture of microbial activity and we suggest that the exometabolome may also contain pertinent information for studying microbial interaction networks. Gas chromatography coupled to mass spectrometry is the most commonly used technique in metabolomics studies. It allows a wide range of metabolites to be detected but requires the derivatisation of compounds prior to detection. This type of non-targeted analysis can introduce biases to the detection and quantification of the different metabolites, particularly at the extraction and derivatisation steps. The aims of this study, therefore, were to quantify the sources of variability and to test the sensitivity of the GC metabolic profiling approach to small environmental changes such as shifts in temperature. The temperature sensitivity of metabolic profiles was compared with that of catabolic profiles obtained using Biolog® microplates. Analytical variability was compared with biological variability by incubating bacterial strains isolated from soil with fructose at 20 °C and by replicating each step of the protocol (incubation, extraction and derivatisation). For both the endo- and the exometabolome, more than 70% of the total variability was of biological origin and principal components analysis clearly separated the strains along the first ordination axis. The endometabolome distinguished bacterial strains at the species level only, whereas separation was evident at the species and group level with the exometabolome. Temperature had a significant but differential effect on the metabolite production of the bacterial strains whilst their catabolic profiles remained relatively unaffected. The exometabolome was more sensitive to temperature shifts than the endometabolome, suggesting that this pool may be of interest for studies in environmental functional ecology.     

Quantitative analysis of procarbazine, procarbazine metabolites and chemical degradation products with application to pharmacokinetic studies

Quantitative analytical methods are described for the analysis of the anticancer drug procarbazine and eight known metabolites including those known to have cytotoxic activity. A direct sample insertion mass spectrometric assay for procarbazine and the urinary excretion product, N-isopropyl-terephthalamic acid, has been developed. This method employs stable isotope labeled variants in a procedure that minimizes analytical errors that may be encountered in the quantitation of the chemically unstable parent drug. A liquid chromatographic method is described for the analysis of seven known procarbazine metabolites. Use of these methods is demonstrated by the analysis of procarbazine metabolism during incubation in a 9000-g rat liver homogenate preparation. Procarbazine disappearance and metabolite appearance are also monitored in rat plasma following intraperitoneal administration of a 150 mg/kg bolus dose. Applications to patient pharmacokinetics is demonstrated using the liquid chromatographic assay to follow the appearance of active procarbazine metabolites on the first and fourteenth day of an oral 250 mg/kg/day course of therapy of a patient being treated for cancer.     

Ecological significance of bacterial polyphosphate metabolism in sediments

The purpose of this study was to find theoretical evidence that bacteria, in particular those capable of polyphosphate (polyP) metabolism, are directly implicated in sediment phosphorus (P) dynamics and control P metabolism of freshwater ecosystems. The specific attributes and functional role of such bacteria were investigated on successive levels of ecological organization: individual microorganism, microbial community, freshwater ecosystem. The results of this systematic approach have been formulated as a number of hypotheses.

1. PolyP metabolism is the mechanism which enables individual polyP bacteria to survive and grow under the fluctuating redox conditions characteristic of their habitat at the sediment-water interface.
2. PolyP metabolism together with anaerobic Mn and/or Fe respiration is the mechanism that confers upon polyP bacteria the advantage required to fill a unique ecological niche within the microbial community to which they belong.
3. To the freshwater ecosystem as a whole bacterial polyP metabolism is a homeostatic mechanism which limits P availability and makes ecosystem productivity self-correcting as a function of oxygen availability. Bacterial polyP pools in the sediment are vital components of the P cycle. It was suggested that the impact of this bacterial mechanism should be tested with regard to the eutrophication issue.

     

Bacterial metabolism of long-chain <Emphasis Type="Italic">n</Emphasis>-alkanes

Degradation of alkanes is a widespread phenomenon in nature, and numerous microorganisms, both prokaryotic and eukaryotic, capable of utilizing these substrates as a carbon and energy source have been isolated and characterized. In this review, we summarize recent advances in the understanding of bacterial metabolism of long-chain n-alkanes. Bacterial strategies for accessing these highly hydrophobic substrates are presented, along with systems for their enzymatic degradation and conversion into products of potential industrial value. We further summarize the current knowledge on the regulation of bacterial long-chain n-alkane metabolism and survey progress in understanding bacterial pathways for utilization of n-alkanes under anaerobic conditions.     

Anaerobic Degradation and Transformation of p-Toluidine by the Sulfate-Reducing Bacterium Desulfobacula toluolica

The ability of the strictly anaerobic sulfate-reducing bacterium Desulfobacula toluolica (strain Tol2) to cometabolically degrade p-toluidine (p-methylaniline) while using toluene as the primary source of carbon and energy has been studied. This organism has been shown to modify and degrade toluidine in dense cell suspensions when no other source of carbon and energy is added. The metabolism led to the formation of a variety of metabolites. From these metabolites a biphenyl-like compound as well as phenylacetic acid have been identified by means of HPLC/MS techniques. The probable conversion of p-toluidine to p-aminophenylacetic acid and phenylacetic acid as dead end products suggested that this organism initiates p-toluidine degradation by the carboxylation of the methyl group. If this could be validated in further experiments, it would be the first time that a toluidine was carboxylated at the methyl moiety by an anaerobic, sulfate-reducing bacterium. Received: 6 March 1998 / Accepted: 3 April 1998     

Genome mining for ribosomally synthesized and post-translationally modified peptides (RiPPs) in anaerobic bacteria

Background

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a diverse group of biologically active bacterial molecules. Due to the conserved genomic arrangement of many of the genes involved in their synthesis, these secondary metabolite biosynthetic pathways can be predicted from genome sequence data. To date, however, despite the myriad of sequenced genomes covering many branches of the bacterial phylogenetic tree, such an analysis for a broader group of bacteria like anaerobes has not been attempted.

Results

We investigated a collection of 211 complete and published genomes, focusing on anaerobic bacteria, whose potential to encode RiPPs is relatively unknown. We showed that the presence of RiPP-genes is widespread among anaerobic representatives of the phyla Actinobacteria, Proteobacteria and Firmicutes and that, collectively, anaerobes possess the ability to synthesize a broad variety of different RiPP classes. More than 25% of anaerobes are capable of producing RiPPs either alone or in conjunction with other secondary metabolites, such as polyketides or non-ribosomal peptides.

Conclusion

Amongst the analyzed genomes, several gene clusters encode uncharacterized RiPPs, whilst others show similarity with known RiPPs. These include a number of potential class II lanthipeptides; head-to-tail cyclized peptides and lactococcin 972-like RiPP. This study presents further evidence in support of anaerobic bacteria as an untapped natural products reservoir.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-983) contains supplementary material, which is available to authorized users.