Metabolic adaptations of Pseudomonas aeruginosa during cystic fibrosis chronic lung infections.

Pseudomonas aeruginosa forms chronic infections in the lungs of cystic fibrosis (CF) patients, and is the leading cause of morbidity and mortality in patients with CF. Understanding how this opportunistic pathogen adapts to the CF lung during chronic infections is important to increase the efficacy of treatment and is likely to increase insight into other long-term infections. Previous studies of P. aeruginosa adaptation and divergence in CF infections have focused on the genetic level, both identifying characteristic mutations and patterns of gene expression. However, these approaches are not sufficient to fully understand the metabolic changes that occur during long-term infection, as metabolic regulation is complex and takes place on different biological levels. We used untargeted metabolic profiling (metabolomics) of cell supernatants (exometabolome analysis, or metabolic footprinting) to compare 179 strains, collected over time periods ranging from 4 to 24 years for the individual patients, representing a series of mostly clonal lineages from 18 individual patients. There was clear evidence of metabolic adaptation to the CF lung environment: acetate production was highly significantly negatively associated with length of infection. For amino acids, which are available to the bacterium in the lung environment, the tendency of isolates to evolve more efficient uptake was related to the biosynthetic cost of producing each metabolite; conversely, for the non-mammalian metabolite trehalose, isolates had significantly reduced tendency to utilize this compound with length of infection. However, as well as adaptation across patients, there was also a striking degree of metabolic variation between the different clonal lineages: in fact, the patient the strains were isolated from was a greater source of variance than length of infection for all metabolites observed. Our data highlight the potential for metabolomic investigation of complex phenotypic adaptations during infection.

Differences in strategies to combat osmotic stress in Burkholderia cenocepacia elucidated by NMR-based metabolic profiling.

AIMS: To investigate mechanisms of osmotic tolerance in Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc) of closely related strains, which is of clinical as well as environmental importance. METHODS AND RESULTS: We employed NMR-based metabolic profiling (metabolomics) to elucidate the metabolic consequences of high osmotic stress for five isolates of B. cenocepacia. The strains differed significantly in their levels of osmotic stress tolerance, and we identified three different sets of metabolic responses with the strains least impacted by osmotic stress exhibiting higher levels of the osmo-protective metabolites glycine-betaine and/or trehalose. Strains either increased concentrations or had constitutively high levels of these metabolites. CONCLUSIONS: Even within the small set of B. cenocepacia isolates, there was a surprising degree of variability in the metabolic responses to osmotic stress. SIGNIFICANCE AND IMPACT OF THE STUDY: The metabolic responses, and hence osmotic stress tolerance, vary between different B. cenocepacia isolates. This study provides a first look into the potentially highly diverse physiology of closely related strains of one species of the Bcc and illustrates that physiological or clinically relevant phenotypes are unlikely to be inferable from genetic relatedness within this species group.

An NMR-based metabonomic approach to the investigation of coelomic fluid biochemistry in earthworms under toxic stress.

The endogenous metabolites of the coelomic fluid of the earthworm Eisenia veneta were characterised using high-resolution one-dimensional and two-dimensional 1H nuclear magnetic resonance spectroscopy. Signals from common organic acids, such as acetate, fumarate, malonate, malate, formate, and succinate, were identified together with adenosine and nicotinamide mononucleotide. The potential use of this information as a baseline dataset for future toxicological or physiological studies was demonstrated by a metabonomic analysis: a series of earthworms were dosed with the model compound 3-fluoro-4-nitrophenol, and toxic effects followed by multivariate analysis of the spectral data of the coelomic fluid. Relative concentrations of acetate and malonate were decreased in the dosed worms compared to the controls.

Development of QSARs to investigate the bacterial toxicity and biotransformation potential of aromatic heterocylic compounds.

A series of aromatic heterocyclic and hydrocarbon compounds were tested for toxicity and biotransformation potential against two contrasting lux-marked whole-cell microbial biosensors. Toxicity was determined by inhibition of light output of a Pseudomonas fluorescens construct that expresses lux constitutively. Biotransformation was tested by increase in light output of P. fluorescens HK44 (pUTK21), which expresses lux when in the presence of a metabolic intermediate (salicylate). The data were then modelled against physical/chemical properties of the compounds tested to see if quantitative structure-activity relationships (QSARs) could be derived. Toxicity was found to be accurately predicted by log Kow (R2 = 0.95, Q2 = 0.88), with the basic (pyridine-ring containing) heterocycles modelled separately. The biotransformation data were best modelled using lowest unoccupied molecular orbital (LUMO) energies (R2 = 0.90, Q2 = 0.87).

Metabolites and metals in Metazoa--what role do phytochelatins play in animals?

Phytochelatins are sulfur-rich metal-binding peptides, and phytochelatin synthesis is one of the key mechanisms by which plants protect themselves against toxic soft metal ions such as cadmium. It has been known for a while now that some invertebrates also possess functional phytochelatin synthase (PCS) enzymes, and that at least one species, the nematode Caenorhabditis elegans, produces phytochelatins to help detoxify cadmium, and probably also other metal and metalloid ions including arsenic, zinc, selenium, silver, and copper. Here, we review recent studies on the occurrence, utilization, and regulation of phytochelatin synthesis in invertebrates. The phytochelatin synthase gene has a wide phylogenetic distribution, and can be found in species that cover almost all of the animal tree of life. The evidence to date, though, suggests that the occurrence is patchy, and even though some members of particular taxonomic groups may contain PCS genes, there are also many species without these genes. For animal species that do possess PCS genes, some of them (e.g. earthworms) do synthesize phytochelatins in response to potentially toxic elements, whereas others (e.g. Schistosoma mansoni, a parasitic helminth) do not appear to do so. Just how (and if) phytochelatins in invertebrates complement the function of metallothioneins remains to be elucidated, and the temporal, spatial, and metal specificity of the two systems is still unknown.

Comparison of response of six different luminescent bacterial bioassays to bioremediation of five contrasting oils.

The performance of six different bioluminescent bacteria for the assessment of oil bioremediation was compared. Three contained lux genes linked to promoters from hydrocarbon degradation pathways: Pseudomonas fluorescens HK44 (pUTK21), Escherichia coli HMS174 (pOS25) and E. coli DH5 alpha (pGEc74, pJAMA7), responding to naphthalene, isopropylbenzene and octane, respectively. The other three expressed lux constitutively: E. coli HB101 (pUCD607) and P. putida F1 (pUCD607) are genetically engineered, while Vibrio fischeri is naturally bioluminescent and was included to facilitate comparison with previous work. Five different oils (four crude oils plus diesel) were spiked into soil, and the progress of remediation was followed over a period of 119 d by monitoring both hydrocarbon disappearance and changes in the microbial response to soil extracts. The octane bioassay was the only one of the hydrocarbon-responsive bacterial assays to show any appreciable response, with up to 20-fold induction by light crude oils. Heavy crude oil and diesel elicited a much weaker response. The metabolic (lux constitutively expressed) bioassays showed that there was a general increase in toxicity over the course of the experiment, although toxicity to E. coli HB101 (pUCD607) appeared to be decreasing by the final sampling point. The metabolic bioassay response was much less variable between the different oils than for the first three, catabolic, strains.

Microbial communities in different soil types do not converge after diesel contamination.

AIMS: To study the comparative effect of diesel addition and simulated bioremediation on the microbial community in three different soil types. METHODS AND RESULTS: Three different soils were amended with diesel and bioremediation treatment simulated by addition of nutrients. The progress of bioremediation, and the effect on the indigenous microbial communities, was monitored using microbiological techniques. These included basal respiration, sole carbon source utilization patterns using both a commercially-available substrate set and a set designed to highlight changes in hydrocarbon-utilizing bacteria, and phospholipid fatty acid (PLFA) profiling. The development of active hydrocarbon-degrading communities was indicated by the disappearance of diesel, increases in soil respiration and biomass, and large changes in the sole carbon source utilization patterns and PLFA profiles compared with control soils. However, comparison of the relative community structure of the three soils using PLFA profiling showed that there was no tendency for the community structure of the three different soil types to converge as a result of contamination. In fact, they became more dissimilar as a result. Changes in the sole carbon source utilization patterns using the commercially-available set of carbon sources indicated the same result as shown by PLFA profiling. The specially selected set of carbon sources yielded no additional information compared with the commercially-available set. CONCLUSIONS: Diesel contamination does not result in the development of similar community profiles in different soil types. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that different soils have different inherent microbial potential to degrade hydrocarbons, a finding that should be taken into account in impact and risk assessments. Following the development of the microbial community and its recovery is a useful and sensitive way of monitoring the impact and recovery of oil-contaminated soils.

Investigating the specificity of regulators of degradation of hydrocarbons and hydrocarbon-based compounds using structure-activity relationships.

Microbial biosensors which have genes for bioluminescence coupled to genes that control hydrocarbon degradation pathways can be used as reporters on the specificity of regulation of those pathways. Structure-activity relationships can be used to discover what governs that specificity, and can also be used to separate compounds into different groups depending on mode of action. Published data for four different bioluminescent biosensors, reporting on toluene (two separate biosensors), isopropylbenzene, and octane, were analyzed to develop structure-activity relationships between biological response and physical/chemical properties. Good QSARs (quantitative structure-activity relationships) were developed for three out of the four biosensors, with between 88 and 100 per cent of the variance explained. Parameters found to be important in controlling regulator specificity were hydrophobicity, lowest unoccupied molecular orbital energies, and molar volume. For one of the biosensors, it was possible to show that the biological response to chemicals tested fell into three separate classes (non-hydrocarbons, aliphatic hydrocarbons, and aromatic hydrocarbons). A statistically significant QSAR based on hydrophobicity was developed for the fourth biosensor, but was poor in comparison to the other three (44 per cent variance explained).

Metabolism of 4-fluoroaniline and 4-fluorobiphenyl in the earthworm Eisenia veneta characterized by high-resolution NMR spectroscopy with directly coupled HPLC-NMR and HPLC-MS.

1. Little is known about metabolism of xenobiotics by earthworms, despite their importance in soil ecotoxicity testing. Normal earthworms and earthworms treated with antibiotics to ensure inhibition of gut microflora were exposed to two model xenobiotic compounds, 4-fluoroaniline and 4-fluorobiphenyl, to determine which metabolites were produced, and whether the pattern of metabolism was affected by the presence of microbial transformation ability. 2. (19)F-NMR spectroscopy detected the number and relative proportions of metabolites and directly coupled HPLC-(1)H-NMR spectroscopy and HPLC-MS then identified the metabolites. 3. Despite uptake, no metabolism of 4-fluorobiphenyl was observed at any stage, which appears to be a consequence of the lack of oxidative Phase I metabolic activity of the earthworms towards this substrate. In contrast, 4-fluoroaniline exhibited dose-dependent metabolism. At high doses (leading to mortality within 24 h) one predominant metabolite was observed, which was identified as the N-beta-glucoside conjugate. At lower dose levels, the predominant metabolite was the gamma-glutamyl conjugate, although the glucoside and another as yet unidentified metabolite were also detected. 4. The inhibition of gut microflora did not have any influence on metabolism. The study represents the first evidence for glucoside and glutamyl conjugation as a pathway for xenobiotic metabolism in earthworms.