Time-Resolved Metabolic Footprinting for Nonlinear Modeling of Bacterial Substrate Utilization

Behrends, Volker; Ebbels, Timothy M. D.; Williams, Huw D.; Bundy, Jacob G.

doi:10.1128/aem.01742-08

Cited by 52 publications

(74 citation statements)

References 61 publications

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“…In agreement with previous studies (6,8), there was a set of compounds-asparagine, aspartate, alanine, and glutamate (and, in the case of oASM and fASM, also glutamine)-that were taken up early on in growth (Table 1). For these "early-uptake" compounds, the t 1/2 values increased from LB to fASM to oASM, i.e., quickest uptake from LB and slowest from oASM.…”

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confidence: 76%

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Direct Assessment of Metabolite Utilization by Pseudomonas aeruginosa during Growth on Artificial Sputum Medium

Behrends

Geier

Williams

et al. 2013

Appl Environ Microbiol

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bWe grew Pseudomonas aeruginosa in LB and artificial sputum medium (ASM) (filtered and unfiltered) and quantified metabolite utilization and excretion by nuclear magnetic resonance (NMR) spectroscopy (metabolic footprinting or extracellular metabolomics). Utilization rates were similar between media, but there were differences in excretion-e.g., acetate was produced only in unfiltered ASM.T he opportunistic pathogen Pseudomonas aeruginosa is the major cause of morbidity and mortality for cystic fibrosis (CF) patients (1). In order to better understand P. aeruginosa infection, it is important to create model conditions that aim to mimic those in the CF lung, such as media designed to mimic the nutritional environment of sputum from CF patients. These include artificial sputum medium (ASM) (2), a complex medium made up of porcine mucin, DNA, and amino acids, and synthetic cystic fibrosis medium (SCFM) (3), a defined medium containing mainly amino acids and lactate. In addition, many studies use a more-conventional rich medium, like lysogeny broth (LB) (4), which is based on tryptone and yeast extract. All of these media support growth of P. aeruginosa to high cell numbers, but the question remains how the differences in composition alter the physiology and metabolism of the inoculated bacteria and, hence, the relevance to CF infections. Generally, approaches to validating this have depended on indirect means to assess metabolism-for instance, profiling gene transcription (3). However, transcriptional data are often insufficient to capture the actual metabolic changes that occur as a result of perturbing a bacterial cell. Because of this, we wanted to examine the actual changes in metabolite utilization by P. aeruginosa in a comparison of three different complex media, LB, ASM (oASM, prepared according to its original recipe [2]), and a filtered version of ASM (fASM, prepared according to the method of Kirchner et al. [5]). We have previously investigated metabolite utilization in SCFM in depth (6), so we did not include this medium again here.We grew wild-type P. aeruginosa PA14 under aerobic conditions in batch culture for 24 h (growth conditions described in detail in reference 6) and sampled 0.6 ml of culture at nine time points (before inoculation, hourly from 2 to 8 h, and then at 24 h). Of these samples, 0.1 ml was used for measuring the optical density (OD) for LB and fASM samples (oASM is turbid and the OD cannot be used to monitor cell growth). The supernatants were then analyzed by nuclear magnetic resonance (NMR) spectroscopy as detailed in Behrends et al. (7). The resulting spectra were assigned and integrated for each medium. In total, we were able to assign 19 compounds detectable across all media, of which 18 were able to be quantified (Table 1) (histidine was detected but not integrated here because of pH-induced resonance frequency shifts between spectra) and normalized to compound levels in the uninoculated media. Four further metabolites were able to be identified in at least two media. Finally...

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“…Four further metabolites were able to be identified in at least two media. Finally, the metabolite concentrations were fitted using nonlinear (sigmoid) models, a process which allows between-media comparisons of uptake and excretion time for individual compounds (6).…”

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Direct Assessment of Metabolite Utilization by Pseudomonas aeruginosa during Growth on Artificial Sputum Medium

Behrends

Geier

Williams

et al. 2013

Appl Environ Microbiol

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“…For example, unidentified metabolite 15 was released only in media with MEBM or unidentified metabolite 133 was released only in the media with SE (Table S1). These released metabolites may be a result of overflow metabolism 15,17 due to large levels of specific metabolites taken up from the media modifying thermodynamic driving forces or imposing bottlenecks on reactions in the metabolic network of Synechococcus. Thermodynamic driving forces may also be responsible for the release of some metabolites to a certain level or their consumption if their levels cross a certain threshold (e.g.…”

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“…17 More recently, metabolic footprinting was used to study pleiotropic effects of specific mutations, 18 investigation of metabolic impact of heterologous protein production, 19 and optimization of amino acid composition of growth media to support microbial growth.…”

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Untargeted metabolic footprinting reveals a surprising breadth of metabolite uptake and release by Synechococcus sp. PCC 7002

2011

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Cyanobacteria are important primary producers in diverse ecosystems, yet little is known about the extent of their metabolic interactions with the environment. We have used an integrated, untargeted metabolic footprinting approach to systematically evaluate the uptake and release of metabolites between a model marine cyanobacterium Synechococcus sp. PCC 7002 and different growth media. It was found that 47 out of 202 detected metabolites were consumed, and an additional 55 metabolites were released by the cells. Surprisingly, Synechococcus was found to uptake a great diversity of metabolites dominant in and specific to its own metabolite extract including histidine betaine (hercynine), g-glutamyl phenylalanine and a hexosamine-based trisaccharide. This provides Synechococcus a mechanism to benefit from the lysis of part of their population (i.e. due to environmental stress or predation). Additionally, stable isotope probing was used to show that adenine and glutamate are actively metabolized following uptake. A significant turnover of glucosylglycerol, a cyanobacterial compatible solute, as opposed to a negligible turnover of the hexosamine-based trisaccharide were also observed using stable isotope probing. The untargeted metabolic footprinting approach used in this study is generally applicable to investigate metabolic interactions of microorganisms with the environment and may prove useful to construct microbial community foodwebs.

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“…Using iNMR's integration tool (Tools → integrator), integrate the relevant resonances. For a list of resonances useful for quantifi cation of LB and SCFM, see [ 10 ]. A preliminary assignment of the resonances is very useful for this step.…”

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Metabolic Footprinting: Extracellular Metabolomic Analysis

Behrends

Williams

Bundy

2014

Methods in Molecular Biology

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Uptake and excretion of nutrients is an integral part of a cell's physiology. Using analytical chemistry techniques, metabolite uptake and excretion from the culture medium can be quantified. As cellular metabolism changes throughout growth, additional information is available if transient and growth phase-dependent changes are monitored. Here, we describe time-resolved metabolic footprinting (TReF), a technique which employs nuclear magnetic resonance spectroscopy and nonlinear curve fitting to understand and visualize metabolite utilization of P. aeruginosa.

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Time-Resolved Metabolic Footprinting for Nonlinear Modeling of Bacterial Substrate Utilization

Cited by 52 publications

References 61 publications

Direct Assessment of Metabolite Utilization by Pseudomonas aeruginosa during Growth on Artificial Sputum Medium

Direct Assessment of Metabolite Utilization by Pseudomonas aeruginosa during Growth on Artificial Sputum Medium

Untargeted metabolic footprinting reveals a surprising breadth of metabolite uptake and release by Synechococcus sp. PCC 7002

Metabolic Footprinting: Extracellular Metabolomic Analysis

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