• Figure 8.
    Download figureOpen in new tabFigure 8. Model of microbe-microbe metabolite exchange.

    Bolded are metabolites increased due to microbe-microbe interactions.

    DOI: http://dx.doi.org/10.7554/eLife.18855.053

  • Table 1.

    Summary of volatiles detected using GC-MS. Relative abundance of volatiles in the co-culture (S. cerevisiae and A. malorum grown together) compared to the separate-culture mixture (S. cerevisiae and A. malorum grown separately, and their quantities added in during analysis). GC-MS captured volatiles with XAD-4 beads suspended above the cultures during growth; subsequently, beads were methanol-extracted (n = 6 experiments, Table 1—source data 12). Quantification is based on two experiments in which a linear regression was computed with standards (Table 1—source data 34). Quantification is based on beads suspended above the cultures between 84 and 96 hr of culture growth.

    DOI: http://dx.doi.org/10.7554/eLife.18855.018

    Table 1—source data 1.Extracted ion chromatograms of five metabolites detected by gas chromatography-mass spectrometry (GC-MS) in Table 1.

    Extracted ion chromatograms of the five metabolites detected by gas chromatography- mass spectrometry (GC-MS). (A) Schematic depicting the experimental setup (B-F) Representative extracted ion chromatograms from one replicate (out of three total) of one experiment (out of 3–4 total) of m/z values corresponding to major metabolites identified in the experimental conditions along with appropriate standards. Acetic acid (B), isoamyl alcohol (C), isoamyl acetate (D) isobutanol (E), and ethanol (F) were identified as the five major metabolites in the co-culture (S. cerevisiae and A. malorum). Isoamyl alcohol (C), ethanol (E), and isobutanol (F) were identified as the major metabolites in S. cerevisiae grown alone. Extracted ion chromatograms were constructed using the m/z value in the title of each graph. For acetic acid and isobutanol, the m/z value used corresponds to the molecular weight of the molecule. For ethanol, the m/z used corresponds to the molecular weight minus one (hydrogen). For isoamyl alcohol, the m/z used corresponds to the loss of the hydroxyl group (depicted), which may have picked up hydrogen and been lost as water. For isoamyl acetate, the m/z value corresponds to the molecule shown within the graph. In all cases, figures showing the complete mass spectra between the metabolite and standard are found in Table 1—source data 2. Microorganisms were grown 72–96 hr.

    DOI: http://dx.doi.org/10.7554/eLife.18855.019

    Download source data [table-1—source-data-1.media-10.pdf]
    Table 1—source data 2.Representative spectra of metabolites in Table 1.

    Representative spectra of acetic acid (A-B), isoamyl alcohol (C-E), isoamyl acetate (F-G), ethanol (H-J) and isobutanol (K-L) in standard and experimental samples. Standard concentrations are denoted on individual graphs. All mass spectra are one replicate (out of 3–4 experiments with three replicates per experiment).

    DOI: http://dx.doi.org/10.7554/eLife.18855.020

    Download source data [table-1—source-data-2.media-11.pdf]
    Table 1—source data 3.Linear regression of metabolites using GC-MS in Table 1.

    Estimation of volatile quantity using GC-MS. Separate experiments are graphed in panels (A-E) and (F-J). (A-E) Data points represent the value of a single replicate per concentration for each standard. The abundance of a single m/z value at a specific retention time was chosen for each standard. The values were fitted with a linear regression and the equation was used to estimate the concentration of the five metabolites in the experimental samples from the same experiment. (F-J) Data points represent the mean ± SEM of three replicates for a given concentration for each standard. The abundance of a single m/z value at a specific retention time was chosen for each standard. The values were fitted with a linear regression. The equation was used to estimate the concentration of the five metabolites in the experimental samples from the same experiment. When applicable an equation was calculated when the line was forced to go through X,Y = 0,0; these equations were used to calculate the concentrations of isoamyl alcohol, isoamyl acetate, and isobutanol.

    DOI: http://dx.doi.org/10.7554/eLife.18855.021

    Download source data [table-1—source-data-3.media-12.pdf]
    Table 1—source data 4.Raw spectral abundance data as a function of concentration used for linear regressions in Table 1—source data 3.

    DOI: http://dx.doi.org/10.7554/eLife.18855.022

    Download source data [table-1—source-data-4.media-13.xlsx]

    Identity

    Standard confirmation

    Relative quantification (co-culture: separate-culture mixture)

    Ethanol

    Y

    5.0–12.6-fold reduced

    Isobutanol

    Y

    7.3–24.7-fold reduced

    Isoamyl acetate

    Y

    unique to co-culture

    Isoamyl alcohol

    Y

    3.6–6.4-fold reduced

    Acetic acid

    Y

    unique to co-culture

  • Table 2.

    Estimated concentrations of key metabolites in the co-culture using SPME GC-MS. Estimated concentrations of differentially concentrated or unique metabolites in the co-culture. Linear regression equations (Lin. reg. eqs. 1 and 2) were estimated from individual experiments in which peak areas of different concentrations of metabolites were fitted with a linear regression (Table 2—source data 2, 3, 5 and 6). Normalized peak areas correspond to the specified metabolites in co-cultures containing S. cerevisiae and A. malorum. Separate estimates were derived from a normalized peak area estimated from a single experiment (co-culture and standard samples were from a run with similar internal standard signal) or from the mean normalized peak area estimated from all experiments (co-cultures were run over four days, standards were run on two days). The final estimated concentration was an average of all estimated concentrations (n = 4 estimates (two from each standard regression equation times two estimates of the normalized peak area), except for methyl acetate, n = 2 estimates). The estimated concentrations (except acetoin) were added to the co-culture containing A. pomorum adhA (Figure 4C). *Ethyl acetate, acetic acid, and acetoin concentrations were estimated from standards (Table 2—source data 1 and 4).

    DOI: http://dx.doi.org/10.7554/eLife.18855.029

    Table 2—source data 1.Extracted ion chromatograms of differentially emitted or unique metabolites in the co-culture in Table 2.

    Extracted ion chromatograms of differentially emitted or unique metabolites in the co-culture according to solid phase microextraction gas chromatography-mass spectrometry (SPME GC-MS). Specific metabolites are displayed above each panel. For each panel, the left-most plot compares the co-culture containing S. cerevisiae and A. malorum to S. cerevisiae grown alone, A. malorum grown alone, or media (AJM [apple juice medium]); the right-most plot compares the co-culture containing S. cerevisiae and A. pomorum wild-type to the co-culture containing S. cerevisiae and A. pomorum adhA, since A. pomorum adhA is required for Drosophila co-culture preference (Figure 5A). The two plots within the same panel contain the same standard. The y-axis for each plot is the ion current for a m/z value that discriminates the metabolite of interest over a specific retention time window. The following m/z values were chosen for each metabolite based on standards or, in the cases of putative and unknown metabolites (I and J) were chosen from the experimental groups: (A) m/z 74.04 (B) m/z 88.08 (C) m/z 73.03 (D) 87.05 (E) 74.02 (F) 104.04 (G) 60.05 (H) 88.05 (I) 101.06 (J) 101.06. Each panel is one representative replicate of 1 experiment (out of 3–5 total replicates in three experiments).

    DOI: http://dx.doi.org/10.7554/eLife.18855.030

    Download source data [table-2—source-data-1.media-18.pdf]
    Table 2—source data 2.Linear regression of metabolites in defined metabolite mixtures in Table 2.

    Normalized peak areas corresponding to metabolites in a defined metabolite mixture (from SPME GC-MS). A linear regression was calculated to quantify the metabolites in the co-culture. Each concentration is from one replicate. A-E and F-I are two separate experiments. Linear regression was used to estimate the concentration of the metabolites in the co-culture containing S. cerevisiae and A. malorum (Table 2) and to complement the co-culture containing A. pomorum adhA (Figure 4C).

    DOI: http://dx.doi.org/10.7554/eLife.18855.031

    Download source data [table-2—source-data-2.media-19.pdf]
    Table 2—source data 3.Peak area as a function of concentration used to estimate metabolite concentrations in co-cultures in Table 2.

    DOI: http://dx.doi.org/10.7554/eLife.18855.032

    Download source data [table-2—source-data-3.media-20.xlsx]
    Table 2—source data 4.Extracted ion chromatograms of various m/z values used in.

    DOI: http://dx.doi.org/10.7554/eLife.18855.033

    Download source data [table-2—source-data-4.media-21.xlsx]
    Table 2—source data 5.Peak areas as a function of metabolite concentration used in linear regression in Table 2—source data 2A–E.

    DOI: http://dx.doi.org/10.7554/eLife.18855.034

    Download source data [table-2—source-data-5.media-22.xlsx]
    Table 2—source data 6.Peak areas as a function of metabolite concentration used in Table 2—source data 2F–I.

    DOI: http://dx.doi.org/10.7554/eLife.18855.035

    Download source data [table-2—source-data-6.media-23.xlsx]

    Metabolite

    Lin. Reg. eq. 1

    Lin. Reg. eq. 2

    Normalized peak area (single experiment)

    Normalized peak area (Average, All experiments)

    Estimated concentration (%)

    Isobutyl acetate

    Y = 4151X − 0.1319

    Y = 3252X − 0.07251

    0.29

    1.16

    0.00023

    Isoamyl acetate

    y = 8158X

    Y = 7800X

    0.78

    3.8

    0.00026

    2-Phenethyl acetate

    Y = 5129X −0.04011

    Y = 6972X −0.2013

    1.2

    1.9

    0.00028

    2-Methylbutyl acetate acetate

    Y = 8995X − 0.05042

    Y = 8087X−0.1307

    0.56

    3.1

    0.00023

    Methyl acetate

    Y = 75.22X+0.004457

    NA

    0.018

    0.040

    0.00033

    Ethyl acetate

    NA

    NA

    NA

    NA

    ~0.02*

    Acetic acid

    NA

    NA

    NA

    NA

    ~3.0*

    Acetoin

    NA

    NA

    NA

    NA

    ~0.01*