Supplementary Figures (doc 2100K)

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Supplementary Figures (doc 2100K)
Supplementary figures S1-10.
Supplementary Figure S1. OA is excreted by yeast lackingorotidine-5'-phosphate
decarboxylase (ura3p) activity, which inhibits the growth of R. etli CE3. A) Wild-type S.
cerevisiae Σ1278b (wt) does not affect bacterial growth; the S. cerevisiae Σ1278h ura3 mutant
inhibits R. etli CE3 growth. B) The genetic confirmation of the nature of PΣA. Yeast knock-out
(in the BY4741 background) strains were tested against R. etli CE3 to identify the genes
involved in PΣA biosynthesis. The yeast mutants unable to synthesize OA did not inhibit the
growth of R. etli CE3. Approximately 1 x 106 cells of each yeast strain were placed on the PY-D
plates inoculated with R. etli CE3 (OD600nm = 0.05). C) The HPLC profile of the extract of S.
cerevisiae Σ1278h cultures. The fractions were tested against R. etli CE3 using a diffusion assay
in PY-D plates. D) The activity of PΣA against R. etli CE3 is identical OA activity. OA (50 µg)
and PΣA (50 µg) were placed on the PY-D plates inoculated with R. etli CE3 cells.
Supplementary Figure S2. C4-dicarboxylates concentrations (plotted as the sum of malate,
succinate, fumarate and α-ketoglutarate) in the supernatants of cocultures at 12 hours. Coculture
of S. cerevisiae BY4741 ura3Δ (Re.+Sc.ura3Δ) and coculture of S. cerevisiae cyc2 mutant in the
BY4741 background (Re.+Sc. ura3Δ/cyc2Δ). Panel show the means ±S. E. M. (n= 3).
Supplementary Figure S3. The CIMS mass spectrum of standard OA (A) was identical to the
PΣA spectrum (B), and both contain a molecular ion peak at m/z 157 (M+H)+ with characteristic
fragments at m/z 113 (M+H) - CO2, m/z 139 (M+H) - H2O, m/z 69 (C3H2NO + H)+ and m/z 41
(C3H2NO) – HCN. In both spectra, we observed a protonated molecular dimer m/z 313 (2M +
H)+.
Supplementary Figure S4. Inhibition of OA effects by C4-dicarboxylates and purines. A) C4dicarboxylates compete with OA for the available DctA permease and block OA transport. R. etli
CE3 cells were grown in PY-D medium supplemented with H2O (negative control), 100 µg/ml
OA and 100 µg/ml OA plus 100 µg/ml C4-dicarboxylate tested as inhibitors. Succinate (Suc),
Malate (Mal), α-ketoglutarate (α-keto), fumarate (Fum) and cis-aconitate (Cis-ac). B) R. etli CE3
cells were grown in PY-D medium supplemented with H2O (negative control), 100 µg/ml OA
and 100 µg/ml OA plus 1.5 mM/ml purine tested as inhibitors. C.F.U. were determined at 24
hours of culture. Panels show the means ±S. E. M. (n= 3).
Supplementary Figure S5. The growth of R. etli wt and OAR variants in liquid medium. A)
Growth in PY-D medium with 100 µg/ml OA. B) Growth in PY-D medium with 50 µg/ml FOA.
Optical densities were determined at 24 hours of culture. The panels show the means ±S. E. M.
(n= 3).
Supplementary Figure S6. R. etli CE3 mutants pyrE and pyrF accumulated OA in the culture
medium. (A) OA excretion by R. etli pyrE and pyrF mutants inhibited the growth of wt R. etli.
(B) OA accumulation was prevented by supplementation of the medium with uridine (25 µg/ml).
A-B) ~1.2 x 107 cells of each strain were placed on the PY-D plates inoculated with wt R. etli
(OD600nm = 0.05). C) OA concentrations in culture medium of wt R. etli and pyrE and pyrF
mutants. The strains were grown in PY-D medium, and OA was quantified by HPLC. No OA
accumulation was detected in the medium with the wild-type strain. These strains were
spontaneous mutants isolated from cocultures. The panel show the means ±S. E. M. (n= 3).
Supplementary Figure S7. The expression of L-glutamine synthetase (GlnA and GlnII) in R.
etli was affected by OA. A) Two GlnA electrophoretic entities were identified by twodimensional gel electrophoresis and were designated GlnA-a and GlnA-b. In the cells exposed to
50 µg/ml OA, GlnA-a was up-regulated and GlnA-b down-regulated. The sum of the abundance
of both entities is similar in both conditions. B) L-glutamine synthetase II was down-regulated in
cells exposed to OA. The plots illustrate the statistical analysis of changes in protein abundance.
The proteins were separated by 2-DE using ampholytes of pH 3–10 and were identified by
peptide mass mapping via MALDITOF-MS. The data are the means ± S. E. M. (n= 3). C)
Adenylylation state of GSI. D) GSI specific activity. E) GSII specific activity. GS was measured
via its transferase activity. The data are the means ± S. E. M. (n= 3).
Supplementary Figure S8. In cocultures with yeast, R. etli pyrE and pyrF mutants were less
competitive than dctA mutants. The relative fitness of the test genotype was determined relative
to R. etli dctA-(OAR-A1 strain) (initial ratio 1:1) in the presence of S. cerevisiae Σ1278h in
culture medium with 20 µg/ml uridine (open symbols) and without uridine (filled symbols).
Fitness is given as the difference in Malthusian parameters (m). A fitness of zero indicates that
the two genotypes are equally fit. The competitive fitness was determined during a single 48hour growth period. OAR-A1 strain (carrying a deletion of 150 nucleotides of dctA) and the
competing isolate were differentiated by PCR of dctA gene. The panel show the means ±S. E. M.
(n= 3).
Supplementary Figure S9. A) In coculture, after 48 hours, R. etli alkalinized the culture media.
R. etli CE3 monoculture (Re.), S. cerevisiae Σ1278 monoculture (Sc.) and R. etli CE3- S.
cerevisiae Σ1278h coculture (Re.+Sc.). To identify the cause of the extinction of yeast, we
performed experiments in which it was evaluated the effect of coculture supernatants (CS) in
yeast viability. (B) Yeast cells of 7 days old monocultures were growth in filter-sterilized 7 days
old cocultures supernatants. The yeasts were grown in: native CS which has a pH of 8.3 (CS pH
8); Buffered CS contained 150 mM MES pH 5.5 (CS pH 5.5); Buffered CS contained 150 mM
MES pH 5.5 (CS pH 5.5) supplemented with 15 mg/ml yeast extract (CS pH 5.5 + Nutrients).
Control is cells growing in supernatants of 7 days old yeast cultures (Control). The viability was
determined by standard dilution plate counts and expressed as the percentage of the colonyforming units at time 0 h. Data are means ±SD of three independent experiments.
Supplementary Figure S10. The detection of melanin production in R. etli wild-type cells,
constructed mutants and OAR variants with a mutation in the rpoN1gene. The strains were grown
for 3 days in PY-D supplemented with L-tyrosine plus Cu2SO4 and incubated at 30 ºC before
SDS (1%) solution (Hawkins & Johnston, 1988).

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