Extracellular DNA secreted in yeast cultures is metabolism-specific and inhibits cell proliferation

Extracellular DNA (exDNA) can be actively released by living cells and different putative functions have been attributed to it. Further, homologous exDNA has been reported to exert species-specific inhibitory effects on several organisms. Here, we demonstrate by different experimental evidence, including 1H-NMR metabolomic fingerprint, that the growth rate decline in Saccharomyces cerevisiae fed-batch cultures is determined by the accumulation of exDNA in the medium. Sequencing of such secreted exDNA represents a portion of the entire genome, showing a great similarity with extrachromosomal circular DNA (eccDNA) already reported inside yeast cells. The recovered DNA molecules were mostly single strands and specifically associated to the yeast metabolism displayed during cell growth. Flow cytometric analysis showed that the observed growth inhibition by exDNA corresponded to an arrest in the S phase of the cell cycle. These unprecedented findings open a new scenario on the functional role of exDNA produced by living cells.


Figure S6.
Examples of exDNA nucleotide reads mapped to the S288C S. cerevisiae reference genome using both "unique" and "multiple" approaches.
Figure S1.(A) Glucose feeding profiles in EFC and LFC fed-batch cultures.(B) Residual glucose concentrations in the medium of EFC and LFC.

Figure S2 .
Figure S2. 1 H NMR spectrum in D2O of the yeast nutrient medium before inoculation.

Figure S3 .
Figure S3.Details of the 1 H NMR spectra in D2O at different hours.

Figure S4 .
Figure S4.Electrophoretic mobility in agarose gel (1% w/v) of the DNA obtained by direct REPLIg amplification of EFC and LFC supernatants.

Figure S5 .
Figure S5.(A) Effect of different concentrations of DNA amplified from EFC supernatant on yeast growth in batch cultures.(B) Electrophoresis on 10% acrylamide gel of calf thymus DNA, DNA obtained by REPLIg amplification of EFC, and fish sperm DNA.

Figure S7 .
Figure S7.BLAST based alignment of three different reads mapping on the rDNA region (chromosome XII) versus the GenBank nucleotide database.

Figure S8 .
Figure S8.BLAST based alignment of three different reads mapping on the rDNA region (chromosome XII) versus the GenBank nucleotide database eliminating all possible hits matching versus "Saccharomyces" derived sequences, i.e., excluding all possible sequences referred to this term in the screened database.

Figure S1 .
Figure S1.(A) Glucose feeding profiles in EFC and LFC fed-batch cultures.(B) Residual glucose concentrations in the medium of EFC and LFC.

Figure S4 .
Figure S4.Electrophoretic mobility in agarose gel (1% w/v) of the DNA obtained by direct REPLIg amplification of EFC and LFC supernatants.Lane 1 from the left, 1Kb Plus DNA ladder; lane 2 lambda DNA; lane 3 and 4, DNA from EFC; lane 5 and 6, DNA from LFC.

Figure S5 .
Figure S5.(A) Effect of different concentrations of DNA amplified from EFC supernatant on yeast growth in batch cultures.Null effects of heterologous DNAs from fish sperm and calf thymus are shown, as well as after pre-treatment of the amplified DNA with DNase.Data refer to means and standard deviations of 3 replicates.(B) Electrophoresis on 10% acrylamide gel of calf thymus DNA, DNA obtained by REPLIg amplification of EFC, and fish sperm DNA.DNase at different concentrations completely digested both calf thymus and EFC DNAs.

Figure S7 .
Figure S7.BLAST based alignment of three different reads mapping on the rDNA region (chromosome XII) versus the GenBank nucleotide database.The three (A, B and C) most diverging sequences, i.e., those with the greatest number of mismatches, were selected.

Figure S8 .
Figure S8.BLAST based alignment of three different reads mapping on the rDNA region (chromosome XII) versus the GenBank nucleotide database eliminating all possible hits matching versus "Saccharomyces" derived sequences, i.e., excluding all possible sequences referred to this term in the screened database.Results for the same three sequences reported in Figure S7 (A, B and C) are shown.

Table S1 .
Results of two inhibition tests performed with S. cerevisiae CEN.PK1-2C treated with RNA extracted from either the same strain or human Hematopoietic Progenitor Cells (HPC).

Table S2 .
List of reagents, yeast strains and software used in this work.Results of Illumina sequencing of exDNA purified from growth media of EFC, EFC 6h and LFC and the comparison with eccDNA fromMøller et al. (2015).See attached Excel file ("Supplemental Dataset S1.xlsx").

Table S2 .
List of reagents, yeast strains and software used in this work.