Saccharomyces cerevisiae Requires CFF1 To Produce 4-Hydroxy-5-Methylfuran-3(2H)-One, a Mimic of the Bacterial Quorum-Sensing Autoinducer AI-2

Abstract

Quorum sensing is a process of cell-to-cell communication that bacteria use to orchestrate collective behaviors. Quorum sensing depends on the production, release, and detection of extracellular signal molecules called autoinducers (AIs) that accumulate with increasing cell density. While most AIs are species specific, the AI called AI-2 is produced and detected by diverse bacterial species, and it mediates interspecies communication. We recently reported that mammalian cells produce an AI-2 mimic that can be detected by bacteria through the AI-2 receptor LuxP, potentially expanding the role of the AI-2 system to interdomain communication. Here, we describe a second molecule capable of interdomain signaling through LuxP, 4-hydroxy-5-methylfuran-3(2H)-one (MHF), that is produced by the yeast Saccharomyces cerevisiae Screening the S. cerevisiae deletion collection revealed Cff1p, a protein with no known role, to be required for MHF production. Cff1p is proposed to be an enzyme, with structural similarity to sugar isomerases and epimerases, and substitution at the putative catalytic residue eliminated MHF production in S. cerevisiae Sequence analysis uncovered Cff1p homologs in many species, primarily bacterial and fungal, but also viral, archaeal, and higher eukaryotic. Cff1p homologs from organisms from all domains can complement a cff1ΔS. cerevisiae mutant and restore MHF production. In all cases tested, the identified catalytic residue is conserved and required for MHF to be produced. These findings increase the scope of possibilities for interdomain interactions via AI-2 and AI-2 mimics, highlighting the breadth of molecules and organisms that could participate in quorum sensing.IMPORTANCE Quorum sensing is a cell-to-cell communication process that bacteria use to monitor local population density. Quorum sensing relies on extracellular signal molecules called autoinducers (AIs). One AI called AI-2 is broadly made by bacteria and used for interspecies communication. Here, we describe a eukaryotic AI-2 mimic, 4-hydroxy-5-methylfuran-3(2H)-one, (MHF), that is made by the yeast Saccharomyces cerevisiae, and we identify the Cff1p protein as essential for MHF production. Hundreds of viral, archaeal, bacterial, and eukaryotic organisms possess Cff1p homologs. This finding, combined with our results showing that homologs from all domains can replace S. cerevisiae Cff1p, suggests that like AI-2, MHF is widely produced. Our results expand the breadth of organisms that may participate in quorum-sensing-mediated interactions.

Keywords: autoinducer; collective behavior; interspecies; quorum sensing; signaling; yeast.

FIG 1

S. cerevisiae produces MHF, an AI-2 mimic. (A) Diagram showing the structure of DPD and relevant interconversions among molecules. (B) Light output by the V. harveyi TL-26 reporter strain in response to S. cerevisiae culture fluids containing yeast AI-2 mimic in PBS (squares) and in water (triangles). (C) Chromatogram depicting fractionation of yeast AI-2 mimic preparations. The area containing the active fraction is enlarged in the inset. The chromatograms show absorption at 214 (green), 254 (blue), and 280 (red) nm. The arrow depicts the peak containing the activity. mAU, milli-absorbance units. (D) Light output from the V. harveyi TL-26 reporter strain in response to a titration of the active 8- to 9-min fraction from C. (E) Structure of MHF. RLU denotes relative light units, which are bioluminescence/OD₆₀₀ of the reporter strain, and the dotted line-labeled Max AI-2 refers to the activity from 125 nM DPD. In B and D, error bars represent standard deviations of biological replicates, n = 3.

FIG 2

MHF agonizes the LuxP receptor with a nanomolar EC₅₀. (A) Quantitation of MHF levels in yeast AI-2 mimic preparations from S. cerevisiae concentrated to OD₆₀₀ of 25, 50, or 100 using integration under HPLC peaks (black) or activity from the V. harveyi TL-26 reporter strain (white). (B) Light output by the V. harveyi TL-26 reporter strain in response to DPD (black) or MHF (red). The table shows the EC₅₀ values. RLU as in Fig. 1. In A, error bars represent standard deviations of technical replicates, n = 3. In B, error bars represent standard deviations of biological replicates, n = 3.

FIG 3

Cff1p is required for S. cerevisiae to produce MHF. (A) Normalized light output from the V. harveyi TL-26 reporter strain in response to culture fluids from the mutants in the S. cerevisiae deletion library. Each point represents the reporter response to a fluid made from a unique yeast mutant. Dotted lines labeled +2SD and −2SD show two standard deviations above and below the mean, respectively. (B) Light output from the V. harveyi TL-26 reporter strain in response to culture fluids from the putative hit S. cerevisiae mutants from A. (C) Light output from the V. harveyi TL-26 reporter strain in response to culture fluids from WT S. cerevisiae (squares) and the cff1Δ (circles) and rps1bΔ (triangles) mutants. (D) Portion of an HPLC trace from fractionation of yeast AI-2 mimic preparation made from cff1Δ S. cerevisiae. The chromatograms show absorption at 214 (green), 254 (blue), and 280 (red) nm. The arrow shows the expected elution time for MHF based on WT S. cerevisiae results (see Fig. 1C). (E) Light output from the V. harveyi TL-26 reporter strain in response to cell-free fluids made from cff1Δ S. cerevisiae that produced either a HALO control (designated “V”), Cff1p-HALO (designated WT), or Cff1p-E44A-HALO (designated E44A). Normalized RLU in A are RLU of the given sample divided by the average RLU from all plates assayed on a single day. RLU and Max AI-2 as in Fig. 1. In B, C, and E, error bars represent standard deviations of biological replicates, n = 3. In B and E, 10% (vol/vol) of cell-free fluid was added in each case.

FIG 4

Cff1p homologs restore MHF production to cff1Δ S. cerevisiae. (A) Light output from the V. harveyi TL-26 reporter strain in response to cell-free fluids from cff1Δ S. cerevisiae expressing CFF1 homologs from B. cinerea (green), T. versicolor (red), S. cerevisiae (black), and a vector control (gray). (B) Light output from the V. harveyi TL-26 reporter strain in response to cell-free fluids prepared from cff1Δ S. cerevisiae carrying the designated CFF1 homologs and alleles. The vector control is designated V. Cell-free fluids were added at 10% (vol/vol). (C) Alignment of putative Cff1p homologs, trimmed to the first and final amino acids of S. cerevisiae Cff1p. Only one species per genus is shown. Alignment was performed using Clustal Omega (79). Negatively charged residues are shown in red, small hydrophobic residues in orange, aromatic hydrophobic residues in yellow, polar uncharged residues in green, and positively charged residues in blue. Shown above the alignment are consensus sequences for regions containing amino acids that are conserved in >75% of the aligned proteins. Arrow designates location of conserved glutamate residue. RLU and Max AI-2 as in Fig. 1. In A and B, error bars represent standard deviations of biological replicates, n = 3.

FIG 5

Organisms from all domains contain Cff1p homologs. (A) Venn diagram displaying the numbers of bacterial species containing LuxS and/or Cff homologs. (B) Bacterial phylogeny distribution showing species containing potential Cff homologs. Black bars represent species possessing only Cff. Gray bars represent species possessing both LuxS and Cff. (C) Light output from the V. harveyi TL-26 reporter strain in response to cell-free fluids prepared from S. cerevisiae expressing CFF1 and cff homologs. Bars are colored according to the groups in Fig. 4C and Fig. S7, as follows: green, Bacteria; red, Ascomycota; blue, Basidiomycota; black, other. The vector control is designated V. Cell-free fluids were added at 10% (vol/vol). RLU and Max AI-2 are as in Fig. 1. In C, error bars represent standard deviations of biological replicates, n = 3.