Article pubs.acs.org/JAFC
Quorum-Sensing Kinetics in Saccharomyces cerevisiae: A Symphony of ARO Genes and Aromatic Alcohols Martina Avbelj,*,† Jure Zupan,† Luka Kranjc,† and Peter Raspor*,†,‡ †
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia ‡ Faculty of Health Sciences, University of Primorska, Polje 42, 6310 Izola, Slovenia ABSTRACT: The kinetics of quorum sensing in Saccharomyces cerevisiae were studied using a mini-fermentation platform. The quorum-sensing molecules were monitored using our previous HPLC approach that is here supported by quantitative real-time PCR analysis of the quorum-sensing genes. We thus initially confirm correlations between peak production rates of the monitored quorum-sensing molecules 2-phenylethanol, tryptophol, and tyrosol and peak expression of the genes responsible for their synthesis: ARO8, ARO9, and ARO10. This confirms the accuracy of our previously implemented kinetic model, thus favoring its use in further studies in this field. We also show that the quorum-sensing kinetics are precisely dependent on the population growth phase and that tyrosol production is also regulated by cell concentration, which has not been reported previously. Additionally, we show that during wine fermentation, ethanol stress reduces the production of 2-phenylethanol, tryptophol, and tyrosol, which opens new challenges in the control of wine fermentation. KEYWORDS: quorum sensing, wine fermentation, 2-phenylethanol, tryptophol, tyrosol, real-time PCR, ARO genes
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INTRODUCTION Microorganisms do not live solitary lives. They interact and form communities that coexist and interact in any given habitat. For successful interactions, and sometimes for core survival, microorganisms need to communicate. Quorum sensing is a major mechanism of microbial communication that allows unicellular organisms to determine their population density, in order to promote changes in population behavior. This is achieved by secretion of small hormone-like molecules known as autoinducers, or quorum-sensing molecules. The concentrations of quorum-sensing molecules increase during population growth, and once they reach a threshold level, this activates a signal transduction cascade that directs the expression or repression of target genes, which results in collective behavioral adaptation.1−3 Furthermore, different quorum-sensing molecules in bacteria and unicellular eukaryotes show great diversity, such as the chemical identities of the molecules used and the behaviors that are regulated by these messengers. However, all quorum-sensing systems have in common the need for a molecular machinery to synthesize, sense, and respond to secreted communication molecules.4 In fungi, quorum sensing was initially discovered in the yeast Candida albicans, where two antagonistic quorum-sensing molecules, farnesol and tyrosol [2-(4-hydroxyphenyl)ethanol], have been shown to inhibit and promote, respectively, the formation of germ tubes.5 In the model organism Saccharomyces cerevisiae, the aromatic alcohols 2-phenylethanol and tryptophol have been shown to be quorum-sensing molecules under lownitrogen conditions and, thus, to regulate the induction of pseudohyphae formation according to the local population density.4,6 2-Phenylethanol, tryptophol, and tyrosol are synthesized from the amino acids phenylalanine, tryptophan, and tyrosine, © 2015 American Chemical Society
respectively, by chemical degradation, through three-step biochemical reductions via the Ehrlich pathway. A first transamination step produces an α-keto-acid analogue of the amino acid, a decarboxylation step then yields an aldehyde, and a final reduction step converts the aldehyde into a primary alcohol.7−11 The transamination is catalyzed by aromatic aminotransferases I and II, which are encoded by the genes ARO8 and ARO9, respectively, whereas the decarboxylation step is catalyzed by the aromatic decarboxylase encoded by the gene ARO10 and by pyruvate decarboxylases. ARO8 and ARO9 gene transcription is induced by the presence of phenylalanine, tryptophan, and tyrosine in the growth medium, as the enzymes aromatic aminotransferases I and II are active with these three amino acids as amino donors.13 The quorum-sensing mechanism in S. cerevisiae is regulated by the availability of ammonia in the culture medium and by a feedback signal from the end products 2-phenylethanol and tryptophol.4,12 Both of these control mechanisms act on the first two reaction steps, transamination and decarboxylation.4 The production of the quorum-sensing molecules 2-phenylethanol and tryptophol in S. cerevisiae was shown to be regulated by cell density. A high cell density up-regulated the expression of the ARO9 and ARO10 genes and, thus, stimulated the production of aromatic alcohols.12 This aromatic alcohol production can also be autostimulated by tryptophol. This activates the transcription factor Aro80p and, consequently, the expression of the ARO9 and ARO10 transaminase and decarboxylase genes, thus resulting in a positive feedback loop.4,13,14 Consequently, yeast cells at high population density Received: Revised: Accepted: Published: 8544
July 10, 2015 September 7, 2015 September 14, 2015 September 14, 2015 DOI: 10.1021/acs.jafc.5b03400 J. Agric. Food Chem. 2015, 63, 8544−8550
Article
Journal of Agricultural and Food Chemistry
Determination of the 2-Phenylethanol, Tryptophol, and Tyrosol Concentrations. The monitoring and kinetics analyses for 2-phenylethanol, tryptophol, and tyrosol were performed as described previously.15 Briefly, high-performance liquid chromatography (HPLC) analysis was performed using a reverse phase column (XBridge Phenyl, 5 μm, 4.6 mm × 150 mm; Waters) and H2O/ acetonitrile (80:20, v/v) as the mobile phase. Prior to the HPLC analysis, 1 mL of the samples was centrifuged (initially 1500g for 5 min, then 6500g for 5 min) and filtered through 0.2 μm filters (Phenex, Phenomenex, Italy). 2-Phenylethanol, tryptophol, and tyrosol were detected using a fluorescence detector (Shimadzu RF551, Japan), at their optimal wavelengths of 255/285, 280/368, and 275/315 nm, respectively. Quorum-Sensing Kinetics. We described the quorum-sensing kinetics with the recently implemented parameter of “production rate”, which was calculated on the basis of the data from the measurements of the cell concentrations and the quorum-sensing molecule concentrations. The production rate of the quorum-sensing molecules (RQSM) on the interval (t1, t2) was calculated as
produce more aromatic alcohols per cell than yeast cells at low population density.4,12 In the present study, we aimed to correlate our previously implemented quantitative kinetics model to monitor the aromatic alcohols 2-phenylethanol, tryptophol, and tyrosol in S. cerevisiae15 and the expression of the ARO8, ARO9, and ARO10 genes. Additionally, we were interested in whether, and to what extent, the parameters to which yeast are exposed during wine fermentation affect the production of these quorum-sensing molecules. Of the multiple stresses that can affect yeast vitality during fermentation, here ethanol stress was selected, with examination of the possible effects on 2phenylethanol, tyrosol, and tryptophol production. Thus, after ethanol addition to the medium to create stress, the relative expression levels of the ARO8, ARO9, and ARO10 genes were compared to the kinetics of the production of these quorumsensing aromatic alcohols.
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MATERIALS AND METHODS
R QSM( t1+ t2 ) = 2
Strains and Inoculum Preparation. This study was performed with the wine-yeast strain S. cerevisiae ZIM 1927, which was originally isolated from the must of ‘Malvasia’ wine grapes. The strain was obtained from the Collection of Industrial Microorganisms (ZIM) at the Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia. The preparation of the inoculum and the fermentation were carried out using MS300 synthetic must, as described previously,16 with some modifications. The medium contained 100 g/L glucose and 100 g/L fructose, and no anaerobic factors were used. It contained also 460 mg/L ammonium chloride, a mixture of 19 amino acids (612.6 mg/L L-proline, 505.3 mg/L L-glutamine, 374.4 mg/L L-arginine, 179.3 mg/L L-tryptophan, 145.3 mg/L L-alanine, 120.4 mg/L L-glutamic acid, 78.5 mg/L L-serine, 759.2 mg/L L-threonine, 48.4 mg/L L-leucine, 44.5 mg/L L-aspartic acid, 44.5 mg/L L-valine, 37.9 mg/L L-phenylalanine, 32.7 mg/L isoleucine, 32.7 mg/L L-histidine, 31.4 mg/L L-methionine, 18.3 mg/L L-tyrosine, 18.3 mg/L L-glycine, 17.0 mg/L lysine, 13.1 mg/ L L-cysteine), mineral salts (750 mg/L KH2PO4, 500 mg/L K2SO4, 250 mg/L MgSO4·7H2O, 155 mg/L CaCl2·2H2O, 200 mg/L NaCl, 4 mg/L MnSO4·H2O, 4 mg/L ZnSO4, 1 mg/L CuSO4·5H2O, 1 mg/L KI, 0.4 mg/L CoCl2·6H2O, 1 mg/L H3BO3, 1 mg/L NaMoO4·2H2O), and vitamins (20 mg/L myo-inositol, 2 mg/L nicotinic acid, 1.5 mg/L calcium panthothenate, 0.25 mg/L thiamin HCl, 0.25 mg/L pyridoxine HCl, 0.003 mg/L biotin). The medium supports 300 mg of assimilable nitrogen, as provided by 460 mg/L ammonium chloride (corresponding to 120 mg of nitrogen) as well as by the mixture of 19 amino acids (corresponding to 180 mg of nitrogen). The inoculum was prepared aerobically in 100 mL Erlenmeyer flasks on a rotary shaker at 220 rpm constant shaking, at 22 °C. After 48 h of cultivation, the inoculum was centrifuged at 1500g and resuspended in 5 mL of fresh MS300 medium. The concentration of the yeast cells was determined by automatic counting of viable cells using ImageJ software, as described previously.15 The yeast suspensions were diluted with MS300 to obtain two different initial concentrations, 1.2 × 107 cells/mL and a 20-fold dilution for 6 × 105 cells/mL, with 1.5 mL of the inoculated medium distributed in four repetitions into perforated 2 mL microcentrifuge tubes. Fermentation Conditions. The mini-fermentations were carried out at 22 °C, as described previously.15 Throughout the 42 h of the mini-fermentations, four of the microcentrifuge tubes were sampled every 2 h, to determine the concentrations of viable cells and quorumsensing molecules (i.e., 2-phenylethanol, tryptophol, tyrosol), and to collect cell biomass for RNA analysis. Determination of Viable Cell Concentrations. The viable cell concentrations were determined as described previously.15 Briefly, 20 μL of the cell suspensions was collected from each fermenter and diluted 1:1 (v/v) with methylene blue. The stained suspension was transferred to a Bürker−Türk hemocytometer, and bright-field microscope images were recorded using an attached camera. The viable cells were then counted using ImageJ software.
(cQSM(t2) − cQSM(t1))
(
c t1 + c t 2 2
) × (t
2
[fmol/cells/h]
− t1)
(1)
where t is time, cQSM is the concentration of the quorum-sensing molecule, c is the concentration of the cells, and dcQSM/dt ≈ (cQSM(t2) − cQSM(t1))/(t2 − t1). To plot RQSM, curve fitting for the concentration of cells and the quorum-sensing molecules was performed, from which 100 data points were generated. These data points from the fitted curves were then used in eq 1. RNA Isolation. From samples with an initial cell concentration of 1.2 × 107 cells/mL, the total RNA was isolated after 0, 4, 12, 16, 24, and 32 h of cultivation; in samples with an initial cell concentration of 6 × 105 cells/mL, total RNA was isolated after 0, 18, 26, 30, 34, and 38 h of cultivation. RNA was isolated using the commercial Qiagen RNeasy mini kits, as described by the manufacturer. Briefly, cell lysates were prepared using 350 μL of lysis buffer (RLT; Qiagen GmbH) containing β-mercaptoethanol (10 μL/L RLT buffer), acid-washed glass beads (diameter = 420−600 μm; Sigma), and a homogenizer (Bullet Blender Storm, Next Advance). After this, the samples were centrifuged (12000g, 10 min) and 900 μL of supernatant was transferred to new centrifuge tubes. To remove the residual DNA, oncolumn DNase digestion with the RNase-free DNase set (Qiagen) was performed. Total RNA was eluted in 50 μL of RNase-free water (Qiagen) and stored at −80 °C until use. The quantity and purity of the total RNA were measured with a spectrophotometer (Lambda Bio, PerkinElmer), and visualized by agarose gel electrophoresis. Reverse Transcription and cDNA Synthesis. The reverse transcription reaction was performed using commercial Superscript VILO cDNA synthesis kits (Invitrogen), according to the manufacturer’s instructions, with some modifications. For each single reaction, we combined 2 μL of 5-fold concentrated VILO reaction mix, 1 μL of 10-fold concentrated Superscript enzymatic mix, the RNA, and diethylpyrocarbonate-treated water, to a final reaction volume of 10 μL. The RNA concentrations in the samples were determined spectrophotometrically, and the starting RNA concentration was normalized to 650 ng. The reaction mixtures were successively incubated for 10 min at 25 °C, for 60 min at 42 °C, and for 5 min at 85 °C. Primer and Probe Design. The primers and probes for the ARO8, ARO9, and ARO10 target genes and the ACT1, 18S rRNA, TEF1 reference genes were designed with the Primer 3 software package.17 The oligonucleotides and probes used were designed in silico, using the NCBI database. The probes were labeled at their 5′end using 6-carboxyfluorescein (FAM) and at their 3′-end using 5′tetramethylrhodamine-5(6)-carboxamide (TAMRA). Controls. Each sample was analyzed as three biological repetitions and two technical repetitions. To check for residual DNA, each RNA sample was also subjected to a cDNA synthesis reaction without addition of the reverse transcriptase enzyme (NoRT control). When a 8545
DOI: 10.1021/acs.jafc.5b03400 J. Agric. Food Chem. 2015, 63, 8544−8550
Article
Journal of Agricultural and Food Chemistry
Figure 1. Concentrations of viable cells (circles) and production of 2-phenylethanol, tryptophol, and tyrosol (histograms, as indicated) over 42 h of mini-fermentations with S. cerevisiae at the initial cell concentrations of 6 × 105 cells/mL (top) and 1.2 × 107 cells/mL (bottom).
Figure 2. Production rates of 2-phenylethanol (dashed line), tryptophol (dash-dotted line), and tyrosol (dotted line) over 42 h of minifermentations with S. cerevisiae at the initial cell concentrations of 6 × 105 cells/mL (top) and 1.2 × 107 cells/mL (bottom). The production rates were calculated from concentrations of aromatic alcohols and cells presented in Figure 1. ΔCt of >4 between the sample and its respective NoRT control was obtained, the DNA contamination level was considered negligible. Negative control templates consisted of the master mixture with sterile water as the template. Real-Time Quantitative PCR. Real-time quantitative PCR was carried out with the ABI PRISM 7900 HT Sequence Detection System, using the SDS 3.2 application software (Applied Biosystems). For each reaction, 5 μL of EXPRESS qPCR SuperMix Universal was mixed with 0.02 μL of passive reference dye (ROX), 2.25 μL of 125 nM probe, the optimal oligonucleotide concentration (300 or 600
nM), and nuclease-free water, to give a final volume of 8 μL. The 8 μL reaction mixture and 2 μL of diluted cDNA (final concentration = 1 ng/μL) were transferred to each well of 384-well optical reaction plates (MicroAmp, Applied Biosystems). The following cycle profile was used: one cycle at 50 °C for 15 min, one cycle at 95 °C for 2 min, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. The Ct values obtained by real-time PCR were quantified using a relative standard curve. For this purpose, a stock solution of cDNA was prepared, from which 2-fold serial dilutions were made. 8546
DOI: 10.1021/acs.jafc.5b03400 J. Agric. Food Chem. 2015, 63, 8544−8550
Article
Journal of Agricultural and Food Chemistry
Figure 3. Relative expression of the ARO8, ARO9, and ARO10 genes (as indicated) and correlation with the production rates of 2-phenylethanol (dashed line), tryptophol (dash-dotted line), and tyrosol (dotted line) over 42 h of mini-fermentations with S. cerevisiae at the initial cell concentrations of 6 × 105 cells/mL (top) and 1.2 × 107 cells/mL (bottom). Analysis of the Relative Expression Levels of ARO8, ARO9, and ARO10. To analyze the variability of the housekeeping genes used, the software program geNormPLUS was used.18 This program calculates first the gene stability measure M (which must be
