Prediction of flocculation ability of brewing yeast inoculates by flow cytometry, proteome analysis, and mRNA profiling

Authors


Abstract

The ability of brewing yeast to flocculate is an important feature for brewing of qualitatively good beer. Flocculation involves two main cell wall structures, which are the flocculation proteins (flocculins) and mannans, to which these flocculins bind. Unfortunately, in practice, the flocculation ability may get lost after several repitches. Flow cytometry was employed to analyze glucose and mannose structures of the cell surface by application of fluorescent lectins. Validation of the expression of the flocculin genes Lg-FLO1, FLO1, FLO5, and FLO9 was carried out using microarray techniques. SDS-PAGE, western blot, and ESI-MS/MS analyses served to isolate and determine yeast cell flocculins. Mannose and glucose labeling with fluorescent lectins allowed differentiating powdery and flocculent yeast cells under laboratory conditions. Using microarray techniques and proteomics, the four flocculation genes Lg-FLO1, FLO1, FLO5, FLO9, and the protein Lg-Flo1p were identified as factors of major importance for flocculation. The expression of the genes was several times higher in flocculent yeast cells than in powdery ones. Flow cytometry is a fast and simple method to quantify the proportions of powdery and flocculent yeast cells in suspensions under defined cultivation conditions. However, differentiation under industrial conditions will require mRNA and protein expression profiling. © 2008 International Society for Advancement of Cytometry

Flocculation is an important feature of bottom-fermenting yeast strains. In nature, flocculation helps yeast cells to survive in an environment with rare nutrient supply. Some cells will eventually die in the flocs and so nourish their surrounding counterparts (1). In breweries, after several repitches, however, brewing yeast strains may lose their ability to sediment at the end of the fermentation. Currently, no techniques are available to reliably predict changed flocculation characteristics of yeast cells.

Genetic alterations like chromosome deletion, complete or partial deletions in the gene Lg-FLO1 were proposed reasons for declining flocculation (2). Also, physiological reasons and extrinsic parameters were suspected to influence the flocculation ability (3). The widely accepted lectin theory (4) suggests that two binding partners are necessary for flocculation. These are flocculation proteins (flocculins) that interact with the nonreducing termini of α-(1, 3)-linked mannan side-branches, both located in the cell walls (5). Flocculins are lectin-like proteins. From the complete sequence of the yeast genome, 33 genes are now known to be associated with flocculation (6, 7). They are located at the ends of chromosomes close to the telomeres (7, 8). FLO1, Lg-FLO1, FLO5, FLO9, FLO10, FLO8, and FLO11 are all members of the same multi-gene family and were chosen for further analyses as they are the dominant genes. The former five genes encode cell wall-linked proteins involved in flocculation. The lager yeast gene Lg-FLO1 was isolated from a bottom-fermenting yeast and is homologous to FLO1. The corresponding proteins differ in the regions responsible for sugar recognition. For Flo1p, that is tryptophan 228 and for Lg-Flo1p threonine 202 and their surrounding amino acids (9). FLO8 encodes a transcription factor required for flocculation (10), whereas FLO11 encodes a cell surface glycoprotein responsible for the formation of pseudohyphae, sliding motility, and adhesion to agar and plastic surfaces (11).

For brewers, loss of flocculation is a problem because they can hardly predict after which repitch their yeasts are too powdery for further usage, i.e., inoculation of new fermentation tanks. Powdery yeast cells may show accelerated aging and affect the quality of the beer. The other problem with yeasts becoming nonflocculent is the additional effort and the extra costs for filtering the beer. Powdery yeasts are only advantageous for the production of diet beer. As they do not sediment, they can ferment the sugar completely within the wort. However, after one fermentation, these yeasts are usually disposed. Up to now, traditional tests like the Helm's test and its modifications (12, 13) are used for testing flocculation ability. Thinking of an alternative, we hypothesized that direct determinations of the cellular mannose residues or flocculin contents would give dynamic and intra-population-resolved information about the flocculation capability of a beer production strain. This study made use of the fluorescent lectins ConcanavalinA-Alexa-Fluor®-350 (ConA-Alexa-Fluor) and Pisum-sativum-agglutinate-FITC (PSA-FITC) to analyze the flocculation ability of yeast cells. The two proteins are known to bind to different extents to mannose and glucose residues in the cell wall. For ConA, the binding constant to D-mannopyranoside is more than two times stronger than for PSA, hence, mannose is the preferred binding partner for ConA. Although both lectins show a similar binding constant with D-glucopyranoside, PSA was described to preferably bind to glucan (14, 15). Furthermore ConA, unlike PSA, was reported to inhibit flocculation (4). The use of antibodies as marker molecules for flocculins is another possibility to obtain information on the cell's flocculation ability. Up to now, only the flocculin Flo1p was highlighted this way, although with an antibody against a heterologous Flo1p synthesized in E. coli by Bony et al. (16). In this study, a combination of transcriptome and proteome analysis was applied to identify yeast flocculin molecules in their natural cellular backgrounds. The transcriptome analysis focused on the verification of mRNA expression of the flocculin genes. The proteomic approach relied on SDS-PAGE and western blot quantification followed by flocculin identification via mass spectrometry to validate the transcription analysis. Using the combination of these techniques the relevant flocculin molecules were identified and qualified as markers for flocculent lager yeast strains.

As differentiation between flocculent and powdery yeasts is an essential precondition for process optimization and economization in brewing processes, four different brewing yeast strains were used to monitor their specific flocculation ability under different micro-environmental conditions by using flow cytometry, which is widely used in industrial biotechnology (17, 18), transcription analysis, and proteomics.

MATERIALS AND METHODS

Microorganisms and Culture Conditions

Four brewing yeast strains of S. cerevisiae, known as bottom-fermenting yeasts and obtained from the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB, Germany), were used in this study. For better clarity, the strains were renamed (original names in parentheses): flocculent yeast strains FY1 (HS 10) and FY2 (HS 34), powdery yeast strains PY1 (HS 12) and PY2 (HS 37). These strains were either batch-cultured in 300 ml Erlenmeyer flasks with 75 ml Reader medium pH 5.4 (in g/l: 3 (NH4)2SO4, 0.7 MgSO4 · 7H2O, 1 KH2PO4, 0.16 K2HPO4, 0.5 NaCl, 0.4 Ca(NO3)2 · 4H2O, 5 yeast extract; 2% glucose) at 30°C and shaken at 200 rpm or on hops medium pH 4.8–5.4 (200 g/l malt extract; Merck, Darmstadt, Germany; 2 g/l hops pellets; Brewferm, Beverlo, Belgium) at 12°C and shaken at 120 rpm. Hops medium cultured cells were used for microarray and proteome analysis. Cells of FY2 and PY1 were also cultivated under brewing-like conditions as static culture on Reader medium (10% maltose, 12°C) in 1 l Duran®flasks. Inoculation of the fermentation flask occurred by sequenced adding (every 2.5 h) of 200 ml precooled (4.5°C) and aerated medium to 6.5 ml yeast suspension of a preparatory culture as it is done in breweries. After the inset of fermentation samples were taken for lectin staining and ethanol analysis at different time points.

Lectin Staining

For staining of the mannose and glucose structures of the cell surface, yeast cells were harvested after 16 h growth on Reader medium by centrifugation for 5 min with 3,214g at 20°C. The pellet was washed once with PBS buffer, pH 7.2 (in g/l: 1.18 Na2HPO4 · 2H2O, 0.22 NaH2PO4, 8.5 NaCl), resuspended in PBS buffer, and diluted to an optical density of OD700nm = 0.03–0.035 (d = 5 mm; 3.15 × 106 cells ml−1). All dye solutions were freshly prepared each day. All staining procedures were carried out in the dark and on ice. About 1.5 ml of the diluted cell suspensions were stained with 7.5 μl Pisum-sativum-agglutinate-FITC conjugate solution (PSA-FITC; 2.6 mol FITC/mol lectin, Sigma-Aldrich Chemie GmbH, Munich, Germany; stock solution 1 mg conjugate/ml PBS buffer) for 25 min, followed by 10 min incubation with 37.5 μl ConcanavalinA-Alexa-Fluor®-350 conjugate solution (ConA-Alexa-Fluor; 5 mol Alexa Fluor® 350/mol lectin, MoBiTec, Göttingen, Germany; stock solution 1 mg conjugate/ml bidest. water). After incubation the sample was immediately analyzed by flow cytometry.

Flow Cytometry

Flow cytometric measurements were carried out using a Particle Analyzing System PAS III (Partec, Münster, Germany) as described elsewhere (19). PSA-FITC fluorescence was measured using the combination of a TK 500 dichroic mirror and an interference filter EM 520. For measuring ConA-Alexa-Fluor fluorescence, a TK 460 dichroic mirror and an EM 452 band pass filter was used. The fluorescence and scatter signals were recorded linearly in all channels. Measurement of the events was triggered by the FSC signal. A total of 20,000 cells were analyzed in each sample at a rate of 200 cells per second. Alignment was based on the optimized signal from Flow-Check-Fluorespheres (Beckmann-Coulter, Krefeld, Germany) and data were evaluated using Summit V 3.1 (Cytomation, Carpinteria, CA).

Microscopy

Stained cells were observed using a fluorescence microscope (Axioscope HBO 100, Carl Zeiss, Jena, Germany) and image analysis (camera: DXC-9100P, Sony, Japan; software: Openlab 3.1.4., Improvision, Lexington, MA). The fluorescence filters used were: Zeiss filter set 02 for Alexa Fluor® 350 fluorescence (excitation G 365, BS 395, emission LP 420), Zeiss filter set 09 for FITC fluorescence (excitation BP 450–490, BS 510, emission LP 515).

Microarray Analysis

Yeast cells used for microarray analysis were harvested and centrifuged for 5 min with 3,214g at 4°C. Afterward, the pellet was directly frozen and stored at −20°C. For RNA isolation, 2 × 108–5 × 108 cells were disrupted under liquid nitrogen using a mortar and pestle. RNA isolation followed immediately using the RNeasy® Midi Kit (Qiagen, Hilden, Germany) with a concomitant DNA digestion on the column using the RNase-Free DNase Set (Qiagen, Hilden, Germany). RNA concentration and quality was determined as described elsewhere (20, 21).

The microarrays were printed at the Institute of Technical Chemistry (Leibniz University Hannover, Germany). Synthetic 70mer oligonucleotides (Operon, Cologne, Germany) were spotted on aldehyde modified glass slides (VSS25, CEL Associates, Pearland, TX) using an Affymetrix 417 arrayer. 158 genes were chosen coding for flocculins as well as proteins of cell cycle, various metabolic pathways, and glucose transporters of S. cerevisiae (20) and spotted in triplicates.

In this study, the expression status of the flocculin genes Lg-FLO1, FLO1, FLO5, FLO9, and FLO10 was followed. Hybridization was performed in a dye-swap design. During reverse transcription, 6 μg of purified total RNA was converted into either fluorescein (Fl) or biotin (B) labeled cDNA. The Fl and B labeled cDNAs of two yeast samples were hybridized simultaneously to the same array in one experiment. After hybridization, the unbound and nonspecifically fixed cDNA was removed from the array by stringent washing. Specifically bound Fl- and B-labeled cDNAs were sequentially detected with a series of conjugate reporter molecules according to the TSA process, ultimately with Tyramide-Cy3 and Tyramide-Cy5. The array obtained through this process was subsequently scanned for the two distinct fluorescent dyes (Cy3 and Cy5), showing a possible mRNA expression of flocculin genes in flocculent or powdery yeast strains. The scanning process of the hybridized chips included a six-fold scan of each chip at different settings, altering both PMT and laser power settings. For this last experimental step, the Axon 4000B scanner was used. The following primary analysis served as a quantification method and was performed with the Gene Pix Pro 6.0™ software tool. The secondary analysis was subsequently conducted using the data from the primary analysis. Therefore, data from different scans were first normalized by accounting for the overall intensities of the respective scans. Two replicates of each gene were tested for outliers. Outliers amongst the gene replicates were eliminated according to the outlier test by Nalimov. A t-test was applied to detect differences in gene expression between the sample groups. The independent Student's t-test was used to determine the statistical significance of the differences between the staining patterns. All P-values were two-tailed, and differences were considered significant for P-values ≤ 0.05. Summary data are expressed as mean and standard error of mean (SEM).

Proteome Analysis

Protein extraction and purification

For proteome analysis, a cell pellet was washed threefold with lysis buffer, pH 7.4 (50 mM Tris-HCl, 4.5 mM EDTA, 1 mM Pefabloc) for 5 min at 3,214g and 4°C. Afterward, the cells were resuspended in lysis buffer and stored at −20°C. Mechanical cell disruption was done in a bead mill (Retsch, Haan, Germany) using reaction tubes filled with 0.5 ml 0.75–1 mm sized glass beads, 1 ml yeast cell suspension, and 0.5 ml lysis buffer for 1.5 h at 4°C. Afterward, the supernatant was transferred to a new reaction tube. The glass beads were washed twice with 200 μl lysis buffer. The collected supernatants were centrifuged for 10 min at 18,000g at 4°C. The supernatant represented the cytosolic fraction, whereas the pellet represented the membrane and cell wall fraction, which was further treated to extract the cell wall bound proteins. To this end, it was washed twice (10 min, 18,000g, 4°C) with cell wall washing buffer, pH 4.0 (20 mM triethanolamine, 0.4 M KCl, 1 mM MgCl2, 1 mM Pefabloc). Subsequently, an enzymatic digestion with 1 U phospholipase D (PLD, Sigma-Aldrich Chemie GmbH, Munich, Germany) dissolved in 150 μl PLD buffer, pH 8, according to Mann et al. (22), was performed. After incubation in the dark for 16 h at 37°C, the solution was centrifuged (30 min, 18,000g, 21°C), and its supernatant was used for further investigations. Protein concentrations of the cytosolic and cell wall protein fraction were determined according to Lowry et al. (23) with modifications of Holtzhauer and Hahn (24). Dilutions of bovine serum albumin (BSA) were used as a standard.

SDS-PAGE of Extracted Proteins

Before SDS-PAGE, 100 μg of protein was precipitated with 5-fold ice cold acetone. The pellets were air-dried and mixed with sample buffer (25), incubated for 5 min at 60°C, and loaded on SDS-gels (4% stacking gel, 9% running gel). The electrophoresis was carried out with 20 mA and 3 W per gel. Afterward, the gels were stained with colloidal Coomassie Brilliant Blue G-250 (26) and dried in a stream of unheated air.

Western Blotting

Proteins from unstained gels were blotted on nitrocellulose membranes as described elsewhere (27) using a TE 22 tank transfer unit (Amersham Biosciences, Piscataway, NJ). The blotting was carried out for either 2 h (cytosolic proteins) or overnight (PLD cell wall proteins) with constant 100 V. Immunochemical staining was done as described by Benndorf et al. (28). A primary polyclonal anti-peptide antiserum from rabbit (Pineda Antibody Service, Berlin, Germany) was used to target all four flocculins (Lg-Flo1p, Flo1p, Flo5p, and Flo9p). For immunization, the peptide sequence NH2-TTNEQSVSSKMNSAT-CONH2 was chosen, as it is a conserved region present in all four flocculin proteins. As purification of the antibody failed because of its binding strength to the affinity chromatography column, the whole antiserum was used to incubate the western blot at a dilution of 1:1,000 for 12 h at 4°C. The binding of the secondary antibody, anti-rabbit IgG whole molecule peroxidise conjugate (dilution 1:5,000; Sigma-Aldrich Chemie GmbH, Munich, Germany), was detected using a PIERCE ECL Western Blotting Substrate Kit (Perbio Science Deutschland GmbH, Bonn, Germany). The Fluorchem™ 8900 camera and the corresponding software AlphaEaseFC (both Alpha Innotech, San Leandro, CA) were used for the detection of the emitted chemiluminescence.

Identification of Proteins by Nano-LC-ESI-MS

For the identification of the flocculation proteins on the SDS-PAGE, the bands of interest were excised and digested overnight with trypsin (29). The extracted peptides were separated by reversed-phase nano-LC (LC1100 series, Agilent Technologies, Palo Alto, CA; Chip 40 nL trap 75 μm × 150 mm, 5 μm C-18SB-ZX; solvents: 0.1% formic acid, gradient from 0 to 55% acetonitrile increased linear within 30 min), and analyzed by MS/MS (LC/MSD TRAP XCT mass spectrometer, Agilent Technologies, Palo Alto, CA) as described elsewhere (28). Database searches were carried out with MS/MS ion search (MASCOT, Matrix Science, London, UK) against a self created sub-database containing Lg-Flo1p, Flo9p, Flo5p, Flo1p, different keratins and trypsin. Subsequent parameters were selected: tryptic digestion, allowance of up to one missed cleavage site, and allowance of global modifications carbamidomethyl at cysteines and oxidized methionine, which were given as variable modifications. The search was restricted to peptides containing charge state two or three and was conducted with following accuracies: peptide tolerance of ±1.2 Da and MS/MS tolerance of ±0.8 Da. Individual ion scores >2 indicate identity or extensive homology (P < 0.05). Presentation of mass spectra was done with the software DataAnalysis for LC/MSD Trap Version 3.3 Build 146 (Bruker Daltonic GmbH, Bremen, Germany).

RESULTS

Differentiation of Flocculent and Powdery Yeasts via Lectin Staining and Flow Cytometry

Flow cytometric analysis of flocculent and powdery yeast strains was performed by application of the fluorescent lectins ConA-Alexa-Fluor and PSA-FITC (Fig. 1). The flocculent yeast strain FY2 and the nonflocculent powdery yeast strain PY1 were harvested at the early stationary growth phase after growth on Reader medium. Optimal staining conditions to obtain differentiation of FY2 and PY1 were elaborated separately for the two sub-species (see SI 1 and SI 2). Optimal fluorescent lectin concentrations for reliable staining were 80 μg PSA-FITC and 20 μg ConA-Alexa-Fluor per 3.15 × 106 cells ml−1, respectively. In view of possible economic routine application, 50 μg PSA-FITC and 10 μg ConA-Alexa-Fluor per 3.15 × 106 cells ml−1 were used during following calibration steps. The optimal staining durations for both strains were 20 min for PSA-FITC and 25 min for ConA-Alexa-Fluor, respectively. Further optimization was necessary when lectins were applied in combination. Best results were obtained when application of 5 μg PSA-FITC per 3.15 × 106 cells ml−1 for 25 min was followed by incubation with 25 μg ConA-Alexa-Fluor for 10 min. PSA-FITC was applied first to mask glucose residues of the cell wall. Differentiation of the cells harvested at the exponential growth phase was nevertheless impossible. Moreover, when cells were grown under brewing-like conditions (Reader medium with maltose instead of glucose as additional carbon and energy source at 12°C) a clear differentiation of powdery and flocculent cells was also not feasible (not shown). However, the differentiation of the two yeast strains was successful when treating early stationary phase cultures. In this phase, powdery and flocculent yeast cells showed increased binding of ConA-Alexa-Fluor, whereas only the flocculent strain showed also increased PSA-FITC binding (Fig. 2). Differentiation of the two sub-species was thus successful when cultivated under carbon limited conditions during the stationary growth phase, whereas routine application to industrial cultures will require more discriminative tests.

Figure 1.

Early stationary phase yeast cells of PY1 were stained for 60 min with either 10 μg/ml ConA-Alexa-Fluor (A) or 50 μg/ml PSA-FITC (B; bars 5 μm). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 2.

Dot plots of 20,000 yeast cells harvested after 16 h growth in batch culture on Reader medium supplemented with glucose. The cells were double-stained with PSA-FITC and ConA-Alexa-Fluor. Powdery yeast strain PY1 (A) flocculent yeast strain FY2 (B) and a suspended mixture of both (C) are represented.

Gene Expression Analysis using Microarrays

DNA microarray experiments were performed to identify differentially regulated genes in flocculent and powdery strains. Flocculation requires flocculins and mannose/glucose residues as the complementary binding partners. Therefore, the expression of the flocculin genes in the powdery yeast strains PY1 and PY2 versus the flocculent yeast strains FY1 and FY2 was of special interest. The mRNA was isolated from yeast strains harvested during either exponential or the stationary growth phase, reversely transcribed into cDNA, and labeled with biotin and fluorescein. Hybridization of the labeled samples (e.g. FY2 and PY1, harvested while growing at identical rates) onto the microarray (combined with a dye swap) was followed by the application of the signal-enhancing tyramid kit. Comparison confirmed that only the flocculent strains FY1 and FY2 strongly expressed the flocculin-encoding genes Lg-FLO1, FLO1, FLO5, and FLO9, whereas the powdery strains PY1 and PY2 showed nearly no expression (Fig. 3 and Table 1). In contrast, only very low expression levels of the flocculation gene FLO10 were found regardless of the growth phase. Expression of FLO10 was not found in the powdery strains and only faint in the flocculent strains.

Figure 3.

Low-density microarray with the hybridized cDNAs of the strains FY1 (labeled with Cyanin 5—red) and PY2 (labeled with Cyanin 3—green), both from the exponential phase. It is clearly visible that cells of FY1 show a higher expression of the flocculation genes than cells of PY2.

Table 1. Overview of flocculin genes expressed and regulated in the flocculent strains FY1 and FY2 and the powdery strains PY1 and PY2
STRAINYEAST ORFNAME/FUNCTIONSTRENGTH REGULATIONEXPRESSION
  1. Expressed and regulated genes are marked with one to three + according to the respective strengths. If there was no expression or regulation detectable, this was marked with /.

FY1LgFLO1Lg-FLO1++++++
YAR050WFLO1++++
YHR211WFLO5++++
YAL063CFLO9+++++
FY2LgFLO1Lg-FLO1++++++
YAR050WFLO1/++
YHR211WFLO5++++++
YAL063CFLO9++++++
PY1LgFLO1Lg-FLO1/+
YAR050WFLO1/+
YHR211WFLO5/+
YAL063CFLO9//
PY2LgFLO1Lg-FLO1/+
YAR050WFLO1//
YHR211WFLO5//
YAL063CFLO9//

It was therefore observed (Table 1) that expression levels varied between the different strains. The powdery yeast PY2 showed no expression of the genes FLO1, FLO5, and FLO9 and only very low expression of Lg-FLO1. Cells from the powdery yeast PY1 did not express FLO9, whereas low expression levels of the three other flocculin genes were detected. However, all four flocculin genes were highly expressed in the flocculent yeast FY1 although the extent of upregulation varied somewhat between the genes. Similar results were obtained for FY2. Although FLO1 was only intermediately expressed, high expression was observed again for Lg-FLO1, FLO5, and FLO9 compared to PY1 or PY2.

The whole dataset can be accessed on the Platform ID GSE12369 in the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) database.

Proteome Analysis

SDS-PAGE of cytosolic proteins and phospholipase D (PLD) extracted cell wall proteins was performed to verify the presence of the flocculins. Analysis via 2D-GE was not possible because of the strong glycosylation of the proteins (16), which probably prevents the migration of the proteins into the second dimension (results not shown). Cells of all four yeast strains, harvested either at the exponential or the stationary growth phase, were investigated. PLD was used to extract cell wall associated flocculins. Besides their presence in the cytosol, flocculation proteins are known to be attached to the cell wall via glycosylphosphatidylinositol anchors (16). PLD is able to cleave these anchors and to release the flocculation proteins (22).

Semi-quantitative data about flocculin contents of the cytosolic (exponential and stationary growth phase) and cell wall fractions (stationary growth phase) were obtained by western blotting (Fig. 4). A polyclonal rabbit peptide antiserum against the four flocculation proteins Lg-Flo1p, Flo1p, Flo5p, and Flo9p was applied. As a result, the flocculation proteins were detected in the cell wall fractions of FY1 and FY2, harvested in the stationary phase. The same results were obtained with regard to the cytosolic fractions of these two strains for both the exponential and stationary grown cells. In contrast, no labeling was observed for PY2 grown under both cultivation conditions, neither within the cytosolic nor the cell wall fractions. PY1 showed weak signals in the cytosolic and cell wall fractions of stationary grown cells. The cytosolic fractions of FY1 and FY2 of stationary grown cells showed two bands of different molecular weight, each. The respective upper bands remained in the stacking gel region, whereas the respective lower bands represented proteins with a molecular weight of ∼250 kDa. Furthermore, PLD extracted cell wall proteins (stationary phase) contained different amounts of flocculins. FY1 appeared to contain higher amounts of flocculins than strain FY2.

Figure 4.

Western blot analysis of (A) cytosolic proteins from exponentially growing cells, (B) cytosolic proteins from stationary cells and (C) cell wall PDL extracted proteins from stationary cells. For detection of flocculins, an antiserum against the four flocculins Lg-Flo1p, Flo1p, Flo5p, and Flo9p and chemiluminescence (exposure 5 min) was used. All bands lower than 85 kDa were also visible using the preimmunization serum (0; cytosolic proteins from exponential growth phase cells from strain FY2). Arrows mark bands which present flocculins. S, stacking gel; R, running gel.

An SDS-PAGE of the same samples was used to separate the proteins according to their molecular weight. The resulting bands were stained, cut, tryptically digested, and analyzed by ESI-MS/MS. Using this procedure, the identity of the flocculin Lg-Flo1p was verified (Fig. 5). This protein was found in the cytosolic fractions of FY1, PY1 and PY2 during the exponential growth phases and in PY1 and PY2 during the stationary growth phases. In the cell wall fractions, Lg-Flo1p was detected in PY1 and PY2, during both exponential and stationary phases. Lg-Flo1p was also detected in the cell wall fractions of FY1 and FY2 during the stationary phases and of FY1 in the exponential phase. These data are of qualitative nature.

Figure 5.

Nano-LC-ESI-MS/MS fragment spectrum of a 14 amino acid long peptide belonging to the protein Lg-Flo1p. The 1690.8654 Da peptide that covers the amino acids 179 to 192 of Lg-Flo1p had a score of 74.2. The peptide covers 1.33% of Lg-Flo1p, which has a theoretical mass of 109.4 kDa.

DISCUSSION

This study was performed to provide a basis for the differentiation between flocculent and powdery yeast strains. The determination of loss in flocculation ability is of high economic interest for breweries. Powdery yeast cells stay suspended within the brewing reactor even after completion of the secondary fermentation. This evokes increased cell death and spoils the flavor of the beer. Additionally, much more filter material is consumed, which is expensive and not environmentally friendly. Furthermore, the elevated need to filter the beer causes deadlocks (cessation) in the refilling processes of the brewing reactors which, in turn, decreases the efficiency and productivity of the brewery.

Flocculation ability has always been in the focus of brewers. Simple flocculation tests that are used in the laboratories of breweries are relatively imprecise and provide very limited information about the yeast cells and slurries. We chose to directly address the presence of molecules known to be involved in successful flocculation. These are the mannose structures on the yeast cell surface and the flocculins present in cytoplasm and cell wall.

Flow cytometry has proven its usefulness for monitoring and optimizing brewing processes (19, 30), for example, via the analysis of the yeast cell cycle (31). As flocculent yeast cells are assumed to possess somewhat higher amounts of mannose residues than powdery ones (32), ConA was used as the main marker for flocculent yeasts. However, the two strains, PY1 and FY2, were not differentiated by using ConA-Alexa-Fluor alone. Because PSA is known to bind preferentially to glucose residues (15), it was primarily added to favor binding of ConA to mannose residues. This combination allowed differentiation of early stationary growth phase cells of FY2 and PY1, although preferential binding of ConA-Alexa-Fluor to the flocculent strain was not confirmed. The reason for this might be growth or age-dependent expression of sugar residues at the yeast cell surface as it was described by de Nobel et al. (33) or other unknown strain-dependent cell wall peculiarities. It was also described that flocculent yeasts may expose strain-dependent amounts of mannose residues on the cell surface (32, 34). Also, the influence of Ca2+ and Mn2+ ions on the staining procedure was tested (not shown) because lectin conformation and binding is known to be influenced by bivalent cations (4). But, in this study, both ions did not improve differentiation of FY2 and PY1. In conclusion, differentiation of flocculent and powdery yeast strains was not generally feasible under laboratory bench-top growth conditions, particularly not when cells entered less active growth stages. There might be several reasons for such behavior. It is known that the composition of the cell wall is drastically influenced by environmental conditions like temperature, nutrients, and the cell's stage in the cell cycle (35). Most likely, these factors influence the kinds and quantities of sugar residues exposed at the cell surface, thereby exerting effects of lectin binding.

As realizable quantification of mannose residues connected to the cell wall was not feasible under scale up conditions, we focused on the flocculins. We found high expression levels of several flocculins by using a low density microarray tagged with sequences of the following genes: Lg-FLO1, FLO1, FLO5, FLO9, and FLO10. In fact, flocculation genes were highly up-regulated in the flocculent yeast strains FY1 and FY2 compared to the powdery ones. All printed flocculins except FLO10 showed high expression in the flocculent yeasts. It might be that FLO10 is metastably epigenetically silenced through the action of HDACs Hstp1 and Hstp2, as was reported by Halme et al. (11). The up-regulation of the flocculation genes Lg-FLO1, FLO1, FLO5, and FLO9 in flocculent yeast strains, even under different growth conditions, is strongly indicative of their important role in the flocculation process.

As these results underline the relevance of flocculins, it is quite surprising that nearly no studies were available which tried to isolate these proteins. A reason for this might be the unusual structure of the molecules which prevents an easy identification within the proteome. Flocculins are large proteins with molecular weights ranging from 110 to 160 kDa, but due to additional glycosylations (16, 36), molecular weights over 200 kDa can be reached, as was observed in our study where flocculins were detected at ∼250 kDa. Intensive glycosylation of the flocculins might have structural functions (16). 2D-GE was found to be unsuccessful because the usual range of protein verification and differentiation of this technique lies between 15 kDa and 150 kDa (37).

In our study, ESI-MS/MS and semi-quantitative western blot analyses revealed that Lg-Flo1p was the dominant flocculin in the two lager yeast strains FY1 and FY2. Using an antiserum, it was clearly shown that only the two flocculent strains possessed the molecular ability to develop the feature of flocculation. Already, the cytosolic samples from exponentially growing FY1 and FY2 contained flocculins. These findings support the statement by Bony et al. (38) that flocculins are processed in the cytosol. Differences in the cell wall flocculin contents of the two flocculent yeast strains were detected. However, SDS-PAGE and ESI-MS/MS clearly showed the presence of Lg-Flo1p in nearly all investigated strains both in the cytoplasm as well as in the cell wall. This was unexpected because only the flocculent yeast strains showed dominant expression of this gene in the microarray experiments. The discrepancy might be explained by the higher sensitivity of the ESI-MS/MS analysis that can detect smaller amounts of protein than western blot analysis.

To our knowledge, this is the first time that Lg-Flo1p was identified and highlighted in a yeast strain. Until now, the gene was merely described (9).

CONCLUSION

We conclude that the protein Lg-Flo1p can be used as a marker molecule in further studies. Although flow cytometry offered a fast and simple method to quantify the amount of flocculent yeast in a defined mixed suspension under limited cultivation conditions, precise and quantitative differentiation of powdery from flocculent yeast cells under industrial conditions required the application of mRNA and protein expression profiles analyses.

Acknowledgements

The authors wish to thank Martin Pähler from the working group “Chip technology” at the Institute of Technical Chemistry of the Leibniz University Hannover. The authors also want to thank Helga Engewald, Christine Süring, and Michaela Risch for their excellent technical assistance.

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