Arneborg, Nils

Gori, Klaus

Jespersen, Lene

Knudsen, Peter Boldsen

Nielsen, Kristian Fog

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Downloaded from orbit.dtu.dk on: Dec 19, 2017Production of alcohol-based quorum sensing molecules and their role in biofilmformation and sliding motility of the dairy-important yeast Debaryomyces hanseniiGori, Klaus; Knudsen, Peter Boldsen; Nielsen, Kristian Fog; Arneborg, Nils; Jespersen, LenePublication date:2011Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Gori, K., Knudsen, P. B., Nielsen, K. F., Arneborg, N., & Jespersen, L. (2011). Production of alcohol-basedquorum sensing molecules and their role in biofilm formation and sliding motility of the dairy-important yeastDebaryomyces hansenii. Poster session presented at 4th Conference on Physiology of Yeast and FilamentousFungi, Rotterdam, Netherlands.D E P A R T M E N T   O F   F O O D   S C I E N C EF A C U L T Y   O F   L I F E   S C I E N C E S U N I V E R S I T Y   O F   C O P E N H A G E NProduction of alcohol-based quorum sensing molecules      and their role in biofilm formation and sliding motility of the dairy-important yeast Debaryomyces hanseniiKlaus Gori1*, Peter Boldsen Knudsen2, Kristian Fog Nielsen2, Nils Arneborg1 and Lene Jespersen11)Department of Food Science, Food Microbiology, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark.2)Department of Systems Biology, Center for Microbial Biotechnology, Technical University of Denmark, Søltofts Plads, Building 221, DK-2800 Kgs. Lyngby, Denmark.*Correspondence to: Klaus Gori, Tel.: +45 35333284, E-mail: klg@life.ku.dkAimThe aim of the present study was to examine aromatic alcohols including phenylethanol, tyrosol and tryptophol and the sesquiterpene farnesol for their potential quorum sensing effects in the dairy-important yeast Debaryomyces hanseniiIntroductionMany yeasts are dimorphic, which means that they are able undergo a transition from a unicellular yeast form to a filamentous form or vice-versa. The yeast-to-mycelium Production of alcohols at standard conditionsD. hansenii (CBS 767, D18335 and MD02) were found to produce increasing concentrations of phenylethanol and tyrosol in the μM range during growth at standard conditions (Figure 1A). After 120 h of fermentation, the concentrations of phenylethanol and tyrosol were found to be 9.77-12.6 μM and 3.01-8.42 μM, respectively. Concerning tryptophol, this alcohol was in general not detected at standard conditions. For comparison, the concentrations of phenylethanol, tyrosol and tryptophol were 293, 119 and 11.4 μM for C. albicansA Debaryomyces hansenii (CBS767)    B  Debaryomyces hansenii (CBS767)   Aromatic alcohols  (µM) 161412108 6420 7  6  5  4  3  2  1  0Growth ln(OD600nm(t)/OD600nm(0))  Aromatic alcohols (µg/g dry wt) 6000 5000 4000 3000 2000 10000 7  6  5  4  3  2  1  0Growth ln(OD600nm(t)/OD600nm(0))          0           24          48          72          96         120          0           24          48          72          96         120 Time (h) Time (h)   Debaryomyces hansenii (D18335)      Debaryomyces hansenii (D18335)   16 7 6000 7transition can be induced by environmental factors e.g. temperatures below or over the optimal range, limited availability of oxygen, high osmolality, pH or substrate limitation including nitrogen starvation. In addition, quorum sensing (the process by which microorganisms communicate by signalling molecules in a cell density dependent manner) has reported to be involved in the morphogenesis of yeasts. The sesquiterpene farnesol has been found to be a signalling molecule inhibiting both the yeast-to-hyphal shift and biofilm formation of Candida albicans. In addition, the aromatic alcohol tyrosol has been shown due to its promotion of hyphal development to shorten lag phase time of C. albicans. Furthermore, the aromatic alcohols phenylethanol and (CBS 8758) and 31.4, 22.8 and 0.198 μM for S. cerevisiae (1171) (results not shown). The fact that alcohols were primarily determined from the end of exponential phase indicates that these alcohols are potential quorum sensing molecules in D. hansenii. When calculated as μg/g dry wt, D. hansenii (CBS 767 and D18335) produced increasing concentrations of phenylethanol and tyrosol, whereas  D. hansenii (MD02) produced constant concentrations of the two alcohols (Figure 1B). The fact that D. hansenii (CBS 767 and D18335) produced increasing concentrations of phenylethanol and tyrosol during stationary phase indicates that these alcohols besides being produced as potential quorum sensing Aromatic alcohols  (µM) 1412108 6420  6  5  4  3  2  1  0Growth ln(OD600nm(t)/OD600nm(0))  Aromatic alcohols (µg/g dry wt)  5000 4000 3000 2000 10000 6  5  4  3  2  1  0Growth ln(OD600nm(t)/OD600nm(0))       0           24          48          72          96         120                  0           24          48          72          96         120 Time (h) Time (h)   Debaryomyces hansenii  (MD02)      Debaryomyces hansenii (MD02)   atic alcohols  (µM) 161412108  7  6  5  4  3 Growth m(t)/OD600nm(0))  atic alcohols /g dry wt) 6000 5000 4000 3000 7  6  5  4  3 Growth m(t)/OD600nm(0)) Fold inductions/repressionsBased on concentrations in μM Phenylethanol Tyrosoltryptophol have been found to be signalling molecules in Saccharomyces cerevisiae, where they stimulate pseudohyphal growth. Furthermore, phenylethanol was found to stimulate invasive growth. Addition of tryptophol resulted in an even greater invasive growth, whereas tryptophol alone did not have an effect on invasive growth. C. albicans does also produce phenylethanol and tryptophol, but if a function, it is different, from what is observed in S. cerevisiae.Material and methodsThree strains of Debaryomyces hansenii (type strain CBS767 and the dairy isolates D18335 and MD02 were included. Figure 1. Production of phenylethanol and tyrosol (shown in bars) in Debaryomyces hansenii(CBS767, D18335 and MD02) at standard conditions.    phenylethanol and    tyrosol. Furthermore, growth curves are shown.    Growth (ln(OD600nm(t)/OD600nm(0)), t refers to the time.molecules also are products used for survival of these two D. hansenii strains.Alcohol production at different environmental conditionsThe aromatic alcohol production for the three D. hanseniistrains varied with different environmental conditions. Table 1 shows the changes in alcohol production at different environmental conditions for D. hansenii (CBS767) after 120 h of fermentation. Similar results were obtained for D. hansenii (D18335 and MD02) (results not shown). Addition of aromatic amino acids (the precursors of aromatic alcohol production) increased aromatic alcohol production in abundance.  Phenylethanol production increased 22-fold by Table 1. Effects of environmental conditions on phenylethanol and tyrosol production in Debaryomyces hansenii (CBS767). Fold inductions/repressions are based on alcohols concentrations in μM determined after 120 h of fermentation. Aroma 6420 2  1  0Glln(OD600nAroma(µg/ 2000 10000 2  1  0Gln(OD600nm      0           24          48          72          96         120                  0           24          48          72          96         120 Time (h) Time (h)Aromatic amino acid (50 μg/ml)Phenylalanine 22 ± 2.3 2.7 ± 0.60Tysosine 1.5 ± 0.18 87 ± 15Tryptophan 2.4 ± 0.75 5.3 ± 0.10Ammonium sulfate50mM 0.78 ± 0.011 nd100mM 0.82 ± 0.23 nd500mM 0.77 ± 0.069 0.46 ± 0.000881000mM 0.86 ± 0.075 1.7 ± 0.285000mM 0.63 ± 0.11 0.30 ± 0.1637000mM 0.63 ± 0.015 0.32 ± 0.042NaClCandida albicans (CBS8758) and Saccharomyces cerevisiae(CBS1171) were included as reference strains. At standard conditions, yeast cultures were grown in yeast nitrogen base (YNB) without amino acids and ammonium sulfate supplemented with 2% glucose and 5 mM L-Proline (pH 4.3) at 25ºC with shaking at 120 rpm. At appropriate time intervals, cell-free supernatants were prepared and analysed by their content of phenylethanol, tyrosol and tryptophol by ESI+ with the triple quadrupole operating in multiple reaction monitoring mode with two transitions per compound. Quantification was done using 2H5-1-phenylethanol as internal standard (10 ng/mL) and using peak areas as response factor relative to 2H5-1-phenylethanol. Farnesol was determined in a addition of phenylalanine, whereas tyrosol production increased 87-fold by the addition of tyrosine. Addition of tryptophane resulted in tryptophol concentrations between 4.20-7.50 μM. Furthermore, alcohols were not only increased by their own precursors e.g. tryptophane increased tyrosol production by more than 5.0-fold. Production of phenylethanol and tyrosol was generally decreased by increasing concentrations of ammonium, NaCl, pH and temperature values. Contrary to phenylethanol, tyrosol production were found to be autosimulated by addition of phenylethanol and tryptophol, as these alcohols increased tyrosol production by more than 3.8-fold. OD600nm 0.03OD600nm 0.1OD600nm 0.5OD600nm 1.02% (w/v) 0.59 ± 0.0035 0.63 ± 0.0644% (w/v) 0.74 ± 0.067 0.66 ± 0.146% (w/v) 0.51 ± 0.015 0.48 ± 0.0978% (w/v) 0.34 ± 0.047 0.23 ± 0.0051pH5.0 0.80 ± 0.063 0.80 ± 0.00506.0 0.87 ± 0.27 1.8 ± 0.157.0 nd 0.31 ± 0.118.0 nd nd9.0 nd ndTemperature15°C 0.85 ± 0.55 0.81 ± 0.43Aromatic alcohols (1000 μM)similar fashion applying farnesal as internal standard.Biofilm formation was measured by the crystal violet method. Sliding motility was measured by inoculation of yeast cells in the centre of YPD plates with 0.3% agar, and growth was followed for 14 days. To induce pseudohyphal growth, yeast cells were restreaked on SLAD agar. Plates were incubated at 25ºC for 10 days. Colony morphology photographed under a microscope. Biofilm formation, sliding motility and pseudohyphal growthAmong D. hansenii strains investigated, especially D.hansenii (CBS767) was capable of biofilm formation (Figure 2a). Phenylethanol, tyrosol and tryptophol did in general not increase biofilm formation of D. hansenii (CBS767), whereas farnesol concentrations as low as 500 μM completely inhibited D. hansenii (CBS767) in formation of biofilm (Figure 2b). Also concerning sliding motility, D. hansenii (CBS767) shown a particular ability as this strain overgrew YPD plates with low agar content (Figure 3). The correlation between biofilm formation and sliding motility suggests an intimate connection of the two phenotypes. Addition of phenylethanol, A 0.500  0.400  0 300 m    Phenylethanol - 3.8 ± 0.38Tyrosol 1.2 ± 0.18 -Tryptophol 1.2 ± 0.20 5.0 ± 0.94tyrosol and tryptophol as low as 1000 μM resulted in more filamentous growth pattern D. hansenii (CBS767) (Figure 3). Contrary, only D. hansenii  (MD02) was able of pseudohyphal growth on low-ammonium agar plates (Figure 4).ConclusionsThe present study shows that D. hansenii produces alcohol-based quorum sensing molecules previously reported for C.albicans and S. cerevisiae. However, alcohol yields are highly dependent on growth conditions including the availability of aromatic amino acids, ammonium sulphate, NaCl, pH and temperature. Several physiological traits nd not detectableB 10 μM farnesol50 μM farnesol100 μM farnesol500 μM farnesol1000 μM farnesol.   0.200  0.100 0     CBS 767                 D18335                MD02 0.300  0.250  0 200 OD590nm  YPD without  aromatic alcohols  YPD with 1000 µM tyrosol YPD with1000 µM phenylethanol YPD with1000 µM tryptophol CBS 767         D18335         including biofilm formation and sliding motility of D. hanseniiseems to be regulated by alcohol-based quorum sensing molecules. Knowledge on quorum sensing properties of dairy-relevant microorganisms including D. hansenii may be used in optimisation of the cheese ripening processes.AcknowledgementsThis work was supported by The Danish Dairy Research Foundation (Danish Dairy Board) and The Danish Ministry of Food, Agriculture and Fisheries. Janne Benjaminsen, Søren Nørgaard Kristiansen and Kenneth Hansen are thanked for excellent technical assistance.Figure 2. A: Biofilm formation of D. hansenii (CBS767, D18335 and MD02) after 24 h of incubation. B: Influence of farnesol on biofilm formation of D. hansenii (CBS767).Figure 3. Sliding motility of D. hansenii (CBS767, D18335 and MD02) on YPD with 0.3% agar. Figure 4. Pseudohyphal formation of D. hansenii(MD02) on low-ammonum agar plates. .   0.150  0.100  0.0500        12 h                                   24 h  OD590nmMD02        

Production of alcohol-based quorum sensing molecules and their role in biofilm formation and sliding motility of the dairy-important yeast Debaryomyces hansenii

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Production of alcohol-based quorum sensing molecules and their role in biofilm formation and sliding motility of the dairy-important yeast Debaryomyces hansenii

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