Phenomic analysis and cell wall remodulation of <i>Saccharomyces cerevisiae</i> flor strains in the presence of different nitrogen sources

Flor yeasts of Saccharomyces cerevisiae are the main actors in the biological ageing of Sherry wines by developing an air-liquid biofilm, called velum at the surface of wine at the end of fermentation. The air-liquid biofilm occurs due to a complex mechanism which is similar to many microbial biofilms. The velum phenotype is mainly regulated by the expression of a highly O-glycosylated cell wall protein Flo11 which by turn contributes to the hydrophobicity and electrostatic charge of the cell wall which are crucial for unspecific interactions and adhesion capacity of yeasts. In this study we characterized the interactions of flor yeasts with a series of nitrogen compounds known for their hydrophobicity and/or charge. We find that, different flor yeast strains characterized by different degree of functional FLO11 are diversely affected in terms of biofilm formation and adhesion capacity when interact with different nitrogen sources. The positive role of the synthetic hexapeptide PAF26 in enhancing the biofilm formation was first discovered. On the contrary, cationic amino acids were able to inhibit biofilm formation and adhesion ability. Results together highlight on the involvement of the Flo11p in hydrophobic and electrostatic interactions and open to new investigations, addressed to the control of microbial adhesion and biofilm formation

L-Histidine Inhibits Biofilm Formation and <i>FLO11</i>-Associated Phenotypes in <i>Saccharomyces cerevisiae</i> Flor Yeasts

<div><p>Flor yeasts of <i>Saccharomyces cerevisiae</i> have an innate diversity of <i>FLO11</i> which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three <i>S. cerevisiae</i> flor strains handling <i>FLO11</i> alleles with different expression levels. <i>S. cerevisiae</i> strain S288c was used as the reference strain as it cannot produce FLO11p. The flor strains generally metabolized amino acids and dipeptides as the sole nitrogen source, although with some exceptions regarding L-histidine and histidine containing dipeptides. L-histidine completely inhibited growth and its effect on viability was inversely related to <i>FLO11</i> expression. Accordingly, L-histidine did not affect the viability of the Δ<i>flo11</i> and S288c strains. Also, L-histidine dramatically decreased air–liquid biofilm formation and adhesion to polystyrene of the flor yeasts with no effect on the transcription level of the <i>FLO11</i> gene. Moreover, L-histidine modified the chitin and glycans content on the cell-wall of flor yeasts. These findings reveal a novel biological activity of L-histidine in controlling the multicellular behavior of yeasts.</p></div

CFW staining of <i>S. cerevisiae</i> strains in the absence and presence of L-histidine.

<p>The A9, M23, V80, 3238-32, 3238-32Δ<i>flo11</i> and S288c strains (5×10⁶ cell/mL) were incubated for 2 h in flor medium without or with 10 mM L-histidine. After incubation, the samples were stained with 25 µM CFW for 5 min and observed by fluorescence microscopy. Bright-field differential interference contrast (DIC) and CFW images of the same field are shown. All of the images were captured under the same acquisition parameters and therefore reflect actual differences in CFW staining.</p

Loss of adhesion in presence of L-histidine and other dipeptides.

<p>Adhesion is expressed as OD₅₇₀ and was measured using crystal violet dye after 48 h incubation of 5×10⁶ cell/mL of the <i>S. cerevisiae</i> strains in flor medium without (Ctrl) and with 10 mM L-histidine or the L-histidine–containing peptides.</p

Biofilm formation of flor strains is inhibited by L-histidine.

<p>(<b>A</b>) Biofilm formation at the air-liquid interface in 24-well microtiter plates for strains A9, M23, V80, 3238-32, 3238-32Δ<i>flo11</i> and S288c after 5 days of static incubation in 1.5 mL flor medium at 30°C in the absence (Ctrl) and presence of 10 mM of L-histidine (L-his) and L-histidine–containing dipeptides. The biofilm is visualized as opaque floating material at the top of each well. (<b>B</b>) Dry weight determinations of the biofilms formed by the strains in (A) without (Ctrl) and with treatment with 10 mM L-histidine and the L-histidine–containing dipeptides (as indicated). Data are means +SD of three replicate treatments. (<b>C</b>) CFU recovery after plating on YPD agar using serial dilutions of a duplicate of all the strains/L-histidine and strains/dipeptides combinations.</p

<i>Saccharomyces cerevisiae</i> strains used in this study.

<p><i>Saccharomyces cerevisiae</i> strains used in this study.</p

L-histidine effect on cell growth is <i>FLO11</i>-dependent, and related to pH and cell-surface charge.

<p>The histogram and the line graph illustrate the cell growth and the cell surface net charge, respectively, of the 3238-32 and 3238Δ<i>flo11</i> strains. Cells (10⁴ cells/mL) were incubated in SC minimal medium. The media were buffered at pH 3, 4, 5 and 6 using 0.1 M citric acid monohydrate and 0.2 M sodium phosphate. Cells were grown in 96-well microplates, statically at 30°C for 48 h. Growth was monitored measuring the OD₆₀₀ in a SPECTROstar nano-microplate spectrophotometer (BMG Labtech, Germany). Cell surface net charge (Z-potential) was measured under the same conditions using the Zetasizer Ver. 6.20 (Malvern Instruments Ltd) after 48 h of treatment. Data are means ±SD of three replicates. A one-way analysis of varience (ANOVA) was performed and followed by Tukey honestly significant difference test (P<0.05). The analyses were performed independently for growth and cell surface charge data and values with the same letter/symbol are not statistically different.</p

Modulation of cell-wall glycans of <i>S. cerevisiae</i> strains in the absence and presence of L-histidine.

<p>Cell-wall glycan levels (as arbitrary fluorescent units) without (Ctrl) and with 10 mM L-histidine treatment for A9, M23, V80, 3238-32, 3238-32Δ<i>flo11</i> and S288c strains (5×10⁶ cell/mL) in flor medium after 2 h. Data are means +SD from three replicate samples, of the fluorescence intensity of ConA-FITC bound to cell-wall glycans of 20.000 cells/sample. Multiple comparison analysis was conducted. Bars with the same letters are no statistically different (95% confidence).</p

Strains growth rate inhibition and Lag phase delay in the presence of different L-histidine concentrations.

<p>The average of Log-phase specific growth rate was calculated by the DMFit software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112141#pone.0112141-Baranyi1" target="_blank">[26]</a>, and specific growth rate inhibition (%) and Lag-phase delay (h) of L-histidine treated cells were calculated in respect to control cells. Negative values in parentheses represent results with no growth rate inhibition. Values with the same letter are not statistically different (Multiple comparison analysis; 95% confidence). Minus symbol (-) represent a complete Lag-phase delay.</p><p>Strains growth rate inhibition and Lag phase delay in the presence of different L-histidine concentrations.</p