Nitric oxide increases biofilm formation in Saccharomyces cerevisiae by activating the transcriptional factor Mac1p and thereby regulating the transmembrane protein Ctr1

Abstract

Background: Biofilms with immobilized cells encased in extracellular polymeric substance are beneficial for industrial fermentation. Their formation is regulated by various factors, including nitric oxide (NO), which is recognized as a quorum-sensing and signal molecule. The mechanisms by which NO regulates bacterial biofilms have been studied extensively and deeply, but were rarely studied in fungi. In this study, we observed the effects of low concentrations of NO on biofilm formation in Saccharomyces cerevisiae. Transcriptional and proteomic analyses were applied to study the mechanism of this regulation.

Results: Adding low concentrations of NO donors (SNP and NOC-18) enhanced biofilm formation of S. cerevisiae in immobilized carriers and plastics. Transcriptional and proteomic analyses revealed that expression levels of genes regulated by the transcription factor Mac1p was upregulated in biofilm cells under NO treatment. MAC1 promoted yeast biofilm formation which was independent of flocculation gene FLO11. Increased copper and iron contents, both of which were controlled by Mac1p in the NO-treated and MAC1-overexpressing cells, were not responsible for the increased biofilm formation. CTR1, one out of six genes regulated by MAC1, plays an important role in biofilm formation. Moreover, MAC1 and CTR1 contributed to the cells' resistance to ethanol by enhanced biofilm formation.

Conclusions: These findings suggest that a mechanism for NO-mediated biofilm formation, which involves the regulation of CTR1 expression levels by activating its transcription factor Mac1p, leads to enhanced biofilm formation. The role of CTR1 protein in yeast biofilm formation may be due to the hydrophobic residues in its N-terminal extracellular domain, and further research is needed. This work offers a possible explanation for yeast biofilm formation regulated by NO and provides approaches controlling biofilm formation in industrial immobilized fermentation by manipulating expression of genes involved in biofilm formation.

Keywords: Biofilm; CTR1; MAC1; Nitric oxide; Saccharomyces cerevisiae.

Fig. 1

Biofilms formed under treatment with NO donors. a Growth curve of free cells in the presence of different concentrations of SNP during immobilized fermentation. b After 24 h of fermentation, the culture broths from the different groups were observed. c Biofilms formed on cotton fibers after fermentation imaged by SEM. d Biofilms formed in 96-well plates for 24 h in the presence of NO donors, SNP and NOC-18, and the scavengers PTIO, NaNO₂, and NaNO₃. The wells were washed twice with PBS (200 μL) to remove free cells and stained with 1% crystal violet. Biofilm formation was measured at 570 nm after solubilizing crystal violet in acetic acid. The values are the means and standard deviations of three independent experiments. ***p < 0.001, **p < 0.01, *p < 0.05 by Student’s t-test

Fig. 2

a Comparison of the transcriptomes and proteomes of cells that formed biofilms under control conditions and under treatment with 200 μM SNP.  Venn diagram showing the overlap between the transcriptomes and the proteomes in the up- and downregulated groups, respectively. The number of genes/proteins in each part is indicated. b Expression levels of MAC1 and its downstream genes/proteins. c qRT-PCR results. Relative expressions of MAC1 and downstream genes in SNP-treated biofilm cells compared with the control

Fig. 3

a Biofilms of the WT and five mutants formed in 96-well plates under control conditions and with SNP treatment. b Photographs of biofilms formed on plastics by WT, ∆MAC1, ∆CTR1, +pMAC1, +pCTR1, and +p MAC1 CTR1. c Relative expression of MAC1 and downstream genes in ∆MAC1, ∆CTR1, +pMAC1, +pCTR1, and +pMAC1 CTR1, compared with WT. d Plate-wash tests of the WT, ∆MAC1, ∆CTR1, +pMAC1, +pCTR1, and +pMAC1 CTR1. Photos of pre- and post-washed strains were taken. The values are the means and standard deviations of three independent experiments. ***p < 0.001, **p < 0.01, *p < 0.05 by Student’s t-test

Fig. 4

Biofilms formed in 96-well plates measured after adding different concentrations of (a) copper (b) and iron. c Intracellular copper and iron contents in biofilm cells were detected. The values are the means and standard deviations of three independent experiments. ***p < 0.001, **p < 0.01, *p < 0.05 by Student’s t-test

Fig. 5

a Change of glucose concentration during fermentation in 10% (v/v) ethanol in free and biofilm fermentation. b SEM images of biofilms formed on cotton fibers by WT, ∆MAC1, ∆CTR1, +pMAC1, +pCTR1, and +pMAC1 CTR1 after 33 h of fermentation in 10% ethanol. c Kinetics of batch fermentation of the three strains in immobilized cultures