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Expression profile of genes involved in hydrogen

sulphide liberation by _Saccharomyces
cerevisiae_ grown under...

Article in Nature Precedings January 2009

DOI: 10.1038/npre.2008.2736.1 Source: OAI

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EVALUATION OF THE EFFECTS OF NITROGEN SUPPLY ON YEAST CELL PHYSIOLOGY, TRANSCRIPTOME,

PROTEOME AND METABOLOME FOR OPTIMIZING THE PRODUCTION OF ECONOMICALLY IMPORTANT
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 Expression profile of genes involved in hydrogen sulphide liberation by
Saccharomyces cerevisiae under different nitrogen concentrations
Mendes-Ferreira A.1, Barbosa C. 1, del Olmo M. 2, Mendes-Faia A. 1 and Leo C.3

1IBB-Centro de Gentica e Biotecnologia- Universidade de Trs-os-Montes e Alto Douro, Vila Real, Portugal.
2Departament de Bioqumica i Biologia Molecular, Universitat de Valncia, Valncia, Spain.
3Instituto de Investigao em Cincias da Vida e Sade (ICVS), Escola de Cincias da Sade, Universidade do Minho, Braga, Portugal

BACKGROUND
HOW IS NITROGEN LINKED TO H2S PRODUCTION ?
Hydrogen sulphide (H2S) is a secondary metabolite produced by yeasts
during alcoholic fermentation, and its amounts largely depends on the
yeast strain, media composition and fermentation conditions. In
Saccharomyces cerevisiae, sulphide is the product of the Sulphate
Reduction Sequence (SRS) pathway and acts as an intermediate in the
biosynthesis of sulphur-containing amino acids. The biosynthesis of
sulphur amino acids requires nitrogen-containing carbon precursors
derived from the intracellular nitrogen pool and sulphide from the
sulphate reduction pathway.

AIMS
Nature Precedings : doi:10.1038/npre.2008.2736.1 : Posted 30 Dec 2008

The present work intends to establish a relationship between nitrogen availability and H2S liberation with expression levels of genes involved in the SRS
pathway. Our ultimate goal is to elucidate molecular mechanisms underlying H2S production in Saccharomyces cerevisiae associated to nitrogen deficiency, in
order to minimize its incidence in alcoholic beverage production.

GENOME-WIDE ANALYSIS PHENOTYPING ANALYSIS OF H2S FORMATION

IN THREE S. cerevisiae STRAINS

Fermentation profiles and hydrogen sulphide liberation (histograms) by the yeast strains PYCC4072, UCD522, EC1118 grown in synthetic grape juice
medium at 20C, containing an initial nitrogen concentration of 66 (A), 267 (B) and 402 mg N l-1 (C). Data points are the mean from triplicate fermentations
SD (smaller error bars are hidden behind the data symbols).

Overview of the experimental design used for genome wide expression studies with S. cerevisiae
PYCC4072 in synthetic grape-juice medium (panel A) , under different nitrogen concentrations (CF-
control fermentation, 267 mg of N l-1); LN-low nitrogen fermentation, 66 mg of N l-1) and RF- re-fed
fermentation, 66 + 200mg of N l-1). Sulphate Reduction pathway (panel B) and transcript profiles of
genes involved in SRS pathway, obtained by macroarray analysis in S. cerevisiae PYCC4072 (panel ON GOING WORK
C) grown under different nitrogen conditions. The expression diagram, show, from top to bottom,
relative expression levels of each gene and, from left to right, comparisons between time points.
CF24, sample from the control fermentation carried out with 267 mg N l-1 at 24h after inoculation
From the above experiments, cells were collected for RNA extraction and qRT-PCR assays are
when the amount of assimilable nitrogen remaining in the medium was 178 mg l-1; RF80, sample
from the re-fed fermentation (66+200 mg N l-1 at 72h supplied as diammonium phosphate) collected
being conducted using five MET genes as well as SSU1, encoding a plasma membrane sulphite
8h after addition, at 80h, when the amount of assimilable nitrogen present in the medium was 154
mg l-1; CF48, CF96, LN24, LN48, LN80, LN96, LN144, RF96 and RF144 are samples from the
pump involved in sulphite metabolism and required for efficient sulphite efflux.
control (CF), the low nitrogen fermentation (LN) conducted with 66 mg N l-1 and from the re-fed
fermentation (RF) collected throughout the experiments. In all these last time points the nitrogen was C
low or even absent. A
Threshold cycle

y = -3.46x + 21.05 r2 = 0.996

PCR Efficiency for MET3 : 94.4%

FINAL REMARKS Log cDNA

Definitive conclusions cannot be made at this time regarding

B
the relationship between H2S liberation and the expression
levels of the genes under study. However this preliminary Dye: SBG1
c(t) 23.32
PYCC4072 267-72h
Dye: SBG1 Standard curve generated for MET3 using series dilution of a template amplified with iCycler real
c(t) 26.24
PYCC4072 267-120h
time system (panel A). Amplification plots for qRT-PCR using MET3 primers (panel B). Gene
work does suggest that mRNA abundance of these genes expression analysis of six genes on S. cerevisiae PYCC4072 cells collected at 72 and 120h from
the experiment conducted with 267 mg N l-1 (panel C).
were higher at 72h than at 120h. Additional assays are
required to elucidate this event.
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This work was partially supported by FCT project PTDC/AGR-ALI/71460/2006