Description of the mechanisms underlying

JO U R N A L OF P ROTE O MI CS 9 6 ( 20 1 4 ) 1 3–2 8

Available online at www.sciencedirect.com

ScienceDirect
www.elsevier.com/locate/jprot

Description of the mechanisms underlying geosmin
production in Penicillium expansum using proteomics
Marc Behr, Tommaso Serchi, Emmanuelle Cocco, Cédric Guignard, Kjell Sergeant,
Jenny Renaut, Danièle Evers⁎
Centre de Recherche Public-Gabriel Lippmann, Département Environnement et Agro-biotechnologies, Belvaux, Luxembourg

AR TIC LE I N FO

ABS TR ACT

Article history:

A 2D-DIGE proteomics experiment was performed to describe the mechanism underlying the

Received 20 March 2013

production of geosmin, an earthy-smelling sesquiterpene which spoils wine, produced by

Accepted 24 October 2013

Penicillium expansum. The strains were identified by sequencing of the ITS and beta-tubulin
regions. This study was based on a selection of four strains showing different levels of geosmin
production, assessed by GC–MS/MS. The proteomics study revealed the differential abundance of

Keywords:

107 spots between the different strains; these were picked and submitted to MALDI-TOF–TOF MS

Geosmin

analysis for identification. They belonged to the functional categories of protein metabolism,

Penicillium expansum

redox homeostasis, metabolic processes (glycolysis, ATP production), cell cycle and cell signalling

Proteomics

pathways. From these data, an implication of oxidative stress in geosmin production may be

Oxidative stress

hypothesized. Moreover, the differential abundance of some glycolytic enzymes may explain the

MVA pathway

different patterns of geosmin biosynthesis. This study provides data for the characterisation of

MEP pathway

the mechanism and the regulation of the production of this off-flavour, which are so far not
described in filamentous fungi.
Biological significance
Green mould on grapes, caused by P. expansum may be at the origin of off-flavours in wine. These are

characterized by earthy–mouldy smells and are due to the presence of the compound geosmin. This
work aims at describing how geosmin is produced by P. expansum. This knowledge is of use for the
research community on grapes for understanding why these off-flavours occasionally occur in
vintages.
© 2013 Elsevier B.V. All rights reserved.

1.

Introduction

Wine is a product for which organoleptic quality is primordial.
Wine aroma results from the contribution of volatile compounds originating from the grape microflora and winemaking
practices [1]. Although in most cases aroma compounds confer
a special, variety-specific, positive characteristic to a wine,

several grape-derived aroma compounds may alter wine aroma
in a negative way. Over the last years, winegrowers have
observed organoleptic defects in wine characterized by mushroom, mouldy, camphoric or earthy odours [2]. The risk for such
defects is high when grapes are infected by rots. Indeed, they
are produced by Botrytis cinerea (causal agent of grey mould),
Penicillium expansum (causal agent of green mould), a wide-

Abbreviations: ITS, internal transcribed spacer (of the rDNA); PTV, programmed temperature vaporization; MEP pathway,
methylerythritol phosphate pathway; MVA pathway, mevalonate pathway; PH-like, pleckstrin homology-like
⁎ Corresponding author at: Centre de Recherche Public-Gabriel Lippmann, Département Environnement et Agro-biotechnologies, 41, rue du Brill, 4422
Belvaux. Luxembourg. Tel.: + 352 47 02 61 441; fax: + 352 47 02 64.
E-mail addresses: (M. Behr), (T. Serchi), (E. Cocco),
(C. Guignard), (K. Sergeant), (J. Renaut), (D. Evers).
1874-3919/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
/>

14

JO U R N A L OF PR O TE O MI CS 96 ( 20 1 4 ) 1 3 –28

spread filamentous fungus responsible of fruit decay including
grapes [3], or a combination of both [4]. The compounds
responsible for defects associated with mushrooms are C8
alcohols and ketones such as 1-octen-3-ol and 1-octen-3-one
and they have been reported to be metabolites of various fungi;
in oenology, 1-octen-3-ol has been associated with the presence
of B. cinerea on grapes [5]. As for the earthy smell, La Guerche et
al. [2] identified the responsible molecule as (−)-geosmin. The
olfactory perception threshold for geosmin in wines is 50 ng/L
[6]. According to La Guerche et al. [7], geosmin originates from
the metabolism of P. expansum on grapes pre-contaminated by
B. cinerea; thus, B. cinerea would induce the production of
geosmin by P. expansum.
Little is known about the biosynthesis of geosmin by
P. expansum. Although several papers report about geosmin
production in Actinobacteria, certain Cyanobacteria, Myxobacteria and higher Fungi, little is known about the genes
implicated in geosmin biosynthesis [8]. As suggested by
Bentley and Meganathan [9], geosmin would be derived
from a sesquiterpenoid precursor and would be synthesized
from farnesyl pyrophosphate. The use of a geosmin overproducing strain of Streptomyces citreus producing also high
levels of the sesquiterpene alcohol germacra-1-E,5E-dien11-ol (germacradienol), indicated that this compound might
be a precursor of geosmin [10]. Later studies showed that
germacradienol production was indeed the committed step
in geosmin production [11]. According to Gust et al. [3]
presenting a study on Streptomyces, sesquiterpene synthase

would be involved in an early step in geosmin biosynthesis.
The functional characterization of six sesquiterpene synthases
in a basidiomycete called Coprinus cinereus has been described
[12]. As previously said, concerning P. expansum, few data are
available on geosmin synthesis in must or culture medium.
Dionigi [13] has studied the impact of copper sulphate addition
in Czapek medium on the biosynthesis of geosmin. Recently,
a cytochrome P450 monooxygenase gene, gpe1, which may
intervene during the transformation of farnesyl pyrophosphate
to geosmin, has been described [14].
Proteomics techniques are used more and more to unravel
proteins implicated in different pathways [15]. Here we report a
comparative proteomic study aimed at identifying differentially
expressed proteins in four P. expansum strains producing geosmin
in different amounts in culture medium. Some proteins putatively implicated in geosmin production were identified and their
possible implication in geosmin biosynthesis is discussed.

2.

Material and methods

2.1.

Cultivation of strains

The potential of geosmin production was assessed in triplicate on
fourteen Penicillium strains isolated in vineyards in the
luxembourgish part of the Moselle valley. Picking was done on
mature berries between 2007 and 2010. Cultures were conducted
on malt-agar medium in Petri dishes at 25 °C, based on initial

monoconidia production. The same conditions were applied for
the assessment of geosmin production and the proteomic studies:
50 mL of malt-peptone media were inoculated with 50 μL of the
conidia suspension (106 conidia/mL) in a 250 mL Erlenmeyer

flask, plugged with cotton and placed at 120 rpm, at room
temperature, during 3 days. Geosmin quantification was done in
triplicate and protein extraction was done in quadruplicate.

2.2.

DNA extraction

Pure isolates were sub-cultured in Potato Dextrose Broth (PDB)
during one week at 25 °C on a rotary shaker set at 120 rpm.
The subsequent biomass was lyophilised prior to grinding
with metallic beads. DNA was extracted with the DNeasy
Plant Mini Kit (Qiagen, Hilden, Germany), following instructions of the manufacturer. DNA concentration was quantified
with the NanoDrop (ND 1000, Thermo Scientific, Waltham,
MA). The samples were stored at − 20 °C.

2.3.

Molecular identification of strains

All the strains were identified with four sets of primers: β-tubulin
1 BT1 (a and b), β-tubulin 2 BT2 (a and b) [16], ITS1–ITS4 and ITS
U5–ITS R2 [17]. The PCR reaction mixture contained 10 μL of
Finnzymes Taq Phusion-buffer Mastermix HF (Thermo Scientific, Waltham, MA); 1 μL of each primer (10 μM); 1 μL of DNA
(100 ng/μL) and 7 μL of UltraPure™ DNase/RNase-free distilled

water (Invitrogen, Paisley, UK) for a final volume of 20 μL.
Amplification was performed on a Biometra T-professional
thermocycler (Biometra, Goettingen, Germany) using the following programme: an initial denaturation at 98 °C (2 min) followed
by 30 cycles with denaturation at 98 °C (15 s), annealing at 67 °C
(β-tubulin) or 63 °C (ITSU5–ITSR2) or 64 °C (ITS1–ITS4) during
20 s and elongation at 72 °C during 20 s (β-tubulin, ITS1–ITS4) or
10 s (ITSU5–ITSR2); the final elongation was performed at 72 °C
during 10 min. The size, quality and quantity of the amplicons
were checked on 3% w/v agarose gel stained with ethidium
bromide (1 h; 100 V) under UV transillumination.
PCR products were diluted in UltraPure™ DNase/RNasefree distilled water (Invitrogen, Paisley, UK) to reach a
concentration of 10–20 ng/μL. The sequencing PCR was
achieved with these dilutions using Big Dye products (Applied
Biosystems, Carlsbad, CA): 5 × sequencing buffer (2 μL), Big
Dye sequencing RR-100 (2 μL), primer 10 μM (0.32 μL), 1 μL of
the diluted PCR amplicons and 14.68 μL of UltraPure™ DNase/
RNase-free distilled water (Invitrogen, Paisley, UK) for a
total volume of 20 μL. Both strands of each amplicon were
sequenced. The PCR programme consisted in an initial
denaturation at 96 °C (1 min) followed by 25 cycles of denaturation at 96 °C (10 s) and elongation at 60 °C (4 min). The
PCR products were cleaned with the BigDye Xterminator
Purification kit (Applied Biosystems, Carlsbad, CA). Sequencing was done on the Applied Biosystems 3130 Genetic
Analyzer (Applied Biosystems).

2.4.

Geosmin measurement

Post culture geosmin abundance of fourteen strains was measured on sterile-filtered media (cellulose acetate membrane,
0.2 μm, Sartorius, Goettingen, Germany). When it was required,

the cultivation medium was diluted up to 50 fold with fresh
sterile malt-peptone broth. 2-Ethoxy-3-isopropylpyrazine (IPEP)
(TCI, Tokyo, Japan) was used as internal standard. Geosmin
standard [(±)-Geosmin solution, 100 μg/mL in methanol] was


JO U R N A L OF P ROTE O MI CS 9 6 ( 20 1 4 ) 1 3–2 8

obtained from Sigma-Aldrich (St Louis, MO). The determination
of geosmin was performed by GC–MS/MS using a Trace GC Ultra
coupled to a TSQ Quantum XLS tandem mass spectrometer
(Thermo Scientific, Waltham, MA). Geosmin was preconcentrated using Headspace Solid-Phase MicroExtraction
(HS-SPME) on a DVB/CAR/PDMS fibre (Supelco, SigmaAldrich, St. Louis, MO). The extraction was fully automated
using a PAL Combi-xt autosampler with the following
programme: incubation at 70 °C during 5 min, adsorption at
70 °C during 20 min and desorption in the GC injector during
2 min. Injection was done in PTV splitless mode running the
following programme: 56 °C during 0.05 min — ramp of
14.5 °C/s until 270 °C and hold at 270 °C for 17 min. The
column was an Rxi-5Sil MS (20 m ∗ 0.18 mm ∗ 0.18 μm,
Restek, Bellefonte, PA) and the GC oven was programmed as
follows: 50 °C during 2 min, ramp of 15 °C/min until 100 °C
and 30 °C/min until 250 °C (hold for 4.7 min). Retention times
of geosmin and IPEP were respectively 8.38 and 10.07 min. A
first, semi-quantitative approach, based on the calculation of
the geosmin to internal standard ratio, was used to select
four strains within the fourteen, i.e. P4, P8, P21 and P23
(Fig. 1). P4 and P8 were selected as low geosmin producers
whereas P21 and P23 were considered as high geosmin
producers. These four strains were identically resubmitted to

cultivation and geosmin assessment to obtain quantitative data
(using a calibration curve with geosmin standard). The case of P8
was ambiguous: its production during the second round was
much higher than during the first one.

2.5.

Protein extraction and quantification

Unless stated otherwise, all reagents used for extraction and
subsequent separation were purchased from GE Healthcare

15

(GE Healthcare, Little Chalfont, UK). Mycelia which were used
for protein extraction were the same than those produced for
geosmin assessment. Mycelia were removed from growing
medium and dried on cellulose acetate membrane. Afterwards mycelia were ground in liquid nitrogen and proteins
were precipitated by addition of ice cold TCA/Acetone/DTT
(20%/79.9%/0.1% v/v) overnight at − 20 °C. The mixture was
centrifuged at 30,000 g for 45 min at 4 °C. The precipitate was
washed three times with rinsing buffer (TCA 20%, acetone 80%
v/v) and dried in vacuo. Pellets were solubilised in 1 mL of
lysis buffer (Urea 7 M, Thiourea 2 M, CHAPS 4%, TRIS 30 mM
and proteases inhibitors) for about 30 mg of mycelium.
Quantification of the extracted proteins was achieved by the
Bradford method using a DU 800 spectrophotometer
(Beckman Coulter, Brea, CA).

2.6.

Labelling of proteins and 2D electrophoresis

Prior to labelling, the pH of each sample was checked and, if
necessary, adjusted to pH 8.5. 30 μg of proteins were labelled
and separated by 2D-DIGE as reported previously [18]. Briefly,
240 pmol of dyes were incubated with the proteins for 30 min
on ice in the dark. The reaction was then stopped by addition
of 1 μL of 10 mM lysine solution and incubation on ice for 10
additional minutes. Samples were labelled either with Cy3
or Cy5; the internal standard, constituted by an equal amount
of each sample, was labelled with Cy2. Dye swap was
performed by labelling, in each group, 2 biological replicates
with Cy3 and the other 2 biological replicates with Cy5. After
labelling, one Cy3 labelled sample was combined with one Cy5
labelled sample and with the Cy2 labelled standard: the
mixture was diluted to 450 mL with a solution containing
Urea 7 M, Thiourea 2 M, CHAPS 4%, TRIS 30 mM, 9 μL of

Fig. 1 – Preliminary semi-quantitative assessment of geosmin production of 14 strains of Penicillium expansum by GC–MS/MS.
Values represent the geosmin/internal standard ratio ± SD. n = 3 biological replicates.


16

JO U R N A L OF PR O TE O MI CS 96 ( 20 1 4 ) 1 3 –28

Bio-Lyte pH 3–10 ampholyte buffer (Bio-Rad, Hercules, CA)
and 2.7 μL of destreak reagent (GE Healthcare) and traces of
bromophenol blue. 24 cm pH 3–10 non-linear strips (Readystrip

IPG, BioRad) were passively rehydrated at room temperature
overnight and focused at 20 °C in an Ettan IPGphor III (GE
Healthcare) system until reaching approximately 100 kVh.
Following the first dimension, equilibration of the strips was
carried out firstly during 15 min in the equilibration solution
(provided by Serva, Heidelberg, Germany) containing 1% w/v
DTT and then in equilibration solution supplemented with 2.5%
w/v iodoacetamide solution (w/v) for 15 min. The second
dimension was obtained by a run on a 12.5% pre-cast
polyacrylamide gel (Serva, Heidelberg, Germany). Obtained
gels were scanned at a spatial resolution of 100 μm with a
9400 Typhoon (GE Healthcare) using the following wavelengths:
excitation at 488 nm, 532 nm, and 633 nm (Cy2, Cy3 and Cy5,
respectively) and emission at 520 nm, 610 nm and 670 nm (Cy2,
Cy3 and Cy5, respectively). Images of the gels were analysed by
DeCyder 2D Differential Analysis v.7.0 software (GE Healthcare).
Maps were calibrated, to obtain experimental molecular weight
and isoelectric point estimations, using the Fusarium reference
map which was produced earlier in our laboratory [19].
Highlighted proteins of interest (fold change ±1.3; t-test ≤0.05)
were picked, trypsin digested for 6 h at 37 °C and then spotted
on MALDI disposable targets by the Ettan Spot Handling
workstation (GE Healthcare). Identification of the proteins was
carried out using an AB SCIEX TOF/TOF 5800 System (AB SCIEX,
Framingham, MA). MS spectra were internally calibrated using
trypsin autocleavage signals. In MS/MS mode an external
calibration using fragmentation products of Glu-fibrinopeptide
was done. For MS, the recorded spectrum was the accumulation
of 1500 shots, for MS/MS this was 3000. Spectra were acquired
using an automated approach defined in the MALDI software

(TOF/TOF Series Explorer™ V4.1.0, AB Sciex); for each spot the 8
highest peaks in the raw MS spectrum were selected for
fragmentation after exclusion of common contaminants, for
instance peaks from trypsin autocleavage products or keratin.
Proteins were identified by searching with the MASCOT
algorithm version 2.3 (Matrix Science, www.matrixscience.
com, London, UK) against the NCBI database (updated to the
18th of January 2013, with 1,585,852 sequences belonging to
“Other Fungi”), using ProteinPilot™ Software version 4.0 (AB
SCIEX). Searches were carried out allowing a mass window of
100 ppm for the precursor and 0.5 Da for fragment ion masses.
The search parameters allowed maximum two missed cleavages; carbamidomethylation of cysteine as fixed modification;
oxidation of methionine and oxidation of tryptophan (single
oxidation, double oxidation and kynurenine) as variable modifications. Proteins with probability-based MOWSE scores
(p ≤ 0.05) were considered to be successfully identified.

3.

Results

3.1.

Identification of the strains

Consensus sequences have been produced and compared to
reference strains through BLAST. Each strain has been
successfully amplified and sequenced by each primer set.
They were all identified as P. expansum with an E value of 0

and 100% of identity. The amplicon sizes were 484 bp (BT1),

477 bp (BT2), 716 bp (ITS1–4) and 280 bp (ITS U5-R2) (Behr et
al., Journal International des Sciences de la Vigne et du Vin,
accepted manuscript).

3.2.

Geosmin production of the strains

In order to select the appropriate strains for the proteomic
investigations, fourteen isolates were submitted to a preliminary geosmin assessment (Fig. 1). Based on this screening,
four strains, namely P4, P8, P21 and P23, were selected. P4 and
P8 were supposed to be low geosmin producers, while P21 and
P23 exhibited a much higher production during the preliminary screening. During the second assessment (Fig. 2), a
similar geosmin production was measured with the exception
of P8, showing a production comparable to P21 and P23.
Malt-peptone broth was found to be a very efficient medium
for the induction of geosmin production. The determination
of geosmin content is made easier by the liquid form of
the medium, which allows a direct extraction by HS-SPME,
without the extraction step required by culture on solid media
[2]. As compared to similar studies realised on other medium
(grape juice or malt-agar), the production was much higher.
Indeed, Morales-Valle et al. [4] have described a maximum
concentration around 600 ng/L; La Guerche et al. [20] have
reported, under the same conditions, a maximum concentration of 500 ng/L, while we have reached an average concentration superior to 5000 ng/L for P23. Usually, concentrations
reached in wine are lower, generally around 100 ng/L, up to
300 ng/L [21,22]. Despite the first objective to select two
strains of each phenotype, we decided to keep P8 for the
proteomic study, since the examination of such a profile may
be interesting.

3.3.

Differentially expressed proteins

107 proteins were differentially expressed and were used
to do a Principal Component Analysis (PCA, Fig. 3) and a
Hierarchical Clustering (HC, Fig. 4). A typical gel with
differentially expressed proteins is shown (Fig. 5). Information
concerning the fold-change of the proteins is presented in
Table 1. In supplementary data S1, a summary of all relevant
information for each identified protein, such as possible

Fig. 2 – Quantitative assessment of geosmin production (ng/L
of media ± SD) for the strains used in the proteomic study.
n = 3 biological replicates.


JO U R N A L OF P ROTE O MI CS 9 6 ( 20 1 4 ) 1 3–2 8

involvements of the proteins in metabolism as suggested by
KEGG database is
reported. In supplementary data S2, detailed information
about the identification of the differentially expressed proteins, including peptide mass fingerprinting (PMF) and MS/MS
fragmentation data can be found.

3.3.1.

Clustering of the strains

Only the proteins which have exhibited a fold change of at
least ± 1.3 and a p value below 0.05 and resulted in a single
identification are presented in Table 1. Most of the proteins
were presenting multiple isoforms with sometimes different
behaviours in their relative fold-change. It is clear that the two
high geosmin producing strains P21 and P23 can be distinguished neither in the PCA nor in the HC, while the low
geosmin producer P4 is well separated from the other strains.
The strain P8 was not consistent in its production of geosmin,
so that we could not clearly put it in one of the two categories,
and this can be seen in the proteomic profile, since in the PCA
and in the HC this strain was separated from high producers
and the low producer. Hereafter, the classification of the
differentially regulated proteins into different functional
categories will be discussed.

3.3.2.

Proteins involved in metabolic processes

Several proteins involved in metabolic processes were found to
be differentially abundant between the four strains. Some of the
proteins are involved in carbohydrate catabolism, including
enolase and phosphoglycerate kinase (PGKA). Spots containing
these proteins did not show a uniform trend within the groups.
Several of them were more abundant in the strain P4 (3 isoforms
of phosphoglycerate kinases PGKA), while those containing
enolase were less abundant. Fourteen isoforms of enolases
were found; nine of them were found in their full length
(spots n° 1638, 1643, 1655, 1656, 1663, 1667,1674, 1680 and

17

1686; 43 kDa, pI from 4.89 to 5.20) while the others were found
to be degradation/processing products of enolase. Spot n°
2340 contains the original N-terminus, in all the spots (spots
n° 2767, 2768, 2800, 3174 and 4580) the original C-terminus
was identified. The peaks were extracted from the spectra
and used for classification with Speclust (p.
lu.se/speclust.html) [23]. With the peaks-in-common tool,
those peaks that were unique to each spectrum were isolated
and studied. Because the spectra were generally of low
intensity, no differences between the molecular forms at
the same molecular weight could be found nor could cleavage
sites resulting in the observed forms be discerned. The nine
isoforms presenting an intact form did not display significant
differences. Four degraded forms were more abundant in the
geosmin producing strains.
Importantly, acetyl-CoA C-acetyltransferase, the enzyme
which constitutes the beginning of secondary metabolism
starting from acetyl-CoA [24] was less abundant in P4.
This enzyme catalyses the transfer of an acetyl group into
acetyl-CoA, producing acetoacetyl-CoA. Acetyl-CoA is mainly
produced via pyruvate from the glycolysis, or by the β-oxidation
of the fatty acids. Acetoacetyl-CoA is the starting point for
the synthesis of farnesyl diphosphate, the common molecule
of pathways leading to sesquiterpenoid products, including
geosmin. An overview of the glycolytic, methylerythritol
phosphate (MEP) and mevalonate (MVA) pathways is shown
(Fig. 6). Expressions of the ATP synthase enzymes were also
changing from one isoform to the other: two were much more

abundant in P4 and six were less abundant. Other proteins
related to ATP synthesis were also differentially abundant: two
cytochrome C oxidases (subunit 5a) were less abundant in P4.
Cytochrome C oxidase is involved in the ATP production by the
mitochondrial respiratory chain. Two enzymes may be considered both as metabolic and linked to phytopathogenicity: serine
carboxypeptidase and proteinase A. They are involved in

Fig. 3 – PCA analysis of the strains (n = 4 biological replicates) used in proteomic studies. Left panel: score plot of spot maps.
Right panel: loading plot of proteins.


18

JO U R N A L OF PR O TE O MI CS 96 ( 20 1 4 ) 1 3 –28

Fig. 4 – Hierarchical clustering of four strains of Penicillium expansum. Red, yellow, green and blue dots represent P4, P8, P21 and
P23 respectively. Pearson correlation coefficient was used in order to achieve the clustering.

nitrogen metabolism and also in the lysis of molecules
encountered during infection of vegetal host cells (destructuration of cell wall, reaction to PR proteins) [25,26]. The three
spots corresponding to proteinase A were less abundant in P4,
as were two spots containing serine carboxypeptidases. In
contrast, three other spots containing serine carboxypeptidases were significantly more abundant in P4.

3.3.3.

Proteins involved in protein synthesis and folding

Enzymes involved in protein synthesis and folding were also
a major point of differentiation between the groups. One

protein involved in DNA transcription (a nucleic acid binding
protein) was more abundant in P4. Concerning protein
synthesis, ribosomal protein S2 (RPS2) and one translation
elongation factor containing a glutathione S-transferase

Fig. 5 – Representative gel of Penicillium expansum proteome. Whole protein extracts were labelled with CyDyes and separated
in first dimension by 24 cm 3–10 non-linear strips and in second dimension by 12.5% polyacrylamide precast gels. The
numbers of picked spots are reported. The presented image is the standard (Cy2 channel) of the gel which was used as master
gel in the experiment. Additional images, one representative of each experimental group, are presented in supplementary
material S3.


19

JO U R N A L OF P ROTE O MI CS 9 6 ( 20 1 4 ) 1 3–2 8

Table 1 – Differentially abundant proteins identified by MALDI-MS in the four Penicillium expansum strains. Theor.
MW/pI — theoretical molecular weight (expressed Da) and isoelectric point (expressed in pH units), Exp MW/pI — experimental
molecular weight (expressed in Da) and isoelectric point (expressed in pH units); P8/P4 column and followings: fold change relative to
the reported strains with the corresponding p-value are reported: positive values (reported in green) when the numerator is
up-regulated – negative values (reported in red) when the denominator is up-regulated.
UniProt
access.
No

Protein
Existance
(UniProt)

Spot

No

Theor.
MW/pI

Exp
MW/pI

Protein name /
function

799

63209/
4.52

66680/
4.53

Amidase family
protein

K9GF02

Predicted

1638

47250/
5.26

43255/
5.15

Enolase BAC82549

B6H602

1643

47250/
5.26

43322/
5.2 0

Enolase BAC82549

1655

47250/
5.26

47055/
4.95

1656

47250/
5.26

1663

P8/P4

P21/P4

P23/P4

P21/P8

P23/P8

P23/P21

–1.32;
0.014

1.04;
0.75

1.12;
0.053

1.38;
0.036

1.48;
0.0027

1.07;
0.43

Inferred
from
homology

1.00;
0.89

–1.19;
0.21

–1.51;
0.028

–1.19;
0.15

–1.52;
0.018

–1.27;
0.21

B6H602

Inferred
from
homology

1.00;
0.96

–1.21;
0.11

–1.43;
0.036

–1.22;
0.087

–1.43;
0.029

–1.18;
0.31

Enolase BAC82549

B6H602

Inferred
from
homology

1.32;
0.10

–1.26;
0.080

–1.29;
0.10

–1.66;
0.0036

–1.70;
0.0073

–1.02;
0.72

43054/
5.05

Enolase BAC82549

B6H602

Inferred
from
homology

1.04;
0.67

–1.29;

0.15

–1.74;
0.0018

–1.34;
0.10

–1.82;
0.00098

–1.35;
0.16

47250/
5.26

42392/
4.73

Enolase BAC82549

B6H602

Inferred
from
homology

1.28;
0.047

–1.12;
0.17

–1.08;
0.50

–1.44;
0.0036

–1.39;
0.044

1.04;
0.88

1667

47250/
5.26

42656/
5.30

Enolase BAC82549

B6H602

Inferred
from

homology

1.05;
0.42

–1.12;
0.28

–1.30;
0.017

–1.18;
0.11

–1.37;
0.0034

–1.15;
0.25

1674

47250/
5.26

42195/
4.86

Enolase BAC82549

B6H602

Inferred
from
homology

1.09;
0.31

–1.18;
0.16

–1.43;
0.035

–1.29;
0.057

–1.56;
0.016

–1.21;
0.26

1677

47250/
5.26

41869/

4.61

Enolase BAC82549

B6H602

Inferred
from
homology

1.52;
0.0047

1.09;
0.29

1.06;
0.51

–1.39;
0.0097

–1.43;
0.0 093

–1.03;
0.71

1680

47250/
5.26

42064/
4.83

Enolase BAC82549

B6H602

Inferred
from
homology

1.17;
0.12

–1.22;
0.084

–1.39;
0.024

–1.43;
0.0098

–1.63;
0.0043

–1.14;

0.31

1686

47250/
5.26

42260/
4.78

Enolase BAC82549

B6H602

Inferred
from
homology

1.20;
0.21

–1.20;
0.23

–1.47;
0.054

–1.44;
0.015

–1.77;
0.0072

–1.23;
0.18

1693

44107/
5.98

41869/
5.51

Phosphoglycerate
kinase pgkA

B6H903

Predicted

–1.12;
0.41

–1.50;
0.015

–1.47;
0.029

–1.34;
0.076

–1.31;
0.12

1.02;
0.93

2340

47250/
5.26

30182/
4.81

Enolase BAC82549

B6H602

Inferred
from
homology

1.46;
0.0096

1.72;
0.00080

1.62;
0.045

1.18;
0.15

1.11;
0.72

–1.06;
0.61

2361

34281/
5.44

29948/
4.99

B6GYI7

Inferred
from
homology

1.06;
0.54

1.50;
0.018

1.34;
0.11

1.41;
0.033

1.26;
0.20

–1.12;
0.50

2497

35821/
8.44

28366/
5.73

Malate
dehydrogenase

B6HDG8

Inferred
from

homology

–1.05;
0.60

1.22;
0.088

1.24;
0.022

1.29;
0.083

1.31;
0.039

1.01;
0.82

2540

35821/
8.44

27202/
5.43

Malate
dehydrogenase

B6HDG8

Inferred
from
homology

–1.43;
0.058

1.32;
0.10

1.23;
0.17

1.88;
0.027

1.76;
0.056

–1.07;
0.76

2767

47250/
5.26

23768/
4.53

Enolase BAC82549

B6H602

Inferred
from
homology

1.41;
0.024

1.45;
0.045

1.53;
0.0042

1.02;
0.95

1.08;
0.54

1.06;
0.65

2768

47250/
5.26

23731/
4.58

Enolase BAC82549

B6H602

Inferred
from
homology

1.22;
0.16

1.42;
0.0023

1.40;
0.00023

1.16;
0.23

1.15;
0.21

–1.02;
0.86

2769

41175/
5.93

23548/
4.65

Acetyl–CoA C–
acetyltransferase

B6HV94

Inferred
from
homology

1.16;
0.43

1.41;
0.011

1.49;
0.00089

1.22;

0.22

1.29;
0.096

1.06;
0.47

2787

44107/
5.98

23621/
7.33

Phosphoglycerate
kinase

B6H903

Inferred
from
homology

–1.07;
0.66

–1.96;
0.012

–2.07;
0.0034

–1.83;
0.023

–1.93;
0.0078

–1.06;
0.86

2794

44107/
5.98

23548/
7.20

Phosphoglycerate
kinase

B6H903

Inferred
from
homology

–1.05;
0.78

–1.72;
0.020

–1.71;
0.019

–1.64;
0.017

–1.63;
0.016

1.01;
0.97

2800

47250/
5.26

23402/
4.49

Enolase BAC82549

B6H602

Inferred
from
homology

–1.64;
0.058

2.41;
0.00071

2.04;
0.029

3.95;
0.0012

3.34;
0.0080

–1.18;
0.41

2812

27620/
5.45

23221/
4.75

Triosephosphate
isomerase

K9GAV3

Inferred
from
homology

–1.02;
0.74

1.46;
0.024

1.47;
0.0024

1.50;
0.20

1.50;
0.049

1.00;
0.88

Glucose / TCA cycle metabolism

Glyoxysomal and

mitochondrial malate
dehydrogenase


20

JO U R N A L OF PR O TE O MI CS 96 ( 20 1 4 ) 1 3 –28

Table 1 (continued)
Spot
No

Theor.
MW/pI

Exp
MW/pI

Protein name /
function

UniProt
access.
No

Protein
Existance
(UniProt)

P8/P4

P21/P4

P23/P4

P21/P8

P23/P8

P23/P21

2917

44107/
5.98

22165/
7.22

Phosphoglycerate
kinase

B6H903

Inferred
from
homology

1.03;
0.73

–1.62;
0.020

–1.48;
0.066

–1.68;
0.0031

–1.53;
0.022

1.10;
0.65

3131

44142/
6.08

19823/
6.61

Phosphoglycerate
kinase

K9H8W2

Inferred

from
homology

–1.34;
0.064

–1.56;
0.010

–1.68;
0.0056

–1.17;
0.40

–1.26;
0.23

–1.08;
0.65

3174

47250/
5.26

19307/
4.53

Enolase BAC82549

B6H602

Inferred
from
homology

1.27;
0.25

–1.28;
0.12

–1.24;
0.082

–1.63;
0.041

–1.58;
0.031

1.03;
0.72

4042

35821/
8.44

12389/
5.94

Malate
dehydrogenase

B6HDG8

Inferred
from
homology

–1.26;
0.061

1.17;
0.040

1.14;
0.46

1.48;
0.0096

1.44;
0.073

–1.02;
0.76

4318

36149/
6.23

10642/
4.40

Glyceraldehyde–3–
phosphate
dehydrogenase

B6HI59

Inferred
from
homology

1.40;
0.020

1.62;
0.00053

1.59;
0.0056

1.16;
0.23

1.13;
0.40

–1.02;
0.82

4580

47250/
5.26

9269/4
.95

Enolase BAC82549

B6H602

Inferred
from
homology

1.32;
0.0010

1.52;
0.00022

1.79;
1.2e–006

1.15;
0.047

1.36;
0.00016

1.18;
0.010

4703

35821/
8.44

28944/
5.68

Malate
dehydrogenase

B6HDG8

Inferred
from
homology

–1.05;
0.64

1.92;
0.0042

1.65;
0.0027

2.02;
0.0070

1.74;
0.0077

–1.16;
0.41

ATP synthesis
382

69037/
4.47

83112/
4.51

Lysophospholipase1

K9G2P1

Predicted

–1.21;
0.25

2.93;
8.9e–005

3.17;
2.9e–005

3.56;
0.00036

3.85;
0.00020

1.08;
0.50

1335

55237/
5.25

50357/
4.25

F0F1 ATP synthase
subunit beta

B6HI25

Inferred
from
homology

–8.15;
3.7e–006

–7.52;
1.1e–006

–7.94;
7.2e–005

1.08;
0.41

1.03;
0.95

–1.06;
0.63

1437

55237/
5.25

47770/
4.28

F0F1 ATP synthase
subunit beta

B6HI25

Inferred
from
homology

–5.00;
0.00021

–2.75;
0.0023

–3.08;
0.0061

1.82;
0.022

1.62;
0.19

–1.12;
0.54

1525

55237/
5.25

46311/
4.74

F0F1 ATP synthase
subunit beta

B6HI25

Inferred
from
homology

1.33;
0.053

1.37;
0.036

1.36;
0.0072

1.03;
0.85

1.02;
0.78

–1.01;
0.97

1561

55237/
5.25

44274/
4.83

F0F1 ATP synthase
subunit beta

B6HI25

Inferred
from
homology

1.41;
0.00026

1.09;
0.56

–1.03;
0.65

–1.29;

0.056

–1.45;
0.0042

–1.13;
0.45

2254

53763/
5.44

31668/
4.66

F0F1 ATP synthase
subunit beta

Q0CFC5

Inferred
from
homology

1.60;
0.0014

1.25;
0.013

1.18;
0.25

–1.28;
0.017

–1.35;
0.046

–1.06;
0.54

2257

55237/
5.44

31718/
4.72

F0F1 ATP synthase
subunit beta

B6HI25

Inferred
from
homology

1.42;
0.00044

1.24;
0.14

1.14;
0.26

–1.15;
0.27

–1.25;
0.071

–1.09;
0.61

2976

55237/
5.26

21455/
5.27

F0F1 ATP synthase
subunit beta

B6HI25

Inferred
from
homology

–1.32;
0.013

–1.13;
0.20

–1.09;
0.34

1.17;
0.11

1.22;
0.044

1.04;
0.63

3910

55237/
5.25

13162/
4.49

F0F1 ATP synthase
subunit beta

B6HI25

Inferred
from
homology

1.46;
0.051

1.39;
0.021

1.46;
0.0055

–1.05;
0.72

1.00;
0.93

1.05;
0.50

3919

16650/
8.44

13020/
4.40

F0F1 ATP synthase
subunit beta

B6HSK3

Inferred
from
homology

2.99;
6.3e–007

–1.06;
0.15

1.02;
0.98

–3.18;
3.1e–007

–2.94;
0.00011

1.08;
0.64

3924

17127/
6.73

13020/
6.10

Ribose 5–phosphate
isomerase B

B6HSH9

Predicted

–2.27;
0.0039

–1.85;
0.013

–2.26;
0.0034

1.23;
0.36

1.01;
0.97

–1.22;
0.36

4169

17966/
6.17

11662/
4.35

Cytochrome c oxidase
subunit 5a

B6HI83

Predicted

1.73;
0.00023

1.16;
0.071

1.32;
0.0052

–1.48;
0.0014

–1.30;
0.012

1.14;
0.14

4177

17966/
6.17

11411/
4.41

Cytochrome c oxidase
subunit 5a

B6HI83

Predicted

1.43;
0.018

1.36;
0.021

1.27;
0.12

–1.06;
0.64

–1.13;
0.37

–1.07;
0.55


21

JO U R N A L OF P ROTE O MI CS 9 6 ( 20 1 4 ) 1 3–2 8

Table 1 (continued)
Spot
No

Theor.
MW/pI

Exp
MW/pI

Protein name /
function

UniProt
access.
No

Protein
Existance
(UniProt)

P8/P4

P21/P4

P23/P4

P21/P8

P23/P8

P23/P21

Protein metabolism

509

67583/
5.08

77749/
4.87

Serine
carboxypeptidase

B6HNT3

Predicted

–2.09;
0.00011

–1.35;
0.047

–1.38;
0.019

1.55;
0.020

1.51;
0.012

–1.02;
0.91

512

67583/
5.08

77388/
4.83

Serine
carboxypeptidase

B6HNT3

Predicted

–2.26;
0.0014

–1.52;
0.015

–1.45;
0.013

1.49;
0.032

1.55;
0.014

1.04;
0.66

524

67583/
5.08

76197/
4.79

Serine
carboxypeptidase

B6HNT3

Predicted

–1.90;
0.0057

–1.23;
0.23

–1.23;
0.24

1.54;
0.0065

1.54;
0.012

1.00;
0.97

983

67583/
5.08

60005/
4.06

Serine
carboxypeptidase

B6HNT3

Predicted

–1.09;
0.44

1.34;
0.027

1.38;
0.0077

1.46;
0.0083

1.50;
0.0020

1.03;
0.70

1189

56761/
4.55

54250/
4.47

Protein Disulfide
Isomerase (PDIa)

K9GF84

Inferred
from
homology

–1.39;
0.050

–1.00;
0.94

–1.13;
0.41

1.39;
0.018

1.23;
0.064

–1.13;
0.24

1539

61928/
4.99

45598/
4.56

Carboxypeptidase Y,
putative

K9GGQ9

Predicted

1.54;
0.0085

1.57;
0.0086

1.56;
0.0078

1.02;
0.87

1.01;
0.91

–1.01;
0.95

1555

40215/
4.53

45316/
4.50

UV excision repair
protein RAD23

B6HH40

Predicted

–1.01;
0.94

1.38;
0.027

1.44;
0.016

1.40;
0.029

1.45;
0.017

1.04;
0.59

1900

24487/
5.24

37502/
4.68

hypothetical protein
PDIP_62460

K9FL12

Predicted

1.45;
0.0082

1.23;
0.11

1.28;
0.011

–1.18;
0.23

–1.13;
0.28

1.04;
0.65

1927

32517/
4.79

37155/
4.60

Ribosomal protein S2
(RPS2)

B6H2I7

Inferred
from
homology

1.44;
0.012

1.66;
0.0049

1.77;
0.00045

1.16;
0.23

1.23;
0.021

1.06;
0.44

1984

28352/
5.79

35853/
5.78

Nucleic acid binding
protein

B6HQE9

Predicted

–1.20;
0.26

–1.56;
0.068

–1.87;
0.0035

–1.30;
0.23

–1.56;
0.024

–1.20;
0.55

2104

43589/
5.08

34169/
4.48

Proteinase A –
saccharopepsin

B6H445

Inferred
from
homology

–1.43;
0.13

1.32;
0.18

1.51;
0.052

1.90;
0.0019

2.17;
9.3e–005

1.14;
0.30

2702

25049/
4.44

24785/
4.38

Elongation factor, C –
terminal, alpha helical
domain of the GST
family

B6H4G8

Inferred
from
homology

2.00;
0.10

3.35;
0.0040

3.51;
0.0025

1.68;

0.098

1.75;
0.066

1.05;
0.76

2879

43589/
5.08

22617/
4.53

Proteinase A –
saccharopepsin

B6H445

Inferred
from
homology

1.77;
0.00049

1.58;
0.00077

1.48;
0.0031

–1.12;
0.25

–1.20;
0.11

–1.07;
0.47

3247

43589/
5.08

18429/
4.45

Proteinase A –
saccharopepsin

B6H445

Inferred
from
homology

1.80;
0.022

1.62;
0.0057

1.86;
0.00071

–1.11;
0.69

1.03;
0.72

1.15;
0.23

4706

78775/
5.58

16975/
6.56

Oligopeptidase

B6HF99

Predicted

1.10;
0.45

–1.27;
0.11

–1.50;
0.0098

–1.39;
0.016

–1.65;
0.00094

–1.19;
0.065

Protein folding

940

69692/
5.03

62378/
5.02

Heat shock 70kDa
protein 1/8

B6HPY0

Inferred
from
homology

1.03;
0.69

1.41;
0.024

1.36;
0.036

1.36;
0.024

1.32;
0.038

–1.03;
0.81

1302

61986/

5.61

51144/
4.84

Chaperonin HSP60

B6H9L7

Inferred
from
homology

1.40;
0.021

1.33;
0.071

1.54;
0.0095

–1.05;
0.61

1.10;
0.29

1.15;
0.22

2355

66776/
5.22

29948/
4.66

Heat shock protein
70

F2SIL3

Inferred
from
homology

1.58;
0.0072

1.96;
0.00035

2.27;
0.042

1.24;
0.087

1.44;
0.38

1.16;
0.88

2683

67030/
5.32

24978/
4.46

Heat shock 70kDa
protein 1/8

B6HVA2

Inferred
from
homology

1.65;
0.011

1.75;
0.0012

1.76;

0.0043

1.06;
0.56

1.06;
0.66

1.00;
0.95

(continued on next page)


22

JO U R N A L OF PR O TE O MI CS 96 ( 20 1 4 ) 1 3 –28

Table 1 (continued)
Spot
No

Theor.
MW/pI

Exp
MW/pI

Protein name /
function

UniProt
access.
No

Protein
Existance
(UniProt)

P8/P4

P21/P4

P23/P4

P21/P8

P23/P8

P23/P21

2888

73741/
4.81

22407/
4.66

Heat shock 70kDa

protein 5

B6H0S5

Inferred
from
homology

1.42;
0.0096

–1.09;
0.16

–1.06;
0.34

–1.54;
0.014

–1.51;
0.0097

1.02;
0.79

2901

22046/
4.50

22303/
4.35

Hsps_p23–like protein

B6HKR2

Predicted

1.55;
0.023

1.22;
0.11

1.52;
0.044

–1.27;
0.080

–1.02;
0.86

1.25;
0.17

3319

67030/
5.32

18005/
5.10

Heat shock 70kDa
protein 1/8

B6HVA2

Inferred
from
homology

–1.04;
0.89

2.17;
0.0070

1.95;
0.032

2.27;
0.0028

2.04;
0.018

–1.11;
0.56

3367

19425/
5.89

17482/
5.21

Peptidyl–prolyl cis–
trans isomerase
NIMA–interacting 1
Rotamase

B6H262

Predicted

–1.49;
0.052

–1.36;
0.066

–1.58;
0.036

1.09;

0.55

–1.06;
0.75

–1.16;
0.35

3430

69862/
5.04

16870/
5.05

Heat shock 70 kDa
protein

K9H0L4

–1.06;
0.46

1.43;
0.0070

1.52;
0.00024

1.52;
0.0050

1.61;
0.00037

1.06;
0.35

3673

18109/
6.91

14763/
6.85

Peptidyl–prolyl cis–
trans isomerase

B6HAJ7

Inferred
from
homology

–1.32;
0.034

–1.36;

0.018

–1.68;
0.0026

–1.03;
0.76

–1.28;
0.059

–1.23;
0.074

4605

17531/
6.43

9084/4
.87

Chaperonin, putative

K9GM71

Inferred
from
homology

1.65;
0.029

1.62;
0.00096

2.08;
0.00061

–1.02;
0.94

1.26;
0.24

1.28;
0.12

Inferred
from
homology

Redox homeostasis
Inferred
from
homology
Inferred
from
homology
Inferred

from
homology

1.02;
0.89

1.57;
0.0055

1.61;
0.00099

1.54;
0.012

1.58;
0.0035

1.03;
0.74

–1.02;
0.89

1.45;
0.018

1.32;
0.095

1.48;
0.0087

1.35;
0.063

–1.10;
0.49

1.04;
0.64

1.43;
0.020

1.35;
0.044

1.37;
0.011

1.29;
0.032

–1.06;
0.57

Q4WPF8

Inferred

from
homology

–1.13;
0.34

1.23;
0.027

1.11;
0.22

1.40;
0.035

1.25;
0.25

–1.12;
0.27

Superoxide
dismutase

K9G7B6

Inferred
from
homology

–1.57;
0.092

–2.48;
0.0032

–1.88;
0.012

–1.57;
0.18

–1.19;
0.65

1.32;
0.22

14924/
5.34

Peroxiredoxin 5,
atypical 2–Cys
peroxiredoxin

K9GMX2

Predicted

–2.22;

0.00058

–2.07;
0.00037

–2.25;
6.9e–005

1.07;
0.61

–1.02;
0.98

–1.09;
0.50

10126/
4.77

Thioredoxin

K9F7X4

Inferred
from
homology

–2.64;
0.022

–3.50;
0.0024

–4.36;
0.00085

–1.33;
0.47

–1.65;
0.17

–1.24;
0.26

800

79912/
5.37

66165/
5.54

Catalase

B6H9T9

812

79912/
5.37

66992/
5.76

Catalase

B6H9T9

819

79912/
5.37

66784/
5.88

Catalase

B6H9T9

2407

40468/
8.64

29124/
5.13

Cytochrome c
peroxidase,
mitochondrial

3051

31795/
5.64

20767/
6.37

3665

19342/
8.67

4412

12078/
5.31

Cell cycle/cell signaling

289

60397/
4.9

86936/

4.46

Pleckstrin homology–
like domain protein

B6HV58

Predicted

–1.52;
0.0051

–1.30;
0.084

–1.35;
0.15

1.17;
0.32

1.13;
0.82

–1.04;
0.72

306

60397/

4.9

86131/
4.53

Pleckstrin homology–
like domain protein

B6HV58

Predicted

–1.64;
0.0029

–1.60;
0.012

–1.89;
0.0020

1.02;
0.94

–1.15;
0.21

–1.18;
0.30

307

60397/
4.9

85997/
4.67

Pleckstrin homology–
like domain protein

B6HV58

Predicted

–1.57;
0.0097

–1.69;
0.018

–2.01;
0.018

–1.08;
0.50

–1.28;
0.19

–1.19;
0.40

309

60397/
4.9

86131/
4.59

Pleckstrin homology–
like domain protein

B6HV58

Predicted

–1.76;
0.0013

–2.56;
0.0063

–3.16;
0.0022

–1.45;
0.095

–1.80;
0.025

–1.24;
0.54

314

60397/
4.9

85864/
4.63

Pleckstrin homology–
like domain protein

B6HV58

Predicted

–1.66;
0.0012

–1.98;
0.0087

–2.58;
0.0015

–1.19;
0.26

–1.55;
0.029

–1.30;
0.32


23

JO U R N A L OF P ROTE O MI CS 9 6 ( 20 1 4 ) 1 3–2 8

Table 1 (continued)
Spot
No

Theor.
MW/pI

Exp
MW/pI

Protein name /
function

UniProt
access.
No

Protein
Existance
(UniProt)

P8/P4

P21/P4

P23/P4

P21/P8

P23/P8

P23/P21

319

60397/
4.9

85598/
4.56

Pleckstrin homology–
like domain protein

B6HV58

Predicted

–1.62;
0.0041

–2.27;
0.0042

–3.08;
8.2e–005

–1.40;
0.093

–1.90;
0.0014

–1.36;
0.22

497

60397/
4.9

77990/
4.66

Pleckstrin homology–
like domain protein

B6HV58

Predicted

–1.34;
0.059

–1.51;
0.044

–1.79;
0.0050

–1.13;
0.35

–1.34;
0.030

–1.18;
0.39

505

60397/
4.9

76909/
4.74

Pleckstrin homology–
like domain protein

B6HV 58

Predicted

–1.81;
0.0038

–1.35;
0.056

–1.51;
0.027

1.34;
0.074

1.20;
0.28

–1.12;
0.47

582

60397/
4.9

74444/
4.58

Pleckstrin homology–
like domain protein

B6HV58

Predicted

–1.65;
0.0013

–1.92;
0.012

–2.13;
0.0034

–1.17;
0.31

–1.29;
0.10

–1.11;
0.70

595

56106/
4.88

74560/
4.62

Immunogenic protein

A2Q9H3

Predicted

–1.54;
0.0080

–1.68;
0.016

–2.09;
0.00033

–1.09;
0.57

–1.36;
0.057

–1.25;
0.32

1017

41394/
4.75

59173/
4.39

CDC/Septin GTPase
family protein

K9H369

Predicted

–1.46;
0.053

1.13;
0.39

1.11;
0.29

1.66;
0.020

1.63;
0.011

–1.02;
0.95

1029

41394/
4.75

59081/
4.42

CDC/Septin GTPase
family protein

K9H369

Predicted

–1.57;
0.0085

1.11;
0.40

1.07;
0.26

1.73;
0.0063

1.68;
0.0021

–1.03;
0.83

1044

41394/
4.75

58443/
4.45

CDC/Septin GTPase
family protein

K9H369

Predicted

–1.75;
0.0017

1.09;
0.40

1.07,
0.30

1.92;
0.0026

1.88;
0.0013

–1.02;
0.89

1045

41394/
4.75

58172/
4.48

CDC/Septin GTPase
family protein

K9H369

Predicted

–1.40;
0.028

1.15;
0.27

1.12;
0.17

1.62;
0.011

1.56;
0.0035

–1.03;
0.82

1786

32603/
4.94

39472/
4.44

Chitosanase of
glycosyl hydrolase

B6GXD4

Predicted

2.08;
0.087

6.49;
5.2e–005

7.28;
2.9e–005

3.11;
0.0066

3.49;
0.0040

1.12;
0.46

1892

32603/
4.94

37386/
4.44

Chitosanase of
glycosyl hydrolase

B6GXD4

Predicted

1.01;
0.88

1.65;
0.056

2.18;
0.0092

1.63;
0.088

2.15;
0.022

1.32;
0.067

2027

32603/
4.94

35192/
4.44

Chitosanase of
glycosyl hydrolase

B6GXD4

Predicted

–1.01;
0.97

1.46;
0.025

1.68;
0.0032

1.47;
0.021

1.69;
0.0025

1.15;
0.24

2822

25180/
8.00

23330/
6.69

26S proteasome non–
ATPase regulatory
subunit Nas2,
putative

K9FWG5

Predicted

–1.29;
0.0036

–1.46;
0.0054

–1.57;
0.00019

–1.13;
0.20

–1.22;
0.015

–1.07;
0.52

3080

20138/

4.65

20258/
4.42

Uncharacterized
protein

K9G5T5

Predicted

1.84;
0.062

1.81;
0.014

1.87;
0.0013

–1.01;
0.93

1.02;
0.77

1.03;
0.78

3138

20138/
4.65

19670/
4.42

Uncharacterized
protein

K9G5T5

Predicted

1.98;
0.013

1.87;
0.0015

2.14;
9.0e–005

–1.06;
0.91

1.09;
0.50

1.15;
0.16

3143

20138/
4.65

19609/
4.46

Uncharacterized
protein

K9G5T5

Predicted

1.78;
0.016

2.39;
0.0027

2.82;
0.0017

1.35;
0.19

1.59;
0.073

1.18;
0.48

3236

20138/
4.65

18863/
4.42

Uncharacterized
protein

K9G5T5

Predicted

2.00;
0.00089

1.78;
0.0046

2.00;
0.00040

–1.12;
0.31

–1.00;
0.95

1.12;
0.23

3492

19032/
6.85

16228/
4.39

Mismatched base
pair and cruciform
DNA recognition
protein, putative

K9GE01

Predicted

–1.13;
0.40

1.43;

0.015

1.48;
0.0010

1.62;
0.038

1.67;
0.020

1.03;
0.71

1.89;
0.018

2.00;
0.0056

1.49;
0.10

1.58;
0.043

1.06;
0.70

Secondary metabolites biosyntesis

2805

25629
/5.72

23257/
5.76

isoepoxydon
dehydrogenase

A1XDS5

Inferred
from
homology

1.27;
0.30

(continued on next page)


24

JO U R N A L OF PR O TE O MI CS 96 ( 20 1 4 ) 1 3 –28

Table 1 (continued)
Spot

No

Theor.
MW/pI

Exp
MW/pI

Protein name /
function

UniProt
access.
No

Protein
Existance
(UniProt)

P8/P4

P21/P4

P23/P4

P21/P8

P23/P8

P23/P21

Unknown function

1395

24487/
5.24

48442/
4.47

hypothetical protein
PDIP_62460

K9FL12

Predicted

–1.05;
0.76

1.46;
0.041

1.40;
0.017

1.53;
0.046

1.47;
0.028

–1.04;
0.82

1414

24487/
5.24

47622/
4.5

hypothetical protein
PDIP_62460

K9FL12

Predicted

–1.05;
0.64

1.64;
0.010

1.63;
0.00023

1.72;
0.030

1.71;
0.012

–1.00;
0.92

3137

20550/
9.17

/7.19

Conserved
hypothetical protein

B6H779

Predicted

–1.79;
0.039

–6.19;
0.00027

–6.58;

1.4e–006

–3.46;
0.011

–3.69;
0.0028

–1.06;
0.97

3365

20550/
9.17

17267/
7.43

Conserved
hypothetical protein

B6H779

Predicted

–1.92;
0.019

–4.48;

2.0e–005

–4.73;
0.00012

–2.33;
0.014

–2.46;
0.017

–1.06;
0.69

3722

16830/
5.80

14446/
5.04

Uncharacterized
protein

K9GK67

Predicted

1.25;

0.040

–1.13;
0.46

–1.26;
0.094

–1.41;
0.079

–1.58;
0.00078

–1.11;
0.76

4060

13188/
5.64

12331/
4.87

YjgF_YER057c_UK1
14 (unknown
function)

B6HKJ1

Predicted

1.52;
0.015

1.23;
0.0055

1.38;
0.00011

–1.24;
0.17

–1.10;
0.55

1.13;
0.062

4094

12308/
4.95

12123/
4.63

RNA binding protein,

putative

K9FZG7

Predicted

–1.09;
0.47

–1.56;
0.017

–1.70;
0.00032

–1.42;
0.073

–1.55;
0.0092

–1.09;
0.63

domain were less abundant in P4. Six enzymes playing a role in
protein folding were found: several HSPs 70 (chaperonin), a HSP
60 (chaperonin) and a HSP-p23-like (co-chaperonin of HSP 90
involved in protein folding and degradation) were less abundant
in P4 on one side, and a rotamase containing a peptidylprolyl
isomerase (PPIase) domain was more abundant on the other side.

HSPs 60 are ATP-dependent chaperones exclusively located in
the mitochondria. They are involved in the folding of the proteins
of this organelle, as well as in the maintaining of mitochondria
nuclei [27]; moreover these proteins are also involved in the
protection of proteins containing a Fe–S cluster against reactive
oxygen species (ROS). HSPs 70 are one of the main components
of the protein folding machinery, they aid in forming the native
conformation of proteins [26] but are likewise involved in
proteasomal degradation [28]. They are found in cytosol,
endoplasmic reticulum and mitochondria and are activated by
ATP or by nucleotides through the nucleotide exchange factors
(NEFs), the nature of which depends on the subcellular location
of the HSPs 70.

3.3.4. Proteins involved in cell cycle and cell signalling
pathways
Some proteins relative to cell cycle and cell signalling pathways
were found to be differentially abundant in the investigated
strains. Nine spots corresponding to a pleckstrin homology-like
(PH) domain containing protein were found. These spots were all
more abundant in P4. According to Fugelstad et al. [29], such
proteins may bind F-actin and play a role in the regulation of the
cell wall biosynthesis. PH-like containing proteins are also
involved in cell communication and host protein targeting [30].
Concerning cell cycle, and more specifically cell division, a
spot corresponding to a chitosanase, an enzyme which might

hydrolyse chitosan of the cell wall during division, was found to
be less abundant in P4. Liu et al. [31] reported that chitosanase
reduces the phytopathogenicity of Fusarium solani. Indeed, the

decrease of the disease index has confirmed the lower
relative expression level of CSN1, the gene responsible for
the chitosanase synthesis. The reasons why fungi produce an
enzyme which might disfavour themselves remain elusive.

3.3.5.

Redox homeostasis

Proteins involved in the regulation of redox homeostasis were
detected and significantly differed from one group to the
other. Three spots corresponding to catalase were significantly less abundant in P4, while three others corresponding
to peroxiredoxin 5, thioredoxin and superoxide dismutase
were more abundant. Catalases were presumably associated
with Cat 1 of Aspergillus fumigatus (80% of identity with
XP_748550.1). This large-size subunit catalase is highly produced in conidia, i.e. during germination and initiation of
growth [32]. For phytopathogenic fungi, these developmental
stages mainly occur during the infection of the host. A higher
catalase activity is necessary to counteract the reaction of
the plant. As for peroxiredoxins, they are activated by low
concentrations of peroxides, which oxidise the active-site
cysteine to sulfenic acid [33].

3.3.6. Unknown hypothetical proteins and proteins with
unknown functions
Four spots containing unknown hypothetical proteins (spots
n° 1395, 1414, 3137 and 3365) were differentially abundant.
They were presenting high levels of homology with hypothetical proteins of Aspergillus and Penicillium species. No
conserved domains were found by BLAST algorithm. The

JO U R N A L OF P ROTE O MI CS 9 6 ( 20 1 4 ) 1 3–2 8

25

Fig. 6 – Glycolytic, mevalonate and non-mevalonate pathways leading to the production of geosmin. Green and red arrows
represent respectively enzymes that are more or less abundant in geosmin producing strains (P8, P21 and P23). Names of
differentially abundant proteins are indicated. PEP: phosphoenolpyruvate. MEP: methylerythritol phosphate pathway. MVA:
mevalonate pathway.

available knowledge on these proteins is too low to allow us
even to speculate on their potential role in the biosynthesis
of geosmin.
Three additional proteins were found (spots n° 3722, 4060
and 4094) but their roles are not yet really understood. Spot n°
4060 was less abundant in P4 whereas spot n° 4094 was more
abundant in P4 as compared to the second group.

4.

Discussion

In the perspective of linking geosmin biosynthesis to the
previously described results, several points have to be taken
into account. Geosmin is a molecule of the sesquiterpenoid
family, which is part of the secondary metabolism encountered in fungi. The synthesis of such molecules is highly
energy-consuming and thus requires high quantities of
energy storage molecules such as ATP or NADPH. Production
of ATP via oxidative phosphorylation leads to the formation
of ROS, susceptible to damage cellular organelles and

biological molecules, which is generally counteracted by
detoxifying enzymes, such as catalase and peroxiredoxins
deputed to keep the redox homeostasis. Regulation of fungal
secondary metabolism is fine-tuned by several external
factors, including pH, temperature and nutrition [34], thus it
may be hypothesized that geosmin production has a high
impact on the behaviour of the cell regarding redox homeostasis. Changes in secondary metabolism in general, and in

geosmin production particularly, can be considered as both a
cause and a consequence of oxidative stress. As the culture
conditions were the same for all the isolates, the achieved
results can be considered as intrinsic and constitutive
differences between the strains.
Investigation on the enzymes of the glycolytic pathway
(Fig. 6) has revealed in P4 an increased abundance of
phosphoglycerate kinase, one of the flux-determining enzymes
regulating the glycolysis, suggesting that the ADP/ATP ratio is in
favour of ATP production. Considering that spots containing the
full length chain of enolase were less abundant in P4, we may
consider that phosphoglycerate produced by PGK is dispatched
to other pathways than glycolysis, i.e. production of ribulose
biphosphate or glyoxylate. As a result, production of acetyl-CoA
by the glycolytic pathway may be lower in P4. Acetyl CoA is the
first molecule of the MVA pathway, which leads to the
biosynthesis of isopentenyl diphosphate (IPP), a precursor of
farnesyl diphosphate (FPP). Finally, FPP is the substrate for
sesquiterpenoid production, including geosmin. It must be
noted that pyruvate can also be transformed into IPP via the
methylerythritol phosphate pathway (MEP). The pool of pyruvate may be higher in geosmin producing strains due to the
higher enolase abundance.

Enolase was identified in several spots at a molecular weight
far below the expected for the intact polypeptide chain,
suggesting degradation or processing of the protein as was
previously postulated for enolase in Penicillium chrysogenum [35]
and in more distantly related fungi [36]. Different and


26

JO U R N A L OF PR O TE O MI CS 96 ( 20 1 4 ) 1 3 –28

contradictory hypotheses have been proposed for the observed
degradation. Incubation with PMSF indicated that the fragmentation could be related to proteasome activity [37], even
proposing enolase, a proteasome interacting protein, as substrate or regulator of proteasome activity. In an older study a
contradictory result was obtained. Larsen et al. [38] did a
detailed mapping of the different spots and found a specific
cleavage site in some instances. However the proposed cleavage
sequence K/RxA only appears infrequently in the P. chrysogenum
protein on which our identification is based, and will at best
only explain part of the observed fragmentation, furthermore
no known protease with this specificity was found. Despite the
fact that the protease responsible for this degradation remains
elusive, the same fragmentation of enolase was previously
observed during the switch from a respiratory to a fermentative
metabolism [36].
Larsen et al. [38] hypothesize that degradation of the
enzymes involved in the latest stages of the glycolytic pathway
(including enolase) might be a way of surviving unfavourable
conditions, as was previously suggested [39].
Several differentially expressed enzymes were related to

peptide and protein metabolism. The second group (P8, P21
and P23) has shown a significantly higher abundance of three
proteinases (endopeptidase) and two serine carboxypeptidases
(exopeptidase). The combination of the actions of these classes of
enzymes allows to catabolise entire proteins into small polypeptides and finally single amino acids. Catabolism of proteins thus
appears to be higher in the high-geosmin-producer group.
Blomberg et al. [30] have reported that proteins containing a
PH-like domain are involved in, but not limited to, controlling
and running enzymatic activities or regulation of nuclear
transport. All the ten spots which were identified as PH-like
protein together with the protein rotamase, a protein the
function of which is to optimise protein folding, were more
intense in P4, thus indicating a higher transcriptional activity. In
contrast to these observations, the elongation factor 1-beta and
the 40S ribosomal protein S0 were less abundant in P4. Therefore
the protein synthesis does not exhibit a clear tendency allowing
discriminating P4 from P8, P21 and P23.
Under normal conditions, HSPs play major roles in protein
folding, disaggregation and degradation [27]. When the culture
conditions become more stressful, their increased concentrations in the cell may indicate that they are part of the response
to the caused imbalance. HSP 70 is up-regulated during cellular
response to stress, especially oxidative stress, as described by
Jamieson et al. [40] and reviewed by Mayer and Bukau [28]. It is
also reported that the SSA1 gene, encoding an isoform of HSP 70,
is induced by oxidising agents such as H2O2 [41]. Oxidative
conditions may distort the conformation of many macromolecules, including nucleic acids and proteins, which thus require
both protection/reaction against oxidative species (e.g. catalase,
superoxide dismutase) and reinforcement of molecules dedicated to maintaining structural conformation of the macromolecules like HSPs. HSP 60 has been described as protector of
enzymes containing a Fe–S cluster, avoiding release of free iron
in the cell, as free iron catalyses oxidative reaction [42]. HSP 60 is

an important enzyme due to its localisation in the mitochondria, which is the main site of cellular ROS production via the
electron transport chain. Observation of the fold change values
of ATP synthase indicates that in P4 five out of seven of these

proteins were less abundant. Two cytochrome c oxidases were
also less abundant in P4. This may finally agree with the
high quantity of ATP needed for the production of secondary
metabolites. Qin et al. [3] report that the expression of HSP 60 is
higher in P. expansum culture submitted to borate causing an
oxidative stress. Mitochondrial ATP production may cause a
comparable oxidative mechanism. Interestingly, HSP 60 and
HSP 70 intervene successively for the protein folding inside the
mitochondria [42]. We have also detected a significant fold
change in the expression of a HSP-p23 like protein, which is a
co-chaperone of HSP 90 [43]. This HSP-complex is involved in
the protection of the nucleic acids against oxidative stress that
could result in the loss of chromosome fragments [44]. This
study shows that H2O2 causes major loss of chromosome
fragments by inhibiting HSP 90 activity, in a comparable way
as benomyl, a fungicide obstructing microtubule synthesis.
Importance of HSP 90 in cell viability and protection against
oxidative stress is also proved by the targeting of this protein by
antifungal agents as geldanamycin or radicicol [45].
The detoxification of ROS is done by a group of enzymes that
degrade these molecules and that have the potential to scavenge
the free radicals that are formed during the process, preventing
the formation of damage on biomolecules. One of the key actors
in this defensive mechanism is the protein family of the
peroxiredoxins, which constitute the first step of the cellular
response to oxidative stress. Its synthesis occurs when a limited

concentration of oxidant species is encountered in the cell [46],
i.e. at H2O2 concentration below 10 μM [32]. Moreover, H2O2
might activate other cellular pathways: 2-cys peroxiredoxin has
a chaperone activity and prevents ROS-linked damage to DNA in
Saccharomyces cerevisiae [47]. Another way by which the cell can
react to oxidative stress, and in particular to H2O2, is overexpressing catalase, which catalyses the degradation of this
dangerous molecule. This enzyme exhibits a Km of 20–200 mM,
which is 1000 to 10000 fold higher than the Km of peroxiredoxin
[48]. All together, the expression patterns of these anti-oxidative
enzymes may suggest that P4 has a more favourable redox
status. The abundance of catalase, the most efficient enzyme for
ROS detoxification, was lower. At the same time, peroxiredoxin
was more abundant, which may indicate a slight oxidative
stress. Indeed, catalytic cysteine in its hyperoxidised form
(mechanisms explained in [33]) becomes catalytically inactive,
even if it is still participating to ROS signalling and having a
chaperone activity [49], pushing the cell to adapt its response
to oxidative aggression with faster and more powerful
systems.

5.

Conclusion

By comparing the proteome profile of P. expansum strains with a
different geosmin production under identical culture conditions,
107 proteins were found to be differentially abundant. Functional
classification has revealed that enzymes involved in redox
homeostasis, protein folding and in the first steps of secondary
metabolism may have an important role in the biosynthesis of

geosmin. P4 may present a lower oxidative status as compared to
P8, P21 and P23 regarding the differential abundance of catalase
and peroxiredoxin. Other enzymes related to glycolysis, protein
metabolism, cell cycle and ATP production have also been


JO U R N A L OF P ROTE O MI CS 9 6 ( 20 1 4 ) 1 3–2 8

described and analysed. All together, these results constitute a
partial explanation of i) the reasons and ii) the mechanism of
geosmin production and provide a first insight in the comprehension of geosmin synthesis by filamentous fungi.
Supplementary data to this article can be found online at
/>
Acknowledgements
All the authors would like to thank Sébastien Planchon for his
valuable technical assistance and support.

REFERENCES

[1] Ugliano M, Bartowsky EJ, McCarthy J, Moio L, Henschke PA.
Hydrolysis and transformation of grape glycosidically
bound volatile compounds during fermentation with three
Saccharomyces yeast strains. J Agric Food Chem
2006;54:6322–31.
[2] La Guerche S, Dauphin B, Pons M, Blancard D, Darriet P.
Characterization of some mushroom and earthy off-odors
microbially induced by the development of rot on grapes. J
Agric Food Chem 2006;54:9193–200.
[3] Qin G, Tian S, Chan Z, Li B. Crucial role of antioxidant proteins
and hydrolytic enzymes in pathogenicity of Penicillium

expansum. Mol Cell Proteomics 2007;6:425–38.
[4] Morales-Valle H, Silva LC, Paterson RRM, Venancio A,
Lima N. Effects of the origins of Botrytis cinerea on earthy
aromas from grape broth media further inoculated with
Penicillium expansum. Food Microbiol 2011;28:1048–53.
[5] Darriet P, Pons M, Henry R, Dumont O, Findeling V,
Cartolaro P, et al. Impact odorants contributing to the
fungus type aroma from grape berries contaminated by
powdery mildew (Uncinula necator); incidence of enzymatic
activities of the yeast Saccharomyces cerevisiae. J Agric Food
Chem 2002;50:3277–82.
[6] Boutou S, Chatonnet P. Rapid headspace solid-phase
microextraction/gas chromatographic/mass spectrometric
assay for the quantitative determination of some of the main
odorants causing off-flavours in wine. J Chromatogr A
2007;1141:1–9.
[7] La Guerche S, Chamont S, Blancard D, Dubourdieu D, Darriet
P. Origin of (−)-geosmin on grapes: on the complementary
action of two fungi, Botrytis cinerea and Penicillium expansum.
Antonie Van Leeuwenhoek 2005;88:131–9.
[8] Gust B, Challis GL, Fowler K, Kieser T, Chater KF. PCR-targeted
Streptomyces gene replacement identifies a protein domain
needed for biosynthesis of the sesquiterpene soil odor
geosmin. Proc Natl Acad Sci U S A 2003;100:1541–6.
[9] Bentley R, Meganathan R. Geosmin and methylisoborneol
biosynthesis in Streptomycetes: Evidence for an isoprenoid
pathway and its absence in non-differentiating isolates. FEBS
Lett 1981;125:220–2.
[10] Pollak FC, Berger RG. Geosmin and related volatiles in
bioreactor-cultured Streptomyces citreus CBS 109.60. Appl

Environ Microbiol 1996;62:1295–9.
[11] Cane DE, Watt RM. Expression and mechanistic analysis of
a germacradienol synthase from Streptomyces coelicolor
implicated in geosmin biosynthesis. Proc Natl Acad Sci U S A
2003;100:1547–51.
[12] Agger S, Lopez-Gallego F, Schmidt-Dannert C. Diversity of
sesquiterpene synthases in the basidiomycete Coprinus
cinereus. Mol Microbiol 2009;72:1307–8.

27

[13] Dionigi CP. The Effects of copper-sulfate on geosmin
biosynthesis by Streptomyces tendae, Streptomyces albidoflavus,
and Penicillium expansum. Water Sci Technol 1995;31:135–8.
[14] Siddique MH, Liboz T, Bacha N, Puel O, Mathieu F, Lebrihi A.
Characterization of a cytochrome P450 monooxygenase gene
involved in the biosynthesis of geosmin in Penicillium
expansum. Afr J Microbiol Res 2012;6:4122–7.
[15] Bhadauria V, Banniza S, Wang LX, Wei YD, Peng YL.
Proteomic studies of phytopathogenic fungi, oomycetes and
their interactions with hosts. Eur J Plant Pathol
2010;126:81–95.
[16] Glass NL, Donaldson GC. Development of primer sets
designed for use with the PCR to amplify conserved genes
from filamentous ascomycetes. Appl Environ Microbiol
1995;61:1323–30.
[17] La Guerche S, Garcia C, Darriet P, Dubourdieu D, Labarère J.
Characterization of Penicillium species isolated from grape
berries by their internal transcribed spacer (ITS1)
sequences and by gas chromatography–mass spectrometry

analysis of geosmin production. Curr Microbiol
2003;48:405–11.
[18] Haas B, Serchi T, Wagner DR, Gilson G, Planchon S, Renaut J,
et al. Proteomic analysis of plasma samples from patients
with acute myocardial infarction identifies haptoglobin as
a potential prognostic biomarker. J Proteomics
2011;75:229–36.
[19] Pasquali M, Serchi T, Renaut J, Hoffmann L, Bohn T. 2D DIGE
reference map of a Fusarium graminearum nivalenol producing
strain. Electrophoresis 2013;34:505–9.
[20] La Guerche S, De Senneville L, Blancard D, Darriet P. Impact of
the Botrytis cinerea strain and metabolism on (−)-geosmin
production by Penicillium expansum in grape juice. Antonie
Van Leeuwenhoek 2007;92:331–41.
[21] Darriet P, Pons M, Lamy S, Dubourdieu D. Identification and
quantification of geosmin, an earthy odorant contaminating
wines. J Agric Food Chem 2000;48:4835–8.
[22] Franc C, David F, de Revel G. Multi-residue off-flavour
profiling in wine using stir bar sorptive extraction–thermal
desorption–gas chromatography–mass spectrometry. J
Chromatogr A 2009;1216:3318–27.
[23] Alm R, Johansson P, Hjernø K, Emanuelsson C, Ringnér M,
Häkkinen J. Detection and identification of protein isoforms
using cluster analysis of MALDI-MS mass spectra. J Proteome
Res 2006;5:785–92.
[24] Roze LV, Chanda A, Linz JE. Compartmentalization and
molecular traffic in secondary metabolism: a new
understanding of established cellular processes. Fungal
Genet Biol 2011;48:35–48.
[25] Ramirez-Zavala B, Mercado-Flores Y, Hernandez-Rodriguez

C, Villa-Tanaca L. Purification and characterization of a
serine carboxypeptidase from Kluyveromyces marxianus. Int J
Food Microbiol 2004;91:245–52.
[26] ten Have A, Espino JJ, Dekkers E, Van Sluyter SC, Brito N, Kay J,
et al. The Botrytis cinerea aspartic proteinase family. Fungal
Genet Biol 2010;47:53–65.
[27] Panaretou B, Zhai C. The heat shock proteins: their roles as
multi-component machines for protein folding. Fungal
Biology Reviews 2008;22:110–9.
[28] Mayer M, Bukau B. Hsp70 chaperones: cellular functions
and molecular mechanism. Cell Mol Life Sci
2005;62:670–84.
[29] Fugelstad J, Brown C, Hukasova E, Sundqvist G, Lindqvist A,
Bulone V. Functional characterization of the pleckstrin
homology domain of a cellulose synthase from the
oomycete Saprolegnia monoica. Biochem Bioph Res Co
2012;417:1248–53.
[30] Blomberg N, Baraldi E, Nilges M, Saraste M. The PH superfold:
a structural scaffold for multiple functions. Trends Biochem
Sci 1999;24:441–5.


28

JO U R N A L OF PR O TE O MI CS 96 ( 20 1 4 ) 1 3 –28

[31] Liu H, Zhang B, Li C, Bao X. Knock down of chitosanase
expression in phytopathogenic fungus Fusarium solani
and its effect on pathogenicity. Curr Genet
2010;56:275–81.

[32] Hansberg W, Salas-Lizana R, Dominguez L. Fungal catalases:
function, phylogenetic origin and structure. Arch Biochem
Biophys 2012;525:170–80.
[33] Wood ZA, Schröder E, Robin Harris J, Poole LB. Structure,
mechanism and regulation of peroxiredoxins. Trends
Biochem Sci 2003;8:32–40.
[34] Fox EM, Howlett BJ. Secondary metabolism: regulation and
role in fungal biology. Curr Opin Microbiol 2008;11:481–7.
[35] Jami MS, Garcia-Estrada C, Barreiro C, Cuadrado AA,
Salehi-Najafabadi Z, Martin JF. The Penicillium chrysogenum
extracellular proteome. Conversion from a food-rotting strain
to a versatile cell factory for white biotechnology. Mol Cell
Proteomics 2010;9:2729–44.
[36] Kobi D, Zugmeyer S, Potier S, Jaquet-Gutfreund L.
Two-dimensional protein map of an “ale”-brewing yeast
strain: proteome dynamics during fermentation. FEMS Yeast
Res 2004;5:213–30.
[37] Trabalzini L, Paffetti A, Scaloni A, Talamo F, Ferro E, Coratza
G, et al. Proteomic response to physiological fermentation
stresses in a wild-type wine strain of Saccharomyces cerevisiae.
Biochem J 2003;370:35–46.
[38] Larsen MR, Larsen PM, Fey SJ, Roepstorff P. Characterization
of differently processed forms of enolase 2 from
Saccharomyces cerevisiae by two-dimensional gel
electrophoresis and mass spectrometry. Electrophoresis
2001;22:566–75.
[39] Godon C, Lagniel G, Lee J, Buhler JM, Kieffer S, Perrot M, et al.
The H2O2 stimulon in Saccharomyces cerevisiae. J Biol Chem
1998;273:22480–9.

[40] Jamieson DJ, Rivers SL, Stephen DWS. Analysis of
Saccharomyces cerevisiae proteins induced by peroxide and
superoxide stress. Microbiol 1994;140:3277–83.
[41] Stephen DWS, Rivers SL, Jamieson DJ. The role of the YAP1
and YAP2 genes in the regulation of the adaptive oxidative
stress responses of Saccharomyces cerevisiae. Mol Microbiol
1995;16:415–23.
[42] Cabiscol E, Bellí G, Tamarit J, Echave P, Herrero E, Ros J.
Mitochondrial Hsp60, resistance to oxidative stress, and the
labile iron pool are closely connected in Saccharomyces cerevisiae. J
Biol Chem 2002;277:44531–8.
[43] Felts SJ, Toft DO. p23, a simple protein with complex
activities. Cell Stress Chaperon 2003;8:108–13.
[44] Chen G, Bradford WD, Seidel CW, Li R. Hsp90 stress
potentiates rapid cellular adaptation through induction of
aneuploidy. Nature 2012;482:246–50.
[45] Nicola A, Andrade R, Dantas A, Andrade P, Arraes F, Fernandes L,
et al. The stress responsive and morphologically regulated hsp90
gene from Paracoccidioides brasiliensis is essential to cell viability.
BMC Microbiol 2008;8:158–65.
[46] Vivancos A, Jara M, Zuin A, Sanso M, Hidalgo E. Oxidative
stress in Schizosaccharomyces pombe: different H2O2 levels,
different response pathways. Mol Genet Genomics
2006;276:495–502.
[47] Morgan BA, Veal EA. Functions of typical 2-Cys
peroxiredoxins in yeast peroxiredoxin systems. In: Flohé L,
Harris JR, editors. Netherlands: Springer; 2007.
[48] Díaz A, Valdés VJ, Rudino-Pinera E, Horjales E, Hansberg W.
Structure–function relationships in fungal large-subunit
catalases. J Mol Biol 2009;386:218–32.

[49] Edgar RS, Green EW, Zhao Y, van Ooijen G, Olmedo M, Qin X,
et al. Peroxiredoxins are conserved markers of circadian
rhythms. Nature 2012;485:459–64.