Accepted Manuscript
Title: Sample preparation and 2-DE procedure for protein expression profiling of black
microcolonial fungi
Authors: Daniela Isola, Gorji Marzban, Laura Selbmann, Silvano Onofri, Margit
Laimer, Katja Sterflinger
PII:
S1878-6146(11)00049-3
DOI:
10.1016/j.funbio.2011.03.001
Reference:
FUNBIO 159
To appear in:
Mycological Research
Received Date: 1 October 2010
Revised Date:
1 March 2011
Accepted Date: 1 March 2011
Please cite this article as: Isola, D., Marzban, G., Selbmann, L., Onofri, S., Laimer, M., Sterflinger, K.
Sample preparation and 2-DE procedure for protein expression profiling of black microcolonial fungi,
Mycological Research (2011), doi: 10.1016/j.funbio.2011.03.001
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Sample preparation and 2-DE procedure for protein expression profiling
of black microcolonial fungi
Daniela Isolaa, Gorji Marzbanb, Laura Selbmanna, Silvano Onofria, Margit
a
RI
PT
Laimerb, Katja Sterflingerc
Department of Ecology and Sustainable Economic Development, University of Tuscia,
Largo dell’Università snc, 01100 Viterbo, Italy.
University of Applied Life Sciences Vienna, Department of Biotechnology, Plant
SC
b
Biotechnology Unit, Muthgasse 18, 1190 Vienna, Austria
University of Applied Life Sciences Vienna, Department of Biotechnology, Austrian
M
AN
U
c
Center of Biological Resources and Applied Mycology, Muthgasse 18, 1190 Vienna,
Austria
Corresponding author. Daniela Isola Tel. +39 0761357138; fax +39 0761357751
TE
D
E-mail address: [email protected]
Gorji Marzban E-mail address: [email protected]
Margit Laimer E-mail address:
[email protected]
EP
Laura Selbmann E-mail address: [email protected]
Silvano Onofri E-mail address: [email protected]
AC
C
Katja Sterflinger E-mail address: [email protected]
ACCEPTED MANUSCRIPT
Sample preparation and 2-DE procedure for protein expression
profiling of black microcolonial fungi
Daniela Isolaa, Gorji Marzbanb, Laura Selbmanna, Silvano Onofria, Margit
a
Department of Ecology and Sustainable Economic Development, University of
Tuscia, Largo dell’Università snc, 01100 Viterbo, Italy.
University of Applied Life Sciences Vienna, Department of Biotechnology, Plant
SC
b
RI
PT
Laimerb, Katja Sterflingerc
Biotechnology Unit, Muthgasse 18, 1190 Vienna, Austria
University of Applied Life Sciences Vienna, Department of Biotechnology, Austrian
M
AN
U
c
Center of Biological Resources and Applied Mycology, Muthgasse 18, 1190 Vienna,
Austria
Corresponding author. Daniela Isola Tel. +39 0761357138; fax +39 0761357751
TE
D
E-mail address: [email protected]
Gorji Marzban E-mail address: [email protected]
Margit Laimer E-mail address:
[email protected]
EP
Laura Selbmann E-mail address: [email protected]
Silvano Onofri E-mail address:
[email protected]
AC
C
Katja Sterflinger E-mail address: [email protected]
1
ACCEPTED MANUSCRIPT
Abstract
The ecology and stress adaptation of black rock inhabiting fungi in hot and cold
extreme
environments
is
not
yet
well
understood.
Two-dimensional
gel
electrophoresis (2-DE) is a promising tool to study the protein expression profiling
and the metabolic status of microorganisms under stress conditions. The sample
RI
PT
preparation has been shown to be the bottleneck for high resolution protein
separation in 2-DE. For this purpose conditions must be optimized to obtain reliable
and reproducible results. In addition, due to a multilayered and strongly melanized
cell wall of black microcolonial fungi, special protocols for cell disruption and
SC
processing are required. In the present study, the protocol for protein extraction was
established and optimized for the black yeast Exophiala jeanselmei MA 2853. The
same protocol was successfully examined also for the meristematic fungus
M
AN
U
Coniosporium perforans MA 1299. Among the three procedures evaluated,
trichloroacetic acid (TCA) precipitation, TCA/acetone precipitation, and phenol
extraction combined with methanol/ammonium acetate precipitation, the latter
showed to be the best method for black yeasts and meristematic fungi. Penicillium
TE
D
chrysogenum was used as reference strain.
inhabiting fungi
AC
C
Introduction
EP
Keywords: black meristematic fungi, black yeast, protein extraction method, rock
With respect to their prominent morphological characteristics, black fungi are a group
of melanized, slow growing filamentous or yeast-like fungi also called black yeast,
meristematic and microcolonial fungi.
Black fungi, originally described as inhabitants of living and dead plant material
(Sterflinger 2005), could be also isolated from hypersaline waters (Zalar et al. 1999),
acidic environments (Baker et al. 2004; Selbmann et al. 2008), from rock in the hot
and cold deserts (Friedmann 1982; Staley et al.1982) and recently even in more
temperate climates (Ruibal et al. 2005; Sert et al. 2007). Meristematic fungi and black
yeasts were also found in human environments (de Hoog et al. 1997; Matos et al.
2002) as pathogens or opportunists (de Hoog et al. 1999). Although black fungi show
2
ACCEPTED MANUSCRIPT
many different ecologies, they share a number of universally present characters like
strong melanization, thick and multilayered cell wall and exopolysaccharides (EPS)
production (Sterflinger 2005) resulting in an extraordinary ability to tolerate chemical
and physical stresses.
Ecology, survival limits and phylogeny were extensively investigated in black fungi
RI
PT
showing their enormous heat and acidity tolerance, their ability to cope with high
levels of UV radiation and even radioactivity as well as halophilic conditions
(Gorbushina et al. 2008; Onofri et al. 2008; Wember et al. 2001). Whereas the
molecular phylogeny and taxonomy of black fungi has been extensively studied since
SC
1997 when first species of black fungi were described based on DNA sequencing
data (de Hoog et al. 1999; Sterflinger et al. 1998), the molecular mechanisms
underlying stress tolerance and adaptation are still not clarified.
M
AN
U
To date, protein expression profiling using a 2-DE approach has not been performed
in physiological changes and stress response investigations in black fungi. 2-DE is a
powerful tool to analyse proteins regulating stress tolerance and pathogenicity, which
may give new insights onto adaptation to the environment and evolution of black
fungi. Protein expression profiles are additionally suitable to characterize states of
dormancy, activity and growth related to different ecological conditions.
TE
D
Sample preparation is one of the most crucial steps for high resolution separation of
proteins in a 2-DE gel and it is expected to be difficult for black yeasts and
meristematic fungi due to their extraordinary thick multilayered cell walls.
EP
Furthermore, exopolysaccharides and melanins, extensively produced by these fungi,
may bind proteins causing artefacts and streaking (Marzban et al. 2008).
AC
C
However, an optimized protocol for sample preparation might guarantee the integrity
of proteins after cell disruption, and efficiently remove contaminants which may
interfere with the 2DE-gel separation. A complete removal of interfering compounds
is achieved with protein precipitation but each additional step must be carefully
considered as major cause for protein loss, chemical modification, degradation and
non-specific protein binding to surfaces (Marzban et al. 2008).
The present study was carried out in order to answer the following main questions:
(1) Which protocol delivers the optimal protein extract from black fungi? (2) What is
the optimal composition for equilibration solutions, by which the resolubilization and
transfer of proteins in the first and second dimension can be guaranteed? (3) Can the
procedure, after optimization for one single species, be applied to other species of
3
ACCEPTED MANUSCRIPT
meristematic fungi and black yeasts? Finally we examined variations in the protein
profile after a drastic environmental change (temperature decrease from 28°C to
1°C).
Materials and methods
RI
PT
Fungal strains and culture conditions.
Penicillium chrysogenum MA 3995, the opportunistic human pathogen Exophiala
jeanselmei MA 2853, and the meristematic fungus Coniosporium perforans MA 1299
were obtained from the ACBR culture collection (Austrian Center of Biological
SC
Resources and Applied Mycology, http://www.biotec.boku.ac.at/acbr.htm).
P. chrysogenum, a well-known filamentous fungus, was chosen as control due to its
ubiquitous, worldwide distribution. It is a mesophilic fungus with a great adaptation
M
AN
U
ability to different environmental conditions, even to high salt concentrations (Tresner
& Hayes 1971) and oxidative stress (Emri et al. 1998). All strains were grown on malt
extract agar (MEA, Applichem GmbH, Darmstadt, Germany) at 28°C for four weeks.
A suboptimal condition was also tested for C. perforans. In detail the fungus, grown in
optimal condition, was exposed for one week at low temperature (1± 1°C). Biomass,
TE
D
composed for P. chrysogenum predominantly by mycelium, was harvested by
scratching the material from the plates using a scalpel, and immediately frozen and
stored at -80°C until extraction.
EP
Extraction protocols
Frozen biomass (400 mg wet weight) was transferred into ice-cold 2 ml O-ring screw-
AC
C
capped microfuge tube, then thawed on ice and washed twice with saline solution
(NaCl 0.9%) as follows: tubes were filled up with saline solution, mixed carefully with
the biomass by short vortexing, then centrifugated at 16000 xg for 2 minutes at 4°C.
The supernatant was removed and about 400 mg of acid-washed 0.5 mm Ø glass
beads (Biospec, Bartlesville, OK) were added. The protein extractions were
performed using three different protocols:
A) Washed samples were homogenized at 5.0 m/s for a total of 4 min divided into 30
s increments using FastPrep FP120 instrument (Thermo Savant, Holbrook, N.Y) and
800µl of lysis buffer I (20 mM Tris-HCl, pH 7.6, 10 mM NaCl, 0.5 mM sodium
deoxycholate, protease inhibitor cocktail 1 pill for 80 ml of buffer (Complete, Roche))
4
ACCEPTED MANUSCRIPT
Between stages sample tubes were maintained in an ice bath for at least 1 min to
avoid warming. After homogenization, tubes were centrifuged at 16000 xg for 10 min
at 4°C. The supernatant was transferred to a pre-we ighed 1.5 ml microfuge tube and
proteins were precipitated adding ice-cold trichloroacetic acid (Sigma-Aldrich,
Steinheim, Germany) to a final concentration of 20% (v/v). After incubation of the
RI
PT
tube on ice for 20 min, a crude pellet was collected by centrifugation at 6000 ×g for
20 min at 4°C. The supernatant was discarded and th e pellet was washed three
times with 500 µl ice-cold acetone. After each washing cycle with acetone, the
protein pellet was collected by centrifugation at 850 ×g for 1 min at 4°C. Following the
SC
last acetone wash, the pellet was dried by vacuum centrifugation at room
temperature for 1 min using Savant SpeedVac Concentrator ISS110 (Thermo
Scientific, Waltham, MA), and resuspended in Modified Sample Buffer (MSB; 2 M
7
M
urea
(Merck,
Darmstadt,
Germany),
M
AN
U
thiourea,
Cholamidopropyl)dimethylammonio)-1-propanesulfonate
(CHAPS;
4%
3-((3-
Sigma-Aldrich,
Steinheim, Germany), 1% dithiothreitol (DTT; Serva Electrophoresis GmbH,
Heidelberg, Gemany), 2% (v/v) ServalythTM carrier ampholytes, pH 2–11 (Serva)
according to the final pellet weight (i.e., for a 5–15 mg pellet, 500 µl MSB was used
2008).
TE
D
and for a 16–30 mg pellet, 750 µl MSB) and stored at -80°C until use (Chandler et a l.
B) The resulting pellet was transferred in a 15 ml polypropylene centrifuge tube and
mixed with 5 ml of cold precipitation solution containing trichloroacetic acid (Sigma-
EP
Aldrich, Steinheim, Germany) 10 % (v/v) in acetone (Merck, Darmstadt, Germany),
and incubated overnight at -20°C. The pellet was ob tained by cold centrifugation at
7834 xg for 30 minutes, incubated for one hour at -20°C in cold acetone, then rinsed
AC
C
in acetone and centrifuged at 7834 xg for 10 minutes. The acetone was discarded
and the pellet dried at -20°C.
C) 1200 µl of lysis buffer II (50mM Tris-HCl, pH 8.5; 5mM EDTA, 100mM KCl, 1%
Poly- (vinylpolypyrrolydone) (PVPP), 30% Sucrose) were added before cell disruption
performed using the same procedure seen before. Tubes contents were transferred
in a 15 ml polypropylene centrifuge tube and 3 ml of tris–buffered phenol solution, pH
8.0 (Sigma- Aldrich, Steinheim, Germany) was added and mixed at least for 15 min
at room temperature. The phenolic phase was separated after centrifugation at 7834
xg for 10 min at 4°C and transferred to a new pre-w eighed tube. Subsequently, five
times volume of cold 0.1M ammonium acetate in methanol was added to collect
5
ACCEPTED MANUSCRIPT
phenolic phase. After overnight precipitation (-20°C) the protein pellet was obtained
by centrifugation at 7834 xg for 30 minutes at 4°C and washed with cold methanol
(absolute) and then with cold acetone (80% v/v). Finally the pellet was dried at -20°C
and resolved in 500 µl of MSB.
RI
PT
Protein determination
The Bradford protein Assay (Bradford 1976) was performed to determine the
concentration of protein in fungal extracts. Reactions were carried out in microtiter
plates according to the manufacturer instructions. A standard curve was established
SC
using serial dilutions from 0.8 to 100 µg/ml of bovine serum albumin (BSA). The
resulting optical density (OD) at 595 nm was analyzed with a plate reader (Magellan;
M
AN
U
Tecan Austria, Grödig, Austria).
SDS- PAGE gel
In a total volume of 25 µl, equal amounts of protein extract (4 µg) were added to SDS
Sample Buffer consisting in 0.5M Tris-HCl pH 6.8, Glycerol 85%, 10% (w/v) SDS,
and 0.1% bromophenol blue, a pH indicator that is blue when pH is ≥ 4.6 and turns
his colour in yellow when the pH is ≤ 3.0. Samples were then incubated for 10 min at
TE
D
90°C, shacked at 500 rpm using a Thermomixer compac t (Eppendorf, Hamburg,
Germany) and loaded into a precasted 4-20% Tris –Glycine gel 1.0 mm x 12 well
(Invitrogen, Carlsbard, CA- USA). The run was performed at 125V and 13 mA. Page
EP
Ruler pre-stained protein ladder 4-20% Trys-Gly-SDS PAGE (Fermentas, Vilnius,
Lithuania) was used as MW marker and protein bands were visualized by silver
AC
C
staining (Rabilloud 1992).
Two-dimensional gel electrophoresis
Electrophoresis in the first dimension was performed using 13 cm IPG
TM
DryStrip pH
3-10 NL (GE Healthcare Bio- Sciences AB, Uppsala, Sweden). IPG-strips were
passively loaded and rehydrated in a total volume of 250 µl for 16 hours at room
temperature with 20 µg of proteins extract in rehydration buffer (8 M urea, 2% (w/v)
CHAPS, 10 mM DTT, 2% (v/v) Servalyte, 0.1% bromophenol blue). The iso-electric
focusing (IEF) was performed using a Protean IEF cell system (Bio-Rad
Hartfordshire, USA) at 20°C for 14000 V-hr.
Equilibration of strips was carried out using two different protocols:
6
ACCEPTED MANUSCRIPT
Equilibration protocol I) The IPG strips were incubated 2 times each for 15 minutes
under gentle shaking in 10 ml of solution that contains 50 mM Tris- HCl pH 8.8, 2%
SDS, urea 6M, and 30% (w/v) glycerol. DTT is incorporated in the first equilibration
time (1%) and iodoactamide (IAA) (4%) in the second time (Görg et al. 1987).
Equilibration protocol II) The IPG strips were incubated for 12 minutes with
RI
PT
equilibration solution II (urea 6M, 50 mM Tris- HCl pH 8.4, glycerol 30% (v/v), sodium
dodecyl sulfate (SDS) 2% (w/v), DTT 2% (w/v)) and 5 minutes in urea 6M, 50 mM
Tris- HCl pH 6.8, glycerol 30% (v/v), SDS 2% (w/v), IAA 2.5 % (w/v) and a trace of
SC
bromophenol under gentle agitation (Bjellqvist et al. 1993).
Equilibrated IPG strips were loaded onto 12% polyacrylamide gels (14 cm × 14 cm)
prepared with electrophoresis buffer (0.375 M Tris, 0.1% SDS and glycine). Second
M
AN
U
dimension was performed using the Perfect Blue twin gel system (PeqLab GmbH,
Erlangen, Germany). The chamber was attached via plastic tubing to a circulating
water bath at 4°C (type CBN 8-30, Heto, Birkerød, D enmark) and the run was
performed at 160 V and variable mA.
IPG strips used to test the influence of environmental condition on protein expression
TE
D
profile, were loaded onto 10% polyacrylamide gels (14 cm × 14 cm).
Statistical and Image analysis
Extraction efficiency was analysed by two way ANOVA followed by a Bonferroni post
test. Proteins visualized by ammoniacal silver staining (Merril et al. 1982) were
EP
captured and processed using ImageMasterTM 2D Platinum version 5.0 (Amersham
Bioscience, Swiss Institute of Bioinformatics, Geneva, Switzerland). The spots were
AC
C
quantified using the volume criterion (optical density expressed as spot volume) and
only intensities greater than two-fold were considered.
Results
Comparison of protein extractions
Different protein extraction procedures showed different protein yields. TCA
precipitation obtained 1.04 ± 0.09 and 0.57 ± 0.07 (µg proteins/mg biomass) from P.
chrysogenum and E. jeanselmei respectively (Fig. 1). TCA/acetone and phenolbased precipitation resulted in a more efficient protein extraction than TCA alone and
yielded in, at least, a double quantity of protein with a high statistical significance (P <
0.001). However, independently from the procedure used, the amounts of proteins
7
ACCEPTED MANUSCRIPT
obtained from P. chrysogenum were always significantly higher (P < 0.001) than
those extracted from E. jeanselmei.
SDS-PAGE analyses revealed that the majority of bands is common in all samples
(Fig. 2). TCA/acetone extract presents some differences resulting particularly rich in
proteins with molecular weight higher than 70 kDa and lower than 25 kDa. In addition
RI
PT
both TCA/acetone extracts (P. chrysogenum and E. jeanselmei) turned the colour of
the SDS sample buffer containing bromophenol, from blue to yellow, indicating a pH
decrease ( pH ≤ 3.0).
2-DE analysis reconfirmed the results of SDS-PAGE regarding the size distribution of
SC
separated proteins. The number of detected protein spots were 185 for E. jeanselmei
(Fig. 3A) extracted by the TCA procedure, 207 by TCA/acetone precipitation (Fig. 3B)
and 211 by phenol-based procedure (Fig. 3C). TCA/acetone extraction gel delivered
M
AN
U
2D gels with decreased spot resolution (Fig. 3B) and the majority of protein spots
appeared in the lower pI (isoelectric point) range. The comparison of the focused IPG
strips of E. jeanselmei and P. chrysogenum, showed that TCA/acetone protein
extracts resulted in a more limited migration than other methods as the bromophenol
front could not reach the acidic end of IPG strip (Fig. 4).
TE
D
Influence of equilibration solution on protein separation.
After iso-electric focusing proteins are fixed in IPG gel matrix, due to immobilized pH
gradients, stronger than in carrier ampholyte gels. A prolonged equilibration time and
EP
the composition of buffers are therefore crucial for efficient protein transfer from the
first to the second dimension. The protocols used differed mainly in pH and exposure
time. As shown in Fig. 5B, best results were gained using equilibration protocol II. 2-
AC
C
DE gel of P. chrysogenum obtained by precipitation with TCA allowed the detection
of approximately 50 spots when strips were equilibrated using protocol I and
approximately
400
with
protocol
II.
Since
gels
were
stained
together
misinterpretations tied to over- or under-staining could be excluded.
Influence of environmental conditions on protein expression profile
Equilibration protocol II was also tested for the meristematic rock fungus C.
perforans. Results showed that the protocol is appropriate to be applied to P.
chrysogenum as well as to C. perforans.
8
ACCEPTED MANUSCRIPT
In a first approach C. perforans, grown under optimal conditions (MEA, 28°C),
yielded 465 detected protein spots in 10% acrylamide 2-DE gel (Fig. 6B), which was
numerically quite similar to P. chrysogenum grown under optimal conditions (Figs 6A,
479 protein spots); by contrast, their spot distributions were completely different so
that it was not possible to match common spots confidently. C. perforans after a one-
RI
PT
week incubation to suboptimal conditions (MEA, 1± 1°C) expressed a significantly
decreased number of proteins (271 detected protein spots; Fig. 6C). In detail, 281
spots detected in optimal condition were lost at low temperature (Figs 7A, B), the
majority of them having pIs from neutral to acidic and molecular weights within 10
SC
and 55 KDa. On the other hand, new spots were exclusively detected under
suboptimal conditions (87 spots red highlighted in Fig. 7B). It was also possible to
observe variations among the 187 common protein spots in their volume ratios. Sixty-
exposed to low temperatures.
Discussion
M
AN
U
four resulted to be down-regulated and 29 up-regulated when C. perforans was
Recently, fungal proteomics has become an interesting field of study as evidenced by
Kim et al. (2007). Despite the increasing number of publications, a unique and
TE
D
defined protocol cannot be adopted for all fungal organisms. Moreover, every
extraction method has its physiochemical limitations that determine a specific and
reproducible protein loss (Carpentier et al. 2005; Marzban et al. 2008). The focus of
EP
the present study was to find the best protocol for separation of the highest number
of proteins from black microcolonial fungi and, at the same time, providing the best
resolution in 2-DE gel electrophoresis.
AC
C
Two way ANOVA statistical analysis, followed by a Bonferroni post test, confirmed
the high significant (P value <0.0001) influence of the methodologies used on the
proteins extraction efficiency as well as the influence of the fungal strain (P.
chrysogenum vs E. jeanselmei) in the amounts of the total soluble proteins.
Concerning black microcolonial fungi, the extraordinary thick, multilayered and
melanin encrusted cell wall which characterize these organisms may be related to the
reduced release of intracellular proteins, thus finding an effective protocol for protein
extraction is of high priority.
TCA-based extraction is a quick procedure, compared to phenol-based extraction
method, was performed very fast, but its efficiency was significantly lower. The main
9
ACCEPTED MANUSCRIPT
obstacle was in resolubilization of pellets resulting in low reproducibility which was
already reported by Chen & Harmon (2006). The TCA/acetone protocol, as reported
from other authors (Bhadauria et al. 2007), demonstrated to be more efficient than
TCA precipitation protocol and yielded higher amount of proteins. On the other hand,
for black fungi, the extract resulted in a dark pellet showing incapability to clean up
RI
PT
the impurities, causing artefacts and streaking in the subsequent isoelectric focusing
(IEF) process. In addition, extracts from P.chrysogenum and E. jeanselmei were
characterized by a low pH (≤ 3.0), which can be referred to a poor removal of TCA
residues resulting, also in this case, in IEF troubles. All samples extracted with the
SC
TCA/acetone method, in fact, did not complete the charge migration during isoelectric
focusing (bromophenol front did not reach the acidic end of the IPG strip).
M
AN
U
The 2-D protein pattern obtained with TCA, TCA/acetone and phenol-based
extraction, in combination with methanol/ammonium acetate precipitation, showed
only few but relevant differences. Discrepancies seems to be originated from methodspecific protein loss, inactivation of proteolytic or other modifying enzymes,
partitioning of proteins from the aqueous phase (e.g. phenol-based method) and
different precipitation/dissolving capacity. In particular TCA/acetone extracts resulted
TE
D
in 2-D gels with a higher number of protein spots with molecular weight range above
70 kDa and under 25 kDa. The other two extraction procedures are also
characterized by low spot resolution due to troubles in IEF.
EP
By contrast, phenol extraction resulted in high amounts of soluble proteins and good
reproducibility in both first and second dimension gels. Phenol-based extraction
efficiently solubilizes membrane proteins, removes nucleic acids and minimizes
AC
C
proteolysis (Hurkman & Tanaka 1986). Furthermore, phenol works as dissociating
agent decreasing the interactions of proteins with other compounds and
demonstrating a high clean–up capacity (Carpentier et al. 2005).
Even if phenol extraction has been always regarded as time consuming, toxic and
laborious (Faurobert et al. 2007), results obtained in the present study demonstrated
its high efficiency and capability to deliver the best results.
The optimization of protein extraction method and IPG strips equilibration allowed to
establish protein maps for different black fungal species exposed to different
conditions. In detail, through a proteomic approach, we were able to detect
10
ACCEPTED MANUSCRIPT
differences tied to the organisms and to their respective metabolic state (Figs 6A, B).
Additinally, alterations in the proteomic expression profile could be unravelled under
a particular stressing conditions. In fact, C. perforans incubated at two different
temperatures expressed (Figs 6B, C) almost half of the detected protein when the
fungal cultures were incubated under suboptimal temperatures, indicating enormous
RI
PT
physiological changes.
According to our results proteomic approach allows the qualitative and quantitative
measurements of large numbers of proteins which directly influence cellular
biochemistry, and reflects the metabolic state of organisms during growth,
SC
development and response to environmental factors. Preliminary proteomic analyses
performed in our study will allow us a better understanding of environmental
processes such as biodeterioration and biodegradation in which black fungi play a
M
AN
U
substantial role. Presented methodologies will be also useful to characterize different
black fungi with respect to their metabolic stress regulation and resistance.
The future task of our studies will remain in the identification of proteins involved in
stress tolerance which will give new insights into the ecology of these organisms;
Acknowledgements
TE
D
which are the most stress resistant eukaryotes known to date.
The authors acknowledge Carex for Student Projects' Support
References
EP
knowledge grant” 2009.
"Transfer of
AC
C
Bhadauria V, Zhao WS, Wang LX, Zhang Y, Liu JH, Yang J, Kong LA, Peng YL,
2007. Advances in fungal proteomics. Microbiological Research 162: 193—200.
Baker BJ, Lutz MA, Dawson SC, Bond PL, Banfield JF, 2004. Metabolically active
eukaryotic communities in extremely acidic mine drainage. Applied Environmental
Microbiology 70: 6264-6271.
Bjellqvist B, Pasquali C, Ravier F, Sanchez JC, Hochstrasser D, 1993. A
nonlinear wide-range immobilized pH gradient for two-dimensional
electrophoresis and its definition in a relevant pH scale. Electrophoresis 14: 13571365.
Bradford MM. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Analitical Biochemistry 72: 248–254.
Carpentier SC, Witters E, Laukens K, Deckers P, Swennen R, Panis B, 2005.
Preparation of protein extracts from recalcitrant plant tissues: an evaluation of
11
ACCEPTED MANUSCRIPT
different methods for two-dimensional gel electrophoresis analysis. Proteomics 5:
2497–2507.
Chandler JM, Treece ER, Trenary HR, Brenneman JL, Flickner TJ, Frommelt JL,
Oo ZM, Patterson MM, Rundle WT, Valle OV, Kim TD, Walker GR, Cooper CR Jr,
2008. Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei.
Proteome Science 6:17.
RI
PT
Chen S, Harmon AC, 2006. Advances in plant proteomics. Proteomics 6: 5504–
5516.
de Hoog GS, Beguin H, Batenburg-van de Vegte WH, 1997. Phaetotheca
triangularis, a new meristematic black fungus from humidifier. Antonie van
Leeuwenhoek 71: 289–295.
SC
de Hoog GS, Zalar P, Urzì C, de Leo F, Yurlova NA, Sterflinger K, 1999.
Relationships of dothideaceous black yeasts e meristematic fungi based on 5.8S
e ITS2 rDNA sequence comparison. Studies in Mycology 43:31–37
M
AN
U
Emri T, Pócsi I, Szentirmai A, 1998. Glutathione metabolism and protection
againsoxidative stress caused by peroxides in Penicillium chrysogenum. Free
Radical Biology and Medicine 23: 809-814.
Faurobert M, Pelpoir E, Chaïb J, 2007. Phenol extraction of proteins for proteomic
studies of recalcitrant plant tissues In: Thiellement H, Zivy M, Damerval C,
Méchin V, (eds), Methods in Molecular Biology, Plant Proteomics: Methods and
Protocols, Humana Press Inc., Totowa, NJ 335: 9-14.
Friedmann EI, 1982. Endolithic microorganisms in the Antarctic cold desert.
Science 215: 1045-1053.
TE
D
Gorbushina AA, Kotlova ER,. Sherstneva OA, 2008. Cellular responses of
microcolonial rock fungi to long-term desiccation and subsequent rehydration.
Studies in Mycology 61: 91-97.
Görg A, Postel W, Weser J , Günther S, Strahler JR, Hanash SM, Somerlot L,
1987. Electrophoresis 8: 122-124.
EP
Hurkman WJ, Tanaka CK, 1986. Solubilization of plant membrane proteins for
analysis by two-dimensional gel electrophoresis. Plant Physiology 81: 802-806
AC
C
Kim Y, Nandakumar MP, Marten M, 2007. Proteomics of filamentous fungi.
Trends in Biotechnology 25: 395 – 400.
Marzban G, Herndl A, Katinger H, Laimer M, 2008. Mapping of fruit allergens by
2D electrophoresis and immunodetection. Expert Review Proteomics 5: 61-75
Matos T, de Hoog GS, de Boer AG, Haase G, 2002. High prevalence of the
neurotrope Exophiala dermatitidis and related oligotrophic black yeasts in sauna
facilities. Mycoses 45: 373- 377.
Merril CR, Goldman D, Van Keuren ML, 1982. Electrophoresis 3: 17-23.
Onofri S, Barreca D, Selbmann L, Isola D, Rabbow E, Horneck G, de Vera JPP,
Hatton J, Zucconi L, 2008. Resistence of Antarctic black fungi e cryptoendolithic
communities to simulated space e Mars conditions. Studies in Mycology 61: 99–
109.
Rabilloud T, 1992. A comparison between low background silver diammine and
silver nitrate protein stains. Electrophoresis 13: 429-439.
12
ACCEPTED MANUSCRIPT
Ruibal C, Platas G, Bills GF, 2005. Isolation and characterization of melanized
fungi from limestone formation in Mallorca. Mycological Progress 4: 23- 38.
Selbmann L, de Hoog GS, Gerrits van den Ende AHG, Ruibal C, De Leo F,
Zucconi L, Isola D, Ruisi S, Onofri S, 2008. Drought meets acid: three new genera
in a Dothidealean clade of extremotolerant fungi. Studies in Mycology 61: 1–20.
Sert HB, Sümbül H, Sterflinger K, 2007. Microcolonial fungi from antique marbles
in Perge/Side/Termessos (Antalya/Turkey). Antonie Leeuwenhoek 91: 217–227.
RI
PT
Staley JT, Palmer F, Adams B, 1982. Microcolonial fungi: common inhabitants on
desert rocks? Science 215: 1093–1095.
Sterflinger K, 1998. Temperature and
NaCl- tolerance of rock-inhabiting
meristematic fungi. Antonie van Leeuwenhoek 74: 271–281.
SC
Sterflinger K, 2005. Black Yeasts and meristematic fungi: ecology, diversity and
identification. In: Seckbach J, (eds), The yeast handbook. Biodiversity and
ecophysiology of yeasts; Springer-Verlag Berlin and Heidelberg GmbH & Co:
501– 514.
M
AN
U
Tresner HD, Hayes JA, 1971. Sodium chloride tolerance of terrestrial fungi.
Applied Microbiology 22: 210-213.
Wember VV, Zhdanova NN, 2001. Peculiarities of linear growth the melanin
containing fungi Cladosporium sphaerospermum Perz. and Alternaria alternata
(Fr.) Keissler. Mikrobiolohichnyĭ zhurnal 63: 3-12.
AC
C
EP
TE
D
Zalar P, de Hoog GS, Gunde- Cimmerman N, 1999. Trimmatostroma salinum, a
new species from hypersaline water. Studies in Mycology 43: 57-62.
13
ACCEPTED MANUSCRIPT
Fig 1. Efficiency of protein extraction procedures applied to P. chrysogenum
MA 3995 and E. jeanselmei MA 2853: TCA method, TCA/acetone and phenolic
extraction.
Fig 2. Evaluation of extraction and precipitation protocol using SDS-PAGE for
RI
PT
E. jeanselmei MA 2853. A) TCA precipitation; B) TCA/acetone precipitation; C)
phenolic extraction.
Fig 3. Evaluation of extraction and precipitation protocol using 2DE for E.
SC
jeanselmei MA 2853. A) TCA precipitation; B) TCA/acetone precipitation; C)
phenolic extraction.
M
AN
U
Fig 4. IEF performed on E. jeanselmei A) TCA precipitation; B) TCA/acetone
precipitation; C) phenolic extraction. IEF performed on and P. chrysogenum D)
TCA/acetone precipitation. Arrows highlighted the migration front of protein
extracts.
Fig 5. Evaluation of equilibration buffers. Silver stained 2DE 12 % acrylamide gel
TE
D
of P.chrysogenum MA 1299. A) Equilibration protocol I; B) Equilibration protocol II.
IEF separation was realized on 13 cm strips pI 3-10.
Fig 6. Comparison of 2DE gel protein expression profiles. A) P. chrysogenum
EP
grown at optimal conditions (479 spots); B) C. perforans under optimal conditions
(465 spots) and C) C. perforans incubated for one week at 1 ±1°C (271 spots). IEF
AC
C
separation was realized on 13 cm strips pI 3-10 and resolution of high molecular
weight proteins was optimized using SDS-PAGE 10%- acrylamide gels.
Fig 7. Alteration of protein expression profile under different environmental
conditions. A) C. perforans grown at optimal conditions and B) C. perforans
incubated for one week at 1 ±1°C. Common spots, hig hlighted in green, were 187
and unmatched spots, in red.
14
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT