proteomic analysis of the extremely thermoacidophilic archaeon picrophilus torridus at ph and...

RESEARCH ARTICLE

Proteomic analysis of the extremely thermoacidophilic

archaeon Picrophilus torridus at pH and temperature

values close to its growth limit

Andrea Th .urmer1, Birgit Voigt2, Angel Angelov1,3, Dirk Albrecht 2, Michael Hecker2

and Wolfgang Liebl1,3

1 Institute of Microbiology and Genetics, Georg-August-Universit .at, Gottingen, Germany2 Institute of Microbiology, Ernst-Moritz-Arndt-Universit .at, Greifswald, Germany3 Department of Microbiology, Technische Universit .at M .unchen, Freising, Germany

Received: January 4, 2011

Revised: August 2, 2011

Accepted: September 13, 2011

The thermoacidophilic archaeon Picrophilus torridus belongs to the Thermoplasmatales order

and is the most acidophilic organism known to date, growing under extremely acidic

conditions around pH 0 (pHopt 1) and simultaneously at high temperatures up to 651C. Some

genome features that may be responsible for survival under these harsh conditions have been

concluded from the analysis of its 1.55 megabase genome sequence. A proteomic map was

generated for P. torridus cells grown to the mid-exponential phase. The soluble fraction of the

cells was separated by isoelectric focusing in the pH ranges 4–7 and 3–10, followed by a two

dimension (2D) on SDS-PAGE gels. A total of 717 Coomassie collodial-stained protein spots

from both pH ranges (pH 4–7 and 3–10) were excised and subjected to LC-MS/MS, leading to

the identification of 665 soluble protein spots. Most of the enzymes of the central carbon

metabolism were identified on the 2D gels, corroborating biochemically the metabolic

pathways predicted from the P. torridus genome sequence. The 2D master gels elaborated in

this study represent useful tools for physiological studies of this thermoacidophilic organism.

Based on quantitative 2D gel electrophoresis, a proteome study was performed to find pH- or

temperature-dependent differences in the proteome composition under changing growth

conditions. The proteome expression patterns at two different temperatures (50 and 701C)

and two different pH conditions (pH 0.5 and 1.8) were compared. Several proteins were up-

regulated under most stress stimuli tested, pointing to general roles in coping with stress.

Keywords:

Acid habitation / Heat shock proteins / Microbiology / Stress / Two-dimensional gel

electrophoresis

1 Introduction

The ability to cope with environmental stress is essential

for microorganisms, especially for those thriving in

extreme environments. Certain Archaea, such as members

of the Thermoplasmatales order, occupy niches that are

extreme in two respects: low pH and elevated temperature

[1]. With their ability to grow at pH values around pH 0

(pHopt 1) and at temperature up to 651C (Topt 601C),

Picrophilus torridus and the closely related P. oshimaeare the most extreme representatives within this group [2].

Picrophilus torridus was first isolated from a dry solfataric

field in northern Japan [2]. Normally, thermoacidophilic

organisms manage to maintain an intracellular pH close to

neutral [3]. In contrast, an unusually low intracellular pH of

4.6 has been measured in Picrophilus cells [4]. The high

specialization for growth in extremely acidic habitats is

also evident from the inability of P. torridus to grow at pH

values above 4.0.Abbreviation: ED pathway, Entner–Doudoroff pathway

Correspondence: Professor Wolfgang Liebl, Department of

Microbiology, Technische Universit .at M .unchen, Emil-Ramann-

Str. 4, D-85350 Freising, Germany

E-mail: [email protected]

Fax: 149-8161-715475

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2011, 11, 4559–4568 4559DOI 10.1002/pmic.201000829

Maintaining pH homeostasis involves complex

genetic and metabolic strategies. One of the best-studied

organisms with respect to acid stress survival is Escherichiacoli, for which three clearly defined acid resistance

systems have been described. Two of these systems require

extracellular amino acids and depend on intracellular amino

acid (glutamate, arginine) decarboxylation reactions with

concomitant proton consumption and subsequent anti-

porter-driven export of the decarboxylation products in

exchange for importing new amino acid substrates [5–7]. In

contrast, apart from the interpretation of the genome

sequence data surprisingly, little is known about the

mechanisms and regulation of acid stress response in the

most extreme acidophile genus, Picrophilus. Here we

present the first proteomic studies of P. torridus. The

basic metabolic pathways of this organism could be

deduced from its genome sequence [8]. However, the

proteome is highly dynamic and flexible and gives

each organism a defined protein pattern that is imposed

upon the cell by environmental stimuli, e.g. pH and

temperature. The use of proteomic studies makes it possible

to identify gene products that are differentially

expressed under specific conditions [5]. The extreme envir-

onment in which P. torridus exists and its unusual

intracellular pH suggest that selective pressure may have

led to the evolution of proteins with characteristics

that are significantly different from those of organisms

growing in less extreme environments. In theory, differ-

ences at the level of amino acid composition, amino acid

sequence or three-dimensional structure, including surface

properties such as charge or hydrophobicity distribution,

etc. might be important. Comparative analysis of the

P. torridus proteome deduced from its genome sequence

with the proteomes of acidophiles and neutrophiles failed to

show significant peculiarities in the isoelectric point

distribution or amino acid composition [9]. In addition, or

alternative to possible adaptations at the polypeptide

sequence or structure level, special adaptations in metabo-

lism may have evolved [8], including specific acid resistance

mechanisms which may be regulated in response to the

environmental conditions. The application of proteomics

methods is an appropriate way to investigate the changes in

the protein expression pattern of P. torridus cells in response

to environmental stress condition. Picrophilus torridus with

its small genome of 1537 ORFs is well suited for the reso-

lution of soluble proteins via two-dimensional (2D) gel

electrophoresis.

In an initial proteomic investigation, we created a 2D-

PAGE-based proteomic map of all accessible soluble

proteins of P. torridus at optimal growth conditions. The

master gels obtained were then used as tools for physiolo-

gical studies of this thermoacidophilic organism. Using the

2D gel electrophoresis technique, we examined the meta-

bolic responses of P. torridus to temperature and pH shock

by the analysis of the differential protein expression

patterns.

2 Materials and methods

2.1 Cultivation and harvesting of cells

Picrophilus torridus (DSM 9790) cultures were grown aero-

bically at 50 rpm in an incubator at 601C. Cells were grown

in Brocks medium [10] with following stocks: 100� Stock

(130 g (NH4)2SO4, 25 g MgSO4 � 7H2O, 2 g FeCl3 � 6H2O),

200� stock (56 g KH2PO4, 0.36 g MnCl2 � 4 H2O; 0.044 g

ZnSO4 � 7H2O, 0.01 g CuCl2 � 2H2O, 0.006 g VoSO4 � 5H2O,

0.002 g CoSO4 � 7H2O, 0.006 g Na2MoO4 � 2H2O, 0.0009 g

Na2B4O7, 5 mL 50% H2SO4), 1000� stock (70 g

CaCl2 � 2H2O). For 1-L P. torridus growth medium with pH

1, 968.5 mL deionized water was autoclaved with 5.5 mL of

100% H2SO4. Ten milliliters of 100� stock, 5 mL of 200�

stock, 1 mL of 1000� stock and 10 mL of 10% yeast extract

were added. Cells were harvested by centrifugation at

4000 rpm at 41C for 10 min and the cell pellets were resus-

pended and washed twice in 50 mM sodium acetate

(pH 4.6). The washed pellets were then stored at �201C

until analysis.

2.2 Experimental design

2.2.1 Master gel

Picrophilus torridus cells, grown at pH 1 and 601C, were

collected in the exponential growth phase. All soluble

proteins were extracted and a 2D gel electrophoresis was

performed (see Sections 2.3 and 2.4). To get an overview

over a broad range of all soluble proteins, two different pH

ranges (IPG-strips 4–7 and 3–10, GE Healthcare, Uppsala,

Sweden) were used in the first dimension.

2.2.2 Stress experiments

A 1-L P. torridus culture was grown at pH 1 until exponential

phase and then split into three 330 mL cultures. In the first

experiment, these three pH 1 cultures were incubated at 50

and 701C and at the optimal growth temperature 601C. At 5,

35 and 125 min, a sample of 100 mL was collected. In a

second experiment, the three split cultures were incubated

at 601C. For two of the cultures, the medium was adapted to

pH 0.5 and 1.8 before the cells were incubated. The cells

then were incubated for 2 h, and at 10, 40 and 130 min,

100 mL of samples were collected. In each set-up, the total

experimental time was 120 min, but the pH adjustment of

the media took 5 min longer than the temperature shift,

accounting for the 5-min differences in the time points.

Stress experiments were performed in two biological repli-

cas, and the abundance rates were derived from these two

independent experiments. P. torridus has a doubling time of

almost 10 h. Preceding growth experiments were done and

growth rates were determined from pH 0.3 to 2.5 and 45 to

4560 A. Th .urmer et al. Proteomics 2011, 11, 4559–4568


751C. Under the stress conditions of this study, only a slight

change of the doubling time was observed (data not shown).

All samples were taken in the exponential growth phase.

Thus, we could exclude that the differences in the protein

profiles observed at sub-optimal growth conditions might be

caused by different growth phases and not by temperature

or pH stress.

2.3 Protein extraction

For the protein extraction, the rapid method of Wessel and

Flugge based on a defined methanol–chloroform–water

mixture was used with slight adaptations [11].

Cells of a 100-mL P. torridus sample were lysed by pH

triggered cell lysis using 50 mM Tris pH 8.5 containing

1 mM PMSF. This cell extract was spun at 3500 rpm for

5 min using an Eppendorf 5424 R benchtop centrifuge to

collect large debris and non-lysed cells. The supernatant was

treated with a mixture of methanol, chloroform and H2O at

a ratio of 1:4:1:3 v/v/v/v. After mixing rigorously the sample

was spun for 8 min at 10 000 rpm. The upper phase was

carefully aspirated. The proteins were pelleted by adding

300 mL of methanol and spinning the sample for 10 min at

13 000 rpm. The pellet was air-dried and then resuspended

with sample buffer [8 M urea, 2% CHAPS, 1% dithiothreitol

(DTT), 35 mM Tris, 0.5% IPG buffer] and the total protein

concentration was determined by the Bradford method,

using bovine serum albumin as the standard. The sample

was adjusted to contain 350 mg protein in a volume of 170mL

and was mixed with an equal volume of rehydration solution

(8 M urea, 2% CHAPS, 0.5% IPG buffer, 0.005% bromo-

phenol blue) and subjected to 2D electrophoretic separation

(see Section 2.4).

2.4 2D gel electrophoretic separation and protein

detection

For isoelectric focusing, ImmobilineTM DryStrip gels (IPG

strips, 18 cm; pH 3–10 or 4–7, GE Healthcare) were used.

Sample application was performed automatically by in-gel

rehydration and isoelectric focusing in a one-step procedure

as described by Gorg et al. [12] with slight adaptations. The

strips were rehydrated for 16 h at 20 V on an IPGphor cell

(GE Healthcare). Isoelectric focusing was performed with

three steps with fixed voltages (3 h at 100 V, 1 h at 200 V, 1 h

at 500 V), followed by a 1-h gradient to 8000 V and a final

step of 6 h at 8000 V. For the second dimension, the strips

were incubated twice for 15 min in equilibration solution

(6 M urea, 50 mM Tris, pH 8.8, 30% glycerol, 2% SDS and

0.005% bromophenol blue), the first time with 2% DTT and

the second time with 2.5% iodoacetamide. The strips were

then applied to 1.5 mm thick SDS-polyacrylamide (12%)

gels, which were subjected to electrophoresis for 15 min at

15 mA, followed by 8 h at 60 mA. Proteins were detected by

the very sensitive (detection limit 10 ng) Coomassie colloidal

staining [13]. Proteins were identified by MS as described by

Voigt et al. [14] with a specific P. torridus sequence database.

2.5 Imaging and statistical analysis

Images were scanned using a ScanMaker 1000XL (Microtek,

Evestar GmbH, Willich, Germany) with a resolution of 300

dots per inch (dpi). The transparency mode was used to

obtain a grayscale image. For the analysis of the gels

(including calculation of the protein abundance rate with

normalization and mean), the Delta 2D software (Decodon

GmbH, Greifswald, Germany) was used [15]. Images were

loaded and processed into the Delta 2D program following

the software manual instructions. For the quantification, all

detected spots were normalized as described by Berth et al.

[16], and the ten most intensive spots on each gel were

excluded to avoid hiding weaker expressed spot. For

comparison of the protein patterns during different stress

conditions, each gel of a stress condition was warped to the

gel image of the reference (gel image at optimal growth

conditions) at three time points (temperature stress 5, 35

and 125 min, pH stress 10, 40 and 130 min, in each set-up

the total experimental time was 120 min, but the pH

adjustment of the media took 5 min longer than the

temperature shift, accounting for the 5-min differences in

the time points). Significant up-regulation occurred if the

ratio of a spot of one experiment compared with its refer-

ence was at least 2. Ratios were determined by the

comparison of the % volume of a spot in a 2D gel of the

stress condition to the % volume of the same spot in the

reference gel. The numbers given for the % volume repre-

sents the relative portion of one spot of the total protein

present on the complete gel. Each experiment was

confirmed in two independent biological experiments. The

mean of the ratios of the same spot from the two experi-

ments was calculated (Supporting Information Tables S1a–d

and S2).

2.6 In silico analysis

2.6.1 Generation of a virtual proteome map

A in silico proteome map of P. torridus was generated using

the web-tool JVirGel version 2.0 (http://www.jvirgel.de/

index.html) [17].

2.6.2 Discovery of sequence motifs

For the analysis of the upstream DNA regions of the up-

regulated proteins, multiple sequence alignments were

done. The 50-bp upstream region of the coding sequence of

each up-regulated protein was loaded to the MEME database

Proteomics 2011, 11, 4559–4568 4561


(http://meme.sdsc.edu/meme/intro.html) [18]. MEME

calculates the significance of conserved sequence motives

from a multiple sequence alignment.

3 Results and discussion

3.1 Generation and analysis of a proteomic map of

P. torridus

Virtual maps of the proteome of P. torridus were generated

for pH 4–7 and 3–10, respectively using the web tool JVirGel

V2.0 (http://www.jvirgel.de/index.html) (Supporting Infor-

mation Fig. S1A and B). This virtual map showed the main

pI regions that were also described in other bacteria [19–24].

The 723 (pH 4–7) and 1345 (pH 3–10) visible spots,

including intracellular and membrane proteins, correspond

to 47 and 87% of the theoretical proteome, respectively. This

is similar to the findings of Majumder et al. [22] and showed

that the theoretical molecular weight and isoelectric point

distribution of the P. torridus proteins are not significantly

different from those of organisms growing in less extreme

environments. Only small differences in the overall amino

acid composition of the complete genome-derived proteome

were observed [8]. Unfortunately, only few three-dimen-

sional structures of P. torridus proteins are available for

detailed analysis.

The soluble fraction of P. torridus cells was separated by

isoelectric focusing in the pH ranges of 4–7 and 3–10 in the

first dimension, followed by a 2D separation on 12% SDS-

PAGE gels. We were able to detect in total 717 Coomassie

collodial-stained protein spots in both gels (pH 4–7 and

3–10, Supporting Information Fig. S2A and B). Six hundred

and sixty-five protein spots in both gels could be identified

with LC-MS/MS analysis (Supporting Information Tables

S3a–b and S4). Analysis revealed that 99 of these protein

spots could only be identified on the gel pH 3–10, whereas

64 protein spots were unique on gel pH 4–7. We also

identified 35 spots that were visible as a single spot after

Coomassie collodial staining but resulted in two sequences

after MS/MS analysis (Supporting Information Table S3a

and b). The assignment of corresponding protein annota-

tions to the identified spots was derived from the reference

genome sequence of P. torridus (NCBI accession number:

NC_005877.1; Supporting Information Table S5a and b).

Further analysis revealed that the 665 identified spots

represent 251 different gene products in both gels. Proteins

with the same annotation that string together on the gel are

supposed to be a result of posttranslational modification

(PTM). These proteins are visible as chains of spots but

represent the same proteins (compare Supporting Infor-

mation Fig S1A spot nos. 5–11). These proteins presumably

change their running characteristics due to their possible

PTM and occur as highly similar isoforms. Isolelectric

heterogeneity is a common phenomenon in the 2D gel

electrophoresis method and has been described in several

other studies [22, 25–28]. One reason for PTM is the

carbamylation by urea or deamidation [26]. The number of

the identified 251 different proteins is equivalent to 16% of

the predicted 1537 ORFs deduced by genome sequence

analysis [8]. This number is in the same range (3–21%)

found in other proteomic studies [22, 25, 29–32]. For

example, Barry et al. [29] generated a proteomic map for

another thermoacidophile, Sulfolobus solfataricus, and were

able to identify proteins corresponding to 11% of the ORFs

that were predicted from the genome sequence. Thirty-five

percent (pH 3–10) and 26% (pH 4–7) of the visible spots on

the virtual gels (Supporting Information Fig. S1A and B) are

predicted membrane proteins.

Despite the complexity of the membrane proteins and

general problems to grasp these proteins in 2D protein

electrophoresis, we were able to separate a few of them on

the 2D gel. Based on the membrane prediction of the JVir-

Gel tool, 85 of the 251 identified proteins are predicted

membrane-associated or even transmembrane proteins.

This corresponds to 33% of our identified proteins.

The identified proteins of the P. torridus master gel were

sorted into different functional categories according to their

annotation derived from the genome sequence (NCBI

Reference sequence: NC_005877.1). The largest number of

the identified proteins (18%) was classified into the energy

metabolism category, followed by hypothetical proteins,

others, those involved in protein synthesis, amino acid

metabolism, energy conservation and nucleotide metabo-

lism (Fig. 1). Groups with low numbers of identified

proteins (1–3%) were those involved in RNA metabolism,

transport, regulation, cofactor metabolism, fatty acid meta-

bolism and protein degradation.

The functional category distribution of the proteins from

the P. torridus proteomic map was compared with the

theoretical proteome obtained from genome sequence

analysis and with the virtual proteome generated with the

JVirGel web tool. This comparison gives information about

the relative number of proteins from the different functional

categories (and hence an indication about their importance)

under the given growth conditions (Fig. 2). Due to the fact

that the virtual proteome under the chosen parameters

(Supporting Information Fig. S2) represents 87% (pH 3–10)

of the theoretical proteome deduced from the genome

sequence, we could only find slight decreases in the number

of protein synthesis and the category of hypothetical

proteins. The largest discrepancy was found in the hypo-

thetical, energy metabolism and protein synthesis categories

when comparing the theoretical proteome to the number of

proteins of the P. torridus master gel. Indeed, the proportion

of expressed proteins belonging to the hypothetical proteins

category was merely 63% of the proportion of the hypothe-

tical ORFs deduced from genome data alone. The under-

representation of hypothetical proteins in the master gel

observed by us has also been reported for other proteomic

studies [29, 33]. This observation may be due to a low-level

abundance of proteins from this group that are used under



these growth conditions and/or false ORF predictions, but it

also confirms that the function of the majority of the

abundant soluble proteins is already known. Conversely, the

18% of the energy metabolism category, as well as the 11%

of the protein synthesis category, were found to be twofold

overrepresented in the P. torridus obtained master gel

compared with its proportion observed from the genome

sequence or the virtual 2D gel, indicating the essential role

of these proteins in the exponential growth phase. In

accordance, it is conceivable that due to its extreme envir-

onment combining pH values near 0 and simultaneously

high temperature and its need to avoid over-acidification of

the cytoplasm under these conditions, P. torridus has a high

energy demand which must be met by an active central

carbon metabolism and an efficient respiratory apparatus.

A map with an overview of the central metabolism of

P. torridus which was based on the genome sequence has

been constructed by F .utterer et al. [8]. With the 2D master

Figure 2. Comparison of the distribution of the different functional classes of proteins identified in this study (dark gray) to the theoretical

proteome deduced from P. torridus genome sequence (mid gray) and to a virtual proteomic map generated with JVirGel 2.0 (light gray).

Figure 1. Distribution of the

identified proteins according to

their functional categories. Two

hundred and fifty-one different

proteins were identified by LC-

MS/MS.

Proteomics 2011, 11, 4559–4568 4563


gels and the proteomic analysis presented here, we tried to

validate this map under physiological conditions. We could

identify most proteins that are involved in the basic meta-

bolic pathways such as glycolysis and the tricarboxylic acid

(TCA) cycle (Fig. 3). From the genome sequence, it was

predicted that P. torridus most likely catabolizes glucose via a

non-phosphorylated variant of the Entner–Doudoroff (ED)

pathway, because the genes for all steps have been assigned

[8]. Except for the fructose-1,6-bisphosphate aldolase, all

genes required for the Embden–Meyerhof–Parnas (EMP)

pathway are also present. Therefore, it was assumed that a

non-classical fructose-1,6-bisphosphate aldolase may be

present in P. torridus, and the EMP pathway might also be

functional, at least for gluconeogenesis [8]. With the iden-

tified spots of the proteomic map, we could identify only

proteins belonging to the non-phosphorylated ED pathway

(Fig. 3). This result confirms the assumption that this

pathway is most likely used in P. torridus to catabolize

glucose, which is in line with the observation that this is the

usual mode of glucose breakdown in thermoacidophilic

archaea [34, 35]. In agreement, it has recently been shown

by 13C-labeling experiments that in P. torridus glucose is

Figure 3. Overview of experi-

mentally detected protein spots

corresponding to enzymes,

which are involved in the

central metabolic pathways of

P. torridus.



metabolized exclusively via the ED pathway [36]. Except for

2-methylcitrate synthase we could identify all proteins of the

TCA cycle and the 2-methylcitrate pathway for the oxidation

of propionyl CoA. For the pathway of the oxidative phos-

phorylation reaction, we could also detect a few of the

enzymes (PTO0995-998, PTO1133, PTO1119, PTO1123,

PTO0487, PTO0849, PTO0500) that are associated with the

different complexes of the respiratory chain (Fig. 3).

Comparative proteome analysis of P. torridus cells

grown under different conditions

The proteomic map of P. torridus described here allowed us

to perform experiments aimed at detecting changes in

protein expression triggered by exposing cells to stress

conditions. After growing P. torridus cells under optimal

growth conditions (pH 1.0 and 601C) until early exponential

phase, the cultures were split and subjected to high-

temperature (701C) and low- temperature (501C) stress and

the changes in protein expression were analyzed after 5, 35

and 125 min. Similarly, the effect of pH stress was investi-

gated by exposing P. torridus cells to low (0.5) and high (1.8)

pH and performing quantitative 2D PAGE of samples

collected after 10, 40 and 130 min. The most pronounced

changes in protein expression were observed during the pH

stress experiment. Both low and high pH stress elicited a

strong reaction which is reflected by the high numbers of

about 30 up-regulated proteins each under both pH stress

conditions (Table 1). The alteration in protein expression

occurred immediately after exposure to pH stress (10 min),

and the number of up-regulated proteins decreased when

the incubation was continued (130 min). In contrast, the

response in protein abundance after heat shock (701C)

seemed to be more prolonged, i.e. the first changes were

detectable after 35 min and the number of altered proteins

increased with time. Incubation of P. torridus at 501C yielded

the least changes in differential proteins expression – a total

of 10 proteins were found to be up-regulated only after

125 min of incubation (Table 1). The fact that merely a few

changes were detected in the proteome pattern after lower-

ing the growth temperature to 501C indicates that only few

adaptive changes suffice to adjust cellular metabolism to a

moderately reduced growth. It also indicates that 501C is

still in or close to the physiological scope of P. torridus.A major part of the proteins that were found to be

up-regulated under the different stress conditions belonged

to the functional categories of protein synthesis (18

proteins), other (17 proteins) and energy metabolism (12

proteins; Table 1). Surprisingly, the largest number of

significantly up-regulated proteins (21) was found to belong

to the category of proteins with unknown function (hypo-

thetical), especially in the low pH exposure experiment. This

observation is further emphasized by the fact that the group

of hypothetical proteins was found to be underrepresented

in the proteome–genome data set comparison (Fig. 2).

These findings point out the importance of research on

hypothetical proteins and the determination of their func-

tion to get a better understanding on processes that are

potentially based on these hypothetical proteins. On the

other hand, none of the stress stimuli used in these

experiments resulted a change in the expression of proteins

Table 1. Numbers of protein spots that were at least twofold up-regulated under the different stress conditions sorted by functionalcategory

Functional category Number of proteins

Temperature stress pH stress

501C 701C pH 0.5 pH 1.8

t5 t35 t125 t5 t35 t125 t10 t40 130 t10 t40 t130 Total

Protein degradation 0 0 0 0 1 0 0 1 0 0 2 0 4Protein synthesis 0 0 3 0 2 4 3 1 1 3 0 1 18Nucleotide metabolism 0 0 0 0 0 0 0 0 0 1 0 0 1Energy metabolism 0 0 1 0 2 2 0 1 1 1 4 0 12Other 0 0 0 0 2 3 2 3 1 0 4 2 17Amino acid metabolism 0 0 2 0 0 0 0 0 0 0 0 0 2Fatty acid metabolism 0 0 0 0 0 0 0 1 0 0 0 0 1Energy conservation 0 0 1 0 1 1 1 1 0 2 2 1 10DNA metabolism 0 0 1 0 0 2 0 1 1 0 0 1 6Hypothetical 0 0 2 0 0 2 6 3 0 3 3 2 21Regulation 0 0 0 0 1 0 1 0 0 0 0 0 2Cofactor metabolism 0 0 0 0 0 1 0 1 1 1 1 0 5RNA metabolism 0 0 0 0 0 0 0 0 0 0 0 0 0Transport 0 0 0 0 0 0 0 0 0 0 1 0 1Total 0 0 10 0 9 15 13 13 5 11 17 7 100

Proteomics 2011, 11, 4559–4568 4565


of the RNA metabolism category. Additionally, temperature

stress failed to induce proteins from the nucleotide,

fatty acid metabolism and transport functional categories

(Table 1). Only one protein of the nucleotide metabolism

(pH 1.8) and one protein of the transport and the

fatty acid metabolism category (pH 0.5) were changed.

Considering the fact that proteins of these categories

have the lowest abundance compared with all proteins

of the genome (Fig. 2), the findings may result in such

a low detection during the stress experiments. On the other

hand, the proteins from the transport and fatty acid

metabolism category are often associated with the

membrane. Therefore, the small number of up-regulated

proteins of these categories may also be due to the low

number of membrane proteins that we could resolve on our

proteomic map.

A closer look at the individual proteins indicates that

there are ORFs that are specifically expressed during a

single stress condition, while others are up-regulated under

more than one stress condition. Several proteins were found

to be up-regulated by all of the stress stimuli tested except

lowering the temperature by 101C, indicating their potential

role as universal stress proteins. For example, SSU riboso-

mal protein (PTO1280), the TENA/THI-4 family protein

(PTO1372), the succinyl-CoA synthetase (PTO0893) and the

thioredoxin reductase (PTO0734) were all up-regulated in

response to the stimuli high and low pH and high

temperature. Other proteins were only up-regulated under

two conditions and some were specific for one stress

stimulus (Fig. 4). Homologs of most of these proteins have

been reported to be up-regulated under stress conditions in

other organisms [37–40]. Therefore, we assume that we

could detect a general stress response in P. torridus under

the chosen conditions.

In many regulatory pathways, groups of genes are regu-

lated by a shared DNA-binding regulatory protein such as a

sigma factor in bacteria [41–44]. However, with the analysis

of the 50 bp upstream regions of the up-regulated candidate

heat/pH shock genes identified in this proteomic study

using the MEME tool (http://meme.nbcr.net/meme4_6_1/

intro.html), we could not identify a significant consensus

binding motif that may indicate a common regulation (data

not shown).

4 Concluding remarks

In conclusion, with this study we have established the

methods for 2D electrophoretic separation and identification

of soluble Picrophilus proteins. The master gels and

accompanying information shown here can be used as tools

for physiological studies of this thermoacidophile. As an

example, we have used the 2D-PAGE-based proteomic map

to analyze differential protein abundances in P. torridus cells

Figure 4. Comparative analysis of the stress experiments to detect proteins that were universally up-regulated in all experiments.

Proteins that are up-regulated in all experiments except temperature drop to 501C, proteins that are up-regulated at both pH stress

experiments. Proteins that are up-regulated at low pH and high temperature, protein that is up-regulated at high pH and

high temperature. Two proteins that are up-regulated during low pH and low temperature. Protein that is up-regulated in both

temperature experiments. Diagram shows an intersection of all three harvesting points.



after subjecting growing cells to shifts of pH and tempera-

ture. As a next step, it would be of particular interest to

analyze the roles of selected hypothetical proteins found to

be up-regulated under low- or high-pH shock conditions by

gene knock-out studies. Unfortunately, however, genetic

modification techniques are not yet available for the most

extreme thermoacidophilic organisms known on earth.

Financial support by the German Federal Ministry ofEducation and Research (BMBF) in the framework of theGenoMik programme is gratefully acknowledged.

The authors have declared no conflict of interest.

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