The proteomic alterations of Thermoanaerobacter tengcongensis cultured at different temperatures.

Abstract

Thermoanaerobacter tengcongensis, one of many thermophilic organisms, survives harsh living conditions in temperatures ranging from 50 to 80 degrees C. In this comprehensive analysis, we present a robust approach, 2-DE and MALDI-TOF MS, to compare and identify the bacterial proteins responding to the temperature stress. In total, 164 spots of 2-DE were found with the significant changes in spot volume at three culture temperatures, 55, 75, and 80 degrees C, respectively; furthermore, 87 unique proteins were characterized by MS. Our results reveal that the electrophoretic images of the bacterial proteins, extracted from two culture temperatures (55 and 75 degrees C), had similar patterns; however, the bacteria cultured at 80 degrees C had dramatically decreased their spot volumes. Additionally, the temperature-sensitive proteins are broadly divided into two groups: specific expression at certain temperatures and consistent changes of expression responsive to temperature. For instance, three proteins closely related with redox regulation, dihydrolipoamide acyltransferase, NADH:ubiquinone oxidoreductase, and ferredoxin, were only detected in the bacteria cultured at 55 degrees C. Whereas, two chaperonins, GroES and GroEL, were found to show a consistent increase during the elevated temperatures with the determinations, either by MS or Western blot. The proteomic information, thus expedites our understanding of the molecular mechanisms regarding how thermophilic bacteria adapt to the alterations in living environment.

Full text

Page 1

RESEARCHARTICLE
The proteomic alterations of Thermoanaerobacter
tengcongensis cultured at different temperatures
Jingqiang Wang1, 2, Caifeng Zhao1, 2, Bo Meng1, 2, Junhua Xie1, 2, Chuanqi Zhou1, 2,
Xishu Chen1, 2, Kang Zhao1, 2, Jianmin Shao1, 2, Yanfen Xue3, Ningzhi Xu1, 2,
Yanhe Ma3*and Siqi Liu1, 2, 4
1Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, China
2Beijing Proteomics Institute, Beijing, China
3The Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
4The Department of Medicine, University of Louisville, Louisville, KY, USA
Thermoanaerobacter tengcongensis, one of many thermophilic organisms, survives harsh living
conditions in temperatures ranging from 50 to 807C. In this comprehensive analysis, we present
a robust approach, 2-DE and MALDI-TOF MS, to compare and identify the bacterial proteins
responding to the temperature stress. In total, 164 spots of 2-DE were found with the significant
changes in spot volume at three culture temperatures, 55, 75, and 807C, respectively; further-
more, 87 unique proteins were characterized by MS. Our results reveal that the electrophoretic
images of the bacterial proteins, extracted from two culture temperatures (55 and 757C), had
similar patterns; however, the bacteria cultured at 807C had dramatically decreased their spot
volumes. Additionally, the temperature-sensitive proteins are broadly divided into two groups:
specific expression at certain temperatures and consistent changes of expression responsive to
temperature. For instance, three proteins closely related with redox regulation, dihydrolipoamide
acyltransferase, NADH:ubiquinone oxidoreductase, and ferredoxin, were only detected in the
bacteria cultured at 557C. Whereas, two chaperonins, GroES and GroEL, were found to show a
consistent increase during the elevated temperatures with the determinations, either by MS or
Western blot. The proteomic information, thus expedites our understanding of the molecular
mechanisms regarding how thermophilic bacteria adapt to the alterations in living environment.
Received: April 12, 2005
Revised: August 16, 2006
Accepted: January 26, 2007
Keywords:
GroEL / GroES / MALDI-TOF MS / Temperature-dependence / T. tengcongensis
Proteomics 2007, 7, 1409–1419
1409
1Introduction
Thermoanaerobacter tengcongensis, an anaerobic, Gram-nega-
tive, rod-shaped bacterium, is an extremely thermophilic
eubacterium found in the Tengchong hot springs, Yunnan
province, China [1]. Its genomic structure has been
sequenced, paving a way to explore the underlying molecular
mechanisms of this bacterium surviving in harsh conditions
[2]. Recently, we have reported the proteomic analysis of
T. tengcongensis at an optimal culture condition [3]. At 757C,
we experimentally determined approximately 46% of the
theoretical proteome using an integrated proteomic strategy,
combining three different methods, shotgun digestion plus
HPLC with IT MS/MS (shotgun-LC/MS), 1-D SDS-PAGE
plus HPLC with IT MS/MS (1-DE-LC/MS), and 2-DE plus
MALDI-TOF MS (2-DE-MALDI-TOF MS).
Correspondence: Dr. Siqi Liu, Beijing Genomics Institute, Chi-
nese Academy of Sciences, Beijing Airport Industrial Zone B-6,
Beijing 101300, China
E-mail: siqiliu@genomics.org.cn
Fax: 186-10-8048-5324
Abbreviation: PFOR, pyruvate ferredoxin oxidoreductase
* Additional corresponding author: Dr. Yanhe Ma,
E-mail: mayanhe@sun.im.ac.cn
DOI 10.1002/pmic.200500226
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com

Page 2

1410
J. Wang et al.Proteomics 2007, 7, 1409–1419
A number of questions, poorly annotated by genomic
data have been addressed properly through the proteomic
information of T. tengcongensis. For instance, a key enzyme
involved in glycolysis, glucose kinase, was missed in the
annotation from the genomic data. By the combination of
two sets of data from proteomic determination as well as
functional domain screening, two candidates of glucose
kinase, TTE0090 and TTE1961, were verified [3], and fur-
thermore their catalytic activity was confirmed in the
recombinant forms (data not shown). In T. tengcongensis ge-
nome, there is no gene annotated as pyruvate dehy-
drogenase or pyruvate decarboxylase, thus, the formation of
acetyl-CoA, which is a fundamental element for controlling
anaerobic fermentation, is unlikely to be catalyzed by the
two enzymes. With careful analysis of proteomics, a large
amount of proteins of pyruvate ferredoxin oxidoreductases
(PFORs), which can catalyze the conversion of pyruvate to
acetyl-CoA, were detected in high abundance, and also the
abundant expressions of phosphate transacetylase and ace-
tate kinase, which catalyze acetyl-CoA to acetyl-P and acetyl-
P to acetate, respectively, were identified with all the three
proteomic approaches [3]. On the basis of the observation,
we proposed a model that pyruvate formation is a fork in
the metabolic pathway, and PFOR regulates the generation
of the final metabolites in T. tengcongensis, either acetate or
ethanol.
The expression profiles of T. tengcongensis at optimal
temperature offer the information extending our knowledge
on how this bacterium survives at high temperature. An
interesting question is, moreover, how this bacterium could
grow within a wide range of temperatures from 50 to 807C
[1]. How does T. tengcongensis adapt to the environment with
alternated temperatures? How are the bacterial proteins
expressed at different temperatures? Obviously, the prote-
omic data at optimal temperature cannot respond to these
questions. The temperature regulations of gene expression
in mesophilic system are different from thermophilic bac-
teria [4]. Nocker et al. [5] proposed that repression of heat-
shock gene expression (ROSE) in Bradyrhizobium japonicum
was a novel regulation system that functions by a cis-acting
element positioned in the 50-untranslated region. The ribo-
some-binding site is protected by the formation of stem-loop
from the 30-region of ROSE, but elevated temperature is able
to disrupt the secondary structure. In some of the hyper-
thermophilic bacteria, heat-shock proteins are only synthe-
sized above the temperature thresholds. For instance, Pyro-
coccus furiosus has a single copy of a short heat-shock protein
gene, whose expression is induced by heat shock regulated
by a putative heat-shock regulator (Phr) [6]. There remain so
many mysteries to be explored: e.g. how thermophilic pro-
teins are regulated and function at varied temperatures.
Since the individual bacteria follow different mechanisms of
expression and regulation, therefore, the information
regarding the temperature-dependent proteins is naturally
required in detail for better understanding of the survival
mechanisms of T. tengcongensis.
In this communication, we report a proteomic survey for
T. tengcongensis cultured at three different temperatures, 55,
75, and 807C, respectively. Using the techniques of 2-DE, 164
spots were found to show significant changes in spot inten-
sities at the different culture temperatures. A total of 87
unique proteins were characterized by the approach of MS.
Out of these temperature-dependent proteins, some corre-
late well with the growth rate of this bacterium. The prote-
omic information thus expedites our knowledge for the mo-
lecular mechanisms of how T. tengcongensis survives in hot
springs.
2 Materials and methods
2.1 Materials
All chemicals, IPG strips, and ampholytes employed for
electrophoresis were purchased from Amersham Bio-
sciences(Uppsala, Sweden). All chemicals ofanalytical grade
were from Sigma (St. Louis, MO, USA). All HPLC grade
solvents were from J. T. Baker (Phillipsburg, NJ, USA).
CHCA was purchased from Bruker Daltonics (Bremen, Ger-
many). Modified trypsin (sequence grade) was obtained from
Promega (Madison, WI, USA). The components of culture
media were from Oxoid (Basingstoke, England).
2.2 Bacterial culturing conditions
T. tengcongensis strain MB4T(T = type strain) was activated
and cultured in the modified MB medium at 757C, as
described earlier, until its OD at 600 nm reached to 0.5 [1].
The activated bacteria were transferred into the medium
prewarmed at 55, 75, and 807C, respectively, and cultured
under the indicated temperatures. To monitor the growth
curves of bacteria, the cultured bacteria were withdrawn at
various time intervals followed by the measurement of
OD600 nm. When the bacteria grew to the middle stage of log
phase, the cells were harvested and washed with isotonic
buffer containing 10 mM Tris-HCl, pH 7.0, and 250 mM
D-sorbitol. The cell pellets were collected after centrifugation
at 30006g for 15 min at 47C. The growth curves of bacteria
were fitted to a regression equation
A = A01 a/[1 1 exp(L 2 t)/b]
In this equation, A represents OD values at 600 nm, t is the
time forcell culture, A0isthe constant ODat lag phase, and L
is the time reaching to the middle stage of log phase.
2.3 Protein extraction
The cell pellets of T. tengcongensis collected from different
culture temperatures were ground to a fine powder by a
metal pulverizer immersed in liquid nitrogen. The T. teng-
congensisproteins were precipitated in precooled10% TCA in
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com

Page 3

Proteomics 2007, 7, 1409–1419
Microbiology
1411
acetone containing 10 mM DTTand incubated at 2207C for
3 h followed by centrifugation at 350006g for 15 min. The
precipitates were washed with precooled acetone containing
10 mM DTT, 1 mM PMSF, and 2 mM EDTA, and dried by a
Speedvac. The dried pellets were finally suspended in lysis
buffer containing 8 M Urea, 4% CHAPS, 10 mM DTT, 0.5%
ampholyte (pH 3.0–10.0), 1 mM PMSF, 2 mM EDTA, and
20 mM Tris-HCl, pH 8.5, followed by sonication for 10 min
and centrifugation at 400006g for 30 min. Protein con-
centration was measured by Bradford assay.
2.4 2-DE
The T. tengcongensis proteins (1.3 mg/gel) were rehydrated
overnight with the commercial IPG strips (Amersham Bio-
sciences), 24 cm with a linear range of pH 4.0–7.0. Electro-
focusing was carried out for 70 k?Vh using IPGphor
(Amersham Biosciences) at 207C following the manu-
facturer’s instruction (Amersham Biosciences). Prior to the
second dimension, the IPG strips were equilibrated by two
equilibration steps [3]. The electrophoresed strips were
loaded and run on 12% polyacrylamide Laemmli gels
(26620 cm2) using the Ettan DALT II system with a pro-
grammable power control, 0.5 h at 2.5 W per gel, then at
15 W per gel until the dye front reached the gel bottom. The
separated proteins were visualized by CBB R250 staining.
2.5 Gel image analysis
The 2-DE images were acquired with an Image Scanner
(Amersham Biosciences)ina transmissionmode.The image
analysis was carried out by the combination of manual visu-
alization and software analysis with Image Master Elite ver-
sion 4.1 (Amersham Biosciences). To have comparable data
for quantitative analysis, several key parameters in the image
analysis were fixed as constant [3]. The average spot intensity
was normalized to the total spot volume with a multi-
plication factor of 100.
2.6 In-gel digestion
The spots on 2-DE gels were carefully excised, successively
destained, and dehydrated with ACN. The proteins were
reduced with 10 mM DTT in 25 mM ammonium bicarbo-
nate at 567C for 1 h and alkylated by 55 mM iodoacetamide
in 25 mM ammonium bicarbonate in the dark at room tem-
perature for 45 min in situ. Finally, the gel pieces were thor-
oughly washed with 25 mM ammonium bicarbonate in
water/ACN (50:50) and completely dried by Speedvac. Pro-
teins were digested in 10 mL of modified trypsin solution
(1 ng/mL in 25 mM ammonium bicarbonate) with overnight
incubation at 377C. The digestion reaction was stopped by
1 mL of 10% TFA.
2.7 Mass spectrometric analysis
Gel spot digestions were desalted by Poros R2 and mixed
with 0.6 mL of matrix solution consisting of CHCA (12 mg/
mL) in 70% ACN with 0.1% TFA. The slurry was applied
onto the target well, dried at room temperature and sub-
jected into a Bruker AutoFlex MALDI-TOF MS (Bruker Dal-
tonics). The mass spectrometer was operated under 19 kV
accelerating voltage in the reflectron mode and the m/z
range of 600–4000. The monoisotopic peptide masses
obtained from MALDI-TOF MS were analyzed by m/z soft-
ware. Mass spectra were internally calibrated with peptides
arising from trypsin autoproteolysis at m/z = 842.509 and
2211.105 to reach a typical mass measurement accuracy of
100 ppm.
Some digestive products from 2-DE spots were analyzed
by LC-MS/MS using an LCQ DecaXPIT MS (Thermo Finni-
gan, San Jose, CA, USA) for further confirmation of the
amino acid sequences. After capillary RP HPLC, the sepa-
rated peptides were subjected to IT MS with 3.2 kV of spray
voltage and 1507C at the heated desolvation capillary. The
m/z range of 400–2000 was scanned in 1.2 s, and ions were
detected with a high energy Conversion Dynode detector.
2.8 Protein identification using database search
algorithm
Database searches were conducted using MASCOTsoftware
1.9 (Matrix Science) against the protein database of T. teng-
congensis generated by our institute with one fixed protein
modification, carboxamidomethylation of cystein residues,
and two variable modifications, methionine oxidations and
N-terminal pyroglutamylation. Positive identification was
achieved with the quality criteria that 100 ppm mass accu-
racy and maximally one or two missed cleavage sites
matched with a significant probability score (above 47), and
nearly all the dominant signals of the spectrum were
assigned to the protein or mixture of proteins identified. The
LC-MS/MS data were converted into DTA-format files which
were further searched for proteins with TurboSEQUEST
(Thermo Finnigan) or MASCOT.
2.9 Generation of polyclonal antibodies against
GroEL and GroES
The expression vectors of GroEL (TTE0580) and GroES
(TTE0579) were generated by insertion of the two interested
genes into pET28a, a bacterial expression vector with His-tag
at the N-terminus. The recombinant proteins of GroEL and
GroES were expressed in Escherichia coli BL-21 strain after
IPTG inducement, and purified with Ni21affinity chroma-
tography. The antibodies against GroEL and GroES were
produced by immunization of rabbit with the purified
recombinant proteins followed by three boosts. Using pro-
tein A affinity resin, the antibodies in rabbit sera were par-
tially purified.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com

Page 4

1412
J. Wang et al.Proteomics 2007, 7, 1409–1419
2.10 Western blot analysis
The extracted proteins from T. tengcongensis(20 mg) were first
separated by 2-DE (pH 4.0–7.0, 7.0 cm), and transferred onto
a PVDFmembrane. After blocking the nonantigen sites with
TTBS buffer (0.1 M Tris pH 7.9, 0.15 M NaCl, 0.1% Tween
20, and 5% nonfat milk) at 377C for 60 min, the primary
antibody against GroEL or GroES was incubated with the
blotted PVDF membrane at 47C for overnight. The immuno-
recognized antibodies were captured by the secondary
antibody against rabbit IgG conjugated with alkaline phos-
phatase. Finally, the immunoreactivespots werevisualized by
color developing using nitro-blue tetrazolium chloride (NBT)
and 5-bromo-4-chloro-30-indolylphosphate p-toluidine salt
(BCIP) as the substrates.
2.11 Statistical analysis
The average values of the parallel experiments are given as
the mean 6 SD. Comparison of differences among the
groups was carried out by Student’s t-test. Significance was
defined as p,0.05.
3 Results
3.1 The growth curves of T. tengcongensis at
different culture temperatures
When T. tengcongensis was cultured in modified MB medium
with pH 7.5, it could survive at the temperature range of 50–
807C. This bacterium, activated at the optimum temperature
followed by reculturing at three different temperatures, grew
at very different rates as depicted in Fig. 1. T. tengcongensis
recultured at 757C went to the log phase in approximately
8 h, and reached the plateau within 18 h; however, it took a
much longer time to adapt to the alternative temperature,
either at 55 or 807C. The lag phase at 557C for T. tengcongensis
almost extended to 20 h (Fig. 1), whereas, this phase at 807C
was even slower after adaptation of 50 h (Fig. 1). Upon the
growth curves, two facts merit attention. First of all, the cell
intensities of T. tengcongensis at saturation stage in both tem-
peratures, at 55 and 757C, are quite close even though the
bacteria has a longer lag period at 557C, whereas the bacteria
cultured at 807C displayed significantly low OD values at
600 nm. The other phenomenon is that the death of bacterial
cells obviously exceeds the growth, once T. tengcongensis
reaches the growth plateau at 807C because OD600 nmdrops
down significantly after the plateau; the bacteria cultured at
55 or 757C, however, retain the saturation status with stable
OD values for some time. Therefore, an interesting question
arising from the growth curves is whether the physiological
behavior of T. tengcongensis at 807C is significantly different
from that of the cells cultured at a relatively low temperature
in the same medium.
Figure 1. The growth curves of T. tengcongensis at different cul-
ture temperatures. The sample collections and the curve fittings
are described in detail in Section 2. The symbols “D”, “u”, and
“s” represent the bacteria cultured at 80, 55, and 757C, respec-
tively.
3.2 The proteomic profiles of T. tengcongensis at
different culture temperatures by 2-DE
According to our previous study [3], most of the proteins of
T. tengcongensis expressed under optimal temperature were
distributedaroundpI regionsof4.0–7.0correspondingtothe
range of molecular masses from 10 to 100 kDa. We therefore
employed narrow pH strips of 4.0–7.0 and 12% polyacryl-
amide for a better separation of the T. tengcongensis proteins
cultured at different temperatures. On the basis of the 2-DE
images shown in Fig. 2, the outline of the expression profiles
is established with a satisfactory resolution, at least for the
relatively abundant and soluble proteins of T. tengcongensis;
moreover the patterns of 2-DE spot distribution from all of
three samples are similar but the number of 2-DE spots at
807C is significantly decreased (Fig. 2). In order to obtain a
reliable result from 2-DE images for quantitative analysis,
three parallel 2-DEs were run for each sample. The total
number of 2-DE spots from the three samples were statisti-
cally counted: 314 6 10 at 557C, 327 6 10 at 757C, and
230 6 6 at 807C. Although the bacteria took 20 h to adapt to
the change of culture temperature from 75 to 557C, the cell
intensity was comparable with those cultured at 757C, once
they adapted to the living environment (Fig. 1). The observa-
tion was further supported by the results of 2-DE images be-
cause the expression profiles of the bacterial proteins
extracted from the two culture temperatures (55 and 757C)
have been revealed in similar patterns, even though the
intensities of some spots were different on the gels. Inter-
estingly, the low protein yield correlates with the small value
of cell intensity at 807C, indicating that the protein expres-
sion is globally inhibited under such high temperature. In
contrast to most of the bacterial proteins, a few of them were
found to show overexpressionat 807C, which are reasoned to
perform the stabilizing functions for this bacterium surviv-
ing at high temperature.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com

Page 5

Proteomics 2007, 7, 1409–1419
Microbiology
1413
3.3 Analysis of 2-DE images to define the
temperature-dependent bacterial proteins
To identify these bacterial proteins which are sensitive to the
culture temperatures, it is necessary to conduct a careful
analysis of 2-DE images. The image comparison was based
upon the following procedure: (i) the intensities of 2-DE
spots from T. tengcongensis cultured at certain temperature
were normalized as a reference; (ii) the spot volume differ-
ences over three-folds between any two temperature treat-
ments were considered to be significant; (iii) the spots with
significant changes were analyzed throughout the three cul-
ture temperatures.
With careful analysis of 2-DE image using a combination
of software and manual, 67 spots were detected with signifi-
cant changes in their spot volumes between 55 and 757C, 118
spots were found between 75 and 807C, whereas 142 spots
were confirmed between 55 and 807C. Moreover, while set-
ting the 2-DE image of bacterial proteins at 757C as a refer-
ence, the gel spots affected by the adaptation at 557C were
unbiased both in up-regulated and in down-regulated spots,
numbering 38 and 29, respectively (Table 1). However, the
down-regulated spots caused by the adaptation at 807C were
much more than up-regulated spots (93 vs. 25) (Table 1),
suggesting that the adaptiveprocessofthis bacteriumat high
temperature may be mainly attributed to the inhibition of
protein expression or the enhancement of protein degrada-
tion.
In the next step of 2-DE image analysis, some variable
spots during temperature alterations were focused on a
quantitative comparison, in which two types of temperature-
dependent spots are specifically interesting, some spots
found only at a certain temperature and some with consistent
changes throughout all the three temperatures. Table 2 sum-
marizes that the patterns of the 2-DE spots resulted from the
image comparisons. The unique 2-DE spots detected at 55
and807Cbacterialextracts,respectively,aresimilar (16vs.17),
but only 11 such spots are found in the bacteria cultured at
757C. Except for a few of the spots, such as spot 148, most of
the unique spots display low staining intensities with varied
patterns of distribution on 2-DE (Fig. 2). For reasons
unknown, the sample preparation of the bacteria cultured at
807Cwasnot assatisfactory asatothertemperaturesresulting
in its 2-DE gels with less sharp resolution, especially at the
range of high molecular mass around 43–66 kDa (Fig. 2C).
Comparison of these different spots with quantitative
changes in spot volume which are shared only at two tem-
peratures, the spots (47 spots) shared with 55 and 757C, are
much higher than those (nine spots) shared at 75 and 807C,
indicating that the protein expression at 807C is quite differ-
ent from the other two temperatures somehow (Table 2).
Looking deeply forspotswith alterationsinspot volumesover
the entire range of culture temperatures, the number of the
spots with increased spot volumes is significantly lower than
those with decreased spot volumes (12 vs. 26) following the
culture temperature rising upwards (Table 2). In Fig. 3, there
Figure 2. Separation of the T. tengcongensis proteins by 2-DE
approach. (A) The 2-DE image of the proteins extracted from the
T. tengcongensis cultured at 557C; (B) The 2-DE image of T. teng-
congensis proteins at 757C; and (C) The 2-DE image of T. teng-
congensis proteins at 807C. The spots with qualitative and quan-
titative changes in spot volume due to temperature alternations
are labeled accordingly.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com

Page 6

1414
J. Wang et al.Proteomics 2007, 7, 1409–1419
Table 1. Quantitative comparison of 2-DE spots for the protein extracts of T. tengcongensis cultured at different temperatures and the
identification rates for these spots using MALDI-TOF MS
Comparison
(7C)
Up-regulated Down-regulated
Different
spots
Identified
spots
Identified
rate (%)
Identified
proteins
Different
spots
Identified
spots
Identified
rate (%)
Identified
proteins
55 vs. 75
80 vs. 75
55 vs. 80
38
25
100
33
24
88
89
96
88
30
20
67
29
93
42
26
83
41
90
89
98
17
59
24
Table 2. The distribution of the 2-DE spots with significant changes in spot volumes at different culture temperatures
DistributionSpots Spots number
Only at 557C
Only at 757C
Only at 807C
Only at 55 and 757C
16
11
17
47
133, 134, 135, 136, 137, 138, 139, 141, 142, 143, 144, 145, 146, 147, 148, 149
4, 5, 8, 16, 19, 45, 69, 72, 73, 116, 118
150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166
1, 2, 3, 6, 7, 10, 15, 18, 19, 20, 23, 24, 28, 30, 31, 33, 38, 39, 46, 48, 59, 65, 75, 82, 86,
88, 89, 91, 93, 96, 99, 100, 101, 102, 104, 105, 107, 110, 114, 115, 117, 119, 120,
121, 127, 129, 130, 132
42, 44, 64, 68, 78, 94, 95, 106, 131
9, 13, 14, 25, 43, 54, 60, 63, 67, 79, 111, 113
Only at 80 and 757C
Increased expression following the
rise in temperature
Decreased expression level following
the rise in temperature
Irregular change
9
12
2611, 12, 32,36, 37, 47, 49, 52, 53,71, 74, 77, 84, 85,87, 90, 92, 97, 103,108,109,112,
122, 124, 126, 128
17, 21, 22, 26, 27, 29, 34, 35, 40, 41, 50, 51, 55, 56, 57, 61, 62, 66, 70, 76, 80, 81, 83,
98, 123, 125
26
Total spots with significant changes164
are three typical examples demonstrating the consecutive or
irregular development of 2-DE spot volumes during altera-
tions of the adaptive temperatures. With rise in temperature,
the intensity 0.324 of spot 13 at 557C developed to 1.487 at
807C (Fig. 3A). In contrast to this spot, the spots 11 and 12,
with their intensities of 2.114 and 1.12 at 557C dropped
down to 0.47 and 0.22 at 807C, respectively (Fig. 3B). The
spots with irregular changes in spot volumes are shown in
Fig. 3C, where the spots 80 and 81 with relatively low spot
intensities 0.296 and 0.285, respectively, at 557C, developed
into the highest values at 757C and the intensity values
dropped down to 0.228 and 0.236, respectively, at 807C. Some
spots did not change their spot volumes during the elevated
temperature, and a typically consistent spot is shown in
Fig. 3D. Taking these comparisons together on the basis of
the spot volumes and the total spot numbers from 2-DE
images, the conclusions are consistent, i.e. the protein
expression of T. tengcongensis is attenuated dramatically at
807C.
3.4 Identification of 2-DE spots by MALDI-TOF MS
Identification of the temperature-dependent 2-DE spots was
carried out mainly by MALDI-TOF MS. Stringent criteria
were adopted to ensure the accuracy of protein identification:
(i) the identified protein must rank at the top two hits with at
least five matched sequences and (ii) a total coverage of over
15%.
As mentioned above, if the quantity of the digestive
products was available, double examinations, using two
approaches of MS, MALDI-TOF MS, and LC-MS/MS, were
conducted in protein identification for the same gel spot to
achieve more reliable mass data for protein identification.
The spot 13 was identified using two methods of MS. Fig. 4
illustrates the verification result for this spot using MALDI-
TOF MS and LC-MS/MS. By MALDI-TOF MS, 31 peptide
fragments matched to the protein of GroEL (HSP60
TTE0580) (see Supporting Table S1); whereas the spectrum
of LC-MS/MS detected 33 partial amino acid sequences
matched to the protein of GroEL. The protein identification
with two different measurements of mass techniques
strengthens the searching results and reduces the errors
generated from different mass machines as well as peptide
search engines.
A total of 164 spots corresponding to the alterations of
culture temperatures were picked up from all the three 2-DE
gels. On an average, two qualified MALDI-TOF MS spectra
were collected for each protein identification. A total of 147
spots matched the correct proteins (89.6% of identification
rate); ofthese, 87 unique proteinswere assigned.The protein
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com

Page 7

Proteomics 2007, 7, 1409–1419
Microbiology
1415
Figure 3. Three examples with significant changes of spot vol-
ume on 2-DE during temperature alterations. (A) Spot 13, GroEL
(HSP60 TTE0580), with increased spot volumes following the ris-
ing culture temperature; (B) Spots 11 and 12, Rubrerythrin
(TTE0487), with declined spot volumes following rising culture
temperature; (C) Spots 80 and 81, Peroxiredoxin (TTE0270), dis-
play the irregular changes in spot volumes during elevated tem-
perature. Detailed information is listed in Table 2 and Supporting
Table S1. (D) Spot C maintains almost consistent spot volumes
during evaluated temperature.
Figure 4. Identification of GroEL (HSP60TTE0580)by MALDI-TOF
MS and LC-MS/MS. (A) MALDI-TOF MS spectrum of the tryp-
sinized product from the 2-DE spot 13 of T. tengcongensis cul-
tured at 757C, 31 peptide fragments matched to this protein
(details are described in Supporting Table S1); (B) LC-MS/MS
spectrum, the parent ion 1049.021(RT = 41.0 min) was selected
for MS/MS analysis and the tandem mass spectrum was col-
lected corresponding to the amino acid sequence, LLIIADDVE-
GEALATLVVNK, by analyzing b- and y-ions derived from the
peptide ions (details are described in Supporting Table S2).
identification rates from MALDI-TOF MS are summarized
in Table 1. The identification results in detail are listed in
Supporting Tables S1 and S3.
3.5 Classification of the temperature-dependent
T. tengcongensis proteins
In Table 2, there are 16, 11, and, 17 unique 2-DE spots spe-
cifically found from the bacteria cultured at 55, 75, and
807C, respectively. Of these spots, 11, 8, and 16 were identi-
fied to be proteins by MALDI-TOF MS, respectively (Sup-
porting Tables S1 and S3). Checking these temperature-de-
pendent proteins carefully, two types of these unique pro-
teins are intriguing: (i) a specific isoform is expressed at a
certain temperature, but alternative forms are found at
other temperatures, such as peroxiredoxin (TTE0270)
detected at 807C with slightly acidic shift of pI but found at
the other two temperatures as well. Most of the unique
spots identified are attributed to this group (69%); (ii) A
protein only exists at a certain temperature. This type of
temperature-dependent protein is not present in large
number but may play active roles in adaptation process.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com

Page 8

1416
J. Wang et al.Proteomics 2007, 7, 1409–1419
Five T. tengcongensis proteins only detected in the bacteria
cultured at 557C are dihydrolipoamide acyltransferase
(TTE0690), NADH:ubiquinone oxidoreductase (TTE0890),
ferredoxin (TTE0892), phosphomannomutase (TTE0731),
and trypsin-like serine protease (TTE2568); two proteins
specifically favorable at 807C are N-acetylmannosamine-6-
phosphate epimerase (TTE2420) and UDP-glucose pyr-
ophosphorylase (TTE0732); and two proteins, S-layer
homology protein (TTE2411), and predicted dehydro-
genases (TTE0199) are uniquely expressed at 757C (Table 3).
A total of 38 2-DE spots appearing at all the three tem-
peratures were confirmed to be stress-responsive, either
increasing or decreasing consistently in spot volume (Table
2), and further all were identified as T. tengcongensis proteins
(Supporting Tables S1 and S3). These proteins are broadly
divided into two groups. (i) The modified forms of protein
are responsive to temperature stress, in which they have one
or more forms with consistent changes in spot volume cor-
respondingtotemperature changes, whereasotherformsare
expressed insensitively to culture temperatures. For exam-
ple, the spots 36 and 37, glutamate dehydrogenase
(TTE2203), display a consistent attenuation of spot volume
due to rising temperature; however, the other forms located
at spots31 and 35 lack this trend. (ii) The unique proteins are
stress-responsive for all the temperatures examined in this
study, implying that temperature really triggers the expres-
sionsofthese proteins.Atotal of 18 proteins are considerably
present in this group (Table 3). Further classifying this
group, most of them (11 proteins), including three hypo-
thetical proteins, have attenuated their expression with ele-
vated temperature. A typical
(TTE0487), which is widely distributed in anaerobic sulfate-
reducing bacteria and a nonheme iron protein that has the
ability to catalyze the reduction of hydrogen peroxide to
water. On the 2-DE gels, five spots shared with similar pI
values and variable sizes of molecular mass, ranging from 20
to 80 kDa, were identified as this protein. A chaperon pro-
tein, HSP70 (TTE0890), was also confirmed to have a low-
ered expression with increasing temperature. The other
seven proteins in this group were found to significantly
increase their expressionsduring elevated temperature, such
asdipeptidylaminopeptidases/acylaminoacyl-peptidases
(TTE1551), histidinol phosphatase and related hydrolase
(TTE1963), signal recognition particle GTPase (TTE1462),
GroES (HSP10 TTE0579), GroEL (HSP60 TTE0580), iron-
regulated ABC-type transporter SufB (TTE2672), and hypo-
thetical protein (TTE0418). Two spots were identified as
GroES, whereas more than ten spots of GroEL on the 2-DE
gels. Interestingly, GroEL expression is not only enhanced
quantitatively due toelevatedtemperature, but alsoincreased
its modified forms (Fig. 2). It is believed that the complex of
GroEL and GroES, formed as a homo-oligomeric double tor-
oid consisting of 14 GroEL monomers and a single ring cap
of seven GroES monomers, allows folding intermediates to
avoid misfolding polypeptide chains thereby preventing
aggregation. The intensity ratios of GroEL to GroES on the
sample is rubrerythrin
2-DE gels are 7.01 at 557C, 8.63 at 757C, and 10.36 at 807C,
respectively, and these values well correlate with the theoret-
ical prediction.
3.6 Immunoblot analysis for two temperature-
sensitive proteins in T. tengcongensis
Although the stringent criteria were adopted to ensure the
accuracy of protein identification, the conclusions drawn
from the determinations with MS are strengthened by other
analytical approaches. Therefore, we selected two typical
temperature-sensitive proteins (Table 3), GroEL and GroES,
as the targets examined by immunoblot analysis. As shown
in Fig. 5A, the anti-GroEL antibody recognizes several spots
on the 2-DE transblotted PVDFmembrane, 5 spots at 557C, 7
spotsat757C,and12spotsat807C.Inaddition,somedegraded
products, mainly located around 30–55 kDa with multiple pI
values, are detected due to rising culture temperature, espe-
ciallyat807C.Obviously,thepatternsofimmunopositivespots
recognizing anti-GroEL antibody are well matched with these
spotsdeterminedby2-DEandMS,asdescribedabove.
Similar observations were obtained using anti-GroES
antibody to define the temperature-sensitive 2-DE spots. On
2-DE images, spots 111 and 113 (Fig. 2), identified to be
GroES, have significantly increased their spot volumes with
elevated temperature. As compared to lower temperature,
the intensities of the two immunoreactive spots recognized
by anti-GroES antibody are intensified dramatically at higher
temperatures (Fig. 5B). For instance, with rising culture
temperature from 75 to 807C, on the 2-DE images, the
intensities of the spots 111 and 113, changed 2.485- and
2.414-folds, respectively, the corresponding 2-DE Western
blot intensity changes were 2.435- and 2.131-folds, respec-
tively. Therefore, the data of 2-DE Western blot offer addi-
tionally solid data to support the conclusions drawn from
proteomic determinations.
Figure 5. Identification of the temperature-sensitive proteins of
T. tengcongensis with Western blot. (A) Anti-GroEL antibody; (B)
Anti-GroES antibody.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com

Page 9

Proteomics 2007, 7, 1409–1419
Microbiology
1417
Table 3. Classification of the temperature-dependent T. tengcongensis proteinsa)
Identified protein Gene_ID Spots Trend (7C)Functional classification
The protein identified only at certain temperatures
Dihydrolipoamide acyltransferases
NADH:ubiquinone oxidoreductase 24 kD subunit
Ferredoxin
Phosphomannomutase
Trypsin-like serine protease
Predicted dehydrogenases and related proteins
S-layer homology domain
PutativeN-acetylmannosamine-6-phosphate epimerase
UDP-glucose pyrophosphorylase
TTE0690
TTE0890
TTE0892
TTE0731
TTE2568
TTE0199
TTE2411
TTE2420
TTE0732
2
1
1
1
1
1
1
1
1
55
55
55
55
55
75
75
80
80
Energy production and conversion
Energy production and conversion
Energy production and conversion
Carbohydrate transport and metabolism
PTM, protein turnover, and chaperones
General function prediction only
Unclassified
Carbohydrate transport and metabolism
Cell envelope biogenesis, outer membrane
The proteins with the consistent changes of spot volumes during alternations of culturing temperatures
Rubrerythrin
HSP70 class molecular chaperones involved in cell morphogenesis
Diadenosine tetraphosphate (Ap4A) hydrolase
Methylglyoxal synthase
Translation elongation factor Ts
Flagellar motor switch protein
Ni21-binding GTPase
Transcription regulator AbrB
Dipeptidyl aminopeptidases/acylaminoacyl-peptidases
Histidinol phosphatase and related hydrolases of the PHP family
Signal recognition particle GTPase
Co-Chaperonin GroES (HSP10)
Chaperonin GroEL (HSP60 family)
Iron-regulated ABC-type transporter SufB
TTE0487
TTE0898
TTE0693
TTE0906
TTE1408
TTE1441
TTE0130
TTE0106
TTE1551
TTE1963
TTE1463
TTE0579
TTE0580
TTE2672
5
1
1
2
2
1
2
1
1
1
1
2
3
1
Decreased
Decreased
Decreased
Decreased
Decreased
Decreased
Decreased
Decreased
Increased
Increased
Increased
Increased
Increased
Increased
Energy production and conversion
Cell division and chromosome partitioning
Nucleotide transport and metabolism
Carbohydrate transport and metabolism
Translation, ribosomal structure and biogenesis
Cell motility and secretion
PTM, protein turnover, and chaperones
Unclassified
Amino acid transport and metabolism
Amino acid transport and metabolism
Cell motility and secretion
PTM, protein turnover, chaperones
PTM, protein turnover, chaperones
General function prediction only
a)
The hypothetical T. tengcongensis proteins, five spots in total, were not included.
4Discussion
Thermophilic organisms usually grow optimally at 50–807C,
and T. tengcongensis is a typical member of this family.
Understanding of the adaptations, how thermophilic bac-
teria survive at extreme temperatures is a challenging re-
search activity [7]. Although great strides have been made
along this line of inquiry over the past several years, a
detailed knowledge corresponding to thermostability in
thermophiles is to be explored yet. A lot of studies to date
have focused on amino acid sequence variations that are
proposed to retain the thermodynamic stability of thermo-
philic proteins [8]. In fact, similar comparisons between
thermophilic and mesophilic proteins have demonstrated
40–85% sequence similarity; moreover, these proteins share
the same mechanisms in catalytic reactions and super-
impose the 3-D structures [9]. To gain an insight into the
cellular protection against a variety of environmental
insults, therefore, research effort in this field will not only
consider the intrinsic regulations but also need to investi-
gate the extrinsic factors such as PTMs and expression
levels of proteins. Olson et al [10] reported that the glyco-
sylation of b-glucanases expressed in E. coli and in
Saccharomyces cerevisiae had positive effects on thermo-
stability and the thermostabilization level was dependent
more on the location of the carbohydrate moiety on the pro-
tein than on the extent of glycosylation [10]. In this study, the
data from 2-DE and MS have revealed the that most rela-
tively abundant proteins of T. tengcongensis are expressed
consistently throughout the three culturing temperatures
because of the similarity of the 2-DE images found in the
bacterial proteins extracted from different cultures. Com-
pared to the 2-DE images from bacteria cultured at the three
temperatures, there are 164 spots with significant changes
in spot volume. Furthermore, 147 spots were verified as
bacterial proteins by determinations of MALDI-TOF MS
with only 87 unique proteins, indicating that a protein may
have multiple forms responding to temperature alterations.
These protein isoforms recognized by the shifts in the values
of pI or apparent molecular mass are not necessarily in
accordance with a downward or upward trend of tempera-
ture. Six spots with significant changes in spot volume were
identified as glutamate dehydrogenase (TTE2202 and
TTE2203), whereas this protein did not show a consistent
pattern of expression following temperature changes. The
spots 35, 40, and 41 have the highest volumes at 757C, and
spots 36 and 37 decrease their spot volumes due to rising
temperature, however, spot 31 was not found at 807C. Based
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com

Page 10

1418
J. Wang et al. Proteomics 2007, 7, 1409–1419
upon our observations, it is hard to conclude which mod-
ification forms favor the thermostability. Nevertheless, these
data have evidenced that the PTMs and expression levels of
bacterial proteins may offer active functions for the survival
of microbes in harsh environment.
Of these stress-responsive proteins, six are the enzymes
involved in redox regulation (Table 3 and Supporting Table
S3), and four of them, rubrerythrin (TTE0487), dihydro-
lipoamide acyltransferase (TTE0690), NADH:ubiquinone
oxidoreductase(TTE0890),
decline protein expression during rising culture tempera-
ture. Moreover, except rubrerythrin the other three proteins
are uniquely expressed at 557C. Rubrerythrin is a nonheme
iron protein isolated from anaerobic sulfate-reducing bac-
teria [11]. Recently, it has been implicated in oxidative stress
protection in anaerobes. In Clostridium thermoaceticum, a
thermophilic Gram-positive, Das et al [12] proved that this
thermophile contained gene encoding rubrerythrin, which
was present in a cluster with four additional genes encoding
a rubredoxin oxidoreductase, a rubredoxin, a high-molecu-
lar mass rubredoxin, and a type of flavoprotein. Therefore,
the expressions of these cluster genes were likely co-
regulated to perform a cooperative role in oxidative stress
protection. On 2-DE gels, rubrerythrin in T. tengcongensis,
protein levels significantly attenuated during elevated tem-
perature, were located within a wide range of molecular
masses and a narrow range of pI, suggesting that there
were the neutral modifications in this protein. Two spots
corresponding to dihydrolipoamide dehydrogenase were
only identified at 557C. This enzyme is annotated to a com-
ponent of pyruvate dehydrogenase complex. In our previous
report, we pointed out that T. tengcongensis did not have
pyruvate dehydrogenase and pyruvate decarboxylase thereby
impossibly formed pyruvated dehydrogenase complex with
difficulty [3]. Thus PFORs with abundant expression were
suggested to be the main catalyst for the pyruvate metabo-
lism in T. tengcongensis. However, the T. tengcongensis ge-
nome indeed contains a gene encoding 2-oxo acid dehy-
drogenase (TTE1668). Among cellular systems, the 2-oxo
acid dehydrogenase multienzyme complexes, consisting of
2-oxo acid dehydrogenase (E1), dihydrolipoamide acyl-
transferase (E2), and dihydrolipoamide dehydrogenase (E3),
occupy key positions for redox regulation [13]. During the
reaction, the catalytic action of the 2-oxo acid dehydrogenase
complexes directly influences the NADH/NAD1ratio and
involves the important biological SH/S–S compounds [14].
Two other enzymes uniquely detected at 557C, NADH:ubi-
quinone oxidoreductase(TTE0890)
(TTE0892), have been extensively studied for their roles and
involvement in redox reactions as well [15–17]. Taking to-
gether, the capacity of antioxidant in T. tengcongensis cul-
tured at high temperature, especially at 807C, is reasoned to
be weakening due to low expression of these redox-regu-
lated proteins. Hence the attenuation capacity of anti-
oxidative stress may partially explain the poor growth of this
bacterium at 807C.
and ferredoxin (TTE0892),
andferredoxin
The products of the groES and groEL genes of E. coli form
an operon that was first defined by mutations affecting the
morphogenesis of several bacteriophages [18]. The two genes
code for 10 and 60 kDa of acidic polypeptides, respectively,
found at high intracellular levels. Furthermore, their expres-
sions increased with temperature through a positive tran-
scriptional control exerted by the rpoH (s32) gene product
[19]. The experiments in vitro demonstrated that both pro-
teins were essential at all the temperatures as to prevent
protein aggregation by binding unfolded or partially-folded
proteins [20]. The GroES and GroEL proteins have been
found in a wide distribution in number of thermophilic bac-
teria [21, 22]. In T. tengcongensis, the groE genes have been
sequenced, revealing two ORFs of 285 and 1623 bp, sepa-
rated by 13 bp. The first ORF encodes a 94 amino acid (aa)
10 kDa GroES homologue and the second encodes a 540
amino acid (aa) 60 kDa GroEL homologue, which exhibit 47
and 55% sequence identity with GroES and GroEL from
E. coli, respectively. An inverted repeat (IR, CIRCE) region,
preceded by TTAGCACTC-N9-GAGTGCTAA consensus
promoters, was identified in the upstream of groES, in which
a protein coded by hrcA gene is an ideal regulator to associate
with [23]. The proteomic determinations described above
strongly demonstrate that both proteins are temperature-
sensitive and exhibit overexpression in response to elevated
temperatures. GroES was verified only by two spots which
had apparent values of pI and molecular mass similar to the
theoretical prediction under all the temperatures examined
in this experiment, whereas, GroEL was shown to have the
different patterns in electrophoretic behavior and in PTMs.
First of all, at the region of 60 kDa that is in accordance with
GroEL theoretic molecular mass, GroEL was detected in
multiple spots with varying values of pI. With rising tem-
perature, more GroEL spots appeared on 2-DE gels but these
new spotsat high temperature carriedmore negative charges
and shifted to the acidic direction. On the other hand, the
degraded products of GroEL were significantly detected at
807C, in which the extent of molecular mass loss was limited
to within 15 kDa and pI values shifted to the acidic side. The
chemical modification of GroEL has been reported with two
possibilities in PTM. In E. coli, some GroELs were found in
phosphorylated forms; interestingly, the modification was
temperature dependent and reversible. The GroEL phospho-
rylation was induced by heat and dephosphorylated at low
temperatures [24]. The modification of cysteine residues in
GroEL was also studied in detail [25]. According to the
observation of Martin, Cys138is a reactive and accessible
residue in GroEL. After modification of Cys138with N-ethyl-
maleimide, the resulting NEM-GroEL affected the structural
and functional integrity of the GroE complex [26]. For
instance, NEM-GroEL had an increased ATPase activity and
bound nucleotides with higher affinity, and the affinity of
NEM-GroEL for GroES was reduced compared with the
unmodified GroEL. Under high temperature, oxidation of
reactive cysteine forming cysteine sulfenic acid or cysteine
sulfinic acid may result in the pI shifts of GroEL.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com

Page 11

Proteomics 2007, 7, 1409–1419
Microbiology
1419
In summary, the global determination of the thermo-
responsive proteins in T. tengcongensis paves a path to access
the molecular mechanisms of thermophilic survival. Three
regulations, protein modifications, redox capacity, and
expression of heat-shock proteins, are likely to affect the
growth of this bacterium at the temperature range of 55–
807C. Detailed investigations are necessary in understanding
further the networks and the chemical structures of these
temperature-dependent proteins responding to thermo-
stress.
This work was supported by grants of National Basic
Research Program of China
2001CB210501).
(2004CB719605 and
5 References
[1] Xue, Y., Xu, Y., Liu, Y., Ma, Y. et al., Int. J. Syst. Evol. Microbiol.
2001, 51, 1335–1341.
[2] Bao, Q., Tian, Y., Li, W., Xu, Z. et al., Genome Res. 2002, 12,
689–700.
[3] Wang, J., Xue, Y., Feng, X., Li, X. et al., Proteomics 2004, 4,
136–150.
[4] Vieille, C., Zeikus, G. J., Microbiol. Mol. Biol. Rev. 2001, 65, 1–
43.
[5] Nocker, A., Krstulovic, N. P., Perret, X., Narberhaus, F., Arch.
Microbiol. 2001, 176, 44–51.
[6] Vierke, G., Engelmann, A., Hebbeln, C., Thomm, M., J. Biol.
Chem. 2003, 278, 18–26.
[7] Adams, M. W., Annu. Rev. Microbiol. 1993, 47, 627–658.
[8] Martin, A., Sieber, V., Schmid, F. X., J. Mol. Biol. 2001, 309,
717–726.
[9] Bauer, M. W., Kelly, R. M., Biochemistry 1998, 37, 17170–
17178.
[10] Olson, S. T., Frances-Chmura, A. M., Swanson, R., Bjork, I. et
al., Arch. Biochem. Biophys. 1997, 341, 212–221.
[11] Fournier, M., Zhang, Y., Wildschut, J. D., Dolla, A. et al., J.
Bacteriol. 2003, 185, 71–79.
[12] Das, A., Coulter, E. D., Kurtz, D. M., Ljungdahl, L. G., J. Bac-
teriol. 2001, 183, 1560–1567.
[13] Bunik, V. I., Eur. J. Biochem. 2003, 270, 1036–1042.
[14] Bunik, V., Westphal, A. H., de Kok, A., Eur. J. Biochem. 2000,
267, 3583–3591.
[15] Menon, S., Ragsdale, S. W., Biochemistry 1997, 36, 8484–
8494.
[16] Pieulle, L., Charon, M. H., Bianco, P., Bonicel, J. et al., Eur. J.
Biochem. 1999, 264, 500–508.
[17] Flemming, D., Schlitt, A., Spehr, V., Bischof, T. et al., J. Biol.
Chem. 2003, 278, 47602–47609.
[18] Tilly, K., McKittrick, N., Georgopoulos, C., Murialdo, H., Prog.
Clin. Biol. Res. 1981, 64, 35–45.
[19] Tilly, K., Spence, J., Georgopoulos, C., J. Bacteriol. 1989,
171, 1585–1589.
[20] Lund, P. A., Essays Biochem. 1995, 29, 113–123.
[21] Cross, S. J., Ciruela, A., Poomputsa, K. et al., Biochem. J.
1996, 316, 615–622.
[22] Truscott, K. N., Scopes, R. K., Gene 1998, 217, 15–23.
[23] Segal, G., Ron, E. Z., FEMS Microbiol. Lett. 1996, 138, 1–10.
[24] Sherman, M. Y., Goldberg., A. L., Nature 1992, 357, 167–169.
[25] Mendoza, J. A., Horowitz, P. M., J. Protein Chem. 1992, 11,
589–594.
[26] Martin, J., J. Biol. Chem. 1998, 273, 7351–7357.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.com