JOURNAL OF BACTERIOLOGY, June 2005, p. 4207–4213
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 187, No. 12
Defense against Protein Carbonylation by DnaK/DnaJ and Proteases
of the Heat Shock Regulon
Åsa Fredriksson,1Manuel Ballesteros,2Sam Dukan,3and Thomas Nystro ¨m1*
Department of Cell and Molecular Biology, Microbiology, Go ¨teborg University, Box 462, 405 30 Go ¨teborg, Sweden,1
Centro Andaluz de Biologia del Desarrollo (CABD), University “Pablo de Olavide,” Ctra Utrera km1,
ES-41013 Seville, Spain,2and Laboratoire de Chimie Bacte ´rienne, CNRS-UPR9043,
31 Chemin Joseph Aiguier, 13402 Marseille, France3
Received 21 January 2005/Accepted 8 March 2005
Protein carbonylation is an irreversible oxidative modification that increases during organism aging and
bacterial growth arrest. We analyzed whether the heat shock regulon has a role in defending Escherichia coli
cells against this deleterious modification upon entry into stationary phase. Providing the cell with ectopically
elevated levels of the heat shock transcription factor, ?32, effectively reduced stasis-induced carbonylation.
Separate overproduction of the major chaperone systems, DnaK/DnaJ and GroEL/GroES, established that the
former of these is more important in counteracting protein carbonylation. Deletion of the heat shock proteases
Lon and HslVU enhanced carbonylation whereas a clpP deletion alone had no effect. However, ClpP appears
to have a role in reducing protein carbonyls in cells lacking Lon and HslVU. Proteomic immunodetection of
carbonylated proteins in the wild-type, lon, and hslVU strains demonstrated that the same spectrum of proteins
displayed a higher load of carbonyl groups in the lon and hslVU mutants. These proteins included the ?-subunit
of RNA polymerase, elongation factors Tu and G, the E1 subunit of the pyruvate dehydrogenase complex,
isocitrate dehydrogenase, 6-phosphogluconate dehydrogenase, and serine hydroxymethyltranferase.
Protein carbonylation has become a commonly used biomar-
ker of severe oxidative damage to proteins, and many diseases
have been associated with this modification, including Parkin-
son’s disease, Alzheimer’s disease, cancer, cataractogenesis,
diabetes, and sepsis (6, 17). Carbonylation increases with the
age of cells, organelles, and tissues of various species and has
been linked to age-dependent deterioration of specific en-
zymes, e.g., the aconitase and the adenine nucleotide translo-
cator ANT (29, 30). Carbonyl derivatives can be formed by a
direct metal-catalyzed oxidative attack on the amino acid side
chains of proline, arginine, lysine, and threonine. The quanti-
tatively most important products of the carbonylation reaction
are glutamic semialdehyde from arginine and proline and ami-
noadipic semialdehyde from lysine (21). Compared to other
oxidative modifications, carbonyls are relatively difficult to in-
duce and are irreversible modifications (6).
The levels of carbonylated proteins increase rapidly as Esch-
erichia coli cells enter stationary phase as a result of carbon/
energy (3, 9) or nitrogen (3) starvation. This modification has
been associated with the reduced plating efficiency of station-
ary phase cells (7). Specifically, using an in situ detection of
protein carbonyls in single cells and a density gradient centrif-
ugation technique to separate culturable and nonculturable
cells of the same chronological age it was demonstrated that
proteins of the nonculturable cell population exhibited mark-
edly higher levels of irreversible carbonylation (7). Apart from
the fact that the RpoS and OxyR regulons and the superoxide
dismutases and catalases are important in mitigating protein
carbonylation (10), little is known about the function and iden-
tity of cellular defenses against this oxidation. Since heat shock
genes have a role in cellular resistance against oxidative stress
(14, 24) and are increasingly expressed during oxidant expo-
sure (2, 26), we tested whether the heat shock regulon is in-
volved in attenuating stasis-induced carbonylation. We report
that several members of this regulon, both chaperones and
proteases, are key factors in the cellular defense against this
deleterious oxidative modification.
MATERIALS AND METHODS
Chemicals and reagents. Detection of carbonylated proteins was performed
using the chemical and immunological reagents of an OxyBlot oxidized protein
detection kit (Intergen Company). Anti-GroEL and anti-DnaK mouse mono-
clonal antibodies were purchased from Stressgen Bioreagents (Biosite). Anti-
DnaK mouse polyclonal antibodies and anti-?32 mouse monoclonal antibodies
were from Neoclone. Anti-mouse immunoglobulin G peroxidase conjugates,
trypsin, and isopropyl-?-galactopyranoside (IPTG) were from Sigma. The chemi-
luminescence blotting substrate (ECL?) was obtained from Amersham Corp.,
and the Immobilon-P polyvinylidene difluoride (PVDF) membrane was from
Millipore. Bicinchoninic protein assay reagents were purchased from Pierce.
The precast polyacrylamide gels used for one-dimensional (1-D) electrophoresis
were either Criterion 10% Tris–HCl (Bio-Rad) or NuPAGE 12% Bis–Tris gel
(Invitrogen). The ampholines (Resolyte 4-8) used for two-dimensional electro-
phoresis were from BDH (VWR International). The LIVE/DEAD BacLight
bacterial viability kit was from Molecular Probe. All chemicals and reagents were
used according to instructions provided by the manufacturer.
Bacterial strains, plasmids, and growth conditions. The E. coli K-12 strains
and plasmids used in this study are listed in Table 1. Cultures were grown
aerobically at 37°C in minimal M9 medium (18) supplemented with thiamine
(10 mM), all 20 amino acids, and glucose (0.2%) in Erlenmeyer flasks in a rotary
shaker. When appropriate, the medium was supplemented with carbenicillin (100
?g/ml), tetracycline (20 ?g/ml), kanamycin (50 ?g/ml) or spectinomycin (100
?g/ml). To overproduce Hsps, IPTG was added to the exponentially growing
cultures at an optical density at 420 nm (OD420) of 0.05 to induce expression
from the plasmid pKV1278 (?32), pBB535 (DnaK/DnaJ), or pBB541 (GroES/
GroEL). Early stationary phase samples were collected 30 min after exponential
* Corresponding author. Mailing address: Department of Cell and
Molecular Biology, Microbiology, Medicinaregatan 9C, 413 90 Go ¨te-
borg, Sweden. Phone: 46 31 7732582. Fax: 46 31 7732599. E-mail:
growth (measured as OD420) ceased due to glucose depletion in the medium. For
protein stability measurements, cells were grown exponentially at 37°C and at an
OD420of 0.1 and IPTG was added to induce expression from the plasmid
pKV1278 (?32). After 3 h, protein synthesis was blocked by addition of specti-
nomycin (400 ?g/ml).
General methods. Crude cell extracts were obtained using a 20 K French
pressure cell (Spectronic Instruments) for all experiments. Derivatized protein
extracts were processed for resolution on two-dimensional polyacrylamide (2-D)
gels by the methods of O?Farrell (20) with modifications (25). Isoelectric focus-
ing was performed as described in reference 25. Gel electrophoresis using 11.5%
sodium dodecyl sulfate–polyacrylamide gels and immunoblotting with mouse-
monoclonal or mouse-polyclonal antibodies was carried out according to stan-
dard procedures. For detection, the ECL?blotting kit was used with horseradish
peroxidase-conjugated anti-mouse immunoglobulin G as secondary antibody.
Blots were subsequently exposed in a charge-coupled device camera (FUJIFILM
Image Reader LAS-1000Pro). For quantitative analyses of the blots, Image
Gauge 3.46, Science Lab 99 software was used.
Protein identification. Proteins separated on two-dimensional gels were blot-
ted onto PVDF membranes and used for immunodetection of carbonylated
proteins. The same membranes were stained with Coomassie brilliant blue after
exposure in the charge-coupled device camera and were used as references when
matching the carbonylated proteins to the spots on the 2-D gels. The matched
protein spots were subsequently cut out and used for mass-spectrometric anal-
ysis. Samples were analyzed using a matrix-assisted laser desorption ionization-
linear reflection mass spectrometer (Micromass, Manchester, UK) in reflection
mode. Tryptic digest (0.5 ?l) was mixed with 0.5 ?l matrix solution (12 mg/ml
?-cyano-4-OH-cinnamic acid in acetonitrile/water [1:1]–0.1% trifluoroacetic
acid) directly on the matrix-assisted laser desorption ionization probe and al-
lowed to dry at ambient conditions. ACTH (18-39) MH?2465.199 was used as
an external and tryptic autodigest MH?2211.105 as an internal lock mass.
Monoisotopic mass values were used for peptide mass mapping using MASCOT
(available at www.matrixscience.com) against the nr database at the National
Center for Biotechnology Information. All identifications had a score equivalent
to a 95% level of confidence. When necessary, samples were purified and con-
centrated with ZipTips containing C18resin according to the manufacturer’s
instructions. The peptides eluted in 3.5 ?l of acetonitrile/water (1:1) containing
0.1% formic acid were then analyzed by electrospray ionization mass spec-
trometry/mass spectrometry (MS/MS) on a quadrupole time-of-flight ultima
atmospheric pressure ionization apparatus (Micromass, Manchester, UK).
Fragment ion data were acquired by nanoflow electrospray using argon as the
collision gas. The MS/MS data were used as input for protein identification by
MASCOT (www.matrixscience.com). No restrictions in species, molecular
weight, or pI were applied when searching against the nr database of the
National Center for Biotechnology Information. Proteins identified all had a
score equivalent to a 95% confidence level or higher.
Viability assays. Cells collected after 3 h in stationary phase were serially
diluted in M9 medium lacking glucose and amino acids prior to plating onto solid
LB medium in the presence and absence of the appropriate antibiotic selection.
To induce expression from plasmid pBB535 (ÅF38), IPTG was added at an
OD420of 1.5 before transition into stationary phase. Viability was also de-
termined by microscopic analyses using BacLight LIVE/DEAD methodology,
whereby living cells fluoresce in green and dead cells in red when excited at 590
Carbonylation assays. Analyses of carbonylated proteins were performed us-
ing the chemical and immunological reagents of an OxyBlot oxidized protein
detection kit. The carbonyl groups in the protein side chains were derivatized to
2,4-dinitrophenylhydrazone (DNP-hydrazone) by reaction with 2,4-dinitrophe-
nylhydrazine. The DNP-derivatized proteins were analyzed immunochemically
on one- or two-dimensional Western blots or directly dot blotted onto PVDF
membranes as described previously (9). In general, 1 ?g of protein was loaded
into the slot-blot apparatus and 10 ?g onto 1- or 2-D gels.
RESULTS AND DISCUSSION
Overproduction of ?32and DnaK/DnaJ prevents stasis-in-
duced oxidation of proteins. Previous studies show that certain
proteins are specifically susceptible to stasis-induced oxidation
(9), and we determined the specificity of protein carbonylation
at the time when this is maximal upon growth arrest (early
stationary phase; see Fig. 1). A number of carbonylated pro-
teins, some of which have not previously been shown to be
carbonylated, were identified by mass spectrometry (Table 2).
FIG. 1. Protein carbonylation during growth and in stationary
phase. Relative protein carbonylation levels (black squares) and the
optical density of the wild-type culture (MG1655?lac) (filled circles)
are shown during growth and growth arrest caused by glucose deple-
tion. Carbonyl levels were determined by one-dimensional Western
blot immunoassays and quantified using Image Gauge software. Car-
bonyl levels were related to that obtained during exponential growth,
which was assigned a value of 1.0. The arrow indicates the time at
which samples were obtained for identification of carbonylated pro-
teins (see Table 2). The experiment was repeated at least three times.
Representative results are shown, and there was always less than 15%
variation between experiments.
TABLE 1. E. coli strains and plasmids used in this work
Strain Plasmid GenotypeOrigin
MG1655 ??ilvG-rfb-50 rph-1
MG1655 ?lac ?hslVU::Tetr
MG1655 ?lac ?clpP::Camr
MG1655 ?lac ?lon146::Tetr
MG1655 ???lac ilvG-rfb-50 rph-1
4208THE HEAT SHOCK REGULON AND PROTEIN CARBONYLATIONJ. BACTERIOL.
The identification suggests that several different processes, in-
cluding, e.g., information transfer (transcription and transla-
tion), central metabolism (glycolysis and TCA cycle), protein
folding, and fatty acid biosynthesis, are subjected to stasis-
induced deterioration. Some of the identified carbonylated
proteins have been demonstrated previously to be targets for
the DnaK chaperone, i.e., these proteins are prone to aggre-
gate in the absence of DnaK (19). Based on these results and
the fact that aberrant proteins are more susceptible to carbony-
lation than the native counterparts (3, 8), we entertained the
idea that stationary phase carbonylation of proteins may, in
part, be a consequence of stasis-induced mistranslation (3)
overwhelming the heat shock chaperones. If so, elevated levels
of heat shock chaperones could partly counteract the effect
of such mistranslation and attenuate stasis-induced carbonyla-
We determined whether overproduction of the Hsp tran-
scription factor ?32mitigated protein carbonylation in cells
entering a growth-arrested state. To confirm that overproduc-
tion of ?32resulted in significantly elevated concentrations of
Hsps, we checked the levels of DnaK and GroEL (Fig. 2A). As
seen in Fig. 2A, overproduction of the heat shock regulon
drastically reduced (3.5-fold lower than the control) stasis-
induced carbonylation. In fact, carbonylation did not increase
upon growth arrest in cells with ectopically elevated Hsp levels.
Next, we determined the effect of overproducing the DnaK/
DnaJ chaperone system alone. DnaK/DnaJ overproduction
markedly reduced the levels of other Hsps, such as GroEL
(Fig. 2B), consistent with the role of DnaK/DnaJ as negative
modulators of the Hsp regulon. Nevertheless, despite the re-
duced levels of other Hsps, overproduction of the DnaK/DnaJ
chaperones counteracted protein carbonylation to the same
extent as overproduction of ?32(Fig. 2B). This was due not to
a reduced production of proteins that happen to be carbony-
lation sensitive but rather to a reduction in such proteins’
carbonylation load. For example, the carbonyl level of elon-
gation factor EF-Tu, when normalized to the total levels of
EF-Tu present, was substantially reduced by DnaK/DnaJ over-
production (Fig. 3A). As depicted (Fig. 3B), the total amount
of carbonylated proteins was generally lower upon DnaK/DnaJ
overproduction, indicating a general protective function of this
Overproduction of the GroEL/GroES chaperone system af-
fected carbonylation marginally (Fig. 2C), suggesting that the
DnaK/DnaJ system is more important in the cellular defense
against this oxidative modification. This is consistent also with
the fact that DnaK/DnaJ overproduction reduced carbonyla-
tion despite a concomitantly reduced level of GroEL (Fig.
2B). A role for DnaK in protecting against protein oxidation
is in line with results demonstrating that aerobic growth of
E. coli on ethanol depends on DnaK protection of a mutant
ethanol oxidoreductase (AdhE) against metal-catalyzed ox-
idation (11), but a global role for DnaK/DnaJ in mitigating
stasis-induced carbonylation has not been reported previ-
Defense against carbonylation damage requires the Lon and
HslVU (ClpQY) proteases. Oxidized aconitase has been shown
to be recognized and degraded by mitochondrial Lon in mam-
malian cells (4). Since the bacterial Lon protease is a member
of the heat shock regulon, we investigated whether attenuated
carbonylation caused by Hsp overproduction resulted, in part,
FIG. 2. Effects of overproducing the Hsp regulon on protein oxi-
dation. Severalfold change in the levels of DnaK, GroEL, and car-
bonylated proteins as a consequence of overproducing ?32(ÅF41) (A),
DnaK/DnaJ (ÅF38) (B), and GroEL/GroES (ÅF64) (C) in cells en-
tering a growth arrested state (30 min after growth ceased) is shown.
“1” on the y axis means no change compared to wild type, while “2”
means a twofold increase and “?2” means a twofold decrease. The
cells were grown with (100 ?M) and without IPTG prior to starvation.
Protein and carbonyl levels were determined by one-dimensional
Western blot immunoassays and quantified using Image Gauge soft-
ware. All levels were related to that obtained in the control culture
without overproduction, which was assigned a value of 1.0.
TABLE 2. Proteins subjected to stasis-induced carbonylation at the
time when carbonyl content is maximal. Proteins in bold have
been demonstrated to be substrates for DnaK (19).
RNA polymerase ?-subunita
Phosphoribosyl formylglycinamide synthase
E1 component of pyruvate dehydrogenase
Protein chain elongation factor G
Molecular chaperone DnaK
E2 component of pyruvate dehydrogenase
Polyamine-induced protein precursor
Molecular chaperone GroEL
Protein chain elongation factor Tu
Succinyl CoA ligase
High-affinity zinc uptake system protein precursor
aProteins that have been demonstrated to be substrates for DnaK (19) are
shown in boldface characters.
VOL. 187, 2005FREDRIKSSON ET AL.4209
from increased degradation of carbonylated proteins. To ap-
proach this, we blocked protein synthesis with spectinomycin in
cells with and without overproduced levels of ?32and subse-
quently analyzed protein carbonyl levels at time intervals. As
depicted (Fig. 4A), protein carbonyl levels decreased when ?32
had been overproduced, in stark contrast to the vector control
results. This provided the impetus to analyze carbonylation
levels in mutants lacking Lon and other Hsp proteases. Dele-
tion of Lon increased protein carbonylation upon entry of cells
into stationary phase, and deletion of the HslVU (ClpQY)
protease had an even more pronounced effect (Fig. 5). How-
ever, a clpP deletion alone did not affect total protein carbonyl
levels (Fig. 5). It is possible that increased levels of ?Sin the
clpP mutant (22) may mask a possible role for ClpP in degrad-
ing carbonylated proteins, since ?S-dependent genes have been
shown to counteract protein oxidation (10). However, the clpP
mutations had no significant effect on total protein carbonyl
levels even in a background lacking rpoS (not shown). In con-
trast, the lack of ClpP further elevated carbonylation in cells
lacking Lon, suggesting that ClpP has a role in the defense
against protein carbonylation but that Lon can fully compen-
sate for the absence of ClpP (Fig. 5). In addition, HslVU
appears to partly compensate for the lack of Lon and ClpP
since carbonylation was further increased by introducing an
hslVU deletion (Fig. 5).
Proteomic immunodetection of carbonylated proteins in the
wild-type, lon, and hslVU strains demonstrated that the in-
creased levels of protein carbonyls were, for the most part,
nonspecific in the sense that the same spectrum of oxidized
proteins exhibited a higher load of carbonyl groups in both
mutants (Fig. 6). These results are consistent with previous
results demonstrating that the function and substrate recogni-
tion of Lon and HslVU overlap (28). Several proteins being
increasingly carbonylated in the mutants were identified by
mass spectrometry; they included the ?-subunit of RNA poly-
merase (RpoB), elongation factors Tu and G (EF-Tu, EF-G),
the E1 component (AceE) of the pyruvate dehydrogenase
complex, isocitrate dehydrogenase (Idh), 6-phosphogluconate
dehydrogenase (Gnd), and serine hydroxymethyltranferase
(GlyA) (Fig. 6). Interestingly, GroEL and DnaK were two of
only a few proteins whose carbonylation load did not increase
by deleting lon or hslVU (Fig. 6). At present, it is not clear
whether carbonylated proteins are targets for proteases be-
cause they contain oxidation cues for protease recognition or
whether oxidation-induced unfolding is the primary cause of
increased attack by heat shock proteases.
Overproduction of DnaK/DnaJ does not prolong the average
life span of stationary phase cells. The DnaK/DnaJ system is
not expected to repair or refold carbonylated proteins, since
carbonylation is an irreversible modification. Reduced car-
bonylation in cells overproducing this chaperone system may
instead result from (i) a reduced abundance of aberrant pro-
teins and/or (ii) an increased DnaK/DnaJ-dependent solubil-
ity of carbonylated proteins. The former of these suggestions
stems from data demonstrating that aberrant forms of proteins
are more susceptible to oxidative carbonylation than their na-
tive counterpart (8). Thus, any condition reducing the levels of
FIG. 3. Effects of overproducing DnaK/DnaJ (ÅF38) on EF-Tu
carbonylation during glucose starvation (A). Open squares, no IPTG;
gray squares; 100 ?M IPTG; black squares, 250 ?M IPTG. Carbonyl
levels are normalized to EF-Tu levels. (B) General pattern and level of
carbonylated proteins (equal amounts of total cellular proteins were
loaded/well) in growth-arrested (2.5 h after cell division ceased) wild-
type (ÅF38) cells harboring the PA1/lacO-1-dnaK dnaJ construct. Cells
were grown with (250 ?M) and without IPTG prior to starvation.
Arrow indicates EF-Tu. Protein and carbonyl levels were determined
by one-dimensional Western blot immunoassays and quantified using
Image Gauge software. All experiments were repeated at least three
times. Representative results are shown, and there was always less than
15% variation between experiments.
FIG. 4. Relative stability of carbonylated proteins determined by
one-dimensional Western blot immunoassays in cells with different
levels of ?32. Protein synthesis was inhibited with spectinomycin during
entry to stationary phase (30 min after growth ceased) in cells con-
taining either the empty vector control (ÅF42; open squares) or the
Ptrc-rpoH construct (ÅF41) grown in the presence (black squares) or
absence (gray squares) of 100 ?M IPTG.
4210 THE HEAT SHOCK REGULON AND PROTEIN CARBONYLATIONJ. BACTERIOL.
such aberrant proteins, such as increased ribosomal proof-
reading (3) or elevated levels of DnaK/DnaJ (this work),
may be expected to also reduce cellular carbonyl levels. The
second idea is based on the fact that carbonylated proteins
are susceptible to proteolysis as long as they remain soluble
(13). However, heavily carbonylated proteins are prone to
aggregate and such high-level molecular aggregates escape
proteolysis (4). Increased levels of DnaK/DnaJ may keep
carbonylated proteins in a soluble, protease-susceptible
form and thereby contribute to their degradation by, e.g.,
Lon and HslVU. It has been suggested that the decline in
proteosomal activity during aging (1, 5) may be closely con-
nected to a gradual accumulation of proteolysis-resistant
aggregates of carbonylated proteins that bind and inhibit
proteosomal function (13). Thus, it may be worth consider-
ing that the effects of overproducing Hsp70 (DnaK family
proteins) in retarding aging (31) and delaying the onset of
age-related protein aggregation (14) in eukaryotes may be
linked to its role in reducing the level of carbonylated and
aggregation-prone proteins. However, overproduction of
DnaK/DnaJ did not extend the average life span of station-
ary phase cells (Fig. 7A and B). Both plating efficiency and
membrane integrity were lost with the same rate, or at a
higher rate, in cells with ectopically elevated levels of DnaK/
DnaJ (Fig. 7). Thus, it may be that increased carbonylation
is a diagnostic marker of deterioration (7) but is not the
limiting factor in stationary phase survival of E. coli cells.
Alternatively, it is possible that the reduced concentration
of other important proteins of the heat shock regulon re-
sulting from DnaK/DnaJ overproduction masks potential
benefits of reducing carbonylation. In addition, elevating the
levels of the entire complement of heat shock proteins by
overproducing ?32is not informative with respect to stasis
survival since it drastically reduces expression, by sigma
factor competition (15), of ?S- and ?E-dependent genes,
which are essential for stasis survival. Thus, while it is clear
that mutant cells lacking specific members of the heat shock
regulon (e.g., DnaK, ClpP, HslVU, and Lon) (16, 23, 27) die
at an accelerated rate in stationary phase, it is uncertain
FIG. 5. Severalfold change in the levels of carbonylated proteins in
wild-type (MG1655?lac) cells and in cells lacking the proteases Lon
(ÅF66), HslVU (ÅF49), and ClpPX (PhB1907), ClpPX and Lon
(PhB1465), and ClpPX, Lon, and HslVU (PhB1466). Protein extracts
were from exponentially growing cells (open bars) and cells from an
overnight stationary phase sample (grey bars). Carbonyl levels were
determined by a slot-blot immunoassay and quantified using Image
Gauge software. All levels were related to that obtained for the wild
type during exponential growth, which was assigned a value of 1.0.
FIG. 6. Proteomic immunodetection of carbonylated proteins in wild type (MG1655?lac), ?lon (ÅF66), and ?hslVU (ÅF49) cells entering a
growth-arrested state due to glucose starvation. The major target proteins of carbonylation were identified by mass spectrometry and included the
?-subunit of RNA polymerase (RpoB), elongation factors Tu and G (EF-Tu, EF-G), the E1 component (AceE) of the pyruvate dehydrogenase
complex, isocitrate dehydrogenase (Icd), 6-phosphogluconate dehydrogenase (Gnd), and Serine hydroxymethyltranferase (GlyA). The boxed
proteins are GroEL and DnaK. The experiment was repeated three times, and the patterns of carbonylated proteins were the same in each
experiment. Representative results are shown.
VOL. 187, 2005FREDRIKSSON ET AL. 4211
whether the level of heat shock proteins constitutes a bot-
tleneck in stationary phase survival of wild-type E. coli cells.
We thank B. Bukau, C. Gross, K. Gerdes, M. Kanemori, S. Gottes-
mann, T Yura, J. Urbonavicius, P. Bouloc, and M. P. Mayer for
providing strains, plasmids, and information necessary for this work,
Thomas Larsson for help with the mass-spectrometric analyses, and
also the people in the Nystro ¨m laboratory for valuable comments on
This work was sponsored by grants from the Swedish Natural Re-
search Council and the Foundation for Strategic Research in Sweden
and an award from the Go ¨ran Gustafsson Foundation for Scientific
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