From the Département de Biochimie
Médicale, Centre Médical Universitaire, Université de
Genève, 1, rue Michel-Servet, 1211 Genève 4, Switzerland
and the § Department of Molecular Biology, International
Institute of Molecular and Cell Biology, United Nations Educational,
Scientific and Cultural Organisation-Polish Academy of
Sciences, 4, Trojdena Street, 02-109 Warsaw, Poland
Received for publication, May 5, 2000, and in revised form, December 1, 2000
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ABSTRACT |
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DjlA is a 30-kDa type III membrane protein
of Escherichia coli with the majority, including an extreme
C-terminal putative J-domain, oriented toward the cytoplasm. No other
regions of sequence similarity aside from the J-domain exist between
DjlA and the known DnaK (Hsp70) co-chaperones DnaJ (Hsp40) and CbpA. In
this study, we explored whether and to what extent DjlA possesses DnaK co-chaperone activity and under what conditions a DjlA-DnaK interaction could be important to the cell. We found that the DjlA J-domain can
substitute fully for the J-domain of DnaJ using various in vivo functional complementation assays. In addition, the purified cytoplasmic fragment of DjlA was shown to be capable of stimulating DnaK ATPase in a manner indistinguishable from DnaJ, and, furthermore, DjlA could act as a DnaK co-chaperone in the reactivation of chemically denatured luciferase in vitro. DjlA expression in the cell
is tightly controlled, and even its mild overexpression leads to induction of mucoid capsule. Previous analysis showed that
DjlA-mediated induction of the wca capsule operon required
the RcsC/RcsB two-component signaling system and that wca
induction by DjlA was lost when cells contained mutations in either the
dnaK or grpE gene. We now show using
allele-specific genetic suppression analysis that DjlA must interact
with DnaK for DjlA-mediated stimulation of capsule synthesis.
Collectively, these results demonstrate that DjlA is a co-chaperone for
DnaK and that this chaperone The Hsp70 family of molecular chaperones, of which DnaK is the
major member in Escherichia coli, is regulated by
interaction with co-chaperones that function together as a chaperone
machine. In E. coli, DnaJ has long been recognized as a
partner protein and key regulator of DnaK ATPase activity, together
with the nucleotide exchange factor, GrpE (1). The general mechanism of
the Hsp70 chaperones, including DnaK, is that binding and release of
protein substrates is tightly coupled to their ATPase cycle (2, 3).
The Hsp40 family of molecular chaperones is defined by a short,
~70-amino acid residue signature sequence, termed the J-domain, which
helps direct interaction with partner Hsp70 chaperones (4-6). Indeed,
the J-domain of DnaJ is absolutely essential for its interaction with
DnaK and is specifically required for stimulation of the ATPase
activity (1, 7-9). A highly conserved HPD tripeptide, located in an
exposed loop of the J-domain, is critical for co-chaperone function
because mutations in these residues severely compromise the stimulation
of ATP hydrolysis, not only in E. coli, but in many other
organisms (5, 10, 11).
The E. coli genome codes for two Hsp40 proteins, DnaJ and
CbpA, and two putative J-domain proteins, DjlA and Hsc20. DnaJ and its
close orthologs are characterized by four domains: an N-terminal J-domain, a region rich in glycine and phenylalanine, a zinc finger domain, and a C-terminal domain thought to be involved in substrate binding (4, 5). CbpA is 39% identical to DnaJ at its entire length and
55% at its J-domain (12). CbpA is a multicopy suppressor of
dnaJ mutations and is thus a functional ortholog of DnaJ
(13). A cbpA null mutation shows a synthetic phenotype with
a dnaJ null mutation, the doubly mutant strain becoming
hypersensitive for growth above 37 °C and below 16 °C (14). Hsc20
possesses only 20% sequence similarity to the DnaJ J-domain and is
unable to interact productively with DnaK (15, 16). By sequence
alignment methods, DjlA possesses a J-domain signature sequence at its
extreme C terminus with 31% sequence identity (17) but otherwise
possesses no sequence similarity to other regions of DnaJ and CbpA.
Many Hsp70 co-chaperones have only the J-domain signature in their sequence (5). Thus, DjlA could represent a third DnaK co-chaperone in
E. coli, but under what circumstances is unclear. A clue to a potential role for DjlA in the cell emerged in previous studies when
we and others (17-20) showed that DjlA overexpression could trigger
the synthesis of a colanic acid polysaccharide capsule. In E. coli, a colanic acid capsule can be induced by environmental stress such as osmotic shock, low temperature, and desiccation (21-23). Because a colanic acid capsule is not normally observed at
37 °C, or under physiological conditions prevailing within the host
intestinal tract, it has been suggested that this adaptive response may
help cells to survive conditions outside the host (24). Interestingly,
no link has been shown between colanic acid capsule production and
pathogenicity (25, 26), although a recent study suggests a role for
colanic acid in the architecture of biofilms (27).
The major structural genes for colanic acid synthesis in E. coli K-12 are found in the wcaABCDE locus, formerly
called cps (28-30). Detailed analysis of colanic acid
induction has demonstrated that the wza-wca
operon (hereby referred to as wca) is regulated by the
RcsC/RcsB two-component histidine kinase signaling system (24, 31, 32).
Induction of wca by DjlA is dependent on both RcsC and RcsB,
suggesting that DjlA modulated the activity of this phosphorelay sensor.
Previous genetic analyses of parameters affecting capsule induction
revealed that activation of a wcaB-lacZ reporter by DjlA was
abrogated in strains containing mutations in genes encoding the
molecular chaperones DnaK and GrpE but was not adversely affected by
null mutations in genes encoding DnaJ, CbpA, HtpG (Hsp90), or Hsc66
(Hsp70 homolog) (17, 18). In the present work, we have used both
biochemical and genetic analyses to address whether DjlA is a
bona fide DnaK co-chaperone and whether direct DjlA-DnaK interaction is necessary for wca activation.
Bacterial Strains--
WKG190 is MC4100, araD139
Plasmid Constructions--
Plasmid pWCS19 (37), a pTrcHisA
vector (Invitrogen) contains the gene encoding for the DnaK(R167H)
mutant (a kind gift from Dr. Carol Gross and Dr. Won-Chul Suh,
University of California at San Francisco). The wild type
dnaK+ version of pWCS19, pKG9, was prepared by
the QuickchangeTM method (Stratagene) using the primers
5'-CTGGAAGTAAAACGTATCATCAACGAAC-3' and
5'-GTTCGTTGATGATACGTTTTACTTCCAG-3'. The dnaK+
wild type sequence was verified and tested for functional
complementation of the dnaK103 mutant strain CG800, which is
otherwise unable to form colonies at 42 °C and cannot support growth
of bacteriophage Polymerase Chain Reaction Construction of J-domain
Chimeras--
Plasmid pWKG50 (17) expression phagemid was mobilized
with VCSM13 (Stratagene) in strain CJ236 (40) and mutagenized to introduce the D235N mutation using the method of Kunkel (40) and
the oligonucleotide DjlA D235N: 5'-CGCCACCAGCTTATTGGGATGGTGTTC-3'. The
resulting plasmid, pWKG55, was sequence verified. Plasmids pWKG90 and
pWKG100 (33) encode E. coli dnaJ and dnaJ12,
respectively, with a phenotypically silent H71T mutation that was
engineered to introduce a KpnI site at the carboxyl junction
of the J-domain. Plasmids pWKG90 and pWKG100 were digested with
EcoRI-KpnI to remove the E. coli
J-domain and then exchanged with a 220-base pair polymerase chain
reaction fragment derived from the plasmid templates pWKG50 (djlA), pWKG54 (djlAH233Q), or pWKG55
(djlAD235N) (17) that had been digested with the same
enzymes. Primer sets 5'-GGGAATTCACCATGGAAGATGCCTGTAATG-3', and 5'-CCGGTACCTTTAAACCCTTTCTGCTGCTT-3' were used to
amplify the djlA J-domains and introduced the appropriate
EcoRI and KpnI restriction sites. All constructs
were sequence verified using appropriate primers. Plasmids pKG1,
pKG2, and pKG3 contain djlA, djlA(H233Q), and
djlA(D235N) J-domains, respectively, in the pWKG90 vector background. Plasmids pWKG4, pWKG5, and pWKG6 contain the same respective djlA J-domains in the pWKG100 vector background.
Bacteriophage and Colony Forming Assays--
Bacteriophage
laboratory stocks Protein Purifications--
E. coli DnaJ protein was
prepared from lysates of strain WGK190 containing pWKG90, a
L-arabinose inducible promoter vector driving expression of
wild type DnaJ (33) essentially as described (41). E. coli
GrpE was prepared from strain DA262 containing plasmid pOD1, a
L-arabinose inducible promoter vector driving expression of
wild type GrpE, and using methods essentially as described (42).
E. coli DnaK was purified from strain B178 (43) containing
dnaK+ in pTTQ19 (Amersham Pharmacia Biotech)
using the described procedures (44).
DjlA Purification--
E. coli DjlA soluble
cytoplasmic fragment was purified from strain WKG190 harboring the
vector pWKG52 (17), in which the DNA segment encoding the N-terminal
31-amino acid transmembrane-spanning region had been removed. The use
of a host strain devoid of both DnaJ and CbpA assured that no other
J-domain-containing protein will contaminate the DjlA preparation.
Full-length DjlA encoded by the chromosomal copy of djlA is
expressed poorly in this genetic background, in addition to being
membrane-associated, and thus not detectable by immunoblot analysis in
the soluble fraction (17). For purification of DjlA cytoplasmic
fragment, a fresh overnight culture of pWKG52 in WKG190 was diluted
1:100 into 12 liters of LB broth supplemented with 50 µg/ml
ampicillin and grown at 30 °C with vigorous shaking. At an
A600 = 1.0, L-arabinose inducer was
added to a final concentration of 0.1% (w/v) and shaking continued for
an additional 3 h at 30 °C. Cells were harvested at 10,000 rpm
for 10 min in a Sorvall GSA rotor and resuspended in a minimal volume
of 50 mM Tris-HCl, pH 7.6, 10% (w/v) sucrose. All
subsequent steps were performed at 4 °C. Cells were lysed with 250 ml of buffer A (50 mM Tris-HCl, pH 7.2, 1 M
NaCl, 2 mM MgCl 2, 2 mM
DTT,1 0.4 mg/ml lysozyme) for
60 min on ice. To aid lysis and to reduce viscosity, the cell
suspension was briefly heat shocked in a 45 °C immersion bath for 3 min then sonicated with 20 2-s pulses. The lysate was then centrifuged
at 25,000 rpm for 1 h 45 min in a Beckman 35 Ti rotor. Ammonium
sulfate (0.226 g/ml) was added slowly with stirring to the cleared
supernatant, and the precipitate was collected after centrifugation at
25,000 rpm for 1 h in a 35 Ti rotor. The pellet was resuspended
and dialyzed against 2 liters of buffer B (50 mM HEPES, pH
7.6, 150 mM KCl, 2 mM DTT, 0.05% Triton X-100,
10% (v/v) glycerol). Insoluble material was removed by centrifugation
at 20,000 rpm for 30 min in a 35 Ti rotor. The supernatant was then
applied to a fast flow Q Sepharose column (2.5 × 20 cm) that had
been pre-equilibrated with buffer B. The void volume was collected, and
DjlA fractions were pooled and dialyzed against 2 liters of buffer C
(50 mM potassium phosphate, pH 6.8, 150 mM KCl,
2 mM DTT, 0.05% Triton X-100, 10% (v/v) glycerol). The
dialysate was applied to a P-11 phosphocellulose column (2.5 × 20 cm) that had been pre-equilibrated in buffer C. The column was washed
with 5 column volumes of buffer C, then developed with a 0.15-0.6
M KCl linear gradient in buffer C (800 ml). The 27-kDa DjlA
cytoplasmic fragment eluted first, whereas the truncated 25-kDa
fragment was well separated, eluting at higher KCl. The two peak
fractions were pooled separately, dialyzed against buffer D (50 mM Tris-HCl, pH 7.6, 150 mM KCl, 2 mM DTT, 0.05% Triton X-100, 10% (v/v) glycerol), and
concentrated separately by application on an HTP hydroxyapatite
(Bio-Rad) column (1.5 × 15 cm) pre-equilibrated in buffer D. Bound proteins were eluted with a 0-300 mM linear potassium phosphate, pH 7.2, gradient. Peak fractions were pooled, dialyzed against storage buffer E (50 mM HEPES, pH 7.6, 150 mM KCl, 2 mM DTT, 10% (v/v) glycerol), then
quickly frozen in liquid nitrogen and stored at Protein Sequencing--
For N-terminal protein sequencing,
sample aliquots were resolved in 12% (w/v) SDS-polyacrylamide gels,
electroblotted to polyvinylidene difluoride membranes (Bio-Rad), and
bands were visualized by staining with 0.1% Amido Black in 25% (v/v)
2-propanol, 10% (v/v) acetic acid. Bands were excised, rinsed in
sterile water, dried, and applied directly to an Applied Biosystems
model 473 Sequencer equipped with a blot cartridge.
Protein Concentration Determination--
Protein concentrations
were determined using published or derived molar extinction
coefficients (45, 46) or by Bio-Rad dye reagent assay using bovine
serum albumin as a standard.
Steady-state ATPase Assay--
The assay was performed
essentially as described previously (1). The standard reaction
conditions were 50 mM HEPES, pH 7.6, 40 mM KCl,
50 mM NaCl, 7 mM magnesium acetate, 2 mM DTT, 300 µM disodium ATP, 1 µM DnaK, 1 µM GrpE, and DnaJ or DjlA
titrated over the range 0-1 µM.
[ Luciferase Refolding Assay--
Reactivation of denatured
firefly luciferase was performed essentially as described (47) with
minor modifications. Briefly, luciferase (Promega) at a concentration
of 2.5 µM was denatured for 90 min at 22 °C in a
solution containing 40 mM Tris-HCl, pH 7.4, 6 M
guanidinium hydrochloride, 5 mM DTT, 50 mM KCl,
and 15 mM MgCl 2. The denatured luciferase was
diluted to a final concentration of 80 nM into a reaction
mixture containing 40 mM Tris-HCl pH 7.4, 50 mM
KCl, 5 mM DTT, 15 mM MgCl 2, 100 µg/ml creatine kinase, 20 mM creatine phosphate, 0.015%
bovine serum albumin, 5 mM ATP, 1 µM GrpE,
and 1 µM DnaK. All components were assembled on ice. Renaturation was initiated by adding either DnaJ or DjlA Luciferase Aggregation Assay--
The kinetics of luciferase
aggregation were followed by measuring light scattering at 320 nm with
a Uvikon 940 spectrophotometer using a thermostated cell holder
essentially as described (48).
Immunoblot Analysis--
Anti-DjlA antibodies, the kind gift
of Dr. David Clarke and Dr. I. B. Holland (Institut de
Génétique et Microbiologie, Université de Paris Sud),
were used at a dilution of 1:20,000. Goat anti-rabbit horseradish
peroxidase-conjugated IgG secondary antibodies were used at a dilution
of 1:10,000. Blots were developed with enhanced chemiluminescent
reagents according to the manufacturer's recommendations (Amersham
Pharmacia Biotech).
The DjlA J-domain Can Functionally Replace the J-domain of DnaJ in
Vivo--
Inspection of DjlA J-domain sequence alignment of residues
208-271 (the native C terminus) revealed 31% identity and 44%
similarity to the DnaJ J-domain, including the highly conserved HPD
tripeptide (17, Fig. 1). To determine
whether the J-domain of DjlA was capable of functional interaction with
DnaK, we first constructed plasmids coding for chimeric DnaJ proteins.
Conditional expression of the DnaJ chimeras in a dnaJ cbpA
double null strain permits measurement of J-domain activity (33).
The djlA region encoding the putative DjlA J-domain
corresponding to amino acids 206-271 (the native C terminus) was
amplified by polymerase chain reaction, and chimeras were constructed
as described under "Experimental Procedures." The plasmids were
transformed into strain WKG190 at the permissive temperature of
30 °C, then tested in a colony forming assay. The results are shown
in Fig. 2. We observed that the chimeric
plasmid pKG1 complemented for bacterial growth at 14 °C or 40 °C
as efficiently as wild type DnaJ in the presence, but not in the
absence, of the L-arabinose inducer. In contrast, the
vector alone did not complement for bacterial growth nor did either of
the chimeric plasmids encoding for mutant DjlA J-domains, pKG2 and
pKG3. Similar results were observed using plasmids pKG4, pKG5, and
pKG6, which encode the truncated DnaJ12 derivatives (data not
shown).
The failure of the DjlA H233Q and D235N mutant J-domain chimeras to
complement was not caused by altered steady-state protein expression
levels or stability, as judged by immunoblot analysis or inspection of
Coomassie Blue-stained SDS gels. We observed that the levels of the
DjlA J-domain replacement chimeras were about 10-fold lower than DnaJ
but were of comparable level when compared with each other
(compare the second and fourth lanes from the
left, Fig. 3). We conclude
that the mutations in DjlA altering the highly conserved HPD
tripeptide were disrupting the otherwise productive J-domain
interaction with DnaK.
Restoration of Bacteriophage Purified DjlA Cytoplasmic Fragment Can Stimulate DnaK ATPase in
Vitro--
A well studied consequence of DnaJ-DnaK interaction
in vitro is the ability of DnaJ to stimulate the
intrinsically weak DnaK ATP hydrolysis rate. Because our in
vivo assays indicated that the DjlA J-domain could functionally
replace the DnaJ J-domain and therefore was likely to interact directly
with DnaK, we examined whether the purified cytoplasmic fragment of
DjlA (32) might by itself engage DnaK and stimulate its ATPase, as
predicted if indeed DjlA could function as a DnaK co-chaperone.
Plasmid pWKG52 encodes DjlA lacking the transmembrane-spanning region
of wild type DjlA. It was used to overexpress and purify the
cytoplasmic fragment of DjlA from a strain lacking both dnaJ and cbpA to ensure the absence of contaminating DnaJ
activity. The steps of purification are depicted in Fig.
4. Upon cell lysis, approximately half of
the DjlA is cleaved rapidly, resulting in the formation of a doublet
suggesting the removal of a short stretch of amino acids. The
proteolytic activity has been suggested previously to be an artifact of
the preparation method (19). Using a variety of lysis methods,
including freeze-thaw and detergent lysis as well as a panel of
protease inhibitors, we also noted this cleavage except when whole cell
lysates were prepared by direct solubilization in reducing SDS sample
buffer. Both DjlA products are immunologically reactive with anti-DjlA
antiserum and copurifiy during the early chromatographic steps.
However, the full-length cytoplasmic fragment can be easily separated
from the smaller species, using gradient elution by phosphocellulose
P-11 chromatography.
N-terminal protein sequence analysis of the purified smaller
proteolytic cleavage fragment revealed that cleavage occurs between residues Arg-39 and Lys-40 of the full-length protein, thus resulting in the removal of 8 residues from the cytoplasmic fragment of DjlA. The
full-length cytoplasmic fragment was used for all subsequent studies.
Highly purified DnaK, DnaJ, GrpE, and DjlA Purified DjlA Cytoplasmic Fragment Possesses Co-chaperone
Activity--
As an additional test of DnaJ co-chaperone activity, we
asked whether the purified DjlA cytoplasmic fragment could act as a
co-chaperone, together with DnaK and GrpE in the in vitro
reactivation of chemically denatured firefly luciferase. Because this
assay has been shown to be critically dependent upon DnaJ (47-50) to promote the efficient refolding of denatured luciferase, we expected that if DjlA possessed the requisite co-chaperone activity, it could
fully or partially replace DnaJ in this assay. The results of a
representative kinetic analysis of luciferase reactivation are
presented in Fig. 6A.
We observed that DjlA Purifed DjlA Cytoplasmic Fragment Cannot Act as a Chaperone Alone
but Can Help DnaK to Prevent Luciferase Aggregation--
DnaJ has been
described as a bona fide chaperone because it can bind alone
to various denatured substrates and help protect them from aggregation
(47-50). To test whether the purified DjlA cytoplasmic fragment alone
behaved as a bona fide chaperone, we chose to study the
kinetics of aggregation of denatured luciferase. When DnaJ alone is
used in this type of assay, it has been shown to protect luciferase
partially from aggregation, whereas DnaK alone is unable to prevent
aggregation. However, DnaK and DnaJ together, in the presence of ATP,
can efficiently protect luciferase from aggregation, provided that the
chaperone proteins are supplied in near stoichiometric amounts. The
results of such an experiment are depicted in Fig.
6B.
Our results showed that either DjlA or DnaJ can cooperate with DnaK in
the presence of ATP to prevent luciferase aggregation. However,
although DnaJ alone could partially protect luciferase from aggregation
as expected, we did not observe any such activity for the DjlA Allele-specific Suppression Analysis Shows that DjlA-DnaK
Interaction Is Necessary for wca Transcriptional Activation--
The
E. coli dnaJ236 allele, encoding DnaJ with a point
mutation (D35N) in the highly conserved HPD tripeptide of the
J-domain, is temperature-sensitive for bacterial growth above 42 °C
and cannot support replication of bacteriophage
We exploited this genetic analysis to study DjlA-DnaK interaction.
Specifically, we reasoned that if direct interaction between DjlA and
DnaK were necessary for wca transcriptional activation through the RcsC/RcsB two-component system, then engineered point mutations in the DjlA J-domain should abolish wca
activation. Importantly, if analogous allele-specific suppression were
possible with another J-domain-DnaK pair from the same organism, then
simultaneous coexpression of defective DjlA(D235N) with DnaK(R167H)
should restore wca-lacZ reporter activity. The
allele-specific interaction would also predict that simultaneous
coexpression of DjlA(H233Q) with DnaK(R167H) would not restore
wca-lacZ reporter activity.
Plasmids encoding wild type DnaK(pKG8) or DnaK (R167H)(pKG7) under
the control of the pTrc promoter were constructed.
Combinations of DjlA and DnaK expression plasmids, or parental vectors
pBAD22 and pWKG59 alone, were transformed into the SG20781
wcaB10-lacZ reporter strain and the activation of
the wca operon analyzed. The results are depicted in Fig.
7. The overexpression of DjlA upon
addition of the L-arabinose inducer resulted in a strong induction of the wca operon, as judged by the increased
levels in
When plasmids pWKG54 and pWKG55, containing the mutation H233Q, or
D235N alone were expressed under conditions identical to wild type
DjlA, no wca induction was observed, indicating that mutations within the djlA J-domain HPD tripeptide had
abolished DjlA's effect on capsule synthesis. Furthermore, control
experiments revealed that steady-state protein levels of wild type DjlA
and the two mutant derivatives were indistinguishable under the
conditions of the assay (data not shown).
A significant activation of wca-lacZ reporter was observed,
however, when plasmid pWKG55 was coexpressed with pKG7
dnaK(R167H), but not with pKG8 dnaK+.
In contrast, no significant activation of wca-lacZ was
observed when pWKG54 was coexpressed with pKG7 or pKG8, indicating that the observed wca-lacZ activation was indeed the consequence
of allele-specific genetic interaction between DjlA(D235N) and
DnaK(R167H). We conclude that DjlA and DnaK must interact directly to
elicit wca activation after DjlA overexpression.
The major finding of this work is the demonstration that DjlA
represents a third regulatory DnaK co-chaperone in E. coli. Several lines of evidence presented here clearly demonstrate that (a) the J-domain of DjlA is indeed functional, as judged by
its ability to replace the J-domain of DnaJ in several in
vivo assays; (b) purified DjlA cytoplasmic fragment can
productively interact with DnaK in vitro, thus behaving as a
bona fide co-chaperone for DnaK; (c) DjlA does
not apparently possess intrinsic chaperone activity alone as judged by
its inability to protect luciferase from aggregation; and
(d) direct DjlA-DnaK interaction is necessary for
wca activation as revealed by genetic allele-specific
suppressor analysis.
Our assay for J-domain function in E. coli, using chimeric
proteins, has revealed a surprisingly broad tolerance for J-domains from a wide range of sources with limited sequence homology. In contrast to our findings, analogous experimental use of J-domain chimeras in Saccharomyces cerevisiae and SV40 virus shows
that these systems have a much more strict tolerance for interchanging J-domains (52-54). At the molecular level, specificity determinants for Hsp70-Hsp 40 interaction are largely unknown, but they are thought
to be mediated largely by the J-domain.
Some features of the DjlA J-domain provide some new insights into
J-domain-DnaK interaction. The DjlA J-domain possesses a longer loop
between helices II and III as well as a truncation of the residues
corresponding to the majority of the region helix IV, including most of
the QKRAA motif (see Fig. 1). It is noteworthy that the loop region
between HPD and the start of helix III shows considerable sequence and
length heterogeneity among J-domains that function in E. coli and thus is unlikely to be by itself a crucial specificity
determinant for interaction with DnaK. The QKRAA motif, comprising the
helix IV region of DnaJ J-domain, has been described as a possible site
of interaction with DnaK (57). Because the DjlA J-domain lacks this
motif, yet can functionally replace the DnaJ J-domain in
vivo and in vitro, helix IV cannot be essential for
directing interaction with DnaK. In agreement with this conclusion, a
DnaJ derivative entirely lacking helix IV can fully substitute for DnaJ
in vivo.3 Of
course, these findings do not exclude the possibility that helix IV
plays some as yet undefined modulatory role in the biology of DnaJ
in vivo.
Despite possessing a functional J-domain, DjlA is clearly not a
functional ortholog of DnaJ and CbpA. Previous work showed that DjlA
could not adequately complement DnaJ function in a strain lacking both
dnaJ and cbpA, even when expressed to the same
levels of DnaJ which could functionally complement in this test system (17, 20). Additionally, overexpression of the DjlA cytoplasmic fragment
could not support bacterial growth of the dnaJ cbpA double null strain above 39 °C nor at temperatures below 20 °C, where this strain also exhibits a cold-sensitive phenotype (17, 20). Furthermore, DjlA cannot replace either DnaJ or CbpA in supporting bacteriophage The precise cellular role of DjlA has not been determined yet. A clue
to a possible function emerged when it was shown that overexpression of
DjlA could lead to the induction of a colanic acid capsule (17-19).
Our observation that DjlA Activation of the wca operon by DjlA-DnaK interaction in
E. coli implies a chaperone machine capable of modulating
either directly or indirectly the RcsB/RcsC phosphotransfer signaling pathway, a role for chaperones which has not been described previously in bacteria. However, a GroEL chaperonin homolog found in the intracellular symbiotic bacterium of aphids has been described as a
potential histidine kinase with phosphotransferase activity, although
no direct evidence exists for its role in signaling to date (58). In
contrast, it is well known in eukaryotes that chaperones actively
participate in signaling mechanisms involving the steroid receptor
family (59), and a variety of kinases including double strand
RNA-activated kinase (PKR) in influenza infection (60),
c-Raf1 (61), and members of the Src tyrosine kinase family
(62). Future studies should address the extent of chaperone involvement, if any, in bacterial signaling systems.
co-chaperone pair is implicated directly,
or indirectly, in the regulation of colanic acid capsule.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ara714 dnaJ::Tn 10-42
cbpA::kan (33), SG20781 is MC4100,
wcaB10::lac-Mu-imm
(34),
CG800 is MG1655, thr::Tn 10 dnaK103
(35), MC4100
(argF-lac)U169 araD139 rpsL150
deoC1 relA1 ptsF25 flbB5501 rbsR (36).
at any temperature. The wild type and mutant
dnaK genes were re-engineered for DnaK expression using a
p15A-derived replicon that is compatible in cells harboring the
ColE1-derived pBAD vectors (38). Briefly, pACYC184 (39) was digested
with HincII, and the 3,178-base pair fragment was purified
and self-ligated in the presence of NsiI linkers
(5'-CATGCATG-3'). The resulting plasmid, pWKG59, was then digested with
HindIII and NsiI, and the 2,556-base pair
ori p15A-camr fragment, containing
the ori of replication, was ligated with the 3,509-base pair
HindIII-NsiI fragment containing
lacIq, pTrc promoter, and downstream
dnaK sequence from pWCS19, or pKG9, to yield plasmid pKG7
(dnaKR167H) or pKG8 (dnaK+), respectively.
b2cI
, a clear
plaque former, and
dnaJ+ transducing phage were prepared
from liquid lysates following infection of the host strain MC4100 using
Luria-Bertani (LB) broth supplemented with 50 mM
MgSO4. 5-ml lysates were treated with chloroform to ensure
cell lysis and debris removed by low speed centrifugation.
Bacteriophage were serially diluted in SM buffer (10 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, 50 mM NaCl). Plaque forming assays and spot test viability
assays were performed essentially as described previously (33).
80 °C until use.
-32P]ATP, specific activity of 3,000 Ci/mmol
(Amersham Pharmacia Biotech) was used as tracer in the reaction
mixture. Aliquots were removed from the reaction at the specified
times, spotted on polyethyleneimine thin layer chromatography plates
(Merck), and resolved with a 1:1 solvent mixture 1 M formic
acid, 1 M LiCl. Plates were dried and autoradiographed, and
the region corresponding to spots was excised and counted in a Beckman
liquid scintillation counter. Pilot assays were used to determine ATP
concentrations necessary for the concentration of DnaK used in the
standard assay. For determination of kinetics, linear regression
analysis was applied and data points accepted only when correlation
coefficients exceeded 0.98. In the analysis presented, all data were
obtained in the linear range of the assay, and each data point
represents the average rate derived from regression lines compiled from
five independent experiments. Data points shown at the zero time point should be considered as subject to a relatively large experimental error because of the sample preparation time.
TM (0.4 µM each). The resulting luciferase activity was measured
at different time points after incubation at 22 °C by withdrawing
aliquots and using the Promega luciferase assay kit (E1500) followed by liquid scintillation counting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
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Fig. 1.
Panel A, schematic representation of the
E. coli J-domain protein family. G/F, region rich
in glycine and phenylalanine; Zn, zinc finger domain;
cross-hatching indicates a conserved region in DnaJ and CbpA
thought to be involved in substrate binding; TM,
transmembrane region. Panel B, J-domain sequence alignment
by CLUSTAL analysis. Helical structural elements are shown above the
alignment and are derived from the NMR structure of the E. coli J-domain (55, 56). Identical residues are depicted in
black shading, and conserved amino acid substitutions are
indicated in gray. Note that the C-terminal residue of the
DjlA J-domain terminates in a region corresponding to the middle of
helix IV with only limited sequence similarity to DnaJ J-domain helix
IV residues.
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Fig. 2.
The J-domain of DjlA can substitute for that
of DnaJ. Bacterial viability assay was monitored in strain WKG190
using chimeras that contained the wild type or mutated J-domain of DjlA
engineered to replace the J-domain of DnaJ precisely. Shown is a
representative set of complementation tests for bacterial growth at the
indicated temperature on LB agar plates containing 50 µg/ml
ampicillin and 33 mM L-arabinose inducer. Only
the origin of the relevant J-domains is indicated at the top
of the figure, the rest of the protein being always that of DnaJ.
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Fig. 3.
The various protein chimeras are produced at
approximately equal levels. Immunoblot analysis of whole cell
lysates of WKG190 carrying various chimeric DjlA J-domain constructs in
the pWKG90 dnaJ expression plasmid backbone is shown under
steady-state growth conditions at 30 °C in the presence of 33 mM L-arabinose inducer. Only the relevant
J-domains present in the plasmid vector constructions are indicated at
the top of the figure.
Plaque Formation by J-domain
Chimeras--
As a second, independent assay of J-domain function, we
exploited the dependence of bacteriophage
upon the host bacterial chaperones DnaK, GrpE, and DnaJ for plaque formation. Table
I shows the results of a bacteriophage
plaque forming assay performed on strain WKG190, harboring the
indicated dnaJ or dnaJ12 expression plasmids. The
dnaJ+ transducing bacteriophage served as a
control because it was expected to form plaques with or without
L-arabinose, whereas
b2cI
was expected to grow only on plates where both the
L-arabinose inducer and a complementing plasmid encoding
dnaJ or dnaJ12 were present. We observed that
pKG1 and pKG4 could complement as efficiently as pWKG90 or pWKG100 as a
source of DnaJ for bacteriophage
plaque formation. Plasmids pKG2
and pKG5 could not complement for bacteriophage
plaque formation in
this assay, consistent with the observation that the well characterized
analogous dnaJ259 mutation (encoding for the corresponding
H33Q change) cannot support bacteriophage
growth either when in
single chromosomal copy or when expressed from an
L-arabinose-inducible vector (7, 33). In contrast, pKG3 but
not pKG6 allowed the formation of small turbid plaques in the presence
of the L-arabinose inducer, indicating partial complementation for bacteriophage
growth. It should be noted that
the
b2cI
bacteriophage employed
in this study forms very clear plaques on the wild type host, thus the
appearance of very small turbid plaques is most likely caused by
extremely low bacteriophage progeny yields. Control experiments using a
pBAD expression plasmid carrying dnaJ encoding the
equivalent mutation (D35N) did not support the growth of
b2cI
bacteriophage under any
conditions tested (data not shown). Taken collectively, these results
show that DjlA harbors a bona fide J-domain that is capable
of replacing the J-domain of DnaJ in two independent in vivo
assays of DnaJ function.
In vivo complementation assay for bacteriophage plaque formation on
E. coli strain WKG190 using engineered chimeric J-domain expression
vectors
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Fig. 4.
Overexpression and purification of
DjlA TM mutant protein. A Coomassie
Blue-stained 10% SDS-polyacrylamide gel is shown, indicating the
successive chromatographic steps in the purification of DjlA
TM
protein from strain WKG190 containing plasmid pWKG52. Whole cell
extracts were loaded as markers of induction either with (+) or without
(
) 6.6 mM L-arabinose inducer. Aliquots of
proteins at steps of purification: ammonium sulfate precipitate
(AmS), fast flow Q-Sepharose void volume (Q),
phosphocellulose gradient elution fraction (P11), and
hydroxyapatite (HPT) chromatography. Note the separation of
the 27-kDa cytoplasmic DjlA protein fragment from its N-terminal
proteolyzed fragment lacking eight amino acids.
TM were prepared and used
to analyze steady-state ATP hydrolysis rates in vitro. The
results are shown in Fig. 5. We found
that DjlA
TM could stimulate DnaK ATPase activity in a manner
indistinguishable from DnaJ under the range of protein concentrations
tested.
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Fig. 5.
DjlA TM can stimulate
DnaK ATPase activity. Stimulation of the ATPase activity of DnaK
by DnaJ (
) and DjlA
TM (
) in the presence of 1 µM
DnaK, 1 µM GrpE, and 100 µM ATP is shown.
The percentage of hydrolyzed ATP/min is plotted as a function of the
final DnaJ or DjlA
TM concentration in the reaction mixture.
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Fig. 6.
DjlA can function as a co-chaperone for DnaK
in the refolding of chemically denatured luciferase. Panel
A, representative plot of reactivation kinetics of guanidine
hydrochloride-denatured firefly luciferase dependent upon DnaJ or
DjlA TM. Denatured luciferase was diluted into a refolding reaction
mixture in the presence of DnaK and GrpE (
); in the presence of
DnaK, GrpE, and DnaJ (
); or in the presence of DnaK, GrpE, and
DjlA
TM (*). The percentage of reactivated luciferase is plotted as a
function of time. 100% refers to the luciferase activity without prior
denaturation. Panel B, a representative plot of a luciferase
aggregation protection assay. Denatured luciferase alone (
), in the
presence of DnaK (×), DjlA
TM (
); DnaJ (
), DnaK and DnaJ
(
), DnaK and DjlA
TM (
) is shown. Optical densities were
measured at 320 nm and at 30-s intervals for 10 min. The percentage was
normalized to luciferase aggregation obtained when no chaperones were
added.
TM could fully replace DnaJ in the refolding of
guanidinium hydrochloride-denatured luciferase and that the rate and
degree of reactivation were comparable between DnaJ and DjlA, using our
experimental conditions. Control reactivation of luciferase in the
absence of added DnaJ, or DjlA
TM co-chaperone revealed no
significant reactivation of luciferase and could not be distinguished
from the spontaneous refolding rate observed in the absence of added
chaperones or DnaK alone. We conclude that the cytoplasmically oriented
fragment of DjlA can indeed function as a DnaK co-chaperone.
TM
fragment. Consistent with this result, we were unable to detect binding
of DjlA
TM to denatured luciferase using a sensitive enzyme-linked
immunosorbent assay (data not shown). DnaK alone had no activity in
this assay, thus reinforcing the interpretation that the two pairs of
proteins, DnaJ-DnaK, or DjlA
TM-DnaK, must collaborate to
prevent luciferase aggregation fully. These results suggest that
DjlA
TM does not have intrinsic chaperone activity but can act as a
co-chaperone for DnaK. In a broader context, this result is consistent
with the observation that DjlA
TM cannot support the replication of
bacteriophage
either in vivo (17) or in
vitro,2 nor can DjlA or
DjlA
TM fully substitute for DnaJ for bacterial growth at high or low
temperature (17, 18, 20). Thus, DjlA is clearly distinct from DnaJ and
likely plays a more specific and restricted role in the cell.
at any temperature (39). An allele-specific suppressor of dnaJ236(D35N), but
not dnaJ259(H33Q), was isolated in the dnaK gene,
dnaK(R167H), changing a residue in a solvent exposed cleft
in the ATPase domain (39, 51). The dominant dnaK(R167H)
suppressor carried on a multicopy plasmid restored productive
interaction with DnaJ(D35N) and permitted restoration of bacterial
growth above 42 °C as well as bacteriophage
growth.
-galactosidase activity. Control experiments showed that
cells harboring the pKG8dnaK+ or
pKG7dnaK(R167H) expression plasmids alone did not show any significant wca operon activation.
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Fig. 7.
Allele-specific suppression analysis.
-Galactosidase activity was measured in the reporter strain SG20781
(wcaB10-lacZ) harboring the indicated plasmid
combinations after L-arabinose-induced expression of DjlA
and its J-domain mutant derivatives H233Q and D235N in the presence of
compatible plasmid replicons encoding pKG7 (DnaK), pKG8 (DnaK R167H),
or empty vector (pACYC) as described under "Experimental
Procedures." The data are reported in Miller units as the mean of at
least three independent determinations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
growth under any conditions tested.
TM, the DjlA 27-kDa cytoplasmic fragment,
can function as a co-chaperone, in a manner identical to
that of DnaJ, now strongly suggests that once produced in the cell in
its native form, membrane-anchored DjlA can engage and activate DnaK.
How might a DjlA-DnaK chaperone interaction participate in the
regulation of wca activation? Because it is known that many
members of the J-domain family bind specific substrates (5), it is
possible that DjlA acting as a co-chaperone helps to direct substrate
(or substrates) in the vicinity of DnaK. In this way, the DnaK
chaperone machine may act at a specific site. Other possibilities,
including that DjlA exerts indirect effects, cannot be excluded, and
must await further analysis. In this context, it is interesting that
computer data base searches reveal the existence of hypothetical
proteins sharing strong sequence similarity to DjlA in many pathogenic
Gram-negative bacteria. The high degree of sequence similarity observed
suggests that these putative DjlA orthologs may act as regulatory
co-chaperones for cognate Hsp70s in these organisms as well.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Won-Chul Suh and Carol Gross for the gift of plasmid pWCS19 and for kindly communicating their results prior to publication, Drs. David Clarke and I. B. Holland for the gift of anti-DjlA antibody, Drs. Severine Frutiger and Graham Hughes for aid in protein sequencing through the University of Geneva Faculty of Medicine core services facility, and Françoise Schwager for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported by the Canton of Geneva, Grants FN-31-47283-96 and 7PLPJ048480 from the Swiss National Science Foundation (to C. G.), and Grant from the Polish State Committee for Scientific Research (to M. Z.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Division des Maladies Infectieuses, Hôpital Universitaire de Genève, 24, rue Micheli-du-Crest, 1211 Genève 4, Switzerland. Tel.: 41 22 372 9819; Fax: 41 22 372 9830; E-mail: William.Kelley@medecine.unige.ch.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M003855200
2 A. Wawrzynow, unpublished data.
3 W. L. Kelley, unpublished data.
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ABBREVIATIONS |
---|
The abbreviation used is: DTT, dithiothreitol.
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REFERENCES |
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