Báo cáo khoa học: Direct CIII–HflB interaction is responsible for the inhibition of the HflB (FtsH)-mediated proteolysis of Escherichia coli r32 by kCIII docx

Direct CIII–HflB interaction is responsible for the
inhibition of the HflB (FtsH)-mediated proteolysis
of Escherichia coli r
32
by kCIII
Sabyasachi Halder
1
, Subhamoy Banerjee
1,2
and Pradeep Parrack
1
1 Department of Biochemistry, Bose Institute, Kolkata, India
2 Madhav Institute of Technology & Science, Gwalior, India
Development of the temperate coliphage k depends on
a set of phage-specified regulatory proteins that inter-
act with host target proteins [1–4]. Among several
effects on host protein synthesis, k provokes the over-
production of some bacterial proteins induced in the
heat shock response. This effect depends on early gene
expression encoded by the leftward (p
L
) transcription
unit of k [5,6]. CIII is a k-specific regulatory protein
from the p
L
operon with a potential for host inter-
action. It is a small 54-residue protein that favors the
lysogenic response to infection by stabilizing kCII, the
transcriptional factor that favors lysogeny and is
responsible for primary control of the lambda develop-
mental decision for lysis or lysogeny [4,7–9]. In the

absence of CIII, CII is rapidly degraded by the ATP-
dependent host metalloprotease HflB (FtsH). The
molecular mechanism for CIII-mediated inhibition of
the proteolysis of CII by HflB involves CIII–HflB
interaction [10,11]. CIII itself is a substrate of HflB
[12]. It has also been reported that CIIIC, the central
helical domain of CIII, is resistant to HflB proteolysis,
and is a more effective inhibitor than the full-length
protein [11].
Apart from promoting lysogeny by lambda, CIII
protects Escherichia coli r
32
, the heat shock-specific
sigma factor, which is also a substrate of HflB [13,14].
This effect generates a heat-shock response in the cell
[15,16]. Maximal production of CIII prolongs the
heat-induced synthesis of E. coli heat shock proteins
even at low temperature [17]. The half-life of r
32
( 2 min) is increased approximately fourfold upon
overproduction of CIII, resulting in an overproduction
of heat shock proteins and rapid inhibition of cell
growth [15]. The molecular mechanism of the CIII-
mediated inhibition of proteolysis of r
32
by HflB is
unknown.
We tried to address this issue by studying
the effects of CIII and CIIIC on the proteolysis of
Keywords

antiproteolytic activity; heat shock; lysogeny;
kCII; r
32
Correspondence
P. Parrack, Department of Biochemistry,
Bose Institute, P-1 ⁄ 12, C.I.T. Scheme VIIM,
Kolkata 700 054, India
Fax: +91 33 2355 3886
Tel: +91 33 2569 3227
E-mail:
(Received 28 April 2008, revised 14 July
2008, accepted 28 July 2008)
doi:10.1111/j.1742-4658.2008.06610.x
The CIII protein of bacteriophage lambda exhibits antiproteolytic activity
against the ubiquitous metalloprotease HflB (FtsH) of Escherichia coli,
thereby stabilizing the kCII protein and promoting lysogenic development
of the phage. CIII also protects E. coli r
32
, another substrate of HflB. We
have recently shown that the protection of CII from HflB by CIII involves
direct CIII–HflB binding, without any interaction between CII and CIII
[Halder S, Datta AB & Parrack P (2007) J Bacteriol 189, 8130–8138]. Such
a mode of action for kCIII would be independent of the HflB substrate. In
this study, we tested the ability of CIII to protect r
32
from HflB digestion.
The inhibition of HflB-mediated proteolysis of r
32
by CIII is very similar
to that of kCII, characterized by an enhanced protection by the core CIII

peptide CIIIC (amino acids 14–41 of kCIII) and a lack of interaction
between r
32
and CIII.
Abbreviation
GST, glutathione S-transferase.
FEBS Journal 275 (2008) 4767–4772 ª 2008 The Authors Journal compilation ª 2008 FEBS 4767
r
32
by HflB in vitro. Our results show that both CIII
and CIIIC inhibit the HflB-mediated proteolysis
of r
32
in vitro, and that CIIIC is a more effective
inhibitor. We also found that there is no interaction
between CIII and r
32
. From these results, we suggest
that the inhibition of r
32
by CIII is due to a direct
CIII–HflB interaction.
Results
In vitro proteolysis of r
32
by HflB requires ATP
The proteolysis of r
32
by HflB is very rapid and
requires the DnaK–DnaJ–GrpE chaperone machine

in vivo [18–20]. However, this proteolysis is much
slower in vitro, because of a lack of chaperone machin-
ery. The in vitro proteolysis requires ATP [14]. We
examined the proteolysis of purified r
32
-C-his (1.2 lm)
by glutathione S -transferase (GST)–HflB (0.8 lm) in
the absence or presence of ATP (5 mm). Digestion
after specified time intervals was assayed by 11%
SDS ⁄ PAGE (Fig. 1). It is clear that the HflB-mediated
degradation of r
32
could not proceed without ATP. In
this respect, r
32
resembles kCII.
Inhibition of HflB-mediated proteolysis of r
32
by
CIII and by CIIIC
The effect of CIII and CIIIC on the HflB-mediated
proteolysis of r
32
was checked by treating r
32
-C-his
(1.2 lm) with GST–HflB (0.8 lm) in the presence of
CIII (100 lm) for varying time intervals (Fig. 2A). It
was found that r
32

was partially protected in the pres-
ence of CIII, and  65% of r
32
remained undigested
after 80 min, compared with  40% for the control
(r
32
alone, Fig. 2C).
The inhibitory action of CIIIC on the proteolysis of
r
32
by HflB was also assayed in the presence of CIIIC
(60 lm) instead of CIII (Fig. 2B). It is clear that in this
Fig. 1. ATP-dependence of proteolysis of r
32
by HflB. (Upper)
Band corresponding to r
32
on 11% SDS ⁄ PAGE, in the presence or
absence of 5 m
M ATP. Numbers on the top of each lane indicate
the time of digestion in min. (Lower) Densitometric scan showing
the amount of r
32
-C-his (1.2 lM) remaining after proteolysis with
GST–HflB (0.8 l
M). Each data point represents the mean value from
three identical experiments.
A
B

C
Fig. 2. Inhibition of HflB-mediated proteolysis of r
32
by CIII or
CIIIC. The bands corresponding to r
32
-C-his (1.2 lM) remaining
after proteolysis with GST–HflB (0.8 l
M) in the absence or
presence of (A) His
6
–CIII (100 lM) or (B) CIIIC (60 lM) are shown.
Numbers on the top of each lane indicate the time of digestion in
min. Samples were run on (A) a 15% SDS ⁄ PAGE, or (B) a 17.5%
SDS ⁄ PAGE. (C) A densitometric scan of the above bands showing
the amount of r
32
remaining after proteolysis, for r
32
alone ( )or
in the presence of CIII (
) or CIIIC (d). Each data point represents
the mean value from three identical experiments.
Protection of r
32
from HflB by kCIII S. Halder et al.
4768 FEBS Journal 275 (2008) 4767–4772 ª 2008 The Authors Journal compilation ª 2008 FEBS
case the proteolysis, with  85% of r
32
remaining

undigested after 80 min (Fig. 2C), was inhibited more
effectively than with intact CIII.
The above experiments on the inhibition of the pro-
teolysis of r
32
by HflB were also carried out in the
presence of varying amounts (up to 200 lm) of CIII or
CIIIC (Fig. 3). In this case, proteolysis was terminated
after 40 min. Stronger inhibition by CIIIC is also evi-
dent from these experiments, with  95% of r
32
remaining undigested in the presence of 40 lm CIIIC
(Fig. 3, lower).
r
32
interacts with HflB but does not interact
with CIII
The interaction between r
32
and HflB was tested in an
in vitro GST pull-down assay. GST-tagged HflB was
bound to glutathione-Sepharose beads followed by the
addition of r
32
. The proteins were analyzed on an
11% SDS ⁄ PAGE and visualized by western blotting
with anti-his as the primary antibody. The same exper-
iment was repeated with GST protein as a negative
control. It was observed that r
32

co-eluted with GST–
HflB but not with GST protein (Fig. 4A), implying
that r
32
interacts specifically with GST–HflB.
The interaction between r
32
and CIII was assayed
in an in vitro Ni
2+
-nitrilotriacetic acid pull-down
assay. The Ni
2+
-nitrilotriacetic acid bound His
6
–CIII
was mixed with r
32
(with the 6· histidine tag removed)
and incubated at 4 °C for 4 h. The Ni
2+
-nitrilotriace-
tic acid beads were washed with 1· NaCl ⁄ P
i
and eluted
by boiling with 1· sample buffer. Proteins were ana-
lyzed on 17.5% SDS ⁄ PAGE (Fig. 4B). It was observed
that r
32
did not co-elute with CIII, implying that r

32
does not interact with CIII.
Discussion
Phage protein CIII works as an antiprotease against
E. coli HflB and protects kCII by directly binding to
the protease [10,11], without any detectable interaction
with the substrate kCII [11]. Thus, the protection of
Fig. 3. Inhibition of HflB-mediated proteolysis of r
32
by different
concentrations of CIII or CIIIC. The bands corresponding to r
32
-C-
his (1.2 l
M) remaining after proteolysis with GST–HflB (0.8 lM) for
40 min in the presence of His
6
–CIII or CIIIC (up to 200 lM) are
shown in the upper panels. Numbers on the top of each lane indi-
cate the concentration of His
6
-CIII or CIIIC (in lM). Lane C indicates
the control lane showing undigested r
32
. The samples were run on
a 15% (upper) or 17.5% (middle) SDS ⁄ PAGE. (Lower) Densitomet-
ric scan of the above bands depicting the amount of r
32
remaining
after proteolysis in the presence of CIII (

) or CIIIC (d). Each data
point represents the mean value from three identical experiments.
A
B
Fig. 4. In vitro binding of HflB-r
32
and CIII-r
32
. (A) Interaction
between GST–HflB and r
32
-C-his was tested by GST pull-down fol-
lowed by 11% SDS ⁄ PAGE, and immunoblotting with anti-His Ig.
Lane 1, r
32
-C-his (control); lane 2, fraction pulled down with GST–
HflB; lane 3, fraction pulled down with GST. (B) Absence of interac-
tion between His
6
–CIII and r
32
(without the histidine tag) as
obtained from Ni-nitrilotriacetic acid pull-down, followed by 17.5%
SDS ⁄ PAGE and Coomassie Brilliant Blue staining. Lane 1, His
6
-CIII
alone; lane 2, r
32
alone; lane 3, fraction pulled down.
S. Halder et al. Protection of r

32
from HflB by kCIII
FEBS Journal 275 (2008) 4767–4772 ª 2008 The Authors Journal compilation ª 2008 FEBS 4769
CII by CIII works at the protease level, rather than at
the substrate level. Nevertheless, competition between
CII and CIII for interaction with HflB also influences
the proteolysis of CII [11], because CIII itself is a sub-
strate of HflB [12]. CIIIC, the central region of CIII, is
not a substrate of HflB, and acts as a better inhibitor
for digestion of CII by HflB, than CIII [11]. Is this
mode of antiproteolytic action of CIII a general mode
of CIII activity, or does it apply only for CII? We
examined the mode of the inhibitory action of CIII on
proteolysis of the heat shock sigma factor r
32
, another
substrate of HflB. Like proteolysis of CII, r
32
proteol-
ysis requires ATP. Both CIII and CIIIC inhibit this
proteolysis, with CIIIC exhibiting stronger inhibition
both as a function of time (Fig. 2) or as a function of
concentration (Fig. 3). Under the conditions of our
experiment, near-total inhibition by CIIIC could be
observed, whereas in the presence of CIII, only partial
inhibition was achieved. As in the case of CII [10,11],
CIII appears to work as an inhibitor for the proteoly-
sis of r
32
through direct interaction with the protease,

characterized by a lack of interaction between r
32
and
CIII (Fig. 4). As for CII, competition between r
32
and
CIII for binding to HflB would also decide the extent
and efficiency of protection of r
32
by CIII, because
both are HflB substrates. The relative binding affinity
for r
32
–HflB interaction and CIII–HflB interaction
would play an important role in the inhibition of
proteolysis of r
32
by HflB.
Interestingly, both CII and r
32
are cytosolic sub-
strates for HflB. In addition, HflB also acts on several
membrane-associated substrates, for which the mecha-
nism of proteolysis is probably somewhat different
[21]. Whether k CIII would act as an inhibitor for such
substrates (e.g. SecY, YccA, Foa) remains to be seen.
However, the biological connection between kCIII and
such substrates is poor, and it is unlikely that CIII
would have any role in the proteolysis of substrates
like SecY or YccA by HflB. We think that the primary

role of CIII is associated with k lysogeny. In this
respect, the antiproteolytic activity of CIII needs to be
short-lived, made possible by the fact that CIII is also
an HflB substrate. CIII, however, acts via direct inter-
action with HflB, which may be enabled by the bind-
ing of its central helical region to the substrate-entry
cleft of HflB [22], as pointed out previously [11]. This
probably makes CIII a general inhibitor for the cyto-
solic substrates of HflB, accounting for its protection
of r
32
.
The intriguing question that follows is why would a
lambda protein stabilize the E. coli heat shock sigma
factor? r
32
is an unstable protein [23,24] with a rapid
turnover during normal growth and a transient
stabilization during heat shock, followed by rapid deg-
radation [18,24]. The level of r
32
in E. coli cells is
tightly controlled, through the interactions of the
DnaK–DnaJ–GrpE machinery and by HflB-mediated
degradation. Interestingly, these latter proteins that
promote the degradation of r
32
are themselves pro-
duced as a result of a stress response [13,25], being
transcribed from heat shock promoters involving r

32
.
They may be part of mechanisms that allow the bacte-
rium to respond quickly to changing nutritional and
environmental conditions. When a temperate virus like
lambda takes up the lysogenic pathway in response to
stressed conditions of the host, the phage functions must
closely follow host conditions so that a correct
developmental decision can be taken. During the estab-
lishment of lysogeny, stabilisation of r
32
by kCIII may
lead to elevated production of the bacterial protease lon
that is transcribed from heat shock promoters [26] and
degrades the phage protein N [27], indirectly helping the
lysogenic response. It is known that k lysogeny is
reduced in lon mutants [27,28]. Alternatively, inhibition
of HflB by kCIII would lead to increased levels of r
32
,
causing elevated production of the heat shock proteins
DnaJ, DnaK and GrpE which promote the replication
of k [29,30]. Such an event would serve to keep the
lysogenized cell prepared for stress-induced induction
while degradation by HflB is compromised. Various
pathways, sometimes even antagonistic [31], are known
to regulate the concentration of r
32
in E. coli. Lambda
could be taking advantage of this regulatory network

and act at multiple levels of host–virus interactions.
Experimental procedures
Materials
Various fine chemicals, reagents and enzymes were obtained
from Sigma-Aldrich (New Delhi, India), USB (Cleveland,
OH, USA), Merck Limited (Mumbai, India) and Sisco
Research Laboratory (Mumbai, India). Resins, primers and
columns were used as described in Halder et al. [11].
Purification of proteins and peptides
Purification of r
32
was carried out according to Chattopad-
hyay and Roy [32] and Sambrook et al. [33]. The NUT-21
strain containing pUHE 211-1 was grown at 30 °Cin1L
of 2XYT medium [34] with 100 lgÆmL
–1
ampicillin and
50 lgÆmL
–1
kanamycin. At A
600
 1, isopropyl thio-b-d-
galactopyranoside was added to a final concentration of
0.5 mm. Cells were grown for a further 20 min and poured
into cold tubes. All subsequent steps were performed at
4 °C. After centrifugation at 1900 g for 10 min, the cell
Protection of r
32
from HflB by kCIII S. Halder et al.
4770 FEBS Journal 275 (2008) 4767–4772 ª 2008 The Authors Journal compilation ª 2008 FEBS

pellet was resuspended in 18 mL of ice-cold buffer L
(50 mm phosphate buffer, pH 7.9, 300 mm KCl, 50 mm iso-
leucine, 50 mm phenylalanine) with 20 lgÆmL
)1
of phenyl
methanesulfonyl fluoride and disrupted by sonication. The
cell lysate was centrifuged for 45 min at 14 500 g. The
supernatant was loaded onto a 3-mL Ni
2+
-nitrilotriacetic
acid-agarose column pre-equilibrated with buffer L at a
rate 0.4 mLÆmin
–1
. The column was subsequently washed
with 40 mL of buffer L followed by 10 mL of buffer L plus
15 mm imidazole. Nickel-bound proteins were eluted with
30 mL of 15–150 mm imidazole gradient in buffer L. Pure
fractions of r
32
proteins were dialyzed against two changes
of 1 L of 50 mm phosphate buffer, pH 7.9, containing
300 mm KCl and 50% glycerol.
HflB was overexpressed as a GST fusion protein from
plasmid pAD101 containing the hflB gene cloned in vec-
tor pGEX4T and was purified with a glutathione-Sepha-
rose column (GSTrap FF; Amersham Biosciences,
Sweden). His
6
–CIII was obtained by overexpressing
recombinant plasmid pAB905 containing the cIII gene

and purified with Ni-nitrilotriacetic acid column as
described previously [11]. A 28-residue peptide, CIIIC,
was chemically synthesized and purified by reverse-phase
C
18
column (hypersil) by water–acetonitrile gradient as
stated previously [11].
Measurements of inhibition of proteolysis
by HflB
The activity of CIII (or CIIIC) was measured by its ability
to inhibit HflB-mediated proteolysis of r
32
[14]. Reaction
mixtures (50 lL) were prepared by taking 60 pmol of r
32
in buffer P (50 mm Tris ⁄ acetate, pH 7.2, 100 mm NaCl,
5mm MgCl
2
,25lm Zn-acetate, 1.4 mm b-mercaptoetha-
nol) containing 5 mm ATP. CIII or CIIIC (up to 200 lm)
was also added. Proteolysis was carried out by adding HflB
(0.8 lm) followed by incubation at 37 °C for specified time
intervals. Reactions were stopped by the addition of 5 mm
EDTA and SDS ⁄ PAGE loading buffer, followed by heating
in a boiling water-bath for 5 min. The amount of r
32
that
remained after proteolysis was analyzed after SDS ⁄ PAGE
of the samples followed by Coomassie Brilliant Blue stain-
ing and quantitation in a gel documentation system (Bio-

Rad Gel Doc 1000).
Removal of the histidine tag
To get a native r
32
protein without the C-terminal 6· histi-
dine tag, r
32
( 100 lg) was mixed with thrombin (1 unit)
and incubated overnight at 22 °C. The protein mixture was
then passed through a 1 mL benzamidine column (Amer-
sham) for removal of thrombin. Finally, the native protein
was separated from the mixture by treatment with Ni
2+
–
nitrilotriacetic acid beads. The flow-through contained only
native proteins, devoid of the histidine tag.
In vitro binding assay
The interaction between HflB and r
32
was studied by in vitro
GST pull-down assay. A 50 lL aliquot of bound GST–HflB
(25 lg) was mixed with 30 lgofr
32
in buffer P. The final
volume was made up to 500 lL and incubated overnight on
a rotating machine at 4 °C. Before binding, 5% of the input
solution was kept aside separately and was used as a control
for comparison. The proteins were analyzed in an 11%
SDS ⁄ PAGE and by western blotting using anti-his Ig. As a
negative control, the same experiment was also performed

with GST protein replacing GST–HflB.
The interaction of histidine-tagged proteins with other
proteins was examined by in vitro Ni
2+
-nitrilotriacetic acid
pull-down assay. Purified His
6
–CIII was immobilized at
4 °C for 1 h on Ni
2+
-nitrilotriacetic acid beads in buffer A
containing 10 mm imidazole and washed with buffer P.
Fifty microliters of His
6
–CIII bound bead ( 25 lg) was
mixed with 30 lg r
32
. The final volume was made up to
500 lL and incubated on a rotating machine at 4 °C for
4 h. The beads were then washed three times with buffer P
and resuspended in SDS gel loading buffer for elution of
proteins from the beads. The eluted proteins were separated
by 17.5% SDS ⁄ PAGE and visualized using Coomassie
Brilliant Blue staining.
Acknowledgements
The authors would like to thank Professor Siddhartha
Roy, Indian Institute of Chemical Biology, Kolkata,
for the gift of the E. coli strain NUT-21 containing
pUHE211-1 that was used to purify the r
32

protein.
S.H. was supported by fellowships from CSIR, India,
and by Bose Institute. Subhamoy Banerjee is a summer
student at the Bose Institute, from Madhav Institute
of Technology & Science, Gwalior, India.
References
1 Echols H (1980) Bacteriophage k development. In The
Molecular Genetics of Development (Leighton TJ & Lo o mis
WF, eds), pp. 1–16. Academic Press, New York, NY.
2 Herskowitz I & Hagen D (1980) The lysis–lysogeny
decision of phage k: explicit programming and respon-
siveness. Annu Rev Genet 14, 399–445.
3 Friedman DI, Olson ER, Georgopoulos C, Tilly K,
Herskowitz I & Banuett F (1984) Interactions of bacte-
riophages and host macromolecules in the growth of
bacteriophage k. Microbiol Rev 48, 299–325.
4 Echols H (1986) Bacteriophage k development: tempo-
ral switches and the choice of lysis or lysogeny. Trends
Genet 2, 26–30.
5 Drahos DJ & Hendrix RW (1982) Effect of bacterio-
phage lambda infection on synthesis of groE protein
S. Halder et al. Protection of r
32
from HflB by kCIII
FEBS Journal 275 (2008) 4767–4772 ª 2008 The Authors Journal compilation ª 2008 FEBS 4771
and other Escherichia coli proteins. J Bacteriol 149,
1050–1063.
6 Kochan J & Murialdo H (1982) Stimulation of groE
synthesis in Escherichia coli by bacteriophage lambda
infection. J Bacteriol 149, 1166–1170.

7 Hoyt MA, Knight DM, Das A, Miller HI & Echols H
(1982) Control of phage lambda development by stabil-
ity and synthesis of cII protein: role of the viral cIII
and host hflA, himA and himD genes. Cell 31, 565–573.
8 Rattray A, Altuvia S, Mahajna G, Oppenheim AB &
Gottesman M (1984) Control of phage lambda cII activ-
ity by phage and host functions. J Bacteriol 159, 238–242.
9 Banuett F, Hoyt MA, Mcfarlane L, Echols H & Her-
skowitz I (1986) HflB, a new Escherichia coli locus regu-
lating lysogeny and the level of bacteriophage lambda
CII protein. J Mol Biol 187, 213–224.
10 Kobiler O, Rokney A & Oppenheim AB (2007) Phage
lambda CIII: a protease inhibitor regulating the lysis–
lysogeny decision. PLoS ONE 2, e363.
11 Halder S, Datta AB & Parrack P (2007) Probing the
antiprotease activity of kCIII, an inhibitor of the Esc-
herichia coli metalloprotease HflB (FtsH). J Bacteriol
189, 8130–8138.
12 Herman C, Thevenet D, D’Ari R. & Bouloc P (1997)
The HflB protease of Escherichia coli degrades its inhib-
itor lambda CIII. J Bacteriol 179, 358–363.
13 Herman C, Thevenet D, D’Ari R & Bouloc P. (1995)
Degradation of r
32
, the heat shock regulator in Escheri-
chia coli, is governed by HflB. Proc Natl Acad Sci USA
92, 3516–3520.
14 Tomoyasu T, Gamer J, Bukau B, Kanemory M, Mori
H, Rutman AJ, Oppenheim AB, Yura T, Yamanaka K,
Niki H et al. (1995) Escherichia coli FtsH is a mem-

brane bound, ATP-dependent protease which degrades
the heat-shock transcription factor r
32
. EMBO J 14,
2551–2560.
15 Bahl H, Echols H, Straus DB, Court D, Crowl R &
Georgopoulos CP (1987) Induction of the heat shock
response of E. coli through stabilization of r
32
by the
phage kCIII protein. Genes Dev 1, 57–64.
16 Kornitzer D, Altuvia S & Oppenheim AB (1991) The
activity of CIII regulator of lambdoid bacteriophages
resides within a 24-amino-acid protein domain. Proc
Natl Acad Sci USA 88, 5217–5221.
17 Ang D, Chandrasekhar GN, Zylicz M & Georgopoulos
CP (1986) Escherichia coli grpE gene codes for heat
shock protein B25.3, essential for both lambda DNA
replication at all temperatures and host growth at high
temperature. J Bacteriol 167, 25–29.
18 Straus D, Walter W & Gross CA (1990) DnaK, DnaJ,
and GrpE heat shock proteins negatively regulate heat
shock gene expression by controlling the synthesis and
stability of r
32
. Genes Dev 4, 2202–2209.
19 Tatsuta T, Joo DM, Calendar Y, Akiyama Y & Ogura
T (2000) Evidence for an active role of the DnaK chap-
erone system in the degradation of r
32

. FEBS Lett 478,
271–275.
20 Tomoyasu T, Ogura T, Tatsuta T & Bukau B (1998)
Levels of DnaK and DnaJ provide tight control of heat
shock gene expression and protein repair in E. coli. Mol
Microbiol 30, 567–581.
21 Ito K & Akiyama A (2005) Cellular functions, mecha-
nisms of action, and regulation of FtsH protease. Annu
Rev Microbiol 59, 211–231.
22 Bieniossek C, Schalch T, Bumann M, Meister M, Meier
R & Baumann U (2006) The molecular architecture of
the metalloprotease FtsH. Proc Natl Acad Sci USA 103,
3066–3071.
23 Grossman AD, Straus DB, Walter WA & Gross CA (1987)
Sigma 32 synthesis can regulate the synthesis of heat
shock proteins in Escherichia coli. Genes Dev 1, 179–184.
24 Tilly K, Spence J & Georgopoulos C (1989) Modulation
of stability of the Escherichia coli heat shock regulatory
factor r
32
. J Bacteriol 171, 1585–1589.
25 Yura T, Nagai H & Mori H (1993) Regulation of the
heat-shock response in bacteria. Annu Rev Microbiol 47,
321–350.
26 Gottesman S (1984) Bacterial regulation: global regula-
tory networks. Annu Rev Genet 18, 415–441.
27 Maurizi MR (1987) Degradation in vitro of bacterio-
phage lambda N protein by Lon protease from Escheri-
chia coli. J Biol Chem 262, 2696–2703.
28 Walker JR, Usser CL & Allen JS (1973) Bacterial cell

division regulation: lysogenization of conditional cell
division lon
)
mutants of Escherichia coli by Bacterio-
phage Lambda. J Bacteriol 111, 1326–1332.
29 Liberek K, Georgopoulos C & Zylicz M (1988) The role
of the Escherichia coli DnaK and DnaJ heat shock pro-
teins in the initiation of bacteriophage k DNA replica-
tion. Proc Natl Acad Sci USA 85, 6632–6636.
30 Johnson C, Chandrasekhar GN & Georgopoulos C
(1989) Escherichia coli DnaK and GrpE heat shock pro-
teins interact both in vivo and in vitro. J Bacteriol 171,
1590–1596.
31 Morita MT, Kanemori M, Yanagi H & Yura T (2000)
Dynamic interplay between antagonistic pathways con-
trolling the r
32
level in Escherichia coli. Proc Natl Acad
Sci USA 97, 5860–5865.
32 Joo DM, Ng N & Calendar R (1997) A r
32
mutant
with a single amino acid change in the highly conserved
region 2.2 exhibits reduced core RNA polymerase affin-
ity. Proc Natl Acad Sci USA 94, 4907–4912.
33 Chattopadhyay R & Roy S (2002) DnaK–sigma 32
interaction is temperature dependent: implication for
the mechanism of heat shock response. J Biol Chem
277, 33641–33647.
34 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular

Cloning, A Laboratory Manual, Vol. 3 (2nd edn). Cold
Spring Harbor Laboratory Press, Cold Spring Harbor,
NY.
Protection of r
32
from HflB by kCIII S. Halder et al.
4772 FEBS Journal 275 (2008) 4767–4772 ª 2008 The Authors Journal compilation ª 2008 FEBS