From the Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Received for publication, December 26, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Upon infection to the Escherichia
coli cell, the genome of bacteriophage When bacteriophage It is known that the intracellular concentration of CII is controlled
at the levels of not only transcription but also protein stability. CII
is short-lived, having a half-life of about 2 min in The remaining mutation (hflB29) of Gautsch and Wulff (5)
defined the hflB gene (14), now known to be identical with
ftsH (15). Its product acts as the primary proteolytic
enzyme against CII (12, 13). FtsH is a zinc metalloprotease, which is
membrane-bound and ATP-dependent (reviewed in Ref. 16). Its
cytoplasmic domain includes an evolutionarily conserved AAA ATPase
domain (reviewed in Ref. 17). Proteolytic substrates of this enzyme
include both soluble and membrane proteins. The actions of FtsH against
soluble and membrane-bound substrates are differentially affected by
the HflKC complex (18). Some substrates may require additional factors for optimal proteolysis. For instance, Plasmids--
pKH198 and pKH191 carry ftsH and
hflK-hflC, respectively, under the lac promoter
(11). pKH256 is a derivative of pTWV228 (a pBR322-based lac
promoter vector from Takara Shuzo) carrying ftsH. pKH479,
carrying cII under the lac promoter, was
constructed from pSTD240 (20) by cleavage, filling in, and religation
of the BamHI site to eliminate the in-frame
lacZ
pKH394 (Fig. 1A) is a derivative of pACYC184 with its
tet gene controlled by the
pKH402, pKH421, pKH422, pKH427, pKH428, pKH429, pKH430, and pKH431 were
derivatives of pMW118 or pMW119 (pSC101-based lac promoter
vectors from Nippon Gene) carrying the chromosomal segments shown in
Fig. 2. pKH441 (hflD+) contained the
1.7-kilobase pair (kb)1
BglII fragment from pKH422, which was cloned into the
BamHI site of pTWV228. pKH449, carrying gst-hflD
under the tac promoter, was constructed by amplifying
hflD from pKH441 with the BamHI sites at the
primer ends and cloning it into pGEX-4T-3 (Amersham Pharmacia Biotech).
pKH453 (hflD33), pKH454 (hflD11), pKH455
(hflD13), pKH456 (hflD24), pKH452
(hflD28), pKH457 (hflD45), and pKH458 (hflD48) contained the 1.7-kb BglII genomic
fragment from each mutant strain, which was cloned into pMW118.
Isolation of hflD Mutants--
pKH394 was introduced into cells
of AD16 (21) that had been treated with 40 µg/ml
N-methyl-N'-nitro-N-nitrosoguanidine
(22). Tetracycline-resistant mutants (which appeared at a frequency of
~4 × 10 Construction of the hflD::tet Strain--
A
blunt-ended 1.5-kb XbaI-AvaI tet
fragment of pACYC184 was inserted into hflD
(BseRI site) within the 7.2-kb SalI fragment, originally from pKH422 but now on pTWV228 (this plasmid was named pKH443). The 7.4-kb PvuII-XhoI fragment of pKH443
was then introduced into FS1576 (recD E. coli Two-hybrid Assay (25)--
A cII fragment
was amplified from ptac-cIIY42, using primers
5'-CGGGATCCTCAGAACTCCATCTGGATTTG-3' and
5'-AACTGCAGGGATGGTTCGTGCTAACAAACGC-3' (with BamHI and
PstI recognition sequences) and cloned into the BamHI- and PstI-treated pT25 (25). An
hflD fragment was amplified from pKH441 using primers
5'-GCAGGTACCTGCAACTCCGGGGTTAAATGAGC-3' and
5'-GAGGGTACCGATGGCAAAGAATTACTATGAC-3' (with a KpnI
recognition sequence) and cloned into the KpnI site of pT18
(25). These plasmids were then introduced into strain DHP1
( Purification of the HflD Protein--
Strain TYE024 (26) was
transformed with pKH449 and grown in 3 liters of L-glucose medium
(0.1%), ampicillin (50 µg/ml) at 37 °C. Synthesis of GST-HflD was
induced with 1 mM
isopropyl-thio- Purification of the CII Protein--
Strain BL21(DE3) (27) was
transformed with pETcII and grown as described above with a 30-min
induction. Soluble fractions in 50 mM Hepes·NaOH
(pH 8.0) containing 10% glycerol, 1 M NaCl, and 10 mM 2-mercaptoethanol were applied to a Ni-NTA-agarose
column, washed, and eluted with 50 mM Hepes·NaOH (pH 8.0)
containing 10% glycerol, 200 mM NaCl, 250 mM
imidazole, and 10 mM 2-mercaptoethanol. His6-CII was treated with bovine plasma thrombin (0.5 mg/ml) at 4 °C for 12 h with concomitant dialysis against 50 mM Hepes·NaOH (pH 8.0) containing 10% glycerol, 100 mM NaCl, and 1 mM DTT. The sample was finally
purified by a Mono S HR 5/5 column (Amersham Pharmacia Biotech) with
elution with 100-500 mM NaCl gradient in the same buffer.
Other Methods and Materials--
Hfl phenotypes were assessed by
measuring lysogenization frequency of Isolation of a New Class of Mutations that Elevates
CII-dependent Transcription--
In the original mutant
isolation, only one hfl allele (hflB29) was
isolated at the hflB locus (4, 5), raising a question as to
whether genes involved in
To isolate a new class of hfl mutations, pKH394 was
introduced into mutagenized cells, and tetracycline-resistant mutants were selected. Among 15 mutants, three had mutations at 69 min, where
ftsH is located, and five had mutations at 95 min, where hflK-hflC are located. The remaining seven
mutations, named hflD33, hflD11,
hflD13, hflD24, hflD28,
hflD45, and hflD48, were examined further. After
mutant cells had been cured of the plasmids, they were examined
for an Hfl phenotype. They indeed failed to support growth of Identification of hflD--
To map one of the mutations,
hflD33, we isolated a Tn5 insertion (termed
zcg-2002::Tn5) that was P1
co-transducible with hflD33 at a frequency of about 50%
(Fig. 2). We then cloned a chromosomal segment together with the kan region of the Tn5
insertion. Sequence analysis of one such plasmid (pKH402) showed that
the Tn5 insertion was located at 26.2 min on the E. coli genome. In P1 transduction, zcg-2002::Tn5 was co-transducible with
dsbB::cat (located at 26.6 min; 32) at
a frequency of about 40%, but hflD33 was not. Thus, the
order of
hflD33-zcg-2002::Tn5-dsbB
was suggested. Although some E. coli K-12 strains possess a
defective prophage e14 (33), in this chromosomal region (Fig. 2), the
strains we used in this study did not contain e14 (data not shown).
The clone 7F9 of the E. coli genomic library (34) covers
this region, from which we subcloned several DNA fragments into lac promoter vectors (Fig. 2). pKH422, pKH428, and pKH429
complemented the hflD33 mutant, allowing the growth of
The remaining six hfl mutations also proved to be P1
co-transducible with zcg-2002::Tn5. We
determined the nucleotide sequence for each of the mutant
hflD genes (Table I).
Four of them (hflD24, hflD28, hflD45,
and hflD48) contained a non-sense mutation, whereas two
(hflD33 and hflD11) contained a missense mutation
within the hflD open reading frame. The remaining
mutant (hflD13) had a base change in the putative
Shine-Dalgarno sequence for hflD. We thus identified
hflD as a new gene affecting the CII function of
bacteriophage HflD Down-regulates
We then examined whether overexpression of hflD could
suppress the Hfl phenotypes associated with other classes of
hfl mutations. When a plasmid overexpressing hflD
15-30-fold was introduced into the
hflD::tet, the
zgj-525::IS1A and the
Identification of the HflD Protein and Its Peripheral Association
with the Membrane--
The wild-type and the mutant hflD
genes were expressed in the hflD::tet
strain, and their products were examined by immunoblotting, using
antiserum against an HflD synthetic peptide. A protein with an apparent
molecular mass of 23 kDa was detected for the wild-type gene (Fig.
3A, lane 1). This
product was missing for the non-sense mutants (Fig. 3A,
lanes 4, 5, and 8) except for
hflD45, which produced a smaller fragment (Fig.
3A, lane 7). The hflD33 missense mutation also gave a band of faster electrophoretic mobility (Fig. 3A, lane 6), suggesting its instability. The
hflD13 mutation markedly lowered the expression level of
HflD (Fig. 3A, lane 2), consistent with a lowered
translation initiation. In wild-type cells without plasmid, HflD was
detected as a faint band at the 23-kDa position (data not shown).
Upon cell fractionation, HflD was recovered from the membrane fraction
(Fig. 3B, lane 3), which is consistent with a
previous report (35). It was extractable with 0.1 M NaOH
(Fig. 4B, lane 4),
in contrast to the ATP synthase F0 a subunit
used as an integral membrane protein control (Fig. 3B,
lane 5). HflD was inaccessible to externally added
proteinase K (Fig. 3C, lane 2), unless the spheroplasts were broken by a detergent (Fig. 3C, lane
3). Thus, HflD is peripherally associated with the cytosolic
surface of the cytoplasmic membrane.
HflD Interacts with CII--
We purified CII and HflD (Fig.
4A, lanes 2 and 5). HflD was purified
also as an N-terminal glutathione S-transferase (GST) fusion (Fig. 4A, lane 3), which was
functional as its expression complemented the
hflD::tet strain with respect to the
Hfl phenotype (data not shown). No activity to degrade CII was detected
for the purified HflD preparations, arguing against its being a
CII-degrading protease. We failed to detect any interaction between
HflD and FtsH (data not shown).
When GST-HflD and CII were mixed in vitro and subjected to
affinity isolation using a glutathione-Sepharose column, they were co-eluted by 20 mM glutathione (Fig. 4B,
lane 8). CII alone did not bind to the column (Fig.
4B, lane 2). Thus, HflD has an ability to bind to
CII. Independent evidence for the HflD-CII interaction was obtained by
cross-linking experiments. HflD was partially converted by treatment
with 3, 3'-dithiobis-(succinimidyl) propionate, a primary
amine-reactive homobifunctional cross-linker, to a form expected for a
dimer (~41 kDa; Fig. 5, lanes
4 and 5). When a mixture of HflD and CII was treated
similarly, an additional product was observed at ~32 kDa (Fig. 5,
lanes 8-10). This new band most likely represented an
HflD-CII cross-linked product, because it possessed both the HflD (Fig.
5) and the CII (data not shown) antigenicity. CII itself, which did not
react with the anti-HflD, produced homodimeric, trimeric, and
tetrameric products upon cross-linking (data not shown). A tetrameric
state of CII was reported previously (37).
To examine whether HflD and CII interact mutually in vivo,
the E. coli two-hybrid system (25) was used. CII was fused
to the N-terminal domain of adenylate cyclase from Bordetella
pertussis, and HflD was fused to the independently cloned
C-terminal domain of this enzyme. In the presence of both plasmids, the
expression of lacZ, which is dependent on cyclic AMP, the
product of adenylate cyclase, was increased about 3-fold over the
control. This result suggests that HflD and CII interact with each
other in vivo.
HflD Enhances CII Degradation in Vivo but Interferes with CII
Degradation in Vitro--
A purified preparation of
FtsH-His6-Myc, in detergent-solubilized states, can degrade
CII in the presence of ATP (Ref. 12; Fig.
6A, open circles).
When increasing concentrations of HflD were added to the reaction,
degradation of CII was increasingly inhibited (Fig. 6A).
Thus, HflD binding to CII results in the protection of the latter from
FtsH-mediated proteolysis. This observation, however, was in apparent
contradiction with the in vivo ability of HflD to
down-regulate
We then examined the in vivo effects of the hflD
mutations as well as of HflD overproduction on the stability of CII as
it was expressed from a plasmid. CII was degraded with an initial half-life of about 3 min in wild-type cells (Fig. 6B,
open squares). It was stabilized in the
hflD::tet cells although not
completely; the initial half-life was prolonged to ~10 min (Fig.
6B, closed squares). This residual degradation,
observed in the absence of HflD, should have been FtsH-mediated,
because loss-of-function mutations of ftsH effectively
stabilizes CII (12, 13). In contrast, overproduction of HflD
destabilized CII; the initial half-life became ~1 min (Fig.
6B, open circles). These results indicate that
HflD contributes positively to the degradation of CII in
vivo.
The CII protein of Although HflD is not absolutely required for FtsH-mediated degradation
of CII, it significantly stimulates the degradation in vivo
(Fig. 6B). HflD is not itself a protease. It is not simply an activator of FtsH either, because its in vitro effect on
the FtsH-dependent CII degradation is negative (Fig.
6A). The HflD effect on protein degradation is specific for
CII, as in vivo degradation of Under the in vitro reaction conditions, HflD-binding to CII
resulted in an inhibition of CII degradation. In this regard HflD does
not appear to be a specificity-enhancing factor like SspB (40). How can
an in vivo stimulator act as an in vitro
inhibitor? Although FtsH is membrane-associated in vivo, it
was in the solubilized states in vitro. CII is predominantly
DNA-bound in vivo (37), but no DNA was involved in the
in vitro reaction. These differences between the in
vivo and in vitro reaction conditions may explain the
observed discrepancy. Thus, a conclusion that HflD·CII complex is a less favored substrate of FtsH than free CII holds only for the
in vitro reactions, in which every component has been
solubilized. We propose that HflD acts in vivo to change
localization of CII from DNA to the membrane. Then, in the in
vivo situation, the HflD·CII complex might be a better substrate
than the DNA-bound form of CII. The reason for this may be 2-fold.
First, HflD will bring CII to the membrane surface where
the active site domain of the FtsH protease resides (41), increasing
the frequency of the CII-FtsH contacts. Second, the DNA-bound state of
CII may be less susceptible to proteolysis than the dissociated state even though the latter might always be in the HflD-bound form in
vivo.
Although the affinity of CII to HflD seems to be higher than to FtsH
under the in vitro conditions used, the relative affinities could change by modification such as phosphorylation of a component (42). It is tempting to speculate that HflD is a part of the host
system that controls the activity and the level of CII in response to
environmental conditions. It should also be noted that an additional
factor could participate in vivo, in conjunction with HflD,
to stimulate CII degradation, although we do not understand why such a
factor, if it does exist, escaped our mutant selection.
In gel filtration in the presence of a detergent, HflD migrated in two
peaks (data not shown). Cross-linking experiments identified a
cross-linked dimer of HflD. Thus, HflD may partly be in a
dimeric or larger homocomplex. Cross-linking in the presence of CII
yielded an HflD·CII complex, whereas CII alone produced tetrameric
homo-cross-linking (data not shown for the CII cross-linking). These
results suggest that HflD is in equilibrium between the monomeric and
oligomeric states and that the monomeric HflD may interact with CII.
Although HflD is hydrophilic overall, some segments of it could form
amphipathic Although our results indicate that HflD enhances degradation of CII by
bringing it to the vicinity of membrane-bound FtsH, it might
participate in lysogeny control also by sequestering CII from the
target promoter (43). In this latter respect, it could be regarded as
similar to RseA, a membrane-bound anti-sigma E factor (44). The role
played by HflD in the uninfected host cell is an interesting subject
which will be left for future studies.
either replicates to
form new progenies (lytic growth) or integrates into the host
chromosome (lysogenization). The
CII protein is a key determinant
in the lysis-lysogeny decision. It is a short-lived transcription
activator for the
genes essential for lysogeny establishment. In
this study, we isolated a new class of hfl (high frequency
lysogenization) mutants of E. coli, using a new selection
for enhancement of CII-stimulated transcription. The gene affected was
termed hflD, which encodes a protein of 213 amino acids. An
hflD-disrupted mutant indeed showed an Hfl phenotype,
indicating that HflD acts to down-regulate lysogenization. HflD is
associated peripherally with the cytoplasmic membrane. Its interaction
with CII was demonstrated in vitro using purified proteins
as well as in vivo using the bacterial two-hybrid system. Pulse-chase examinations demonstrated that the HflD function is required for the rapid in vivo degradation of CII, although
it interfered with FtsH-mediated CII proteolysis in an in
vitro reaction system using detergent-solubilized components. We
suggest that HflD is a factor that sequesters CII from the target
promoters and recruits it to the membrane where the FtsH
protease is localized.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
infects to the Escherichia coli
cell, it undergoes either lytic growth or lysogenization (1). The establishment of
lysogenization requires sufficient accumulation of
the repressor protein, CI, before the lytic cycle is initiated. Transcription of the cI gene from the pRE promoter is
positively regulated by the CII protein, which also activates the pI
promoter for the int gene expression required for the
prophage integration, as well as the pAQ promoter for the antisense RNA
that inhibits the synthesis of the Q protein required for late lytic
gene expression. Thus, increased activity of CII leads to preferential lysogenization.
-infected
wild-type cells (2, 3). Host hfl (high
frequency lysogenization) mutations that
stabilize CII have been studied. The mutation first isolated by Belfort
and Wulff (4) and an additional five mutations isolated by Gautsch and
Wulff (5) were mapped at the hflA locus of the chromosome,
which was later shown to comprise three genes, hflX,
hflK, and hflC (6). The hflA mutations
affect either hflK or hflC (7, 8), but the function of hflX is unknown (6). HflK and HflC are membrane proteins, forming a binary complex, HflKC (8-10). Although HflKC was
once believed to be a protease degrading CII (10), we showed that it is
not a protease; rather, it associates with the true protease, FtsH
(11-13), and somehow modulates the activity of the latter (11, 12).
Both HflK and HflC have a signal-anchor sequence at the N terminus,
which is followed by the main body of protein exposed to the
periplasmic space (8, 12).
32 requires the
DnaK chaperone for its degradation in vivo (19). Thus, FtsH may be subject to modulation by multiple factors that interact either with the substrates or FtsH itself. In this study, we
addressed the question of whether any additional host factors exist
that affect the
CII protein with respect to its stability or
function. A new factor, termed HflD, was thus identified as a negative
regulator of
lysogenization. HflD is a peripheral membrane protein,
which interacts with CII. It is positively required for the degradation
of CII in vivo. We propose that HflD holds CII on the
membrane surface, away from the target promoters but close to FtsH.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cII fusion. pETcII, which carries
his6-cII under the T7 promoter, was provided by Amos B. Oppenheim (Hadassah Medical School, The Hebrew University).
pI promoter and
cII and lacZ
placed within the cat
transcription unit. It was constructed as follows. First, three DNA
fragments were amplified with appropriate restriction sequences
(underlined) at the ends. Fragment 1 contained the
pI
promoter (template,
+; primers,
5'-CGGATGGGAGTAAGCTTATTGCTAAACTGG-3', and
5'-CGACGAACTGTTTCAAAGCTTCTTGGACGTC-3' with the
HindIII site). Fragment 2 contained the
cy42-mutated
cII gene (template,
ptac-cIIy42 (12); primers,
5'-ACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAATCTAAGGAAATACTTACATATGGTTCG-3' and
5'-GGGTTTTCCCAGTCAGTACTTTGTTAACCGACGGCCAGTGCC-3'
with the ScaI and the HpaI sites). Fragment 3 contained lacZ
(template, pTWV228; primers,
5'-TTAAGTGAGCGGTTAACAATTTCACACAGGAAACAGC-3', and
5'-CTGGCAAGTGTAGCGTTAACGCTGCGCGTAACCACC-3' with the
HpaI site). Then, Fragment 1 (HindIII-digested),
fragment 2 (ScaI-digested), and fragment 3 (HpaI-digested), respectively, were cloned successively into
the HindIII site of pACYC184 within its tet
promoter, into the ScaI site down stream of cat,
and into the HpaI site within the cloned fragment 2.
4) were selected on
L-tetracycline medium (12.5 µg/ml)-agar (23) at 37 °C. From
four independently mutagenized pools, we saved a total of 15 mutants
that were Hfl. We isolated a transposon insertion named
zcg-2002::Tn5 that was co-transducible
with the hflD33 mutation at about 50%, by a combination of
random Tn5 transposition and P1 transduction (18). Using
this transposon, each hflD mutation was introduced into
strain AD16 by P1 transduction. The isogenic strains thus constructed
were AK2035 (hflD33), AK2036 (hflD11), AK2037
(hflD13), AK2038 (hflD24), AK2039
(hflD28), AK2040 (hflD45), AK2041
(hflD48), and AK2033 (hflD+).
; Ref.
24), and tetracycline (6.25 µg/ml)-resistant recombinants were
selected. One of them was confirmed for the disrupted hflD gene by polymerase chain reaction, and its
hflD::tet marker was P1-transduced into
AD16, yielding AK2149.
cya).
-D-thiogalactoside (IPTG) and 1 mM cyclic AMP for 3 h. Membrane fractions (11) were
solubilized with 0.1% Nonidet P-40 in 140 mM NaCl, 2.7 mM KCl, 10.1 mM
Na2HPO4, 1.8 mM
K2HPO4 (pH 7.3), 1 mM
dithiothreiotol (DTT) and subjected to glutathione-Sepharose 4B column
chromatography (Amersham Pharmacia Biotech). After washing, bound
proteins were eluted with 20 mM reduced glutathione in 100 mM Hepes·NaOH (pH 8.0) containing 10% glycerol, 0.1%
Nonidet P-40, and 1 mM DTT. GST-HflD was then treated with
bovine plasma thrombin (1 mg/ml, Sigma) at 4 °C for 10 h, and
the products were purified by a Mono S HR 5/5 column (Amersham
Pharmacia Biotech) with washing with 50 mM Hepes·NaOH (pH
8.0) containing 10% glycerol, 0.1% Nonidet P-40, and 1 mM
DTT and elution with 0-1 M NaCl gradient in the same buffer.
+ (12) as well as
by examining bacterial growth after infection with
c17 phage (12).
Transduction using P1vir was done by the established
procedures (22). Pulse-chase and immunoprecipitation (28),
immunoblotting (8), and cross-linking (11) were done essentially as
described previously. Rabbit antiserum against HflD was prepared using
a synthetic peptide for residues 76-91 and affinity-purified using the
immobilized peptide. Anti-CII serum was prepared using a synthetic
peptide for residues 77-93. Antisera against a proton-ATPase subunit,
F0 a (29), and GroEL (30) were described previously.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
lysogenization control have been all
identified. We revisited this question using a "modernized" approach. A plasmid (pKH394) was constructed in which the tetracycline resistance gene (tet) was placed under the control of a
CII-controlled promoter, pI (Fig.
1A). This plasmid also
contained the cII gene itself and the lacZ
gene, which are expressed by read-through transcription from the
chloramphenicol resistance gene (cat). The cII
gene contained the silent cy42 base substitution (31), which
reduces the CII binding to the reverse-oriented pRE promoter within
cII (31), thus minimizing complication due to the presence of two CII-responding promoters of different orientations. Model experiments showed that the
zgj-525::IS1A mutation, which reduces the expression level of ftsH (21), indeed conferred
tetracycline resistance to the pKH394-bearing cells (Fig.
1B).
View larger version (25K):
[in a new window]
Fig. 1.
Plasmid pKH394 used for isolation of
hfl mutants. A, a schematic
representation of pKH394. Original pACYC184 parts are shown by
solid arrows, and the fragments inserted in this work are
shown by hatched line arrows. B, Hfl phenotype
can be scored by tetracycline resistance. Cultures of AK519
(wild-type)/pKH394 and
AK525(zgj-525::IS1A)/pKH394 were
spotted on L agar containing 12.5 µg of tetracycline/ml and
incubated at 37 °C for 24 h.
c17
mutant phage (14).
View larger version (29K):
[in a new window]
Fig. 2.
The chromosomal region around
hflD and the plasmids covering this region. Shown
at the top is the 26-min region of the E. coli chromosome. A
cryptic prophage, e14 (not present in the strains used in this study),
and zcg-2002::Tn5 are indicated by
open and hatched bars, respectively. 7F9 is a phage clone described by Kohara et al. (34); this segment is
enlarged. Open reading frames are shown by arrows indicating
the directions of transcription. Regions carried in the plasmids
constructed in this study are also shown. Abbreviations for the
restriction sites are: E, EcoRI; H,
HindIII; L, SalI; C,
ScaI; B, BglII; M,
SmaI.
c17. pKH429 carried ycfC/orf-23
(GenBankTM accession number X59307; Ref. 35) as the
sole intact chromosomal gene, which we designated hflD
hereafter (Fig. 2). The complementation ability of pKH429 was
IPTG-dependent. In contrast, pKH422 and pKH428 complemented
hflD33 IPTG independently. It was suggested then that
trmU, hflD, and purB comprise a single
transcription unit (35).
.
hfl gene status and lysogenization frequency of
Lysogenization--
The hflD
gene is expected to encode a protein of 213 amino acids (35). Its
homologs exist in several bacterial species including Hemophilus
influenzae, Vivrio cholerae, and Pseudomonas
aeruginosa (GenBankTM accession numbers,
respectively, I64155, B82237, and D83317). No physiological role has
been assigned for this dispensable gene (35). We also constructed a
strain in which chromosomal hflD was disrupted by
tet. No obvious growth phenotype was observed for any of the
hflD mutants without
infection. Four non-sense (hflD24, hflD28, hflD45, and
hflD48) and one missense (hflD33) mutation as
well as the tet disruption of hflD increased the
lysogenization frequency of
by 25-50-fold (Table I,
Experiment I), whereas zgi-525::IS1A
did so by about 100-fold (Table I, Experiment II). One missense
mutation (hflD11) and the noncoding region mutation (hflD13) gave ~5-fold increases in the lysogenization
frequency. When hflD was overexpressed from a plasmid in a
wild-type strain, the
lysogenization frequency was lowered by
10-fold (Table I, Exp. III). These results indicate that the
hflD gene product actively participates in keeping the
lysogenization frequency low.
hflK-hflC::kan strains, these bacteria were converted to be completely sensitive to
c17 (data not shown). Thus, HflD overproduction channels the cell to a more
lysis-oriented status.
View larger version (55K):
[in a new window]
Fig. 3.
Identification and cellular localization of
HflD. A, anti-HflD immunoblotting of wild-type and
mutant proteins. Strain AK2149
(hflD::tet) was transformed with either
pKH429 (lane 1), pKH453 (lane 2), pKH454
(lane 3), pKH455 (lane 4), pKH456 (lane
5), pKH452 (lane 6), pKH457 (lane 7), or
pKH458 (lane 8). Cells were grown in L-ampicillin medium at
37 °C and induced with 1 mM IPTG and 3 mM
cyclic AMP for 3 h. Total cellular proteins were separated by
SDS-PAGE and subjected to immunoblotting using antiserum against an
HflD synthetic peptide. The asterisk indicates a nonspecific
background. B, fractionation of HflD. Strain AD202 (36)
carrying pKH441 (plac-hflD) was grown as described in
A. Cell lysate was prepared by sonication in 50 mM Hepes·NaOH (pH 8.0) containing 10% glycerol, 50 mM NaCl, 1 mM DTT, 10 mM EDTA, and
100 µg/ml lysozyme and clarified by low speed centrifugation
(lane 1). It was fractionated into the 182,000 × g 1-h supernatant (lane 2) and pellets
(lane 3). The pellets, suspended in the same buffer, were
then mixed with an equal volume of 0.2 N NaOH and
centrifuged (102,000 × g for 30 min) to separate
peripheral (supernatant (S), lane 4) and integral
(pellets (P), lane 5) membrane components.
Proteins in each fraction were separated by SDS-PAGE for visualization
of HflD (upper panel) and F0 a
(lower panel) by immunoblotting. C, proteinase K
accessibility test (8). Spheroplasts were prepared from AD202/pKH441
and incubated with (lanes 2 and 3) or without
(lane 1) 1 mg/ml proteinase K (PK) at 0 °C for
1 h. The samples for lane 3 received 1% Triton X-100
(Triton). Proteins were separated by SDS-PAGE for detection
of HflD (upper panel) and GroEL (a cytosolic control;
lower panel) by immunoblotting.
View larger version (52K):
[in a new window]
Fig. 4.
Purification of HflD and its in
vitro association with CII. A, shown are
Coomassie Brilliant Blue-stained SDS-PAGE profiles of the purified
preparations of His6-CII (lane 1), CII
(lane 2), GST-HflD (lane 3), GST-HflD after
cleavage with thrombin (lane 4), and HflD (lane
5). B, purified CII alone (1.4 µg (120 pmol),
lanes 1-4) and a mixture of CII (1.4 µg) and GST-HflD
(11.7 µg (240 pmol), lanes 5-8) were incubated in 300 µl of 50 mM Tris·HCl (pH 8.1) containing 10% glycerol,
50 mM NaCl, 0.1% Nonidet P-40, and 1 mM DTT at
0 °C for 30 min and applied to a glutathione-Sepharose column (load,
lanes 1 and 5; flow-through, lanes 2 and 6). The column was washed (lanes 3 and
7) and eluted with 20 mM glutathinone in the
same buffer (lanes 4 and 8). Proteins were
separated by SDS-PAGE and stained with Coomassie Brilliant Blue.
View larger version (74K):
[in a new window]
Fig. 5.
Cross-linking of HflD and CII. Purified
HflD (5.8 µg or 250 pmol, lanes 1-5) as well as a mixture
of HflD (5.8 µg) and CII (2.8 µg or 250 pmol, lanes
6-10) in 40 µl of 50 mM Hepes·NaOH (pH 8.0)
containing 10% glycerol, 0.1% Nonidet P-40, and 1 mM DTT
were treated with the solvent (dimethyl sulfoxide) alone (lanes
1 and 6) or with 3,3'-dithiobis-(succinimidyl)
propionate (DSP) at 3.1 µg/ml (lanes 2 and 7), 6.3 µg/ml (lanes 3 and 8),
12.5 µg/ml (lanes 4 and 9), and 25.0 µg/ml
(lanes 5 and 10) at 4 °C for 1 h. Proteins were
separated by SDS-PAGE using the Weber and Osborn (38) system and
visualized by anti-HflD immunoblotting.
lysogenization.
View larger version (17K):
[in a new window]
Fig. 6.
Contrasting effects of HflD on in
vitro and in vivo degradation of CII.
A, effects of purified HflD on FtsH-mediated in
vitro degradation of CII. Proteolysis of CII (1.86 µg or 160 pmol) by detergent-solubilized and purified FtsH-His6-Myc
(1.48 µg or 20 pmol) was carried out as described previously (11) in
a total volume of 60 µl and in the presence of ATP (open
circles). Added to the reaction mixture was HflD at 0.9 µg (40 pmol; closed circles), 1.8 µg (80 pmol; closed
squares), 3.7 µg (160 pmol; closed triangles), and
7.4 µg (320 pmol; closed diamonds). Reaction was at
37 °C for 0, 0.5, 1, and 2 h. CII in each sample was visualized
by immunoblotting, and the relative intensities were determined by a
lumino-image analyzer (LAS1000, Fuji Film). Values relative to the
initial amount of CII are shown. B, stability of CII
in cells with defective or overproduced HflD. Plasmid pKH479 expressing
CII was introduced into strains AD16 (wild-type; open
squares), AK2149 (hflD::tet;
solid squares), AD16/pTWV228 (vector control; closed
circles), and AD16/pKH441 (overexpressing hflD;
open circles). Plasmid-encoded genes were induced at
37 °C with 1 mM IPTG and 3 mM cyclic AMP for
10 min and pulse-labeled with [35S]methionine for 30 s followed by chase for the indicated time periods. Radioactive CII was
immunoprecipitated, separated by SDS-PAGE, and quantitated using a
phosphorimager (BAS1800 , Fuji Film). Values relative to those at the 0 min chase point are shown for each sample.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
offered a classical example in which
stability control of a regulatory protein serves as a developmental switch. Several factors affect the stability of CII. These include FtsH
(HflB), the primary enzyme responsible for proteolysis (12, 13), and
the HflKC complex, which modulates the proteolytic function of FtsH
against different classes of substrates (11, 12, 18). The
-encoded
CIII protein also affects CII stability, as it interferes competitively
with the rapid degradation of CII (3, 14, 39). In this study, we
identified a new host element, HflD, which directly interacts with CII
and functions to reduce the lysogenization frequency of
.
32 or SecY was
not affected by the hflD disruption (data not shown). We
have shown that HflD directly interacts with CII both in
vivo and in vitro.
-helices. In particular, 8 leucines and 1 isoleucine can
be aligned on one side of an
helix for the
Leu85-Leu120 interval, and the amphipathic
nature seems to continue up to Tyr138. These regions could
mediate membrane association, CII association, and/or dimerization of HflD.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank A. B. Oppenheim for plasmids and comments on the putative amphipathic helix in HflD, D. Ladant for the E. coli two-hybrid system, H. Mori for discussion, and K. Mochizuki for technical support.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from CREST (Core Research for Evolutional Science and Technology), Japan Science and Technology Corporation (JST), and the Ministry of Education, Science and Culture, Japan.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.
Supported by a Japan Society for the Promotion of Science (JSPS)
Research Fellowship for Young Scientists. Present address: Dept. of
Cell Biology, National Inst. for Basic Biology, Nishigounaka 38, Myoudaiji-cho, Okazaki 444-8585, Japan.
§ To whom correspondence should be addressed. Fax: +81-75-771-5699; E-mail: kito@virus.kyoto-u.ac.jp.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M011699200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
kb, kilobase pair(s);
IPTG, isopropyl-thio--D-thiogalactoside;
DTT, dithiothreitol;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | McAdams, H. H., and Shapiro, L. (1995) Science 269, 650-656[Medline] [Order article via Infotrieve] |
2. | Gottesman, S., Gottesman, M., Shaw, J. E., and Pearson, M. L. (1981) Cell 24, 225-233[Medline] [Order article via Infotrieve] |
3. | Hoyt, M. A., Knight, D. M., Das, A., Miller, H. I., and Echols, H. (1982) Cell 31, 565-573[Medline] [Order article via Infotrieve] |
4. | Belfort, M., and Wulff, D. L. (1971) in The Bacteriophage Lambda (Hershey, A. D., ed) , pp. 739-742, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
5. |
Gautsch, J. W.,
and Wulff, D. L.
(1974)
Genetics
77,
435-448 |
6. | Noble, J. A., Innis, M. A., Koonin, E. V., Rudd, K. E., Banuett, F., and Herskowitz, I. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10866-10870[Abstract] |
7. | Banuett, F., and Herskowitz, I. (1987) J. Bacteriol. 169, 4076-4085[Medline] [Order article via Infotrieve] |
8. |
Kihara, A.,
and Ito, K.
(1998)
J. Biol. Chem.
273,
29770-29775 |
9. | Zorick, T. S., and Echols, H. (1991) J. Bacteriol. 173, 6307-6310[Medline] [Order article via Infotrieve] |
10. | Cheng, H. C., Muhlrad, P. J., Hoyt, M. A., and Echols, H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7882-7886[Abstract] |
11. | Kihara, A., Akiyama, Y., and Ito, K. (1996) EMBO J. 15, 6122-6131[Abstract] |
12. |
Kihara, A.,
Akiyama, Y.,
and Ito, K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5544-5549 |
13. | Shotland, Y., Koby, S., Teff, D., Mansur, N., Oren, D. A., Tatematsu, K., Tomoyasu, T., Kessel, M., Bukau, B., Ogura, T., and Oppenheim, A. B. (1997) Mol. Microbiol. 24, 1303-1310[Medline] [Order article via Infotrieve] |
14. | Banuett, F., Hoyt, M. A., McFarlane, L., Echols, H., and Herskowitz, I. (1986) J. Mol. Biol. 187, 213-224[Medline] [Order article via Infotrieve] |
15. | Herman, C., Ogura, T., Tomoyasu, T., Hiraga, S., Akiyama, Y., Ito, K., Thomas, R., D'Ari, R., and Bourec, P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10861-10865[Abstract] |
16. | Schumann, W. (1999) FEMS Microbiol. Rev. 23, 1-11[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Neuwald, A. F.,
Aravind, L.,
Spouge, J. L.,
and Koonin, E. V.
(1999)
Genome Res.
9,
27-43 |
18. | Kihara, A., Akiyama, Y., and Ito, K. (1998) J. Mol. Biol. 279, 175-188[CrossRef][Medline] [Order article via Infotrieve] |
19. | Tilly, K., Spence, J., and Georgopoulos, C. (1989) J. Bacteriol. 171, 1585-1589[Medline] [Order article via Infotrieve] |
20. |
Akiyama, Y.,
Kihara, A.,
Mori, H.,
Ogura, T.,
and Ito, K.
(1998)
J. Biol. Chem.
273,
22326-22333 |
21. | Kihara, A., Akiyama, Y., and Ito, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4532-4536[Abstract] |
22. | Miller, J. H. (1972) Experiments in Molecular Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
23. | Davis, R. W., Bostein, D., and Roth, J. R. (1980) Advanced Bacterial Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
24. |
Stahl, F. W.,
Kobayashi, I.,
Thaler, D.,
and Stahl, M. M.
(1986)
Genetics
113,
215-227 |
25. |
Karimova, G.,
Pidoux, J.,
Ullmann, A.,
and Ladant, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5752-5756 |
26. |
Yoshihisa, T.,
and Ito, K.
(1996)
J. Biol. Chem.
271,
9429-9436 |
27. | Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130[Medline] [Order article via Infotrieve] |
28. | Taura, T., Baba, T., Akiyama, Y., and Ito, K. (1993) J. Bacteriol. 175, 7771-7775[Abstract] |
29. | Futai, M., Noumi, T., and Maeda, M. (1989) Annu. Rev. Biochem. 58, 111-136[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Akiyama, Y.,
Ogura, T.,
and Ito, K.
(1994)
J. Biol. Chem.
269,
5218-5224 |
31. | Wulff, D. L., Beher, M., Izumi, S., Beck, J., and Mahoney, M. (1980) J. Mol. Biol. 138, 209-230[Medline] [Order article via Infotrieve] |
32. |
Kobayashi, T.,
Kishigami, S.,
Sone, M.,
Inokuchi, H.,
Mogi, T.,
and Ito, K.
(1997)
Proc. Natl. Adad. Sci. U. S. A.
94,
11857-11862 |
33. | Brody, H., and Hill, C. W. (1988) J. Bacteriol. 170, 2040-2044[Medline] [Order article via Infotrieve] |
34. | Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508[Medline] [Order article via Infotrieve] |
35. | Green, S. M., Malik, T., Giles, I. G., and Drabble, W. T. (1996) Microbiology 142, 3219-3230[Abstract] |
36. | Akiyama, Y., and Ito, K. (1990) Biochem. Biophys. Res. Commun. 167, 711-715[Medline] [Order article via Infotrieve] |
37. |
Ho, Y.,
Lewis, M.,
and Rosenberg, M.
(1982)
J. Biol. Chem.
257,
9128-9134 |
38. |
Weber, K.,
and Osborn, M.
(1969)
J. Biol. Chem.
244,
4406-4412 |
39. | Herman, C., Thévenet, D., D'Ari, R., and Bouloc, P. (1997) J. Bacteriol. 179, 358-363[Abstract] |
40. |
Levchenko, I.,
Seidel, M.,
Sauer, R. T.,
and Baker, T. A.
(2000)
Science
289,
2354-2356 |
41. | Tomoyasu, T., Yamanaka, K., Murata, K., Suzaki, T., Bouloc, P., Kato, A., Niki, H., Hiraga, S., and Ogura, T. (1993) J. Bacteriol. 175, 1352-1357[Abstract] |
42. |
Becker, G.,
Klauck, E.,
and Hengge-Aronis, R.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6439-6444 |
43. | Ho, Y. S., Mahoney, M. E., Wulff, D. L., and Rosenberg, M. (1988) Genes Dev. 2, 184-195[Abstract] |
44. |
Ades, S. E.,
Connolly, S. E.,
Alba, B. M.,
and Gross, C. A.
(1999)
Genes Dev.
13,
2449-2461 |