Archives of Biochemistry and Biophysics 493 (2010) 175–183

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Archives of Biochemistry and Biophysics

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HflD, an Escherichia coli protein involved in the k lysis–lysogeny switch,

impairs transcription activation by kCII
Pabitra K. Parua, Avisek Mondal, Pradeep Parrack *
Department of Biochemistry, Bose Institute, P-1/12, C.I.T. Scheme VIIM, Kolkata 700 054, India

a r t i c l e i n f o a b s t r a c t

Article history: The CII protein of bacteriophage lambda is the key regulator for the lytic–lysogenic choice of the viral life-
Received 6 August 2009 cycle. An unstable homotetrameric transcription activator of the three phage promoters pE, pI and paQ,
and in revised form 16 October 2009 kCII is stabilized by kCIII and destabilized by the host protease, Escherichia coli HflB (FtsH). In addition,
Available online 22 October 2009
other E. coli proteins HflK, HflC and HflD also influence lysogeny by acting upon CII. Among these, HflD
(22.9 kDa), a peripheral membrane protein that is exposed towards the cytoplasm, interacts with CII
Keywords: and decreases the frequency of lysogenization of k by stimulating the degradation of CII. In this study,
Lysis–lysogeny decision
we show that in addition to helping CII degradation, HflD inhibits the DNA binding by CII, thereby inhib-
Bacteriophage lambda
kCII
iting CII-dependent transcription activation. From biochemical, biophysical and modelling studies we
CII–HflD interaction also suggest that HflD–CII interaction takes place through the Cys31-accessible surface area of mono-
Transcription inhibition meric HflD, which binds to tetrameric CII as a 1:1 complex.
Ó 2009 Elsevier Inc. All rights reserved.

Introduction The cellular concentration of CII determines which pathway the

When the temperate coliphage k infects Escherichia coli, it can
follow either the lytic or the lysogenic pathway, depending upon
the conditions of infection and conditions of growth [1–3]. The
choice of pathways is influenced by several phage proteins as well
as host proteins [4,5]. The most important regulator for the lytic/
lysogenic decision is kCII, an unstable homotetrameric transcrip-
tion factor that activates the three k promoters pE, pI and paQ, lead-
ing to products that help establish the lysogenic pathway [6–13].
kCII is a small (4  97 aa) protein that assumes an all-helix struc-
ture with each monomer comprising four a helices (a1–a4) and
a disordered C-terminal end (16 residues) [14–17]. The a4 helix
(residues 64–77) from each subunit is responsible for the tetramer-
ization of the protein, formed by a four-helix bundle [14,15]. The
disordered C-terminal tail is the target for the proteolysis of CII
by E. coli HflB (also known as FtsH) and makes CII unstable, a prop-
erty essential for its function [18–20]. The three-dimensional crys-
tal structure of the CII–DNA complex has revealed that CII binds
DNA as a tetramer (dimer of dimers), where monomer A of the
AB dimer and monomer C of the CD dimer make nearly identical,
sequence-specific interactions with every base pair of the TTGC di-
rect repeats of DNA via the recognition helix (a3) [15].
* Corresponding author. Address: Department of Biochemistry, Bose Institute, P-
1/12, C.I.T. Scheme VIIM, Kolkata 700 054, West Bengal, India. Fax: +91 33
23553886.
E-mail address: pradeep@bic.boseinst.ernet.in (P. Parrack).
phage will follow. The level of CII depends upon kCIII, which stabi-
lizes CII by its antiproteolytic action against the host protease HflB
[21–24]. It also depends upon other host factors, viz. HflK, HflC and
HflD [5,25–26], which destabilize CII, thereby affecting the lysis–
lysogeny decision [4]. Among the latter, HflD (213 amino acids)
is a peripheral membrane protein of E. coli that is exposed to the
cytosol [26]. The 640 bp hflD gene, previously known as ycfC
[27], is located at 23.7 min in the E. coli genome, upstream of purB
and downstream of asuE (trmU) [28]. The purB gene encodes succi-
nyl-AMP (S-AMP) lyase required for purine metabolism. Despite its
location within the pur operon, ycfC was not found to play any role
in purine biosynthesis, neither is it essential for the growth of
E. coli [28]. Random mutational studies identified some point and
deletion mutations within this gene that increased the lysogeniza-
tion frequency of k [26]. Subsequently, ycfC was named hflD, a new
hfl (high frequency of lysogenization) gene. Its gene product HflD
interacts with CII both in vivo and in vitro [26] and stimulates the
in vivo degradation of CII, thereby decreasing the lysogenization
frequency of k [26]. However, HflD inhibits the HflB-mediated deg-
radation of CII in vitro [26]. The reason behind these contradictory
roles of HflD is not known. It was proposed that HflD sequesters CII
from its target promoters (pE, pI and paQ) and recruits it to mem-
brane-bound HflB for rapid degradation [26]. Nevertheless, no
information is available about the regions of CII or of HflD that
are involved in CII–HflD interaction. The mechanism of action of
HflD remains unresolved. The three-dimensional structure of HflD
(YcfC), deposited in the protein databank in 2004 (PDB ID: 1QZ4
and 1SDI) remains unpublished.

0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2009.10.010
176 P.K. Parua et al. / Archives of Biochemistry and Biophysics 493 (2010) 175–183

In this study, we have explored how HflD interacts with CII, and
examined the effect of HflD on CII-activated transcription. Through
biochemical and biophysical experiments supplemented with
modelling studies we provide some newer aspects of CII–HflD
interactions, which may be helpful in understanding the mecha-
nism of action of HflD.
Materials and methods
Bacterial strains and plasmids
Commonly used E. coli strains and plasmids were obtained from
commercial sources as mentioned in the text. All other strains,
plasmids and primers used in this study are shown in Table 1.
Construction of plasmids
For expression of HflD with a 6-histidine tag (His6-HflD), the
hflD gene was removed from pKH449 [26] by digestion with BamHI
and cloned into pET28a vector (Novagen) at the BamHI site. The
resulting plasmid was named pKP509.
pKP07 was prepared to replace the T7 promoter of pET28a by
lac promoter. The lac promoter-operator region (lacP/O; 122 bp)
was PCR amplified using LacF and LacR as the forward and the re-
verse primers, and pUC19 (Fermentas) as the template, and was
cloned into pET28a at the BglII and XbaI sites. pKP109 (carrying
lacZ under the control of pE) was constructed by cloning pE
(298 bp) upstream of lacZ at the XbaI site into pSD5B [29] vector
in the correct orientation.
pKP219 (contains lacZ under pE and cII under lacP) and pKP419
(contains lacZ under pE and cIIA, encoding residues 1–82 of cII, un-
der lacP) were constructed for in vivo transcription assays. First the
cII (or cIIA) gene was PCR amplified using CIIF and CIIR (or CIIF and
CIIAR) as forward and reverse primers and pAB305 [30] as tem-
plate, and cloned into pKP07 at the NdeI and BamHI sites down-
stream of lacP/O, to obtain pKP106 (or pKP108). The lacP/O-cII
(500 bp) (or lacP/O-cIIA, 450 bp) cassette was then removed
from pKP106 (or pKP108) by digestion with BglII and BamHI and
cloned into the pKP109 vector at the BglII site. pKP917 (carrying
hflD under lacP/O) was constructed for in vivo transcription assays.
hflD was first removed from pKH449 by digestion with BamHI and
cloned into BamHI-digested pKP07 under lacP/O, to obtain pKP107.
The lacP/O-hflD cassette (830 bp) was then removed from
pKP107 by digestion with BglII and BamHI and cloned into
pQE30 (Qiagen) at the BamHI site. Prior to sub-cloning of the
lacP/O-hflD cassette into pQE30, the plasmid was modified by
removing its existing T5 promoter-lacO region by digestion with
XhoI and EcoRI followed by end-filling with Klenow polymerase
and self-ligation. The pKP917 was digested by BamHI and self-li-
gated. The resultant plasmid that contained only the lacP/O region
(without any downstream gene) was named pKP908 and was used
as the empty control vector for in vivo studies.
Mutation and cloning of the hflD gene
The specific mutation (W181A) within the hflD gene was car-
ried out by PCR-directed site specific mutagenesis [31a]. In the
primary step, two separate PCRs were performed taking pKP509
as template. In one, PDF was used as the forward primer and
PRW181A as the reverse primer. In the other, RD and PFW181A were
used as the reverse and the forward primers, respectively. To am-
plify the complete mutated gene, secondary PCR was carried out
using the two overlapping fragments obtained from the primary
PCR as the template and PDF and RD as the forward and the re-
verse primers. The resulting 640 bp PCR product was then
cloned into pKP07 at the NdeI and XhoI sites and named

Table 1
Bacterial strains, plasmids and primers used.

Strains and plasmids Genotype/description Protein expressed Reference/Source

E. coli strains
AK525 Dpro-lac thi/F0 lacIq ZM15 Y+ pro+ zgj-460::Tn5 zgj-525::IS1A – Kihara et al. [42]
Plasmids
pKP07 lacP/O – This study
pKP109 pE-lacZ – This study
pKP219 pE-lacZ & lacP-cII His6-CII This study
pKP917 lacP-hflD His6-HflD This study
pKP908 lacP – This study
pKP804 lacP/O-hflD (W181 is substituted by A) His6-HflDW181A(mHflD) This study
pKP509 T7P-hflD – This study
pAB305 T7P-cII CII (tagless) Datta et al. [30]
pSD5B containing lacZ gene and p15A replicon – Jain et al. [29]
pKH449 carrying gst-hflD under the tac promoter GST-HflD Kihara et al. [26]
pKP108 lacP/O-cIIA (expressing truncated CII containing 1–82 residues) His6-CIIA This study
pKP419 pE-lacZ & lacP-cIIA His6-CIIA This study
pAB412 T7P-cII His6-CII Halder et al. [24]

Primers (restriction sites are underlined, mutation sites are in bold)

PDFB: 50 -GCAGGGATCCGTGGCAAAGAATTAC-30 (BamHI)
RD: 50 -CGCCCTCGAGTCACAACTCCGGGG-30 (XhoI)
PDF: 50 -CGGCATATGGTGGCAAAG-30 (NdeI)
PFW181A: 50 -GCCGCCGTGCTCGCGCACCAGGTCGGC-30 (Mutation forward primer)
PRW181A: 50 -GCCGACCTGGTGCGCGAGCACGGCGGC-30 (Mutation reverse primer)
LacF: 50 -GCGGAGATCTGCGCAACGCAATTAATG-30 (BglII)
LacR: 50 -GTGGTCTAGAAGCTGTTTCCTGTGTG-30 (XbaI)
PEF: 50 -GCGGTCTAGAGGGCATCAAATTAAACC-30 (XbaI)
PER: 50 -TATTTCTAGACGCGCCAATCGAGCCATG-30 (XbaI)
CIIF: 50 -CAGGCCGTCATATGGTTCGTGCAAAC-30 (NdeI)
CIIR: 50 -GCTAACGGGATCCTCAGAACTCCATC-30 (BamHI)
CIIAR: 50 -GGCGGGATCCTTTTTTATTGGTGAGAATCGC-30 (BamHI)
 P.K. Parua et al. / Archives of Biochemistry and Biophysics 493 (2010) 175–183
pKP804, which was used for overexpression of His6-HflDW181A,
henceforth called mHflD.
Overexpression and purification of proteins
Escherichia coli BL21 (DE3) (Invitrogen) cells carrying pKP509 or
pKP804 were grown in a 750 ml fresh Luria–Bertani medium in
presence of kanamycin (50 lg/ml) at 37 °C till A590 of the culture
was between 0.4 and 0.5. The culture was then induced by
300 lM isopropyl-b-D-thiogalactopyranoside (IPTG) at 16 °C for
20 h. Bacterial cells were recovered by centrifugation at 5000g
for 7 min at 4 °C. The cell pellet was suspended in lysis buffer
(20 mM Tris–HCl, 500 mM NaCl, 5 mM MgCl2, 10% glycerol,
10 mM imidazole, 0.5% nonidet P-40, 10 mM b-mercaptoethanol,
1 mM phenylmethylsulfonyl fluoride and 200 lg/ml lysozyme,
pH 8.0). The cells were then lysed by sonication followed by centri-
fugation at 17,400g for 20 min at 4 °C. The supernatant was loaded
onto a Ni2+–NTA column (pre-equilibrated with the lysis buffer).
After loading, the column was consecutively washed with wash
buffers I (20 mM Tris–HCl, 500 mM NaCl, 5% glycerol and 25 mM
imidazole, pH 8.0) and II (100 mM NaP, 100 mM NaCl, 5% glycerol,
pH 8.0). Elution of the protein (His6-HflD or mHflD) was accom-
plished with the elution buffer (100 mM NaP, 100 mM NaCl, 5%
glycerol and 250 mM imidazole, pH 8.0).
Native CII (without a histidine tag) was obtained by over-
expressing CII from the recombinant plasmid pAB305 in BL21
(DE3) cells and purification by two steps of ion-exchange chroma-
tography as described earlier [30].
GST-HflD, His6-CII and its truncated variant His6-CIIA required
for in vitro interaction studies were overexpressed and purified
by the following method. BL21 (DE3) cells carrying pKH449 were
induced by 500 lM IPTG at A600  0.5–0.6 for 4 h at 37 °C. Cells
were harvested by centrifugation at 5000g at 4 °C for 7 min and
suspended in the lysis buffer (1 phosphate buffered saline, 10%
glycerol, 10 mM b-mercaptoethanol, 1 mM phenylmethylsulfonyl
fluoride, 0.5% nonidet P-40 and 200 lg/ml lysozyme, pH 7.4). Cells
were lysed by sonication followed by centrifugation at 17,400g for
20 min at 4 °C to collect the clear supernatant, which was applied
to a Glutathione SepharoseTM 4B column (GE Healthcare Bio-Sci-
ences AB, Sweden) pre-equilibrated with the lysis buffer. The col-
umn was washed with 10 times the bead volume of wash buffer
(1 phosphate buffered saline, 10% glycerol, 0.5% nonidet P-40,
pH 7.4). GST-HflD was eluted from the column with elution buffer
(100 mM NaP, 100 mM NaCl, 5% glycerol, 10 mM reduced glutathi-
one, pH 7.0).
His6-CII was overexpressed within E. coli BL21 (DE3) cells from
pAB412 and purified as described in Parua et al. [31b]. His6-CIIA
was overexpressed and purified in an identical fashion, from
pKP108 in BL21 (DE3) cells.
Size-exclusion chromatography
Size-exclusion chromatography was carried out to identify the
oligomeric state of HflD. Here, 1–2 mg/ml of the recombinant
His6-HflD was applied onto a SuperdexÒ75 column. Prior to sam-
ple loading, the column was pre-equilibrated with buffer D
(100 mM NaP, 100 mM NaCl, 5% glycerol, 1 mM EDTA, pH 8.0).
The flow rate was maintained at 0.5 ml/min.
Glutaraldehyde crosslinking
Glutaraldehyde crosslinking was used to further assess the olig-
omeric state of HflD as well as the stoichiometry of interaction be-
tween CII and HflD, as described in [23]. 0.1% of glutaraldehyde
was used to crosslink 8 lM of His6-HflD in buffer D (see above)
at 4 °C for 20 min. Reactions were terminated by the addition of
177
200 mM of glycine, followed by the addition of 1 SDS–PAGE
gel-loading buffer and boiling. The protein samples were electro-
phoresed on a 13.5% SDS–PAGE followed by electroblotting onto
a polyvinylidene difluoride (PVDF; Schleicher and Schuell) mem-
brane. Proteins on the membrane were visualized by immunoblot-
ting with mouse anti-His monoclonal IgG.
For measurement of stoichiometry, 8 lM each of CII and His6-
HflD was incubated together for 10 min at 25 °C followed by the
application of 0.1% glutaraldehyde to the reaction mixture. Cross-
linking was carried out at 4 °C for the specified time interval (2,
4, 8 and 12 min) as mentioned above. Reactions were terminated
and subjected to 12–15% gradient SDS–PAGE followed by electro-
blotting onto PVDF membranes and were visualized by immuno-
blotting with anti-CII polyclonal antibody (obtained from Keith
Shearwin, University of Adelaide, Australia).
Cysteine accessibility of HflD
The number of surface-accessible cysteine residues of His6-HflD
was determined following the DTNB (5,50 -dithiobis-2-nitrobenzoic
acid) method mentioned in [32]. Briefly, 100 ll of DTNB solution
was added to 900 ll of protein solution (10 lM) in 100 mM Na-
phosphate buffer (pH. 8.0) at room temperature (25 °C) with a final
protein:DTNB molar ratio of 1:30. Absorbance at 412 nm was mea-
sured after the OD value reached saturation. The number of free
cysteine residue(s) was determined from the changes in absor-
bance at 412 nm as the number of TNB (2-nitro-5-thiobenzoate)
molecules released due to the reaction between accessible –SH
group(s) of the protein and DTNB, using the following equation:
n ¼ ðA412 =TNB Þ=½P
where n is the number of surface-accessible cysteine residues, A412
is the measured absorbance at 412 nm, [P] is the concentration of
protein and eTNB is the molar extinction coefficient of TNB, for which
a value of 13,650 M1cm1 [33] was used.
Tryptophan fluorescence measurement
The tryptophan fluorescence of CII in the presence of mHflD was
measured by titrating 4 lM of CII with increasing concentrations of
mHflD (up to 180 nM) in buffer P (20 mM Tris–HCl, 200 mM NaCl,
1 mM EDTA, pH 8.0), in a Hitachi F-3000 spectrofluorimeter.
kex = 295 nm and kem = 343 nm were used, with bandwidths of
5 nm on both sides.
In vivo b-galactosidase assays
The extent of transcription activation by CII or CIIA in the pres-
ence or absence of HflD was quantified by measuring the b-galac-
tosidase expressed from a lacZ reporter gene, according to Miller
[34]. Escherichia coli AK525 cells carrying a pair of plasmids
expressing b-galactosidase and either HflD, CII, CIIA or both CII/
CIIA and HflD (for details, see Table 2) were used as hosts. The cells
were allowed to grow at 37 °C in 5 ml Luria–Bertani medium sup-
plemented with 100 lg/ml ampicillin and 50 lg/ml kanamycin,
followed by induction by 300 lM IPTG at A590  0.6–0.7 for
60 min and immediate transfer onto ice. The absorbance at
590 nm was recorded. 0.5 ml of these cultures were separately
added to 0.9 ml of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4,
10 mM KCl, 1 mM MgSO4, 50 mM b-mercaptoethanol, pH adjusted
to 7.0). The cells were lysed by the addition of 10 ll of 1% SDS,
20 ll of chloroform and vortexing for 10 s. The tubes containing
the reaction mixtures were then placed in a 28 °C water bath for
5 min. Reactions were initiated by the addition of 0.2 ml of ortho
2-nitrophenyl-b-D-galcatopyranoside (ONPG) (4 mg/ml, dissolved
in Z buffer) at 28 °C. Reactions were stopped by the addition of
178 P.K. Parua et al. / Archives of Biochemistry and Biophysics 493 (2010) 175–183
Table 2
Effects of HflD on in vivo transcription activation by CII.
E. coli plasmids (His)6-protein b-Galactosidase activity
strain carried expressed (Miller Units)a
AK525 pKP109 & pKP908 – 0.2 (±0.3)
pKP109 & pKP917 HflD 0.4 (±0.4)
pKP219 & pKP908 CII 47.9 (±6.5)
pKP219 & pKP917 CII & HflD 6.1 (±3.2)
pKP419 & pKP908 CIIA 52.8 (±7.8)
pKP419 & pKP917 CIIA & HflD 10.2 (±4.3)
a
All values shown are the average of three independent experiments. The
respective standard deviations are indicated within brackets.
0.3 ml of 1 M Na2CO3 after sufficient yellow colour had developed.
The absorbances at 420 and 550 nm were then recorded. The unit
of b-galactosidase activity was calculated from
Miller Units ¼ 1000  ðA420  1:75  A550 Þ=ðt  V  A590 Þ
where t is the time of reaction (in min) and V is the volume (in ml)
of the culture used in the assay [34] .
In vitro transcription assays
The effect of His6-HflD on CII-mediated transcription from pE
was quantitated by in vitro transcription assays as described in
[17]. Reactions were performed in a 20-ll reaction volume in tran-
scription buffer (40 mM Tris–HCl, 0.1 M potassium glutamate,
1 mM DTT,1 20 mM MgCl2; pH 8.0) using the 540 bp DNA fragment
[17] as the template, which contained the promoters pE (CII-depen-
dent, would generate a 144-base run-off transcript) and poop (CII-
independent, would give a 77-base terminated transcript). The tran-
script from the latter promoter was used as an internal positive con-
trol. In all reactions, DNA and E. coli RNA polymerase were used at 5
and 60 nM, respectively. The experiment was carried out in two
ways. In the first, 250 nM of CII and a specific concentration of
His6-HflD (0, 50, 100, 250 or 500 nM) were incubated at 37 °C for
10 min before RNA polymerase and template DNA were added. This
was followed by further incubation at 37 °C for 10 min. Transcription
was initiated by the addition of nucleotide mix (0.1 mM each of ATP,
GTP, CTP; 0.01 mM of UTP and 5 lCi of [a-32P] UTP and 1 lg of hep-
arin). The reactions were terminated after 20 min by the addition of
5 ll of formamide stop dye. In the second, CII was mixed at a con-
centration of 250 nM with RNA polymerase and template DNA,
and reactions were incubated at 37 °C for 20 min followed by initia-
tion of transcription by the addition of NTP mix. After 4 min, His6-
HflD was added at concentrations of 0, 50, 100, 250 and 500 nM
and the reactions were further incubated at 37 °C for an additional
16 min. Finally, reactions were terminated by the addition of the
stop dye. Transcripts were resolved by electrophoresis in a 10% (w/
v) polyacrylamide–7 M urea gel, which was dried and autoradio-
graphed at 20 °C.
DNA binding studies
DNA binding studies for CII in the presence of His6-HflD were
carried out by fluorescence anisotropy measurements [35]. In this
study, a 20 bp oligonucleotide containing the recognition sequence
for CII (pE promoter) was used. The sense (50 -fluorescein-TCGT
TGCGTTTGTTTGCACG-30 ) and anti-sense (50 -CGTGCAAACAAACGC
AACGA-30 ) strands were purchased from BioTechdeskTM as HPLC-
purified DNA. The strands were mixed with each other to a final
concentration of 100 lM of each in a buffer containing 20 mM
1
Abbreviations used: DTT, dithiothreitol; HTH, helix-turn-helix; CRP, cyclic AMP
receptor protein.
Tris–HCl, 100 mM NaCl and 1 mM EDTA, pH 8.0. The oligonucleo-
tide mixture was then heated on a boiling water bath for 15 min
and allowed to cool slowly to room temperature. The concentra-
tion of the double-stranded DNA was estimated from its optical
density at 260 nm. This DNA was used at a final concentration of
10 nM in a total volume of 2.5 ml. The titration of DNA by CII in
presence of His6-HflD was performed in the assay buffer containing
20 mM Tris–HCl, 100 mM potassium glutamate, 10% glycerol and
1 mM EDTA, pH 8.0. The experiment was carried out in two ways:
(a) 150 nM of His6-HflD was mixed with the DNA which was ti-
trated with increasing concentrations of CII, or (b) CII and His6-
HflD were pre-incubated at equimolar concentrations (up to
900 nM) of each for 1 h at 4 °C and was then added to DNA. Mea-
surements were carried out in a Hitachi F-4500 spectrofluorimeter
with excitation and emission wavelengths set at 494 and 520 nm,
respectively. Bandwidth on each side was 5 nm. Anisotropy was
measured by scanning the sample for 2 min at each titration point
and recording the averaged value of the anisotropy.
In vivo stability of CII in presence of HflD
In vivo stability of His6-CII in the presence of His6-HflD was car-
ried out following the protocol of [36]. Here, E. coli XL1 Blue or
AK525 cells carrying the pair of plasmids pKP219 and pKP908 or
pKP219 and pKP917 was used as the host. Cells were grown till
A590 = 0.6–0.7 in Luria–Bertani medium supplemented with ampi-
cillin (100 lg/ml) and kanamycin (50 lg/ml) at 37 °C, followed by
induction with 300 lM of IPTG for 60 min and treatment with
spectinomycin (100 lg/ml) to inhibit protein synthesis. 500 ll ali-
quots of cell culture were collected at different time intervals after
the addition of spectinomycin. Cells were harvested by centrifuga-
tion at 6600g at 4 °C for 8 min and resuspended in 1 gel-loading
buffer. Samples were then electrophoresed on a 15% SDS–poly-
acrylamide gel followed by electroblotting using Mini Trans-BlotÒ
Cell (Bio-Rad, USA) onto a PVDF membrane. Proteins were visual-
ized by immunoblotting with rabbit polyclonal anti-CII antibody.
The blot pattern was subjected to densitometric analyses using
the volume analysis routine of the Molecular Analyst software
(Bio-Rad, USA).
Possible HflD–CIIA interactions in vitro
GST pull-down assays were carried out to check possible inter-
actions between HflD and CIIA, according to Halder et al. [24]. Fifty
micrograms of purified GST-HflD was immobilized on Glutathione
SepharoseTM 4B beads at 4 °C for 30 min in buffer B (50 mm NaP,
100 mM NaCl and 5% Glycerol, pH 7.0). To the same beads, 50 lg
of purified His6-CII or His6-CIIA was added and incubated for
30 min at 4 °C with mixing by continuous rotation. The beads were
then washed thrice with buffer B. Bound GST-HflD was eluted by
the elution buffer (buffer B plus 10 mM of reduced glutathione).
Each fraction (loading, each wash and elution) was analysed by
15% SDS–PAGE. Proteins were then electroblotted onto a PVDF
membrane and visualized by immunoblotting using polyclonal
anti-CII IgG.
Modelling and computation
To get an idea about the possible interacting region(s) of CII and
HflD, molecular docking between HflD and CII was carried out
using the GRAMM docking program in a low resolution matching
mode [37–39]. The molecular structures of HflD (1SDI) and CII
(1XWR) from the Protein Data Bank were used. The parameters
of docking are shown in Supplementary Table S1. The best model
structures having the minimum energy were used to obtain
 P.K. Parua et al. / Archives of Biochemistry and Biophysics 493 (2010) 175–183 179

information on the binding interaction between HflD and CII, and

on intermolecular hydrogen bond formation.

Results

Accessibility of cysteine residues of HflD

Upon incubation of HflD with DTNB, an increase in A412 was ob-

served, which reached a saturation value of 0.14 at 45 min. This va-
lue was used to calculate the number of accessible cysteine(s) in
HflD. It was found that HflD had one (0.95) surface-accessible
cysteine residue. From the three-dimensional crystal structure of
HflD it was observed that of the two cysteines in HflD, Cys31
was surface accessible, while Cys16 was buried.

Oligomeric state of HflD

The oligomeric state of HflD was examined both by glutaralde-

hyde crosslinking and size-exclusion chromatography. The anti-
His antibody immunoblotting profile of His6-HflD crosslinked with
glutaraldehyde (Fig. 1a) showed that in the presence of glutaralde-
hyde, a weaker band corresponding to the molecular weight of the
His6-HflD dimer (48 kDa) appeared, in addition to the monomer
band observed in the absence of the crosslinker. The size-exclusion
chromatogram of His6-HflD (Fig. 1b) clearly showed that recombi-
nant HflD existed mostly as a monomer with a small amount of di-
mer. Fig. 1c shows a plot of the Rf value against the logarithm of
molecular weight obtained from gel-filtration. In this plot, the
positions of the His6-HflD dimer (46.70 kDa) and monomer
(24.20 kDa) are shown.

Interaction between CII and HflD: effects on the conformations of CII

and HflD Fig. 1. Oligomeric state of HflD. (a) Glutaraldehyde crosslinking. 13.5% SDS–PAGE
showing HflD alone (lane 1) or crosslinked for 5 or 10 min (lanes 2 and 3). Anti-his
It is known that HflD interacts with CII both in vivo and in vitro antibody was used for immunoblotting. Lane 4 shows molecular weight markers
[26]. Whether this interaction causes any structural change in (97, 66, 43, 30 and 20 kDa). Arrows indicate dimeric (Di) and monomeric (Mono)
HflD. (b) Elution profile of His6-HflD. HflD peaks corresponding to dimeric (A) and
either of the proteins is not known. The possible structural
monomeric (B) His6-HflD are indicated. A SuperdexÒ75 column was used. (c) A plot
change(s) in HflD and in CII were examined by various means, re- of Rf against the logarithm of molecular weight for the four standard proteins
sults of which are presented below. (Albumin, 67 kDa; Ovalbumin, 43 kDa; Chymotrypsinogen A, 25 kDa; Ribonuclease
A, 13.7 kDa) obtained by size-exclusion chromatography. The linear fit is shown
along with its equation. The positions of dimeric (s) and monomeric (h) His6-HflD
Effect of CII on cysteine accessibility of HflD are shown along with molecular weight standards ().
HflD has two cysteines (Cys16 and Cys31), of which only one
(most likely, C31) is available to react with DTNB. Does the binding
of CII change the accessibility of this cysteine? DTNB titration of
His6-HflD in the presence of CII was carried out to examine this.
It may be noted that CII has no cysteine residue; therefore DTNB
would react only with the accessible cys of HflD. In the presence
of different concentrations of CII (up to 20 lM), His6-HflD (5 lM)
was treated with DTNB and the number of accessible cysteines
was determined for each concentration of CII present. The final
concentration of DTNB was 150 lM. These results are shown in
Fig. 2. It was found that with increasing concentrations of CII, the
number of accessible cys in HflD decreased drastically from 1
to 0.2. Is this reduction due to a molecular crowding effect by
CII as its concentration was increased? To exclude this possibility,
the accessibility of cys in HflD was also measured in the presence
of PEG 6000, a straight chain polymer of a simple repeating unit
H(OCH2CH2)nOH that induces macromolecular crowding of solutes
in aqueous solution [40]. Increasing concentrations of PEG 6000
did not affect the cysteine accessibility in HflD (Fig. 2). To examine
if the effect of CII on HflD was due to a specific interaction between
the proteins, E. coli CRP, a protein that is not expected to interact Fig. 2. Number of accessible cysteine residues in HflD. The number of modified cys
residues in HflD was determined by DTNB treatment, as a function of different
with CII, was chosen. The accessibility of the cysteine residues of concentrations of kCII (h) or PEG 6000 (j). The number of accessible cysteines per
CRP, a homodimer having two accessible cysteines per dimer monomer of E. coli CRP as a function of varying kCII concentrations is also shown
[32], was measured in the presence of the same concentrations (s).
180 P.K. Parua et al. / Archives of Biochemistry and Biophysics 493 (2010) 175–183
of CII as for HflD. No effect of CII on the number of accessible cys
residues of CRP was observed (Fig. 2). Therefore, it is clear that
the observed decrease of cysteine accessibility of HflD upon addi-
tion of CII was due to a specific interaction between the two pro-
teins, which could be due to either of the following: (a) HflD
interacted with CII through its cysteine accessible surface area or
(b) as a result of the interaction with CII, structural changes oc-
curred within HflD causing the surface exposed cysteine to be
inaccessible.
Quenching of tryptophan fluorescence of CII
Possible structural changes within CII due to its interaction with
HflD was also monitored by examining the intrinsic tryptophan
fluorescence of CII in the presence of mHflD, an HflD mutant de-
void of any tryptophan residue (verified by the abolition of intrin-
sic tryptophan fluorescence of HflD, data not shown). It was
observed that the relative fluorescence intensity of CII increased
with increasing concentrations of mHflD, reaching saturation at
130 nM (Fig. 3). This result indicates that binding of HflD caused
a conformational change in CII, leading to an increase in the quan-
tum yield(s) of one or more of the three tryptophan residues in CII.
Glutaraldehyde crosslinking
To determine the stoichiometry of interaction between CII and
HflD, His6-HflD and native CII (without any tag) were subjected
to glutaraldehyde crosslinking (Fig. 4a). It was observed that be-
sides the monomer and higher order oligomers of CII, another
higher molecular weight protein band corresponding to a molecu-
lar weight of 70 kDa appeared. This band, which was recognized
by anti-CII polyclonal antibody, did not appear when CII was cross-
linked alone (Fig. 4b), indicating that it corresponded to a CII–HflD
complex. The molecular weights of CII and His6-HflD are 11 and
24 kDa, respectively. Therefore, the probable molar ratio of HflD
and CII in the complex could be 1:4 or 2:2. Thus, one molecule of
HflD may complex with four monomers of CII giving rise to a
molecular weight of 68 kDa for the complex. Alternatively, a
2:2 complex comprising two molecules of both CII and HflD may
result in the higher molecular weight band (70 kDa). Since in
solution CII exists predominantly as a tetramer and HflD as a
monomer, the first possibility, i.e. interaction of CII tetramer with
HflD monomer is more likely. This result also suggests that CII re-
tains its native tetrameric state in the presence of HflD.
Fig. 3. Effect of addition of mHflD on the intrinsic tryptophan fluorescence of kCII. A
plot representing the change in relative fluorescence intensity [(F0  F)/F0, where F0
and F are the observed fluorescence intensity at 343 nm in the absence and
presence of mHflD, respectively] as a function of the concentration of mHflD
(kex = 295 nm).
Fig. 4. Stoichiometry of interaction between CII and His6-HflD from glutaraldehyde
crosslinking. (a) CII (8 lM) and His6-HflD (8 lM) were crosslinked and run on a 12–
15% gradient SDS–PAGE, followed by electroblotting onto a PVDF membrane and
immunoblotting by anti-CII antibody. Lane 1: CII without crosslinker; lanes 2–5: a
mixture of CII and HflD, crosslinked for 2, 4, 8 and 12 min, respectively, on ice; lane
6: protein molecular weight standards (85, 48, 34, 26 and 19 kDa). The band
corresponding to CII–HflD complex (70 kDa) is shown. The bands corresponding
to monomer (Mono), dimer (Di), trimer (Tri) and tetramer (Tetra) of CII are
indicated. (b) CII alone was subjected to crosslinking followed by immunoblotting
with anti-CII-antibody. Lane 1: protein molecular weight standards (85, 48, 34,
26 kDa); lane 2: CII without crosslinker; lane 3: CII crosslinked for 10 min on ice.
Effect of HflD on transcription activation by CII
The effect of HflD on CII-mediated transcription activation from
pE was examined both in vitro and in vivo. The rate of in vitro tran-
scription activation of pE decreased with increasing concentrations
of HflD, when CII was pre-incubated with His6-HflD (Fig. 5). Inter-
estingly, the maximum inhibition (35–40%) occurred around 60–
70 nM of HflD, which was 25% of the concentration of CII (250 nM
of monomers) used in the reaction. This fact is consistent with an
interaction of monomeric HflD with tetrameric CII with a 1:1 stoi-
chiometry, as indicated in the previous section. However, HflD had
no effect on in vitro transcription from pE when His6-HflD was
added after initiation (Fig. 5).
The in vivo effect of HflD on CII-dependent transcription activa-
tion from pE was monitored by measuring the amount of b-galac-
tosidase expressed from lacZ as a reporter gene under the control
of pE, the results of which are shown in Table 2. When CII was ex-
pressed alone, the unit of b-galactosidase activity was observed to
be 47.9. This activity was solely due to the action of CII on pE up-
Fig. 5. Effect of HflD on CII-dependent transcription activation in vitro. Amount of
transcripts from pE and poop were quantified using densitometry. The ratio of the
two (taken as 100% when HflD was absent) was plotted against the concentration of
HflD added (up to 500 nM). Results for HflD pre-incubated with CII (250 nM) before
initiation of transcription (j) or when HflD was added after 4 min of initiation of
transcription by 250 nM CII (s) are shown. Each data point represents the average
value from three independent experiments.
 P.K. Parua et al. / Archives of Biochemistry and Biophysics 493 (2010) 175–183
Fig. 6. Binding of CII to pE from fluorescence anisotropy measurements. Fluores-
cence anisotropy was plotted for a 20-bp ds DNA (10 nM) containing pE and labelled
by fluorescein at the 50 end of the sense strand in presence of increasing
concentrations of CII alone (j) or an equimolar mixture of CII–HflD (4). Results
from a similar experiment when DNA was pre-incubated with 150 nM HflD before
titration with CII are also shown (s).
stream of the lacZ gene, since the activity measured in the absence
of CII was nominal (0.2). Upon simultaneous overexpression of
HflD with CII, the activity underwent an 87% reduction, decreas-
ing to a value of 6.1. Thus, HflD inhibited CII-dependent transcrip-
tion both in vitro and in vivo.
The results of the in vitro and in vivo transcription studies reveal
that HflD inhibits CII-dependent transcription activation. This may
happen due to either of the following reasons: (a) upon interaction
with CII, HflD may block recognition of its cognate DNA, (b) HflD–
CII interaction disrupts specific CII–RNA polymerase interaction
essential for transcription activation, or (c) the stimulating effect
of HflD on in vivo proteolysis of CII may predominate. To distin-
guish among these possibilities, DNA binding and in vivo proteoly-
sis of CII was carried out in the presence of HflD, as described
below.
Effect of HflD on DNA binding by CII
The effect of HflD on binding of CII at pE was examined by fluo-
rescence anisotropy measurements. When a 50 -fluorescein labelled
20 bp ds DNA containing the pE sequence was titrated with
Fig. 7. In vivo proteolysis of CII. (a) Western blot (using rabbit polyclonal anti-CII antibody) of whole cell (XL1 Blue) lysates for His6-CII at various time points in the presence
(s) or absence (j) of overexpressed His6-HflD after arresting the protein synthesis by spectinomycin (100 lg/ml). The bands were quantified using Molecular Analyst
software (Bio-Rad, USA) and the percentage of intracellular CII remaining was plotted. (b) Similar experiments as in (a) but carried out in AK525 cells (DhflB). Each experiment
was repeated four times.
181
increasing concentrations of CII, a gradual increase in anisotropy
was observed (Fig. 6), indicating binding of CII to the DNA. How-
ever, the anisotropy value remained unchanged when the same
experiment was repeated with an equimolar mixture of CII and
His6-HflD (Fig. 6), indicating a lack of binding. Clearly, CII–HflD
interaction prevented CII from binding to its cognate site on pE.
When His6-HflD (150 nM) was pre-incubated with DNA prior to
addition of CII, an interesting result was obtained. At lower (up
to 150 nM) concentrations of CII, there was no change in anisot-
ropy of DNA. Above 150 nM, the anisotropy increased, though the
rate of increase was less compared to that for CII alone (Fig. 6).
Therefore, it is likely that HflD–CII interaction is stronger than
pE–CII interaction, and binding of HflD to CII inhibits the DNA bind-
ing ability of the latter.
In vivo proteolysis of CII: effect of HflD
As shown above, HflD inhibits the CII-dependent transcription
activation from pE in vivo. To test whether this inhibition resulted
from a stimulation of in vivo CII proteolysis by HflD, the proteolysis
was carried out in the presence of His6-HflD, as shown in Fig. 7.
HflD significantly accelerated (by 25%) the in vivo proteolysis of
CII in wild type XL1 Blue cells (Fig. 7a), while there was no effect
of HflD in mutant AK525 cells that lacked HflB (Fig. 7b). Thus,
the destabilization of CII by HflD occurred only in the presence of
HflB.
From this result one may also conclude that the observed inhi-
bition of CII-mediated transcription from pE by HflD (carried out in
AK525 cells) was not due to the depletion of CII but due to a spe-
cific interaction between CII and HflD that resulted in a loss of DNA
binding by CII.
Interactions between CII and HflD by molecular modelling
The three-dimensional structure of HflD (formerly known as
YcfC), an all-helix protein, has been reported by D. Borek and Z.
Otwinowski (PDB ID: 1QZ4 and 1SDI; unpublished). The crystal
structure of CII in the free state (PDB ID: 1XWR) and as a DNA-
bound complex (PDB ID: 1ZS4) are also available [14,15]. We used
the structure 1XWR to build a reasonable molecular model in order
to unravel the possible interactions between CII and HflD by dock-
ing the two proteins using the GRAMM docking program [38,39] in
a low resolution matching mode. Such a minimum-energy model
of the CII–HflD complex obtained from docking is shown in
182 P.K. Parua et al. / Archives of Biochemistry and Biophysics 493 (2010) 175–183

Fig. 8. Molecular models for CII–HflD interaction (PyMOL view). (a) CII–HflD complex. The four chains (A, B, C and D) of CII (cartoon) and HflD molecule (lines) are shown,
along with the Cys31 of HflD. (b) CII–HflD–pE complex. When the above CII–HflD complex was docked with DNA, the HTH motifs of A and C chains (indicated by white circle)
of CII were unable to interact with the TTGC repeats in DNA within successive major grooves (indicated by red arrow). Instead, only the HTH motif from the B chain of CII
(indicated by white arrow) contacted DNA. In each case, minimum energy structures generated by the GRAMM docking program was used. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8a. The interface residues of both the proteins were identified
using a web-server, ProFace [41], developed for dissecting protein–
protein interfaces and deriving various physicochemical parame-
ters. In this model, HflD was found to interact with CII through
its surface containing the Cys31 residue. As a result of the interac-
tion, Cys31 of HflD was buried. This model is consistent with the
experimental results obtained in this study: (a) decrease of surface
accessibility of a cysteine residue of HflD upon interaction with CII,
(b) interaction of monomeric HflD with tetrameric CII and (c)
retention of the tetrameric nature of CII upon interaction with
HflD. From this model, potential residues that may form intermo-
lecular hydrogen bonds were also identified (Table 3). A prelimin-
ary experiment to test the model was carried out, using a mutant
in which four residues of HflD (Q28, E67, D124 and E128) were
all changed to alanine. In vivo transcription by CII decreased by
10–20% in the presence of this mutant (data not shown), while
for wild type HflD the decrease was 90% (see ‘‘Effect of HflD on
Transcription Activation by CII” section, Table 2).
In the published crystal structures of CII used for the above
docking studies, 17 residues at the disordered C-termini are miss-
ing [14]. Therefore, a possibility remains that HflD may interact
with CII at the C-terminal residues of the latter. Such an interaction
would be missed in our molecular modelling. To explore this pos-
sibility, a C-terminal deleted version of CII (CIIA) that contains res-
idues 1–82 was prepared. Binding of GST-HflD to this mutant was
carried out using GST pull-down assays. As is evident from Fig. 9,
both CII and CIIA showed comparable binding to HflD, supporting
the idea that the C-terminal end of CII was not required for CII–
HflD interaction. This was further vindicated by the results of
in vivo transcription experiments carried out with CIIA alone or
in the presence of HflD (Table 2). The b-galactosidase activity de-
creased from 52.8 to 10.2 (a reduction of 80%) in the presence
of HflD.
However, how HflD blocks the DNA binding of CII and inhibits
transcription activation at pE (both in vivo and in vitro) remains
unresolved from this model. To understand this aspect, another
docking experiment was carried out where the generated model
structure of CII–HflD complex was docked with a 27 bp DNA con-
taining the pE promoter sequence (coordinates of which were ob-
tained from the crystal structure of CII–pE complex; PDB ID: 1ZS4
[15]). The result (Fig. 8b) shows that even at the best position at
which the CII–HflD complex could be docked to DNA, the HTH mo-
tifs in the A and C chains of CII, which are involved in CII–DNA
interactions [14,15], were unable to contact the cognate CII sites
(TTGC) at the major grooves. In the CII–HflD–DNA model
(Fig. 8b), CII was able to interact with DNA only through the HTH
motif of chain B instead of the HTH motifs of A and C chains. Such
an interaction would essentially be weak and insufficient to hold
CII onto DNA tightly. Indeed, in the presence of HflD, CII–DNA
binding was inhibited (Fig. 6). This would explain the observed loss
in transcription activation (Fig. 5 and Table 2) in the presence of
HflD.
Discussion
HflD was identified at the gene level, as an additional locus in
E. coli, mutations to which increased the frequency of lysogeniza-
tion by lambda [26]. Apart from reports that it interacts with CII
and has an effect upon degradation of the latter by HflB [26], little
is known about this protein. Kihara et al. [26] had suggested that

Table 3
Probable intermolecular hydrogen bond forming residues identified from the CII–HflD
complex model structure (Fig. 8a).

Residue of CII Residue of HflD

Glu31 (Chain A) Arg78
Arg7 (Chain B) Thr152
Glu14 (Chain B) Gln28 Fig. 9. In vitro interactions between HflD–CII and HflD–CIIA by GST pull-down
Arg45 (Chain B) Glu67 assays. GST-HflD was immobilized on Glutathione Sepharose beads and purified
Arg69 (Chain C) Gln28 His6-CII or His6-CIIA was applied and incubated to allow binding. After washing,
Arg12 (Chain D) Asp124 GST-HflD was eluted from the beads and all fractions were run on 15% SDS–PAGE
Gln73 (Chain D) Glu128 and visualized by immunoblotting with polyclonal anti-CII antibody. L, W1, W2, W3
and E indicate loading, wash 1, wash 2, wash 3 and elution fractions, respectively.
 P.K. Parua et al. / Archives of Biochemistry and Biophysics 493 (2010) 175–183
HflD may exist as dimers or higher oligomers. In this work, how- Appendix A. Supplementary data
ever, we found that HflD existed predominantly as a monomer,
though a small amount of dimer was also present under our exper- Supplementary data associated with this article can be found, in
imental conditions. It was proposed that HflD sequestered CII from the online version, at doi:10.1016/j.abb.2009.10.010.
its target promoters (pE, pI and paQ) and recruited it to the proxim-
ity of HflB for rapid degradation [26]. Our results show that mono- References
meric HflD interacted with tetrameric CII which retained its
tetrameric nature after the interaction. The accessibility of the [1]
[2]
accessible cysteine residue of HflD (likely to be Cys31, as seen in [3]
the crystal structure of HflD) decreased upon interaction with CII, [4]
indicating that either HflD interacts with CII through its cysteine [5]
[6]
accessible surface or as a result of the interaction, a structural [7]
change is occurring which causes Cys31 to be buried. From the [8]
model structure of the HflD–CII complex, it was found that HflD
[9]
interacted with CII through its Cys31-accessible surface area,
[10]
explaining the decrease in the accessibility of Cys31 of HflD upon
interaction with CII. This study also shows that HflD inhibited [11]
CII-dependent transcription from pE. The down regulation of the [12]
[13]
lysogenization frequency of lambda by HflD could be a result of [14]
this inhibition, rather than by the stimulation of the in vivo prote-
olysis of CII [26]. Both the effects may work together. [15]
We also found that HflD was unable to inhibit CII-dependent [16]
transcription once CII was bound to DNA. This result contradicts [17]
the sequestering activity of HflD proposed by Kihara et al. [26]. [18]
Further, our results suggest that HflD blocked recognition of DNA
by CII due to a higher affinity of CII towards HflD than towards [19]
its cognate DNA site. A structural change in CII upon its interaction
[20]
with HflD, as indicated from fluorescence quenching (see ‘‘Quench-
ing of Tryptophan Fluorescence of CII” section), could be responsi- [21]
ble for the loss of DNA binding by CII in the presence of HflD. As our
[22]
docking results show, a CII–HflD complex would make an effective
[23]
CII–DNA interaction extremely weak. This might be the possible [24]
reason for the inhibitory role of HflD on CII-mediated transcription [25]
activation, and on its effect on lambda lysogeny. [26]
[27]
Conclusions
[28]
We find that HflD, a peripheral membrane protein, acts as a
transcription inhibitor. This is an unusual feature for an inhibitor [29]
[30]
protein that acts through a direct interaction with an activator pro-
tein (in this case, kCII). [31]
All proteins that have an effect on the lysogenization of k (such
as kCIII, HflB, HflKC and HflD) are believed to act through their ef-
fects on the stability of CII. Our results show that in addition, HflD [32]
binds to CII and prevents the binding of the latter to its cognate [33]
DNA site(s), leading to an inhibition of CII-dependent transcription. [34]
This role of HflD makes it a unique component in the k lysis–lysog- [35]
eny switch. [36]
[37]
Acknowledgments [38]
[39]
This work was funded by Institutional Project 5 (Microbial [40]
Genomics) of Bose Institute. Shrihari Sonavane (Bose Institute) [41]
helped in carrying out the molecular docking experiments. Pabitra
Parua was supported by a fellowship from CSIR, India. [42]
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