In the present study, using the extremely pure repressor of temperate Staphylococcus aureusphage /11 CI, we demonstrate that CI is composed of a-helix and b-sheet to a substantial extent
Trang 1capacity of the repressor of temperate
Staphylococcus aureus phage /11
Tridib Ganguly*, Malabika Das*, Amitava Bandhu, Palas K Chanda, Biswanath Jana, Rajkrishna Mondal and Subrata Sau
Department of Biochemistry, Bose Institute, Calcutta, India
The basic regulatory elements that most temperate
phages use for the establishment and maintenance of
their lysogeny are the phage-encoded repressor and the
cognate operator DNA [1–12] A temperate phage
generally enters into the lysogenic life cycle once
its repressor inhibits the transcription of the
phage-specific lytic genes from the early promoter by binding
to the overlapped operator DNA Repressors of the
temperate phages, although varying greatly in size and
in primary sequence level, mostly harbor a DNA
bind-ing domain and an oligomerization domain The size
and type of the operator DNAs also vary from phage
to phage Although some repressors bind to operators
with dyad symmetry [1,5–9] or operators with direct
repeats [10], other repressors bind to asymmetric oper-ators [2,3,11–13] to establish lysogeny Interestingly, the repressor of Vibrio cholerae phage CTX/ binds to extended operators, stopping lytic growth, as well as ensuring lysogeny of this phage [4] Although these regulatory elements have enriched both basic and applied molecular biology enormously, they have not been cloned from most temperate phages or character-ized in any depth
The temperate Staphylococcus aureus phage /11 [14] harbors the cI and cro genes in a divergent orientation
to that in lambdoid phages [1,8] The sequence of the immunity region of /11, however, differs significantly from those of the lambdoid phages and other
temper-Keywords
dimer; major groove; operator; phage /11;
repressor (CI)
Correspondence
S Sau, Department of Biochemistry, Bose
Institute, P1 ⁄ 12 – CIT Scheme VII M,
Calcutta 700 054, India
Fax: +91 33 2355 3886
Tel: +91 33 2569 3200
E-mail: subratasau@gmail.com
*These authors contributed equally to this
work
(Received 20 November 2008, revised 16
January 2009, accepted 21 January 2009)
doi:10.1111/j.1742-4658.2009.06924.x
The repressor protein and cognate operator DNA of any temperate Staph-ylococcus aureus phage have not been investigated in depth, despite having the potential to enrich the molecular biology of the staphylococcal system
In the present study, using the extremely pure repressor of temperate Staphylococcus aureusphage /11 (CI), we demonstrate that CI is composed
of a-helix and b-sheet to a substantial extent at room temperature, pos-sesses two domains, unfolds at temperatures above 39C and binds to two sites in the /11 cI-cro intergenic region with variable affinity The above
CI binding sites harbor two homologous 15 bp inverted repeats (O1 and O2), which are spaced 18 bp apart Several guanine bases located in and around O1 and O2 demonstrate interaction with CI, indicating that these
15 bp sites are used as operators for repressor binding CI interacted with O1 and O2 in a cooperative manner and was found to bind to operator DNA as a homodimer Interestingly, CI did not show appreciable binding
to another homologous 15 bp site (O3) that was located in the same primary immunity region as O1 and O2 Taken together, these results sug-gest that /11 CI and the /11 CI–operator complex resemble significantly those of the lambdoid phages at the structural level The mode of action of /11 CI, however, may be distinct from that of the repressor proteins of k and related phages
Abbreviations
CI, repressor of temperate Staphylococcus aureus phage /11; CTD, C-terminal domain; DMS, dimethyl sulfate; DTNB, 5,5¢-dithiobis-(2-nitrobenzoic acid); NTD, N-terminal domain.
Trang 2ate S aureus phages, such as /PVL, /13, /53, 3A, 77
and /Sa3ms [14–17] By contrast, the 239 amino acid
product of the /11 cI gene shows a moderate
homol-ogy over the entire length of the k repressor
Interest-ingly, although the sequences of the C-terminal ends of
the above S aureus phage repressors are identical, the
sequences of their N-terminal ends vary considerably
[15] The predicted secondary structures of the
repres-sors of S aureus phages show a notable similarity to
that of k repressor, especially at the C-terminal ends
As noted with the C-terminal end of k repressor [1],
the C-terminal ends of the repressors of S aureus
phages may be involved in oligomerization The
N-ter-minal half of /11 repressor carries a putative
helix-turn-helix DNA binding motif similar to that of
lambdoid phages, indicating that this half of the /11
repressor most likely participates in the binding of
operator DNA An N-terminal histidine-tagged form
of the repressor of temperate S aureus phage /11 (CI)
was overexpressed in Escherichia coli and was purified
to some extent [15] An additional 19 kDa protein
was always co-purified at a low level along with the
intact 31 kDa repressor This smaller protein, found
to comprise the N-terminal end fragment of repressor,
was most possibly the result of cleavage of the
repres-sor at its alanine–glycine site The histidine-tagged
repressor, however, was shown to form dimers in
solu-tion and bind to two sites in the /11 cI-cro intergenic
region Two homologous 15 bp inverted repeats with
partial two-fold symmetry, identified in the /11 cI-cro
intergenic region, were suggested to act as operator
sites because synthetic DNA fragments carrying either
repeat showed appreciable binding to CI [15] Little is
known about the structures of /11 CI, its cognate
operators and CI–operator complex, the precise
bind-ing affinity of CI to the two operators, and the
mecha-nism of action of CI In the present study, we report
the purification of /11 CI to near homogeneity and,
for the first time, present evidence for the two-domain
structure, its thermolability and the binding of CI to
two 15 bp operator sites in the cI-cro intergenic region
with variable affinity We also suggest putative tertiary
structures for the domains of both the CI and the
CI–operator complex
Results and Discussion
Purification, physicochemical properties and
structure of CI
To purify CI to homogeneity, we subjected affinity
column chromatography-purified CI [15] to gel
filtra-tion chromatography (for details, see Experimental
procedures), analyzed the resulting protein containing elution fractions by 13.5% SDS⁄ PAGE (Fig 1A) and found that only fractions F2 and F3 (loaded in lanes 2 and 3) contain intact CI with an estimated purity of almost 98% The overall yield of CI was approxi-mately 1 mgÆL)1 of induced E coli culture Because the above highly-purified CI did not show any degra-dation upon storage on ice for more than 1 month and possessed operator DNA binding activity (described below), it was utilized in all the in vitro experiments performed in the present study
To map the possible flexible region or domain struc-ture in CI, we performed a partial proteolysis of CI by trypsin and found that protein fragments I and II were the two major products generated from CI at a very early stage of the enzymatic cleavage (Fig 1B) Both the fragments remained mostly undigested throughout the entire period of digestion Interestingly, limited proteolysis of CI with chymotrypsin also generated a similar digestion pattern (data not shown) Neither of the above fragments interacted with anti-(his Ig) (data not shown), indicating the loss of the N-terminal histi-dine tag from CI immediately after exposure to the enzyme The first three N-terminal end amino acid residues of fragment I were determined to be LVS (corresponding to amino acid residues 156–158 of CI), suggesting that it belonged to the C-terminal end of
CI The fragment I most possibly harbors residues 156–276 of CI, with a molecular mass of 13.3 kDa The fragment II, having a molecular mass of almost 12.14 kDa (as shown by MALDI-TOF analysis), might originate from the N-terminal end of CI because the intensity of fragment I did not decrease with time The N-terminal end sequencing of one of the chymotryp-sin-digested fragments revealed that the junction region between the C-terminal end of the histidine tag and the N-terminal end of the native /11 repressor (which carries both chymotrypsin and trypsin cleavage sites) is exposed to the surface of the CI Taken together, this suggests that the histidine-tagged CI carries two flexi-ble regions: one at the N-terminal end and another almost at the middle of the molecule Tryptic digestion
of CI at the above two regions yielded two extremely folded structures or domains [designated N-terminal domain (NTD) and C-terminal domain (CTD)] of CI where the majority of the thirty four trypsin cleavage sites are buried The two-domain structure of /11 CI monomer therefore approximately resembles that of k
CI and related repressor monomers [1,8] Interestingly, the putative tertiary structure of the CTD of the /11 repressor (Fig 1C), modeled using amino acid residues 119–238 of the native /11 repressor (equivalent to residues 156–275 of fragment I), indeed showed
Trang 3remarkable structural resemblance to the LexA CTDs
(r.m.s.d = 0.46 A˚) [18] and to k CI CTD (r.m.s.d =
1.09 A˚) [19] Similarly, the NTD (Fig 1D) generated
with residues 10–69 of the native /11 repressor
exhib-ited structural similarity to a putative DNA-binding
protein from Bacteriodes fragilis (r.m.s.d = 0.06 A˚)
and to the NTD of k CI (r.m.s.d = 1.43 A˚) [20]
The CD spectrum of /11 CI showed a peak of large
negative ellipticity at 208 nm and 25 C, indicating
the presence of a-helix in CI at room temperature
(Fig 1E) Analysis of the spectrum by CD neural
networks [21] revealed approximately 23.6% a-helix and 18.5% b-sheet in CI at 25C The above CD data are as expected because the NTD and CTD of /11 CI are mostly composed of a-helix and b-sheet (Fig 1C,D) The peaks in the CD spectra of CI at
208 nm, however, were reduced substantially once the incubation temperature of CI was raised above 39C (Fig 1C) The plot of molar ellipticity at 222 nm ver-sus the incubation temperature (Fig 1C, inset) shows that the melting temperature of CI is close to 41C
At this temperature, the concentration ratio of native
1 2 3 4 5 6 7 8
30 25 20 15
30 25 20 15 10
15
10
5 0 –5
–10
–15 –20
2·dmol
25 º & 39 ºC
40 ºC
48 ºC
Temp (ºC)
Tm
25 –14
–10
Ellipticity (222 n
) –6
≈
Wavelength (nm)
Try
I II
– + + + + + + + +
41ºC
E
B
C
Fig 1 Purification and properties of /11 CI (A) The protein-containing elution fractions from different chromatographys were analyzed by 13.5% SDS ⁄ PAGE (for details, see Experimental procedures) Almost 10 lg of protein was loaded in each lane Lane 1, elution fraction from affinity chromatography; lanes 2–8, elution fractions F2 to F8 Molecular masses (kDa) of the marker protein bands are shown to the right of the gel (B) Approximately 4 lg of CI was incubated with 16 ng of trypsin (Try) at 25 C in 20 lL of buffer C and aliquots, withdrawn at the indicated time intervals, were analyzed by Tris–Tricine 15% SDS ⁄ PAGE Molecular masses (kDa) of marker proteins are shown to the right
of the gel For some unknown reason, Try-generated fragments I and II showed a 3–4 kDa higher molecular mass than their actual masses (C) Schematic tertiary structure of CTD of /11 CI The ribbons, helices and tubes represent a-helices, b-sheets and loops, respectively (D) Schematic representation of NTD of /11 CI; notation as in (C) (E) Far-UV CD-spectra of 10 l M repressor in 200 lL of buffer C, were mea-sured at temperatures in the range 25–48 C Spectra obtained at 25, 39, 40 and 48 C are shown The inset shows the plot of the molar ellipticity (h) values at 222 nm (obtained from the above CD spectra) versus the incubation temperatures of CI The melting temperature (T m )
of CI is also indicated.
Trang 4and denatured CI is 1 The data therefore suggest that
the a-helical content of CI, which decreases at
temper-atures above 39C, might be responsible for the
alter-ation of the conformalter-ation of CI, as well as the
reduced operator DNA binding affinity of CI [15] /11
CI, although structurally similar, is more
thermosensi-tive than k repressor [22] The biological significance
of this phenomenon is not known with any certainty
However, we found that the alanine and proline
con-tents in /11 CI are significantly less than that in
k repressor Several studies have demonstrated that a
higher alanine and⁄ or proline content contributes
sig-nificantly to the enhanced thermostability of various
proteins, including phage repressor [22–24]
/11 CI carries three cysteine residues at positions 125,
159 and 207 [14] To obtain clues about the status
(bur-ied versus exposed) of these cysteine residues, we
deter-mined the free sulfhydryl group content in CI by the
5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB) test and
found that the number of free thiols in CI is almost 1.5,
indicating that two cysteine residues are partially
exposed to its surface The putative surface structure of
CTD of /11 CI (data not shown) reveals that cysteine
125 and cysteine 207 are approximately 27% and 30%
surface exposed, respectively, whereas, cysteine 159 is
mostly buried The former two cysteine residues most
likely showed reactivity with DTNB Interestingly, k CI
also harbors three cysteine residues in its CTD, but none
of them are exposed to the surface [25]
Cooperative binding of CI to two sites in the /11
cI-cro intergenic region
To identify the precise location of the repressor binding
sites in the primary immunity region of /11 (Fig 2A),
we performed a DNase I footprinting experiment using
200 nm CI and radioactively labeled O DNA (Fig 2B)
The footprints of both the top and bottom strands of O
DNA reveal that two regions in O DNA became
resis-tant to digestion by DNase I in the presence of CI More
precisely, the )21 to )48 and )52 to )87 regions of
the top strand and)24 to )53 and )58 to )87 regions of
the bottom strand were protected by CI (Fig 3B) The
centers of these two sites harbor the 15 bp O1 and O2,
which are the two putative CI binding sites [15]
Previously, we reported that the binding affinity of CI
to O1 DNA is slightly higher than that to O2 DNA [15]
To determine the relative affinities of the repressor to
O1 and O2 sites more accurately, we again performed
gel shift assays using a repressor of better quality and
smaller O1 and O2 DNA fragments As expected, both
O1(Fig 2C) and O2 (Fig 2D) yielded one shifted
com-plex with increasing CI concentrations From the plot of
percent operator bound versus CI concentration (Fig 2E), the CI concentrations that gave 50% satura-tion of input O1 and O2 DNAs (i.e the apparent equi-librium dissociation constants) were calculated to be almost 32 nm and 120 nm, respectively Thus, CI binds
to O1 nearly four-fold more strongly than to O2 During the course of the present study, we identified eight additional 15 bp inverted repeats in the /11 gen-ome sequence [including one (designated O3) in the /11 cI-cro intergenic region; Figs 2A and 3B], which showed 60% or more identity with O1 The O3 site is located
31 bp upstream of O2 Surprisingly, CI was found to bind to O3 DNA (Fig 2F) at concentrations that are required for its binding to S aureus cspC DNA carrying
no operator (Fig 2G) An additional gel shift assay (Fig 2H) using labeled O DNA and higher CI concen-trations showed that CI does not bind to O3, even in the presence of O1 and O2 The data therefore indicate that binding of CI to O3 is nonspecific in nature Interest-ingly, /11 Cro that neither binds to O1 or O2 demon-strates specific binding to O3 DNA [26]
To determine whether the binding of CI to O1 and O2
is cooperative in nature, we also studied the equilibrium binding of CI to radiolabeled O1O2 DNA by a gel shift assay It was found that the O1O2 DNA formed two shifted complexes (1 and 2) with increasing CI concen-trations (Fig 2I) The complex 1 appears at 3 nm, reaching a maximum at 46 nm and starts disappear-ing at higher CI concentrations By contrast, complex 2
is barely detectable at 10 nm and starts appearing as the predominant form only when the intensity of com-plex 1 declines at more than 50 nm CI Complex 1 was estimated to contain 36% of the labeled O1O2 DNA at 46 nm CI (Fig 2J) Under these conditions, the extent of labeled O1O2 DNA that remained in free form
or was retained in complex 2 was determined to be approximately 30% Using the above data, the cooper-ativity parameter was calculated to be approximately 5 (for details, see Experimental procedures), indicating that binding of CI to O1 causes an approximately five-fold increase of the binding affinity of CI to O2, which is
18 bp away from the former operator (Fig 3B)
Only 15 bp O1 and O2 interact with CI
To confirm that the 15 bp O1 and O2 operators inter-act with CI, we performed the guanine base-specific dimethyl sulfate (DMS) protection assay in the presence⁄ absence of saturating amounts of CI and
32P-labeled O DNA (Fig 3A) Only the guanine base-specific methylation experiment was chosen because both the operators were found to carry more than one guanine base (Fig 3B) The results revealed
Trang 5cl O3
O
O1O2 O2 O3
O1
O1 O2
O2
O3
120 100 80 60 40 20 0
2 1
*
O1
O1
O2
O1
O2
0 15
0 50 100 250 500 1000 0 3 10 25 50 75 100
0 50 100 250 500 1000
0 50 100 250 500
100 125 150 175 200 300 400
30 40 50 60 70 100 125 150
[CI]
[CI]
A
B C
D
E
J G
H
(n M )
[CI] (n M )
[CI] (n M )
[CI]
(n M )
[CI]
(n M )
O1O2
100 75
1 50
25
0
0 15 30 45 60 75 90 105
CI (n M )
CI (n M )
CI (n M )
O
cspC
[CI]
Fig 2 DNA–protein interaction (A) A schematic representation of the primary immunity region of /11 (not drawn to scale) The coding regions of cI and cro genes (divergent arrows), the 15 bp O1, O2 and O3 operator sites (gray boxes) in the cI-cro intergenic region, and the different DNA fragments of the immunity region (black horizontal bars), which were utilized in the gel shift or footprint assays, are shown (B) Autoradiograms of DNase I footprints O DNA labeled (with 32 P) at the top (Top) or bottom (Bottom) strand was incubated with (+) ⁄ with-out ( )) 200 n M CI, digested with DNase I and the resulting DNA fragments were resolved through urea ⁄ 6% PAGE The guanine (G) and adenosine + guanine (A + G) markers were generated from labeled O DNA by standard methods Locations of the 15 bp O1 and O2 sites within the protected regions are indicated by solid bars (C, D, F–I) Autoradiograms of different gel shift assays Each autoradiogram repre-sents the gel shift assay with a specific 32 P-labeled DNA (noted in the left bottom corner) and the indicated amounts of CI All gel shift assays were performed three of four times and only representative data are presented The arrow and asterisk indicate the shifted complex and contaminating band, respectively (E) Using the scanned data from the autoradigrams (C, D), plots of percent operator bound versus repressor concentration were generated Curves O1 and O2 denote the equilibrium binding of CI to O1 and O2 DNA respectively (J) Coop-erative binding: the operator DNA contents in the shifted complexes 1 and 2 and in the unbound labeled O1O2 DNA were determined by scanning the intensities of all the bands shown in the autoradiogram of the gel shift assay (I) and plotted against the respective repressor concentrations Curves 1, 2 and f denote the status of O1O2 DNA concentrations in complexes 1 and 2 and in the unbound state The maxi-mum amount of bound operator in complex 1 was estimated from curve 1 The amounts of operator in complex 2 and in the unbound state
at the condition of maximum bound operator in complex 1 were determined from curves 2 and f, respectively All these values were used for calculation of the cooperativity parameter by a standard method (see Experimental procedures) All curves are best-fit curves.
Trang 6that the intensities of six bottom strand guanine bases
and five top strand guanine bases of O DNA are
decreased notably in the presence of CI The guanines
protected by CI correspond to )41G, )43G, )63G,
)67G, )74G and )76G (bottom strand) and )33G,
)35G, )46G, )56G and )68G (top strand) (Fig 3B)
All the protected guanine bases except )56G are
located in and around O1 and O2 Interestingly,
)35G, )41G and )43G in O1 and )68G, )74G and
)76G in O2 are conserved The )40G in O1 and )73G
in O2, although conserved, most likely do not interact
with the CI The data, however, confirm that 15 bp O1
and O2 DNAs are involved in the binding of CI The
intensities of some top ()53G) and bottom ()36G and
)49G) strand guanine bases were also increased
nota-bly, suggesting that these bases became more exposed
as a result of a conformational change of the operator
DNA upon CI binding The N7 group of guanine,
which is methylated by DMS, is exposed in the major
groove of the DNA helix [1] Therefore, the data also
suggest that the interaction between CI and the
opera-tor DNA may occur through the major groove of the
operator DNA helix
The absence of detectable interaction between O3
and /11 CI (as evident from both the gel shift and
footprint assays) is quite unexpected because the
pri-mary immunity regions of phages k [1], P22 [12], 434
[8], A2 [27], /g1e [28], HK022 [6] and N15 [29] bear more than two CI binding sites Lactococcal phage Tuc2009 [30] and S aureus /Sa3ms [17], however, bear two CI binding sites, similar to that of /11 in the cI-cro intergenic region Transcription of k cI mRNA from PRM, which overlaps OR2 and OR3, was shown
to be positively regulated by k CI [1] At very high concentrations, k CI binds to OR3, which in turn inhibits the expression of k cI transcripts The )35 element of promoter of /11 cI was found to partly overlap with the 15 bp O2 site (data not shown) Taken together, this suggests that the transcription of /11 cI is most possibly regulated by O2 alone and O3
is needed merely to stop the transcription of /11 cI by /11 Cro (which favors the lytic development of /11)
Binding stoichiometry
To determine the CI binding stoichiometry precisely,
we performed glutaraldehyde-mediated crosslinking experiments with CI in the presence⁄ absence of varying amounts of O1 DNA As shown in Fig 4A, dimeric
CI is the predominant form formed in the presence of O1 DNA Although the tetrameric and hexameric forms of CI (formed without O1 DNA) disappeared, a small amount of monomeric CI reappeared in the presence of O1 DNA The reason for the presence of
[CI]
+
*
*
*
*
*
*
Top Bottom
O1
O2
O2
cl
O3
O2
cro
O1
O1
+ –
5'CATTTTCTTACCTCCTTAAATTTACCTATAGTATAACCCAATTATTTTTGGTATTCA GTAAAAGAATGGAGGAATTTAAATGGATATCATATTGGGTTAATAAAAACCATAAGT
ACAAAAAAATACACGAAAAGCAAACTTTTATGTTGACTCAAGTACACGTATCGTGTAT TGTTTTTTTATGTGCTTTTCGTTTGAAAATACAACTGAGTTCATGTGCATAGCACATA
AGTAGGTTTTGTAAGCGGGAGGTGACAACATG TCATCCAAAACATTCGCCCTCCACTGTTGTAC 5'
–130 –120 –110 –100 –90
–80 –70
Fig 3 Interaction of CI with 15 bp operator DNA (A) Autoradiograms of DMS protection footprints O DNA labeled at the top (Top) or bottom (Bottom) strand was incubated with (+) ⁄ without ()) 0.25 l M CI followed by treatment of the reaction mixture with DMS as described
in the Experimental procedures Solid bars indicate the locations of O1 and O2 sites Stars and arrowheads indicate the hypermethylation sites and protected guanine bases, respectively (B) Summary of different footprinting experiments Angled lines at the top and bottom of the DNA sequence (cI-cro intergenic region) indicate the DNase I-protected regions The 15 bp O1 and O2 DNA sequences are surrounded
by a solid box, whereas O3 is surrounded by a broken box The protected guanine bases and hyper-methylated bases detected in the DMS protection experiment are denoted by vertical arrowheads and stars, respectively The start codons of CI and Cro are indicated by angled arrows The first base of the start codon of Cro was considered as +1 and the whole sequence was numbered with respect to +1.
Trang 7operator DNA slightly slowing down the migration of
dimeric CI remains unclear at present
To confirm that dimeric repressor binds to a single
operator, we carried out gel shift assays under
condi-tions (i.e using very high CI and O1 DNA
concentra-tions) that strongly favor the formation of the CI–O1
complex (Fig 4B) The corresponding plot of CI
bind-ing to O1 DNA, as obtained from quantitation of the
gel shift data, is also shown (Fig 4C) It is apparent
that the binding stoichiometry is approximately two
CI monomers per O1 DNA Taken together, the data
suggest that, similar to k CI and Cro [8] /11, CI binds
to 15 bp operator DNA as a homodimer
The CTDs of k CI [1] and LexA [18] (i.e the
struc-tural homologs of /11 CTD) are involved in the
homodimerization of these repressors Sequence
align-ment of /11 CI and LexA revealed that several
resi-dues involved in the dimerization of LexA CTD were
also present in the CTD of /11 CI (data not shown)
The CTDs of two /11 CI monomers may therefore be
responsible for the formation of a dimeric /11 CI [15]
By contrast, the NTD of /11 CI, which harbors a
potential helix-turn-helix DNA binding motif, could
participate in the binding of the dimeric /11 CI to the
major groove of operator DNA helix (Fig 3A) The average size of each DNase I-protected region of oper-ator DNA was found to be approximately 25–27 bp (Fig 2B), suggesting the involvement of at least two adjacent (full) turns of DNA helix in the interaction with /11 CI Thus, two NTDs of dimeric /11 repres-sor may attain a specific conformation in space for easing the interaction of its two HTH motifs to two adjacent major grooves located on the same face of operator DNA helix (Fig 4D) The )33G, )35G, )41G and )43G bases of O1 possibly contact CI from the front, whereas )46G may contact from the back of helix The way that NTD contacts with )46G and other bases on the back of the DNA helix remains unclear at present
Conclusions
The present study provides valuable insights into the basic structures of /11 CI, its cognate operators and the /11 CI–operator complex, and these are found to
be quite similar to those in k and related phage systems Despite structural relatedness, the mechanism
of action of /11 CI does not completely resemble that
GCHO
D
C
O1
O1
CTD CTD
NTD NTD
[CI]
0 0 4 20 40 M 200 150
120
100
70
50
40
30
120
100
80
60
40
20
0
0 1
[CI]/[O1 DNA]
2 3
60
85
kb 0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 1.0 2.0 +
+ + + –
Fig 4 Binding stoichiometry (A) 10% SDS ⁄ PAGE analysis of glutaraldehyde (GCHO) treated CI 4 l M CI was incubated with the indicated amount of O1 DNA prior to treatment with (+) ⁄ without ( )) GCHO Protein marker bands and their respective molecular masses (kDa) are shown to the right of the gel (B) Autoradiogram of the gel shift assay shows the binding of varying concentrations (0.2–2 l M ) of CI to a fixed amount of O1 DNA mix ( 0.1 n M32P-labeled O1 DNA plus 0.4 l M cold O1 DNA) Using the scanned data from the autoradiogram,
a plot of percent O1 bound versus CI concentration was generated (C) (D) The schematic model structure of the CI–O1 DNA complex, developed as based on our present experimental data, reveals that two NTDs (light gray balls) of dimeric CI are pointed towards two adja-cent major grooves of O1 DNA located on the same face of DNA helix CI monomers in dimeric CI contact each other through their CTDs (dark gray balls) The G bases that interact with the NTDs of dimeric CI are circled.
Trang 8of the repressor proteins of the lambdoid phages.
Although k CI requires three operators to regulate the
expression of genes flanking the k cI-cro intergenic
region, /11 CI possibly requires two operators to
regu-late the transcription of genes located on the two sides
of the /11 cI-cro intergenic region Furthermore, the
information gathered in the present study may prove
useful in the construction of S aureus-based expression
vectors that could be induced by a physical inducer
such as temperature
Experimental procedures
Bacterial and phage strains and plasmids
Madison, WI, USA) cells were routinely grown in
Trypti-case soy broth [32] and LB [33], respectively Growth media
were supplemented with appropriate antibiotics if required
The temperate phage /11 and its growth conditions have
been described previously [32] The construction of plasmid
pSAU1201 and pSAU1220 was also described previously
[15] The 269 bp /11 DNA insert in pSAU1201 carrying
the /11 cI-cro intergenic region was designated as O DNA
Plasmid pSAU1220 was utilized for overexpression of /11
CI in E coli
Molecular biological techniques
Plasmid DNA isolation, DNA estimation, digestion of
DNA by restriction enzymes, modification of DNA
frag-ments by modifying enzymes, PCR, purification of DNA
fragments, labeling of DNA fragments with radioactive
materials and agarose gel electrophoresis were carried out
following standard procedures [33] or according to the
(Qiagen, Hilden, Germany; Fermentas GmbH, St
Leon-Rot, Germany; Bangalore Genei P Ltd., Bangalore, India)
polyacrylamide gel and western blotting were performed as
described previously [13,34] DNA from /11 phage particles
was isolated as described previously [32] Sequencing of all
/11 DNA inserts (amplified by PCR) were performed at
the DNA sequencing facility at the University of Delhi,
South Campus (Delhi, India) Sequencing of the N-terminal
ends of all protein fragments was performed using a protein
sequencer (Applied Biosystems, Foster City, CA, USA)
according to the manufacturer’s protocol
Overexpression and purification of /11 repressor
(pSAU1220) and purified by Ni-NTA column
chromatogra-phy, as described previously [15] To further purify the /11 repressor, we loaded almost 2.8 mg of repressor (derived from the above affinity chromatography) onto a 40 mL Sephadex G-50 column (diameter 1.5 cm) pre-equilibrated with buffer C [10 mm Tris–Cl¢ (pH 8.0), 200 mm NaCl,
1 mm EDTA, 5% glycerol] Repressors were eluted at a
frac-tions (marked F1 to F20) were collected and protein esti-mation revealed that only fractions F2 to F8 contained protein Because fractions F2 and F3 contained mainly intact repressor (discussed below), we stored these fractions
on ice until use The concentration of CI was calculated using the molecular mass of monomeric CI
Biochemical and biophysical analysis of /11 repressor
Glutaraldehyde-mediated crosslinking, partial proteolysis and recording of CD spectrum of the repressor were car-ried out as described previously [13,15] Using the molar extinction coefficients for 5-thio-2-nitrobenzoic acid at
sulf-hydryl (-SH) groups in CI in buffer C was determined by DTNB according to a standard procedure [35] MALDI-TOF analysis of protein fragments was carried out using
protocol
Homology modeling Amino acid residues 1–118 and 119–239 of native /11 CI were used to develop 3D model structures of the NTD and CTD of this protein by the First Approach Mode of
structure of E coli LexA CTD (Protein Databank code: 1jhc) was utilized as a template for developing the model structure of the CTD of /11 CI, the X-ray structure of a putative DNA binding protein (Protein Databank code: 3bs3) of Bacteroides fragilis was used as a template for generating the model structure of NTD of /11 CI Using the coordinates of the resulting model structures, molecu-lar visualization, superimposition of the structures, surface structure determination and drawing of Ramachandran plots were carried out by the swiss-pdb viewer (http:// ExPasy.org)
Gel shift assay
was investigated by the standard gel shift assay, as described previously [15] The 154 bp O1O2 DNA fragment
Trang 9(Fig 2A) was synthesized by PCR using pSAU1201 DNA
as a template and primers IIa and pHC1 (Table 1)
Similarly, 90 bp O3 (a third putative operator in the /11
cI-cro intergenic region) DNA was amplified using primers
PCI15 and IIId and pSAU1201 DNA On the other hand,
214 bp cspC DNA was amplified using primers CSP4 and
CSP6 and the chromosomal DNA of S aureus Newman as
a template [36] All three DNA fragments were purified
from agarose gel using the QIAquick Gel Extraction Kit
(Qiagen) The 34 bp O1, and 49 bp O2 DNAs (Fig 2A)
were prepared by mixing and annealing primers PCR11
and PCR21 and IIa and IIb, respectively (Table 1) The
cooperativity parameter for the binding of CI to O1O2
DNA was determined from the scanned data of the
autora-diogram (Fig 2I) according to Monini et al [37] To study
CI binding stoichiometry, a gel shift assay was performed
using essentially the same method (see above), except that
P-labeled O1 DNA The CI preparation used in
the binding stoichiometry experiment was considered to
have 100% activity
DNase I footprinting
footprinting assays, were prepared by standard end labeling
procedures [33] Briefly, to label the bottom strand of O
Finally, the bottom strand labeled O DNA was purified
from an agarose gel To label the top strand of O DNA
amplifi-cation of O DNA by Taq polymerase using pSAU1201 DNA or /11 DNA as a template and the oligonucleotides pHC1 and labeled pHC2 as primers The resulting DNA fragment was purified from an agarose gel
DNase I footprinting was performed according to a stan-dard procedure [5] with some modifications Briefly, 60 nm labeled DNA fragment ( 5000 c.p.m.) was incubated with varying concentrations of CI in 50 lL buffer C for 20 min
and treated with 0.15 units of DNase I for 4 min at room temperature followed by termination of the reactions by the addition of 90 lL of Stop solution [200 mm NaCl, 80 mm EDTA (pH 8.0), 1% SDS, 0.03% glycogen] Cleaved DNA fragments, prepared by sequential passage of each reaction mixture through phenol–chloroform (1 : 1) extraction and ethanol precipitation steps, were resuspended in sequencing gel buffer [98% deionized formamide, 10 mm EDTA (pH 8.0), 0.025% bromophenol blue] Each labeled DNA was treated with DNase I identically in the absence of CI and the recovered DNA fragments were used as controls Finally, both experimental and control DNA fragments
from the identically labeled DNA fragments by standard procedures [38]
DMS protection assay The DMS protection assay was performed as described pre-viously [39] Briefly, 0.5 lm repressor was incubated with
buffer C for 20 min at room temperature followed by the treatment of repressor–operator complexes with 0.2% DMS for 2 min at room temperature After termination of the reaction with DMS stop solution [1.5 m sodium acetate (pH 7.0), 1 m beta-mercaptoethanol], DNA was recovered
by successive passage of the reaction mixture through phe-nol–chloroform (1 : 1) extraction and ethanol precipitation steps in the presence of glycogen The same labeled O DNA was also treated directly with DMS as above in the absence
of CI and the recovered DNA was used as a control The gunaine-specific ladder DNAs, prepared from both control and experimental DNAs by a standard procedure [38], were
Acknowledgements
This work was supported by the financial assistance from the Department of Atomic Energy (Government
of India, Mumbai, India) to S Sau The authors thank Drs P Parrack, R Chattopadhyaya and N C Mandal for critically reading, correcting and modifying the manuscript The authors are extremely grateful to
Table 1 Details of the oligonucleotides used.
Name Sequence (5¢- to 3¢) Purpose
pHC1 GGATCCTAAATCTTCTTGAGTAC Synthesis of O and
O1O2 DNAs pHC2 GAATTCTTGGTTCTATAGTATCTG Synthesis of O DNA
PCR11 GACTCAAGTACACGTATCGTGTATA
GTAGGTTTA
Synthesis of O1DNA
PCR21 AAACCTACTATACACGATACGTGTA
CTTGAGTCA
Synthesis of O1 DNA IIa ATTCAACAAAAAAATACACGAAAAG
CAAACTTTTATGTTGACTCAAGTA
Synthesis of O2 and O1O2 DNAs IIb TACTTGAGTCAACATAAAAGTTTGC
TTTTCGTGTATTTTTTTGTTGAAT
Synthesis of O2 DNA PCI51 GAATTCTCGCTAATTCTTTTTTATC Synthesis of O3 DNA
IIId TTTTTTTGTTGAATACCAAAAATAA
TTGGGTTATACTATAG
Synthesis of O3 DNA CSP4 CATGCCATGGATGAATAACGGTACAG Synthesis of S aureus
cspC DNA CSP6 CTCGAGCATTTTAACTACGTTTG Synthesis of S aureus
cspC DNA
Trang 10Dr C Y Lee (UAMS, Little Rock, AR, USA) for
providing plasmids and strains used in the study
The authors would like to thank Mr A Banerjee,
Mr A Poddar, Mr J Guin and Mr M Das for their
excellent technical help Mr Tridib Ganguly, Ms
Mal-abika Das and Mr Amitava Bandhu received Senior
Research fellowships from the Council of Scientific
and Industrial Research (Government of India, New
Delhi) Mr Palas K Chanda is a recipient of the
Senior Research fellowship of Bose Institute Mr
Bisw-anath Jana received a Junior Research fellowship from
the Department of Biotechnology (Government of
India, New Delhi)
References
1 Gussin GN, Johnson AD, Pabo CO & Sauer RT (1983)
Repressor and Cro Protein: Structure, Function, and
Role in Lysogenization In Repressor and Cro Protein:
Structure, Function, and Role in Lysogenization Lambda
RA, eds), pp 93–121 Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY
2 Hatfull GF (2000) Molecular Genetics of Mycobacteria
ASM Press, Washington, DC
3 Heinrich J, Velleman M & Schuster H (1995) The
tri-partite immunity system of phages P1 and P7 FEMS
Microbiol Rev 17, 121–126
4 Kimsey HH & Waldor MK (2004) The CTX phi
repres-sor RstR binds DNA cooperatively to form tetrameric
repressor-operator complexes J Biol Chem 279, 2640–
2647
5 Koudelka AP, Hufnagel LA & Koudelka GB (2004)
Purification and characterization of the repressor of the
shiga toxin-encoding bacteriophage 933W: DNA
bind-ing, gene regulation, and autocleavage J Bacteriol 186,
7659–7669
6 Carlson NG & Little JW (1993) Highly cooperative
DNA binding by the coliphage HK022 repressor J Mol
Biol 230, 1108–1130
7 Ogawa T & Ogawa H (1988) Organization of the early
region of bacteriophage 80 J Mol Biol 202, 537–550
8 Ptashne M (ed.) (1992) A Genetic Switch Gene Control
Black-well Scientific Publications, Cambridge, MA
9 Dodd IB, Shearwin KE & Egan JB (2005) Revisited
gene regulation in bacteriophage Lambda Curr Opin
Genet Dev 15, 145–152
10 Rousseau P, Betermier M, Chandler M & Alazard R
(1996) Interactions between the repressor and the early
operator region of bacteriophage Mu J Biol Chem 271,
9739–9745
11 van Kaer L, van Montagu M & Dhaese P (1989)
Purifi-cation and in vitro DNA-binding specificity of the
264, 14784–14791
12 Susskind MM & Youderian P (1983) Bacteriophage P22 Antirepressor and its control In Bacteriophage P22
Roberts JW, Stahl FW & Weisberg RA, eds), pp 93–121 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
13 Ganguly T, Bandhu A, Chattoraj P, Chanda PK, Das
M, Mandal NC & Sau S (2007) Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity Virol J 4, 64
14 Iandolo JJ, Worrell V, Groicher KH, Qian Y, Tian R, Kenton S, Dorman AH, Ji H, Lin S, Loh P et al (2002) Comparative analysis of the genomes of the temperate bacteriophages phi 11, phi 12 and phi 13 of
15 Das M, Ganguly T, Chattoraj P, Chanda PK, Bandhu
A, Lee CY & Sau S (2007) Purification and character-ization of repressor of temperate S aureus phage phi11
J Biochem Mol Biol 40, 740–748
16 Kaneko J, Kimura T, Narita S, Tomita T & Kamio Y (1998) Complete nucleotide sequence and molecular characterization of the temperate staphylococcal bacte-riophage phiPVL carrying Panton-Valentine leukocidin genes Gene 215, 57–67
17 Sumby P & Waldor MK (2003) Transcription of the toxin genes present within the Staphylococcal phage phiSa3ms is intimately linked with the phage’s life cycle
J Bacteriol 185, 6841–6851
18 Luo Y, Pfuetzner RA, Mosimann S, Paetzel M, Frey
EA, Cherney M, Kim B, Little JW & Strynadka NC (2001) Crystal structure of LexA: a conformational switch for regulation of self-cleavage Cell 106, 585–594
19 Bell CE, Frescura P, Hochschild A & Lewis M (2000) Crystal structure of the lambda repressor C-terminal domain provides a model for cooperative operator binding Cell 101, 801–811
20 Stayrook S, Jaru-Ampornpan P, Ni J, Hochschild A & Lewis M (2008) Crystal structure of the lambda repres-sor and a model for pairwise cooperative operator bind-ing Nature 452, 1022–1025
21 Bohm G, Muhr R & Jaenicke R (1992) Quantitative analysis of protein far UV circular dichroism spectra by neural networks Protein Eng 5, 191–195
22 Hecht MH, Sturtevant JM & Sauer RT (1984) Effect of single amino acid replacements on the thermal stability
of the NH2-terminal domain of phage lambda repres-sor Proc Natl Acad Sci USA 81, 5685–5689
23 Stearman RS, Frankel AD, Freire E, Liu BS & Pabo
CO (1988) Combining thermostable mutations increases the stability of lambda repressor Biochemistry 27, 7571–7574