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Journal of Bacteriology, February 2004, p . 870-874, Vol . 186,
No . 3
DNA
Binding by the Meningococcal RdgC Protein, Associated with Pilin Antigenic
Variation
Timothy Moore, Gary J . Sharples,
and Robert G . Lloyd*
Institute of Genetics, Queen's Medical Centre, University of Nottingham,
Nottingham NG7 2UH, United Kingdom
Received 29 July 2003/ Accepted 22 October 2003
The RdgC protein of Neisseria gonorrhoeae is required for efficient
pilin antigenic variation, although its precise role has yet to
be established . We demonstrate that the nearly identical RdgC from
Neisseria meningitidis binds DNA with little specificity for
sequence or structure, like the Escherichia coli protein . We
also show that neither protein is able to constrain torsional tension
in relaxed DNA . These data exclude several possible roles for RdgC in
pilin antigenic variation and suggest that RdgC performs a similar
function in both E . coli and the Neisseria spp .
Orthologues of the rdgC gene are found only in the beta and
gamma subdivisions of the Proteobacteria, including Escherichia
coli and the obligate human pathogens Neisseria gonorrhoeae
and Neisseria meningitidis . The E . coli RdgC gene encodes a
DNA binding protein of 34 kDa (14) . Deletion of this
alone causes no obvious phenotype but is highly deleterious in
strains lacking certain enzymes involved in recombination and
replication restart (14, 18) .
The explanation(s) for these effects is uncertain but could imply
that RdgC aids replication processivity .
In N . gonorrhoeae (the gonococcus) rdgC is required for
efficient pilin antigenic variation and plays some role in cell
growth (12) . Pilin antigenic variation allows
N . gonorrhoeae and N . meningitidis (the meningococcus) to
alter the sequence of the main structural component of the type IV
pilus, PilE (5, 15) . It has been
proposed that the expression of variant type IV pili through pilin
antigenic variation promotes adhesion to different tissue types (9,
17) as well as contributing to evasion of the host
immune response (2) . Unfortunately, the molecular
mechanisms and enzymology underlying pilin antigenic variation are
poorly understood (7) . It is known that sequence from one
of numerous silent loci (pilS) is copied to the expression locus,
pilE (5) . Several conserved sequences present in
pilS and pilE are important, including the coding
cys1 and cys2 elements and the Sma/Cla repeat located in
the 3' untranslated region (6, 8,
24) . In terms of proteins involved, genetic studies have
shown a strong dependence on recA as well as the recO,
recQ, and recJ genes . Hence, pilin antigenic variation
appears to utilize a RecF-like pathway for recombination, suggesting
functions targeted at DNA single-strand gaps or at replication forks
(10, 13, 19) .
Recently identified protein RecX also participates in these reactions
and is likely to regulate RecA activity (20,
22) . As noted, RdgC is also important, but its role is
unknown (12) .
A current working hypothesis for pilin antigenic variation utilizes
the common occurrence of circular DNAs, including hybrid pilE-pilS
molecules, in the gonococcus (1, 7) .
Recombination directed by pilin-antigenic-variation-specific factors,
independent of the RecFOR pathway, initiates an exchange between
pilE and pilS loci on one chromosome . Resolution of the
junction(s) formed creates a circular molecule with a hybrid pilE-pilS
locus . This intermediate is then utilized in a second,
RecFOR-dependent, recombination reaction with the pilE locus
on an intact chromosome .
We report here that purified meningococcal RdgC binds DNA in a
sequence- and structure-independent manner and does not introduce
torsional tension in DNA, arguing against a structural role in pilin
antigenic variation .
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Purification of meningococcal RdgC .
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The meningococcal rdgC gene was amplified from strain B16B6
with primers introducing restriction sites (5'-ACAGGAAACCATATGTGGTTCAAGC-3'
and 5'-ATTGGATCCTGGCTGACGGTATAAA-3'; NdeI and BamHI
sites underlined) . These sites were used to insert the product into
pT7-7, yielding pDIM008 . Nucleotide sequencing of rdgC
revealed that the predicted protein sequence differed from that of
the protein of the sequenced serogroup B strain, MC58, by a single
substitution (T288I) but was identical to that of the protein of the
serogroup A strain Z2491 . Compared to gonococcal RdgC there are two
changes (T288I and R231Q); neither is highly conserved (Fig.
1A) . The biochemical activities of meningococcal
and gonococcal RdgC proteins are likely to be identical, and
therefore the proteins are likely to perform the same function in
both Neisseria species .
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FIG . 1 . Production of meningococcal RdgC . (A) Alignment of RdgC C
termini highlighting the two amino acid substitutions between gonococcus
and meningococcus (asterisks) . The position in each protein relative to
the first residue is indicated at the start of each sequence . Residues
identical or functionally similar between the proteins are shaded .
Eco, E . coli; Hin, Haemophilus influenzae;
Pae, Pseudomonas aeruginosa; Ngo, N . gonorrhoeae;
Nme, N . meningitidis B16B6 . (B) Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis analysis involving Coomassie
blue staining summarizing purification of meningococcal RdgC . Lane i,
molecular weight markers; lane ii, crude cell lysate; lane iii, pooled
50 to 80% (NH4)2SO4 cut; lane iv,
pooled fractions from heparin column; lane v, pooled fractions from Q
Sepharose column . The RdgC band is indicated . (C) Gel filtration of
purified meningococcal RdgC protein . Elution profiles of molecular
weight standards (upper profile) and meningococcal RdgC (lower profile)
are shown . The molecular mass of each standard is indicated next to its
peak; interpolation between the elution points of these was used to
estimate the mass of RdgC.
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Meningococcal RdgC was overexpressed in a
 rdgC::Tmr
(14) derivative of E . coli B strain
BL21(DE3) (DIM026) carrying pLysS and pDIM008, following addition of
IPTG (isopropyl-ß-D-thiogalactopyranoside) .
Induced cells were lysed on ice by sonication in buffer A (50 mM
Tris-HCl [pH 7.5], 1 mM EDTA, 1 mM dithiothreitol [DTT]) before the
addition of NaCl to 1 M . RdgC was precipitated from the cleared
lysate with a 50 to 80% ammonium sulfate cut, resuspended in buffer A
plus 0.1 M NaCl, applied to a 10-ml heparin-Sepharose 6 column, and
eluted on a linear gradient of buffer A plus 0.1 to 2 M NaCl .
Fractions eluting between 1.0 and 1.5 M NaCl were pooled, dialyzed
into buffer B (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1 mM DTT) plus 0.1
M NaCl, and applied to a 4-ml Q-Sepharose fast-flow column, and bound
proteins were eluted with a linear gradient of buffer B plus 0.2 to 1
M NaCl . RdgC eluted at approximately 0.5 M NaCl . Peak fractions were
pooled, dialyzed into buffer A plus 0.2 M NaCl and 50% glycerol, and
stored as aliquots at -80°C . The protein concentration was estimated
by a modified Bradford assay (Bio-Rad) with bovine serum albumin as
the standard .
Gel filtration on a Superose-12 column with buffer A plus 0.1 M
NaCl revealed that, like E . coli RdgC, meningococcal RdgC is a
dimer in solution (Fig . 1C) . We have therefore expressed
concentrations of the protein as moles of dimer .
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Meningococcal RdgC binds to DNA .
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Using band shift assays performed as previously described (14)
we tested whether meningococcal RdgC binds DNA (Fig . 2) .
Details of the substrates used are given in Table 1 .
Stable complexes were formed with linear single-stranded DNA (ssDNA)
and double-stranded DNA (dsDNA) and with a variety of branched
molecules designed to mimic intermediates in recombination and
replication . The affinities for these substrates appear broadly
similar, suggesting that meningococcal RdgC, like E . coli RdgC
(14), does not target branch points in DNA (Fig.
2) . With the 50-nucleotide ssDNA only a single
stable complex was formed (Fig . 2A), whereas with
dsDNA, tailed duplexes, and the hairpin, two stable complexes were
formed (Fig . 2B to E) . With the branched fork and junction
structures, three or more complexes were formed (Fig . 2F to
I) . The differences can be explained in terms of the number
of arms available for protein binding, or potentially binding
to different numbers of DNA ends . However, since meningococcal RdgC
is also able to bind to circular plasmid DNA (see below), the latter
possibility is unlikely .
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FIG . 2 . Band shift assays showing binding of meningococcal RdgC to
different DNA substrates . Binding reaction mixtures contained 0.1 nM DNA
and increasing RdgC concentrations: 0, 0.5, 5, 50, and 500 nM . The
substrates are illustrated above each panel . Half arrows, 3' ends . (A)
Fifty-nucleotide ssDNA; (B) 50-bp dsDNA; (C) 5'-tailed duplex; (D)
3'-tailed duplex; (E) hairpin; (F) replication fork with leading strand;
(G) replication fork with lagging strand; (H) replication fork with both
strands; (I) Holliday junction . Details of the substrates used are given
in Table 1.
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| TABLE 1 . Substrates used in DNA binding assaysa
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Binding studies with 25-nucleotide ssDNA and a 25-bp duplex revealed
that, like E . coli RdgC, meningococcal RdgC has a preference
for binding dsDNA over ssDNA (Fig . 3) . However, the difference
in affinity for ss- versus dsDNA on these substrates was less
obvious with than with the E . coli protein . We also note that
the affinity of meningococcal RdgC for both substrates was slightly
higher than that of E . coli RdgC under the conditions used .
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FIG . 3 . Comparison of ss- and dsDNA binding by meningococcal (diamonds)
and E . coli (triangles) RdgC (14) . Binding
curves for RdgC with 25-nucleotide ssDNA (open symbols) and 25-bp dsDNA
(shaded symbols) are shown . A 1 nM DNA substrate was used in each
reaction, with the indicated concentration of RdgC . Data are means of
two experiments.
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We failed to observe substantial differences in affinities for
substrates of different nucleotide sequences (Fig . 2 and
3; data not shown), suggesting that meningococcal
RdgC does not target specific sequences in DNA . In addition, multiple
rounds of selective enrichment for dsDNA sequences preferentially
bound by meningococcal RdgC according to the procedure of Pollock
and Treisman (16) did not generate any enhanced
affinity for the duplex DNA (data not shown) .
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Effect of RdgC on topology of DNA .
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The binding of proteins to DNA can cause distortion at the binding
site, increasing or decreasing the DNA twist . As a consequence,
positive or negative torsional tension is constrained . To probe the
ability of RdgC to twist DNA, pUC19 was relaxed by topoisomerase in
the absence of RdgC and then the relaxation was continued after RdgC
addition . First, a pool of pUC19 was relaxed with vaccinia virus
topoisomerase I (Sigma; 5 U/17.5 ng of pUC19) in VTB1 (50 mM Tris-HCl
[pH 8.0], 100 µg of bovine serum albumin/ml, 2 mM MgCl2, 1
mM DTT) for 3 h at 30°C . One aliquot of the relaxed DNA was
deproteinized by standard phenol-chloroform-indoleacetic acid
(25:24:1) treatment . The remainder of the relaxed DNA was aliquoted
to tubes containing additional topoisomerase (10 U/17.5 ng of pUC19)
and various concentrations of protein . These were incubated for a
further 3 h at 30°C . After secondary incubation, samples were
deproteinized as before . Plasmids were then resolved by
electrophoresis in a 0.9% agarose gel with Tris-borate-EDTA (TBE)
buffer (Bio-Rad; Mini Sub Cell GT; 7 by 10 cm) for 2 h at 45 V . DNA
was visualized under UV light, after being stained in TBE with SYBR
gold .
Both meningococcal and E . coli RdgC induced at most one extra
turn in the plasmid DNA before inhibiting topoisomerase activity
(Fig . 4A and B, compare lanes with 0 and 0.8 µM RdgC) .
We assume that the inhibition of topoisomerase activity by 1.6
µM RdgC correlates with high occupation (75 to 100%) of pUC19 DNA by
the protein . Thus, at 0.8 µM RdgC, a significant proportion of the
plasmid must have been coated, suggesting that each RdgC molecule
introduced a small degree of twist . Additional assays involving
resolution in gels containing chloroquine revealed that a positive
turn had been introduced (data not shown), indicating that RdgC
"overwinds" DNA . In contrast, a plasmid relaxed in the presence of
HU, which is known to constrain negative supercoils in DNA (3),
gained approximately five negative turns prior to inhibition of
topoisomerase activity (Fig . 4C, compare lanes
containing 0 and 0.2 µM HU; data not shown) . The results suggest that
the ability of RdgC to twist DNA was about five times less potent
than that of HU, which has previously been estimated at 1 turn per 12
or 13 dimers bound (23) . We conclude that RdgC
does not introduce considerable torsional tension into DNA upon
binding and is therefore unlikely to constrain supercoils to a
significant degree in vivo .
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FIG . 4 . Topoisomerase-mediated relaxation of plasmid DNA bound by RdgC .
Shown is relaxation in the presence of meningococcal RdgC (A), E .
coli RdgC (14) (B), and E . coli HU (C) .
For all reactions the pUC19 DNA concentration was 1 nM; U, untreated
DNA; Rel, DNA from relaxed pool . Concentrations of protein added to the
reaction after initial relaxation are indicated . Arrows indicate
increasing supercoiling of plasmid DNA.
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The data presented suggest that meningococcus RdgC does not target
specific sequences or structures in DNA to promote pilin antigenic
variation . It also introduces little twist in DNA, arguing against an
architectural role similar the kind seen in many site-specific
recombination reactions, although the possibility that it bends DNA
without inducing torsional tension cannot be excluded .
Given the similarities between meningococcal and E . coli RdgC
proteins now established, it is reasonable to envisage that
they perform similar functions in both species . Since E . coli
lacks an equivalent of pilin antigenic variation, this also argues
against a role unique to that system . An association between RdgC and
RecFOR recombination proteins in E . coli was uncovered by
Moore et al . (14) . In this case, the recFOR genes
are deleterious in the absence of rdgC in a strain which also
carries priA and dnaC212 mutations . This suggests that RdgC
acts to limit a toxic effect of the RecFOR complex, perhaps
during reinitiation of a stalled replication fork . Since multiple
RecF pathway products participate in pilin antigenic variation, a
role for the DNA binding activity of RdgC in these processes is
likely . The gonococcal RecX gene, which regulates RecA activity, is
also required for these reactions (21, 22),
indicating that control of recombinational exchanges involving the
loading and unloading of RecA, RecFOR, and SSB is a critical feature
for generating the recombinants that ultimately lead to pilin
antigenic variation .
We thank Carol Brown and Lynda Harris for excellent technical
support . We also thank Qin Wen for donating junction substrates, Tom
Baldwin for supplying N . meningitidis chromosomal DNA, and
Peter McGlynn for the HU protein .
The work was funded by the Medical Research Council .
* Corresponding author . Mailing address: Institute of Genetics,
University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United
Kingdom . Phone: 44 (0)115 9709406 . Fax: 44 (0)115 9709906 . E-mail: bob.lloyd@nottingham.ac.uk.
Present address: Centre for Infectious Diseases, Wolfson Research
Institute, University of Durham, Stockton-on-Tees TS15 6BH, United
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