The Yersinia enterocolitica pYV virulence plasmid contains multiple intrinsic DNA bends which melt at 37 degrees C.
ABSTRACT Temperature has a pleiotropic effect on Yersinia enterocolitica gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (</=30 degrees C) and virulence plasmid-encoded Yop secretion (37 degrees C). The mechanism by which temperature exerts this change in genetic programming is unclear; however, altered gene expression by temperature-dependent changes in DNA topology has been implicated. Here, we present evidence that the Y. enterocolitica virulence plasmid, pYV, undergoes a conformational transition between 30 and 37 degrees C. Using a simplified two-dimensional, single-gel assay, we show that pYV contains multiple regions of intrinsic curvature, including virF, the positive activator of virulence genes. These bends are detectable at 30 degrees C but melt at 37 degrees C, the temperature at which the cells undergo phenotypic switching. We also show that pACYC184, a plasmid used as a reporter of temperature-induced changes in DNA supercoiling, has a single region of intrinsic bending detected by our assay. Topoisomers of pACYC184, with and without this bend, isolated from Y. enterocolitica were resolved by using chloroquine gels. The single bend has a dramatic influence on temperature-dependent DNA supercoiling. These data suggest that the Y. enterocolitica pYV plasmid may undergo a conformational change at the host temperature due to melting of DNA bends followed by compensatory adjustments in superhelical density. Hence, changes in DNA topology may be the temperature-sensing mechanism for virulence gene expression in Y. enterocolitica and other enteric pathogens.
- SourceAvailable from: Zachary Bent[Show abstract] [Hide abstract]
ABSTRACT: Yersinia enterocolitica biovar 1B maintains two type III secretion systems (T3SS) that are involved in pathogenesis, the plasmid encoded Ysc T3SS and the chromosomally encoded Ysa T3SS. In vitro, the Ysa T3SS has been shown to be expressed only at 26°C in a high-nutrient medium containing an exceptionally high concentration of salt - an artificial condition that provides no clear insight on the nature of signal that Y. enterocolitica responds to in a host. However, previous research has indicated that the Ysa system plays a role in the colonization of gastrointestinal tissues of mice. In this study, a series of Ysa promoter fusions to green fluorescent protein gene (gfp) were created to analyze the expression of this T3SS during infection. Using reporter strains, infections were carried out in vitro using HeLa cells and in vivo using the mouse model of yersiniosis. Expression of green fluorescent protein (GFP) was measured from the promoters of yspP (encoding a secreted effector protein) and orf6 (encoding a structural component of the T3SS apparatus) in vitro and in vivo. During the infection of HeLa cells GFP intensity was measured by fluorescence microscopy, while during murine infections GFP expression in tissues was measured by flow cytometry. These approaches, combined with quantification of yspP mRNA transcripts by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), demonstrate that the Ysa system is expressed in vitro in a contact-dependent manner, and is expressed in vivo during infection of mice.MicrobiologyOpen. 10/2013;
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ABSTRACT: Pathogenic Yersinia species employ the Ysc-Yop type III secretion system (T3SS) encoded by a highly conserved pYV virulence plasmid to export the virulence effectors into host cells. The Ysc-Yop T3SS is tightly regulated by multiple contributing proteins that function at different levels. However, systematic transcriptional regulation analysis of Ysc-Yop T3SS is lacking and the detailed mechanism under this regulation process is still elusive. Aimed at systematically characterizing transcriptional regulations of all T3SS genes in Y. pseudotuberculosis, we amplified 97 non-coding fragments from the pYV plasmid and analyzed transcriptional responses of the T3SS genes under different growth conditions. Transcriptions of T3SS genes were induced at 37°C and genes encoding T3SS effectors were highly induced by further depletion of Ca2+. The temperature induced gene transcription process is mediated by modules encoded on the chromosome, while the Ca2+ depletion-induced process is controlled by the positive regulatory protein LcrF as well as the negative regulatory protein LcrQ. In this process, LcrQ shares the same targets with LcrF and the effect of LcrQ is dependent on the presence of LcrF. Furthermore, over-expression of LcrF showed the same phenotype as that of the lcrQ mutant strain and intracellular amount balance of LcrQ and LcrF is important in T3SS regulation. When the expression level of LcrF exceeds LcrQ, expression of the Ysc-Yop T3SS genes is activated and vice versa. Together, these data support a model in which LcrQ blocks the activation role of LcrF in regulating the transcription of T3SS genes in Yersinia.PLoS ONE 01/2014; 9(3):e92243. · 3.73 Impact Factor
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ABSTRACT: Bacteria exploit different strategies to perceive and rapidly respond to sudden changes of temperature. In Helicobacter pylori the response to thermic stress is transcriptionally controlled by a regulatory circuit that involves two repressors, HspR and HrcA. Here we report that HrcA acts as a protein thermometer. We demonstrate that temperature specifically modulates HrcA binding to DNA, with a complete and irreversible temperature-dependent loss of DNA binding activity at 42°C. Intriguingly, although the reduction of HrcA binding capability is not reversible in vitro, transcriptional analysis showed that HrcA exerts its repressive influence in vivo, even when the de novo repressor synthesis is blocked after the temperature challenge. Accordingly, we demonstrate the central role of the chaperonine GroESL in restoring the HrcA binding activity, lost upon heat challenge. Together our results establish HrcA as a rare example of intrinsic temperature sensing transcriptional regulator, whose activity is posttranscriptionally modulated by the GroESL chaperonine.Molecular Microbiology 04/2014; · 4.96 Impact Factor
Fox and S. A. Minnich
John R. Rohde, Xing-she Luan, Harold Rohde, James M.
Bends Which Melt at 37°C
Plasmid Contains Multiple Intrinsic DNA
The Yersinia enterocolitica pYV Virulence
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on May 31, 2013 by guest
JOURNAL OF BACTERIOLOGY,
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
July 1999, p. 4198–4204 Vol. 181, No. 14
The Yersinia enterocolitica pYV Virulence Plasmid Contains
Multiple Intrinsic DNA Bends Which Melt at 37°C
JOHN R. ROHDE,† XING-SHE LUAN,‡ HAROLD ROHDE, JAMES M. FOX,§
AND S. A. MINNICH*
Department of Microbiology, Molecular Biology, and Biochemistry,
University of Idaho, Moscow, Idaho 83843
Received 6 January 1999/Accepted 30 April 1999
Temperature has a pleiotropic effect on Yersinia enterocolitica gene expression. Temperature-dependent
phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis
(<30°C) and virulence plasmid-encoded Yop secretion (37°C). The mechanism by which temperature exerts
this change in genetic programming is unclear; however, altered gene expression by temperature-dependent
changes in DNA topology has been implicated. Here, we present evidence that the Y. enterocolitica virulence
plasmid, pYV, undergoes a conformational transition between 30 and 37°C. Using a simplified two-dimen-
sional, single-gel assay, we show that pYV contains multiple regions of intrinsic curvature, including virF, the
positive activator of virulence genes. These bends are detectable at 30°C but melt at 37°C, the temperature at
which the cells undergo phenotypic switching. We also show that pACYC184, a plasmid used as a reporter of
temperature-induced changes in DNA supercoiling, has a single region of intrinsic bending detected by our
assay. Topoisomers of pACYC184, with and without this bend, isolated from Y. enterocolitica were resolved by
using chloroquine gels. The single bend has a dramatic influence on temperature-dependent DNA supercoiling.
These data suggest that the Y. enterocolitica pYV plasmid may undergo a conformational change at the host
temperature due to melting of DNA bends followed by compensatory adjustments in superhelical density.
Hence, changes in DNA topology may be the temperature-sensing mechanism for virulence gene expression in
Y. enterocolitica and other enteric pathogens.
It is now evident that DNA is not merely the repository of
genetic information, but that the structure of the molecule
itself influences information access. For example, DNA bend-
ing (curvature), both intrinsic and induced by protein binding,
directly affects transcription. Static or induced bends can de-
termine promoter strength, both positively or negatively, de-
pending on the orientation and distance of the bends relative
to the RNA polymerase binding site. Involvement of such
ternary structures with regulatory regions appears to be a com-
mon trait of most bacterial promoters (reviewed in reference
Intrinsic DNA bending was originally identified with try-
panosome kinetoplast DNA. Kinetoplast DNA fragments with
phased deoxyribosyladenine (dA) and deoxyribosylthymine
(dT) nucleotide tracks display retarded migration in polyacryl-
amide gels (15). Regions of intrinsic bending identified on the
chromosome of Escherichia coli have been predominantly as-
sociated with the 5? regulatory regions of genes (19, 28). The
bacterial histone-like protein H-NS (21, 30) preferentially rec-
ognizes this topological feature, and this interaction (or bind-
ing) can repress gene expression (10, 11, 21). As such, H-NS
has been considered a potential generalized repressor for some
operons, with gene activation occurring by either competitive
displacement of H-NS by an activator protein or displacement
of H-NS by changes in DNA supercoiling induced by some
environmental signal. Thus, supercoiling, intrinsic or induced
bending, and histone-like proteins, individually or synergisti-
cally, can modulate gene expression.
Pathogenic bacteria utilize host environmental cues for vir-
ulence gene activation (reviewed in references 4 and 16). Tem-
perature is the key environmental parameter for virulence
gene induction for the pathogenic Yersinia spp. Shifting Yer-
sinia enterocolitica from ?30 to 37°C has multiple effects on
cell morphology and physiology. Changes after a temperature
upshift include the coordinate repression of flagellum synthesis
(12, 24) and induction of a set of plasmid-encoded virulence
genes (reviewed in reference 7). The virulence plasmid, termed
pYV (plasmid of Yersinia virulence) encodes a type III secre-
tion system (ysc and lcr genes) essential for delivery of addi-
tional plasmid-borne anti-host factors collectively referred to
as Yops (Yersinia outer proteins). Expression of ysc, lcr, and
yop genes requires the trans-acting DNA binding protein VirF,
a member of the AraC family of positive activators (5). VirF
synthesis is induced by elevated temperature (17, 24). How-
ever, experiments in which VirF was artificially overexpressed
from the tac promoter at 30°C failed to induce yop expression
(14). The Yersinia pestis homologue of VirF, LcrF, is constitu-
tively expressed as assayed by lacZ transcriptional fusions, but
yop gene transcription is still dependent upon elevated tem-
perature (9). Therefore, Yop expression requires factors in
addition to VirF.
Several lines of evidence suggest that DNA topology is in-
volved in Yersinia virulence gene expression. Cornelis et al. (6)
showed the role of a putative histone-like protein, YmoA, as
having a repressor-like activity on Yop expression. Yop expres-
sion becomes VirF independent in ymoA mutants, but expres-
* Corresponding author. Mailing address: Department of Microbi-
ology, Molecular Biology, & Biochemistry, University of Idaho, Mos-
cow, ID 83843. Phone: (208) 885-7884. Fax: (208) 885-6518. E-mail:
† Present address: Department of Biochemistry, University of Brit-
ish Columbia, Vancouver, BC V67-1Z3 Canada.
‡ Present address: Shandong Institute of Biology, Jinan 250014, Peo-
ple’s Republic of China.
§ Present address: Laboratory of Persistent Viral Diseases, Rocky
Mountain Laboratories, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Hamilton, MT 59840.
on May 31, 2013 by guest
sion is still enhanced by elevated temperature. Rohde et al.
(24) have shown that temperature-induced modifications in
DNA supercoiling are coincident with the temperature phasing
of two type III secretion systems (flagellum and Yop synthesis)
in Y. enterocolitica. Furthermore, novobiocin (DNA gyrase in-
hibitor)-resistant mutants include a class that is constitutive for
Yop expression, similar to the ymoA mutants mentioned
above. Additionally, Yop genes can also be expressed in Y.
enterocolitica wild-type cells at a low temperature by inducing
supercoiling with subinhibitory levels of novobiocin. There-
fore, modulations in supercoiling and the histone-like protein,
YmoA, show that temperature activation of Yop expression is
partially dependent on changes in DNA topology.
In this report we suggest that a third component of DNA
topology may act in concert with temperature-mediated
changes in supercoiling and YmoA. First we show that the
pYV plasmid contains multiple intrinsic DNA bends. Detec-
tion of intrinsic bending is based upon a rapid single-gel assay
that may have broad application. Secondly, we show that a
single bend in the reporter plasmid pACYC184 dramatically
affects supercoiling levels. Third, the intrinsic bends of plasmid
pYV are sensitive to melting at 37°C but not at 30°C. A model
based on these data taken together is presented to explain
temperature induction of virulence genes by the temperature-
dependent alteration of plasmid architecture.
MATERIALS AND METHODS
Bacteria, plasmids, and growth conditions. Y. enterocolitica 8081 pYV?was
grown in brain heart infusion broth or Luria broth (Difco) at either 25 or 37°C.
Plasmid pACYC184 (Cmr, Tcr) was electroporated into this strain and main-
tained on either chloramphenicol (20 ?g/ml) or tetracycline (20 ?g/ml). Plasmid
pACYC?DraI was constructed by cleaving the pACYC184 plasmid with DraI,
purifying the plasmid backbone, and religating to form a circular molecule. The
ligation mixture was electroporated into E. coli DH5?, and transformants were
selected for Tcrand then screened for chloramphenicol sensitivity. This plasmid
was introduced into Y. enterocolitica by electroporation.
Plasmid extraction and radiolabeling. Plasmid DNA was purified from Y.
enterocolitica and E. coli cultures by using the alkaline lysis procedure as de-
scribed by Sambrook et al. (25). DNA was further extracted with chloroform and
purified by CsCl density gradient centrifugation. Protein was not detectable in
these preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) performed with Coomassie blue staining. A BamHI library of
pYV8081 was constructed with the pBluescript KS? (Stratagene) cloning vector
to facilitate the mapping of specific genes. DNA sequence analysis showed that
part of virB (yscN-U operon), virF, and the N-terminal coding sequence for virC
(yscA-M operon) are contained within BamHI fragment 5 (4). Additionally,
BamHI fragment 6 contains yscV (lcrD) and yopN (lcrE) (data not shown).
Radiolabeling of individual fragments of pYV8081 was accomplished by elec-
troelution of the desired fragment from the pBluescript subclone followed by
radiolabeling with a random priming kit (U.S. Biochemical) according to the
protocol supplied by the manufacturer.
Two-dimensional gel electrophoresis. A mini-protean II slab gel apparatus
(Bio-Rad, Richmond, Calif.) was used to detect DNA intrinsic bends based on a
modification of the procedure reported by Mizuno (19). DNA samples to be
analyzed (ca. 1 to 2 ?g) were digested with the appropriate restriction endo-
nuclease(s), mixed with 1/5 volume bromphenol blue xylene-cyanol marker dye
buffer, and loaded in wells on the left-hand side of a 5% acrylamide nondena-
turing gel. If multiple samples were analyzed on the same gel, a well was skipped
between samples. For the first dimension, the DNA samples were vertically
resolved at 60°C at 10 V per cm. At this temperature, DNA bends melt and the
fragments migrate at their true molecular weights. The gel spacers were then
carefully removed, and the glass-gel sandwich was submerged in a horizontal
electrophoresis chamber (2-liter buffer capacity) maintained at 4°C. The samples
were electrophoresed in the second horizontal dimension again at 10 V per cm
until the xylene-cyanol dye was eluted from the right-hand side of the gel. At 4°C,
the intrinsic bends can reform, and at that time such fragments will display
retarded migration. The gels were stained with ethidium bromide and photo-
graphed. Therefore, in this system, linear molecules form a diagonal line de-
scending from left to right, and nonlinear DNA lags behind this diagonal. For
both dimensions Tris-borate-EDTA (TBE) buffer is used (0.045 M Tris-borate,
1 mM EDTA [pH 8.4]).
For analysis of the Y. enterocolitica pYV (70 kb), the plasmid was first digested
with three restriction endonucleases to generate fragments within the 1- to 10-kb
size range. These fragments were resolved in the first dimension by running both
the bromphenol blue-xylene cyanol marker dyes off the gel. After additional
running, dye was loaded to the well, and electrophoresis was continued until the
xylene cyanol reached the bottom of the gel. For the second dimension, electro-
phoresis was stopped within 2 to 3 h after the xylene cyanol was run off the gel.
The analysis of individual fragments (?1 kb) for DNA bending was conducted
by mixing the fragment with a 1-kb DNA ladder preparation (Gibco/BRL). This
ladder preparation forms a straight diagonal and facilitates identification of bent
DNA. Sufficient resolution is obtained by running the xylene cyanol marker dye
to the bottom of the gel in the first dimension and to the side of the gel in the
second dimension. This method also allows multiple samples to be assayed on the
same gel for comparative purposes.
Southern transfer. DNA resolved on two-dimensional gels was transferred to
nitrocellulose by the method of Southern (27) with the following modifications.
The gels were soaked in denaturant (0.2 N NaOH, 1.5 M NaCl) for 30 min and
then neutralized for 30 min (0.5 M Tris-HCl [pH 7.5], 1.5 M NaCl), followed by
pH equilibration by soaking in 10? SSC (1? SSC is 0.15 M NaCl plus 0.015 M
sodium citrate) for 30 min before being transferred to nitrocellulose. The 1.1-kb
EcoRI fragment (part of the yscN operon and virF) and BamHI fragment 6
(yscVyopN) of pYV8081 were radiolabeled with [?-32P]dATP and used as probes
to identify whether these genes reside on fragments with intrinsic curvature.
Chloroquine agarose gel electrophoresis. Chloroquine gels were used essen-
tially as described by Goldstein and Drlica (8). Y. enterocolitica was transformed
with either pACYC184 or the ?DraI chloramphenocol-sensitive derivative of this
plasmid. Cells were grown in Luria-Bertani broth at both 25 and 37°C to mid-
exponential growth without antibiotic selection, and plasmid DNA was purified
by the alkaline lysis procedure described by Sambrook et al. (25). DNA was
extracted with buffered chloroform-isoamyl-phenol to remove protein and pre-
cipitated. Further purification was achieved by cesium chloride density ultracen-
trifugation. Purified plasmid DNA was loaded into the wells of a horizontal 1%
agarose gel containing 10 ?g of chloroquine/ml and resolved at 4 V per cm for
18 to 24 h in TBE buffer containing 10 ?g of chloroquine/ml. Following electro-
phoresis, the gels were washed 1 to 3 h in distilled water, stained with ethidium
bromide, and photographed.
Two-dimensional gel assay development for detection of
DNA intrinsic bends. The method reported by Mizuno (19) for
detection of bent DNA fragments utilized a two-dimensional
gel system; DNA fragments were first resolved in tube gels at
a high temperature (60°C), and the tube gel was then overlaid
onto a slab gel and electrophoresed at a low temperature
(4°C). Differentiation of bent DNA from linear DNA relied on
temperature differences in migration; linear molecules form a
straight diagonal with bent DNAs lagging behind this line.
Because the same buffer is used for both dimensions, the tech-
nique could be simplified by conducting analysis in the same
gel, thus eliminating the requirement for tube gels in the first
dimension (see Materials and Methods).
To test and standardize this approach, we first analyzed
DNA fragments containing the E. coli proU and ompF pro-
moter regions previously reported to contain intrinsic bends
(21, 26). For each gene, the respective promoter fragments are
retarded in the second dimension of two-dimensional gels
compared to linear molecular weight standards (data not
The Y. enterocolitica serotype 08 virF gene is contained
within BamHI fragment 5 on the pYV plasmid. We have de-
termined that the basic gene order for this region of pYV (Fig.
1) is the same as reported by Cornelis et al. (4) for pYVe227
(serotype 09). As shown in Fig. 2, the BamHI fragment was
digested with RsaI (lane 1) allowing mapping of two bends to
the fragments shown in Fig. 1. In lane 2, the 376-bp RsaIc-d
fragment was mixed with the 1-kb ladder. Lane 1 also shows
additional bent fragments within the 4-kb BamHI fragment 5
localized to virB and the ysc operon. The 269-bp fragment (Fig.
1) includes approximately 70 bp upstream of the virF transla-
tion start site.
Analysis of pYV for intrinsic bending. Purified pYV plasmid
was digested with BamHI, EcoRI, and HindIII, and the frag-
ments were subjected to two-dimensional gel analysis. As
shown in Fig. 2B, this plasmid contains multiple fragments
demonstrating pronounced bending based on retarded migra-
VOL. 181, 1999INTRINSIC DNA BENDS OF pYV 4199
on May 31, 2013 by guest
tion of DNA fragments at low temperature (4°C) in the second
dimension. Subsequent transfer of DNA resolved on this gel to
nitrocellulose and probed with the EcoRI fragment, which
contains part of virB (yscN-U) and virF, determined that the
identified fragment (marked by an arrow) was on a nonlinear
DNA fragment. Likewise, a two-dimensional gel of restricted
pYV DNA (Fig. 3A) was transferred to a membrane and
probed with BamHI fragment 6 (containing yscVyopN) deter-
mined by DNA sequencing. As shown in Fig. 3B, the yscV and
yopN probe hybridizes to fragments that fall to the left of the
diagonal line of noncurved DNA. We conclude that both virF
and a fragment that contains yopN and yscV genes are con-
tained within fragments that display intrinsic bends.
DNA bending affects supercoiling. Our previous analysis of
temperature-regulated gene expression in Y. enterocolitica de-
termined that phenotypic switching between 25 and 37°C was
coincident with thermoinduced modulations of DNA super-
coiling (24). Two observations, cited in the conclusion of this
report, suggested that other parameters contributed to tem-
perature regulation as follows: (i) other environmental condi-
tions that induce DNA supercoiling in Salmonella typhimurium
and E. coli, e.g., increased osmolarity and anaerobiosis, do not
mimic the effects seen with temperature. Therefore, supercoil-
ing alone is insufficient to account for the temperature re-
sponse of Y. enterocolitica. (ii) The mean topoisomer distribu-
tion of extracted pACYC184 from Y. enterocolitica cells
cultured at 25°C rather than 37°C was greater than the theo-
retical prediction based on the paper by Goldstein and Drlica
(8). That is to say, a change of two linking numbers over this
12°C temperature range is predicted, when in actuality we
measured a four to six linking number difference. Therefore,
the superhelical density of plasmids in Y. enterocolitica is de-
termined by factors in addition to those that may be predicted.
To account for this difference, we examined pACYC184 for
regions of intrinsic bends by using the two-dimensional gel
assay described. A Sau3A digest showed a single region of
bending, which we subsequently mapped to the DraI fragment
(bp 3988 to 83, relative to the EcoRI site at position 1). This
fragment is internal to the chloramphenicol acetyltransferase
(cat) gene of the plasmid. Figure 4A shows a two-dimensional
gel of this fragment when pACYC184, digested with DraI, was
mixed with a 1-kb molecular size ladder. This fragment shows
significant retardation using the two-dimensional gel assay.
A DraI deletion of plasmid pACYC184 was constructed and
introduced into Y. enterocolitica 8081. Isogenic strains of Y.
enterocolitica containing pACYC184 and the DraI deletion
construct (pACYC?DraI) were grown at 25 and 37°C, and the
plasmids were purified by cesium chloride density centrifuga-
tion and separated on chloroquine gels to resolve topoisomers.
Topoisomers can be resolved and visualized by using this assay
as a plasmid ladder. Each band differs from adjacent bands by
one linking number, with the more negatively supercoiled
DNA topoisomers migrating more rapidly in the gel. The re-
sults of this experiment are shown in Fig. 4B. The left two lanes
show pACYC184 topoisomers resolved at both temperatures.
Plasmid pACYC184 topoisomers isolated from Y. enteroco-
litica are difficult to separate, as the plasmid appears to be in a
more relaxed state (less supercoiled) as evidenced by slower
migration in the gels. Compared to the same plasmid with the
DraI deletion (right two lanes), it can be seen that removal of
FIG. 1. Genetic and physical map of the Y. enterocolitica 08 pYV region
encoding virF. The virF gene is contained on a 4-kb BamHI fragment subcloned
from pYV. The arrow indicates the direction of virF transcription. When this
fragment is digested with RsaI and EcoRI and subjected to two-dimensional gel
analysis, two fragments show retarded migration. Restriction sites are denoted as
follows: B, BamHI; RI, EcoRI; Rs, RsaI; C, ClaI; and Bt, BstII.
FIG. 2. The Y. enterocolitica virF gene contains intrinsic bends. (A) The pYV
4.0-kb BamHI fragment 5 contains part of virB (yscN-U operon), virF, and the
N-terminal coding region of the yscAM operon. This fragment was digested with
RsaI mixed with the 1-kb ladder for reference and subjected to two-dimensional
gel analysis (lane 1). Several fragments show retarded migration, including
376-bp and 269-bp fragments which map to virF. The 1.1-kb EcoRI fragment
contained within BamHI fragment 5 was subcloned, purified, and then digested
with RsaI. The RsaIc-dfragment (Fig. 1) was electroeluted and mixed with the
1-kb ladder (lane 2). (B) Total pYV plasmid DNA was digested with BamHI,
EcoRI, and HindIII, and the fragments were resolved on a two-dimensional gel.
Multiple DNA fragments show retarded migration. The arrow denotes the DNA
fragment hybridizing to the 1.1-kb EcoRI fragment (containing virF; Fig. 1) by
4200ROHDE ET AL. J. BACTERIOL.
on May 31, 2013 by guest
the bend has a dramatic effect on the overall migration of the
plasmid. Part of this effect can be attributed to the removal of
339 bp of DNA (ca. a 7% change in total size). Whereas the
difference in migration of supercoiled DNA is significant, the
difference in migration of open circles between the two plas-
mids is negligible. Removal of the bend in this plasmid results
in ca. a two linking number increase after a shift from 25 to
37°C as predicted. We conclude that plasmid pACYC184 re-
quires less supercoiling when a bend is present and that this
bend accounts for the previous discrepancy in topoisomer dis-
tribution noted by Rohde et al. (24).
The apparent size of the pACYC184 DraI 339-bp fragment
(sequence determination) when separated on a 5% polyacryl-
amide gel run at 4°C is 490 bp, ca. 1.5 times its actual size (Fig.
5; compare panel A, lane 2 with panel C, lanes 2 and 4). Figure
5 also shows that the DraI fragment shows minimal retardation
in gels run at 37°C (compare panel B, lane 1 to panel A, lane
2) but significant retardation in gels run at 25°C (compare
lanes 1 and 4 in panel B). These data imply that the bend is
maintained at 25°C but melts at 37°C (see next section). This
conformational change may therefore account for the signifi-
cant difference in topoisomer linking number observed over
this temperature range.
DNA bends melt at 37°C. Chan et al. (2, 3) reported that the
mean melting temperature in vitro of intrinsically bent syn-
thetic DNA oligomers containing phased dA or dT nucleotide
tracts was between 37 and 40°C. Because this is the tempera-
ture that induces Y. enterocolitica virulence genes, we utilized
the two-dimensional gel assay to determine if pYV bent DNA
melts within this temperature range. Figure 6 compares a sam-
ple of Y. enterocolitica pYV plasmid digested with three en-
zymes (BamHI, EcoRI, and HindIII) and resolved at 4, 30, and
37°C, respectively, in the second dimension. As can be seen,
the bending is maintained at 30°C but is completely eliminated
FIG. 3. DNA bends associated with pYV. In panel A, purified pYV DNA
was digested with HindIII, EcoRI, and BamHI, and the digestion mixture was
subjected to two-dimensional gel analysis. A Southern blot was prepared on this
gel and probed with radiolabeled pYV BamHI fragment 6, which contains yscV
and yopN. These genes are induced by elevated temperature (37°C) and are
localized to nonlinear DNA fragments that lag behind the linear fragments of the
digest (arrow). The diagonal in panel B was determined by superimposing the
X-ray film over a scaled gel photograph.
FIG. 4. The pACYC184 DraI fragment is bent, and this fragment affects
plasmid supercoiling. (A) Cesium-purified plasmid pACYC184 DNA was di-
gested with DraI to release a 339-bp fragment. The digest was mixed with the
1-kb ladder and analyzed by two-dimensional gel electrophoresis. The DraI
fragment, which falls off the linear diagonal generated by the ladder, shows
retarded migration. (B) Chloroquine gel electrophoresis was conducted on
pACYC184 and pACYC?Dra1 isolated from Y. enterocolitica cells grown at
different temperatures. Lanes 1 and 2, pACYC184 isolated from Y. enterocolitica
grown at 25 and 37°C, respectively; lanes 3 and 4, plasmid pACYC?Dra1 (339-bp
deletion) isolated at the same temperatures. The band across the top in all four
lanes is open-circle DNA, showing little difference in migration, whereas the
deletion derivative topoisomers migrate much further in the gel.
FIG. 5. Temperature affects migration of the pACYC184 DraI fragment.
Cesium-purified pACYC184 was digested with DraI, and the fragments sepa-
rated on 5% acrylamide gels run at different temperatures (60, 37, 25, and 4°C)
along with a set of molecular size markers. The arrows in panels A, B, and C
denote the three reference molecular size markers of 506, 396, and 344 bp. The
size of the DraI fragment is 339 bp, based on DNA sequence analysis. This size
is also indicated in panel A, lane 2, where it can be seen to migrate just below the
344-bp marker (lane 1) when the gel is run at 60°C. A similar size (ca. 339 bp)
is also apparent when this fragment is separated at 37°C (panel B, lane 1)
compared to the molecular size standards (panel B, lane 2). Retardation of the
DraI fragment is apparent in gels run at 25°C (compare panel B lane 4 to lane 1).
In panel C, duplicate digests of the DraI fragment, resolved at 4°C (lanes 2 and
4), migrate with an apparent molecular size of ca. 490 bp relative to the molec-
ular size markers (lanes 1 and 3). Comparison of the migration of the DraI
fragment at 60°C (panel A, lane 2) and 37°C (panel B, lane 1) shows that the
temperature effect on the retardation of this fragment is minimized around 37°C.
VOL. 181, 1999 INTRINSIC DNA BENDS OF pYV4201
on May 31, 2013 by guest