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J Bacteriol, June 1998, p. 2854-2861, Vol. 180, No. 11
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Molecular Characterization of the Gene Encoding the
DNA Gyrase A Subunit of Streptococcus pneumoniae
Delia
Balas,
Esteban
Fernández-Moreira, and
Adela
G.
De La Campa*
Unidad de Genética Bacteriana (Consejo
Superior de Investigaciones Científicas), Centro Nacional de
Biología Fundamental, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain
Received 30 December 1997/Accepted 23 March 1998
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ABSTRACT |
The gene encoding the DNA gyrase A subunit of Streptococcus
pneumoniae was cloned and sequenced. The gyrA gene
codes for a protein of 822 amino acids homologous to the gyrase A
subunit of eubacteria. Translation of the gene in an Escherichia
coli expression system revealed a 92-kDa polypeptide. A
sequence-directed DNA curvature was identified in the promoter region
of gyrA. The bend center was mapped and located between the
35 and 10 regions of the promoter. Primer extension analysis showed
that gyrA transcription initiates 6 bp downstream of an
extended 10 promoter. The possible implications of the bent DNA
region as a regulatory element in the transcription of gyrA
are discussed.
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INTRODUCTION |
DNA topoisomerases control bacterial
DNA topology, which is implicated in the processes of DNA replication,
recombination, and transcription. These physiological effects made the
bacterial type II DNA topoisomerases, DNA gyrase (gyrase) and DNA
topoisomerase IV (topo IV), essential for cell viability. Gyrase is
composed of two A (GyrA) and two B (GyrB) subunits, which are encoded
by the gyrA and gyrB genes, respectively. The
enzyme introduces negative supercoils into DNA by wrapping DNA around
the A2B2 protein complex, cleaving both DNA
strands (which involves the formation of DNA-protein covalent bonds)
and using ATP hydrolysis to pass another portion of DNA through this
break. Resealing of the break results in the introduction of two
negative supercoils (10, 44). The A subunit is required for
the double-stranded breakage and reunion of DNA (41), and
the B subunit is required for energy transduction via ATP hydrolysis
(25, 40). Topo IV is composed of two C (ParC) and two E
(ParE) subunits encoded by the parC and parE genes, respectively. This enzyme is essential for chromosome
partitioning (1, 16). The amino acid sequences of ParC and
ParE are homologous to those of GyrA and GyrB, respectively.
Gyrase and topo IV can be inhibited by different types of drugs
(22). Among them are the fluoroquinolones, a relatively new
class of potent, broad-spectrum antimicrobial agents (45). However, their limited activity against Streptococcus
pneumoniae (the pneumococcus) and the increasing resistance
observed in this species worldwide (2) have led to the
continued search for more active compounds. Several studies have shown
that the primary target for quinolones in gram-negative bacteria is
gyrase, while in the gram-positive bacteria topo IV is the primary
target for most quinolones, although it has been reported that
sparfloxacin targets gyrase in S. pneumoniae
(30). Previous studies on Escherichia coli have
identified quinolone resistance mutations in the GyrA quinolone
resistance-determining region (QRDR), located between amino acid
residues 67 and 106 (48). This region has the highest sequence conservation between GyrA and ParC. Recent studies have identified similar mutations in the analogous region of ParC
(18). However, E. coli parC resistance mutations
are expressed only in the presence of gyrA mutations
(18), and purified E. coli topo IV is less
sensitive to quinolones than E. coli gyrase (18). Likewise, Neisseria gonorrhoeae (4) and
Haemophilus influenzae (11) strains with
low-level resistance contain gyrA mutations, while those
with higher levels of resistance have mutations in both gyrA
and parC. The opposite is true for S. pneumoniae:
mutations altering amino acid residues within the QRDR of ParC confer
low-level resistance; mutations altering both QRDRs of ParC and GyrA
confer high-level resistance (15, 29, 31, 43). For
enterococci, the absence of gyrA mutations in first-step
fluoroquinolone-resistant mutants and their presence in second-step
mutants (19) suggest the possibility that first-step mutants
contain parC mutations, as in the pneumococcus. Moreover,
genetic as well as biochemical evidence shows that in
Staphylococcus aureus, topo IV is also the primary target
for these antimicrobial agents (5, 9).
The genes encoding the two subunits of S. pneumoniae topo IV
have been cloned and sequenced (29, 32). We have also
previously reported the genetic characterization of the pneumococcal
gyrB gene (28) and of a region of the
gyrA gene encoding 127 amino acids which includes the QRDR
(29). We report here on the characterization of the complete
gyrA gene and shown that it is transcribed from a promoter
containing a 10 extended promoter that is located in a region showing
intrinsic DNA bending. This work complements the genetic
characterization of the type II DNA topoisomerases of the pneumococcus
and open new ways for the study of the regulation of gyrA
gene expression.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and DNA manipulations.
The
E. coli strains used for plasmid transformation were DH5
(13) and XL1-Blue (Stratagene). Plasmids used for cloning were pUC18 (47) and pEMBL18+ (7).
Chromosomal DNA from S. pneumoniae wild-type strain R6 was
obtained as described previously (8). Plasmids were prepared from E. coli by the alkaline lysis method or by equilibrium
centrifugation in CsCl-ethidium bromide gradients (37).
Manipulations of DNA, including electrophoresis, and Southern blotting
were carried out by standard methods (37). For in situ
colony hybridization, DNA was radiolabeled with 50 µCi of
[-32P]dCTP (300 Ci/mmol), using the Multiprime DNA
labeling system (Amersham).
PCR amplification and cloning procedures.
PCR amplifications
were performed as described previously (8). Amplification
was achieved with an initial cycle of 5 min of denaturation at 95°C,
15 min of annealing at 55°C (7 min before and 8 min after adding the
enzyme), and 6 min of polymerase extension at 72°C, followed by 20 cycles of 1 min at 95°C, 2 min at 55°C, and 2.5 min at 72°C, with
a final 20 min 72°C extension step and slow cooling at 4°C. For PCR
reactions with oligonucleotides gyrA46 and gyrA435 (see below),
conditions were an initial cycle of 5 min at 95°C and then 60 cycles
of 1 min at 95°C, 1 min at 40°C, and 2.5 min at 72°C. The
synthetic oligonucleotide primers used were gyrAUP1
(5'-gcgctctagaTGGTTTAGAGGCTGAAATAGAC-3'), gyrADOWN (5'-gcgctctagAGTAATATCAGAAATCCTGCTAGG-3'), 512 (8), gyrA46 (29), gyrA172 (29),
gyrA435 (5'-gcgcgtcgACNGATCA(A/G)GATGA(A/G)GTT-3'), and
gyrA607 (5'-gcgcgtcgacGA(T/C)GCNTA(T/C)CTNTT(C/T)TT(T/C)ACNAC-3'). The 5' ends of some of the primers contained sequences including either a PstI (gyrA435), SalI (gyrA607), or
XbaI (gyrAUP1 and gyrADOWN) restriction site (lowercase
letters).
Cloning of the gyrA gene was done by PCR amplifications of
overlapping DNA fragments. The upstream gyrA region was
amplified by using the degenerate primer gyrA172, (Fig.
1 and 2)
and the A-T-rich primer 512. PCR products were hybridized with the
gyrA44-170 probe, isolated from agarose gel slices, cut with
HindIII (a target included both in gyrA172 and in the
chromosomal region upstream of gyrA), and cloned into
pEMBL18+ cut with the same enzyme. Plasmid pGYAN6 was
selected by in situ colony hybridization with the gyrA44-170 probe. Its
chromosomal insert showed the sequence corresponding to the N terminus
of GyrA, and the gyrA172 oligonucleotide at one of its ends (Fig. 1).
To clone the downstream gyrA region, we performed PCR
experiments using three single degenerate primers (Fig. 1 and 2):
gyrA46, gyrA435, and gyrA607. Amplification of R6 DNA by using the
gyrA46 primer, cutting of the PCR products with EcoRV (a
target located within gyrA) (Fig. 1) plus XbaI (a
target included in the primer), and ligation into
XbaI-SmaI-cut pUC18 allowed the isolation of plasmid pGYAN31 (Fig. 1). Its gyrA origin was detected by
hybridization with the gyrA44-170 DNA probe. For the isolation of
plasmids pGYAN1 and pGYAN3, PCR amplification was done with primer
gyrA435, and a gyrA332-488 probe obtained from plasmid pGYAN31 was used
for hybridization. PCR fragments were cut with PstI (a
target included into the gyrA435 oligonucleotide) and cloned into
pEMBL18+ cut with the same enzyme. Isolation of plasmid
pGYAN11 was achieved after a PCR amplification of R6 DNA with
oligonucleotide gyrA607. After cutting of the PCR products with
KpnI-PstI (targets included in the chromosomal
gyrA region [Fig. 1]), bands of the appropriate size that
hybridized with the insert of plasmid pGYAN1 were isolated and cloned
into pEMBL18+, cut with the same enzymes.
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FIG. 1.
Restriction map of the gyrA region of
S. pneumoniae (A) and genetic structure as deduced from the
nucleotide sequence (B). P at the left of the gray arrow indicates the
promoter. The physical maps of the inserts of pertinent plasmids are
also indicated (hatched bars). Bg, BglII; D,
DraI; E, EcoRI; EV, EcoRV; H,
HindIII; K, KpnI; P, PstI; S,
SacI. The DNA probes used in Southern blot experiments are
indicated as bars labeled gyrA44-70 (insert of plasmid pQRDR26) and
gyrA136-485 (insert of pGYAN31). The oligonucleotides used in PCR
experiments are indicated by black arrows (not drawn to scale).
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FIG. 2.
Nucleotide sequence of a 2,685-bp fragment of S. pneumoniae R6 which contains the gyrA gene. The strand
corresponding to the mRNA is shown. Nucleotides and amino acids
(italics) are numbered by taking the first gyrA nucleotide
as nt 1 and the first GyrA residue as residue 1. The 35 and the
extended 10 promoter regions and the putative ribosome-binding site
(RBS) are underlined and in boldface. The first nucleotide of the mRNA
is indicated as +1. The amino acid residues encoded by the degenerate
oligonucleotides used in PCR experiments are also underlined and in
boldface. Other pertinent oligonucleotides and restriction endonuclease
sites are labeled and underlined.
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Circular permutation analysis.
A 533-bp
DraI-EcoRV fragment from plasmid pGYAN6 was
cloned into the SmaI site of plasmid pCY7 (35), a
pBR322 derivative in which the 375-bp EcoRI-BamHI
fragment is present as a tandem repeat separated by a
SacI-SmaI-XbaI-BglII
polylinker. To shorten the size of the insert, the resultant plasmid
was digested with SacI, and a 222-bp SacI
fragment was then cloned into the SacI site of the plasmid
pCY7 polylinker. The orientation of the 222-bp pneumococcal insert into
plasmid pBEND-11 was tested by sequence analysis. Plasmid pBEND-11 was
digested separately with restriction enzymes EcoRI,
HindIII, EcoRV, NheI, and
BamHI to generate a set of 609-bp fragments that differ in
the position of the pneumococcal insert relative to their ends. DNA
samples were fractionated by 5% polyacrylamide gel electrophoresis at
4°C, and the bands were visualized by staining with ethidium bromide.
DNA sequence determination and analysis.
DNA sequencing was
carried out with protocols and materials from the Sequenase system
(U.S. Biochemical). All sequences shown in this report were determined
for both strands of the DNA. DNA and protein sequence comparisons were
done with software from Intelligenetics (PCGENE 6.0). Bending analysis
was performed by the use of the DNASTAR computer program (DNASTAR,
Inc., London, United Kingdom).
RNA purification and analysis.
Cells from a mid-log-phase
culture (optical density at 600 nm of 0.50) of E. coli
XL1-Blue(pGYAN6) were collected and resuspended in 1/20 volume of 20 mM
sodium acetate (pH 5.5)-1 mM EDTA-1 mg of lysozyme per ml. The
suspension was lysed by freezing (70°C) and fast thawing (37°C)
several times. The solution was then made 0.5% sodium dodecyl sulfate
(SDS), and RNAs were extracted three times with phenol (buffered with
20 mM sodium acetate [pH 5.5]) at 70°C and precipitated twice. For
primer extension analysis, cellular RNA (3 to 15 µg) was annealed
with 1 pmol of oligonucleotide gyrA20 (5'-CACTCATGGCGTAGTCG-3')
during slow cooling from 65 to 20°C in 14 µl of 50 mM Tris
(pH 8.3)-75 mM KCl-3 mM MgCl2. The sample was brought to
a final volume of 20.5 µl and incubated for 30 min at 37°C with 8 U
of Moloney murine leukemia virus reverse transcriptase (Gibco-BRL) in
the presence of 100 µM deoxynucleoside triphosphates (except dATP),
10 mM dithiothreitol, 9.75 µM cold dATP, and 15 µCi of
[-32P]dATP (300 Ci/mmol). The products were phenol
extracted, precipitated, and dissolved in 4 µl of formamide dye
solution for storage at 20°C.
Analysis of plasmid-encoded proteins.
The pGEM3Z
vector/BL21(DE3) host cloning system permits the specific synthesis and
labeling of protein products encoded by genes placed under the control
of the phage T7 
10 promoter (39). The cell harbors a
defective 
prophage that contains the T7 RNA polymerase gene under
the control of lacUV5, which can be induced by
isopropyl-
-D-thiogalactopyranoside (IPTG).
One-milliliter cultures of E. coli BL21(DE3) carrying the
desired plasmids were grown at 37°C in M9 or LB medium containing 200 µg of ampicillin. When the culture reached an
A600 of 0.5, cells grown in M9 were induced with
IPTG (1 µmol), incubation proceeded for 30 min, and 200 µg of
rifampin was added. After 90 min, 10 pmol of
[35S]methionine (1,000 Ci/mmol) was added, and
incorporation was terminated 10 min later by chilling the cultures.
Cells grown in LB were induced with IPTG for 25 min. The cells were
centrifuged, suspended, and lysed in sample loading solution for gel
electrophoresis. Proteins were analyzed by electrophoresis in 10%
(wt/vol) polyacrylamide gels, and they were revealed by staining the
gels with Coomassie blue, photography, and autoradiography.
Nucleotide sequence accession number.
The DNA sequence
corresponding to the gyrA gene has been assigned GenBank
accession no. AF053121.
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RESULTS AND DISCUSSION |
Cloning and sequencing the gyrA gene of S. pneumoniae.
We had previously cloned into plasmid pQRDR26
(29) a region of S. pneumoniae R6 encoding GyrA
amino acids 44 to 170 (Fig. 1), which includes the QRDR region.
Attempts to clone the complete gyrA gene from a gt11
library of pneumococcal DNA were unsuccessful (29). In this
work we designed a cloning strategy based on a PCR-walking approach. We
constructed degenerate oligonucleotide primers based on the known amino
acid sequence, designed to account for the codon usage of S. pneumoniae. These primers were used in PCR DNA amplifications,
individually or in combination with an A-T-rich oligonucleotide that
was assumed to prime in any region of the (A-T-rich) pneumococcal
chromosome. PCR products from the gyrA region were
identified through hybridization with specific DNA probes derived from
the previously cloned regions and by the presence of restriction
targets on the chromosomal DNA, as deduced from Southern blot
experiments (Fig. 1). Positive fragments with appropriate sizes were
isolated, digested with restriction endonucleases (target sequences
included in the oligonucleotide primers and/or in the chromosomal
gyrA region), and cloned into E. coli plasmid vectors. Recombinant clones were detected once more by in situ colony
hybridization with the same DNA probe used for the identification of
the PCR products. This PCR-cloning approach allowed the genetic characterization of the pneumococcal gyrA gene (Fig. 2).
Analysis of the nucleotide sequence revealed the presence of a putative
promoter for
gyrA transcription, and the gene was
preceded
by a putative ribosome-binding site (
38). The deduced
product of
gyrA is a protein of 822 amino acid residues that
shows
about 60% identity with the GyrA subunit of
Bacillus
subtilis and
S. aureus and near 50% identity with GyrA
of
E. coli (Fig.
3). The
residues that form the active site of the breakage-reunion
reaction, as
revealed by the crystal structure of this domain
of
E. coli
GyrA (residues 2 to 523) (
26), are conserved in the
pneumococcal GyrA protein (Fig.
3). A FASTA search on the Swiss-Prot
sequence database was performed to select sequences to be used
in the
construction of the protein tree shown on Fig.
4. Only
the 25 more similar full-length
protein sequences that corresponded
to the GyrA and ParC subunits of
gyrase and topo IV were used.
These GyrA and ParC sequences formed
separate groups within the
tree. The identities among
S. pneumoniae GyrA and the other GyrA
proteins varied between 43 and
60%, while identities for ParC
proteins were between 31 and 39%.
S. pneumoniae GyrA and ParC
sequences were 39% identical.
The GyrA and ParC sequences of the
gram-positive bacteria with low G+C
content (
S. pneumoniae,
S. aureus, and
B. subtilis) formed differentiated clusters. These
data are in
agreement with the phylogenetic comparisons of type
II topoisomerases
reported by Huang (
14).
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FIG. 3.
Comparison of the amino acid sequences of the DNA gyrase
A subunits from S. pneumoniae (Spn), B. subtilis (Bsu) (27), S. aureus
(Sau) (21), and E. coli
(Eco) (42). Identical amino acids are boxed.
Residues in E. coli GyrA that form the active site of the
breakage-reunion reaction, including the active Tyr-122 residue that
links to DNA, are indicated by arrows. Residues involved in quinolone
resistance in S. pneumoniae are indicated by circles
(15, 29, 31, 43).
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FIG. 4.
Protein tree of full-length GyrA and ParC subunits from
bacteria. The tree was compiled by using the CLUSTAL multiple sequence
alignment program from PCGENE.
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Identification of the gyrA gene product.
To
identify the protein encoded by gyrA, the complete gene was
cloned into plasmid pGYAN9 (Fig. 1). A PCR amplification of R6
chromosomal DNA with oligonucleotides gyrAUP1 (nucleotides [nt] 77
to 56 of the sequence in Fig. 2) and gyrA DOWN (nt 2548 to 2525) was
performed. PCR products were cut with XbaI, cloned into the
XbaI site of pGEM3Z, which contains the T7 polymerase promoter (39), and established in E. coli
BL21(DE3). When a culture of this strain was induced with IPTG, one
polypeptide, which is not expressed by the pGEM3Z vector, of 92 kDa was
produced and specifically labeled with [35S]methionine
(Fig. 5). This identified the
pneumococcal DNA gyrase A subunit as a 92-kDa polypeptide. This value
is in agreement with the molecular mass (92.096 kDa) predicted from
sequence analysis.
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FIG. 5.
Expression of the GyrA protein. Cultures of E. coli BL21(DE3) containing pGEM3Z (lanes 3 and 5) or pGYAN9 (lanes
4 and 6) were grown in M9 (lanes 5 and 6) or in LB (lanes 3 and 4)
medium and induced with IPTG as described in Materials and Methods.
Samples containing 15 µg of protein were electrophoresed in an
SDS-polyacrylamide gel. Lanes 3 to 6, polypeptides revealed by
Coomassie blue staining. The dried gel was exposed for 8 h to
Kodak X-Omat for autoradiography. Lane 2 shows the autoradiogram of
lane 6. Lane 1, molecular mass protein standards.
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Characterization of a sequence-directed DNA bend in the promoter
region and the transcription start site.
Analysis of the
nucleotide sequence in the region preceding the putative ATG initiation
codon for gyrA revealed the presence of a putative promoter
(Fig. 2). We had previously observed an anomalous mobility of some
restriction fragments carrying the upstream gyrA region.
Because this behavior is normally associated with intrinsic DNA
curvatures, we performed a computer modeling of the promoter region
including nt 128 to 8 of the sequence in Fig. 2. The modeling (Fig.
6A) predicted a bent region located at
position 67, i.e., centered at about the 35 promoter region. Two-dimensional gel electrophoresis in polyacrylamide gels, the first
dimension run at 60°C and the second run at 4°C, showed that the
403-bp HindIII-SacI fragment contained the
promoter region (Fig. 1) separate from the diagonal of noncurved DNA
fragments (not shown), indicating the presence of bent DNA. To map
precisely the center of curvature, we used the circular permutation
assay of Wu and Crothers (46). This method is based on the
observation that the electrophoretic mobility of a DNA fragment in
polyacrylamide gels decreases as the center of the bending approaches
the center of the fragment. Thus, plasmid pBEND-11, which carries a
222-bp pneumococcal insert (including nt 128 to 94 of the sequence in Fig. 2), was cut with a series of restriction enzymes with cutting sites present only once in the EcoRI-BamHI
duplicated fragment to produce a family of fragments of identical size
(609 bp) (Fig. 6B). These molecules, which differ only in the position
of the bending center, were analyzed in a polyacrylamide gel run at
4°C. Their relative mobilities were plotted against the distance
between the 5' end (the EcoRI site) of the duplicated
fragment in the vector and the half point of the fragment generated by
each of the restriction enzymes used (Fig. 6C). In such plots, the
lowest point of the best-fitting curve corresponds to the bending
center (46). In our case, the position of the bend center
mapped at 74 bp from the 5' end of the fragment (nt 128), i.e., at
around nt 55 of the sequence in Fig. 2, between the 35 and 10
extended regions of the putative promoter for gyrA
transcription.
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FIG. 6.
Mapping of the center of curvature by circular
permutation analysis. (A) Computer-generated structure of the DNA
region from coordinates 128 to 8 of the sequence in Fig. 2. Plot is
shown in the YZ plane to show the predicted bend region. (B) Plasmid
pBEND-11 was digested separately with the indicated enzymes (E,
EcoRI; H, HindIII; EV, EcoRV; N,
NheI; B, BamHI) to generate 609-bp fragments
containing the insert at different positions relative to their ends.
These fragments were isolated from agarose gel slices, and mobility was
analyzed by electrophoresis in a 5% polyacrylamide gel run at 4°C.
Plasmid pBR322 digested with HpaII was used as the size
marker (lane Mw). (C) Physical structure of plasmid pBEND-11. The
restriction sites that occur only once within each duplicated region
flanking the insert (box) are indicated. The mobility of each permuted
fragment was determined and plotted against the distance from the 5'
end (EcoRI site) of the duplicated fragment in the vector to
the midpoint of the fragment generated by each of the restriction
enzymes used. The line represents the best fit to the experimental
data, and the arrow points to their minima, which correspond to the
center of curvature (see text for details).
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To map the transcription initiation site of the
gyrA gene, a
17-base oligonucleotide (gyrA20) was used in a primer extension
assay
with RNA extracted from
E. coli XL1-Blue carrying plasmid
pGYAN6 and from the same strain carrying pEMBL18
+ as a
negative control. This yielded an 89-nt runoff product (Fig.
7) mapping to the A (position
31 of the
sequence in Fig.
2) that
is 6 bp downstream from the 3' terminus of a
10 extended promoter
sequence (Fig.
7). Thus, the pneumococcal
gyrA gene is transcribed
from a promoter that shows the
sequence TGAAA(N)
12TATGGTATAAT,
with a
10
extended region (
17). Such
10 extended sites have
been
demonstrated to function in
E. coli (
17,
34), but
extended
10 sites occur rarely in this species (
20).
Extended
10 sites
occur more frequently in gram-positive bacteria
(
12), and they
commonly occur in the pneumococcus
(
36). It has been suggested
that the presence of this kind
of promoter could result in excessive
expression in
E. coli,
probably accounting for difficulties in
cloning pneumococcal DNA
fragments in
E. coli, either by producing
high levels of
toxic proteins or by interfering with vector functions
(
36).
This might explain why we did not find any lambda recombinant
clone
carrying the
gyrA gene when we looked for it in a
gt11
library of pneumococcal DNA (
29). Another factor that can
influence
the strength of the pneumococcal
gyrA promoter is
the presence
of a static curvature centered at the spacer region
located between
the
35 and
10 regions (Fig.
6) (
33).
Interestingly, an extended
10 region, which matches the consensus
TaTGgTATAAT (
36), is
also present in the
E. coli gyrA gene (
42). It is well known
that expression
of the
E. coli gyrA gene is regulated by DNA supercoiling
(
24). The activity of the promoter is stimulated by gyrase
inhibition
(i.e., by DNA relaxation), and the sequence required for
this
response is a 20-bp segment spanning positions
19 to +1 of the
promoter region, which excludes the
35 region (
23).
Whether
that
10 extended sequence is involved in regulation of
expression
of the
gyrA genes by targeting of a specific
factor is unknown,
but it has been suggested that an activator would be
required
for the transcription of
E. coli gyrA
(
6). However, it has
been reported that the
E. coli RNA polymerase sigma 70 subunit
recognizes the extended
10
motif at promoters (
3).
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FIG. 7.
DNA sequence of the 5' region of gyrA and
localization of the transcription initiation site. Sequenase reactions
using plasmid pGYAN6 as the template and gyrA20 as the primer provided
a reference sequence ladder. G, A, T, and C indicate the
dideoxynucleotides used during the sequencing assay. For primer
extension experiments, RNAs obtained from E. coli XL1-Blue
containing either pEMBL18+ (lane 1, 15 µg of RNA) or
pGYAN6 (lane 2, 3 µg of RNA; lane 3, 15 µg of RNA) were used. The
arrow indicates the direction of electrophoresis. The extended 10
region, the first nucleotide of the mRNA (+1), and the putative
ribosome-binding site (RBS) are framed. The double-strand DNA sequence
of the 5' gyrA region and the deduced amino acid sequence
are shown.
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DNA supercoiling is known to influence the curvature of DNA
(
33). If a bend site is located in the promoter region, as
is
the case for the
S. pneumoniae gyrA gene, then DNA
supercoiling
may influence the transcription of that gene by modifying
the
bend. The presence of an intrinsic DNA curvature in the
pneumococcal
gyrA promoter would make this promoter very
sensitive to changes
in supercoiling, allowing the expression of
gyrA to act as a regulator
of DNA supercoiling in the cell.
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ACKNOWLEDGMENTS |
We thank R. Muñoz for construction of plasmid pGYAN31, M. Espinosa for computer bending analysis and critical reading of the
manuscript, and P. A. Lazo for allowing us to use the PCGENE program on his computer.
D.B. has a Beca de Perfeccionamiento from the Fondo de
Investigación Sanitaria, and E. F.-M. has a Beca de
Formación de Personal Investigador from Comunidad Autónoma
de Madrid. This work was supported by grant FIS 97/2026 from Fondo de
Investigación Sanitaria.
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FOOTNOTES |
*
Corresponding author. Mailing address: Unidad de
Genética Bacteriana (Consejo Superior de Investigaciones
Científicas), Centro Nacional de Biología Fundamental,
Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain. Phone:
(341) 91-509-7904. Fax: (341) 91-509-7918. E-mail:
agcampa{at}isciii.es.
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J Bacteriol, June 1998, p. 2854-2861, Vol. 180, No. 11
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
