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Coupling of the biosynthesis and export of the DNA gyrase
inhibitor simocyclinone in Streptomyces antibioticusmmi_6735 1462..1474
Tung B. K. Le,1* Hans-Peter Fiedler,2
Chris D. den Hengst,1Sang Kyun Ahn,3
Anthony Maxwell4and Mark J. Buttner1
Departments of1Molecular Microbiology and4Biological
Chemistry, John Innes Centre, Colney Lane, Norwich
2Mikrobiologisches Institut, Eberhard-Karls-Universität
Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen,
3Department of Biochemistry and Biomedical Sciences,
McMaster University, 1200 Main St. W., Hamilton,
Ontario, Canada L8N 3Z5.
Because most antibiotics are potentially lethal to the
producing organism, there must be mechanisms to
ensure that the machinery responsible for export of
the mature antibiotic is in place at the time of
biosynthesis. Simocyclinone D8 is a potent DNA
gyrase inhibitor produced by Streptomyces antibioti-
cus Tü 6040. Within the simocyclinone biosynthetic
cluster are two divergently transcribed genes, simR
and simX, encoding proteins that resemble the
TetR/TetA repressor–efflux pump pair that cause
tetracyclines. Engineered expression of simX from a
strong, heterologous promoter conferred high level
simocyclinone D8 resistance on Streptomyces liv-
idans, showing that simX encodes a simocyclinone
efflux pump. Transcription of simX is controlled by
SimR, which directly represses the simX and simR
promoters by binding to two operator sites in the
simX–simR intergenic region. Simocyclinone D8 abol-
ishes DNA binding by SimR, providing a mechanism
that couples the biosynthesis of simocyclinone to its
export. In addition, an intermediate in the biosynthetic
pathway, simocyclinone C4, which is essentially inac-
tive as a DNA gyrase inhibitor, also induces simX
expression in vivo and relieves simX repression by
SimR in vitro.
Aminocoumarin antibiotics are active against Gram-
positive bacteria and function principally by inhibiting DNA
gyrase (Maxwell and Lawson, 2003), an essential DNA
topoisomerase found in all bacteria, which catalyses DNA
supercoiling (Nollmann et al., 2007). In addition, a likely
secondary target of these compounds is topoisomerase
IV, which is involved in chromosome decatenation (Hardy
and Cozzarelli, 2003; Oppegard et al., 2009). All four
known aminocoumarins are produced by Streptomyces
species. The first three to be discovered, novobiocin, clo-
robiocin and coumermycin A1, each competitively inhibit
the ATPase activity of the GyrB subunit of DNA gyrase
and exhibit Ki values in the nanomolar range (Gormley
et al., 1996). The most recently identified aminocoumarin
antibiotic, simocyclinone D8 (Schimana et al., 2000)
(Fig. 1), also inhibits DNA gyrase but was unexpectedly
found to have a completely novel mode of action, binding
instead to the GyrA subunit of the enzyme and preventing
its binding to DNA (Flatman et al., 2005; M.J. Edwards
et al., unpubl. data). Simocyclinone D8 is a potent inhibitor
of DNA gyrase supercoiling with an IC50lower than that of
novobiocin (Flatman et al., 2005; Oppegard et al., 2009).
The genus Streptomyces accounts for the production of
approximately two-thirds of the known antibiotics. They
expel these compounds into their environment, typically
the soil, most probably to give them a competitive advan-
tage over other organisms that share the same ecological
niche. Because the antibiotic is often potentially lethal to
the producing organism, there must be mechanisms to
ensure that the machinery responsible for export of the
mature antibiotic is in place at the time of biosynthesis.
This export machinery may be sufficient to confer resis-
tance to the antibiotic, or there may be additional resis-
tance mechanisms. For example, in the case of the
novobiocin, clorobiocin and coumermycin A1 producers,
the principal mechanism of resistance is production of an
aminocoumarin-resistant GyrB subunit (GyrBR), encoded
within the biosynthetic cluster and activated during antibi-
otic production (Thiara and Cundliffe, 1988; 1989; 1993;
Schmutz et al., 2003). The GyrBRsubunit replaces the
sensitive subunit (GyrBs) in the (GyrA)2(GyrB)2heterotet-
ramer during the production phase. In addition, an
Accepted 5 May, 2009. *For correspondence. E-mail tung.le@bbsrc.
ac.uk; Tel. (+44) 1603 450757; Fax (+44) 1603 450778.
Molecular Microbiology (2009) 72(6), 1462–1474 ?
First published online 22 May 2009
© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd
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ParYR, encoded within the clorobiocin and coumermycin
A1 biosynthetic clusters (but not the novobiocin biosyn-
thetic cluster), also confers resistance to these antibiotics
when introduced into a naïve host, presumably by an
analogous mechanism (Schmutz et al., 2003). However,
no DNA gyrase or topoisomerase IV subunits are
encoded within the simocyclinone D8 (sim) biosynthetic
cluster (Galm et al., 2002; Trefzer et al., 2002), leaving
unknown the mechanism of resistance in the producing
organism, Streptomyces antibioticus Tü 6040.
Simocyclinone D8 consists of four different parts, with a
halogenated aminocoumarin at one end connected to an
angucyclic polyketide at the other end via a tetraene linker
and a D-olivose sugar (Galm et al., 2002; Trefzer et al.,
2002) (Fig. 1). Within the sim cluster, among the genes
responsible for the biosynthesis and linking of the four
constituents of the antibiotic, are two divergently tran-
after, simX) (Galm et al., 2002; Trefzer et al., 2002). The
SimR/SimX pair resembles the TetR/TetArepressor–efflux
pump pair that causes widespread resistance to clinically
important tetracyclines in several human pathogens
(Chopra and Roberts, 2001). TetR represses transcription
of the divergently transcribed tetA gene, encoding a
proton-dependent tetracycline efflux pump. Binding of tet-
racycline to the C-terminal domain ofTetR causes it to lose
affinity for its operators, derepressing expression of tetA,
which confers high-level resistance to the drug (Hillen and
Berens, 1994; Kisker et al., 1995; Orth et al., 2000; Ramos
et al., 2005). The similarity of SimR/SimX to TetR/TetA
suggested they might be involved in simocyclinone efflux
and, potentially, in simocyclinone resistance (Galm et al.,
2002; Trefzer et al., 2002).
Here we show that simX encodes a simocyclinone
efflux pump, and that transcription of simX is controlled by
SimR, which directly represses the simX and simR pro-
moters by binding to two operator sites in the simR–simX
intergenic region. We show that simocyclinone D8 abol-
ishes DNAbinding by SimR, providing an intimate mecha-
nism that couples the biosynthesis of simocyclinone to its
export. In addition, we show that an intermediate in the
biosynthetic pathway, simocyclinone C4 (Fig. 1), which
is essentially inactive as a DNA gyrase inhibitor, also
induces simX expression in vivo and relieves DNAbinding
by SimR in vitro, suggesting a potential 'feed-forward'
mechanism (Tahlan et al., 2007) that might ensure
expression of the SimX efflux pump prior to the build-up of
a toxic concentration of the mature, active antibiotic.
SimX encodes a simocyclinone efflux pump
encoded in the sim cluster, SimEX1 (hereafter SimX) and
SimEX2 (Trefzer et al., 2002). To determine if either of
these two proteins is involved in simocyclinone D8 efflux,
we expressed simX and simEX2 from the strong consti-
tutive promoter ermEp* using the integrative, single-copy
vector pIJ10257 (Hong et al., 2005). We introduced these
constructs into the heterologous host S. lividans and com-
pared the susceptibility of the resulting strains to simocy-
clinone D8. The strain carrying ermEp*–simEX2 had an
MIC of 2 mg ml-1, as did S. lividans alone, or S. lividans
containing the parent vector, pIJ10257. In contrast, the
strain carrying the ermEp*–simX construct had an MIC of
are twopump-liketransmembrane proteins
Fig. 1. Structures of simocyclinone D8 and its biosynthetic intermediate, simocyclinone C4, and their IC50s for Escherichia coli DNA gyrase.
Regulated efflux of simocyclinone
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65 mg ml-1. The ermEp*–simX construct did not confer
resistance to the structurally related aminocoumarin DNA
gyrase inhibitor novobiocin, nor to unrelated antibiotics
such as erythromycin, lincomycin, rifampicin, bacitracin
and nisin. These results suggested that simX encodes a
simocyclinone-specific efflux pump. Adjacent to simX is
the divergent gene simR, encoding a potential transcrip-
tional repressor of simX. We also cloned simX into an
integrative vector under the control of its native promoter
and in the absence of simR. This construct (pIJ10461)
only mildly enhanced resistance to simocyclinone D8 in
S. lividans, giving an MIC of 4 mg ml-1.
Mapping the transcription start points of simR and simX
High-resolution S1 nuclease mapping of the simR and
simX promoters was performed using RNA isolated from
the simocyclinone D8 producing organism, S. antibioticus
Tü 6040 (Fig. 2A). A single simR promoter (simRp) was
identified, initiating transcription 20 bp upstream of the
simR ATG start codon, and a single simX promoter
(simXp) was identified, initiating transcription 47 bp
upstream of the simX ATG start codon (Fig. 2A and 3). In
vitro run-off transcription
S. coelicolor RNA polymerase confirmed the presence
and locations of the simR and simX promoters (Fig. 2B).
experimentswith purified
SimR regulates expression of simR and simX
To investigate the regulation of simR and simX, we mea-
sured simR and simX promoter activities in the presence
and absence of SimR, using the integrative luciferase
(luxAB) reporter plasmid pIJ5972 (Aigle et al., 2000;
M. Paget, pers. comm.). Fragments carrying simRp
and simXp were individually cloned into pIJ5972, and
the resulting reporter constructs were introduced into
Fig. 2. A. High-resolution S1 nuclease mapping of the 5′ ends of the simR and simX transcripts using PCR-generated probes and RNA from
the simocyclinone D8 producing organism, S. antibioticus Tü 6040. The most likely transcription start points are indicated by the asterisks. The
G+A Maxam–Gilbert chemical sequencing ladder was generated using the same probe as the one used for S1 nuclease mapping assays.
The size markers are a radiolabelled HinfI digest of FX174 DNA.
B. Run-off transcription from the simR and simX promoters in vitro using purified S. coelicolor RNA polymerase and the templates illustrated
containing the simR–simX intergenic region. Lane numbers correspond to the different templates shown in the illustration below.
T. B. K. Le et al.
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S. lividans in order to probe simR and simX promoter
activities in the absence of SimR. To measure simR and
simX promoter activities in the presence SimR, an inte-
grative plasmid (pIJ10469) carrying simR under the
control of its own promoter was introduced into the strains
already harbouring the promoter-probe plasmids. Trans-
formants were grown on Difco Nutrient Agar to promote
vegetative growth and delay the formation of aerial
hyphae, which may interfere with diffusion of the
luciferase substrate (n-decanal) and with light emission.
Figure 4 shows that simX and simR promoter activities
were repressed 100-fold and 12-fold, respectively, in the
presence of simR.
Purified SimR binds to the simR–simX intergenic region
at two distinct operator sites
The lux reporter data suggested that SimR is a repressor
that regulates its own expression as well as that of simX.
To test this idea, we monitored SimR binding to the simR–
simX intergenic region by electrophoretic mobility shift
assay (EMSA). An N-terminally His6-tagged derivative of
SimR was overexpressed in Escherichia coli and purified
to homogeneity. Increasing concentrations of SimR were
incubated with a radiolabelled probe spanning the simR–
simX intergenic region and the complexes were resolved
on a native gel. Purified SimR bound to the intergenic
region at concentrations as low as 0.23 nM and, as the
concentration of SimR increased, two sets of shifted
protein–DNA complexes became evident, suggesting that
there are two SimR binding sites in the simR–simX inter-
genic region (Fig. 5A).
DNaseI footprinting on both DNA strands was used to
map precisely the SimR operator sites within the simR–
simX intergenic region. Two separate SimR binding sites
were observed, consistent with the two shifted species
seen in the EMSA experiments: the operator closer to
simX was designated OXand the one closer to simR was
Fig. 3. Sequence of the simR–simX intergenic region showing the simRp and simXp transcription start points and putative -10 sequences,
the simR and simX ribosome binding sites (RBS), the extent of the SimR DNaseI footprints on the OX and OR operators, and the imperfect
inverted repeats within the footprints that may represent SimR binding motifs.
Fig. 4. Promoter activities of simR and simX in the presence and absence of simR. simRp–luxAB and simXp–luxAB transcriptional fusions
were created in an integrative luciferase promoter-probe vector (pIJ5972) and assayed in S. lividans, in either the presence or absence of
simR. Plasmid-containing S. lividans strains were grown on Difco Nutrient Agar in single wells of a 96-well microtitre plate (Sterilin) for 3 days.
Each well was inoculated with approximately 5 ¥ 104spores. Plates were exposed to filter paper impregnated with n-decanal for 5 min and
luciferase activities were quantified using a NightOwl camera (Berthold) equipped with WinLight software (Berthold) using a 1 min exposure
time. Values given correspond to the average of three biological replicates from three different spore stocks and standard errors are shown.
Regulated efflux of simocyclinone
© 2009 The Authors
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designated OR(Figs 3 and 6). Within the OXand ORfoot-
prints we identified imperfect inverted repeats that
may represent the binding sequences for the SimR
homodimer. Figure 3 shows these imperfect inverted
repeats, the extent of the SimR DNaseI footprints on the
sequence of the simR–simX intergenic region and the
positions of the simRp and simXp transcription start points
and putative -10 promoter sequences in relation to the
two SimR operators.
SimR binding to the two operators is non-cooperative
In the DNaseI footprinting analysis, OXwas occupied at a
lower concentration of SimR than was OR, suggesting that
OX has a higher affinity for SimR than OR. Competitive
EMSA was used to explore this issue further. In these
experiments, unlabelled fragments containing either OXor
OR were used to compete with a radioactively labelled
simR–simX intergenic fragment containing both OX and
OR. The final concentration of SimR used in the competi-
tive EMSA experiments was set at 20 nM such that all
the labelled probes were in complex with SimR in
the absence of unlabelled competitor DNA. A 1000-fold
excessof unlabelledOX-containing
competed the labelled intergenic probe (no complex for-
mation between SimR and the labelled intergenic probe)
(Fig. 7). However, the same excess of OR-containing frag-
ment could not completely abolish SimR complex forma-
tion with the labelled intergenic probe (Fig. 7), confirming
that SimR binds OXmore tightly than OR.
Using the EMSA data shown in Fig. 5, we determined
the approximate equilibrium dissociation constants (Kds)
for the two complexes to be 1.2 ? 0.4 nM for SimR-OX
and 3.5 ? 1.4 nM for SimR-OR. In order to determine
whether there is cooperativity between SimR binding at
OX and OR, we also determined the Kds of each SimR–
operator complex by EMSA using probes containing only
OXor only OR(data not shown), instead of the full simR–
simX intergenic region. The Kds were found to be
0.9 ? 0.2 nM for SimR-OXand 3.6 ? 0.3 nM for SimR-OR.
The Kds for the two SimR–operator complexes did not
change substantially when the two operators were sepa-
rated, suggesting that SimR binding to its two operators is
Exogenous simocyclinone D8 induces expression of the
SimX efflux pump in vivo
In order to determine whether the simR and simX promot-
ers respond to simocyclinone D8 in vivo, we used the
S. lividans luxAB reporter system to measure simRp and
simXp activities in the presence of SimR and in response
to exogenously added antibiotic. Figure 8A shows the
response curve of simR and simX promoter activity to
Fig. 5. A. Electrophoretic mobility shift assay
showing binding of purified SimR to the
simR–simX intergenic region. Bands
corresponding to protein–DNA complexes
(Bound) and free DNA (Free) are indicated.
The final concentration of SimR is indicated
above each lane.
B. Saturation curve of the data from EMSA
experiments. EMSA data were collected and
analysed on a PhosphorImager (FujiFilm)
using Multi Gauge image analysis software
(FujiFilm). Two independent EMSAs were
carried out [one of which is shown in (A)] and
the mean values calculated. Standard errors
are shown. Saturation curves (saturation
fraction against concentration of protein) were
fitted with SigmaPlot (see Experimental
procedures) to determine Kds. In all EMSA
experiments, SimR was present in molar
excess over the probe.
T. B. K. Le et al.
© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 72, 1462–1474
Page 6
increasing concentrations of simocyclinone D8. Both
promoters were induced by simocylinone D8, suggesting
that the antibiotic can relieve SimR-mediated repression
of both simRp and simXp. The highest concentration
tested was 0.6 mg ml-1because S. lividans is sensitive to
simocylinone D8 (MIC = 2 mg ml-1).
Simocyclinone D8 dissociates SimR from the
simR–simX intergenic region
To determine if SimR responds directly to simocyclinone
D8, we examined the effect of the antibiotic on SimR–
operator complex formation by EMSA(Fig. 9A). The SimR
concentration was held constant and an increasing con-
centration of simocyclinone D8 was introduced into the
binding reaction. As the simocyclinone D8 concentration
increased, there was a progressive decrease in SimR–
DNA complex formation and a concomitant liberation of
free probe; 62.5 mM simocyclinone D8 was sufficient to
dissociate the SimR–DNA complexes almost completely.
This effect was not due to DMSO, the simocyclinone D8
solvent, as equivalent amounts of pure DMSO had no
effect on SimR–DNAcomplex formation (data not shown).
Furthermore, to test specificity, we examined the effect of
simocyclinone D8 on the DNA binding activity of ActR, a
TetR homologue that regulates actinorhodin export in
Fig. 6. DNaseI footprinting analysis of SimR
binding to the simR–simX intergenic region. A
DNA fragment containing the simR–simX
intergenic region, 5′ end-labelled on either the
upper strand (left panel) or the lower strand
(right panel), was exposed to DNaseI in the
presence of increasing concentrations of
SimR. The sequencing ladders were
generated by subjecting the probes to
Maxam–Gilbert G+A chemical sequencing.
Regions protected from DNaseI cleavage
(operators OX and OR) are indicated with
vertical bars and inverted repeats within the
DNaseI protected regions are indicated by
convergent arrows; these features are also
highlighted on the DNA sequence in Fig. 3.
Fig. 7. Competitive electrophoretic mobility
shift assay comparing the binding affinity of
SimR to OX and OR. All lanes contain SimR at
a final concentration of 20 nM and a constant
amount of a radiolabelled simR–simX
intergenic probe that carries both OX and OR.
The fold excess (over the radiolabelled probe)
of a competing unlabelled fragment containing
either OX or OR is indicated above each lane.
Regulated efflux of simocyclinone
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Page 7
Streptomyces coelicolor (see Discussion) and found that
100 mM simocyclinone D8 had no effect on ActR binding
to its cognate operator (data not shown). These results
show that simocyclinone D8 is able to specifically disrupt
SimR–DNA complex formation.
An intermediate in the simocyclinone biosynthetic
pathway induces simX in vivo and dissociates SimR
from its operators in vitro
In addition to simocyclinone D8, we also tested the ability
of simocyclinone C4, a natural intermediate in the D8
biosynthetic pathway, to induce the simR and simX pro-
moters in vivo and to dissociate SimR from its operators in
vitro. Simocylinone C4 lacks the aminocoumarin ring
present in the mature antibiotic (Fig. 1) and is essentially
inactive as a DNA gyrase inhibitor; the simocyclinone
D8 IC50= 0.1 mM,whereas
IC50> 100 mM (M.J. Edwards et al., unpubl. data).
Simocyclinone C4 induced the simR and simX promoters
in vivo, although somewhat more weakly than the mature
thesimocyclinone C4
antibiotic (Fig. 8B). Because simocyclinone C4 is not an
active antibiotic, we were able to test higher concentra-
tions than for simocyclinone D8. Consistent with the in
vivo inductions, simocyclinone C4 also caused SimR to
dissociate from its binding sites in vitro, although again
somewhat more weakly than simocyclinone D8; 250 mM
simocyclinone C4 was sufficient to abolish all complex
formation (Fig. 9B).
This report shows that simX encodes a simocyclinone D8
efflux pump and that the simX promoter is directly
repressed by SimR, which binds to two operator sites in
the simR–simX intergenic region. Simocyclinone D8 abol-
ishes DNA binding by SimR, coupling the biosynthesis of
simocyclinone to its export.
Simocyclinone D8 is a potent inhibitor of DNA gyrase
supercoiling (IC50= 0.1 mM) (Flatman et al., 2005; Oppe-
gard et al., 2009; M.J. Edwards et al., unpubl. data). The
recent structure of the antibiotic bound to the DNA gyrase
Fig. 8. Induction of the simR and simX
promoters in vivo by (A) simocyclinone D8
and (B) the biosynthetic intermediate
simocyclinone C4. S. lividans containing
pIJ10469 (carrying simR under its native
promoter) together with luciferase
promoter-probe plasmids pIJ10465
(simXp–luxAB) or pIJ10466 (simRp–luxAB)
were assayed. Values given correspond to the
average of three biological replicates from
three different spore stocks and standard
errors are shown. For further details, see the
legend of Fig. 4.
T. B. K. Le et al.
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Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 72, 1462–1474
Page 8
A subunit shows that the two moieties at the ends of
simocyclinone D8, the aminocoumarin ring and the
angucyclic polyketide, bind to two separate, well-defined
pockets within the GyrA DNA binding saddle, linked by
theinterveningtetraenelinkerand D-olivosemoieties(M.J.
Edwards et al., unpubl. data). Given the prominence of the
aminocoumarin ring in the overall binding of the antibiotic
to the GyrAsubunit, it is not surprising that the biosynthetic
marin ring (Fig. 1), is essentially inactive as a DNAgyrase
inhibitor (IC50> 100 mM). Importantly, however, although
simocyclinone C4 is not a DNA gyrase inhibitor, it can
efficiently derepress SimR in vivo and in vitro.
The most striking analogy to the research presented
here is the work of Nodwell and colleagues on the regu-
lation of efflux of actinorhodin (Ahn et al., 2007; Hopwood,
2007; Tahlan et al., 2007; 2008; Willems et al., 2008).
Actinorhodin is a blue-pigmented, six-ring polyketide
antibiotic made by S. coelicolor (Bystrykh et al., 1996).
Within the actinorhodin biosynthetic cluster are two
co-transcribed genes, actA and actII-ORF3, encoding
integral membrane proteins implicated in actinorhodin
export (Caballero et al., 1991; Fernandez-Moreno et al.,
1991). Expression of these two genes is regulated by a
TetR-like protein, ActR, the product of the adjacent, diver-
gently transcribed gene. Nodwell and colleagues have
been characterizing ligands that relieve repression by
ActR. Importantly, they showed that, in addition to the
mature six-ring antibiotic, three-ring intermediates from
the biosynthetic pathway also relieve repression by ActR.
From this, they suggested that the ability of actinorhodin
intermediates to relieve repression by ActR might provide
a 'feed-forward' mechanism that would ensure expression
of the ActA efflux pump prior to the build-up of a toxic
concentration of the mature antibiotic (Ahn et al., 2007;
Hopwood, 2007; Tahlan et al., 2007; 2008; Willems et al.,
2008). Similarly, the ability of an inactive simocyclinone
intermediate to relieve repression by SimR might also act
as a biosynthetic checkpoint to ensure feed-forward regu-
lation of simocyclinone export.
As applied to simocyclinone (or actinorhodin) export,
the feed-forward hypothesis is speculative. We have
shown that the pathway intermediate simocyclinone
C4 induces simX expression in vivo when applied
exogenously. However, for a feed-forward mechanism to
operate in the producing organism, simocyclinone C4 or
other SimR-binding pathway intermediates would have to
accumulate in the cytoplasm to a concentration high
enough to trigger simX expression, and the cytoplasmic
concentrations of pathway intermediates are hard to
determine experimentally. However, there are other
examples in the literature where the TetR-like protein
blocks expression not only of the exporter gene, but also
of late biosynthetic genes, and in these cases it seems
that an intermediate in the pathway must be responsible
for inducing expression of these genes (i.e. feed-forward
Fig. 9. Electrophoretic mobility shift assays
showing that (A) simocyclinone D8 and (B) its
biosynthetic intermediate simocyclinone C4
abolish SimR DNA binding activity. All lanes
contain a constant amount of SimR and
radiolabelled simR–simX intergenic fragment
containing both OX and OR. Bands
corresponding to protein–DNA complexes
(Bound) and free DNA (Free) are indicated.
The final concentrations of simocyclinone D8
or simocyclinone C4 are indicated above each
Regulated efflux of simocyclinone
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Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 72, 1462–1474
Page 9
activation), as induction of the late biosynthetic enzymes
is required to generate the mature antibiotic.
In the landomycin A producer, Streptomyces cyanoge-
nus, a TetR-family regulator, LanK, represses expression
of lanJ, encoding a putative landomycin A efflux pump
(Ostash et al., 2008). LanK is derepressed by mature
landomycin A, which carries a hexasaccharide chain, but
also by intermediates in the pathway that carry only a
pentasaccharide or a trisaccharide chain (Ostash et al.,
2008). However, lanJ is co-transcribed with several down-
stream biosynthetic genes involved in late glycosylation
steps. As a consequence, LanK couples production of
intermediates not only to assembly of the export machin-
ery, but also to expression of late biosynthetic enzymes
that attach the final sugars to produce mature landomycin
A (Ostash et al., 2008). A more complex example con-
cerns the biosynthesis of the clinically important antican-
cer agents daunorubicin and doxorubicin made by
Streptomyces peucetius. In this system, binding of
rhodomycin D appears to derepress the TetR-like regula-
tor DnrO, activating a cascade involving two further tran-
scriptionfactors,DnrN and
expression of the resistance genes and of late biosyn-
thetic genes (Otten et al., 1995; Jiang and Hutchinson,
In addition to repressing the simX promoter, SimR also
directly negatively regulates its own expression. In other
systems, negative autoregulation has been shown to
confer specific functions that are absent in systems that
have simple regulation. Many of these studies on the
design principles of genetic circuits that incorporate nega-
tive autoregulation have exploited TetR, the founding
member of the family to which SimR belongs (Becskei
and Serrano, 2000; Rosenfeld et al., 2002; D. Madar and
U. Alon, pers. comm.). These studies show that negative
autoregulatory feedback loops in gene circuits can
provide stability, thereby limiting stochastic fluctuations in
the system (Becskei and Serrano, 2000). In addition,
negative autoregulation can speed the response time of
the transcription network to a stimulus (Rosenfeld et al.,
2002), and it can broaden the dynamic range of the input
signal to which the downstream genes respond (D. Madar
and U. Alon, pers. comm.).
clinone D8 in the producing organism. In the strepto-
mycetes that produce the aminocoumarins novobiocin
(Streptomyces sphaeroides), clorobiocin (Streptomyces
roseochromogenes) and coumermycin A1 (Streptomyces
rishiriensis), expression of an aminocoumarin-resistant
GyrBRgyrase subunit encoded within the biosynthetic
gene cluster is turned on during antibiotic production,
conferring resistance (Thiara and Cundliffe, 1988; 1989;
1993; Schmutz et al., 2003).The absence of an equivalent
intermediates like
DnrI, whichleads to
gyrB resistance gene in the simocyclinone biosynthetic
sequenced, until it was subsequently and unexpectedly
shown that simocyclinone has a completely different mode
of action, binding instead to the GyrA subunit of the
enzyme (Flatman et al., 2005; M.J. Edwards et al., unpubl.
data). However, no GyrARsubunit is encoded within the
sim cluster either (Galm et al., 2002; Trefzer et al., 2002).
Expressing simX from the strong, constitutive promoter,
ermEp*, conferred simocyclinone resistance on the heter-
ologous host S. lividans (MIC 65 mg ml-1instead of
2 mg ml-1for S. lividans alone), showing that SimX can act
as an effective resistance determinant when expressed at
appropriate levels. However, the MIC was only 4 mg ml-1
when simX was expressed from its own promoter in the
absence of SimR. This may reflect additional levels of
simX regulation that have yet to be uncovered. For
example, in addition to its repression by SimR, it is pos-
sible that simX expression in S. antibioticus might also be
activated by a simocyclinone pathway-specific activator
that is absent from S. lividans. Alternatively, it is possible
that SimX-mediated efflux is not the principal simocycli-
none resistance mechanism in S. antibioticus.
Experimental procedures
Bacterial strains, plasmids, oligonucleotides, culture
conditions and conjugal plasmid transfer from E. coli to
Streptomyces spp.
Strains, plasmids and oligonucleotides are described in
Table 1. Unless stated otherwise, S. lividans 1326 was grown
on SFM (Kieser et al., 2000) and S. antibioticus Tü 6040 was
grown on MYMTap [0.4% (w/v) maltose, 0.4% (w/v) Yeast
Extract, 1% (w/v) Malt Extract, supplemented trace elements;
made up in tap water]. For conjugal transfer into Streptomy-
ces (Paget et al., 1999), plasmids were introduced into the
dam dcm hsdS E. coli strain ET12567, which carries the
non-transmissible, oriT-mobilizing plasmid pUZ8002 (Paget
et al., 1999). All cloned PCR fragments were verified by
Fermentation, isolation and purification of simocyclinone
D8 and simocyclinone C4
Simocyclinone D8 was isolated as described by Schimana
et al. (2000). Briefly, S. antibioticus Tü 6040 was fermented in
a complex medium consisting of 2% (w/v) mannitol and 2%
(w/v) soybean meal in a 20 l fermentor, and simocyclinones
were extracted from the mycelium with methanol. Pure
simocyclinone D8 was obtained after reversed-phase HPLC
using Nucleosil-100 C-18 material and 0.01% trifluoroacetic
acid-acetonitrile gradient elution, resulting in a dark yellow
powder after drying. Pure simocyclinone C4 was isolated
using essentially the same procedure, but fermentation was
carried out in a defined medium containing 20% (v/v) glycerol
and 0.15% (w/v) L-arginine to maximize simocyclinone C4
T. B. K. Le et al.
© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 72, 1462–1474
Page 10
production (Theobald et al., 2000). The nature and purity of
the simocyclinone D8 and C4 samples were confirmed by
chromatography-mass spectrometry (Holzenkämpfer et al.,
2002; M.J. Edwards, pers. comm.)
spectroscopy andliquid
Construction of simX and simEX2 expression plasmids
and minimal inhibitory concentration (MIC)
simX and simEX2 were amplified by PCR using primers
carrying NdeI (upstream) and HindIII (downstream) sites [for
simX, simEX1-F-NdeI and simEX1-R-HindIII; for simEX2,
simEX2-F-NdeI and simEX2-R-HindIII (Table 1)]. To express
the simX and simEX2 genes from the ermEp* promoter, the
fragmentswere clonedinto
(Table 1) to generate pIJ10480 and pIJ10481 respectively. To
express simX from its own promoter, a fragment containing
the simX coding sequence and 122 bp of DNA upstream of
the simX translation start codon was amplified by PCR using
the primers intRX-138-F and simR-int-simX-R (Table 1), and
cloned into the SmaI site of pUC19. The resulting construct
was digested with BamHI and EcoRI and the insert was
cloned into BamHI-EcoRI-cut pSET152 to give pIJ10461.
Constructs were transferred into S. lividans by conjugation.
Table 1. Bacterial strains, plasmids and primers used in this study.
Relevant genotype/commentsSource/reference
S. antibioticus Tü 6040
S. lividans 1326
E. coli
ET12567 (pUZ8002)
Environmental isolate; original producer of simocyclinone D8
Wild-type SLP2+SLP3+
Schimana et al. (2000)
Kieser et al. (2000)
ET12567 containing helper plasmid pUZ8002Paget et al. (1999)
FC311 attP-int derived integration vector for the conjugal transfer of DNA
from E. coli to Streptomyces (AprR)
FBT1 attP-int derived integration vector for the conjugal transfer of DNA
from E. coli to Streptomyces (HygR)
Integrative Streptomyces promoter-probe plasmid based on TTA codon-free
derivatives of the luxAB reporter genes
Integrative expression vector based on the strong, constitutive ermE* promoter
pSET152 carrying simX under the control of its own promoter, without simR
pIJ5972 simXp–luxAB
pIJ5972 simRp–luxAB
pMS82 carrying simR under the control of its native promoter
pIJ10257 ermEp*–simX
pIJ10257 ermEp*–simEX2
pET28a derivative expressing His6-tagged SimR
Bierman et al. (1992)
Gregory et al. (2003)
pIJ5972Aigle et al. (2000);
M. Paget, pers. comm.
Hong et al. (2005) pIJ10257
This study
This study
This study
This study
This study
This study
This study
Regulated efflux of simocyclinone
© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 72, 1462–1474
Page 11
For MIC determinations, approximately 5 ¥ 104spores
were added to each well of a 96-well microtitre plate (Sterilin),
containing a twofold simocyclinone D8 dilution series in
SMMS medium (Kieser et al., 2000). The plates were evalu-
ated everyday for 5 days after inoculation, and the MIC was
defined as the lowest concentration at which no growth was
observed. Simocyclinone D8 was dissolved in DMSO with the
final concentration of DMSO in the agar not to exceed 1%
Construction of luxAB reporter plasmids and luciferase
activity measurements
To probe simXp and simRp activities in the absence of SimR,
the promoter regions (500 bp upstream of the translation start
codons) were amplified by PCR using upstream primers car-
rying an EcoRI site and downstream primers carrying a
BamHI site [primers pX-F-EcoRI and pX-R-BamHI for simXp;
pR-F-BamHI and pR-R-EcoRI for simRp (Table 1)]. The
simXp and simRp promoter fragments were cloned into
EcoRI-BamHI-cut pIJ5972, an integrative, Streptomyces
promoter-probe plasmid based on TTAcodon-free derivatives
of the luxAB reporter genes (Aigle et al., 2000; M. Paget,
pers. comm.), to create plasmids pIJ10465 and pIJ10466,
respectively, and these reporter constructs were transferred
by conjugation into S. lividans. To probe simXp and simRp
activities in the presence of SimR, a plasmid carrying simR
under the control of its own promoter (pIJ10469) was trans-
ferred by conjugation into strains already harbouring the
promoter-probe plasmids pIJ0465 and pIJ10466. To con-
struct pIJ10469, simR with its promoter was amplified by
PCR using an upstream primer carrying a HindIII site and a
downstream primer carrying a KpnI site [primers MS82-simR-
F-HindIII and MS82-simR-R-KpnI (Table 1)] and the fragment
was cloned into HindIII-KpnI-cut pMS82.
Plasmid-containing strains were grown on Difco Nutrient
Agar in single wells of a 96-well microtitre plate (Sterilin) for
3 days. Each well was inoculated with approximately 5 ¥ 104
spores. Plates were exposed to filter paper impregnated with
n-decanal for 5 min and luciferase activities were quantified
using a NightOwl camera (Berthold) equipped with WinLight
software (Berthold) using a 1 min exposure time. Values
given correspond to the average of three biological replicates
from three different spore stocks.
Overexpression and purification of SimR
The simR gene was chemically synthesized with codon opti-
mization (Genescript) for expression in E. coli and ligated into
pET28a (Novagen) to give pIJ10490, which was introduced
into E. coli BL21 (DE3) pLys. Recombinant His6-SimR was
purified from a 250 ml LB culture induced with a 0.5 mM final
concentration of IPTG at 30°C for 4 h. The cell pellet was
re-suspended in 20 ml Buffer A [10 mM Tris-HCl, pH 7.8,
150 mM NaCl, 50 mM imidazole, 1¥ Complete Mini, EDTA-
10 microns for three cycles), and the lysate was clarified by
centrifugation. The filtered cell lysate was loaded onto a 1 ml
Ni-loaded Hi-Trap Chelating HD column (GE Healthcare) and
His-tagged SimR was eluted with Buffer B [10 mM Tris-HCl,
sonicated(20 s at
pH 7.8, 150 mM NaCl, 500 mM imidazole] and dialysed into
Storage Buffer [10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 10%
(v/v) glycerol]. The resulting preparation of SimR was 98%
pure as judged by SDS-PAGE and was stored at -80°C.
Electrophoretic mobility shift assays and determination
of dissociation constants (Kds)
The EMSA DNA probe spanning the entire simR–simX inter-
genic region was amplified by PCR using primers intRX-
138-F and intRX-138-R (Table 1) and then 5′ end-labelled
using [g32-P]-ATP and T4 polynucleotide kinase (New
England Biolabs). The competitor DNA carrying only ORwas
amplified using primers intOR-130-F and intOR-130-R, and
the competitor DNA carrying only OX was amplified using
primers intOX-123-F and intOX-123-R (Table 1). Binding of
SimR to DNA was carried out in 20 ml EMSA buffer [20 mM
Tris, pH 8.0, 1 mg poly(dI-dC), 1 mM EDTA, 100 mM NaCl,
0.5 mM DTT, 8% (v/v) glycerol] containing 0.1 nM radiola-
belled DNA(approximately
amounts of SimR. After incubation at 30°C for 5 min, the
binding reaction mixtures were loaded on 5% (w/v) native
polyacrylamide gels and run in TBE buffer at 120 V for
45 min. The effect of simocyclinone D8 and simocyclinone C4
on the ability of SimR to bind DNA was tested by adding the
compounds to the EMSA buffer. The simocyclinone D8 and
simocyclinone C4 stock solutions were made up in 100%
EMSA data were collected and analysed on a Phosphor-
Imager (FujiFilm) using Multi Gauge image analysis software
(FujiFilm). Two independent EMSAs were carried out for each
probe and mean values were calculated. In order to calculate
Kds for SimR binding to each operator when the two operator
sites were separated, saturation curves (percentage of probe
bound against concentration of protein) were fitted using
SigmaPlot (one site saturation model). For Kds of each
operator when the two sites were coupled, a random-order
binding model was used, where Y = [S]/(2(Kd1+ [S])) + [S]/
(2(Kd2+ [S])), in which Y is the fractional saturation, Kd1and
Kd2 are dissociation constant of SimR binding to OX and OR
operators, respectively, and [S] is the concentration of
protein. The fractional saturation was calculated from inten-
sities of EMSA bands in each lane (Fig. 5A) as followed,
Y = (0.5*intensity of middle band + intensity of top band)/
(intensity of all bands in a lane). The equation was then fitted
using SigmaPlot to determine Kds.
8000 c.p.m.)and varying
DNaseI footprinting
Templates for DNaseI footprinting were amplified by PCR
using one unlabelled primer and one primer 5′ end-labelled
using [g32-P]-ATP and T4 polynucleotide kinase (New
England Biolabs). The primers were intRX-138-F and intRX-
138-R (Table 1), the same pair used to generate the simR–
simX intergenic region probe for the EMSA experiments.
DNaseI footprinting assays were performed in 40 ml EMSA
buffer containing approximately 180 000 c.p.m. radiolabelled
DNA and varying amounts of SimR. After incubation at room
temperature for 5 min, 10 ml of DNaseI (10 units in 10 mM
CaCl2) was added and the incubation was continued for a
T. B. K. Le et al.
© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 72, 1462–1474
Page 12
further 60 s. Reactions were stopped by adding 140 ml
DNaseI stop solution (200 mM unbuffered sodium acetate,
30 mM EDTA, 0.15% SDS and 0.1 mg ml-1yeast tRNA), the
samples were precipitated with ethanol, and the pellets were
dried and dissolved in 5 ml Sequencing Loading Dye [80%
(v/v) formamide, 10 mM NaOH, 1 mM EDTA, 0.1% (w/v)
xylene cyanol and 0.1% (w/v) bromophenol blue]. After
heating at 80°C for 3 min and cooling on ice, the samples
were run on a 6% (w/v) polyacrylamide/8 M urea sequencing
gel, which was dried and analysed using a PhosphorImager
(FujiFilm). A G+A sequencing ladder was generated from the
template DNA by chemical sequencing (Maxam and Gilbert,
RNA preparation, S1 nuclease protection analysis and
in vitro run-off transcription
For RNA preparation, approximately 109S. antibioticus
spores were germinated by heat-shock treatment in 5 ml TES
buffer (0.05 M, pH 8) at 50°C for 10 min, then diluted with an
equal volume of double-strength germination medium [1%
(w/v) Difco yeast extract, 1% (w/v) Difco casaminoacids, 0.01
M CaCl2] and incubated with shaking at 37°C for 6 h (modi-
fied from Kieser et al., 2000). Germinated spores were inocu-
lated into NMMP (Kieser et al., 2000) and incubated with
shaking for a further 15 h at 30°C. RNA was prepared essen-
tially as described by Hesketh et al. (2007), but with minor
modifications taken from the Qiagen RNA Extraction Kit pro-
cedure (Qiagen). Probes for S1 nuclease protection analysis
were generated by PCR using a 5′ end-labelled oligonucle-
otide internal to the ORF and an unlabelled upstream primer
[for simXp, primers S1-probeX-F and S1-probeX-R; for
simRp, primers S1-probeR-F and S1-probeR-R; (Table 1)].
Hybridizations were carried out in sodium trichloroacetic acid
buffer at 45°C overnight after denaturation at 65°C for 10 min
(Kieser et al., 2000). A G+A sequencing ladder was gener-
ated by chemical sequencing (Maxam and Gilbert, 1980).
RNApolymerase containing a mixture of sigma factors was
purified as described previously (Buttner et al., 1988) from
S. coelicolor M600 grown to exponential phase in YEME
(Kieser et al., 2000). In vitro transcription was carried out as
described previously (Buttner et al., 1987). Three different
templates for in vitro run-off transcription were generated by
PCR that differed in their left or right ends, to allow the simR
and simX transcripts to be identified unambiguously. Tem-
plates were generated using the following primers: Template
1, Invitro1-F and Invitro1-R; Template 2, Invitro1-F and
Invitro2-R; Template 3, Invitro3-F and Invitro1-R (Table 1).
We thank Ray Dixon, Justin Nodwell, David Hopwood and
Daniel Madar for their comments on the manuscript, Richard
Little, Nick Tucker, Stephen Bornemann and Maureen Bibb
for practical help and advice, Luis Servín-González for the gift
of S. coelicolor RNA polymerase, and Andreas Bechthold for
providing us with cosmids covering the sim cluster. This work
was funded by a John Innes Centre Rotation Studentship (to
T.B.K.L.) and by a grant-in-aid to the John Innes Centre from
the BBSRC.
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© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 72, 1462–1474


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View gay Singles in Kennedale
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Though the Board of Directors of the Big Bear City Community Services District has held the line in terms of rate increases for customers within their service area, the Board will consider an increase to new connection fees. The introduction of ordinances addressing connection fee increases for water, sewer and commercial disposal service will be made at this evening's meeting of CSD, to be held at their offices at 139 E. Big Bear Boulevard at 5:30pm.
Related posts:
- Community Services District Customers Will Have Delinquent Trash Collection, Sewer Use and Water Service Charges Placed on Their Tax Roll
- Community Services District Welcomes Garlands, Gives Bradley a Send-Off; Connection Fee Increases Also Approved in Monday Meeting
- Community Services District Directors Meet This Evening; Other Valley Activities, Meetings Canceled Due to Storm


text:
Who Gets Pinched By Health Overhaul?
The president's promise of expanding coverage to tens of millions of uninsured Americans would come at a heavy personal price for some.
Health coverage under the overhaul plan would be mandatory, with a few exemptions, and those who don't get coverage through their jobs or a government plan like Medicaid would face a penalty if they don't buy insurance themselves.
That's where subsidies are supposed to make a difference, but healthy young people who've long gambled they can get by without coverage and working folks who are just eking out a living may balk anyway. Subsidies may not help them much, if at all, so an insurance mandate could cost these people a pretty penny.
The Wall Street Journal's Vanessa Fuhrmans takes a look at the universal coverage laboratory that is Massachusetts for clues. Three years ago the state made health coverage mandatory and provides subsidies to help people afford it. The law lowered the rate of uninsured to about 3 percent, the lowest in the nation, but there are
Take, for instance, a community college professor who doesn't get insurance at work and whose family-of-three's income is just north of $60,000 a year, too much to quality for help. "I can't use up all of my savings just to buy mandatory insurance," Ron Norton tells the Journal, adding, "it's like penalizing 'the homeless for refusing to buy a mansion."In 2008, he paid a penalty of about $1,000 rather than buy coverage that he considered too expensive and not worth the money.
The Washington Post zeroes in on the financial price young people may have to pay under an insurance mandate.
Even the cheapest plan contemplated by a Senate Finance Committee bill in the works could cost more than $100 a month, which might lead young single people to choose paying a fine that could be less than a $1,000 a year.
The implications for passage of an overhaul loom large. "If you're talking about millions of people who will have to buy insurance by themselves, this could be a difficult political issue," Harvard's Robert Blendon tells the Journal. "Unless subsidies are substantial, you're going to have middle-class resistance to this."
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Friend or foe?
This plant has been happily growing this summer next to my stairs but I cannot figure out what it is. It doesn't seem to be getting any taller (about 8 inches high currently), but it is getting fuller. When it was small, I swore it was a volunteer tomato plant, but now I know it isn't. Any ideas? The stem is thick like a succulent, and the leaves have very little distinctive smell. No flowers...just happy, healthy stems.
Having trudged through acres of it, I can affirm that Conium maculatum has a strong, fetid, unpleasant scent when the leaves are bruised, plus the stems usually have purple blotches. So if, as ellez6 says, the leaves have "very little distinctive smell", that wouldn't correspond with Conium maculatum in my experience.
Looking at photos of Anthriscus, I think the foliage bears some resemblance, plus Anthriscus reputedly has little scent and has been introduced to parts of the U.S.
That's about the extent of my contribution here as I'm not so familiar with umbellifers, especially outside my locale and when they aren't in bloom. So, unfortunately, I can't suggest plausible alternatives.
Maybe check this site > see LINK
Here is a link that might be useful: Conium versus Anthriscus


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Factors related to cycling performance
Date: May 2001
Creator: Naukkarinen, Vesa
Description: There were two primary goals in this investigation. The first goal was to determine if results from field tests (time-trials and a Conconi incremental test) are related to performance in mass-start long-distance bicycle races. The second goal was to investigate inter-relationships among field test variables. The testing variables measured were critical velocity (CV), Conconi anaerobic threshold (AT) velocity, 4mM AT velocity, fatigue index, peak blood lactate, and anaerobic work capacity. Participants were USCF 30 category 1 through 5 cyclists. Participants performed one 20.75 km and two 10.37 km all-out tests in the field. They also performed an incremental test. The tests were performed at one-week intervals. Results from the field tests were compared to recent mass-start racing performance. Results indicated that Conconi AT velocity was related to performance in a 161-km race. There was also a relationship between 4mM AT velocity and CV and between Conconi AT velocity and 4mM AT velocity. It was concluded that field tests might provide information about performance ability in mass-start long-distance bicycle races.
Contributing Partner: UNT Libraries
Use of repeated tests and rolling breath averages affects the precision of quantifying the VO2 response profile in moderate intensity cycling.
Date: August 2006
Creator: Pedini, Daniela Marie
Description: The purpose of this study was to determine whether working in the field of deaf education, as opposed to general education, results in a higher level of technology integration. A secondary goal was to determine if deaf educators who are deaf integrate technology at a higher level than their hearing counterparts. The instrument chosen for this study was the LoTi Technology Use Profile, a tool used to explore the role of technology in the classroom. A total of 92 participates were included in the study of which 48 were regular educators and 44 were deaf educators. The participants were selected from a population pool whereby teachers were presumably pre-disposed to using technology based upon their attendance at a technology training session in the form of a conference or a class. Deaf educators as a whole did not perform as well as general educators on the LoTi scales. Given the fact that the technology-minded general educators who comprised the sample population of this study scored exceptionally high on the LoTi scales, further research is needed to ensure comparability between the two groups. The findings of the current study do suggest, though, that deaf educators who are deaf have the potential to ...
Contributing Partner: UNT Libraries
VO2 response profile in heavy intensity cycling after heavy intensity arm or leg exercise.
Date: December 2006
Creator: Ptak, April Louise
Description: The elevated CO2 levels, elevated temperature, and lower blood pH that may occur during exercise should enhance O2 delivery to the exercising muscles. It was hypothesized that performance of prior exercise (PE) would result in a faster VO2 response, as well as a reduced slow component contribution, in subsequent exercise bouts. Five women (21 ± 1 yr) and 10 men (23 ± 2 yr) performed nine 6-min bouts of heavy intensity cycle ergometer exercise (i.e., above the ventilatory threshold, individually determined by an incremental test). Three bouts were performed without prior heavy exercise (noPE), three were performed 6 min after a 6-min bout of heavy intensity arm cranking (PEA), and three were performed 6 min after a 6-min bout of heavy intensity cycle ergometer exercise (PEL). Breath-by-breath VO2 data from each of the three sets of three tests were combined and fitted to a two-component model, which ignores the cardiodynamic phase. The primary and slow component amplitudes were truncated to reflect actual increases in VO2 in each phase. The effects of PE on the time constant of the primary component were inconsistent. As hypothesized, the amplitude of the slow component was reduced by PE (noPE vs PEA vs. PEL: 25% ...
Contributing Partner: UNT Libraries
The Relationship Among Gender, Gender Role Attitudes, and the Anticipated Commitment to Career, Marriage, Family, and Housework
Date: August 2002
Creator: Hunter, Kate
Description: The relationships between gender, gender role attitudes, and participants' anticipation of future life roles (career, marriage, family, and homecare) were examined. Participants consisted of 297 single college students between the ages of 17-29 years (M = 20). Females reported significantly (p< .01) more egalitarian gender role attitudes than males. Significant results were found for the relationship between gender and anticipated life roles (p< .01) as well as between gender role attitudes and anticipated life roles (career role value, r = .14 and marital role value, r = - .18). The study findings suggest a possible gender conflict for females with more egalitarian gender role attitudes and behavior intentions and their male counterparts.
Contributing Partner: UNT Libraries
Muscular Differences Between Female Power and Endurance Athletes
Date: August 1997
Creator: Akers, Allen (Roy Allen)
Description: The purpose of this study was to compare the torque generating capabilities and fatigue responses of female power athletes, female endurance athletes, and age-matched female non-athletic controls.
Contributing Partner: UNT Libraries
The Effect of Steroid Dose Regimen on the Relationship Between Lower Extremity Muscle Function and Cardiac Function in Post Heart Transplant Patients
Date: May 2001
Creator: Galatas, Mary V.
Description: Differences in cardiovascular/aerobic function in heart transplant patients might be attributed to the rate of corticosteroid withdrawal and/or to skeletal muscle function. This hypothesis was tested among nine male, cardiac transplant recipients. Prednisone dosage was monitored, and isokinetic strength testing was performed at 4 different time periods throughout the first year post-transplantation. Cardiovascular/aerobic measurements were obtained at the fourth time period. Pre-surgery characteristics were obtained from the patient's medical record. Significant Pearson-product moment correlations were only found between muscle function and aerobic function and between pre-surgery characteristics and cardiovascular/aerobic performance. The results of this study show no evidence that rapid reduction of prednisone dosage enhances aerobic function by benefiting skeletal muscle function.
Contributing Partner: UNT Libraries
Oxygen Uptake Kinetics in Severe Intensity Exercise
Date: May 2000
Creator: Blumoff, Sonja
Description: The purpose of this study was to describe mathematically the oxygen uptake kinetics during cycle ergometry, and to examine the effect of intensity on the kinetic responses within the severe domain. Sixteen volunteers performed a series of exercise tests at a range of intensities selected to elicit fatigue in ~3 to 10 min. A simple mono-exponential model effectively described the response across all intensities. There was a positive correlation between the response time and the time to fatigue, demonstrating that the maximal oxygen uptake was achieved faster at higher intensities within the severe domain. Models incorporating two components effectively described the responses only in tests lasting 8 min or more. It was concluded that there is a second, slow component in the oxygen uptake response only at the lower intensities within the severe domain.
Contributing Partner: UNT Libraries
NCAA division I athletes preferences for coaching behaviors.
Date: December 2003
Creator: Barnes, Kelly A.
Description: The purpose of this study was to determine whether coaching behavior preferences of NCAA Division I athletes differ as a function of gender and type of sport. The Coaching Behavior Questionnare (CBQ; Martin & Barnes, 1999) was administered to 195 NCAA Division I athletes. Gender and sport type were the independent variables and the participant's mean scores for the subscales on the CBQ were the dependent variables. Descriptive statistics revealed that, overall, NCAA Division I athletes prefer positive and instructional behaviors more than non-responses or negative behaviors. A 2 (gender) x 3 (type of sport) MANOVA and follow-up discriminant function analysis indicated that coaching behavior preferences differed as a function of gender and type of sport played. Thus, NCAA Division I coaches should consider both individual and situational characteristics when working with their athletes to achieve the desired outcome.
Contributing Partner: UNT Libraries
The effect of mode and intensity on VO2 kinetics in the severe intensity domain
Date: May 2000
Creator: Updyke, Rhonda S.
Description: The purpose of this study was to evaluate the effect of mode and intensity on VO2 kinetics in the severe intensity domain. Seventeen participants completed 3-7 tests each on a cycle ergometer and treadmill. For each test, Tfatigue, VO2max, Tmean response, VO2GAIN, TVO2max and T@VO2max were determined. Linear regression techniques were used to describe the relationship between TVO2max and Tfatigue . VO2max values were higher in running. The VO2 response profile was faster for running than cycling and faster at higher intensities. The faster VO2 response in running may be associated with larger active muscle mass or differences in muscle activation patterns. The faster response at higher intensities may suggest that VO2 response is driven by O2 demand.
Contributing Partner: UNT Libraries
Timed Vs. Untimed Initiation Intervals and the Effects of Confidence on a Golf Putting Task
Date: August 1999
Creator: Sundberg, Jeffrey D.
Description: Low, moderate, and high handicap golfers, while wearing glasses with a blinder on the side, attempted 9 putts differing in length from 5 to 21 ft under timed (less than 3.5s from grounding of club to initiation of back swing) and untimed conditions in a counterbalanced design. Confidence ratings were taken prior to and following each putt. The results revealed a significant condition by handicap group interaction (p=.021). The lower handicap group had a more consistent and lower initiation interval duration than did the moderate and high handicap players. Post interviews determined that 33 of the 35 golfers felt more comfortable in the untimed condition. However, golfers' confidence levels were not significantly different in the untimed condition as compared to the timed condition.
Contributing Partner: UNT Libraries


text: This hotel has easy access and easy parking. The room is acceptable. Triple sheeting is now used and beds were very comfortable. 4 pillows per bed!! Pool and hot tub were the perfect temperature. Cookies, coffee and tea available all the time in lobby. Great breakfast. We had gravy/biscuits/home fries option one day and then scrambled eggs/Canadian bacon/homefries for day... More


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Gilbert Arenas
Gilbert Arenas arrested in California for illegal fireworks posessionComment
Former Washington Wizards star Gilbert Arenas was busted early Thursday morning in California for having more than a dozen crates of illegal fireworks, TMZ says.
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Senior Member
How about a champion passive that allows their summoner to see dotted lines indicating commands given by enemy summoners telling their champions where to move?
I thought of the idea, but I've also seen it before in Command and Conquer: Red Alert 2. Oh, and more recently in Starcraft 2, the Terrans have a 'detector' tower that keeps track of where enemies are (but not where they've been ordered to move).
This would be a champion that never needs wards, but their skills would need long cooldowns because the summoner would be pinging the minimap incessantly for his teammates.....unless part of the effect is to share the knowledge telepathically with all allied summoners....man that would....be....COOL.
If not a passive, perhaps an ultimate passive (R)?
© 2015 Riot Games, Inc. All rights reserved. Riot Games, League of Legends and PvP.net are trademarks, services marks, or registered trademarks of Riot Games, Inc.


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|Property type
|Minimum Stay
Rates and Availability
1)Deer Run is managed by Carolina Cabin Rentals.
2)Price and availability is kept up to the minute at the website.
3)To go directly to this home's page on our website, click on Owner Info tab above and see the link that says, "Click here to See Updated Calendar & Book Online!"
4)Or call us at 828-295-6000
5)Or use the "Request a Quote" link to the right to email an inquiry to Carolina Cabin Rentals.
Experience the peace and quiet of the high country from this beautiful new log home located in Boone North Carolina. This home has just about every amenity you can desire from a Hot Tub on the covered porch overlooking a long range view of the mountains, to the beautiful Marble counters in the kitchen, Pool Table, Dart board, flat screen TV in the game room, large flat screen TV in the living room, and the elegant and luscious professional decorations through out the home with custom fabrics and leather furniture.
This home has 3 terrific bedrooms, each with their own grandeur. The master bedroom with a king bed and large screen TV, the large bedroom on the main floor with twin beds or the queen size bedroom on the main floor with a private sitting porch just of the room. This home is just minutes from the heart of Boone, Blowing Rock, historic Valle Crucis, Grandfather Mountain, and several of the area's ski resorts. Enjoy relaxing on the porch overlooking the mountain view, or enjoy the luxury through-out the home.
1 King bedroom, 1 bedroom with twins, and 1 bedroom with a queen size bed can comfortably accommodate a party of six people. The loft also has a full size futon that can comfortably sleep 2 additional people, if desired.
This property is managed by our property manager, Carolina Cabin Rentals who keeps all of our availability up to date, so please contact them to check availability.
Boone, Banner Elk, Blowing Rock, and Beech Mountain North Carolina are vibrant mountain towns that boasts a multitude of recreational attractions and outdoor activities as well as the cultural interest and diversity of being home to Appalachian State University and Lees McCrae College. Play tennis or go swimming in downtown Blowing Rock, play golf at the Boone Golf Club, or enjoy some of many cultural activities such as Horn in the West, symphonies, Art in the Park, or other artistic offerings. Enjoy some of the finest rainbow trout fishing in the world in the cool, clear waters of the Watauga River, just minutes away from this cottage. Visit the ski slopes of Appalachian Ski Mountain, Beech Mountain Resort or Sugar Mountain. Spend a day exploring the outdoor wildlife refuge and hiking of Grandfather Mountain or the Mountain and Western theme park of Tweetsie Railroad. Or maybe you are looking for extreme adventure and might brave the white water of the High Country's guided rafting tours or head out for some extreme Mountain Biking trails. After a day of activity, enjoy a quiet evening in our comfortable, mountain retreat or partake of some of the area's great variety of casual or fine dining establishments.
Keywords: Log Cabin, Pool Table, Hot Tub, Leather Furniture, Marble Counters, Boone, North Carolina, Views, Mountain Lodge Realty, Blue Ridge Mountains, mountainlodgerealty
|Rate Period
|Weekend Night
Jun 24 2014 - Jun 25 2014
1 night
Flat fee
|Property Damage Protection
Payment is usually accepted in the quoted currency (USD) unless the currency and the amount is specifically agreed in advance with the owner / advertiser.


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The match at Wembley Stadium will celebrate the 150th anniversary of England's Football Association and will be the first time the neighbours have met since the 1995 match in Dublin, which was abandoned when English fans rioted.Wednesday, 29 May, 2013, 5:46am
More than 60,000 people packed into Gelora Bung Karno Stadium in Jakarta on a recent Saturday night to see the national soccer team play. Another 100 million tuned in to television to watch the match, underlining the appeal of soccer in Indonesia where attendance rivals the top English and German soccer leagues.25 May 2013 - 2:42am
How much is a coin toss worth? Can foolishly faking a frivolous flip and flick of a thin metallic disc prove financially tempting? You bet. Five years ago, the going rate was 100,000 yuan (HK$124,000).1 Mar 2013 - 4:53am
Crystal Palace boss Ian Holloway was clear last week about his intimidatory, insensitive and inappropriate intentions towards match officials. Hostile treatment towards match officials inevitably spreads to all levels where ticking time bombs are effectively being seeded.22 Feb 2013 - 4:17am
Oldham banned their players from swapping shirts with Everton's stars after their dramatic 2-2 draw in the FA Cup fifth round as the cash-strapped League One club try to cut costs.18 Feb 2013 - 3:15am
Researchers from the University of Leeds found that some school-age footballers were showing signs of chronic stress, exhaustion and disillusion with the sport given the demands for perfection from coaches, parents and teammates.31 Jan 2013 - 4:26am
Unforeseen troubles for referees lie ahead because of the FA's decision to rescind Manchester City captain Vincent Kompany's red card for what match referee Mike Dean deemed to be serious foul play. In undermining his interpretation, the FA has opened Pandora's Box.25 Jan 2013 - 3:13am
Arsenal manager Arsene Wenger revealed his faith in German economic efficiency by saying it was the reason why defender Per Mertesacker had become the club's debt collector.24 Jan 2013 - 3:09am
After hooligans forced a Bundesliga game to be delayed by throwing lit flares at the weekend, Germany coach Joachim Loew is leading the call for tighter security measures in stadiums.
Last Saturday, hooligans in the Eintracht Frankfurt fan block forced their team's 3-1 defeat at Bayer Leverkusen to be delayed by six minutes after lighting flares and setting off fireworks.24 Jan 2013 - 3:09am
Of all the things we fans enjoy when watching soccer, now is the time to count the number of tackles and savour them.19 Jan 2013 - 5:20am
The debate over nationalism, racism and football hooliganism, the touchpaper topics overshadowing the Euros, sizzled at Camp Wok before the much anticipated opening match in Warsaw. This wooded, gentrified camping site which serves as the SCMP HQ has now been invaded by Polish, German and English-cum-Irish-cum-Scottish fans - 11 in total.9 Jun 2012 - 12:00am
The opening of a new English Premier League season today will thrill football followers around the globe, not least in Hong Kong. British authorities will also be hoping that the distraction will help defuse the mob psychology behind the worst rioting England has seen for decades.13 Aug 2011 - 12:00am
His antics may have sent revellers wild at last year's Hong Kong Sevens, but the man who led security staff a merry dance when he climbed the goalposts at the Hong Kong Stadium has forced organisers to get tough on people who invade the pitch this year.25 Mar 2011 - 12:00am
Sandton's neon lights are ablaze barely a kilometre from Innisfree Park, the green glow from Johannesburg's glitziest and wealthiest district illuminating the sky above. Below is Nelson Mandela Square, where well-heeled South Africans and foreign visitors congregate to create a cultural melting pot beneath the eponymous statue at the centre of the plaza.18 Jun 2010 - 12:00am
After a difficult victory against Scotland last week at Hampden Park stadium in Glasgow, Italy's footballers qualified to take part in the 2008 European Championships.2 Dec 2007 - 12:00am


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Men's basketball: Warriors fall to Wildcats
WAHPETON, N.D. — Speed is a killer, especially for basketball teams just starting to find their legs.
The North Dakota State College of Science ran the floor quickly and efficiently in a 93-67 win over Ridgewater on Wednesday.
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From the critics
Community Activity
CommentAdd a Comment
This book was not very good, in my opinion. Many people are saying it's good, but in my opinion, it is very unrealistic. All the characters are perfect, and it kinda bruised my self-esteem. I mean, Lauren gets everything and anything she asks for! She also seems to be good at everything. I'm not saying I want her to die; it sure would be nice to see her getting something wrong once in a while.
book seventeen.I heart this book.
Oh, plz!I waited 4 AGES 4 the next book! WHY hasn't Jessica written 4 ALL this time?
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text:
Ubuntu Security Notice (C) 2009-2015 Canonical, Inc. / NASL script (C) 2009-2015 Tenable Network Security, Inc.
Synopsis :
The remote Ubuntu host is missing a security-related patch.
Description :
Pavel Polischouk discovered that Pan incorrectly handled certain data
structures. If a user were tricked into viewing malicious nntp data, a
remote attacker could cause a denial of service or possibly execute
arbitrary code with the privileges of the user invoking the program.
Solution :
Update the affected pan package.
Risk factor :
High / CVSS Base Score : 9.3
CVSS Temporal Score : 8.1
Public Exploit Available : false
Family: Ubuntu Local Security Checks
Nessus Plugin ID: 42080 ()
Bugtraq ID: 29421


text:
Bobbitt, Mozelle
Published: Wednesday, December 17, 2003 at 6:01 a.m.
Last Modified: Tuesday, December 16, 2003 at 11:00 p.m.
Mozelle Bobbitt, 79, Mt. Olive Community, died Monday, Dec. 15, 2003.
She is survived by her family.
Funeral will be at 2 p.m. Thursday at Mt. Olive Church. Burial in Mt. Olive Cemetery. Jordan Funeral Home is in charge.
Reader comments posted to this article may be published in our print edition. All rights reserved. This copyrighted material may not be re-published without permission. Links are encouraged.
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Is weight really a good indicator of your overall health and fitness? What about body composition? Body mass index? You may have heard of these terms, but it can be a challenge to know exactly what they mean in terms of your health. Learn which measurements are the most...
Have you ever wondered what health and fitness experts eat to fuel their bodies? From meals to snacks, top health and fitness experts offer insight into their dietary habits, nutrition tips and favorite healthy recipes.
If you eat the same things day after day, you're probably in a food rut, and more likely to jump off the clean-eating ship and onto the junk food highway. Here are five foods you should consider adding to your diet this year—they'll give you energy, boost your immunity and...
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text:
8 of 10 people found the following review helpful
Of a time and place long ago, but enjoyable nonetheless,
Verified Purchase(What is this?)
This review is from: 4 Play [2CD] (Audio CD)
The Fairport Convention of today has evolved and changed somewhat, both in personnel and sound, since these recordings were made.
With Swarb as a lead singer in those days, his remastering of these tracks is as masterly as his violin playing has always been.
The music is of an era long past, but the sound is crisp and the liveliness and joy of that band in those days is there for all to hear.
For completists and newcomers alike, this will enliven any leaden day of the summer of 2012.
Swarb will be appearing at Fairport's Cropredy Convention, August 2012, together with Fairport and many ex-Fairport personnel to help celebrate their 45th anniversary. More power to him and to them.
Tracked by 1 customer
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Showing 1-2 of 2 posts in this discussion
Initial post: 27 Jul 2012 08:38:23 BDT
John says:
Sounds like a good album from your review, so why only 3 stars?
In reply to an earlier post on 27 Jul 2012 16:57:13 BDT
Andy Leslie says:
As I said, it is redolent of a time and place long ago and as such isn't necessarily up to 4 or 5 stars today. 3 stars aren't bad.
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Review Details
(4 customer reviews)
Location: Llandybie, Wales
Top Reviewer Ranking: 61,127


text:
I liked the views and challenges of the course but not so enjoyable as it's very sloppy throughout the course
Whilst I am not a "Golfing Enthusiast" my wife Inge and I have often eaten at the Ilfracombe Golf Club. Their Sunday lunch gives a very wide choice of "carvery meats" and vegetables and yes one should book beforehand. We have always found their Sunday lunches popular and their caterer/chef Dean Richardson and staff always make everyone most welcome. Their...
Made very welcome by caterer, Dean Richardson. Very efficient and friendly service. Good lunchtime snacks, wide menu well cooked and presented. Plenty of it and very reasonably priced. Bar staff had to cater for large funeral party and were astonishing at multi tasking. In spite of peak demand our waiting time was short. Nice well filled omelettes. Look forward to...
We had lunch before playing the course. You get plenty of food and it is well cooked. You need food if you play the course as it is very hilly and challenging
Very nice and friendly club, a warm welcome and beautiful views, it's a bit hilly so buggies are useful
Great course, very friendly and great views. There was plenty of room for the medium to high handicapper although the breeze does make it a good test when it does blow.
What a lovely, friendly place to play golf! The course was immaculate with amazing views from every hole, the staff in the clubhouse were welcoming and well informed and the food in the restaurant was delicious and great value for money.
Great course on the hill. Views from every hole on a course that was in good condition. Great value food in clubhouse with a good atmosphere.
Were recommended this golf course while on holiday in the area. The golf course is great with seaviews, but the bar/ dining room is very dated and the food was very disappointing. This golf club could be so much better with the views, just needs an injection of money. I would play here again and have a drink in the...
Came here with a large group to eat on a Saturday night on a recommendation. No atmosphere - we were the only people there - and pretty tatty surroundings. A basic menu poorly delivered with most of our party sending back the inedible steak. The golf may be fabulous, the catering is not.
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Young players from some of Europe's top football teams are marking 100 years since 1914 Christmas Truce.
Topics: belgium
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Young soccer players from some of Europe's top teams take part in a tournament in Belgium to mark 100 years since the Christmas Truce of 1914.
Topics: history, government-and-politics, world-politics, world-war-1, world-war-2, belgium, european-union
Liverpool is out of the Champions League, after Swiss side Basel denied the 10-man home side at Anfield.
Topics: sport, soccer, champions-league, england, switzerland, turkey, russian-federation, germany, greece, sweden, portugal, italy, belgium
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The Kookaburras let a three-goal lead slip to draw 4-4 with Belgium and remain winless at the Champions Trophy.
Topics: sport, field-hockey, india, australia, belgium
Belgians are united in moves to have potato fries recognised as global cultural heritage and put it on a par with Peking opera and the Argentinian tango.
Topics: popular-culture, community-and-society, food-and-beverage, belgium
The US-led coalition has inflicted serious damage on Islamic State militants but the fight could last years, US secretary of state John Kerry warns.
Topics: unrest-conflict-and-war, terrorism, defence-and-national-security, belgium, united-states, syrian-arab-republic, iraq
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The spouses of G20 leaders have spent the afternoon having a close-up experience with some of Australia's most iconic wildlife.
Topics: human-interest, world-politics, zoos, fig-tree-pocket-4069, china, belgium, turkey
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Free hugs was the selling point for two students running around Hobart's Elizabeth Street Mall this week.
Their motivation was simply the bad weather and a mission to spread a feeling of love.
Topics: relationships, people, human-interest, weather, hobart-7000, belgium, germany
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Race cars, titanium knees, artificial heart models and outlandish lamps made up of dozens of naked human figurines are just some of the products being churned out at one of the world's largest 3D printing factories.
Topics: business-economics-and-finance, markets, offbeat, belgium
Turkey is keeping 24 people under observation at clinics after a suspicious powder was sent to five consulates in Istanbul, the health ministry says.
Topics: unrest-conflict-and-war, terrorism, turkey, canada, belgium, germany
Belgium got its Euro 2016 qualifying campaign off to a flying start with a 6-0 victory over Andorra.
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Euthanasia campaigner Philip Nitschke has called for Australian prisoners jailed with no prospect of release to be given the option of euthanasia.
Topics: euthanasia, crime, prisons-and-punishment, law-crime-and-justice, australia, belgium
US athlete Justin Gatlin celebrates after winning the men's 100m event of the Memorial Van Damme athletics Diamond League meeting in Brussels, on September 5, 2014.
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Justin Gatlin rolled back the years to scorch to a rare 100-200m double at the final Diamond League meet of the season.
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The stone lions which guard the entrance to the Australian War Memorial in Canberra are going on tour to mark the centenary of major battles in World War I.
Topics: world-war-1, unrest-conflict-and-war, library-museum-and-gallery, foreign-affairs, canberra-2600, act, belgium, canada
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The Socceroos suffered their fifth consecutive loss as a slick Belgium outfit recorded a comfortable 2-0 win.
Mile Jedinak, the captain of Australia looks dejected after his teams defeat during International friendly match between Belgium and Australia at Stade de Sclessin on September 4, 2014 in Liege, Belgium.
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Socceroos coach Ange Postecoglou has hit back at a reporter suggesting his team is afraid of opponent Belgium.
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Socceroos star Robbie Kruse is ready to face star-studded Belgium despite not playing a competitive match since January.
Rising Wanderers striker Tomi Juric believes a new work ethic and attitude has him ready to take his Socceroos chance.
Topics: socceroos, a-league, soccer, sport, parramatta-2150, sydney-2000, nsw, australia, belgium, saudi-arabia
Police in several European countries have arrested 12 people and seized millions of euros in assets in a swoop on counterfeit Viagra dealers.
Topics: crime, law-crime-and-justice, austria, cyprus, england, belgium, hungary
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Premier League stars Eden Hazard and Romelu Lukaku have been ruled out of the friendly with the Socceroos.
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European Union leaders have chosen Poland's prime minister Donald Tusk to chair their Council and named Italian Federica Mogherini to run the bloc's foreign relations, as the EU prepares to threaten Russia with new sanctions over Ukraine.
Topics: unrest-conflict-and-war, government-and-politics, world-politics, belgium
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Ukrainian president Petro Poroshenko says there are now thousands of foreign troops and hundreds of tanks in the country, as the European Union warns a "new Cold War" with Russia would be detrimental to all Europe.
Mr Poroshenko, who is in Brussels to discuss the ongoing crisis with EU leaders, said his country is now the subject of foreign military aggression and terrorism.
"Thousands of the foreign troops and hundreds of foreign tanks [are] now on the territory of Ukraine," he said.
European Commission president Jose Manuel Barroso said Ukraine was on the verge of a full-scale war as tougher sanctions against Russia were being considered.
Topics: world-politics, foreign-affairs, government-and-politics, unrest-conflict-and-war, belgium, ukraine, russian-federation
Reigning Europa League champions Sevilla will face former champions Feyenoord in this year's group stage.
Topics: soccer, sport, france, spain, belgium, italy, croatia, england, united-kingdom


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One million of anything is a lofty goal.
But when it comes to selling hunting licenses, it's one that I think is attainable.
The Pennsylvania Game Commission is embarking on a mission to increase its hunting license sales to one million. It's a figure that hasn't been reached since 2004, when more than 1,013,000 resident and non-resident licenses were sold by the agency.
In 2012, the Game Commission sold 943,583 resident and non-resident hunting licenses — marking an increase for the third straight year.
One million? It can be done.
Here's how the PGC hopes to achieve the goal — the agency will launch an ad campaign with radio, online and print media, along with a website, www.gohuntpa.org, that will give users the basics on the current hunting seasons.
The Game Commission hopes to reach one million license sales by 2018. Considering there are already roughly 2.5 million people in the state who consider themselves hunters, adding another 57,000 license buyers to the 2012 total should be no problem.
Heck, why not aim for 1.5 million in license sales by 2020 if that many people already consider themselves hunters?
Before launching the campaign — which will initially target nine counties including Allegheny, Berks, Butler, Cambria, Cumberland, Erie, Lancaster, York and Westmoreland — the agencies conducted several studies to better understand their potential customers.
The biggest one, in my opinion, revealed that 25 percent of adults in Pennsylvania consider themselves hunters. That's where the 2.5 million figure is derived.
It's a strong number, one that appears to contradict the general mindset that hunters are a dying breed.
Another interesting finding is the amount of license buyers who drop in and drop out of the sport each year. According to another PGC study, more than 1.3 million people bought a hunting license during the four-year span from 2009-12. About half of them bought a license all four years, 13 percent made the purchase three out of the four years, 14 percent bought a license two out of the four years and 23 percent bought a license just one of the four years.
That's a lot of people dropping in and out of the sport, and now the PGC wants them to come back and stay.
The aspect that I really like about the new campaign is the Game Commission is going to let the sport of hunting sell itself.
There are three main reasons why people hunt, according to yet another study. They are, in no particular order, to harvest a deer, relax and enjoy the outdoors, and to spend time with family and friends.
Those are the areas that will be the focus of the advertising campaign and that's the right approach.
Still, there is yet another approach that many would like to see that could draw quite a few people back to the sport — increase deer numbers.
This might be the biggest selling point of them all. After all, if people see deer during the season, they stay interested in hunting and are more inclined to buy a license again.
While the agency can still make a few more tweaks to the current deer seasons, they have already made quite a few changes in recent years that should improve deer numbers in some areas.
Sure, more can be done, but let's not overlook the steps that have already been taken.
Other potential selling points include providing more places to hunt — something that is also being done with an increase in the amount of acres purchased for Game Lands — and improve small game hunting. While many will argue, and rightly so, that the small game species simply aren't as plentiful as they were years ago, the PGC has made strides in this area with increases to the pheasant allocation and habitat projects specifically targeting small game species.
Whether you agree or disagree with what the Game Commission does, anyone who enjoys hunting should be supportive of the agency's mission to increase license sales.
Having one million hunters in the state again — that's a goal we can all get behind.