Microbiology (2015), 161, 2243–2255
DOI 10.1099/mic.0.000166
The Fur homologue BosR requires Arg39 to
activate rpoS transcription in Borrelia burgdorferi
and thereby direct spirochaete infection in mice
Laura I. Katona
Correspondence
Laura I. Katona
Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, Stony Brook
University, Stony Brook, NY, USA
[email protected]
Received 21 June 2015
Revised 25 August 2015
Accepted 26 August 2015
Borrelia burgdorferi is the causative agent of Lyme disease. In B. burgdorferi, RpoS controls the
expression of virulence genes needed for mammalian infection. The Fur homologue BosR
regulates the transcription of rpoS and therefore BosR determines, albeit indirectly, the infection
status of the spirochaete. Transcription of rpoS in B. burgdorferi is complex: rpoS can be
transcribed either from an RpoD-dependent promoter to yield a long transcript or from an
RpoN-dependent promoter to yield a short transcript. This study shows that BosR repressed
synthesis of the long transcript while at the same time activating synthesis of the short
transcript. How BosR does this is unclear. To address this, spirochaetes were engineered to
express either BosR or the naturally occurring variant BosRR39K. Mice became infected by the
spirochaetes expressing BosR but not by the spirochaetes expressing BosRR39K. Furthermore,
the spirochaetes expressing BosR activated rpoS transcription during growth in culture whereas
the spirochaetes expressing BosRR39K did not. Thus, BosR’s activation of rpoS transcription
somehow involves Arg39. This arginine is highly conserved in other FUR proteins and therefore
other FUR proteins may also require this arginine to function.
INTRODUCTION
Lyme disease is caused by the spirochaete Borrelia burgdorferi (Benach et al., 1983; Burgdorfer et al., 1982; Steere
et al., 1983). Infection occurs in humans when the spirochaete is transmitted by an infected tick during tick feeding. Normally, however, the spirochaete completes its life
cycle in the wild, passing back and forth between an
Ixodid tick vector and various vertebrate hosts (e.g. mice,
deer). This requires that the spirochaete survive and multiply in two very different environments. To do this,
B. burgdorferi employs a separate set of genes for each of
these environments. Determining how the spirochaete
turns these genes on and off is currently an area of intense
research (Radolf et al., 2012; Samuels, 2011).
In B. burgdorferi, many of the genes required for mammalian infection are under the control of the alternative sigma
factor RpoS (Radolf et al., 2012). Transcription of rpoS
in B. burgdorferi is complex. Under certain growth conditions (i.e. higher temperatures, low cell density), the spirochaete produces a long rpoS transcript from an RpoD
(s 70)-dependent promoter; however, under other growth
Abbreviations: kan, kanamycin resistance; qRT-PCR, quantitative realtime PCR; str, streptomycin resistance.
Seven supplementary tables, nine supplementary figures and
supplementary methods are available with the online Supplementary
Material.
000166 G 2015 The Authors
conditions (i.e. acid pH, higher temperatures, high cell
density), the spirochaete produces a short rpoS transcript
from an RpoN (s N)-dependent promoter (Hübner et al.,
2001; Lybecker & Samuels, 2007; Lybecker et al., 2010;
Samuels, 2011). Translation of the long transcript (but
not the short transcript) also requires the assistance of a
small RNA (Samuels, 2011).
Production of the short rpoS transcript is tightly regulated,
requiring both the alternative sigma factor RpoN (Hübner
et al., 2001) and the Fur homologue BosR (Hyde et al.,
2009; Ouyang et al., 2009). To initiate transcription,
RpoN must first be activated by the response regulator
Rrp2 (Yang et al., 2003) which, in turn, must first be phosphorylated (Xu et al., 2010). The binding site for RpoN
within the rpoS promoter is known (Smith et al., 2007;
Studholme & Buck, 2000). However, even though Rrp2
has a predicted DNA-binding domain, it appears that
Rrp2 acts on RpoN without the benefit of binding DNA
(Blevins et al., 2009; Burtnick et al., 2007; Yang et al.,
2003). In vitro, BosR binds multiple sites within the rpoS
promoter (Ouyang et al., 2011, 2014, 2015). Presumably,
in vivo, BosR must bind one or more of these sites to activate rpoS transcription.
Originally, BosR was annotated as Fur ( ferric uptake regulator) because it showed homology to the FUR family of
DNA-binding proteins (Fraser et al., 1997). Boylan et al.
(2003) renamed it BosR (Borrelia oxidative stress regulator)
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L. I. Katona
and put forward the idea that BosR’s function in
B. burgdorferi was to regulate the oxidative stress response.
Genes which may be regulated by BosR in this fashion
include napA (renamed bicA), sodA and cdr (Boylan
et al., 2003, 2006).
Although BosR may be involved in the oxidative stress
response (Hyde et al., 2009, 2010), it appears its main function is to act as a global regulator. To prepare the spirochaete for mammalian infection, BosR upregulates
transcription of rpoS (Hyde et al., 2009, 2010; Ouyang
et al., 2009, 2011) and also downregulates transcription
of various tick-phase genes (e.g. ospA and ospD), which
need only be expressed in the tick and which if allowed
expression in the mammal could lead to immune clearance
(Shi et al., 2014; Wang et al., 2013). When BosR activates
rpoS, it binds the direct repeat (DR) sequence TAAATTAAAT (Ouyang et al., 2011); however, when BosR represses
ospA and ospD, it binds a variant of the Per box sequence
TTATAAT-ATTATAA (Wang et al., 2013). How BosR
can bind two very different sequences and generate two
very different outcomes is currently unclear.
B. burgdorferi strain B31 was isolated in 1981 from a tick collected on Shelter Island (Burgdorfer et al., 1982). Genome
sequencing of strain B31 identified a fur homologue
(bb0647) and determined that residue 39 of the predicted
protein was an arginine (Fraser et al., 1997). Since then,
clonal isolates of strain B31 have been found that contain a
single nucleotide change in bosR, making residue 39 of
BosR a lysine (unpublished data; Seshu et al., 2004). This
residue is highly conserved as arginine in virtually all FUR
proteins. With BosR, substitution of this arginine with
lysine affects the protein’s ability to bind DNA in vitro
(Seshu et al., 2004). Not known is what effect this substitution has on the protein’s ability to function in vivo
during infection (Hyde et al., 2006; Seshu et al., 2004).
Here, strains of B. burgdorferi were constructed that
expressed either BosR or BosRR39K. Mice became infected
by the strains expressing BosR but not by the strains
expressing BosRR39K. This was, at least in part, because
the spirochaetes expressing BosR activated rpoS transcription while the spirochaetes expressing BosRR39K did not.
The R39K mutation affected the ability of BosRR39K to
bind the rpoS promoter in vitro. It is therefore possible
that BosRR39K failed to activate rpoS transcription because
it could not bind the rpoS promoter in vivo. This study
shows that BosR needs Arg39 to upregulate rpoS and in
so doing support mammalian infection.
METHODS
Bacterial strains and culture conditions. Table S1 (available in the
online Supplementary Material) lists the B. burgdorferi strains used in
this study. Strain B31-A3 was kindly provided by Patricia Rosa and
strain B31-F was kindly provided by Justin Radolf. Spirochaetes were
routinely cultured at 33 uC in BSK-II (Barbour, 1984) or BSK-H
medium (Sigma) supplemented with 6 % rabbit serum. To upregulate
rpoS, spirochaetes were cultured at 37 uC in BSK-II or BSK-H
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medium supplemented with 6 % rabbit serum and adjusted to
pH 6.8. Spirochaetes were enumerated by dark-field microscopy.
bosR disruption mutants and complemented strains. bosR dis-
ruption mutants and complemented strains were constructed as
detailed in the Supplementary Material.
Mouse infections. C3H/HeN mice (Taconic) were infected by
intradermal injection at 8 weeks of age. Ears, heart and bladder were
collected 21 days post-infection, and the tissues were cultivated at
33 uC in BSK-H medium containing 6 % rabbit serum with or
without 50 mg rifampicin ml21. Outgrowth of spirochaetes in these
cultures was assessed by dark-field microscopy at weekly intervals for
up to 1 month at which time the infections were deemed negative if
no spirochaetes were detected. All animal procedures were carried out
according to protocols approved by the Institutional Animal Care and
Use Committee (IACUC) at Stony Brook University.
Quantitative real-time PCR (qRT-PCR). qRT-PCR was carried out
on a model 7500 machine (Applied Biosystems) using the relative
quantification method (DDCt) with flaB as endogenous control. RNA
was extracted with Tri-Reagent LS (Molecular Research Center) and
further purified on an RNeasy spin column (Qiagen). cDNA was
synthesized by using random hexamers with the Superscript III first
strand synthesis system (Invitrogen). qRT-PCR primers (Table S2)
were designed with ABI Primer Express software. ABI Power SYBR
Green PCR master mix was used with 200 nM primers.
Gel shift assays. Gel shift assays were carried out as previously
described (Katona et al., 2004). Briefly, recombinant BosR (or
BosRR39K) was mixed with target DNA in 20 mM Bistris/borate
(pH 7.5) buffer containing 1.5 mM DTT, 1.3 mM MgCl2, 53 mM
KCl, 3.3 % glycerol and 133 mg BSA ml21, incubated for 20 min at
room temperature, and separated on a 6 % nondenaturing gel. The
DNA was stained with SYBR Green I (Molecular Probes) and the gel
was scanned in blue fluorescence mode on a Storm 860 imager
(Amersham-Pharmacia Biotech).
Target DNA. Target DNA was generated by PCR from cloned
sequences. For PrpoS, a 1.6 kb region containing the rpoS gene plus
upstream sequence was PCR-amplified from strain B31-A3 genomic
DNA with primers BB0771-Sph I F and BB0771-Xho I R and inserted
into the Sph I–Xho I site of pST-Blue1 (Novagen), giving pLK43/1.
The PrpoS target DNA was then amplified by PCR from pLK43/1 using
primers BB0771p F1 and BB0771p R1. For BbDR, equal molar
amounts of the synthesized single strands DRF2 and DRR2 were
mixed, heated for 5 min at 95 uC, and slowly cooled to room temperature. Next, the duplex oligonucleotide was inserted into the
Eco RV site of pST-Blue1, giving pLK45/16. The BbDR target DNA
was then amplified by PCR from pLK45/16 using primers T7 promoter and U-19mer. Table S2 lists all primer sequences.
Recombinant BosR and BosRR39K. Recombinant proteins were
prepared as previously described (Katona et al., 2004). Briefly, the
His-tagged proteins were overexpressed in Escherichia coli BL21(DE3)
from pLK1/5 or pLK42/2, purified by zinc chelate chromatography,
and then treated with thrombin to free the N-terminal His tags.
pLK42/2 was constructed by PCR-amplifying the bosRR39K gene
from pLK38/2 using primers FLAF and FRR (Table S2) and inserting
it into the Nde I–Xho I site of pET28a (Novagen). pLK1/5 was from an
earlier study (Katona et al., 2004).
Tricine SDS-PAGE and Western blotting. Tricine SDS-PAGE was
carried out as previously described (Katona et al., 2004). Western
blots of recombinant proteins were probed with mouse anti-His-1
monoclonal antibody (Sigma) to detect the N-terminal His tags.
Western blots of Borrelia lysates were probed with mouse anti-FlaB
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Microbiology 161
BosR requires Arg39 to direct spirochaete infection
CB1 monoclonal antibody (Coleman & Benach, 1989), rat anti-OspC
(kindly provided by Justin Radolf) or rat anti-BosR. Rat anti-BosR
was raised against purified recombinant BosR that retained the Nterminal His tag. All blots were developed with IRDye-conjugated
secondary antibodies (Rockland or LI-COR) and scanned on an
Odyssey infrared imager (LI-COR). Odyssey 2-colour protein markers were used.
Statistics. Fisher’s exact test was used to measure statistical significance in the mouse studies and Student’s t-test was used to measure
statistical significance in the qRT-PCR studies.
RESULTS
bosR disruption mutants are generated in strain
B31-A3
In previous studies, knockouts of bosR were generated in
strains B31-MI (Ouyang et al., 2009), ML23 (Hyde et al.,
2009) and 297 (Ouyang et al., 2011). Strains B31-MI and
297 are infectious in mice (Ouyang et al., 2009, 2011);
however, strain ML23 lacks the lp25 linear plasmid and
therefore is not infectious in mice (Hyde et al., 2009).
Here, strain B31-A3 was chosen for study. Strain B31-A3
is a clonal isolate of strain B31-MI that retains infectivity
(Elias et al., 2002).
Two sets of bosR mutants were generated (Fig. S1a).
A kanamycin resistance (kan) cassette was introduced by
allelic exchange in the forward orientation to yield the
L-series mutants and in the reverse orientation to yield
the K-series mutants. The kan cassette had a flgB promoter
to drive constitutive expression of the kanamycin resistance
gene and a T7 terminator to avoid overexpression of downstream genes. A total of 58 transformants were identified:
28 with the kan cassette in the forward orientation and
30 with the kan cassette in the reverse orientation. Clones
L9 and K18 were selected for further study. Both clones
contained the full complement of linear and circular plasmids originally identified by Elias et al. (2002) in strain
B31-A3 (data not shown). PCR products of a size consistent with the introduction of a kan cassette were obtained
for both clones (Fig. S1b). Sequence analysis of these
PCR products confirmed that bosR was disrupted and
that no unwanted mutations were incorporated.
The complemented strains are engineered to
express either BosR or BosRR39K
Repeated attempts were made to complement the bosR
mutants in trans by introducing a pKFSS1 shuttle vector
harbouring bosR; however, no transformants were identified. Li et al. (2007) succeeded in complementing their
dps mutant by inserting dps with its promoter into the
intergenic region between bb0445 and bb0446. This
approach offered several advantages: the location on the
main chromosome ensured that the gene was present as a
single copy and because the gene was located downstream
of both bb0445 and bb0446, transcriptional read-through
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from the inserted sequences was less of a concern. Here,
their approach was adopted, but with some modifications.
Li et al. (2007) inserted dps downstream of bb0445 and followed it with a streptomycin resistance (str) cassette. This
arrangement, however, made the inserted gene susceptible
to transcriptional read through from promoters upstream
of bb0445. Here, bosR along with its promoter was inserted
downstream of the str cassette and a T7 terminator was
then added to the cassette to prevent read through from
the cassette’s flgB promoter.
Three complemented strains were constructed for each
bosR mutant strain: a mock complement that contained
only the str cassette, a bosR complement that contained
the str cassette plus a WT bosR gene, and a bosRR39K
complement that contained the str cassette plus an R39Kmutated bosRR39K gene (Fig. S2a). Multiple transformants
were identified. A single clone was selected from each set
and assayed by PCR to determine that it contained the
desired insert (Fig. S2b). All of the selected clones were
then screened for plasmid content and found to have the
same profile as strain B31-A3 (data not shown). DNA
sequence analysis confirmed the identity of the inserts
and established that no unwanted mutations were incorporated. Clones KN1 (mock complement), KN107 (bosR
complement) and KN203 (bosRR39K complement) were
from bosR mutant K18. Clones LN1 (mock complement),
LN101 (bosR complement) and LN202 (bosRR39K complement) were from bosR mutant L9.
bosR is expressed at near WT levels in both of the
bosR-complemented strains
Ouyang et al. (2009) reported that the region bb0648bb0647-bb0646 functioned as an operon with transcription
initiating from a promoter upstream of bb0648. Because
genes within an operon can often be transcribed from
their own promoters as well, qRT-PCR analyses were carried out on the bosR-complemented strains to determine
if bosR was being transcribed. For these studies, the spirochaetes were cultured at 33 uC and the RNA was isolated
during early exponential phase, mid-exponential phase
and stationary phase. As expected, the bosR-mutant strains
(L9 and K18) produced no bosR transcript (Table S3).
However, the bosR-complemented strains (LN101 and
KN107) each produced nearly the same level of bosR transcript as the WT strain during all three stages of growth
(Table S3). Therefore, the sequence located immediately
upstream of bosR does appear to function as a promoter
to allow transcription independent of the operon.
qRT-PCR analyses were also carried out on the gene
located immediately downstream of bosR. Compared with
the WT strain, the two bosR-mutant strains each showed
a lower level of expression of bb0646 during early exponential phase and mid-exponential phase, and a much lower
level of expression during stationary phase (Table S3).
This decreased expression of bb0646 was not due to
the loss of bosR expression, because both of the bosR-
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L. I. Katona
complemented strains also showed decreased expression of
bb0646 (Table S3). Interestingly, the orientation of the kan
cassette did not seem to matter: bosR mutant L9 (forward
orientation) and bosR mutant K18 (reverse orientation)
showed the same decreased expression of bb0646 (Table
S3). Thus, if the T7 terminator embedded within the kan
cassette was responsible for terminating transcription of
bb0646 from an upstream promoter, then it did so from
either orientation.
From these data, it was clear that the bosR-mutant strains
showed two defects: loss of bosR expression and reduced
expression of bb0646. The reduced expression of bb0646
due to disruption of bosR is a cis effect since complementation of bosR did not restore bb0646 expression. Neither of
these defects, however, affected the ability of the spirochaetes to grow in culture (Fig. S3 and data not shown).
The complemented strains express comparable
levels of bosR and bosRR39K transcript
Seshu et al. (2004) reported that strain CHP100 encodes a
variant of the bosR gene. This variant, bosRR39K, is also
present in strain B31-F (unpublished observation). Strains
CHP100 and B31-F were both derived from high-passage
strain B31: CHP100 in the Skare laboratory (Hyde et al.,
2006; Seshu et al., 2004) and B31-F in the Radolf laboratory
(Eggers et al., 2002). Other mutations in the bosR gene have
not been identified.
Seshu et al. (2004) further reported that BosRR39K and
BosR did not function the same in vitro or in vivo. However, because their studies were carried out with non-infectious isolates, it remained unclear if BosRR39K, like BosR,
could support infection in mice. Here, spirochaetes were
engineered to express bosR or bosRR39K in the infectious
background of strain B31-A3. According to the qRT-PCR
analyses, both of the bosRR39K-complemented strains
(LN202 and KN203) produced transcript at a level comparable to that of the bosR-complemented strains (Table S3).
Also, both of the bosRR39K-complemented strains
expressed decreased levels of bb0646 (Table S3). Thus, the
bosR- and bosRR39K-complemented strains seemed to
differ only in their expression of bosR versus bosRR39K.
Mice become infected by spirochaetes expressing
BosR but not BosRR39K
Others have reported that bosR-knockouts of B. burgdorferi
are not infectious in mice (Hyde et al., 2009; Ouyang et al.,
2009, 2011). Here, the WT strain B31-A3 was infectious at
a dose of 20 000 spirochaetes per mouse, whereas neither
of the bosR-mutant strains (L9 and K18) was infectious
at this dose or at a higher dose of 100 000 spirochaetes
per mouse (Table 1 and data not shown).
Ouyang et al. (2009) also reported that their bosR-complemented strain regained its infectious phenotype. Here, both
of the bosR-complemented strains (LN101 and KN107)
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were infectious; however, the frequency of infection was
about half that of the WT strain (Table 1). In part, this
may have been because the bosR-complemented strains
expressed slightly less bosR transcript than the WT strain
(Table S3). Also, the defect in bb0646 expression may
have been a contributing factor (Table S3). Shaw et al.
(2012) found that while a bb0646 knockout was infectious
in mice, its ability to infect was attenuated. bb0646 encodes
an enzyme that has lipase activity (unpublished observation; Shaw et al., 2012). It is thus possible that the spirochaete uses this enzyme to help promote infection.
Regardless of the reason for the reduced infectivity of the
bosR-complemented strains, the results still show that
expression of bosR from the bb0445–446 intergenic site
allowed the spirochaete to regain infectivity. In contrast,
Table 1 shows that neither of the bosRR39K-complemented
strains (LN202 or KN203) was infectious at the dose of
20 000 spirochaetes per mouse. Thus, while the bosR-complemented strains infected 10 out of 20 mice, the
bosRR39K-complemented strains infected 0 out of 20
mice (Table 1). According to Fisher’s exact test, there is a
highly significant difference between these two frequencies
of infection (Pv0.001).
Taken together, the data show that under this one set of
infection conditions, the spirochaetes expressing BosR
were capable of infecting mice whereas the spirochaetes
expressing BosRR39K were not.
OspC is detected in spirochaetes expressing
BosR but not BosRR39K
Others have suggested that the reason why their bosRknockout strains were not infectious in mice was, at least
in part, because they failed to produce sufficient OspC
(Hyde et al., 2009; Ouyang et al., 2009). To test this, WT
strain B31-A3, bosR mutant L9, mock complement LN1,
bosR complement LN101 and bosRR39K complement
LN202 were each cultured under conditions previously
shown to upregulate expression of OspC in vitro (Yang
et al., 2000). Under these conditions (37 uC, pH 6.8),
OspC was expressed at high levels in both the WT strain
and bosR complement, but not in the bosR mutant, mock
complement or bosRR39K complement (Fig. 1). The level
of BosR, or BosRR39K, present in the samples was also
determined. The bosR mutant and mock complement
showed no protein; however, the WT strain, bosR complement and bosRR39K complement each showed about
the same level of protein (Fig. 1). Thus, the bosRR39K
complement did not fail to express OspC only because
the BosRR39K was not expressed or not stable. A second
experiment gave similar results (data not shown).
Transcripts of ospC and rpoS are detected by
qRT-PCR in spirochaetes expressing BosR
OspC expression in B. burgdorferi is regulated at the level of
transcription by the alternative sigma factor RpoS
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BosR requires Arg39 to direct spirochaete infection
Table 1. Infectivity of B. burgdorferi clones in mice
Mice were infected by intradermal injection of 26104 spirochaetes.
Strain
A3
L9
K18
LN1
KN1
LN101
KN107
LN202
KN203
Description
No. of cultures positive/total no. of
cultures
WT
bosR mutant of A3
bosR mutant of A3
Mock complement of L9
Mock complement of K18
bosR complement of L9
bosR complement of K18
bosRR39K complement of L9
bosRR39K complement of K18
Skin
Bladder
Heart
17/18
0/8
0/8
0/10
0/10
4/10
6/10
0/10
0/10
17/18
0/8
0/8
0/10
0/10
4/10
6/10
0/10
0/10
17/18
0/8
0/7
0/10
0/10
4/10
6/10
0/10
0/10
No. of mice infected/total no. of mice*
17/18
0/8
0/8
0/10
0/10
4/10
6/10
0/10
0/10
37
25
(Samuels, 2011). Both Ouyang et al. (2009) and Hyde et al.
(2009) reported that their bosR-knockout strains produced
less RpoS than their WT strains during growth in culture.
Ouyang et al. (2009) also reported that their bosR-knockout strain produced less rpoS transcript. Here, the WT
strain, bosR mutant L9 and bosR complement LN101
were each cultured as before and the RNA was isolated
for qRT-PCR during late-exponential phase. The results
in Table 2 show that the level of ospC transcript was greatly
decreased in the bosR-mutant strain compared with the
WT strain (125-fold), thus confirming that BosR was
needed to see upregulation of ospC.
LN202 - bosRR39K complement
LN101 - bosR complement
LN1 - mock complement
L9 - bosR mutant
A3 - WT
*P,0.001 by Fisher’s exact test for the following comparisons: (i) bosR mutants L9 and K18 vs WT A3, (ii) bosR complements LN101 and KN107 vs
mock complements LN1 and KN1, and (iii) bosRR39K complements LN202 and KN203 vs bosR complements LN101 and KN107.
FlaB
OspC
20
25
20
BosR or
BosRR39K
Fig. 1. Expression of OspC, BosR and BosRR39K in bosRmutant and complemented strains. Spirochaetes were cultured
for 6 days (late-exponential phase) at 37 8C in BSK-H medium
(pH 6.8) containing 6 % rabbit serum. Whole-cell lysates were
prepared and subjected to Tricine SDS-PAGE. Each lane
received the lysate from ,66107 spirochaetes. Western blots
were probed with mouse monoclonal antibody CB1 to detect
FlaB, rat anti-OspC antiserum to detect OspC, and rat anti-BosR
antiserum to detect BosR or BosRR39K. FlaB served as the
loading control. Protein size standards (in kDa) appear to the left.
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As noted earlier, Borrelia rpoS can be transcribed from an
RpoD-dependent promoter to yield a long transcript or
from an RpoN-dependent promoter to yield a short transcript (Lybecker & Samuels, 2007; Smith et al., 2007).
Here, two sets of qRT-PCR primers were designed to
allow detection of both transcripts (Figs. 2 and S4). The
first set of primers (Fig. 2, primers 1 and 2) recognized
sites near the 39 end of both long and short transcripts
and therefore yielded data on the level of total rpoS transcript (i.e. the sum of long and short transcripts). The
second set of primers (Fig. 2, primers 3 and 4) recognized
sites near the 59 end of the long transcript (but not the
short transcript) and therefore yielded data on the level
of long rpoS transcript only. qRT-PCR analysis with primers 1 and 2 showed that the level of total rpoS transcript
was decreased twofold in the bosR-mutant strain compared
with the WT strain (Table 2, Experiment I – rpoS). However, qRT-PCR analysis with primers 3 and 4 showed
that the level of long rpoS transcript was increased 2.1
fold in the bosR-mutant strain compared with the WT
strain (Table 2, Experiment I – rpoS-L). Thus, BosR
appears to have repressed long rpoS transcription while
at the same time activated total rpoS transcription
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Table 2. Relative expression levels of rpoS and ospC as determined by qRT-PCR
In Experiment I, spirochaetes were inoculated at an initial density of 250 spirochaetes ml21 and cultured at 37 8C for 6 days (late-exponential
phase) in BSK-II medium (pH 6.8) containing 6 % rabbit serum. In Experiment II, spirochaetes were inoculated at an initial density of 143 spirochaetes ml21 and cultured at 37 8C for 7 days (early stationary phase) in BSK-II medium (pH 6.8) containing 6 % rabbit serum.
Gene
Relative expression level*
Experiment I
bb0771 (rpoS)
bb0771L (rpoS-L)
bbb19 (ospC)
bb0647 (bosR)
bb0646
Experiment II
bb0771 (rpoS)
bb0771L (rpoS-L)
bbb19 (ospC)
bb0647 (bosR)
bb0646
WT
A3
bosR mutant
L9
bosR complement
LN101
bosRR39K complement
LN202
Mock complement
LN1
1.000
1.000
1.000
1.000
1.000
0.490¡0.005
2.103¡0.147
0.008¡0.001
0.000¡0.000
0.132¡0.001
1.328¡0.007
1.130¡0.113
1.208¡0.016
1.277¡0.043
0.123¡0.008
ND D
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
1.000
1.000
1.000
1.000
1.000
0.079¡0.008
0.789¡0.079
0.002¡0.000
0.744¡0.018
0.870¡0.008
0.132¡0.004
1.519¡0.017
0.003¡0.000
0.000¡0.000
1.569¡0.155
*Mean¡SEM of two determinations, each with four replicates. Statistical significance was evaluated by Student’s t-test for the following comparisons. Total rpoS transcript: L9 vs A3 (P50.01), L9 vs LN101 (P,0.01), LN101 vs LN1 (P50.02), LN202 vs LN1 (P50.07) and LN101 vs LN202
(P,0.01). Long rpoS transcript: L9 vs A3 (P50.04), L9 vs LN101 (P50.02), LN101 vs LN1 (P50.01), LN202 vs LN1 (P50.08) and LN101 vs LN202
(P50.23).
DND , Not done.
(Fig. 3a, b). These data do not indicate if BosR acted
directly or indirectly; however, two additional studies
gave similar results (Tables S4 and S5). Taken together,
the combined data (Fig. 4a, b) showed that the increased
expression of total rpoS transcript by the bosR mutant
was highly statistically significant (Pv0.01) while the
decreased expression of the long rpoS transcript by the
bosR mutant was not quite statistically significant
(P50.11).
772
858
771
3 4
1
770
2
cDNA
Long rpoS transcript
1
Short rpoS transcript
2
cDNA
Fig. 2. Diagram illustrating the long and short transcripts of
B. burgdorferi rpoS. Small arrows indicate the sites recognized
by the qRT-PCR primers in the cDNA synthesized from the rpoS
transcripts. Primers 1 and 2 (BB0771 RTF1 and BB0771 RTR1)
allow amplification of both long and short transcripts while primers 3 and 4 (BB0771 RTF3 and BB0771 RTR3) allow amplification of the long transcript only.
2248
Ouyang et al. (2009) previously reported that BosR activated rpoS transcription; however, their qRT-PCR analysis
determined the level of total rpoS transcript and did not
distinguish between the long and short transcripts. Here,
the qRT-PCR analysis showed that BosR activated short
rpoS transcription (Fig. 4a), but not long rpoS transcription
(Fig. 4b). All three studies showed, however, that the WT
strain and bosR mutant strain differed in their expression
of bb0646 (Tables 2, S4 and S5). Thus, the rpoS result is
valid only if it was unaffected by the varied expression of
bb0646. To address this concern, additional studies were
carried using the complemented strains. Experiment I
showed that the bosR-complemented strain mirrored the
behaviour of the WT strain, yet expressed bb0646 at a
level comparable to that of the bosR mutant (Table 2,
Fig. 3a, b). Thus, adopting this strategy of comparing the
complemented strains could circumvent the issues with
bb0646.
Experiment II employed three complemented strains: the
bosR complement LN101 which expressed only BosR, the
bosRR39K complement LN202 which expressed only
BosRR39K, and the mock complement LN1 which
expressed neither BosR nor BosRR39K and thus functioned as the bosR mutant. Each strain was cultured at
37 uC, pH 6.8 to ensure high-level expression of ospC
and upregulation of rpoS. Spirochaetes were harvested
during early stationary phase and the RNA was isolated
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Microbiology 161
BosR requires Arg39 to direct spirochaete infection
(a)
(b)
3.0
Relative expression
Relative expression
1.5
1.0
0.5
0.0
A3
L9
2.0
0.8
1.6
Relative expression
Relative expression
A3
L9
LN101
LN101
LN202
LN1
Day 6
Day 7
Day 8
(d)
1.0
0.6
0.4
0.2
0.0
LN101
LN202
1.2
0.8
0.4
0.0
LN1
(f)
(e)
15
Relative expression
15
Relative expression
1.0
0.0
LN101
(c)
2.0
10
5
0
Day 6
Day 7
Day 8
10
5
0
Fig. 3. Regulation of rpoS transcription by BosR versus BosRR39K. The plots show the relative expression levels for total
rpoS transcript (a, c and e) and long rpoS transcript (b, d and f) as determined by qRT-PCR. (a, b) Experiment I: WT strain
B31-A3 was compared with bosR mutant L9 and bosR complement LN101. (c, d) Experiment II: bosR complement LN101
was compared with bosRR39K complement LN202 and mock complement LN1. (e, f) Experiment III: bosR complement
LN101 (black bars) was compared with bosRR39K complement LN202 (white bars) using spirochaetes harvested during
mid-exponential phase (day 6), late-exponential phase (day 7) and stationary phase (day 8). Error bars show the SEM (n52).
for qRT-PCR analysis. The results of the qRT-PCR analysis (Table 2) showed that the bosR complement produced
333-fold more ospC transcript than the mock complement, confirming that the culture conditions did allow
for high-level expression of ospC. The results also
showed that, as expected, all three complemented strains
expressed similar levels of bb0646 transcript. Finally, the
results showed that the bosR complement produced eightfold more total rpoS transcript than the mock complement
and 1.5-fold less long rpoS transcript than the mock
complement (Table 2, Fig. 3c, d). Therefore, BosR appears
to have both activated total rpoS transcription and
http://mic.microbiologyresearch.org
repressed long rpoS transcription. A second study gave
similar results (Table S6). Student’s t-test was used to
evaluate the data from the three studies which compared
the bosR complement (LN101) with a bosR mutant (L9
or LN1). This analysis showed that the increased
expression of total rpoS transcript by the bosR complement
was highly statistically significant (Fig. 4c; Pv0.01) while
the decreased expression of the long rpoS transcript by the
bosR complement was also statistically significant (Fig. 4d;
P50.03). These data indicate that BosR both activates
total rpoS transcription and represses long rpoS
transcription.
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2249
L. I. Katona
(a)
(b)
1.0
Relative expression
Relative expression
1.0
0.8
0.6
0.4
P < 0.01
0.2
0.0
(c)
P = 0.11
0.2
bosR mutant L9
WT A3
1.0
0.8
0.6
0.4
0.2
0.0
P < 0.01
bosR mutant L9 or
mock complement LN1
Relative expression
Relative expression
0.4
(d)
1.0
0.8
0.6
0.4
P = 0.03
0.2
0.0
bosR complement
LN101
(e)
bosR mutant L9 or
mock complement LN1
bosR complement
LN101
(f)
1.5
1.5
1.0
0.5
P < 0.01
0.0
bosR
complement LN101
bosRR39K
complement LN202
Relative expression
Relative expression
0.6
0.0
bosR mutant L9
WT A3
0.8
1.0
0.5
P = 0.23
0.0
bosR
complement LN101
bosRR39K
complement LN202
Fig. 4. BosR both activates total rpoS transcription and represses long rpoS transcription. The plots show the relative
expression levels for total rpoS transcript (a, c and e) and long rpoS transcript (b, d and f) as determined by qRT-PCR. (a, b)
WT strain B31-A3 was compared with bosR mutant L9 using data taken from Tables 2, S4 and S5 (n53). (c, d) bosR
mutant L9 and mock complement LN1 (both of which are bosR disruption mutants) were compared with bosR complement
LN101 using data taken from Tables 2 and S6 (n53). (e, f) bosR complement LN101 was compared with bosRR39K complement LN202 using data taken from Tables 2, S6 and S7 (n55). Statistical significance was evaluated by Student’s t-test.
P values j0.05 were interpreted to indicate statistical significance. Error bars show the SEM .
Spirochaetes expressing BosR, but not
BosRR39K, activate rpoS transcription
These data indicate that, unlike BosR, BosRR39K did not
activate total rpoS transcription.
The next question was: what effect did BosRR39K have on
rpoS transcription? The results of Experiment II showed
that the bosRR39K complement produced about the same
level of ospC transcript as the mock complement, indicating that, unlike BosR, BosRR39K did not upregulate ospC
(Table 2). These results also showed that the bosRR39K
complement produced 1.7-fold less total rpoS transcript
than the mock complement, and therefore while BosR activated total rpoS transcription, BosRR39K did not (Table 2,
Fig. 3c). A second study gave similar results (Table S6).
Taken together, the combined data from the two studies
gave a P value of 0.53, according to Student’s t-test.
The results for the long rpoS transcript were less clear.
Experiment II showed that the bosRR39K complement produced less long rpoS transcript than the mock complement
(Table 2). However, the second study showed that the
bosRR39K complement produced a similar level of long
rpoS transcript as the mock complement (Table S6).
In neither study was the difference between LN202 and
LN1 statistically significant (Tables 2 and S6). Taken
together, the combined data gave a P value of 0.40, according to Student’s t-test. These data suggest that BosRR39K
did not repress long rpoS transcription; however, further
studies are needed to clarify this point.
2250
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Microbiology 161
BosR requires Arg39 to direct spirochaete infection
BosR, but not BosRR39K, binds the DR sequence
in vitro
Ouyang et al. (2011) reported that recombinant BosR
bound the DR sequence TAAATTAAAT within the rpoS
http://mic.microbiologyresearch.org
244
122
61
30
15
8
4
0
244
122
61
30
15
8
4
0
BosR (nM)
(b)
B. burgdorferi DR
159
319
159
319
80
40
5
0
319
159
80
40
20
10
5
20
BosR (nM)
BosRR39K (nM)
10
(c)
(d)
80
40
20
10
5
0
BosR (nM)
319
159
80
40
20
BosRR39K (nM)
10
Fig. 3(e, f) shows the same rpoS data as Table S7, but
expressed in different form. In the plotted data, all of the
results are expressed relative to the day 6 – LN202 data
point (which is arbitrarily assigned a value of 1). It is
clear from the plotted data that the BosR-expressing spirochaetes increased their expression of total rpoS transcript
during the transition from mid-exponential phase to
stationary phase (Fig. 3e), yet maintained a fairly constant
level of expression of long rpoS transcript (Fig. 3f). Because
the level of long rpoS transcript remained essentially constant, it is clear that the increase in total rpoS transcript
was owing to short rpoS transcript. These data provide
direct evidence that BosR activates short rpoS transcription
while BosRR39K does not.
BosRR39K (nM)
0
Strains LN101 and LN202 were also compared in Experiment II (see Tables 2 and S6). The results in Table 2 show
that, compared with strain LN202, strain LN101 produced
12.7-fold more total rpoS transcript and 1.3-fold more long
rpoS transcript. The results in Table S6 show that, compared
with strain LN202, strain LN101 produced 4.2-fold more
total rpoS transcript and 1.3-fold less long rpoS transcript.
Taken together, the combined data from all three studies
showed that strains LN101 and LN202 differed statistically
in their expression of total rpoS transcript (Fig. 4e,
Pv0.01), but not in their expression of long rpoS transcript
(Fig. 4f, P50.23). Of course, BosR and BosRR39K may both
regulate long rpoS transcription, but by different means.
(a)
5
As expected, strains LN101 and LN202 expressed similar
levels of bb0646 transcript and similar levels of bosR or
bosRR39K transcript (Table S7). Also as expected, strain
LN101 expressed high levels of ospC transcript while
strain LN202 expressed trace levels of ospC transcript
(Table S7). Compared with strain LN202, strain LN101
expressed 4.7-fold more total rpoS transcript on day 6,
5.2-fold more on day 7 and 5.8-fold more on day 8
(Table S7). And, compared with strain LN202, strain
LN101 expressed 1.1-fold less long rpoS transcript on day
6, 1.3-fold less on day 7 and 1.6-fold less on day 8 (Table
S7). Thus, while strains LN101 and LN202 differed greatly
in their production of total rpoS transcript, they differed
less in their production of long rpoS transcript.
promoter from strain 297. The rpoS promoter in strain
B31 differs slightly from that in strain 297 (Fig. S5); however, it still contains all four DR sequences identified in the
297 promoter (Ouyang et al., 2011). To test binding to the
B31 promoter, recombinant BosR and BosRR39K were
prepared in parallel. Both proteins ran with an estimated
mass of 20.5 kDa on SDS-PAGE and appeared free of
major contamination (Fig. S6). The results of gel shift
assays showed that although both proteins bound the
B31 promoter, BosR showed a different pattern of binding
than BosRR39K (Fig. 5a). Because competitor DNA was
not added to the assay, both proteins may have bound
the DNA non-specifically. However, at low concentration
(8 nM), BosR generated three shifted bands while
BosRR39K generated a single shifted band (Fig. 5a). The
0
Experiment III was designed to gain a better picture of how
BosR regulates rpoS transcription. Strains LN101 and
LN202 were grown in parallel under conditions conducive
to upregulation of rpoS. Spirochaetes were harvested
during mid-exponential phase (6 day cultures), late-exponential phase (7 day cultures) and stationary phase (8 day
cultures) and the RNA was isolated and analysed by
qRT-PCR. Table S7 shows the results of this analysis.
Plots of the rpoS data also appear in Fig. 3(e, f).
Fig. 5. Binding of BosR and BosRR39K to the DR sequence
located within the rpoS promoter. (a) Strain B31 rpoS promoter
DNA (270 bp) was assayed at 2.5 nM with BosR or BosRR39K
at the concentrations indicated (0–244 nM). (b) DR sequence
TAAATTAAAT located immediately upstream of the RpoN site
within the rpoS promoter (Fig. S5). (c) BbDR target DNA
(257 bp), which contained the DR insert, was assayed at 2.5 nM
with BosR or BosRR39K at the concentrations indicated
(0–319 nM). (d) Vector control DNA (229 bp), which contained
no insert, was assayed at 2.5 nM with BosR or BosRR39K at the
concentrations indicated (0–319 nM). Arrows indicate the complexes formed between the target DNA and BosR or BosRR39K.
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2251
L. I. Katona
same results were obtained when the two proteins were
assayed together (Fig. S7).
The putative BosR binding site located immediately
upstream of the 224/212 RpoN site contains a perfect DR
sequence in both strains (Fig. S5). This site (Fig. 5b) was
inserted into the Eco RV cloning site of pST-Blue1 and the
insert plus flanking vector sequence was PCR-amplified to
generate BbDR target DNA for testing (Fig. S8). The
vector sequence with no insert was also PCR amplified to
provide control DNA (Fig. S8). The BbDR target DNA was
257 bp. The control DNA was 229 bp. Thus, the insert
(28 bp) accounted for *10 % of the entire length. The
results of the gel shift assay showed that BosRR39K and
BosR both bound the BbDR target DNA (Fig. 5c) and both
also bound the vector control DNA (Fig. 5d). However,
while BosRR39K showed the same pattern of binding for
both targets, BosR showed a different pattern of binding.
Thus, at 10 nM, BosR generated a major shifted band with
the BbDR target DNA (Fig. 5c, arrow) that was not present
in the assay with the vector control (Fig. 5d). These data provide preliminary evidence that BosR bound the BbDR insert
while BosRR39K did not.
DISCUSSION
This study shows that a single nucleotide change in the
bosR gene to generate bosRR39K had the effect of rendering
B. burgdorferi non-infectious in mice. Others have shown
that bosR is required for B. burgdorferi to activate
expression of the alternative sigma factor RpoS (Hyde
et al., 2009; Ouyang et al., 2009). Because RpoS controls
the expression of virulence factors needed for mammalian
infection (Radolf et al., 2012), failure to upregulate rpoS
could account for why the spirochaetes expressing
BosRR39K were non-infectious. Assays carried out to
determine the level of rpoS transcript present in the spirochaetes grown under conditions conducive to rpoS
expression found that while the BosR-expressing spirochaetes did transcribe rpoS, the BosRR39K-expressing spirochaetes did not. Furthermore, the BosR-expressing
spirochaetes produced high levels of ospC transcript and
OspC protein while the BosRR39K-expressing spirochaetes
did not. Transcription of ospC is known to require RpoS
(Radolf et al., 2012). In vivo, OspC is expressed on the surface of the spirochaete at the point when the spirochaete
moves from the gut of the tick into the haemolymph just
prior to invading a mammalian host (Grimm et al., 2004;
Pal et al., 2004; Tilly et al., 2006). Therefore, although
genes other than rpoS may have had a role in making the
BosRR39K-expressing spirochaetes non-infectious, failure
to upregulate rpoS appears to have been a major cause.
Studies published in 2009–2010 established that BosR controls expression of rpoS in B. burgdorferi (Samuels &
Radolf, 2009). Ouyang et al. (2011) identified sites within
the promoter of strain 297 rpoS that bound BosR
in vitro. They observed that these sites contained near
2252
matches to the DR sequence TAAATTAAAT and suggested
that BosR activated rpoS transcription by binding one or
more of these sites in vivo. Later, Ouyang et al. (2014,
2015) redefined this putative BosR binding site as a palindromic 6-1-6 inverted repeat consisting of the sequence
ATTTAA-TTAAAT. They termed this sequence the BosR
box. If BosR does bind this BosR box in vivo, it is still
unclear how this binding activates rpoS transcription.
The preliminary results of the gel shift assays suggested that
only BosR bound the BbDR insert. However, because the
BbDR insert contained both a perfect DR sequence
(tTTTAAATTAAAT) and an imperfect BosR box
(tTTTAAATTAAAT), it is not possible to distinguish
which of these sequences BosR recognized.
The protein families database (Pfam) lists BosR as belonging
to the FUR family of DNA-binding proteins (Finn et al.,
2014). Pfam alignments of FUR proteins (PF01475) show
that BosR’s Arg39 aligns with a highly conserved arginine
present in virtually all FUR proteins. X-ray crystallography
studies show that FUR proteins consist of two domains: an
N-terminal DNA-binding domain and a C-terminal dimerization domain (An et al., 2009; Butcher et al., 2012; Dian
et al., 2011; Gilston et al., 2014; Jacquamet et al., 2009; Lin
et al., 2014; Lucarelli et al., 2007; Makthal et al., 2013; Pohl
et al., 2003; Sheikh & Taylor, 2009; Shin et al., 2011;
Traoré et al., 2006, 2009). The DNA-binding domain contains a winged helix–turn–helix (wHTH) motif that provides
the protein with the means to bind DNA (Pohl et al., 2003).
The wHTH motif contains a three-helix bundle: the conserved arginine that aligns with BosR’s Arg39 is located
near the N-terminal end of a-helix 1 (Pohl et al., 2003). Typically, residues within the recognition helix (a-helix 3) determine the binding specificity of wHTH proteins and therefore
Arg39 would not be expected to be involved in defining the
binding specificity of BosR (Aravind et al., 2005; Huffman &
Brennan, 2002). However, others have suggested that positively charged residues located within the N-terminal
region of FUR proteins are likely to form electrostatic interactions with their target DNAs (Butcher et al., 2012; Lucarelli
et al., 2007; Pecqueur et al., 2006). Recently, Streptococcus
pyogenes PerR was found to require Arg21, Arg26 and
Arg31, as well as other positively charged residues, to bind
DNA in vitro (Lin et al., 2014). Of interest here, Arg31 is
the conserved arginine in PerR that aligns with BosR’s Arg39.
Models of BosR and BosRR39K (Fig. S9) were built using
the S. pyogenes PerR as template (Lin et al., 2014).
In these models, Arg39 and Lys39 were each located at
the base of a pocket in a position adjacent to a-helix 3
(Fig. S9a). The electrostatic surface potential was determined for both proteins and showed that the surfaces
appeared virtually identical (Fig. S9b).
In 2014, Gilston and coworkers published the crystal structure of E. coli Zur bound to DNA (Gilston et al., 2014).
Included in their data was the observation that Zur’s
Arg28 was located sufficiently close to the DNA to allow
an electrostatic interaction to form between the arginine
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Microbiology 161
BosR requires Arg39 to direct spirochaete infection
RpoD
rpoS
DR
RpoD
RpoN
rpoS
BosR
Fig. 6. Model of BosR regulation of rpoS transcription. RpoDdependent transcription from an upstream promoter produces a
long transcript in the absence of BosR (top). RpoN-dependent
transcription from a downstream promoter produces a short transcript in the presence of BosR (bottom). BosR is pictured binding
to all four DR sequences within the rpoS promoter; however,
binding to all four sequences may not be required to allow transcription to proceed from the downstream promoter. The extent
of long versus short transcription is indicated by the line
thickness.
side chain and phosphate backbone. Of interest here, Arg28
is the conserved arginine in Zur that aligns with BosR’s
Arg39.
Because arginine and lysine are both positively charged at
neutral pH, both are capable of forming electrostatic interactions with the phosphate backbone of DNA. However,
arginine differs from lysine in that it more readily inserts
itself into narrowed minor grooves and can therefore be
used for protein–DNA recognition (Rohs et al., 2009).
Narrow minor grooves exist in A-tract DNA. The DR
sequence has two A-tracts: TAAATTAAAT and TAAATTAAAT. The BosR box also has two A-tracts: ATTTAATTAAAT and ATTTAA-TTAAAT. If Arg39 were to insert
itself into one or more of these A-tracts and thereby help
BosR to bind this DNA, then substitution of lysine for
this arginine could potentially cause a loss in binding affinity and/or specificity. Further studies are needed to determine if BosR uses Arg39 to interact with the A-tracts in the
DR/BosR box DNA.
The results of these studies show that BosR’s regulation of
rpoS transcription is more complex than originally imagined. BosR repressed long rpoS transcription while at the
same time activating short rpoS transcription. It is unclear
if these two activities are linked; however, because these
results may provide insight into how BosR controls rpoS
transcription, I offer the following model for consideration.
When BosR is present at low concentration (or when BosR
http://mic.microbiologyresearch.org
is unable to bind the DR/BosR box), RNA polymerase
initiates transcription from an upstream RpoD-dependent
promoter and proceeds unhindered along the DNA to synthesize the long rpoS transcript (Fig. 6, top). However, when
BosR is present at high concentration (or when BosR is able
to bind the DR/BosR box), RNA polymerase still initiates
transcription at the upstream promoter but is blocked by
bound BosR and therefore fails to progress past the DR/
BosR box site. RpoN is now free to bind at the 224/212
site. RNA polymerase initiates transcription at the RpoNdependent promoter and proceeds to synthesize the short
rpoS transcript (Fig. 6, bottom). In this model, the spirochaete employs transcriptional interference (Palmer et al.,
2009, 2011; Shearwin et al., 2005) to control (i.e. close
down) synthesis of the short rpoS transcript. The role of
BosR is to act as a roadblock (King et al., 2003) and thereby
reverse this control.
ACKNOWLEDGEMENTS
I thank Jorge Benach for his support, Christopher Kuhlow for expert
technical assistance and Patricio Mena for help with the animal
studies. I thank Patricia Rosa for supplying strain B31-A3, Justin
Radolf for supplying strain B31-F and anti-OspC antiserum, and
Scott Samuels for supplying plasmids pKFSS1 and pCR2.1 : : PflgBaphI-T7t. I thank the reviewers for help in making this a better
paper. This work was supported, in part, by a NIAID grant from
the National Institutes of Health (AI 027044) awarded to Jorge
L. Benach.
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