JOURNAL OF BACTERIOLOGY, Apr. 2009, p. 2656–2667
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 8
Activation of the Campylobacter jejuni FlgSR Two-Component System
Is Linked to the Flagellar Export Apparatus?
Stephanie N. Joslin and David R. Hendrixson*
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390
Received 3 December 2008/Accepted 28 January 2009
Activation of ?54-dependent gene expression essential for formation of flagella in Campylobacter jejuni
requires the components of the inner membrane-localized flagellar export apparatus and the FlgSR two-
component regulatory system. In this study, we characterized the FlgS sensor kinase and how activation of the
protein is linked to the flagellar export apparatus. We found that FlgS is localized to the C. jejuni cytoplasm
and that His141 of FlgS is essential for autophosphorylation, phosphorelay to the cognate FlgR response
regulator, motility, and expression of ?54-dependent flagellar genes. Mutants with incomplete flagellar export
apparatuses produced wild-type levels of FlgS and FlgR, but they were defective for signaling through the
FlgSR system. By using genetic approaches, we found that FlgSR activity is linked to and downstream of the
flagellar export apparatus in a regulatory cascade that terminates in expression of ?54-dependent flagellar
genes. By analyzing defined flhB and fliI mutants of C. jejuni that form flagellar export apparatuses that are
secretion incompetent, we determined that formation of the apparatus is required to contribute to the signal
sensed by FlgS to terminate in activation of expression of ?54-dependent flagellar genes. Considering that the
flagellar export apparatuses of Escherichia coli and Salmonella species influence ?28-dependent flagellar gene
expression, our work expands the signaling activity of the apparatuses to include ?54-dependent pathways of
C. jejuni and possibly other motile bacteria. This study indicates that these apparatuses have broader functions
beyond flagellar protein secretion, including activation of essential two-component regulatory systems required
for expression of ?54-dependent flagellar genes.
Responding to changing environmental and intracellular
conditions in cells requires efficient communication networks
that can rapidly receive and integrate signals. Two-component
regulatory systems, which are distributed almost ubiquitously
among prokaryotic organisms, allow bacteria to monitor their
intracellular and extracellular environments and react by alter-
ing the expression of appropriate genes. These systems are
typically comprised of a sensor histidine kinase and a response
regulator protein (reviewed in references 46 and 65). The
sensor protein contains a domain usually in the N-terminal
portion that detects a specific signal, commonly through an
interaction with another protein or a small effector molecule.
Activation includes autophosphorylation of the sensor kinase
and a conformational change that allows the transmitter do-
main, usually in the C-terminal portion, to activate a cognate
response regulator via phosphotransfer. Some histidine kinases
also have the ability to function as a phosphatase to remove a
phosphate group from either themselves or their cognate re-
sponse regulators when activity of the regulatory system is not
The largest group of sensor histidine kinases includes those
that are anchored to the cytoplasmic membrane and receive
signals from the extracellular environment, allowing a cell to
respond to external factors such as pH, temperature, or the
presence of specific compounds (reviewed in reference 46).
Since the monitoring of intracellular conditions is also vital to
basic cellular processes, sensor kinases that are activated by
alterations within bacteria have also evolved. These kinases
include a relatively small group of kinases that are membrane
anchored but respond to signals in the cytoplasm or periplasm
and a larger group of soluble, cytoplasmic sensor kinases. Sev-
eral members of the latter group have been characterized, such
as NtrB, a kinase involved in nitrogen metabolism, whose ac-
tivity is controlled by the PII protein (33). Nitrogen starvation
results in uridylylation of PII, which blocks interaction with
NtrB and causes the sensor protein to function as a kinase to
initiate phosphorelay, culminating in phosphorylation of its
cognate response regulator, NtrC. Under nitrogen-replete con-
ditions, PII is deuridylylated and interacts with NtrB, allowing
it to function as a phosphatase instead of as a kinase. Another
example of a cytoplasmic histidine kinase that responds to
intracellular conditions is KinA of Bacillus subtilis. Through
interactions with two different proteins that inhibit the func-
tion of the kinase, KinA is responsive to the energy state of the
bacterium or the ability of the cell to initiate replication (9, 58,
63). Activation of KinA begins a complex regulatory cascade
leading to expression of genes essential for sporulation.
Flagellar assembly and chemotaxis systems also rely on two-
component signaling systems to properly regulate bacterial
motility (6, 62). The CheA kinase receives signals from a num-
ber of membrane-bound methyl-accepting chemotaxis protein
(MCP) receptors (reviewed in references 3, 17, and 18). Motile
bacteria respond via chemotaxis to small molecules that are
attractants or repellants, and many of these effectors are bound
by the periplasmic domains of MCPs. Through interactions of
the cytoplasmic domains of MCPs with the CheA kinase, CheA
is able to integrate and transmit these signals via phosphorelay
* Corresponding author. Mailing address: University of Texas South-
western Medical School, Department of Microbiology, 5323 Harry Hines
Boulevard, Dallas, TX 75390-9048. Phone: (214) 648-5949. Fax: (214)
648-5907. E-mail: firstname.lastname@example.org.
?Published ahead of print on 6 February 2009.
to CheY, ultimately influencing the decision to continue
swimming in a single direction or tumble and change direction.
Campylobacter jejuni is a gram-negative, microaerophilic bac-
terium commonly associated with a number of animals of agri-
cultural significance, especially fowl. While the relationship be-
tween C. jejuni and avian species develops into commensalism,
infection of humans causes gastroenteritis that can range from
very mild enteritis to severe, bloody diarrheal episodes (7, 8). In
both the developed and developing regions of the world, C. jejuni
is responsible for a substantial percentage of cases of bacterial
gastroenteritis (13, 53). In the United States, this bacterium is
believed to be the leading single-species cause of diarrheal dis-
ease, resulting in significant loss in economic productivity (11).
C. jejuni is a highly motile organism owing to the presence of
a single flagellum elaborated from one or both poles of the
bacterium. Motility is critical for promoting optimal interac-
tions between C. jejuni and avian or human hosts. Nonmotile
variants of C. jejuni colonize the gastrointestinal tract of chicks
at levels significantly lower than wild-type motile strains (25,
29, 50, 64, 66), and only motile strains can be recovered after
coinfection of human volunteers with motile and nonmotile
strains (5). In this organism, motility is a highly organized,
regulated, and complex process, relying on the coordination of
over 40 proteins to assemble a complete organelle (26). Al-
though there are some similarities with the well-characterized
regulatory cascades described for Escherichia coli and Salmo-
nella species (12), genetic screens and in silico analyses indi-
cate that there are several differences that distinguish the
flagellar gene transcription and assembly processes in C. jejuni.
While C. jejuni utilizes ?28to activate transcription of the
major flagellin (encoded by flaA) and other minor flagellum-
associated proteins (10, 22, 23, 28, 30, 66), ?54has been shown
or proposed to be involved in transcription of the bulk of
flagellar genes, including those encoding the hook, basal body,
and minor flagellin (26, 28, 30, 66). The use of both ?28and ?54
in these pathways indicates that flagellar gene transcription in
C. jejuni is more similar to the regulatory cascades of species of
Vibrio, Pseudomonas, and Helicobacter than to those of E. coli
or Salmonella species (2, 16, 34, 39, 40, 47, 51, 56, 60).
In a transposon mutagenesis screen, a number of gene prod-
ucts were found to be required for ?54-dependent flagellar
gene transcription in C. jejuni (30). These proteins include
members of the flagellar export apparatus (FEA), FlhF (a
putative GTPase), and the FlgSR two-component system, com-
prised of the FlgS sensor kinase and the FlgR response regu-
lator (30). It was hypothesized that these proteins may act
separately or in concert to integrate signals required to initiate
transcription of ?54-dependent flagellar genes. Previous work
characterized the unusual NtrC-like response regulator FlgR
to understand the means by which this protein functions (35).
Our group has also found that FlgR and FlgS are targets of
phase variation, making FlgSR the only known two-component
regulatory system in which both proteins are subject to this
form of control (25, 27). However, the mechanism by which
FlgS is activated and functions as a sensor kinase remains to be
characterized. Sequence analyses indicate that this protein
appears to contain domains common to many sensor histi-
dine kinases, such as the ATP-binding catalytic domain and
the histidine-containing phosphotransfer domain (61, 65).
Although the homology is somewhat weaker in the N-ter-
minal region of the protein, FlgS is similar to the flagellum-
associated histidine kinases that are required for ?54-depen-
dent flagellar gene expression and motility in species of
Vibrio, Pseudomonas, and Helicobacter (14, 40, 51, 57). How-
ever, the signals that activate any of these kinases for pos-
itively influencing flagellar gene expression are uncharacter-
In this work, we characterized FlgS and the activating signals
that influence its ability to positively regulate flagellar gene
expression and motility in C. jejuni. We first identified the
histidine in the phosphotransfer domain that is autophosphor-
ylated upon activation by FlgS. Through extensive experimen-
tation, we characterized the origin of the signal that influences
FlgS activation. Our research has led us to believe that (i)
activation of FlgSR is dependent on the FEA and (ii) the signal
for FlgS autophosphorylation may lie within the FEA, as for-
mation of this apparatus appears to be necessary to promote
expression of ?54-dependent flagellar genes. Our work expands
previous models of Campylobacter flagellar gene regulation
and motility by characterizing the FlgS sensor kinase and in-
troducing potential mechanisms for activating this protein.
Furthermore, our work suggests that the FEAs of a subset of
motile bacteria that use ?54to control expression of flagellar
genes have broader functions than flagellar protein secretion,
including influencing signaling pathways through two-compo-
nent regulatory systems to activate flagellar gene expression.
MATERIALS AND METHODS
Bacterial strains. C. jejuni strain 81-176 is a clinical isolate from a patient
presenting with gastroenteritis and has been shown to promote commensal
colonization of the chick gastrointestinal tract and to cause disease in human
volunteers (5, 29). C. jejuni was routinely grown on Mueller-Hinton (MH) agar
containing 10 ?g/ml trimethoprim (TMP) under microaerobic conditions (85%
N2, 10% CO2, and 5% O2) at 37°C. When necessary, strains were grown on MH
agar containing 50 ?g/ml kanamycin, 15 ?g/ml chloramphenicol, or 0.5, 1, 2, or
5 mg/ml streptomycin. All C. jejuni strains were stored at ?80°C in a solution of
85% MH broth and 15% glycerol. E. coli strains DH5?, XL1-Blue, and
BL21(DE3)/pLysE were cultured with Luria-Bertani (LB) agar or broth contain-
ing 100 ?g/ml ampicillin or 15 ?g/ml chloramphenicol when required. All E. coli
strains were stored at ?80°C in a solution of 80% LB broth and 20% glycerol.
Construction of mutants. All strains were constructed by using previously
described protocols (28). To construct flgS(H141) mutants, pDRH310 (30) was
subjected to PCR-mediated mutagenesis (45) to mutate the histidine codon at
position 141 to a codon for alanine and then was verified by DNA sequence
analysis. One plasmid, pDRH1276, was recovered and introduced into 81-176
SmrflgS::cat-rpsL (DRH441 ) and 81-176 Smr?astA flgS::cat-rpsL (DRH460
) by electroporation. Mutants were recovered on MH agar containing strep-
tomycin and verified by PCR analysis and DNA sequencing. Mutants used for
further analysis were designated DRH1323 [81-176 SmrflgS(H141A)] and
SNJ947 [81-176 Smr?astA flgS(H141A)].
We replaced native flgR with the flgR?receiverand flgR?CTDalleles (where
receiver indicates the N-terminal receiver domain and CTD indicates the C-
terminal domain) in FEA mutants. For ?fliP, ?flhA, and ?flhB mutants,
flgR::kan-rpsL (pDRH443) was electroporated into strains 81-176 Smr?astA
?fliP (DRH1016), 81-176 Smr?astA ?flhA (DRH979), and 81-176 Smr?astA
?flhB (DRH1734) (30). The resultant strains, 81-176 Smr?astA ?fliP
flgR::kan-rpsL (SNJ158), 81-176 Smr?astA ?flhA flgR::kan-rpsL (DRH1765),
and 81-176 Smr?astA ?flhB flgR::kan-rpsL (DRH1830), were electroporated
with pDRH1855 containing the flgR?receiverallele and pDRH1856 containing the
flgR?CTDallele (35). All transformants were selected on MH agar with strepto-
mycin and verified by PCR and DNA sequencing.
C. jejuni ?fliI mutants were constructed by first cloning the fliI locus into
pUC19 (to generate pDRH1453) and then cloning an SmaI-digested kan-rpsL
cassette (from pDRH427 ) into a PmeI site within the fliI coding sequence
to generate pDRH1506. pDRH1506 was introduced into 81-176 Smr?astA
(DRH461 ) by electroporation, generating 81-176 Smr?astA fliI::kan-rpsL
VOL. 191, 2009FlgSR ACTIVATION BY FEA2657
(DRH2246), which was recovered on MH agar with kanamycin. pDRH1453 was
then used in PCR-mediated mutagenesis (45) to delete a large portion of
the coding sequence of the gene by fusing codon 4 to codon 453, creating
pDRH1843. DRH2246 was then electroporated with pDRH1843 to replace
fliI::kan-rpsL with the ?fliI allele to create 81-176 Smr?astA ?fliI (DRH2257).
Generation of flhB mutants first involved PCR-mediated mutagenesis (45) to
create a point mutation, generating an StuI site in the coding sequence of flhB in
pDRH666 (30) to create pSNJ355. This plasmid was then digested with StuI so
that a cat-rpsL cassette generated by digestion of pDRH265 (28) with SmaI could
be inserted with flhB. The resulting plasmid, pSNJ360, was then introduced into
DRH461 (81-176 Smr?astA [(30]) by electroporation, replacing flhB with
flhB::cat-rpsL to generate SNJ404 (81-176 Smr?astA flhB::cat-rpsL). PCR-me-
diated mutagenesis (45) with pDRH666 was used to generate point mutations
and in-frame deletions within flhB. These mutations and the resulting plas-
mids included flhB(N267A) (pSNJ238), flhB?214-218(pSNJ243), flhB?224-228
(pSNJ236), and flhB?244-253(pSNJ237). These plasmids were introduced into
SNJ404 by electroporation to replace the flhB::cat-rpsL allele with the different
flhB alleles. Mutants were recovered on MH agar with streptomycin. The result-
ing strains included SNJ438 [81-176 Smr?astA flhB(N267A)], SNJ464 (81-176
Smr?astA flhB?214-218), SNJ428 (81-176 Smr?astA flhB?224-228), and SNJ475
(81-176 Smr?astA flhB?244-253). Mutants were verified by PCR and DNA se-
To construct strains containing transcriptional reporters, plasmids pDRH532
(containing flgDE2::nemo), pDRH608 (containing flaA::astA), pDRH610 (con-
taining flaB::astA), and pDRH669 (containing flgD::astA) were electroporated
into C. jejuni to replace the native flgDE2, flaA, flaB, and flgD loci on the
chromosome as previously described (30, 59). All mutants were recovered on
MH agar containing kanamycin and were verified by PCR analysis.
Generation of polyclonal antiserum against C. jejuni proteins. Generation of
polyclonal murine antiserum against the RNA polymerase subunit A (RpoA)
protein of C. jejuni involved first constructing primers with 5? BamHI sites to
amplify the coding sequence from codon 2 through the stop codon of rpoA from
C. jejuni strain 81-176 (31). Ligation of this DNA fragment into the BamHI site
of pQE30 (Qiagen) and transformation into E. coli XL1-Blue allowed recovery
of pDRH2907, which encodes a His6-RpoA fusion protein. To purify the protein,
a 1-liter culture in LB broth was grown to an optical density at 600 nm (OD600)
of 0.5, and then the culture was induced for 4 h with 1 mM isopropyl-?-D-
thiogalactopyranoside (IPTG). The bacteria were disrupted by two passages
through an EmulsiFlex-C5 cell disrupter (Avesin) at 15,000 to 20,000 lb/in2. The
protein was purified under native conditions from the soluble fraction with
Ni-nitrilotriacetic acid agarose according to the manufacturer’s instructions.
Polyclonal murine antiserum was generated in mice by standard procedures
using a commercial vendor (Cocalico Biologicals).
Detection of FlhB in C. jejuni required generation of rabbit polyclonal anti-
serum against the cytoplasmic domain of the protein. Because this portion of
FlhB in Salmonella enterica serovar Typhimurium undergoes autoproteolytic
processing between the asparagine and proline residues at positions 269 and 270
(19, 21, 48), we attempted to create a soluble, more stable protein to immunize
rabbits for antiserum generation. We first used PCR-mediated mutagenesis (45)
with pDRH666 (containing the wild-type flhB allele ) to change the codons
for asparagine and proline at positions 267 and 268, respectively, to codons for
alanines to generate pDRH2339. After construction of pDRH2339, we amplified
a portion of the flhB(N267A P268A) sequence encoding amino acids 209 through
369 that encompasses the predicted entire unprocessed cytoplasmic domain of
the protein. Primers were used in PCR so that in-frame BamHI sites were added
to the 5? end of the amplified product. The DNA was then cloned into BamHI-
digested pGEX-4T-2 (GE Healthcare) in the correct orientation to produce a
glutathione S-transferase (GST)-FlhBcyto(N267A P268A) fusion protein. The
resulting plasmid was designated pDRH2367 and used to transform BL21(DE3).
The resulting strain was grown in 3 liters of LB broth to mid-log phase and then
induced with 25 mM IPTG for 3 h at 37°C. The bacteria were harvested and
disrupted with an EmulsiFlex-C5 cell disrupter (Avesin) at 15,000 to 20,000
lb/in2. The soluble fraction was obtained by removing the insoluble material by
centrifugation at 13,000 rpm for 2 h at 4°C. The soluble material was rocked with
2.4 ml of glutathione Sepharose 4B (GE Healthcare) for 30 min at room tem-
perature. The protein was then purified according to the manufacturer’s instruc-
tions. Despite our attempt to create a more stable, unprocessed version of the
cytoplasmic domain of FlhB fused to GST, about one-third of the recovered
purified protein had a molecular mass of approximately 29-kDa after sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, which
correlates with the size of GST rather than 46 kDa, the predicted size of the
full-length fusion protein. Therefore, this protein was only partially stable. The
purified products were used to immunize rabbits by standard procedures for
antiserum generation using a commercial vendor (Cocalico Biologicals).
Immunoblot analyses of FlgS, FlgR, FlhB, and FlaA proteins. C. jejuni strains
were grown from frozen stocks on MH agar containing appropriate antibiotics at
37°C for 48 h and restreaked 16 h prior to use. SDS-PAGE and immunoblotting
of FlgS and FlgR proteins were performed as previously described with anti-FlgS
Rab11 and anti-FlgR Rab13 rabbit polyclonal antisera, respectively (25). Briefly,
cells were resuspended from 16-h growth plates in MH broth and diluted to an
OD600of 0.8. One-milliliter samples were harvested by centrifugation and
washed once with phosphate-buffered saline. For whole-cell lysates (WCLs), the
pellet was resuspended in 50 ?l 1? Laemmli buffer, and 4 ?l (for FlgR analysis)
or 7 ?l (for FlgS analysis) of each resuspended pellet was loaded onto 10%
For FlgS localization studies, 5-ml portions of cultures of wild-type and mutant
strains at an OD600of 0.8 were prepared as described above, resuspended in 10
mM HEPES (pH 7.4), and broken by sonication. Unbroken cells were removed
by centrifugation at 13,000 ? g for 5 min at 4°C, and the supernatant was
transferred to a new tube and centrifuged at 13,000 ? g for 30 min at 4°C to pellet
the total membrane fraction (outer and inner membrane proteins). The super-
natant contained soluble proteins (cytoplasmic and periplasmic proteins). Vol-
umes representing equivalent cell numbers for the membrane and soluble pro-
teins were analyzed by 10% SDS-PAGE after resuspension and boiling in 1?
Laemmli buffer. For detection of FlgS, anti-FlgS Rab11 antiserum was used at a
dilution of 1:10,000 (25). To detect proteins representative of the cytoplasmic
fraction or inner membrane fraction, we analyzed the location of the RpoA
cytoplasmic protein and the AtpF inner membrane protein by using anti-RpoA
M59 antiserum at a dilution of 1:2,000 and anti-AtpF M3 antiserum at a dilution
of 1:1,000 (4), followed by a goat anti-mouse secondary antibody.
To monitor the stability and location of FlhB proteins, bacteria were grown
and 5-ml samples of cultures of wild-type and mutant strains were prepared and
sonicated as described above. The total membrane fraction, containing inner and
outer membrane proteins, was recovered by centrifugation at 13,000 rpm for 30
min at 4°C. The recovered pellet was suspended in 50 ?l of 1? Laemmli buffer
and loaded onto a 10% SDS-PAGE gel for immunoblot analysis. Primary anti-
FlhB Rab476 antiserum was used at a dilution of 1:1,000 and was rocked with the
membrane overnight at 4°C. The blot was then washed and incubated with a
1:10,000 dilution of goat anti-rabbit secondary antibody for 4 h at room temper-
For analysis of FlaA secretion in C. jejuni strains, bacteria were grown and
resuspended from plates as described above. WCLs from 1-ml portions of cul-
tures of wild-type and mutant strains were prepared as described above. For
recovery of outer membrane proteins, 5-ml cultures of each bacterial strain were
prepared and sonicated, and the unbroken cells were removed by centrifugation
at 13,000 rpm for 5 min at 4°C. Each supernatant was recovered and spun at
13,000 rpm for 30 min at 4°C. The pellet containing insoluble material repre-
senting the total membrane proteins (inner and outer membrane proteins) was
resuspended in 1% N-laurylsarcosine (sodium salt) and incubated for 30 min at
room temperature to solublize the inner membrane proteins. The outer mem-
brane proteins were recovered as the insoluble pellet after centrifugation at
13,000 rpm for 30 min at 4°C. Volumes of sample corresponding to 200 ?l and
700 ?l of bacteria were loaded for analysis of WCLs and outer membrane
proteins, respectively. Immunoblot analysis was performed with a 1:10,000 dilu-
tion of anti-FlaA LLI antiserum (42) and a 1:10,000 dilution of goat anti-rabbit
Motility assays and transmission electron microscopy. To analyze relative
levels of motility, strains were grown on MH agar with TMP from freezer stocks
for 48 h at 37°C under microaerobic conditions and then restreaked and grown
for 16 h prior to use. Cells were resuspended in MH broth to an OD600of 0.8,
and a sterile needle was used to inoculate semisolid MH motility agar as de-
scribed previously (28). Plates were incubated under microaerobic conditions for
24 to 36 h at 37°C and photographed. For transmission electron microscopy, 1-ml
samples of bacteria in MH broth at an OD600of 1.0 were centrifuged at 13,000
rpm for 3 min and then resuspended in 2% glutaraldehyde. After incubation for
1 h on ice, samples were stained with 1% uranyl acetate and visualized with an
FEI Technai G2 Spirit BioTWIN transmission electron microscope.
Arylsulfatase reporter assays. Strains were grown from frozen stocks for 48 h
at 37°C under microaerobic conditions on MH agar with TMP or kanamycin and
restreaked and grown for 16 h prior to the assay. Strains were analyzed for
arylsulfatase activity by a previously described method (30), which was based on
previously established methods (24, 67). Briefly, all strains were resuspended in
phosphate-buffered saline to an OD600of 0.8 to 1.0, washed in arylsulfatase assay
buffer, and incubated with 10 mM nitrophenylsulfate and 1 mM tyramine for 1 h
at 37°C. NaOH was added to terminate the assays, and the amount of nitrophe-
2658JOSLIN AND HENDRIXSONJ. BACTERIOL.
nol present in each sample was determined spectrophotometrically at OD410.
The number of arylsulfatase units produced by each strain was calculated by
comparing the OD410value of each sample to a standard curve obtained using
known nitrophenol concentrations. One arylsulfatase unit is defined as the
amount of enzyme catalyzing the release of 1 nmol of nitrophenol per h per
OD600unit. Each strain was tested in triplicate, and each assay was performed
Purification of FlgS and FlgR proteins. Wild-type His6-FlgR protein was
purified as previously described (35). flgS(H141A) from pDRH1276 was ampli-
fied from codon 2 to the stop codon by PCR using primers that added in-frame
5? and BamHI restriction sites to facilitate cloning into BamHI-digested pQE30
to generate pSNJ960. This plasmid was then transformed into XL1-Blue for
induction and purification of the protein. Wild-type His6-FlgS and His6-
FlgS(H141A) were purified by using previously described protocols (25).
Autophosphorylation of FlgS. FlgS autophosphorylation assays were per-
formed as described previously using purified His6-FlgS or His6-FlgS(H141A) in
the presence of [?-32P]ATP (35, 66). Briefly, 6 pmol His6-FlgS or His6-
FlgS(H141A) was added to a buffer containing 50 mM Tris-HCl (pH 8.0), 75 mM
KCl, 2 mM MgCl2, and 1 mM dithiothreitol. Ten microcuries of [?-32P]ATP was
then added. At each time point, a sample was removed and the reaction was
stopped by addition of an equal amount of 2? SDS-PAGE loading buffer.
Proteins were resolved by 10% SDS-PAGE, and the gels were dried and exposed
to a phosphorimager screen. The screen was read with a Storm 820 phosphor-
imager (Amersham Biosciences), and the data were analyzed using the manu-
FlgR phosphorylation. In vitro phosphotransfer from His6-FlgS proteins to
His6-FlgR was monitored as previously described (35, 66). For each reaction, 6
pmol of His6-FlgR was added to 6 pmol of His6-FlgS or His6-FlgS(H141A) that
had been allowed to autophosphorylate for 2 min as described above. Reactions
were stopped by addition of an equal volume of 2? SDS-PAGE loading buffer,
and the samples were analyzed by SDS-PAGE. After drying, polyacrylamide gels
were analyzed with a phosphorimager.
Real-time RT-PCR. C. jejuni strains 81-176 Smr(DRH212), 81-176 ?flhA
(DRH946), 81-176 ?flhB (SNJ471), and 81-176 ?fliP (DRH1065) were grown
from frozen stocks on MH agar containing appropriate antibiotics at 37°C for
48 h and restreaked 16 h prior to use (28, 30). Bacteria were suspended from the
agar plates in MH broth, and total RNA was extracted from the bacteria with
Trizol reagent (Invitrogen). The RNA was then treated with DNase prior to
analysis. The final concentration of RNA used in a Sybr green PCR master
mixture was 50 ng/?l. Real-time reverse transcription (RT)-PCR was performed
using a 7500 real-time PCR system (Applied Biosystems). Detection of mRNA
for gyrA, encoding DNA gyrase, served as an endogenous control, and the
transcript levels of flgS and flgR in mutants (lacking flhA, flhB, or fliP) were
compared to those in the wild-type strain (DRH212). The following primer pairs
were used for real-time RT-PCR analysis: flgS RT#1 (5?-GCTACAGATATTA
GCGATGAAAAACG-3?) and flgS RT#2 (5?-TAGGATTTCTTATCTCATGT
GCCAAAT-3?), flgR RT#3 (5?-TCAAGCCAAACTTTTAAGAGCTTTG-3?)
and flgR RT#4 (5?-CTATTTTGATGCTTTTCGTACTTCCA-3?), and gyrA F
(5?-CGACTTACACGGCCGATTTC-3?) and gyrA R (5?-ATGCTCTTTGCAG
Transposon mutagenesis. Chromosomal DNA from C. jejuni 81-176 ?astA
?flhA flgDE2::nemo (DRH1021 ), 81-176 ?astA ?flhB flgD::astA (SNJ331),
and 81-176 ?astA ?fliP flaB::astA (DRH1178 ) was purified and subjected to
in vitro random transposon mutagenesis with the darkhelment transposon by
using previously published protocols (27–30). Twelve in vitro transposon mu-
tagenesis reactions were performed with DNA from each strain. Each reaction
mixture contained 2 ?g of chromosomal DNA, 1 ?g of pSpaceball1, and 250 ng
of Himar1 C9 transposase purified from DH5?/pMalC9 (1). After transposition,
the mutagenized DNA was repaired and transformed into each strain as previ-
ously described (28). Transposon mutants were recovered after growth on MH
agar containing chloramphenicol and 5-bromo-4-chloro-3-indolyl sulfate and
then examined for blue or white colony phenotypes.
FlgS is a cytoplasmic protein. Bioinformatic analysis sug-
gests that unlike most sensor kinases that are localized to the
bacterial inner membrane, the C. jejuni FlgS sensor kinase is a
cytoplasmic protein. This protein lacks both a predicted signal
sequence that would target it for secretion and hypothetical
spans of hydrophobic residues that would be indicative of a
protein associated with the inner membrane. To determine if
FlgS is localized to the cytoplasm, we fractionated wild-type C.
jejuni 81-176 Smr(DRH212 ) to obtain a soluble fraction
containing cytoplasmic and periplasmic proteins and an insol-
uble fraction containing proteins associated with the outer and
inner membranes. As shown in Fig. 1, FlgS was found only in
WCLs and the soluble fraction of wild-type bacteria. In a
comparison with the control proteins, FlgS was present in the
same fraction as the soluble cytoplasmic protein RpoA and
absent in the fraction containing the insoluble inner membrane
protein AtpF. Considering both the bioinformatic and bio-
chemical analyses, we concluded that FlgS is a cytoplasmic
protein (Fig. 1).
Autophosphorylation of residue H141 is required for FlgS
activity. It has been shown that the autophosphorylation site of
the NtrB sensor kinase is residue H139 (52). Alignment of FlgS
to NtrB indicated that this phosphorylated residue likely cor-
responds to H141 of FlgS, an amino acid located within the
putative phosphotransfer domain (spanning amino acids 131 to
195) that receives the phosphate group upon autophosphory-
lation of other kinases. To determine if H141 is essential for
FlgS activity as a kinase and for flagellar gene expression, the
wild-type flgS allele of C. jejuni was replaced with flgS(H141A),
which results in production of FlgS with alanine at position 141
instead of histidine. The resulting mutant was first examined
for a potential defect in FlgS stability. We found that while
FlgS(H141A) appears to lack any detectable degradation prod-
ucts, the levels of the FlgS(H141A) protein present in WCLs
and the soluble fraction were about one-half the levels of the
wild-type FlgS (Fig. 1). By comparing the phenotypes of the
wild-type and mutant strains, we found that the flgS(H141A)
mutation affected motility, flagellar biosynthesis, and ?54-de-
pendent flagellar gene expression (Fig. 2 and data not shown).
The nonmotile phenotype of the flgS(H141A) mutant on semi-
solid agar plates at 24 h after inoculation was similar to that
observed for a ?flgS strain in which flgS had been deleted from
the chromosome (Fig. 2A and data not shown) (30). This lack
of motility in the flgS(H141A) mutant correlated with a com-
FIG. 1. Localization and stability of FlgS proteins in C. jejuni. Wild-
type strain C. jejuni 81-176 Smr(DRH212) (WT), 81-176 Smr?flgS
(DRH460), and 81-176 SmrflgS(H141A) (DRH1323) were grown, and
protein samples were obtained from the WCL, the soluble fraction (Sol),
and the insoluble membrane fraction (Mem) after sonication. Anti-FlgS
Rab11 antiserum (?-FlgS) was used to detect FlgS proteins (25). Anti-
RpoA M59 antiserum (?-RpoA) and anti-AtpF M3 antiserum (?-AtpF)
were used to detect the soluble cytoplasmic RpoA protein and the insol-
uble inner membrane protein AtpF, respectively (4).
VOL. 191, 2009FlgSR ACTIVATION BY FEA 2659
plete absence of flagella as analyzed by transmission electron
microscopy (data not shown). We then analyzed expression of
flgDE2- and flaB-astA transcriptional fusions in strains produc-
ing the FlgS(H141A) protein and found that the level of ?54-
dependent flagellar gene expression in an flgS(H141A) mutant
was equivalent to that in a ?flgS mutant (Fig. 2B), indicating
that H141 is critical for proper function of FlgS in C. jejuni.
Since H141 is the predicted site of phosphorylation, we
performed autophosphorylation assays with purified His6-
tagged versions of FlgS and FlgS(H141A). Whereas FlgS
autophosphorylated and accumulated radiolabeled phosphate
over time, FlgS(H141A) remained unphosphorylated (Fig. 3A
and 3B). In previous work, we showed that the FlgR response
regulator is modified by phosphorylation in the presence of
purified FlgS in vitro (35). We performed similar experiments
to determine if phosphotransfer to FlgR was abolished in the
presence of FlgS(H141A). In these experiments, we observed
phosphorelay to FlgR in the presence of wild-type FlgS but not
in the presence of the FlgS(H141A) protein (Fig. 3C), consis-
tent with the hypothesis that autophosphorylation of FlgS on
H141 contributes to phosphotransfer to FlgR. Thus, we believe
that H141A is the most likely site of autophosphorylation and
is essential for proper function of the protein.
Production of FlgS and FlgR is not dependent on the pres-
ence of the FEA. The FEA is a multiprotein complex that
translocates flagellar subunits across the inner membrane for
incorporation into a functional organelle (for a review, see
reference 44). As mentioned above, many of the FEA compo-
nents (e.g., FlhA, FlhB, FliP, and FliR) in addition to FlgS and
FlgR are required for ?54-dependent flagellar gene expression
in C. jejuni (30). We next performed experiments to determine
if the FEA and FlgSR systems are linked together in a regu-
latory cascade that terminates in activation of expression of
?54-dependent flagellar genes. More specifically, we investi-
FIG. 2. Phenotypic analyses of C. jejuni wild-type and flgS(H141A) mutant strains. (A) Motility phenotypes of C. jejuni strains producing wild-type
or mutant FlgS proteins in MH semisolid agar 24 h after inoculation. The strains used included wild-type strain 81-176 Smr(DRH212) (WT), 81-176 Smr
?flgS (DRH460), and 81-176 SmrflgS(H141A) (DRH1323). (B) Arylsulfatase assays for analysis of expression of flaB::astA and flgDE2::nemo in C. jejuni
81-176 derivatives producing wild-type and FlgS mutant proteins. The results are the results of a typical assay in which each strain was tested in triplicate.
The values reported for each strain are the average arylsulfatase activity ? standard deviation relative to the level of expression of each transcriptional
(81-176 Smr?astA flaB::astA) (WT), DRH939 (81-176 Smr?astA ?flgS flaB::astA), and SNJ958 [81-176 Smr?astA flgS(H141A) flaB::astA]. For
expression of flgDE2::nemo, the strains used included wild-type strain DRH533 (81-176 Smr?astA flgDE2::nemo) (WT), DRH936 (81-176 Smr?astA
FIG. 3. Autophosphorylation of FlgS proteins and phosphorelay
to FlgR. (A and B) Analysis of autophosphorylation of His6-FlgS and
His6-FlgS(H141A) over time after incubation of proteins with
[?-32P]ATP. (A) Representative gel analyzed by autoradiography from
an FlgS autophosphorylation assay. (B) Relative quantification of
autophosphorylation of FlgS proteins as determined by densitometry af-
ter autoradiography of gels. Three separate FlgS and FlgS(H141A)
autophosphorylation assays were performed, and the results of these
assays were averaged. The amount of incorporation of32P is expressed
in arbitrary units based on the densitometric analysis. (C) Analysis of
phosphorelay to His6-FlgR from His6-tagged FlgS or FlgS(H141A)
protein. FlgS proteins were preincubated with [?-32P]ATP before ad-
dition of His6-FlgR. A representative gel analyzed by autoradiography
from a phosphotransfer assay is shown. The presence (?) or absence
(?) of FlgR and the FlgS protein used in each reaction are indicated
above the lanes. WT, wild type.
2660 JOSLIN AND HENDRIXSONJ. BACTERIOL.
gated whether the FEA influences the production or activity of
the FlgSR two-component system.
To examine if production of FlgS or FlgR is dependent on
the FEA, we performed an immunoblot analysis of cell lysates
from the wild-type strain and mutant strains lacking flhA, flhB,
and fliP, which encode some of the proteins comprising the
FEA. We observed similar levels of FlgS and FlgR in the
wild-type strain and the FEA mutants (Fig. 4A), indicating that
production of FlgS and FlgR is independent of the FEA. As
additional verification that the FEA does not affect production
of FlgS and FlgR, we compared the levels of the flgS and flgR
mRNA transcripts in mutants lacking flhA, flhB, and fliP to the
levels in the wild-type strain by real-time RT-PCR analysis. We
did not detect significant changes in the levels of the flgS or flgR
mRNAs in the mutant strains compared to wild-type bacteria
(data not shown). Therefore, FEA mutants of C. jejuni appear
to produce normal levels of the FlgS and FlgR proteins but
have defects in signaling pathways for stimulation of ?54-de-
pendent flagellar gene expression.
We next analyzed C. jejuni to determine if the FlgSR system
functions downstream of the FEA in a regulatory cascade to
activate expression of ?54-dependent flagellar genes. Previous
work in our laboratory generated flgR alleles encoding proteins
lacking the N-terminal receiver or C-terminal domain of the
response regulator (35). These proteins were shown to have
partial constitutive activity in the absence of the FlgS sensor
kinase, indicating that FlgR functions downstream of FlgS
(35). We used these flgR alleles (flgR?receiverand flgR?CTD) to
replace wild-type flgR on the chromosome of mutants lacking
flhA, flhB, or fliP to determine if these partially constitutively
active FlgR proteins suppress the phenotype of the FEA mu-
tants for expression of flagellar genes. As shown previously
(30) and in Fig. 4B, flhA, flhB, or fliP mutants containing
wild-type flgR and producing the wild-type protein expressed
40- to 50-fold less of the ?54-dependent flaB- and flgDE2-astA
transcriptional fusions. When flgR in these FEA mutants was
replaced with the flgR alleles encoding FlgR?receiverand
FlgR?CTD, partial restoration of ?54-dependent flagellar gene
expression was observed (Fig. 4B). Although the levels of ex-
pression were not restored to wild-type levels, they were ap-
proximately 5- to 10-fold higher than those in the FEA mutants
that produced wild-type FlgR. These analyses suggest that
FIG. 4. Production of FlgS and FlgR and activity of FlgR proteins in FEA mutants of C. jejuni. (A) Production of FlgS and FlgR proteins in
mutants of C. jejuni lacking one component of the FEA. WCLs of wild-type and C. jejuni mutant strains were prepared for immunoblot analysis.
Anti-FlgS Rab11 (?-FlgS) and anti-FlgR (?-FlgR) Rab13 antisera were used to detect the FlgS (left gel) and FlgR (right gel) proteins (25). The
strains used for analysis included wild-type strain DRH212 (81-176 Smr) (WT), DRH460 (81-176 Smr?flgS), DRH737 (81-176 Smr?flgR),
DRH946 (81-176 Smr?flhA), SNJ471 (81-176 Smr?flhB), and DRH1065 (81-176 Smr?fliP). (B) Arylsulfatase assays for analysis of expression
of flaB::astA and flgDE2::nemo in the C. jejuni 81-176 Smrwild-type strain and mutant strains lacking a component of the FEA and producing
wild-type and FlgR mutant proteins. The results are the results of a typical assay in which each strain was tested in triplicate. The values reported
for each strain are the average arylsulfatase activity ? standard deviation relative to the level of expression of each transcriptional fusion in 81-176
Smr?astA ?flhA, which was defined as 1 arylsulfatase unit. For expression of flaB::astA, the strains used included (from left to right) wild-type
strain DRH665, DRH1049, SNJ112, SNJ273, DRH1827, SNJ109, SNJ1021, DRH1178, SNJ261, and SNJ1015. For expression of flgDE2::nemo, the
strains used included (from left to right) wild-type strain DRH533, DRH1021, SNJ115, SNJ274, DRH1827, SNJ113, SNJ1017, DRH1204, SNJ358,
and SNJ1012. The FEA mutation and the type of FlgR protein produced in each strain are indicated below the graph. WT, wild type.
VOL. 191, 2009FlgSR ACTIVATION BY FEA 2661
FlgSR functions downstream of the FEA and that activation of
FlgSR is dependent in some manner on the FEA of C. jejuni.
Formation of the FEA likely initiates activation of the FlgSR
system. Considering our data, we speculated that the FEA may
contribute an essential signal to activate the FlgSR system to
terminate in expression of ?54-dependent flagellar genes. We
hypothesized that either formation of the FEA or the secretory
activity of the FEA may comprise the signal to activate the
FlgS sensor kinase. If the former hypothesis is correct, it is
possible that positioning one component of the FEA or the
whole FEA complex in the inner membrane may directly pro-
vide the signal sensed directly by the cytoplasmic FlgS protein,
leading to autophosphorylation of the kinase. Alternatively,
formation of the FEA may be required for production of a
downstream signal sensed by FlgS. The latter hypothesis in-
cludes the possibility that the secretory activity of a formed
FEA may influence activation of FlgS. For instance, a negative
regulator that represses activity of FlgS may be present in the
cell before the FEA is competent for secretion, and the secre-
tory activity of the FEA may be required to inactivate or
remove this protein from the cytoplasm, relieving FlgS from
repression and allowing autophosphorylation and phosphore-
lay to FlgR to occur.
To distinguish between these possibilities, we generated mu-
tants with FEA complexes that are predicted to assemble in the
inner membrane but are hindered for secretion of flagellar
substrates. For this approach, we targeted fliI and flhB for
mutation. FliI functions as an ATPase that dissociates export
substrates (e.g., flagellins) from their chaperones in S. enterica
serovar Typhimurium (49, 55). While FliI is not absolutely
required for secretion of flagellar substrates, its absence sub-
stantially reduces the efficiency of this process. Due to the
significant homology between the FliI proteins of C. jejuni and
S. enterica serovar Typhimurium strain LT2 (43% identity and
62% similarity over 424 amino acids), we hypothesize that FliI
serves a similar function in C. jejuni in increasing the efficiency
of secretion of flagellar proteins. Therefore, we deleted fliI
from the C. jejuni genome to create a mutant with possibly
impaired efficiency of FEA-mediated secretion of flagellar pro-
Previous analysis with S. enterica serovar Typhimurium re-
vealed that defined mutations can also be made in flhB so that
the FEA assembles in the inner membrane, but secretion of
substrates through the FEA is reduced or blocked (21). These
mutations include mutations that result in small, in-frame de-
letions and point mutations in the FlhB protein. By aligning
the sequences of the S. enterica serovar Typhimurium and C.
jejuni strain 81-176 proteins (which are 36% identical and 60%
similar across 351 amino acids), we identified regions of the
FlhB protein of C. jejuni that may be deleted or mutated,
resulting in FEA mutants that form but do not secrete
efficiently. To this end, we constructed flhB mutant alleles
that encoded FlhB?214-218, FlhB?224-228, FlhB?244-253, and
FlhB(N267A) mutant proteins. The deletions and mutations in
the C. jejuni FlhB protein correspond to types of domain de-
letions and point mutations resulting in the FlhB?2, FlhB?4,
FlhB?8-9, and FlhB(N269A) proteins of S. enterica serovar
Typhimurium constructed by Fraser et al. (21), respectively.
After construction of fliI and flhB mutants of C. jejuni, we
first analyzed the strains to determine stability of the FlhB
protein produced in each mutant by immunoblot analysis. FlhB
is produced as a 42-kDa protein in S. enterica serovar Typhi-
murium that is cleaved to a 31-kDa protein by autoproteolysis
of the peptide bond between positions N269 and P270 (19, 21,
48). Although flhB of C. jejuni appears to encode a 37-kDa
protein, we predict that similar processing may occur between
N267 and P268, resulting in a 30-kDa FlhB protein. Immuno-
blot analysis of the total membrane fraction of wild-type C.
jejuni revealed that FlhB appeared as the processed 30-kDa
protein (Fig. 5A). In three of the four fliI and flhB mutants, we
observed similar levels of processed FlhB proteins, indicating
that the mutant FlhB proteins were stable. The flhB(N267A)
mutant was expected to produce an FlhB protein that is not
able to undergo autoproteolytic processing. Indeed, we ob-
served only the full-length 37-kDa protein in this mutant (Fig.
5A). In the flhB?244-253mutant, we could not detect any mutant
FlhB protein. The reason for the lack of detection of this
mutant form of FlhB remains unknown, but it may be due to
the method used to generate the anti-FlhB antiserum. The
antigen that was used to make the anti-FlhB antiserum con-
tained amino acids 209 to 367 of FlhB, which form the com-
plete cytoplasmic domain of the protein before processing.
Due to predicted processing of FlhB at position 267 in C.
jejuni, ultimately only a maximum of 58 amino acids (amino
acids 209 to 267) in processed FlhB proteins are the same as
the amino acids in the antigen that was used to generate the
anti-FlhB antiserum. Since FlhB?244-253lacks 10 of the 58
amino acids of the antigen, the epitope that the anti-FlhB
antiserum recognizes may have been destroyed or deleted in
this protein, resulting in its lack of detection. Because the
mutant producing FlhB?244-253stimulated expression of ?54-
dependent flagellar genes (see below), we believe that this
protein is made and is stable but is undetectable with current
We next determined if the secretion of the flhB and fliI
mutants was impaired. To do this, we performed two different
analyses. We first determined if motility was reduced since
motility is directly dependent on FEA-mediated secretion of
flagellar proteins out of the cytoplasm to construct a flagellar
organelle. For all the flhB and fliI mutants, we observed that
the level of motility was ?10% of that of the wild-type strain,
indicating that flagellar motility and presumably secretion
through the FEA had been severely impaired (Fig. 5A).
We next performed a more direct analysis of the secretion
competence of the FEA in the derived mutants by monitoring
FEA-dependent secretion of the FlaA flagellin protein to the
outer membrane of C. jejuni strains. Unlike the situation in S.
enterica serovar Typhimurium, the complete regulatory path-
ways that govern flaA expression in C. jejuni are not completely
understood. In S. enterica serovar Typhimurium, ?28-depen-
dent expression of fliC encoding the major flagellin is re-
pressed in FEA mutants due to cytoplasmic retention of the
anti-?28factor FlgM (32, 38). In C. jejuni, flaA is expressed by
a ?28-dependent promoter (10, 28, 30, 66). However, evidence
for expression of flaA and secretion of the encoded protein via
the FEA to form a truncated flagellum with partial motility has
been obtained for an fliA (encoding ?28) mutant, indicating
that a ?28-independent promoter likely exists (28, 30, 37). Also
unlike the situation in S. enterica serovar Typhimurium, there
is evidence that flaA expression is only moderately decreased
2662 JOSLIN AND HENDRIXSONJ. BACTERIOL.
in certain FEA mutants of C. jejuni 81-176, indicating that
some expression of flaA is independent of the FEA status of
the bacterium (30). Furthermore, any existing translation con-
trols for flaA mRNAs in C. jejuni have not been characterized.
Since evidence that flaA expression and FlaA production are
not entirely dependent on the status of the FEA in C. jejuni, as
they are in other bacteria, we analyzed FEA-dependent secre-
tion of FlaA in our defined flhB and fliI mutants.
We first ensured that flaA was expressed in the mutants by
monitoring expression of flaA::astA in the flhB and fliI mutants.
We found that flaA::astA expression was not defective in three
of the mutants [flhB?214-218, flhB(N267A), and ?fliI]. Rather,
the expression of flaA::astA in these mutants was approxi-
mately twofold higher than that in the wild-type strain (Fig.
6A). Expression of flaA::astA was slightly reduced in the
flhB?244-253mutant, to approximately 75% of that in the wild-
type strain. The remaining mutant, flhB?224-228, expressed
flaA::astA at a level that was 50% less than the level in the
wild-type strain (Fig. 6A). The level of expression of flaA::astA
in this mutant was similar to that in ?flhB or ?fliA (lacking ?28)
mutants. With the exception of the expression in the flhB?224-228
muant, flaA::astA expression in the mutants was mostly intact or
the level was higher than the level in the wild-type strain.
We next monitored FEA-mediated secretion of FlaA by
comparing the levels of FlaA associated with outer membranes
of wild-type and mutant strains of C. jejuni. As shown in Fig.
6B, the flhB?214-218, flhB(N267A), and ?fliI mutants produced
comparable levels of FlaA in WCLs, but reduced levels of the
protein were associated with the outer membrane compared to
the outer membrane of wild-type bacteria. The most severe
mutation was flhB(N267A), which caused complete lack of
FlaA in the outer membrane. The other two mutants, the
flhB?214-218and ?fliI mutants, had approximately two- to five-
fold reductions in the level of of FlaA associated with the outer
FIG. 5. Phenotypic analyses of C. jejuni strains with formed but secretion-impaired FEA complexes. (A) Immunoblot analysis of FlhB proteins
and motility phenotypes of C. jejuni wild-type and flhB or fliI mutant strains. Total membrane proteins were isolated from wild-type and mutant
strains of C. jejuni. Equal amounts of proteins from the strains were analyzed. Anti-FlhB Rab476 antiserum was used to detect the FlhB proteins.
The arrows indicate the positions of the 37-kDa full-length, unprocessed FlhB protein and the 30-kDa processed FlhB protein. The motility
phenotypes of wild-type and mutant strains are indicated below the blot. The diameter of the motile ring around the point of inoculation in MH
semisolid agar was measured after 36 h of incubation at 37°C under microaerobic conditions. The level of motility of each mutants is expressed
relative to the level of motility of the wild-type strain, which was defined as 100%. The strains used for both analyses included (from left to right)
wild-type strain DRH461 (WT), DRH1734, SNJ464, SNJ428, SNJ475, and SNJ438. (B) Arylsulfatase assays for analysis of expression of flaB::astA
and flgDE2::nemo in C. jejuni 81-176 Smrwild-type or mutant strains containing a secretion-impaired FEA. The results are the results of a typical
assay in which each strain was tested in triplicate. The values reported for each strain are the average arylsulfatase activity ? standard deviation
relative to the level of expression of each transcriptional fusion in wild-type strain 81-176 Smr?astA, which was defined as 100 arylsulfatase units.
For expression of flaB::astA, the strains used included (from left to right) wild-type strain DRH665 (WT), DRH1830, SNJ467, SNJ434, SNJ508,
SNJ442, and SNJ422. For expression of flgDE2::nemo, the strains used included wild-type strain DRH533 (WT), DRH1827, SNJ466, SNJ433,
SNJ504, SNJ439, and SNJ457. The type of mutation in the FEA of each strain is indicated below the graph.
VOL. 191, 2009 FlgSR ACTIVATION BY FEA2663
membrane, suggesting that secretion had been impaired in
these mutants. For the flhB?244-253mutant there was about
threefold less FlaA in WCLs, but this mutant completely
lacked FlaA in the outer membrane. Only in one mutant, the
flhB?224-228mutant, did FlaA production appear to be greatly
hindered, similar to a ?flhB mutant.
Considering that four of the five mutants that we created
appeared to have FEAs with greatly diminished secretion abil-
ities, we then analyzed expression of ?54-dependent flagellar
genes in these mutants. In the same four mutants [flhB?214-218,
flhB?244-253, flhB(N267A), and ?fliI], expression of the flaB-
and flgDE2-astA transcriptional fusions was equal to or slightly
higher than the expression in the wild-type strain (Fig. 5B).
These results indicate that completely blocking or hindering
secretion through the FEA did not affect expression of ?54-
dependent flagellar genes. This analysis provided evidence that
formation of the FEA, rather than secretory activity of the
apparatus, is required and may be the key element to activate
the FlgSR system for expression of ?54-dependent flagellar
Only in the mutant that produced the FlhB?224-228protein
did we observe reduced expression of flaB::astA and flgDE2::
nemo comparable to that of the ?flhB mutant (Fig. 5B). Con-
sidering that this mutant also behaved similar to the ?flhB
mutant in terms of expression of flaA::astA and secretion of the
FlaA protein, we believe that, like the ?flhB mutant, this mu-
tant may not form a complete FEA. Thus, this mutant may not
actually be germane to our goal of creating secretion-incom-
petent but correctly formed FEAs. However, if an FEA forms
in this mutant, then our alternative hypothesis that a negative
regulator may be active and inhibit the FlgSR system in a
nonsecreting bacterium may have some credence. To investi-
gate this hypothesis, we performed transposon mutagenesis
with the darkhelment transposon (27) in C. jejuni 81-176 ?astA
?flhA flgDE2::nemo, 81-176 ?astA ?flhB flgD::astA, and 81-176
?astA ?fliP flaB::astA. These mutants do not express the ?54-
dependent transcriptional astA fusions due to the lack of a
complete FEA. Disruption of a gene encoding a putative re-
pressor would allow expression of the transcriptional reporters
in the FEA mutants. Such a transposon mutant could be iden-
tified by recovering mutants on media containing a chromo-
meric substrate for arylsulfatase and observing a switch from a
white colony phenotype to a blue colony phenotype. Despite
screening over 65,000 transposon mutants, we were unable to
identify any mutant with a transposon that disrupted a gene for
such a negative regulator, suggesting that such a gene may not
exist or is an essential gene. Considering these data as a whole,
we propose that FlgSR activation likely depends on proper
assembly of the FEA. While we cannot entirely exclude the
possibility that the secretory activity is required for FlgSR
activation, our results indicating that four of five flhB or fliI
mutants were impaired for secretion but had mutations that
did not affect expression of ?54-dependent flagellar genes, cou-
pled with the results of our transposon mutagenesis screen,
weaken this hypothesis.
Previous studies in our laboratory have found that the pro-
teins of the FEA, the putative FlhF GTPase, and the FlgSR
two-component system are required for full expression of ?54-
dependent flagellar genes in C. jejuni (30, 35). In the current
study, we obtained evidence that links the FEA to stimulation
of the FlgSR two-component regulatory system. We found that
activation rather than production of the FlgSR system is de-
pendent on the FEA. Furthermore, we believe that formation
of the apparatus rather than the secretory function of the
apparatus is key to producing the signal detected by FlgS
leading to its activation and subsequent expression of ?54-
dependent flagellar genes. Analysis of the genomic sequences
of various C. jejuni strains indicates that the consensus ?54-
binding site is in the promoters of most genes that encode the
flagellar proteins that are external to the cytoplasm and likely
secreted by the FEA (20, 31, 54). Because gene expression and
protein production are energetically expensive processes, it is
likely that the introduction of a level of transcriptional control
by the FEA allows C. jejuni to ensure that ?54-dependent
flagellar genes are expressed and the secreted proteins are
produced only after the apparatus has formed.
The flagellar regulatory cascade of C. jejuni appears to bear
some resemblance to the cascades utilized by species of Heli-
FIG. 6. Analysis of flaA expression and FlaA secretion mediated by
the FEA. (A) Arylsulfatase assays for analysis of expression of flaA::astA
in the C. jejuni 81-176 Smrwild-type strain or strains with a secretion-
impaired FEA. The results are the results of a typical assay in which each
strain was tested in triplicate. The values for each strain are the average
arylsulfatase activity ? standard deviation relative to the level of expres-
sion of each transcriptional fusion in wild-type strain 81-176 Smr?astA,
which was defined as 100 arylsulfatase units. For expression of flaA::astA,
the strains used included (from left to right) wild-type strain DRH655
(WT), DRH1070, SNJ365, SNJ427, SNJ1033, SNJ1034, SNJ1038, and
(B) Immunoblot analysis of FlaA production in WCLs and secretion to
the outer membrane of wild-type and FEA mutant strains. WCL and
outer membrane (OM) fractions were isolated from wild-type and mutant
strains of C. jejuni. Anti-FlaA LL-1 antiserum was used to detect the FlaA
proteins (42). The strains used included (from left to right) wild-type
strain DRH212 (WT), DRH724, DRH655, SNJ471, SNJ464, SNJ428,
SNJ475, SNJ438, and DRH2257.
2664JOSLIN AND HENDRIXSONJ. BACTERIOL.
cobacter, Vibrio, and Pseudomonas (2, 16, 34, 39, 40, 47, 51, 56,
60). First, all the cascades are known to require ?54and a
two-component regulatory system with functional similarity to
FlgSR for expression of a subset of flagellar genes. In addition,
Vibrio and Pseudomonas species require the activity of a master
regulator protein to initiate transcription of genes encoding
FEA proteins and these flagellar two-component regulatory
systems (2, 15, 16, 36, 40, 56). However, in C. jejuni and Heli-
cobacter pylori, no master regulator of flagellar biosynthesis has
been found, and one current hypothesis is that the expression
of genes encoding components of the FEA and FlgSR is largely
constitutive (26, 51). In all these bacteria, activation of the
flagellar two-component regulatory system leads to the ?54-
dependent expression of genes encoding flagellar proteins that
are secreted by the FEA (16, 25, 27, 30, 35, 40, 51, 56). Con-
sidering the similarity of the compositions of these flagellar
regulatory cascades, our findings may suggest that the forma-
tion of the FEA could influence ?54-dependent flagellar gene
expression in a number of bacterial species. Further analysis of
each of these organisms is required to determine if this rela-
tionship is shared across multiple genera of motile bacteria.
The analysis presented in this work allowed us to more
precisely clarify the relationship between the FEA and the
FlgSR system in ?54-dependent flagellar gene expression. We
constructed C. jejuni mutants whose mutations impaired FEA-
mediated secretion to determine if formation of the export
apparatus or its secretory activity was required for FlgS acti-
vation. Based on our finding that three of four flhB mutations
and a fliI mutation reduced or blocked secretion of the FlaA
flagellin but did not negatively affect ?54-dependent gene ex-
pression, we concluded that the formation of the FEA in the
inner membrane could be the signal detected by FlgS that
directly leads to activation of the kinase. Alternatively, forma-
tion of the FEA may be indirectly involved by being required
for the production of a downstream activating signal. Although
the data alone do not define the nature of the communication
between the FEA and FlgSR, we have provided a foundation
for future studies to understand activation of the system. Char-
acterization of additional FEA proteins and structures such as
the inner membrane MS ring and the cytoplasmic C ring that
are associated with the FEA (43, 44), along with better re-
agents to detect complete FEA formation, may allow us to
further define the activating signal emanating from this secre-
If our hypothesis that FlgS detects formation of the FEA for
autoactivation is correct, the cytoplasmic localization of FlgS
may provide insight into the origin of the signal relative to the
FEA structure. Since FlgS is a cytoplasmic protein, FlgS may
detect a signal originating on the cytoplasmic face of the inner
membrane-localized FEA complex. For instance, FlgS may
detect a completed FEA structure by monitoring whether cer-
tain proteins with large cytoplasmic domains are in the FEA.
Possible candidates for this type of signal include the cytoplas-
mic domains of FlhA and FlhB. To find evidence supporting
this hypothesis, we attempted to use numerous approaches to
directly detect interactions that may occur between FlgS and
FEA proteins, including affinity chromatography, affinity blot-
ting, and in vivo chemical cross-linking. However, the results of
these assays were inconsistent and inconclusive. New and bet-
ter reagents and protocols have to be developed to extend
these types of analyses. In vivo detection of an FlgS interaction
with a member of the FEA may be difficult, due to the fact that
flagellated C. jejuni assembles only one or two of these secre-
tory apparatuses per bacterium. Thus, the number of interac-
tions of FlgS with the FEA or an FEA component may be
small and the interactions may be temporally transient.
As mentioned above, our results strongly support the hy-
pothesis that formation of the FEA either directly comprises
the signal or is required to produce the signal to activate FlgSR
and expression of ?54-dependent flagellar genes. An alterna-
tive hypothesis that we considered suggested that the secretory
activity of the FEA could be the activating signal, with a cyto-
plasmic repressor hindering the FlgSR regulatory cascade
prior to formation of and secretion by the FEA. However, four
of the five flhB or fliI mutants whose mutations were shown to
hinder or block secretion of flagellar proteins were not af-
fected for ?54-dependent expression of flagellar genes. Only
the flhB?226-230mutant showed decreased expression of
these genes, but analysis of this mutant suggested that it
behaved most like a ?flhB mutant, which does not form a
complete FEA. Thus, we cannot confidently conclude that
the flhB?224-228mutant makes a fully formed but secretion-
incompetent apparatus. Second, our transposon mutagenesis
screen did not reveal any transposon insertions in FEA mu-
tants that relieved repression of expression of ?54-dependent
flagellar genes. These combined results greatly weaken the
hypothesis that the secretory activity of the FEA alone forms
the FlgS-activating signal. Thus, the results of this study
strongly favor the hypothesis that that formation of the FEA is
a requirement for and quite possibly a component of the es-
sential signal for activating the FlgSR system that results in
expression of ?54-dependent flagellar genes.
Our work also suggests a new function in the signaling me-
diated by the FEA in flagellar regulatory cascades. In the
well-characterized pathways observed in E. coli and Salmo-
nella, formation of the FEA ultimately controls the activity of
the alternative sigma factor ?28involved in expression of genes
encoding the major flagellins and some motor proteins (41).
The FEA is responsible for secretion of flagellar proteins
and the anti-? factor, FlgM, which represses the activity of ?28
until the cell has completed formation of the FEA, basal body,
and hook structures required to secrete flagellins to build a
filament (32, 38). In this study, we found that the FEA is
intimately involved in creating a signal that activates the FlgSR
two-component system, leading to activation of ?54. Therefore,
the FEA plays a different role in influencing signaling for
?54-dependent expression of flagellar genes in C. jejuni. This
finding may also be applicable to other motile bacteria that
utilize ?54in flagellar gene regulation and biosynthesis, includ-
ing species of Vibrio, Pseudomonas, and Helicobacter. This
work expands the known mechanisms of regulating flagellar
gene expression and suggests that there are more complex
functions associated with the FEA beyond protein secretion.
Future analyses of FlgS will involve determining the domain
and residues of the protein required for sensing an autoacti-
vating signal. In analyzing the sequence of FlgS, we found that
the central and C-terminal portions of the protein contain the
histidine-containing phosphotransfer domain and the ATP-
catalytic domain (61, 65). These domains are required for
accepting a phosphate group on a conserved histidine and for
VOL. 191, 2009FlgSR ACTIVATION BY FEA2665
ATP hydrolysis, respectively, for autophosphorylation. Indeed,
we found that H141 in the phosphotransfer domain is required
for modification by phosphorylation and for functioning of the
active FlgS to stimulate expression of ?54-dependent flagellar
gene expression. In a comparison of the amino acid sequence
of FlgS to those of other sensor kinases, the predominant
homology with the latter kinases is localized almost exclusively
to the phosphoacceptor and ATP hydrolysis domains. Only
limited homology between the initial 130 amino acids of FlgS
and other sensor kinases is apparent. The sensor kinases that
share the most homology to this region of the C. jejuni FlgS
protein are other FlgS homologues in Campylobacter species
(almost 100% identity), the FlgS orthologue in Helicobacter
species (31 to 37% identity and 57 to 66% similarity), and the
FlrB sensor kinase of Vibrio cholerae (26% identity and 54%
similarity). The N-terminal regions of these proteins have no
obvious motifs that suggest a function or how they may sense
a specific factor. Since these N-terminal domains are unique to
the group of FlgS orthologues, it is likely that this region of
these proteins may function in specifically recognizing the sig-
nal necessary to culminate in expression of ?54-dependent
flagellar genes. Future studies will focus on further character-
izing this domain of the protein.
Previous work in our laboratory focused on understanding
the activation and function of the FlgR response regulator (25,
30, 35). In this study, we describe work that provides a foun-
dation for understanding the activation of the cognate sensor
kinase, FlgS, and how the FEA influences activation of this
two-component regulatory system. To date, we have linked
activation of the FlgSR system to the FEA and have charac-
terized a previously undescribed mechanism for controlling
activation of flagellar gene expression. In addition, FlgSR ap-
pears to be an unusual two-component system in which expres-
sion of both components is controlled by phase-variable mech-
anisms (25, 27), a trait unique among well-characterized
bacterial two-component systems. Thus, there appears to be at
least two mechanisms for controlling ?54-mediated expression
via the FlgSR proteins. Future analyses will focus on further
defining the nature of the activating signal emanating from the
FEA and how it influences expression of ?54-dependent flagel-
This work was supported by NIH grant R01 AI065539, by National
Research Initiative Grant 2006-35201-17382 from the USDA Cooper-
ative State Research, Education, and Extension Service Food Safety
Program, and by start-up funds from the University of Texas South-
western Medical Center. S.N.J. was supported by NIH training grant
T32 AI007520 from the Molecular Microbiology Graduate Program.
We thank Kevin Gardner for helpful discussions regarding auto-
phosphorylation experiments and analyses.
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