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Journal of Bacteriology, September 2004, p . 5956-5960, Vol .
186, No . 17
FlhF,
the Third Signal Recognition Particle-GTPase of Bacillus subtilis, Is
Dispensable for Protein Secretion
Geeske Zanen,1 Haike Antelmann,2 Helga Westers,1,
Michael Hecker,2 Jan Maarten van Dijl,1*
and Wim J . Quax1
Department of Pharmaceutical Biology, University of Groningen, Groningen, The
Netherlands,1 Institut für Mikrobiologie und Molekularbiologie,
Ernst-Moritz-Arndt-Universität Greifswald, Greifswald, Germany2
Received 11 March 2004/ Accepted 8 June 2004
Bacillus subtilis contains three proteins of the signal recognition
particle-GTPase family known as Ffh, FtsY, and FlhF . Here we
show that FlhF is dispensable for protein secretion, unlike Ffh and
FtsY . Although flhF is located in the fla/che operon,
B . subtilis 168 flhF mutant cells assemble flagella and are
motile .
In eukaryotes, prokaryotes, and archaea, a large number of proteins
is transported across membranes in order to fulfill their biological
function . Complex and well-organized protein transport systems have
evolved for membrane translocation of these proteins . Most proteins
that play a role outside the cytoplasm contain a signal peptide,
which directs the (pre)protein to its final destination (2,
23, 28, 29) . Chaperones
and targeting factors recognize this signal peptide and keep
preproteins in an export-competent state before targeting to the
translocation machinery in the membrane . The major machinery for
protein transport is the Sec translocase, which handles preproteins
in an unfolded state (7) .
On the basis of proteomic studies, it has been proposed that the
majority of secretory proteins of the gram-positive bacterium
Bacillus subtilis are targeted to the Sec translocase by the
so-called signal recognition particle (SRP) (9) . This SRP
seems to be involved in preprotein targeting to membranes of
organisms belonging to all three domains of life . The B . subtilis
SRP complex consists of the Ffh (Fifty-four homolog) protein (10),
a small cytoplasmic RNA (scRNA) (15, 16),
and a histone-like protein (HBsu) (17) . Preprotein
targeting by this SRP complex presumably involves the presence of the
SRP receptor-like protein FtsY (18) . Both Ffh and
FtsY belong to the widely conserved family of SRP-GTPases (8) .
Interestingly, B . subtilis and several other bacterial species
(but not Escherichia coli) contain a third gene encoding a
protein belonging to the SRP-GTPase family . In B . subtilis,
this paralogue of Ffh and FtsY was named FlhF (flagellum-associated
protein) because it appeared to be required for the flagellar
assembly and motility of this bacterium (5) .
Specifically, the B . subtilis FlhF protein has 46% identical
residues and conservative replacements in a stretch of 175 residues
with B . subtilis Ffh and 37% identical residues and conservative
replacements in a stretch of 318 residues with B . subtilis FtsY .
As shown by sequence alignments and domain searches, FlhF contains
the conserved N and G domains of the SRP-like GTPases (Fig .
1) . However, it lacks the so-called M domain typical for the
C termini of Ffh-like proteins and contains a basic B domain
instead of the acidic A domain of FtsY-like proteins of bacteria and
yeasts . Notably, the mammalian SRP receptor SR
contains a more basic N-terminal domain, like FlhF of B . subtilis .
Consistent with its proposed function, the flhF gene is
located within the che/fla operon, which encodes the
majority of the chemotaxis and flagellar proteins (11) .
Pandza and coworkers (20) showed that the FlhF
homologue of Pseudomonas putida has a role in polar flagellar
placement and in induction of the general stress response .
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FIG . 1 . Conserved domains in proteins of the SRP-GTPase family . The
SRP-GTPase family members of yeast (SRP54, SR),
E . coli (P48, FtsY_Ec), and B . subtilis (Ffh, FlhF,
FtsY_Bs) are represented schematically . Different domains that
can be distinguished are the acidic A domain; the basic B domain, the
conserved N domain, the M domain involved in RNA and preprotein binding,
and the GTP-binding G domain . The five conserved boxes, G1 to G5, in the
G domain, as defined by Eichler and Moll (8), are
shown.
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On the basis of the similarity between FlhF and Ffh/FtsY, Carpenter
et al . (5) proposed that FlhF might be involved in protein
secretion . Notably, however, FlhF is dispensable for growth and
viability, whereas Ffh and FtsY are essential, like the key
components SecA, SecY, and SecE of the Sec translocase (12) .
This raised the questions of whether and, if so, to what extent FlhF
is involved in protein secretion .
Construction of a B . subtilis 168 flhF mutant.
Since all of our previous studies on protein secretion by B .
subtilis were performed with sequenced strain 168 (13), a
B . subtilis 168 flhF::cat mutant strain was
constructed by transforming B . subtilis 168 with chromosomal
DNA of flhF mutant strain OI2735, which was constructed by
Carpenter et al . (5) (Table 1) . B .
subtilis 168 was transformed as previously described (22) .
Chloramphenicol-resistant transformants were screened by PCR with
primers cat1 (5'-GAT TTA GAC AAT TGG AAG) and cat2 (5'-GAC AAT TCC
TGA ATA GAG) to show the presence of the cat gene (data not
shown) . PCR was carried out with the Pwo DNA polymerase
(Roche) as described previously (26) .
| TABLE 1 . Plasmid and bacterial strains used in this study
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FlhF is not required for protein secretion by B . subtilis strains
168 and DB430. To investigate the involvement of FlhF in protein
secretion, the composition of the extracellular proteome of B .
subtilis 168 flhF::cat was analyzed and compared to
that of parental strain 168 . In addition, similar experiments were
performed with protease-deficient strain DB430 and a DB430 flhF::cat
derivative that was obtained by transformation of strain DB430 with
chromosomal DNA of strain OI2735 . For analysis of their extracellular
proteomes, all strains were grown at 37°C under vigorous agitation
in rich medium . After 1 h of postexponential growth, cells were
separated from the growth medium by centrifugation and proteins
secreted into the growth medium were concentrated by trichloroacetic
acid precipitation . The resulting samples were used for two-dimensional
gel electrophoresis, and protein spots were identified by matrix-assisted
laser desorption ionization-time of flight mass spectrometry
and/or N-terminal sequencing as previously described (1) .
After dual-channel imaging to visualize possible changes in the
extracellular protein composition, no major differences were observed
between the extracellular proteomes of the flhF::cat
mutants and the respective parental strains, 168 and DB430 (Fig.
2A) . Under the conditions tested, some fluctuation
in the levels of prophage-encoded proteins XkdK, XkdG, XkdM, and YolA
was observed in the growth media of flhF mutant strains, as
well as in the media of parental strains 168 and DB430 (Fig.
2B) . Most likely, this reflects fluctuations in the
expression of genes located on the PBSX prophage (xkdK,
xkdG, and xkdM) and the SPß prophage (yolA) .
Unexpectedly, the extracellular accumulation of proteins known to be
required for cell motility, such as FlgK (flagellar hook-associated
protein 1), FliD (flagellar hook-associated protein 2), and the
flagellin Hag, was not affected by the absence of FlhF .
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FIG . 2 . Extracellular proteome of B . subtilis flhF::cat .
The extracellular proteins of the flhF::cat mutant strains
and the respective parental strains 168 and DB430 were separated by
two-dimensional gel electrophoresis, after which dual-channel
fluorescence imaging was used to visualize possible changes in
extracellular protein composition (3) . Protein spots
identified by mass spectrometry and/or N-terminal sequencing are
indicated . Green protein spots are predominantly present in the image of
the extracellular proteins of the parental strain, red protein spots are
predominantly present in the image of the extracellular proteins of the
flhF mutant strain, and yellow protein spots are present in
similar amounts in both images . (A) Extracellular proteomes of B .
subtilis DB430 and DB430 flhF::cat . (B) Variable
extracellular levels of prophage-encoded proteins YolA, XkdM, XkdG, and
XkdK (top to bottom).
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It was previously shown that the absence of some components of the
Sec machinery of B . subtilis, such as SecDF (4) and
SecG (27), has no detectable effect on protein
secretion unless the secretion machinery is challenged with
overproduced secretory proteins . For example, this was shown by
high-level expression of the
-amylase
AmyQ of Bacillus amyloliquefaciens (4) with
plasmid pKTH10 (Table 1) . To study the importance of FlhF for
AmyQ secretion at high levels, the flhF mutant strain and parental
strain 168 were transformed with pKTH10 . After overnight growth
in TY medium (1% Bacto Tryptone, 0.5% Bacto Yeast Extract, 1% NaCl)
supplemented with kanamycin, cells and medium fractions were
separated by centrifugation (2 min, 16,000 x
g, room temperature) . Next, protein samples for sodium dodecyl
sulfate-polyacrylamide gel electrophoresis were prepared as described
previously (25) . After separation by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, proteins were
transferred to a Protran nitrocellulose transfer membrane (Schleicher
& Schuell) as described by Kyhse-Andersen (14) .
AmyQ was visualized with specific antibodies and horseradish
peroxidase-goat anti-rabbit immunoglobulin G conjugates (BioSource
International) . As shown in Fig . 3A, disruption of
the flhF gene affects neither the amounts of AmyQ secreted
into the growth medium nor the amounts of AmyQ present in the cells .
Taken together, these findings demonstrate that FlhF is dispensable
for protein secretion, even when the secretion machinery of B .
subtilis is challenged by the high-level production of a
secretory protein .
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FIG . 3 . Absence of FlhF has no impact on secretion of AmyQ and cellular
levels of Ffh and FtsY . The secretion of overproduced AmyQ (A) and the
intracellular levels of Ffh and FtsY (B) were analyzed by Western
blotting with cellular (c) and/or growth medium (m) fractions of B .
subtilis 168 flhF::cat and parental strain 168 . d,
degradation products of AmyQ.
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Since FlhF is a paralogue of Ffh and FtsY, it is conceivable that the
B . subtilis cell can suppress the effects of the absence of
FlhF by production of Ffh or FtsY at increased levels . To study
possible changes in the levels of Ffh and FtsY in the absence of
FlhF, Western blotting experiments were performed . For this purpose,
overnight cultures of B . subtilis 168 flhF::cat
and parental strain 168 were diluted to an optical density at 600 nm
of 0.05 and grown until 1 h after the transition between exponential
and postexponential growth . Subsequently, cells were collected by
centrifugation and prepared for Western blotting as indicated above .
Ffh and FtsY were visualized with specific antibodies and alkaline
phosphatase-conjugated anti-rabbit immunoglobulin G (BioSource
International) and a standard nitroblue
tetrazolium-5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside
reaction (21) . The results demonstrate that the
intracellular levels of Ffh and FtsY are not affected by disruption
of the flhF gene (Fig . 3B) . Consequently, it
can be concluded that B . subtilis cells do not compensate for
the absence of FlhF by upregulation of the production of Ffh and/or
FtsY . However, complementation of the flhF mutation by the
production of Ffh and/or FtsY at normal levels cannot be ruled out .
FlhF has a minor role in the motility of strain 168. As the
flhF mutation in B . subtilis OI2735 was shown to result
in nonmotility, we verified whether the same would be true for cells
of B . subtilis 168 flhF::cat by using a motility plate
assay . B . subtilis cultures were grown overnight at 37°C
in TY medium . Next, the optical density at 600 nm was measured and
adjusted to 1.0 with fresh TY medium . Subsequently, an aliquot of 2
µl was spotted onto TY plates containing 0.27% agar (supplemented
with 1 mM isopropyl-ß-D-thiogalactopyranoside
[IPTG] when appropriate) . Finally, after incubation for 12 h at 37°C,
the swarming distances of the different strains were compared (Fig.
4A) . Consistent with the report of Carpenter et al .
(5), B . subtilis flhF mutant strain OI2735 is
nonmotile . Remarkably, however, disruption of the flhF gene in
B . subtilis 168 has no effect on cell motility (Fig.
4A) . Moreover, scanning electron microscopy shows
that B . subtilis 168 flhF::cat produces
apparently intact flagella (data not shown) . These observations imply
that there are substantial differences in the genetic backgrounds of
the two flhF mutant strains . To investigate whether genes
downstream of flhF might be involved in this phenomenon, the
motility of B . subtilis 168 with an integrated copy of the
pMutin2 plasmid in the ylxH gene (strain BFA2616; Fig . 4B)
was tested . As shown in Fig . 4A, the motility of
strain BFA2616 was significantly reduced compared to that of B .
subtilis 168 . However, induction of the transcription of genes
downstream of ylxH by activation of the pMutin2-derived Pspac
promoter with IPTG resulted in less severely impaired motility of the
cells . This shows that the YlxH protein has a role in cell motility .
In addition to YlxH, proteins encoded by genes downstream of
the ylxH gene are required for motility . To further investigate
a possible role of FlhF in this process, the BFA2616 flhF::cat
double mutant was constructed by transformation of strain BFA2616
with chromosomal DNA of strain OI2735 . Irrespective of the presence
of IPTG to induce transcription of genes downstream of ylxH,
the motility of cells of the double-mutant strain was more severely
affected than that of strain BFA2616 cells . It has to be noted,
however, that upon incubation of the swarming plates for 24 h some
motility of the BFA2616 flhF::cat double mutant was observed
when it was grown in the presence of IPTG . In contrast, strain
OI2735 displayed no motility at all . The fact that the flhF
single mutation had no effect on motility while the combined flhF
and ylxH mutations affected motility more severely than the
ylxH single mutation indicates that the functions of FlhF and
YlxH overlap at least partly . In this respect, it is interesting that
YlxH has a putative nucleotide binding site, like FlhF . If FlhF and
YlxH act cooperatively, the function of FlhF can be taken over by
YlxH in flhF mutant cells, but the opposite seems not to
occur . Remarkably, integration of the pMutin plasmid into the ylxH
gene appears to result in a polar effect on the expression of
downstream genes, while there is no evidence for polar effects upon
integration of the cat gene into flhF . Nevertheless,
transcriptome analyses with B . subtilis strain 168, as documented
on the JAFAN website (http://bacillus.genome.jp/),
show that the expression profiles of flhF and ylxH, as
well as the surrounding genes, are highly similar under the 10
different growth conditions tested . This strongly suggests that these
genes are part of one operon or regulon . Moreover, studies by West
and coworkers (30) support the idea that the
expression of flhF and ylxH is controlled by one
promoter . Taken together, our observations demonstrate that FlhF has
a minor role in the motility of B . subtilis 168 cells . It is
not clear why disruption of flhF in strain OI2735 results in a
complete block of motility (5) .
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FIG . 4 . Motility assays . (A) Comparison of the motilities of B .
subtilis strains 168, OI2735, 168 flhF::cat, BFA2616
(grown in the absence or presence of 1 mM IPTG), and BFA2616 flhF::cat
(grown in the absence or presence of 1 mM IPTG) after 12 h of incubation
on 0.27% agar plates at 37°C . (B) Schematic representation of ylxH
gene disruption by pMutin2 via Campbell-type integration . lacI,
E . coli lacI gene; ori pBR322, origin of replication of plasmid
pBR322; ApR, ampicillin resistance marker; EmR,
erythromycin resistance marker; t1t2, transcriptional terminators on
pMutin2; Pspac, IPTG-dependent promoter; Pfla/che,
promoter of the fla/che operon; ylxH', 3'-truncated
ylxH gene; 'ylxH, 5'-truncated ylxH gene; flhF,
flhF gene; cheB, cheB gene.
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In conclusion, our combined proteomic and biochemical analyses of
sequenced B . subtilis strain 168 demonstrate that FlhF, the
third SRP-GTPase of this organism, is dispensable for protein
secretion and has a minor role in cell motility . It remains to be
investigated whether FlhF has a role in the biogenesis of membrane
proteins, as shown for its homologues Ffh and FtsY in E . coli
(24) and proposed for gram-positive bacteria (28) .
We thank George Ordal for providing B . subtilis strain OI2735
and Marc Kolkman from Genencor International for providing anti-Ffh
and anti-FtsY antibodies . Jan Jongbloed, Joen Luirink, Rob Meima, and
Bauke Oudega are thanked for helpful discussions .
G.Z . and W.J.Q . were supported by the Stichting Technische Wetenschappen
(BVI.4837), H.W . and J.M.V.D . were supported by the CEU (BIO4-CT98-0250,
QLK3-CT-1999-00413, and QLK3-CT-1999-00917), and H.A . and M.H .
were supported by the Deutsche Forschungsgemeinschaft, the Bundesministerium
für Bildung, Wissenschaft, Forschung und Technologie, and the
Fonds der Chemischen Industrie .
* Corresponding author . Present address: Department of
Molecular Bacteriology, University of Groningen, P.O . Box 30001, 9700 RB
Groningen, The Netherlands . Phone: 31-50-3633079 . Fax: 31-50-3633528 . E-mail: J.M.van.Dijl@med.rug.nl.
Present address: Department of Genetics, Groningen Biomolecular
Sciences and Biotechnology Institute, University of Groningen, 9751
NN Haren, The Netherlands .
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