Báo cáo khoa học: Identification, subcellular localization and functional interactions of PilMNOWQ and PilA4 involved in transformation competency and pilus biogenesis in the thermophilic bacterium Thermus thermophilus HB27 ppt

Identification, subcellular localization and functional
interactions of PilMNOWQ and PilA4 involved in
transformation competency and pilus biogenesis in the
thermophilic bacterium Thermus thermophilus HB27
Judit Rumszauer, Cornelia Schwarzenlander and Beate Averhoff
Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt ⁄ Main, Germany

Keywords
pilus biogenesis; Thermus thermophilus;
transformation competency
Correspondence
B. Averhoff, Molecular Microbiology &
Bioenergetics, Institute of Molecular
Biosciences, Johann Wolfgang Goethe
University Frankfurt ⁄ Main, Campus
Riedberg, Max-von-Laue-Str. 9,
60438 Frankfurt, Germany
Fax: +49 69 79829306
Tel: +49 69 79829509
E-mail:
(Received 10 April 2006, revised 17 May
2006, accepted 23 May 2006)
doi:10.1111/j.1742-4658.2006.05335.x

The natural transformation system of the thermophilic bacterium Thermus
thermophilus HB27 comprises at least 16 distinct competence proteins encoded by seven distinct loci. In this article, we present for the first time
biochemical analyses of the Thermus thermophilus competence proteins
PilMNOWQ and PilA4, and demonstrate that the pilMNOWQ genes are
each essential for natural transformation. We identified three different
forms of PilA4, one with an apparent molecular mass of 14 kDa, which
correlates with that of the deduced protein, an 18-kDa form and a 23-kDa

form; the last was found to be glycosylated. We demonstrate that PilM,
PilN and PilO are located in the inner membrane, whereas PilW, PilQ and
PilA4 are located in the inner and outer membranes. These data show that
PilMNOWQ and PilA4 are components of a DNA translocator structure
that spans the inner and outer membranes. We further show that PilA4
and PilQ both copurify with pilus structures. Possible functions of PilQ
and PilA4 in DNA translocation and in pilus biogenesis are discussed.
Comparative mutant studies revealed that mutations in either pilW or pilQ
significantly affect the location of the other protein in the outer membrane.
Furthermore, no PilA4 was present in the outer membranes of these
mutants. From these findings, we conclude that the abilities of PilW, PilQ
and PilA4 to stably localize or accumulate in the outer membrane fraction
are strongly dependent on one another, which is in accord with an outer
membrane DNA translocator complex comprising PilW, PilQ, and PilA4.

Members of the extremely thermophilic genus Thermus
belong to one of the oldest branches of bacterial evolution and, together with the genus Deinococcus, form
a distinctive group within the Bacteria deserving the
taxonomic status of a phylum [1,2]. Thermus representatives, such as Thermus thermophilus strain HB27,
Thermus thermophilus HB8, Thermus flavus AT62,
Thermus caldophilus, and Thermus aquaticus YT1, exhibit the extraordinary trait of high transformation competence [3,4]. The high transformation frequencies,
together with the high thermotolerance, suggest a

significant impact of the Thermus transformation system on DNA transfer in extreme environments and
therefore on the evolution of life. This is supported by
recent data from comparative genomics and phylogenetic analyses in the thermophilic bacterium T. thermophilus HB27. This strain seems to have acquired
numerous genes from (hyper)thermophilic bacteria and
archaea, suggesting that horizontal gene transfer was
probably decisive in its thermophilic adaptation [5].
Despite the significance of natural transformation systems of thermophiles, information about transformation

Abbreviations
IPTG, isopropyl thio-b-D-galactoside; TFMS, trifluoromethanesulfonic acid; TM, Thermus medium.

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J. Rumszauer et al.

systems of thermophiles and extreme thermophiles is
very scarce.
To get insights into the transformation systems of
thermophilic bacteria, we chose T. thermophilus HB27,
which exhibits the highest transformation frequencies
among the Thermus strains, as a model strain [4]. On
the basis of the complete genome sequence of T. thermophilus HB27, we have identified by directed gene
disruption seven distinct competence gene loci [6–8].
Sequence analyses revealed that several of the deduced
proteins are similar to proteins of the type IV pili and
type II secretion machineries. PilA1, PilA2, PilA3 and
PilA4 are similar to the precursors of the structural
subunits of type IV pili, the prepilins, PilD exhibits
similarities to the prepilin-processing prepilin peptidases, and PilQ is similar to members of the secretin family, which is a large family whose members form
multimeric pores in the outer membranes of Gramnegative bacteria [9–12]. These similarities, together
with the finding that transformation-defective pilA4,
pilD and pilQ mutants, respectively, are devoid of pilus

structures, suggest a functional link between pili
and natural transformation in T. thermophilus HB27,
although the functions of the type IV pili-related competence proteins in the process of DNA uptake are still
unknown.
The pilMNOWQ competence genes are located in a
competence locus comprising five tandemly arranged
analogously orientated genes, pilM, pilN, pilO, pilW,
and pilQ [7]. Mutant studies with T. thermophilus
HB27 mutants, carrying marker insertions in pilM,
pilN, pilO, pilW, and pilQ, respectively, revealed that
the pilMNOWQ cluster is essential for natural transformation and piliation. Owing to the head-to-tail
organization of the genes, potential polar effects of
marker insertions on downstream-located genes of the
pil cluster could not be excluded, and therefore the
question of whether the products of pilM, pilN, pilO
and pilW each play a role in natural transformation
and piliation is still open.
Here, we present the identification of the competence proteins PilM, PilN, PilO, PilW, PilQ and PilA4
in T. thermophilus HB27; the last of these was found
to undergo glycosylation. We show that the individual
proteins of the pilMNOWQ competence cluster are
each essential for natural transformation of T. thermophilus HB27. Furthermore, we present the first information on the subcellular localization of the
PilMNOWQ and PilA4 competence proteins and on
the effect of mutations in distinct competence proteins
on the subcellular localization of other proteins. Taken
together, the data presented here provide the first
insights into the function of the competence proteins
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PilM, PilN, PilO, PilW, PilQ and PilA4 in the DNA

translocator of T. thermophilus HB27.

Results
Heterologous expression and purification of
PilMNOWQ and PilA4
To perform biochemical analyses with the corresponding proteins, pilM, pilN, pilO, pilW, pilQ or pilA4 gene
fragments were fused to malE and the fusion proteins
were produced in Escherichia coli DH5a (Fig. 1). The
fusion proteins were purified on an amylose matrix.
The apparent molecular masses of the chimeric proteins were 82 kDa (MalE–PilM), 57 kDa (MalE–PilN),
60 kDa (MalE–PilO), 60 kDa (MalE–PilW), 72 kDa
(MalE–PilQ), and 51 kDa (MalE–PilA4). These values
correlate nicely with predicted molecular masses of the
fusion proteins. Antisera against the purified fusion
proteins were generated in rabbits and tested by western blotting with purified fusion proteins.
Identification of PilM, PilN, PilO, PilW and PilQ
in crude extracts
The first goal of this study was to identify the individual proteins encoded by the pilMNOWQ competence
gene cluster and pilA4 in T. thermophilus HB27. Therefore, the polyclonal antisera raised against fragment
fusions of PilM, PilN, PilO, PilW, PilQ, and PilA4,
respectively, were applied to T. thermophilus HB27
crude extracts separated by SDS ⁄ PAGE (Fig. 2A–E).
The antisera against PilM, PilN, PilO, and PilQ,

Fig. 1. Organization of pilMNOWQ and generation of gene fragments fused to the gene for maltose-binding protein (malE). The
arrows indicate the directions of transcription. Numbers indicate
base pairs of the complete genes.

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J. Rumszauer et al.

Analyses of novel Thermus competence proteins

A

C

could be due to the amount of PilA4 being below the
detection limit, because of accumulation of PilA4 in
the external medium as pili or attachment to the cell
debris after cell disruption.

B

D

The competence proteins PilMOW are each
required for natural transformation and piliation

E

Fig. 2. Detection of PilM, PilN, PilO, PilW and PilQ proteins in Thermus thermophilus HB27. Thermus thermophilus HB27 wild-type
strain and mutant strains were grown to the exponential growth
phase and subjected to crude extract preparation. The crude
extracts of wild-type (20 lg of protein) and mutant strains (20 lg of
protein each) were analyzed by SDS ⁄ PAGE and western blotting by
using PilM, PilN, PilO, PilW and PilQ antisera. The results presented are the data from one experiment from a series of five independent experiments that gave identical results.

respectively, detected single protein species correlating
with the predicted masses of 42, 23, 21 and 82 kDa.
PilW has a deduced molecular mass of 29.8 kDa,
which is 10.2 kDa lower than the apparent molecular
mass of 40 kDa (Fig. 2D). Since the PilW antibodies
are specific, and incorrect assignment of the start and
stop sites of pilW can also be excluded, this difference
is probably due to post-translational modifications
resulting in a conformational change; alternatively, the
separation in an SDS gel might be affected by the
N-terminal hydrophobic region in PilW.
In contrast, PilA4 could not be detected in the crude
extracts, although the antiserum was found to react
specifically with the purified fusion proteins. This

A

We previously reported that marker insertions in pilM,
pilN, pilO, pilW, pilQ, and pilA4, respectively, resulted
in a defect in natural transformation and absence of
pilus structures. These findings, together with the
organization of these competence genes, suggested that
PilA4 and PilQ are individually essential for transformation and piliation [7,8]. In contrast to PilQ and
PilA4, an individual role of PilM, PilN, PilO and PilW
in natural transformation and piliation cannot be
deduced from these data with confidence, since polar
effects of marker insertions in pilM, pilN, pilO or pilW
exerted on downstream-located genes could not be
excluded, due to their head-to-tail organization [7,8].
To analyze potential polar effects of marker insertions

in pilM, pilN, pilO, and pilW, respectively, on downstream-located genes, we performed immunostaining
with crude extracts of T. thermophilus mutant strains
Tt4 (pilM::kat), Tt5 (pilN::kat), Tt6 (pilO::kat), and
Tt7 (pilW::kat). In crude extracts of mutants Tt4, Tt6,
and Tt7, the proteins encoded by downstream-located
genes, PilN, PilW, and PilQ, respectively, were detected (Fig. 3A–C). Apparently, insertion of the kanamycin cassette in pilM, pilO or pilW has no polar effect
on the downstream-located pilN, pilW or pilQ genes.
Taken together, these results provide clear evidence
that pilM, pilO and pilW are individually essential for
natural transformation and piliation of T. thermophilus
HB27. PilO was not detected in crude extracts of the
pilN mutant (data not shown), whereas genes located
downstream of pilO, such as pilW, were expressed.
This suggests that either biosynthesis or stability of the
PilO protein is impaired in pilN mutants.

B

C

Fig. 3. PilN, PilW and PilQ production in Thermus thermophilus pilM, pilO or pilW mutant strains. Thermus thermophilus HB27 wild-type and
mutant strains were grown to the exponential growth phase and subjected to crude extract preparation. The crude extracts (20 lg of protein) were analyzed by SDS ⁄ PAGE and western blotting by using PilN, PilW and PilQ antisera. The results presented are the data from one
experiment from a series of four independent experiments that gave identical results.

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Analyses of novel Thermus competence proteins

J. Rumszauer et al.

Subcellular localization of PilMNOWQ and PilA4
To determine the subcellular localization of the competence proteins PilM, PilN, PilO, PilW, PilQ, and
PilA4, cells were lysed by sonification, total membranes were separated from the soluble fraction by
ultracentrifugation, and inner and outer membranes
were further separated by N-lauroylsarcosine extraction and subsequent ultracentrifugation. The competence protein PilM is a rather hydrophilic protein,
except for a short region of limited hydrophobicity
close to the N-terminus. To elucidate the subcellular
localization of PilM, we performed western blot analyses of the cell fractions and found that PilM is exclusively localized in the inner membrane (Fig. 4A). In
addition, PilN and PilO are localized exclusively in
the inner membrane. These results, together with the
rather hydrophilic character of PilN and PilO except
for the N-terminal hydrophobic domain, suggest that
PilN and PilO are inner membrane-anchored proteins,
which may mediate recruitment and assembly of DNA
translocator proteins at the inner membrane.
PilW, a Thermus competence protein with no similarities to known proteins, exhibits a hydrophobic
region at the N-terminus. To answer the question of

whether this region is sufficient to mediate membrane
anchoring, cell fractions were subjected to western blot
analyses with PilW antiserum (Fig. 4D). These studies
revealed that PilW is distributed equally between the
inner and outer membranes.
Major amounts of the secretin-like PilQ were detected in the outer membrane, whereas minor amounts of
PilQ were also detected in the inner membrane
(Fig. 4E). The latter might result from transport of
PilQ through the inner membrane to the outer membrane.

Although we could not detect PilA4 in cell-free
extracts, it is clearly detectable in membrane fractions
and found to be distributed equally between the inner
and outer membranes (Fig. 4F). The detection of
PilA4 in the membranes could be due to an accumulation of high PilA4 levels in the membranes or attachment of the PilA4 to the membranes. Interestingly,
PilA4 had an apparent molecular mass of 23 kDa,
which differs significantly from the deduced molecular
mass of 14 kDa. However, since no reaction of the
antiserum was observed with membrane fractions of
the pilA4 mutant, it is evident that the 23 kDa protein
is PilA4, probably in a post-translationally modified
form.

A

B

C

D

E

F

Fig. 4. Cellular localization of PilM, PilN, PilO, PilW, PilQ, and PilA4. Cells were harvested in the exponential growth phase, resuspended in
lysis buffer and disrupted by sonification. Soluble fractions and membrane fractions were separated by ultracentrifugation prior to separation
of inner and outer membrane fractions by N-laurylsarcosine precipitation. The resulting fractions were analyzed by SDS ⁄ PAGE and western
blotting by using specific antisera against: (A) PilM; (B) PilN; (C) PilO; (D) PilW; (E) PilQ; and (F) PilA4. The data are the data from one experiment that was replicated three times with identical results. S, soluble fraction; IM, inner membrane; OM, outer membrane.

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J. Rumszauer et al.

Fig. 5. Analysis of PilA4 glycosylation. Untreated proteins (– TFMS)
and trifluoromethanesulfonic acid (TFMS)-treated proteins (+ TFMS)
were separated by SDS ⁄ PAGE, transferred onto nitrocellulose
membranes, and probed with MalE–PilA4 antibodies.

PilA4 undergoes glycosylation
Structural subunits of type IV pili of Gram-negative
bacteria are known to undergo different post-translational modifications such as glycosylation, and linkage
to a-glycerophosphate or phosphorylcholine [13–17].
Glycosidic bond cleavage by trifluoromethanesulfonic
acid (TFMS) has been shown to be useful for the identification of polysaccharides linked to proteins, since
the effect of TFMS on a glycoprotein is sufficiently
specific that a change in molecular mass after treatment can be ascribed to removal of oligosaccharides.
Post-translational modifications other than glycosylation, such as by sulfate or phosphate, are stable to
TFMS treatment. To address the potential glycosylation of PilA4, we compared TFMS-treated and
TFMS-untreated protein extracts of HB27 wild-type
cells in western blot analyses. These studies revealed
that deglycosylation via TFMS treatment resulted in a
shift of the apparent molecular mass of PilA4 to 18
and 14 kDa (Fig. 5). This change in molecular mass
after TFMS treatment suggests that PilA4 undergoes
glycosylation. The 14 kDa protein species corresponds
to unmodified PilA4 protein, whereas the 18 kDa

PilA4 might carry a further modification resistant to
TFMS treatment.
PilQ and PilA4 copurify with pilus structures
The similarities of PilM, PilN, PilO, PilQ and PilA4 to
type IV pili proteins led to the question of whether
these competence proteins are structural subunits of
the T. thermophilus pilus structures. To address this
question, we purified the pili structures by separating
shear fractions of T. thermophilus HB27 in a discontinuous sucrose gradient. After centrifugation, the
gradient was fractionated and inspected by electron
microscopy. Two fractions (corresponding to  50%
sucrose) contained exclusively the pilus structures

Analyses of novel Thermus competence proteins

(Fig. 6A). Inspection with respect to the presence of
impurities and homogeneities of the pilus fractions
revealed that small lipid vesicles were occasionally present. Close inspection of representative areas revealed
that 90% of the pilus structures were attached to a
globular structure with a diameter of 20 nm at one
end of the pilus structure (Fig. 6B). To determine whether PilA4 is part of the pilus structures, immunogold
labeling of the purified pili was performed with PilA4
antiserum raised against fragments of the native PilA4
protein. Despite many different attempts, we never
observed binding of gold-labeled antibodies to the
pilus (data not shown). This finding suggests that
either PilA4 is not part of the pilus, PilA4 is inaccessible in the native pilus, or the PilA4 antibody does not
recognize the native protein. To address this question,
we analyzed the purified pilus fraction by SDS ⁄ PAGE
and western blotting with PilA4 antibodies. These

studies revealed the presence of the 23 kDa PilA4 protein in the pilus fraction (Fig. 6C). PilM, PilN and
PilW were not detected in the pilus fraction (data not
shown), indicating that these competence proteins are
not structural subunits of the pili. In contrast, PilQ
was detected in the pilus fraction (Fig. 6D), probably
as a result of being torn out of the membrane together
with the pilus during the shearing step.
Influence of PilM, PilN, PilO, PilW, PilQ and PilA4
on the subcellular localization of competence
proteins
In further studies, we addressed possible interactions
between PilM, PilN, PilO, PilW, PilQ and PilA4. To
do this, we examined the influence of each protein on
the subcellular localization of the other proteins. We
separated the inner and outer membrane fractions
of pilM, pilN, pilO, pilW, pilQ and pilA4 mutants,
respectively, from the soluble fractions (periplasm and
cytoplasm) and performed western blot analyses to
detect the competence proteins in the subcellular fractions. Membrane fractions of the T. thermophilus
HB27 wild type were used as controls. First, we compared the relative levels of PilM, PilN, and PilO in
membrane fractions of mutants carrying insertions in
pilM, pilN, pilO, pilW, or pilQ, but found no significant differences (Table 1). In contrast, mutation in
pilQ led to the absence of PilW and PilA4 in the inner
membrane. In addition, pilW mutation resulted in the
absence of PilQ and PilA4 in the outer membrane. The
abilities of PilW, PilQ and PilA4 to stably localize or
accumulate in the outer membrane are strongly
dependent one another, indicating interactions between
PilW, PilQ and PilA4 in structure and assembly.

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J. Rumszauer et al.

A

B

C

D

Fig. 6. Electron microscopy of Thermus thermophilus HB27 pili separated by sucrose density gradient and western blot analyses of purified
pili fractions. Pili were sheared off and separated as described in Experimental procedures. Each fraction was analyzed by electron microscopy. Major amounts of pili were detected in fractions containing  50% sucrose (A). Close inspection of the pili led to the detection of globular structures (indicated by arrows in A) at the base of the pili (B). SDS ⁄ PAGE was stained with Coomassie. Western blot analyses of the
pilus fraction revealed that pili were copurified with PilA4 (C) and PilQ (D).

Table 1. Subcellular localization of the PilM, PilN, PilO, PilW, PilQ and PilA4 competence factors. OM, outer membrane; IM, inner membrane; ++, major amounts present in one of the membranes; +, present; –, absent.
Subcellular localization
PilM

PilN

PilO

PilW

PilQ

PilA4

Strains

IM

OM

IM

OM

IM

OM

IM

OM

IM

OM

IM

OM

HB27 wild type
Mutants
Tt4 (pilM::kat)
Tt5 (pilN::kat)
Tt6 (pilO::kat)
Tt7 (pilW::kat)
Tt8 (pilQ::kat)
Tt20 (pilA4::kat)

+

–

+

–

+

–

+

+

+

++

+

+

–
+
+
+
+
+

–
–
–
–
–
–

+
–
+
+
+
+

–
–
–
–
–

–

+
–
–
+
+
+

–
–
–
–
–
–

+
+
+
–
+
+

+
+
+
–
–
+

+
+
+
+
–
+

++
++
++
–
–
++

+
+
+
+
+
–

+
+
+
–
–
–

Discussion
We recently reported on the identification and characterization of seven distinct competence gene loci in the

genome of T. thermophilus HB27 comprising a total of
16 potential genes of the DNA translocator [6–8].
However, so far, none of the competence proteins has
been detected or analyzed in T. thermophilus HB27,
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and nothing is known with respect to their function in
DNA translocation.
Therefore, in the first part of this study we produced
fragments of the PilM, PilN, PilO, PilW, PilQ and PilA4
competence proteins and raised antisera against these
proteins to visualize the proteins in the T. thermophilus
wild-type strain. This is the first report on the detection
of competence proteins in T. thermophilus HB27.

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J. Rumszauer et al.

An interesting finding was that PilA4 protein undergoes glycosylation. This is a trait of many pili proteins
and has also been detected in pilin-like proteins of
DNA transformation systems [18–20]. The detection of
an 18 kDa PilA4 after TFMS treatment suggests that
PilA4 may undergo a further modification. This has
been shown for the meningococcal pilin; it contains an
a-glycerophosphate substituent attached to Ser93 by a
phosphodiester linkage [17]. It has been suggested that
glycerol residues might serve as a substrate for fatty
acylation and, thereby, be involved in membrane

anchoring of the pilin. Since PilA4 is similar to the
meningococcal pilin and contains several central serine
residues, it is tempting to speculate that PilA4 might
also contain an a-glycerophosphate substituent. Taken
together, the studies clearly show that the 23 kDa
PilA4 protein undergoes glycosylation and that the glycosylated PilA4 protein is active in the DNA translocator.
Where are the PilM, PilN, PilO, PilW, PilQ and
PilA4 competence proteins located in the cell and what
could be their function? Several of the selected proteins
contain only a few or no hydrophobic segments, and
therefore their subcellular localization was not obvious. Here, we show that PilM is exclusively located in
the inner membrane. PilM contains a conserved C-terminal ATPase domain of actin-like ATPases, such as
FtsA and MreB, which are involved in cell division
and cell morphogenesis (for reviews, see [21] and [22]).
FtsA, the only septum protein without a membrane
anchor, is required in bacteria for the assembly and
stabilization of Z-rings comprising tubulin-like FtsZ
filaments [23], whereas MreB has been shown to
perform dynamic motor-like movements extending
along helical tracks [24]. Owing to the similarities of
PilM with members of the actin family, together with
the inner membrane localization of PilM, it is tempting
to speculate that PilM might represent a dynamic
motor protein involved in the assembly of the DNA
translocator complex in the inner membrane. The
Thermus competence proteins PilN and PilO show very
weak similarities to PilN and PilO proteins of
unknown function in type IV pili of Gram-negative
bacteria. Like PilO and PilN of Pseudomonas aeruginosa and Neisseria gonorrhoeae [25–27], the T. thermophilus PilO and PilN proteins each have a hydrophobic
N-terminal domain which may act as an inner or outer

membrane anchor. This is in accordance with their
localization in the inner membrane. PilN and PilO
may mediate recruitment and assembly of DNA translocator proteins at the inner membrane.
We found that the nonconserved PilW is distributed
equally between the inner and outer membranes. PilW

Analyses of novel Thermus competence proteins

is likely to form integral parts of a transmembrane
DNA translocator structure and it may interact via its
hydrophobic N-terminus with other proteins in the
membranes such as PilQ and PilA4. In addition, its
extended hydrophilic C-terminus may interact with
other DNA translocator proteins in the periplasm.
Consistent with this suggestion is our finding that a
pilW mutation results in the absence of PilA4 and PilQ
from the outer membrane. Taken together, our results
indicate that PilW may interact with PilQ and PilA4 in
the outer membrane and that this interaction is
required for biogenesis of the DNA translocator
and ⁄ or is involved in the stabilization of PilQ and
PilA4 proteins in the outer membrane. Moreover, the
absence of any PilW-like proteins in the transformation machineries of mesophilic bacteria, together with
the effect of a pilW mutation on the biogenesis and ⁄ or
stability of PilA4 and PilQ in the outer membrane,
indicate that PilW is a special feature of the transformation machinery in T. thermophilus that is probably
essential for the adaptation of the DNA translocator
to high temperature.
The secretin-like PilQ was detected in sufficient
amounts in the inner and outer membranes. The presence of PilQ in inner membranes is interesting, because

secretin-like proteins of type IV pili and type II protein
translocation machineries are known to form ring-like
structures in outer membranes. The presence of PilQ in
T. thermophilus inner and outer membranes suggests
that the secretin-like PilQ protein is accumulated and
may be assembled into ring-like structures at the inner
membrane prior to transport through the periplasm to
the outer membrane. The secretin-like PilQ protein of
T. thermophilus has a conserved C-terminal part, very
similar to the C-termini of other members of the secretion family, such as PilQ of Myxococcus xanthus [28],
ExeD of Aeromonas salmonicida [29], PilQ of P. aeruginosa [25,30], and PilQ of N. gonorrhoeae [31]. This
C-terminal stretch has been shown to be required for
multimer formation of the corresponding PulD of Klebsiella and PilQ of N. gonorrhoeae [31,32]. Taken
together, the conserved C-terminus of PilQ and its outer
membrane localization are in agreement with our suggestion that Thermus secretin-like PilQ monomers may
form a multimeric ring-like structure acting in the translocation of DNA through the outer membrane or functioning as a scaffold for the DNA translocator spanning
the outer membrane. However, it has to be noted that
the N-terminus of T. thermophilus PilQ does not exhibit
any similarities to conserved N-terminal domains of
secretins that are proposed to mediate interaction with
other proteins not related to type II secretion or type IV
pili biogenesis pathways. Owing to the nonconserved

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J. Rumszauer et al.

N-terminal domain of PilQ, and the colocalization of
PilQ with PilW in inner and outer membranes and the
results from the pilW and pilQ mutant studies, it is
tempting to speculate that the nonconserved PilW protein is implicated in the assembly and stability of PilQ
multimers at the inner membrane and transport of these
subassemblies to the outer membrane.
The presence of the pilin-like PilA4 protein in the
inner and outer membranes suggests that PilA4 may
represent a structural subunit of a DNA translocator
anchored in the inner membrane and extending
through the periplasm and the outer membrane. The
finding that a PilQ mutant no longer has PilA4 in the
outer membrane is in support of a PilQ-comprising
scaffold in the outer membrane guiding the PilA4-consisting translocator through the outer membrane.
The copurification of PilA4 and PilQ with the pilus
structures indicates that both are structural compo-

nents of the pilus. Moreover, it is tempting to speculate that PilQ might form the globular structure at the
pilus base, since it corresponds in diameter with the
PilQ complex of N. gonorrhoeae (15.5–16.5 nm) [33],
P. aeruginosa (18.3 nm ± 1.2 nm) [34] or N. meningitidis (15.5 nm) [33]. In contrast, PilM, PilN and PilO
are essential for transformation and piliation but do
not copurify with the pili, indicating that they may
contribute to the biogenesis of the pilus, the stability
of pilus structures, and ⁄ or inner membrane association
of the pilus. PilW, which we found to be nearly equally
distributed between inner and outer membranes but
not in the purified pilus fraction, may be involved in

inner and outer membrane associations of pilus proteins and ⁄ or stability of the pilus structure.
On the basis of our current knowledge, we propose
a model for the DNA translocation process in T. thermophilus HB27 (Fig. 7).

Fig. 7. Model for DNA uptake in Thermus thermophilus HB27. DNA is bound to a so far unknown DNA-binding protein close to the potential
ring-like structure of secretin-like PilQ proteins in the outermost layer, which comprises S-layer and lipids and does not represent a classic
outer membrane. The DNA is transported through the ring-like structure, the periplasmic space and peptidoglycan by a DNA translocator
comprising pilin-like (PilA4) proteins. PilW is an inner and outer membrane protein that may be essential for assembly, stabilization and piloting of the PilQ ⁄ PilA4-comprising DNA translocator complex, spanning the outer membrane and periplasmic space, whereas PilM, PilN and
PilO are inner membrane proteins that probably form part of the assembly platform and are involved in the assembly of the DNA translocator
complex in the inner membrane. The potential traffic NTPase PilF is essential for transformation and may be implicated in retraction of the
PilA4-comprising DNA translocator transporting the DNA through the periplasmic space. Binding of the DNA to the DNA-binding protein
ComEA on the surface of the inner membrane may be a prerequisite for DNA translocation across the inner membrane, which could be performed through a ComEC-comprising channel. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.

3268

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J. Rumszauer et al.

Analyses of novel Thermus competence proteins

Current studies are underway to answer the question
of whether the pilus structures themselves are implicated in DNA translocation. Future work will purify
different subassemblies of the DNA transporter in
T. thermophilus, and develop assays for its functional
units.

Experimental procedures
Bacterial strains, growth conditions, and DNA

manipulations
Thermus thermophilus HB27 wild-type and mutant strains
were grown at 68 °C under strong aeration in Thermus
medium (TM) containing 4 g of yeast extract, 8 g of
tryptone peptone and 3 g of NaCl per liter, pH 7.5 [4].
Escherichia coli strains were grown in LB medium (0.5%
yeast extract, 1% tryptone peptone, 1% NaCl) at 37 °C.
Recombinant E. coli strains were grown in the presence
of ampicillin (100 lgỈmL)1). Thermus thermophilus HB27
mutants were grown in liquid media with 20 lgỈmL)1 kanamycin or on solid media with 40 lgỈmL)1 kanamycin. DNA
manipulations were perfomed with standard procedures [35].

Generation of antibodies
To avoid toxic effects of overproduced proteins on the
E. coli host cells, PilM, PilN, PilO, PilW, PilQ and PilA4
fragment fusions (Fig. 1) were overproduced. Therefore,
T. thermophilus HB27 pilA4, pilM, pilN, pilO, pilW and
pilQ fragments were amplified from chromosomal DNA of
T. thermophilus HB27 (for primers see Table 2), cleaved

with appropriate restriction enzymes, and cloned into the
overexpression vector pMalc-2X (New England Biolabs
GmbH, Frankfurt a. M., Germany). The plasmid constructs were sequenced with custom-made primers. Maltosebinding protein (MalE) fusion proteins were overproduced
in E. coli DH5a, by isopropyl thio-b-d-galactoside (IPTG)
induction of the tac promoter and purified by immobilized
amylose affinity chromatography performed as recommended by the manufacturer (New England Biolabs GmbH).
Purified fusion proteins were used for immunization of
rabbits.

Western blot analyses

Thermus thermophilus HB27 cells were harvested in the
exponential growth phase, resuspended in Laemmli sample
buffer [36], and boiled for 10 min to lyse the cells.
SDS ⁄ PAGE was performed in 15% (w ⁄ v) acrylamide separating gels [36]. The proteins were electrotransferred onto
nitrocellulose membranes [37] and stained with 0.2%
PonceauS Red for detection of reference proteins, and
membranes were blocked by incubation for 1 h at room
temperature in NaCl ⁄ Pi Tween-20 (140 mm NaCl, 10 mm
KCl, 16 mm Na2HPO4, 2 mm KH2PO4, 0.05% Tween-20)
containing 0.5% skimmed milk powder. Immunodetection
of proteins in total cell lysates or in membrane fractions
was performed with polyclonal PilA4 antiserum (dilution
1 : 5000), PilM antiserum (dilution 1 : 5000), PilN antiserum (dilution 1 : 2500), PilO antiserum (dilution
1 : 2000), PilW antiserum (dilution 1 :10 000) or PilQ antiserum (dilution 1 : 10 000) obtained from Davids Biotechnologie (Regensburg, Germany). ProteinA–horse radish

Table 2. PCR primer sequences. Restriction sites for cloning are underlined. Mismatches are indicated by bold type.

Gene designation
pilM
5¢ ⁄ EcoRI
3¢ ⁄ BamHI
pilN
5¢ ⁄ EcoRI
3¢ ⁄ XbaI
pilO
5¢ ⁄ EcoRI
3¢ ⁄ SalI
pilW
5¢ ⁄ EcoRI
3¢ ⁄ BamHI

pilQ
5¢ ⁄ EcoRI
3¢ ⁄ HindIII
pilA4
5¢ ⁄ EcoRI
3¢ ⁄ SalI

Primer sequence
(5¢- to 3¢)

Size of fragments (bp)

GGG CTT GGA ATT CGG GGC CTC
GAG CTG GTC CGG ATC CGC CTT G

1246

TCG CCG AAT TCG GCC GCC
GCA GGT AGT TCT AGA GGA GCC

426

AGG CAG GAA TTC GCC ACC GTC
TCA GGC GTC GAC AGG CGT TCT

504

GAC CTG GGA TCC CGA GAC CTT G
GTG GTG GAA TTC CCA CCC CCA

658

CAC CTT CCC CGA ATT CCT CGC C
CAC CCT AAG CTT CAC CTA GGG CAC

832

GAC GCG GTG GAA TTC CAG GAG
CAA CGC TAG CGT CGA CGC CAT AC

291

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J. Rumszauer et al.

peroxidase (HRP) conjugate (Bio-Rad, Munchen, Geră
many) as secondary antibody was used in combination with
the BM Chemiluminescence Blotting Substrate Kit (Roche
Diagnostics GmbH, Mannheim, Germany) to develop the
chemiluminescence for visualization on Kodak X-AR film
(Sigma-Aldrich, Saint-Quentin Falavier, France). Molecular
weight markers, peqGOLD Protein-Marker II (10–
200 kDa), were obtained from Peqlab Biotechnologie
GmbH, Erlangen, Germany.

Membrane isolation and subcellular fractionation
Four hundred milliliter cultures were grown at 68 °C in
TM, harvested in the mid-log-phase, washed with 20 mL of
10 mm Tris ⁄ HCl buffer (pH 8.0), and resuspended in 4 mL
of lysis buffer (10 mm Tris ⁄ HCl, 1 mm EDTA, pH 7.8),
containing 40 lgỈlL)1 DNase I and 40 lgỈlL)1 RNase A.
Cells were disrupted by sonification (3 · 5 min pulse), and
5 mm MgCl2 was added immediately afterwards. Intact
cells and cell debris were removed by low-speed centrifugation (13 000 g, 15 min, 4 °C; rotor type JA25.5, Beckman
Coulter, Krefeld, Germany). The resulting crude cell
extracts (supernatants) were subjected to western blot analyses. Soluble and membrane proteins were separated by
ultracentrifugation for 1 h at 120 000 g at 4 °C (rotor type
Ti70, Beckman Coulter). Membrane pellets were washed
and resuspended in 1 mL of 10 mm Tris ⁄ HCl (pH 8.0). To
separate inner and outer membrane fractions, the membranes were repeatedly pushed through a needle
(0.45 · 25 mm) and subsequently incubated for 10 min on
ice in the presence of 2% N-lauroylsarcosine and 10 mm
EDTA (pH 8.0) [38]. After ultracentrifugation of the membranes for 2 h at 120 000 g (4 °C; rotor type Ti70, Beckman Coulter), the outer membrane pellet was washed once
with 10 mL of 10 mm Tris ⁄ HCl (pH 8.0) (120 000 g, 1 h,
4 °C; rotor type Ti70, Beckman Coulter), resuspended in
H2O and stored at ) 20 °C. To precipitate the inner membrane proteins, the supernatant was incubated for 1 h at
) 20 °C with four volumes of cold acetone. The inner membranes were precipitated by centrifugation for 30 min at
16 000 g (0 °C; rotor type JA25.5, Beckman Coulter), resuspended in H2O, and stored at ) 20 °C. Purity of the
membrane fractions and use of the N-laurylsarcosine solubilization method in T. thermophilus HB27 was verified by
western blot analyses of membrane fractions with antibodies directed against the S-layer protein (outer membrane)
(1AE1 antibodies) and with antibodies directed against the
inner membrane-embedded cytochrome c1 (cytochrome bc1
complex) of the T. thermophilus HB27 respiratory chain
(inner membrane).

Pili purification
An 8 L culture of T. thermophilus HB27 was grown in TM
medium without stirring. The culture was harvested in the

3270

exponential growth phase (after 6 h of incubation) and
washed three times with 50 mm Tris ⁄ HCl (pH 7.5) (8000 g,
5 min; rotor type JA10, Beckman Coulter). The cell suspension was pushed twice through a needle (0.45 · 25 mm) to
shear off the pili. After removal of the cells (20 000 g,
3 · 10 min; rotor type JA25.5, Beckman Coulter) the pili
fraction was pelleted via high-speed centrifugation
(120 000 g, 1 h; rotor type Ti70, Beckman Coulter). The
pellet was resuspended in 1 mL of H2O and subjected to
sucrose density gradient centrifugation (30–70% sucrose
gradient) for 24 h at 160 000 g (rotor type Ti70, Beckman
Coulter). The gradient was fractionated into 1.2-mL samples, which were diluted with five volumes of 30 mm
Tris ⁄ HCl, 0.9% (w ⁄ v) NaCl, pH 7.5, centrifuged
(120 000 g, 1 h; rotor type Ti70, Beckman Coulter) and dissolved in H2O, containing phenylmethanesulfonyl fluoride
to inhibit proteinases. Thermus pili were visualized by electron microscopy in samples containing 50% of sucrose.

Deglycosylation assay
Proteins were deglycosylated by treatment with TFMS [39].
Frozen cells (200 mg) of T. thermophilus HB27 were freezedried overnight and resuspended in 2 mL of 5% SDS. The
solution was refrozen before freeze-drying again for 3 h.
The sample was slightly shaken in 2 mL of anisole ⁄ TFMS
(1 : 2) for 3 h at 4 °C. The proteins were incubated
(15 min, on ice) with 5 mL of 1 m sodium carbonate buffer
(pH 9.2) and 22 mL of ethanol (96%) and precipitated by

centrifugation (17 000 g, 5 min; rotor type JA25.5, Beckman Coulter). The pellets were washed in H2O (47 000 g,
20 min; rotor type JA25.5, Beckman Coulter), resuspended
in 200 lL of H2O and stored at )20 °C until the
SDS ⁄ PAGE was performed.

Electron microscopy and immunogold labeling
Negative staining and electron microscopy were performed
as described [40]. For immunogold labeling, the sheared pili
were attached to Formvar-coated, glow-discharged, 0.01%
poly-l-lysine-treated nickel grids. After washing with
NaCl ⁄ Pi buffer (2 mm KH2PO4, 16 mm Na2HPO4, 140 mm
NaCl, 10 mm KCl, pH 7.2) and blocking with NaCl ⁄ Pi buffer containing 0.1% BSA, the grids were incubated for 1 h
with MalE–PilA4 antibodies. The primary antibody was
detected with the secondary antibody goat anti-rabbit and
conjugated with gold (10 nm, Amersham Biosciences, Freiburg, Germany).

Acknowledgements
This work was supported by grants Av9 ⁄ 4-5 and
Av9 ⁄ 5-1 from the Deutsche Forschungsgemeinschaft.
We are grateful to Gerhard Wanner (Ludwig-Maximi-

FEBS Journal 273 (2006) 3261–3272 ª 2006 The Authors Journal compilation ª 2006 FEBS


J. Rumszauer et al.

lians-Universitat, Munchen), Winfried Haase and
ă
ă
Werner Kuhlbrandt (Max-Planck-Institut fur Biophysă

ă
ik, Frankfurt) for the electron microscopy studies. We
also thank Bernd Ludwig (Johann Wolfgang GoetheUniversitat, Frankfurt) for providing antibodies
ă

against cytochrome c, and Jose Berenguer (Universidad Autonomes de Madrid, Spain) for providing antibodies against T. thermophilus HB27 S-layer protein,
which were used to analyze the purity of membrane
fractions.

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