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
Members of the extremely thermophilic genus Thermus
belong to one of the oldest branches of bacterial evolu-
tion and, together with the genus Deinococcus, form
a distinctive group within the Bacteria deserving the
taxonomic status of a phylum [1,2]. Thermus represent-
atives, such as Thermus thermophilus strain HB27,
Thermus thermophilus HB8, Thermus flavus AT62,
Thermus caldophilus, and Thermus aquaticus YT1, exhi-
bit the extraordinary trait of high transformation com-
petence [3,4]. The high transformation frequencies,
together with the high thermotolerance, suggest a
significant impact of the Thermus transformation sys-
tem on DNA transfer in extreme environments and
therefore on the evolution of life. This is supported by
recent data from comparative genomics and phylo-
genetic analyses in the thermophilic bacterium T. ther-
mophilus 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 sys-
tems of thermophiles, information about transformation
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: Averhoff@em.uni-frankfurt.de
(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 enco-
ded 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.
Abbreviations
IPTG, isopropyl thio-b-D-galactoside; TFMS, trifluoromethanesulfonic acid; TM, Thermus medium.
FEBS Journal 273 (2006) 3261–3272 ª2006 The Authors Journal compilation ª2006 FEBS 3261
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. ther-
mophilus 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 peptidas-
es, and PilQ is similar to members of the secretin fam-
ily, which is a large family whose members form
multimeric pores in the outer membranes of Gram-
negative 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 com-
petence 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 trans-
formation 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 compet-
ence 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. thermo-
philus HB27. Furthermore, we present the first infor-
mation 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
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 correspond-
ing 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 pro-
teins 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 west-
ern 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 individ-
ual proteins encoded by the pilMNOWQ competence
gene cluster and pilA4 in T. thermophilus HB27. There-
fore, 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 frag-
ments fused to the gene for maltose-binding protein (malE).The
arrows indicate the directions of transcription. Numbers indicate
base pairs of the complete genes.
Analyses of novel Thermus competence proteins J. Rumszauer et al.
3262 FEBS Journal 273 (2006) 3261–3272 ª2006 The Authors Journal compilation ª2006 FEBS
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
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.
The competence proteins PilMOW are each
required for natural transformation and piliation
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 transfor-
mation 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 down-
stream-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 detec-
ted (Fig. 3A–C). Apparently, insertion of the kanamy-
cin 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.
AB
CD
E
Fig. 2. Detection of PilM, PilN, PilO, PilW and PilQ proteins in Ther-
mus 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 lgof
protein each) were analyzed by SDS ⁄PAGE and western blotting by
using PilM, PilN, PilO, PilW and PilQ antisera. The results presen-
ted are the data from one experiment from a series of five inde-
pendent experiments that gave identical results.
ABC
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 pro-
tein) 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.
J. Rumszauer et al. Analyses of novel Thermus competence proteins
FEBS Journal 273 (2006) 3261–3272 ª2006 The Authors Journal compilation ª2006 FEBS 3263
Subcellular localization of PilMNOWQ and PilA4
To determine the subcellular localization of the com-
petence proteins PilM, PilN, PilO, PilW, PilQ, and
PilA4, cells were lysed by sonification, total mem-
branes were separated from the soluble fraction by
ultracentrifugation, and inner and outer membranes
were further separated by N-lauroylsarcosine extrac-
tion and subsequent ultracentrifugation. The compet-
ence 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 analy-
ses of the cell fractions and found that PilM is exclu-
sively 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 simi-
larities 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 detec-
ted 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 mem-
brane.
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 accumula-
tion of high PilA4 levels in the membranes or attach-
ment 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.
ACB
DFE
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 experi-
ment that was replicated three times with identical results. S, soluble fraction; IM, inner membrane; OM, outer membrane.
Analyses of novel Thermus competence proteins J. Rumszauer et al.
3264 FEBS Journal 273 (2006) 3261–3272 ª2006 The Authors Journal compilation ª2006 FEBS
PilA4 undergoes glycosylation
Structural subunits of type IV pili of Gram-negative
bacteria are known to undergo different post-transla-
tional 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 iden-
tification of polysaccharides linked to proteins, since
the effect of TFMS on a glycoprotein is sufficiently
specific that a change in molecular mass after treat-
ment can be ascribed to removal of oligosaccharides.
Post-translational modifications other than glycosyla-
tion, such as by sulfate or phosphate, are stable to
TFMS treatment. To address the potential glycosyla-
tion 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 discon-
tinuous sucrose gradient. After centrifugation, the
gradient was fractionated and inspected by electron
microscopy. Two fractions (corresponding to 50%
sucrose) contained exclusively the pilus structures
(Fig. 6A). Inspection with respect to the presence of
impurities and homogeneities of the pilus fractions
revealed that small lipid vesicles were occasionally pre-
sent. 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 whe-
ther 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 inaccess-
ible 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 pro-
tein 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 frac-
tions. Membrane fractions of the T. thermophilus
HB27 wild type were used as controls. First, we com-
pared the relative levels of PilM, PilN, and PilO in
membrane fractions of mutants carrying insertions in
pilM,pilN,pilO,pilW,orpilQ, but found no signifi-
cant 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.
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.
J. Rumszauer et al. Analyses of novel Thermus competence proteins
FEBS Journal 273 (2006) 3261–3272 ª2006 The Authors Journal compilation ª2006 FEBS 3265