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

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.. thermophilus HB27 mutants, carrying

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 thermophilusHB27 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.

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,

pilDand 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.

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

E

Fig 2 Detection of PilM, PilN, PilO, PilW and PilQ proteins in

Ther-mus thermophilus HB27 TherTher-mus 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

presen-ted are the data from one experiment from a series of five

inde-pendent experiments that gave identical results.

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.

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 ultracentrifugaextrac-tion 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

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.

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, or pilQ, but found no signifi-cant differences (Table 1) In contrast, mutation in pilQled 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.

We recently reported on the identification and

charac-terization 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,

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

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

Strains

Subcellular localization

Mutants

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

microsco-py Major amounts of pili were detected in fractions containing  50% sucrose (A) Close inspection of the pili led to the detection of glob-ular 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).

An interesting finding was that PilA4 protein

under-goes 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

gly-cosylated PilA4 protein is active in the DNA

translo-cator

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

obvi-ous Here, we show that PilM is exclusively located in

the inner membrane PilM contains a conserved

C-ter-minal 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

Thermuscompetence 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

aerugi-nosaand Neisseria gonorrhoeae [25–27], the T

thermo-philusPilO 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

trans-locator proteins at the inner membrane

We found that the nonconserved PilW is distributed

equally between the inner and outer membranes PilW

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 pilWmutation 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 transforma-tion 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 transfor-mation 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 pres-ence 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 secre-tion family, such as PilQ of Myxococcus xanthus [28], ExeD of Aeromonas salmonicida [29], PilQ of P aerugi-nosa [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 Kle-bsiella 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 sug-gestion that Thermus secretin-like PilQ monomers may form a multimeric ring-like structure acting in the trans-location of DNA through the outer membrane or func-tioning 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

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

pro-tein 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-con-sisting 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 specu-late 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 meningit-idis (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 pro-teins 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 ther-mophilus 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 pilot-ing 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 per-formed through a ComEC-comprising channel dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.

Current studies are underway to answer the question

of whether the pilus structures themselves are

implica-ted 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 lgÆmL)1) Thermus thermophilus HB27

mutants were grown in liquid media with 20 lgÆmL)1

kana-mycin or on solid media with 40 lgÆmL)1kanamycin 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

pilQfragments 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 con-structs were sequenced with custom-made primers Maltose-binding 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 recommen-ded 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 sep-arating 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 anti-serum (dilution 1 : 2500), PilO antiserum (dilution

1 : 2000), PilW antiserum (dilution 1 :10 000) or PilQ anti-serum (dilution 1 : 10 000) obtained from Davids Biotech-nologie (Regensburg, Germany) ProteinA–horse radish

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

Gene designation

Primer sequence

pilM

pilN

pilO

pilW

pilQ

pilA4

peroxidase (HRP) conjugate (Bio-Rad, Mu¨nchen,

Ger-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 lgÆlL)1 DNase I and 40 lgÆlL)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

centrifuga-tion (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

ana-lyses 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

mem-branes 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

mem-branes for 2 h at 120 000 g (4
C; rotor type Ti70,

Beck-man 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

mem-brane proteins, the supernatant was incubated for 1 h at

) 20
C with four volumes of cold acetone The inner

mem-branes were precipitated by centrifugation for 30 min at

16 000 g (0
C; rotor type JA25.5, Beckman Coulter),

re-suspended in H2O, and stored at ) 20
C Purity of the

membrane fractions and use of the N-laurylsarcosine

solu-bilization method in T thermophilus HB27 was verified by

western blot analyses of membrane fractions with

antibod-ies 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

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 suspen-sion 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 sam-ples, 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 dis-solved in H2O, containing phenylmethanesulfonyl fluoride

to inhibit proteinases Thermus pili were visualized by elec-tron 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 freeze-dried 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, Beck-man 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⁄ Pibuffer (2 mm KH2PO4, 16 mm Na2HPO4, 140 mm NaCl, 10 mm KCl, pH 7.2) and blocking with NaCl⁄ Pi buf-fer 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, Frei-burg, 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