Molecular Microbiology (2005)

56

(2) 361ndash370 doi101111j1365-2958200504294x

copy 2005 Blackwell Publishing Ltd

Blackwell Science LtdOxford UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd 2004

2004

56

2361370

Original Article

Unique structure of archaeal lsquohamirsquoC Moissl

et al

Accepted 28 June 2004 For Correspondence E-mailroberthuberbiologieuni-regensburgde Tel (

+

49) 941 943 3182Fax (

+

49) 941 943 2421

The unique structure of archaeal lsquohamirsquo highly complex cell appendages with nano-grappling hooks

Christine Moissl

1

Reinhard Rachel

1

Ariane Briegel

2

Harald Engelhardt

2

and Robert Huber

1

1

Lehrstuhl fuumlr Mikrobiologie und Archaeenzentrum Universitaumlt Regensburg Universitaumltsstrasse 31 D-93053 Regensburg Germany

2

Max- Planck- Institut fuumlr Biochemie Abteilung Molekulare Strukturbio-logie Am Klopferspitz 18 D-82152 Martinsried Germany

Summary

Proteinaceous hair-like appendages known as fim-briae or pili commonly extend from the surface ofprokaryotic cells and serve important functions suchas cell adhesion biofilm formation motility and DNAtransfer Here we show that a novel group of archaeafrom cold sulphidic springs has developed cell sur-face appendages of an unexpectedly high complexitywith a well-defined base-to-top organization It repre-sents a new class of filamentous cell appendages forwhich the term lsquohamusrsquo is proposed Each archaealcell is surrounded by a halo of about 100 hami whichmediate strong adhesion of the cells to surfaces ofdifferent chemical composition The hami are mainlycomposed of 120 kDa subunits and remained stablein a broad temperature and pH range (0ndash70

infininfininfininfin

C 05ndash115) Electron microscopy and cryo-electron tomog-raphy revealed that the hamus filament possessesa helical basic structure At periodic distances threeprickles emanate from the filament giving it the char-acter of industrially produced barbwire At its distalend the hami carry a tripartite barbed grappling hook(60 nm in diameter) The architecture of this molecularhook is reminiscent of man-made fishhooks grapplesand anchors It appears that nature has developed aperfect mechanical nano-tool in the course of biolog-ical evolution which also might prove useful in thefield of nanobiotechnology

Introduction

On their surface prokaryotes synthesize large-sized fila-

mentous structures that have evolved to accomplish spe-cific functions related to their particular environmentWithin the bacterial domain flagella are the most complexstructures Their structure and function for cell movementhave been intensively studied (Leifson 1960 Adler 1966Berry 2001 Morgan and Khan 2001 Bardy

et al

2003)Pili and fimbriae are proteinaceous appendages thatare classified as type I pili type IV pili and as sex pili(Fernaacutendez and Berenguer 2000 Li and Mobley 2001)They play an important role in adhesion biofilm formationconjugative DNA transfer non-flagellar motility and bacte-riophage infection (Soto and Hultgren 1999 Skerker andShapiro 2000)

In contrast to the bacterial domain knowledge aboutdifferent cell surface appendages within the archaea israre The only well-studied organelle is the archaeal fla-gellum which is a unique motility structure and distinctfrom the bacterial equivalent in architecture compositionand mechanism of assembly (Faguy

et al

1996 Jarrell

et al

1996 2001 Cohen-Krausz and Trachtenberg2002) The archaeal flagellar filament shares some fun-damental properties with bacterial type IV pili and showshomologies in protein sequence synthesis glycosylationand structure (Bayley and Jarrell 1998 Cohen-Krauszand Trachtenberg 2002) For a few members of thearchaeal domain the existence of pili-like fibres was men-tioned (Zillig

et al

1983 Leadbetter and Breznak 1996Miroshnichenko

et al

1998) These surface appendageswere described to be simple in architecture their bio-chemistry ultrastructure and function have not beenstudied so far A possible role proposed for the fibres of

Methanobrevibacter

is the association of this archaeonwith the surface of the hindgut epithelium of its host thetermite

Reticulitermis flavipes

(Leadbetter and Breznak1996)

A decade ago the domain archaea was considered tobe confined to specialized environments (Woese

et al

1990 Stetter 1999) However through the use of the 16SrRNA gene as a molecular marker in environmentalmicrobial surveys (Olsen

et al

1986 Pace

et al

1986)archaea have been detected as major constituents ofmore common and less extreme biotopes (DeLong 1992Fuhrman

et al

1992 Rudolph

et al

2001) Despite theirseeming omnipresence their biological properties andtheir obvious ecological significance in the biosphereremain largely obscure

362

C Moissl

et al

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Recently we reported on the discovery of novelarchaea living in close association with bacteria in cold(

ordf

10

infin

C) sulphurous marsh water of the Sippenauer Moornear Regensburg Bavaria Germany (Rudolph

et al

2001) These prokaryotes form a unique string-of-pearls-like macroscopically visible structure tiny whitish pearls(diameter up to 3 mm) are connected to each other bythin white-coloured threads The inner part of each pearlis predominated by archaeal cocci belonging to a singleeuryarchaeal phylotype tentatively designated SM1euryarchaeon (Rudolph

et al

2001) This novel non-methanogenic archaeon represents a deep phylogeneticbranch within the 16S rRNA tree with no close cultivatedor uncultivated relatives (Rudolph

et al

2001) The outerpart of the pearls and the threads are mainly composedof a single phylotype belonging to the genus

Thiothrix

(Moissl

et al

2002) The SM1 euryarchaeon could not begrown in the laboratory so far

We have therefore designed a novel approach to growmicrobial string-of-pearls-like communities fast and reli-ably on large polyethylene nets in nature (Moissl

et al

2003) About once a week the microbial net populationcan be harvested and the SM1 euryarchaeon specificallyseparated by gentle physical methods (Moissl

et al

2003) This technique allowed us to gain biologicalinsights into the lifestyle of low-temperature archaea(Moissl

et al

2003) First ultrastructural and immunolog-ical studies of single SM1 euryarchaeal cocci lead to anunexpected observation about 100 filamentous cellappendages of 2ndash3

m

m in length emanate radially fromthe surface of each cell (Moissl

et al

2003) The numberof fibres was larger than that of any other cell appendagesfrom archaea known and indicated a particular and signif-icant function

Here we report on the detailed analysis of the SM1euryarchaeon surface appendages which turned out to beunique among prokaryotes so far It represents a newclass of cell appendages of high complexity with a well-defined base-to-top organization To emphasize the nov-elty of this structure and its characteristic morphology theterm lsquohamusrsquo (Latin meaning prickle claw hook barb orfishing rod plural lsquohamirsquo) is proposed

Results

Electron microscopy and tomographical reconstruction

Electron microscopy of platinum-shadowed SM1 eur-yarchaeal cells and immuno-FISH showed the existenceof up to 100 pili-like fibres peritrichously distributed on thesurface of each coccus (Fig 1) The fibres were

ordf

1ndash3

m

min length with an average of about 2

m

m and were foundon cells in different stages of growth (Moissl

et al

2003)Negatively stained preparations revealed an extraordi-

nary and novel type of architecture The fibres exhibiteda very complex structure with a well-defined base-to-toporganization (Figs 2 and 3) Basically each fibre (

ordf

7ndash8 nmin diameter) can be divided into two regions (i) The centralpart the

prickle region

is a barbwire-like structured fila-ment with little prickles sticking out in regular intervals of46

plusmn

14 nm (

n

=

53) They are smaller in diameter thanthe filament (

ordf

4 nm) and 30

plusmn

3 nm (

n

=

48) in length onaverage From the cell surface up to 60 of these recurringunits make up one filament (Fig 2) (ii) The distal part iethe 152

plusmn

3 nm (

n

=

55) long

hook region

is composed ofa plain filament with a tripartite end forming a uniquestructure (Figs 2ndash4) The filament is frayed out into threedistinct arms each of which is about 4 nm in diameter and50 nm in length and exhibits a characteristic curvature ofabout 180 degrees such that the end of each arm alwaysfaces back towards the cell Moreover the tips are some-what thickened (

ordf

5 nm) so that the architecture of thehook region is strongly reminiscent of man-made fish-hooks grapples and anchors (Figs 2ndash4)

To appreciate the unique structure of these molecularbarbed grappling hooks and to distinguish them from otherfilamentous cell appendages we introduce the name

hamus

The spatial arrangement of the prickles and hooks were

investigated under conditions preserving their naturalstructure Cells in original marsh water were frozen invitrified ice and investigated by cryo-electron tomographyTomograms from single hami always showed three prick-les sticking out from one site of the central filament a

Fig 1

Electron micrograph of a platinum-shadowed SM1 eury-archaeal coccus About 100 pili-like fibres emanate radially from the surface of the cell

Unique structure of archaeal lsquohamirsquo

363

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

finding that was not obvious from negatively stained prep-arations According to longitudinal and cross-sections ofthe reconstructed hamus (Fig 4A and E) the onsets ofthe prickles are not exactly aligned along the filament axisbut may be distant by

ordf

2ndash5 mn The prickles assume anangle of 45

infin-

60

infin

with respect of the filament axis pointtowards the hook and are distant by about 120

infin

from eachother (Fig 4E) The same angular arrangement applies tothe three arms of the hook suggesting that the centralfilament possesses a threefold regular basic structure Inaccordance to the data presented in Figs 2 and 3 thearms of the hook show the same curvature and the some-what thicker anchor-like tips (Fig 4)

The tomographic reconstructions already suggested ahelical architecture of the hamus filament which was cor-roborated by typical layer line reflections in power spectraof negatively stained preparations (Fig 5D) Because thecomplex hamus structure and the non-ideal arrangementof the prickles prevented straightforward interpretation ofthe layer line data and reliable application of helical recon-struction schemes we used Fourier filtering to suppressobliterating noise The filtered images in Fig 5 reveal thehelical twist of the filament and show that the helicalperiodicity (pitch) correlates with the regularly positionedattachment sites of the prickles The Fourier spectrumcontains particularly strong information at spatial frequen-cies between 146 nm

-

1

and 146 nm

-

1

(Fig 5D) corre-sponding to the distances of repetitive prickle sites

Fig 2

Electron micrograph of a negatively stained fibre (lsquohamusrsquo) in its total length from the SM1 euryarchaeon after cell lysis by addition of 001 SDS (final concentration) showing the prickle and hook region The three enlarged sections reveal the well-defined base-to-top architecture of the hamus

Fig 3

Ultrastructure of hami from the SM1 euryarchaeon negative staining Bar

=

100 nmA Electron micrographs of grappling hooks located at the distal ends of the hami Arrows indicate location of the barbs ( )B Electron micrograph of high level structured SM1 hami The hami show prickles ( ) and grappling hooks ( )

364

C Moissl

et al

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

(46 nm) and of the smallest structural units (46 nm)resolved in the images of hami (Fig 5B and C) The datasuggest that 10 of these units build a recurring segmentof one protofilament and that three protofilaments formthe filamentous core of the hamus Filtering of a distalprickle-free 120 nm long portion of the hamus corrobo-rated the helical arrangement of 46 nm large units in thefilament but did not reveal further structural detail (datanot shown)

The prickles are thinner than the helical filament Thewidth ranges from 35 to 45 nm whereas the central fila-ment is about 7ndash8 nm in diameter (Figs 4 and 5) In accor-dance with the latter the prickles appear to be built fromunits 46 nm in size along the prickle axis Thus it istempting to speculate that they are structurally closelyrelated to the hamus protofilaments The prickles appearto consist of six (five to seven) units each accounting fora length of

ordf

30 nmThe hami from many different cells observed over an

extended period of time and in numerous experimentsalways showed the same basic architecture the samedimensions and proportions The only variable detectedwas the total length of the hami ranging from about 1

m

mup to 3

m

m Taking all the data together we suggest amodel of the hamus structure as illustrated in Fig 4D

Purification of the SM1 hami and biochemical analyses

For biochemical characterization the hami were extracted

from batch cultures of SM1 euryarchaeal cells that hadbeen grown on polyethylene nets in their biotope (Moissl

et al

2003) To isolate the hami these cells were treatedwith different mechanical methods like ultraturrax sonifi-cation or glass beads Electron microscopy of the differenthami preparations revealed that superior results wereobtained by the use of glass beads We could extractsignificant amounts of hami (

ordf

40

m

g protein per hamipreparation) with minimal lysis of cells The preparationsobtained consisted almost exclusively of hami and weretherefore used for most experiments without furtherpurification Alternatively hami were obtained by gentlelysis of the cells by the addition of SDS (001 finalconcentration)

The hami were very resistant against physical andchemical treatment Their structure as judged by electronmicroscopy remained stable for at least 1 h at tempera-tures between 0

infin

C and 70

infin

C and pH values ranging from05 (adjusted with HCl) to 115 (adjusted with NaOH) At80

infin

C or at pH 125 the hami were destroyed and were nolonger detectable in the electron microscope

We found that proteinase K digests the hami of the SM1euryarchaeal cells (Moissl

et al

2003) In agreement withthe proteinaceous structure the hami were also highlysensitive against enzymatic treatment with protease(Subtilisin A) pronase and trypsin The protein composi-tion was analysed by SDS-PAGE experiments Underreducing conditions the hami dissociated to a major sub-unit with an apparent mass of 120 kDa (Fig 6)

Fig 4

Cryo-electron tomography of an SM1 hamusA and B Longitudinal section through the hookprickle region of the three-dimensionally recon-structed hamus (A) before and (B) after denois-ing of the tomographical data The widths of the sections are 110 nm eachC 3D model of the hamus structure as visual-ized by surface rendering of the denoised data setD Model of the hamus with the characteristic dimensions indicatedE Series of cross-sections through the original tomogram perpendicular to the hamus axis The sections are distant by 136 nm from each other and illustrate the onsets and the number of the prickles of the prickle site indicated in (A) The series is displayed in the order from the bottom close to the central filament towards the tips of the prickles oriented towards the hook regionF Projection through the last three sections in (E) illustrating the positions of the filament and the prickles more clearlyG Corresponding projection through four con-secutive sections in the hook region Image size of cross-sections 67 nm

Unique structure of archaeal lsquohamirsquo

365

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Assuming that the hami consist of three protofilamentsthe cumulative length of one repetitive segment is3

yen

46

=

138 nm (or 30 units) whereas the pricklesaccount for about 3

yen

30

=

90 nm (or 20 units) ie

ordf

65of the building blocks in the filament If the protein formingthe prickles were significantly different in size we wouldexpect a second prominent protein band in SDS gels

N-glycosylation of flagella and S-layer proteins is wide-spread within the archaea (Jarrell

et al

2001 Upreti

et al

2003 Eichler 2004) Therefore the 120 kDa proteinwas tested for possible glycosylation using PAS-stainingor digestion with PNGaseF however both assays werenegative

Immunological studies

For immunological experiments we used polyclonal anti-

bodies raised against about 2

yen

10

8

SM1 euryarchaealcells (Moissl

et al

2003) By immuno-FISH we found thatthese antibodies (anti-SM1 serum) target the SM1 hami(Moissl

et al

2003) In the next step we wanted to specifythe target molecules for these antibodies in more detailImmunoblot analysis of total cell extracts and hami prep-arations showed in both cases a strong antibody reactionwith the 120 kDa protein To verify that the identified120 kDa protein is the substantial part of the hami severalSDS-PAGE gels of hami fractions were blotted and the120 kDa protein bands excised collected and used as atemplate for antibody affinity purification The antibodiesobtained showed specific reaction against the 120 kDaprotein in immunoblot studies In the next step the affinity-purified antibodies were used for immunogold labellingElectron microscopy showed that these antibodiesreacted specifically against the hami of the SM1 eur-yarchaeal cells (Fig 7) This specific antibody binding wasconfirmed independently in immuno-FISH experimentsand we conclude that the 120 kDa protein is the majorconstituent of the hami

Fig 5

Fourier filtering of a hamus in the prickle regionA Original image extracted from a micrograph of a negatively stained preparation Bar indicates 20 nmB Nois- reduced filtered image of the same hamus regionC More rigorously filtered image of the central filament illustrating the helical arrangement of structural units D Central region of a power spectrum from the hamus partly shown in A The hamus was optically isolated by a box-like mask of 650 nm in length in the original image before Fourier transformation The arrows indicate particularly strong reflections (layer lines) and the positions of characteristic spatial frequencies (figures denote the real spacings given in nm) The circle indicates the position of data at 2 nm resolution

Fig 6

SDS-PAGE of a crude cell extract of SM1 euryarchaeal cells (B) and of purified SM1 hami after glass bead extraction and centrif-ugation (C) The arrow indicates the 120 kDa hami protein already visible in the crude extract (B) The molecular weight of the protein markers is given in kDa (A)

366

C Moissl

et al

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Cell adhesion studies and hami stability experiments

In order to address the function of the hami we designedadhesion experiments of single SM1 cells (Moissl

et al

2003) using a variety of surfaces with different chemicalproperties (polylysine polyglutamate gelatine bovineserum albumine laminin fibronectine bind-silane) Allcoating materials tested mediated the strong adherenceof these archaea They were no more detachable evenwhen the maximum output power (25 W) of the lsquoopticaltweezersrsquo laser was used (Huber

et al

1995 Huber andStetter 2001)

In further adhesion studies single SM1 euryarchaealcells in suspension were optically trapped in the laserbeam and placed upon single cells which were fixed to abovine serum albumine-coated glass slide All attempts toseparate the cell pairs by the lsquooptical tweezersrsquo failedindicating that strong adhesion forces also occur betweenSM1 euryarchaeal cells

Discussion

In this study we reported on the discovery and analysisof a unique prokaryotic cell surface structure formed bythe uncultivated cold-loving non-methanogenic SM1 eur-yarchaeon in nature (Rudolph

et al

2001 Moissl

et al

2003) These archaeal cocci were entirely covered bysurface appendages of unexpected high complexity witha well-defined base-to-top organization each filamentexhibited a barbwire-like morphology and carried a tripar-tite barbed grappling hook at its distal end Because of

its complex architecture and the obvious distinctivenessfrom all the bacterial and archaeal cell appendages wehave chosen the term lsquohamusrsquo (plural lsquohamirsquo) for this newclass of cell surface structures The hami closely resembleman-made fishhooks grapples and anchors and sponta-neously suggest a function for cell anchoring or adhesionIt appears that one of the most basic life forms of themicrobial world long ago developed a tool that mankindnow uses in technology worldwide a parallel of directinterest to the fields of bionics and biomimetrics(Dickinson 1999 Ball 2001 Sarikaya

et al

2003)In contrast to the hami architecture prokaryotic

appendages like pili or flagella appear more simply in theirfilament structure They form tubes or helical fibres withoutfurther morphological differentiation (Fernaacutendez andBerenguer 2000 Jarrell

et al 2001 Hahn et al 2002)From bacterial flagella the hami also differ significantly infilament diameter with 7ndash8 nm compared with 24 nm(Yonekura et al 2003) in this aspect they are more sim-ilar to bacterial pili and to archaeal pili-like fibres withdiameters of 5ndash6 nm and 3ndash5 nm respectively (Doddemaet al 1979 Leadbetter and Breznak 1996 Mirosh-nichenko et al 1998 Fernaacutendez and Berenguer 2000)Similar to archaeal flagella and bacterial pili (Cohen-Krausz and Trachtenberg 2002) no evidence for theexistence of a central channel was found in the hamifilaments as far as this can be concluded from the tomo-graphical reconstructions It appears unlikely that the hamigrow by the bacterial flagellum mode of assembly It ismore conceivable that new subunits are added to thebase similar as shown for bacterial pili and as postulatedfor archaeal flagella (Jarrell et al 2001 Li and Mobley2001Bardy et al 2003)

However it is currently beyond our imagination how theprickle structures are synthesized or lsquoattachedrsquo to the fila-ment and by what mechanism the regular arrangement iscreated The same applies for the distal section of thehami including the hooks The extraordinary architectureof the hami suggests that the assembly process requiresdistinct coordination and control probably by a multicom-ponent system Another problem poses the stable anchor-ing of hami in the cytoplasmic membrane Besides thenecessary identification of genes and the isolation ofproteins involved the determination of the hamusrsquo basalstructure close to the membrane and inside thecytoplasm eg by cellular cryo-electron tomography(Baumeister 2002) would shed light on these intriguingquestions

The hami appear to consist of one major protein iethe 120 kDa component as revealed by gel electrophore-sis and immunological experiments This finding does notexclude the existence of protein species of the same sizeor of different proteins in low amounts And indeed thePAGE experiments showed further minor components

Fig 7 Immunogold labelling of hami extending from the surface of an SM1 euryarchaeal cell by the use of affinity-purified antibodies To avoid complete gold-covering of the hami a high dilution of the immunogold-labelled antibodies was used in this preparation Elec-tron micrograph negatively stained

Unique structure of archaeal lsquohamirsquo 367

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

However our data strongly suggest that the structuralcomponents of the hamus filament and the prickles areof the same size Whether they are structurally closelyrelated or even identical remains to be elucidated A pre-requisite for a more detailed investigation of this fascinat-ing microbial structure is knowledge of the genome andthe experimental availability of this currently uncultivatedeuryarchaeon as a laboratory culture

In a first glance the structure of the hami appears tobe distantly reminiscent of actin filaments (F-actin) dec-orated with myosin S1 heads (Milligan et al 1990Holmes et al 2003) The quantitative data howevershow the clear differences between both biopolymers (i)the diameter of the filament with 9ndash10 nm for actin and7 nm for the hami (ii) the number of strands two-strandedhelical for actin three-stranded for the hami (iii) the dis-tance of the sites where the arms are anchored to oremanate from the filament (about 55 nm for F-actin46 nm for the hami) and (iv) their molecular composition(two molecules actin plus myosin whereas only one sub-unit constitutes the hami presumably)

A further remarkable feature of the hami is the stabilityof the three-dimensional (3D) structure in a very broadpH and temperature range Amazingly they are even sta-ble at a temperature of 70infinC although they have beensynthesized at and may have been adapted to 10infinC thenatural growth temperature of the SM1 euryarchaeonThe discovery of the hami raises interesting questionsabout possible ecological functions Our attachmentexperiments showed that the hami mediate strong adhe-sion of single cells to each other and to surfaces of differ-ent chemical nature Therefore they are perfectly wellsuited for cell attachment to various organic and inor-ganic materials in nature They may also play a crucialrole in the formation of the microbial string-of-pearls com-munity by initial attachment to the specific bacterial part-ners of the SM1 euryarchaeon in its environment(Rudolph et al 2001 2004 Moissl et al 2003)Because of the highly specific archaealbacterial partner-ship in the pearls one could even suppose that in thefirst step of recognition the hami mediate cellcell com-munication between members of the two prokaryoticdomains

Nanostructured surfaces with dimensions of a fewnanometres exhibit unique physical and chemical proper-ties that can be utilized for many important technologicalapplications (Roukes 2002) The most complex functionalnanoscale structures are built efficiently from biomole-cules in biological systems especially from nucleic acidspolysaccharides and proteins Microorganisms in particu-lar have novel and interesting structures that could beexploited for example bacterial spore coats and the lat-tice-type crystalline arrays of bacterial S-layers (Sleytr andMessner 1983 Ricca and Cutting 2003) In this context

the discovery of the hami could open new windows in theemerging field of nanobiotechnology

Experimental procedures

In situ growth harvesting and specific separation of SM1 euryarchaeal cells

SM1 euryarchaeal cells were grown and harvested asdescribed (Moissl et al 2003) with the following modificationinstead of using Percoll (Moissl et al 2003) which is unsuit-able for further biochemical and electron microscopical stud-ies the SM1 euryarchaeal cells were specifically enriched bydifferential centrifugation at 20 000 g (10infinC 10 min) Thisstep resulted in a brownish cell pellet (consisting almostexclusively of bacteria) and a whitish cell cloud just aboveThis cloud was selectively removed by a pipette and con-sisted of up to 98 SM1 euryarchaeal cells the remainingorganisms were bacteria

Electron microscopy image processing and tomographical reconstruction

SM1 euryarchaeal cells or protein solutions were depositedon a carbon-coated copper grid and platinum shadowed asdescribed (Moissl et al 2003) or negatively stained with 2uranyl acetate pH 45 Samples were examined using aCM12 transmission electron microscope (Philips) operated at120 keV All images were digitally recorded using a slow-scanCCD camera that was connected to a PC running TVIPSsoftware (TVIPS GmbH) The pixel size on the specimen levelwas 056 nm

Noise reduction via Fourier filtering of images from nega-tively stained hami was performed in two variants For bothprocedures the hami were extracted from electron micro-graphs suitably boxed The images of the hami werestretched to give a straight longitudinal axis using the SEM-PER image processing system (Saxton 1996) Fourier trans-forms of hami images including the prickles were thresholdedfor the most prominent reflections according to the respectivepower spectrum The transforms of filaments without theprickles were filtered for the apparent layer lines indicated inthe power spectra in addition

For cryo-electron tomography a 5 ml droplet of an SM1euryarchaeal cell suspension in original marsh water wasapplied on a lacy carbon grid coated with 5 nm colloidal goldparticles After blotting the sample was vitrified by plungefreezing in liquid ethane (Dubochet et al 1988) Data collec-tion was performed at liquid nitrogen temperature using aPhilips CM300 transmission electron microscope equippedwith a field emission gun and a Gatan imaging filter operatedat 300 keV extraction voltage

The tilt series were recorded with a tilt range from -60infin toabout +67infin and an 15infin increment The images were aquiredwith a magnification of 43 975yen at -12 mm defocus under lowdose conditions The pixel size on the specimen level was136 nm in all the tomograms After alignment of the singleprojections using the nanogold particles as markers thereconstruction was performed by weighted backprojectionwith the EM software (Hegerl 1996) For 3D volume render-

368 C Moissl et al

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

ing the resulting tomograms of the hami were denoised bynon-linear anisotropic diffusion (Frangakis and Hegerl 2001)The final 3D visualization was performed by means of theAMIRA software (Visual concepts GmbH)

Isolation and purification of the SM1 hami

For hami preparations and washing steps a KPH buffer withan ionic composition similar to the original marsh water wasused 07 mM NaCl 01 mM MgCl2 16 mM CaSO4 10 mMHepesNaOH pH 65

For the isolation of the SM1 hami SM1 euryarchaeal cellswere either treated by an ultraturrax for 6 min at 4infinC (PolytronPT1200 Kinematica AG) by sonification for 35 min at 4infinCand 35 kHz (Sonorex super 10P Bandelin Electronic) or bythe use of glass beads (017ndash018 mm Braun Biotech Inter-national GmbH) For the glass bead extraction one volumeof glass beads was added to the cell suspension and stirredgently for 18 h at 4infinC The SM1 cells were removed bycentrifugation (34 000 g 30 min) and the hami were col-lected from the supernatant by ultracentrifugation (62 000 g1 h) After resuspension in KPH buffer the efficiency andquality of the different hami preparations were compared byelectron microscopy

SM1 cell extract and whole-cell protein preparation

SM1 euryarchaeal cells were washed twice with KPH After-wards the cells were disrupted by a French press (FrenchPressure Cell Press Aminco) Cells debris was removed bycentrifugation at 20 000 g (4infinC 15 min) To the supernatantthree volumes of ice cold acetone were added and the pro-teins precipitated for 2 h at -20infinC After centrifugation at20 000 g (4infinC 15 min) the resulting pellet was dried in anexsiccator and used for immunoblot analyses

Denaturing SDS-PAGE analysis

Denaturing SDS-PAGE was performed using the buffer sys-tem of Laemmli (1970) at a constant voltage (200 V) in avertical minigel apparatus (mini Protean III Bio-Rad) withgels containing 10 polyacrylamide in the separating gel and3 polyacrylamide in the stacking gel Samples were boiledin sample buffer for 8 min before loading on acrylamide gelsProtein bands were visualized by Coomassie blue staining

Western blot immunoblot and antibody affinity purification

After SDS-PAGE total SM1 cell extracts and hami prepara-tions were blotted onto PVDF membranes (Immobilon F Mil-lipore) using a Trans-Blot SD transfer cell (Bio-Rad) at 15 Vfor 45 min Transfer of proteins was verified by staining themembranes with 02 Ponceau S (wv) in 3 TCA (vv)

For immunoblots the membranes were blocked for 14 h at4infinC by soaking in 5 milk powder (wv) in TBST [0242TrisHCl (wv) 08 NaCl (wv) 01 Tween 20 (vv)pH 76] The membranes were washed three times in TBST(10 min) Afterwards they were transferred into 5 milk pow-

der in TBST containing the anti-SM1 serum and incubatedfor 3 h at 20infinC After three washing steps for 10 min in TBSTthe membranes were incubated for 2 h with a 110 000 dilu-tion of peroxidase-conjugated rabbit anti-chicken IgG (inTBST with 5 powdered milk Dianova GmbH) The mem-branes were washed in TBST and the protein-bound antibod-ies were detected by shaking the membranes in a 4-chlornaphtol solution for 20 min at 20infinC The 4-chlornaphtolsolution was composed of 10 ml of 50 mM TrisHCl pH 7510 ml of 30 H2O2 and 3 mg of 4-chlornaphtol

For affinity purification of the anti-SM1 serum hami protein(120 kDa) blotted onto PVDF membranes was used Themembrane pieces of excised protein bands were collected inan Eppendorf tube containing 5 (wv) powdered milk inTBST for the blocking step (14 h 4infinC) All membrane pieceswere treated with anti-SM1 serum (11000) in TBST (with 5powdered milk) for 3 h at 20infinC and then washed three timesin TBST buffer The pieces were transferred into an Eppen-dorf tube containing 1 ml of 100 mM triethylamine (pH 115)After 15 min the solution was neutralized by adding 01 mlof 1 M TrisHCl (pH 80) The affinity-purified antibodies wereconcentrated and washed by the use of Centricon YM-30centrifugal filter devices (Millipore) according to the sup-plierrsquos instructions

Immunogold labelling for electron microscopy

SM1 euryarchaeal cells in KPH were incubated with affinity-purified antibodies for 2 h at 20infinC The cells were washedand a 1500 solution of a donkey anti-chicken colloidal gold-labelled (12 nm) IgG (Dianova) was added After incubation(2 h 20infinC) the cells were washed twice gently resuspendedin KPH applied onto carbon-coated copper grids and nega-tively stained with uranyl acetate as described

PAS staining and PNGase F treatment of glass bead-separated hami

PAS staining was performed as described (Segrest and Jack-son 1972) using Avidin as a positive control PNGase F(New England BioLabs) studies were performed as describedin the manufacturerrsquos instruction The hami were treated with1 mg ml-1 PNGase F (1 h 37infinC) and analysed by SDS-PAGE Ovalbumin was used as a positive control

Cell adhesion studies and hami stability experiments

Adhesion forces of single cells were investigated by the useof a computer-controlled inverted microscope equipped witha continuously operating neodymium-doped yttrium alumi-num garnet laser (NdYAG laser) The emission wavelengthof the laser is in the near infrared at 1064 nm the maximumoutput power 25 W (Huber et al 1995 Huber and Stetter2001) The laser can be focused to a spot size of less than1 mm in diameter by the use of a high-numerical-aperture oilimmersion objective (100yen) As a consequence of the strongintensity of the laser light optical trapping and manipulationof single cells in mm size in three dimensions is possible(lsquooptical tweezers traprsquo lsquolaser traprsquo Ashkin and Dziedzic1987 Ashkin et al 1987)

Unique structure of archaeal lsquohamirsquo 369

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

For cell adhesion studies glass slides (76 yen 26 yen 1 mmMarienfeld) were coated with polylysine polyglutamategelatine bovine serum albumine laminin fibronectine (each01 wv in KPH final concentration) or with a bind-silanesolution The solution was prepared by adding 15 ml of bind-silane to a mixture of 12 ml of ethanol and 375 ml of aceticacid (10 wv) Ten microlitres from a cell suspension ofSM1 euryarchaeal cells were spotted onto each slide surfaceAfter sedimentation of most of the cells a cover slide wasplaced on top of each droplet The glass slides were placedon the stage of the laser microscope and single adhered cellswere trapped in the laser beam After adjustment of the laserto its maximum output power we attempted to detach thetrapped cells from the solid surfaces into the liquid phase

Next we investigated the stability of isolated hami In oneseries of experiments the hami were incubated for 1 h atvarious temperatures The influence of low and high pH val-ues was studied at 20infinC after 1 h of incubation the pH ofthe samples was re-adjusted to 70 with HCl or NaOH Theeffect of the different treatments was analysed by electronmicroscopy (presence or absence of hami) Enzymatic diges-tion of hami preparations was tested by adding proteinase K(Merck KG) protease (Subtilisin A Sigma-Aldrich ChemieGmbH) pronase or trypsin (Boehringer) The suspensionswere incubated for 1 h at 37infinC (proteinase K protease pro-nase) or 25infinC (trypsin) As a positive control bovine serumalbumine was used The effect of the enzymes was investi-gated by SDS-PAGE (presence or absence of the 120 kDaprotein band)

Acknowledgements

We are indebted to the Government of Bavaria Germany fora sampling permit Financial support from the DeutscheForschungsgemeinschaft (HU 7112) is gratefullyacknowledged

References

Adler J (1966) Chemotaxis in bacteria Science 153 706ndash716

Ashkin A and Dziedzic JM (1987) Optical trapping andmanipulation of viruses and bacteria Science 235 1517ndash1520

Ashkin A Dziedzic JM and Yamane T (1987) Opticaltrapping and manipulation of single cells using infraredlaser beams Nature 330 769ndash771

Ball P (2001) Lifersquos lessons in design Nature 409 413ndash416Bardy SL Ng SYM and Jarrell KF (2003) Prokaryotic

motility structures Microbiology 149 295ndash304Baumeister W (2002) Electron tomography towards visual-

izing the molecular organization of the cytoplasm CurrOpin Struct Biol 12 679ndash684

Bayley DP and Jarrell KF (1998) Further evidence tosuggest that archaeal flagella are related to bacteria typeIV pili J Mol Evol 46 370ndash373

Berry RM (2001) Bacterial flagella flagellar motor InNature Encyclopedia of Life Sciences London NaturePublishing Group [www document] URL httpwwwelsnet [doi 101038npgels0000744]

Cohen-Krausz S and Trachtenberg S (2002) The structureof the archeabacterial flagellar filament of the extremehalophile Halobacterium salinarium R1M1 and its relationto eubacterial flagellar filaments and type IV pili J Mol Biol321 383ndash395

DeLong EF (1992) Archaea in coastal marine environ-ments Proc Natl Acad Sci USA 89 5685ndash5689

Dickinson MH (1999) Bionics biological insight intomechanical design Proc Natl Acad Sci USA 96 14208ndash14209

Doddema HJ Derksen JWM and Vogels GD (1979)Fimbriae and flagella of methanogenic bacteria FEMSMicrobiol Lett 5 135ndash138

Dubochet J Adrian M Chang JJ Homo JC LepaultJ McDowall AW and Schultz P (1988) Cryo-electronmicroscopy of vitrified specimens Q Rev Biophys 21 129ndash228

Eichler J (2004) Facing extremes archaeal surface-layer(glyco) proteins Microbiology 149 3347ndash3351

Faguy DM Bayley DP Kostyukova AS Thomas NAand Jarrell KF (1996) Isolation and characterization offlagella and flagellin protein from the thermoacidophilicarchaea Thermoplasma volcanium and Sulfolobus shiba-tae J Bacteriol 178 902ndash905

Fernaacutendez LA and Berenguer J (2000) Secretion andassembly of regular surface structures in Gram-negativebacteria FEMS Microbiol Rev 24 21ndash44

Frangakis AS and Hegerl R (2001) Noise reductionin electron tomographic reconstructions using nonlinearanisotropic diffusion J Struct Biol 135 239ndash250

Fuhrman JA McCallum K and Davis AA (1992) Novelmajor archaebacterial group from marine plankton Nature356 148ndash149

Hahn E Wild P Hermanns U Sebbel P GlockshuberR Haumlner M et al (2002) Exploring the 3D moleculararchitecture of Escherichia coli type 1 pili J Mol Biol 323845ndash857

Hegerl R (1996) The EM program package a platformfor image processing in biological electron microscopy JStruct Biol 116 30ndash34

Holmes KC Angert I Kull FJ Jahn W and SchroumlderRR (2003) Electron cryo-microscopy shows how strongbinding of myosin to actin releases nucleotide Nature 425423ndash427

Huber R and Stetter KO (2001) Discovery of hyperther-mophilic microorganisms In Methods in EnzymologyAdams MWW and Kelly RM (eds) London AcademicPress pp 11ndash24

Huber R Burggraf S Mayer T Barns SM RossnagelP and Stetter KO (1995) Isolation of a hyperthermo-philic archaeum predicted by in situ RNA analysis Nature376 57ndash58

Jarrell KF Bayley DP and Kostyukova AS (1996) Thearchaeal flagellum a unique motility structure J Bacteriol178 5057ndash5064

Jarrell KF Bayley DP Correia JD and ThomasNA (2001) Archaeal flagella In Nature Encyclopediaof Life Sciences London Nature Publishing Group[www document] URL httpwwwelsnet [doi 101038npgels0000386]

Laemmli UK (1970) Cleavage of structural proteins during

362

C Moissl

et al

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Recently we reported on the discovery of novelarchaea living in close association with bacteria in cold(

ordf

10

infin

C) sulphurous marsh water of the Sippenauer Moornear Regensburg Bavaria Germany (Rudolph

et al

2001) These prokaryotes form a unique string-of-pearls-like macroscopically visible structure tiny whitish pearls(diameter up to 3 mm) are connected to each other bythin white-coloured threads The inner part of each pearlis predominated by archaeal cocci belonging to a singleeuryarchaeal phylotype tentatively designated SM1euryarchaeon (Rudolph

et al

2001) This novel non-methanogenic archaeon represents a deep phylogeneticbranch within the 16S rRNA tree with no close cultivatedor uncultivated relatives (Rudolph

et al

2001) The outerpart of the pearls and the threads are mainly composedof a single phylotype belonging to the genus

Thiothrix

(Moissl

et al

2002) The SM1 euryarchaeon could not begrown in the laboratory so far

We have therefore designed a novel approach to growmicrobial string-of-pearls-like communities fast and reli-ably on large polyethylene nets in nature (Moissl

et al

2003) About once a week the microbial net populationcan be harvested and the SM1 euryarchaeon specificallyseparated by gentle physical methods (Moissl

et al

2003) This technique allowed us to gain biologicalinsights into the lifestyle of low-temperature archaea(Moissl

et al

2003) First ultrastructural and immunolog-ical studies of single SM1 euryarchaeal cocci lead to anunexpected observation about 100 filamentous cellappendages of 2ndash3

m

m in length emanate radially fromthe surface of each cell (Moissl

et al

2003) The numberof fibres was larger than that of any other cell appendagesfrom archaea known and indicated a particular and signif-icant function

Here we report on the detailed analysis of the SM1euryarchaeon surface appendages which turned out to beunique among prokaryotes so far It represents a newclass of cell appendages of high complexity with a well-defined base-to-top organization To emphasize the nov-elty of this structure and its characteristic morphology theterm lsquohamusrsquo (Latin meaning prickle claw hook barb orfishing rod plural lsquohamirsquo) is proposed

Results

Electron microscopy and tomographical reconstruction

Electron microscopy of platinum-shadowed SM1 eur-yarchaeal cells and immuno-FISH showed the existenceof up to 100 pili-like fibres peritrichously distributed on thesurface of each coccus (Fig 1) The fibres were

ordf

1ndash3

m

min length with an average of about 2

m

m and were foundon cells in different stages of growth (Moissl

et al

2003)Negatively stained preparations revealed an extraordi-

nary and novel type of architecture The fibres exhibiteda very complex structure with a well-defined base-to-toporganization (Figs 2 and 3) Basically each fibre (

ordf

7ndash8 nmin diameter) can be divided into two regions (i) The centralpart the

prickle region

is a barbwire-like structured fila-ment with little prickles sticking out in regular intervals of46

plusmn

14 nm (

n

=

53) They are smaller in diameter thanthe filament (

ordf

4 nm) and 30

plusmn

3 nm (

n

=

48) in length onaverage From the cell surface up to 60 of these recurringunits make up one filament (Fig 2) (ii) The distal part iethe 152

plusmn

3 nm (

n

=

55) long

hook region

is composed ofa plain filament with a tripartite end forming a uniquestructure (Figs 2ndash4) The filament is frayed out into threedistinct arms each of which is about 4 nm in diameter and50 nm in length and exhibits a characteristic curvature ofabout 180 degrees such that the end of each arm alwaysfaces back towards the cell Moreover the tips are some-what thickened (

ordf

5 nm) so that the architecture of thehook region is strongly reminiscent of man-made fish-hooks grapples and anchors (Figs 2ndash4)

To appreciate the unique structure of these molecularbarbed grappling hooks and to distinguish them from otherfilamentous cell appendages we introduce the name

hamus

The spatial arrangement of the prickles and hooks were

investigated under conditions preserving their naturalstructure Cells in original marsh water were frozen invitrified ice and investigated by cryo-electron tomographyTomograms from single hami always showed three prick-les sticking out from one site of the central filament a

Fig 1

Electron micrograph of a platinum-shadowed SM1 eury-archaeal coccus About 100 pili-like fibres emanate radially from the surface of the cell

Unique structure of archaeal lsquohamirsquo

363

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

finding that was not obvious from negatively stained prep-arations According to longitudinal and cross-sections ofthe reconstructed hamus (Fig 4A and E) the onsets ofthe prickles are not exactly aligned along the filament axisbut may be distant by

ordf

2ndash5 mn The prickles assume anangle of 45

infin-

60

infin

with respect of the filament axis pointtowards the hook and are distant by about 120

infin

from eachother (Fig 4E) The same angular arrangement applies tothe three arms of the hook suggesting that the centralfilament possesses a threefold regular basic structure Inaccordance to the data presented in Figs 2 and 3 thearms of the hook show the same curvature and the some-what thicker anchor-like tips (Fig 4)

The tomographic reconstructions already suggested ahelical architecture of the hamus filament which was cor-roborated by typical layer line reflections in power spectraof negatively stained preparations (Fig 5D) Because thecomplex hamus structure and the non-ideal arrangementof the prickles prevented straightforward interpretation ofthe layer line data and reliable application of helical recon-struction schemes we used Fourier filtering to suppressobliterating noise The filtered images in Fig 5 reveal thehelical twist of the filament and show that the helicalperiodicity (pitch) correlates with the regularly positionedattachment sites of the prickles The Fourier spectrumcontains particularly strong information at spatial frequen-cies between 146 nm

-

1

and 146 nm

-

1

(Fig 5D) corre-sponding to the distances of repetitive prickle sites

Fig 2

Electron micrograph of a negatively stained fibre (lsquohamusrsquo) in its total length from the SM1 euryarchaeon after cell lysis by addition of 001 SDS (final concentration) showing the prickle and hook region The three enlarged sections reveal the well-defined base-to-top architecture of the hamus

Fig 3

Ultrastructure of hami from the SM1 euryarchaeon negative staining Bar

=

100 nmA Electron micrographs of grappling hooks located at the distal ends of the hami Arrows indicate location of the barbs ( )B Electron micrograph of high level structured SM1 hami The hami show prickles ( ) and grappling hooks ( )

364

C Moissl

et al

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

(46 nm) and of the smallest structural units (46 nm)resolved in the images of hami (Fig 5B and C) The datasuggest that 10 of these units build a recurring segmentof one protofilament and that three protofilaments formthe filamentous core of the hamus Filtering of a distalprickle-free 120 nm long portion of the hamus corrobo-rated the helical arrangement of 46 nm large units in thefilament but did not reveal further structural detail (datanot shown)

The prickles are thinner than the helical filament Thewidth ranges from 35 to 45 nm whereas the central fila-ment is about 7ndash8 nm in diameter (Figs 4 and 5) In accor-dance with the latter the prickles appear to be built fromunits 46 nm in size along the prickle axis Thus it istempting to speculate that they are structurally closelyrelated to the hamus protofilaments The prickles appearto consist of six (five to seven) units each accounting fora length of

ordf

30 nmThe hami from many different cells observed over an

extended period of time and in numerous experimentsalways showed the same basic architecture the samedimensions and proportions The only variable detectedwas the total length of the hami ranging from about 1

m

mup to 3

m

m Taking all the data together we suggest amodel of the hamus structure as illustrated in Fig 4D

Purification of the SM1 hami and biochemical analyses

For biochemical characterization the hami were extracted

from batch cultures of SM1 euryarchaeal cells that hadbeen grown on polyethylene nets in their biotope (Moissl

et al

2003) To isolate the hami these cells were treatedwith different mechanical methods like ultraturrax sonifi-cation or glass beads Electron microscopy of the differenthami preparations revealed that superior results wereobtained by the use of glass beads We could extractsignificant amounts of hami (

ordf

40

m

g protein per hamipreparation) with minimal lysis of cells The preparationsobtained consisted almost exclusively of hami and weretherefore used for most experiments without furtherpurification Alternatively hami were obtained by gentlelysis of the cells by the addition of SDS (001 finalconcentration)

The hami were very resistant against physical andchemical treatment Their structure as judged by electronmicroscopy remained stable for at least 1 h at tempera-tures between 0

infin

C and 70

infin

C and pH values ranging from05 (adjusted with HCl) to 115 (adjusted with NaOH) At80

infin

C or at pH 125 the hami were destroyed and were nolonger detectable in the electron microscope

We found that proteinase K digests the hami of the SM1euryarchaeal cells (Moissl

et al

2003) In agreement withthe proteinaceous structure the hami were also highlysensitive against enzymatic treatment with protease(Subtilisin A) pronase and trypsin The protein composi-tion was analysed by SDS-PAGE experiments Underreducing conditions the hami dissociated to a major sub-unit with an apparent mass of 120 kDa (Fig 6)

Fig 4

Cryo-electron tomography of an SM1 hamusA and B Longitudinal section through the hookprickle region of the three-dimensionally recon-structed hamus (A) before and (B) after denois-ing of the tomographical data The widths of the sections are 110 nm eachC 3D model of the hamus structure as visual-ized by surface rendering of the denoised data setD Model of the hamus with the characteristic dimensions indicatedE Series of cross-sections through the original tomogram perpendicular to the hamus axis The sections are distant by 136 nm from each other and illustrate the onsets and the number of the prickles of the prickle site indicated in (A) The series is displayed in the order from the bottom close to the central filament towards the tips of the prickles oriented towards the hook regionF Projection through the last three sections in (E) illustrating the positions of the filament and the prickles more clearlyG Corresponding projection through four con-secutive sections in the hook region Image size of cross-sections 67 nm

Unique structure of archaeal lsquohamirsquo

365

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Assuming that the hami consist of three protofilamentsthe cumulative length of one repetitive segment is3

yen

46

=

138 nm (or 30 units) whereas the pricklesaccount for about 3

yen

30

=

90 nm (or 20 units) ie

ordf

65of the building blocks in the filament If the protein formingthe prickles were significantly different in size we wouldexpect a second prominent protein band in SDS gels

N-glycosylation of flagella and S-layer proteins is wide-spread within the archaea (Jarrell

et al

2001 Upreti

et al

2003 Eichler 2004) Therefore the 120 kDa proteinwas tested for possible glycosylation using PAS-stainingor digestion with PNGaseF however both assays werenegative

Immunological studies

For immunological experiments we used polyclonal anti-

bodies raised against about 2

yen

10

8

SM1 euryarchaealcells (Moissl

et al

2003) By immuno-FISH we found thatthese antibodies (anti-SM1 serum) target the SM1 hami(Moissl

et al

2003) In the next step we wanted to specifythe target molecules for these antibodies in more detailImmunoblot analysis of total cell extracts and hami prep-arations showed in both cases a strong antibody reactionwith the 120 kDa protein To verify that the identified120 kDa protein is the substantial part of the hami severalSDS-PAGE gels of hami fractions were blotted and the120 kDa protein bands excised collected and used as atemplate for antibody affinity purification The antibodiesobtained showed specific reaction against the 120 kDaprotein in immunoblot studies In the next step the affinity-purified antibodies were used for immunogold labellingElectron microscopy showed that these antibodiesreacted specifically against the hami of the SM1 eur-yarchaeal cells (Fig 7) This specific antibody binding wasconfirmed independently in immuno-FISH experimentsand we conclude that the 120 kDa protein is the majorconstituent of the hami

Fig 5

Fourier filtering of a hamus in the prickle regionA Original image extracted from a micrograph of a negatively stained preparation Bar indicates 20 nmB Nois- reduced filtered image of the same hamus regionC More rigorously filtered image of the central filament illustrating the helical arrangement of structural units D Central region of a power spectrum from the hamus partly shown in A The hamus was optically isolated by a box-like mask of 650 nm in length in the original image before Fourier transformation The arrows indicate particularly strong reflections (layer lines) and the positions of characteristic spatial frequencies (figures denote the real spacings given in nm) The circle indicates the position of data at 2 nm resolution

Fig 6

SDS-PAGE of a crude cell extract of SM1 euryarchaeal cells (B) and of purified SM1 hami after glass bead extraction and centrif-ugation (C) The arrow indicates the 120 kDa hami protein already visible in the crude extract (B) The molecular weight of the protein markers is given in kDa (A)

366

C Moissl

et al

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Cell adhesion studies and hami stability experiments

In order to address the function of the hami we designedadhesion experiments of single SM1 cells (Moissl

et al

2003) using a variety of surfaces with different chemicalproperties (polylysine polyglutamate gelatine bovineserum albumine laminin fibronectine bind-silane) Allcoating materials tested mediated the strong adherenceof these archaea They were no more detachable evenwhen the maximum output power (25 W) of the lsquoopticaltweezersrsquo laser was used (Huber

et al

1995 Huber andStetter 2001)

In further adhesion studies single SM1 euryarchaealcells in suspension were optically trapped in the laserbeam and placed upon single cells which were fixed to abovine serum albumine-coated glass slide All attempts toseparate the cell pairs by the lsquooptical tweezersrsquo failedindicating that strong adhesion forces also occur betweenSM1 euryarchaeal cells

Discussion

In this study we reported on the discovery and analysisof a unique prokaryotic cell surface structure formed bythe uncultivated cold-loving non-methanogenic SM1 eur-yarchaeon in nature (Rudolph

et al

2001 Moissl

et al

2003) These archaeal cocci were entirely covered bysurface appendages of unexpected high complexity witha well-defined base-to-top organization each filamentexhibited a barbwire-like morphology and carried a tripar-tite barbed grappling hook at its distal end Because of

its complex architecture and the obvious distinctivenessfrom all the bacterial and archaeal cell appendages wehave chosen the term lsquohamusrsquo (plural lsquohamirsquo) for this newclass of cell surface structures The hami closely resembleman-made fishhooks grapples and anchors and sponta-neously suggest a function for cell anchoring or adhesionIt appears that one of the most basic life forms of themicrobial world long ago developed a tool that mankindnow uses in technology worldwide a parallel of directinterest to the fields of bionics and biomimetrics(Dickinson 1999 Ball 2001 Sarikaya

et al

2003)In contrast to the hami architecture prokaryotic

appendages like pili or flagella appear more simply in theirfilament structure They form tubes or helical fibres withoutfurther morphological differentiation (Fernaacutendez andBerenguer 2000 Jarrell

et al 2001 Hahn et al 2002)From bacterial flagella the hami also differ significantly infilament diameter with 7ndash8 nm compared with 24 nm(Yonekura et al 2003) in this aspect they are more sim-ilar to bacterial pili and to archaeal pili-like fibres withdiameters of 5ndash6 nm and 3ndash5 nm respectively (Doddemaet al 1979 Leadbetter and Breznak 1996 Mirosh-nichenko et al 1998 Fernaacutendez and Berenguer 2000)Similar to archaeal flagella and bacterial pili (Cohen-Krausz and Trachtenberg 2002) no evidence for theexistence of a central channel was found in the hamifilaments as far as this can be concluded from the tomo-graphical reconstructions It appears unlikely that the hamigrow by the bacterial flagellum mode of assembly It ismore conceivable that new subunits are added to thebase similar as shown for bacterial pili and as postulatedfor archaeal flagella (Jarrell et al 2001 Li and Mobley2001Bardy et al 2003)

However it is currently beyond our imagination how theprickle structures are synthesized or lsquoattachedrsquo to the fila-ment and by what mechanism the regular arrangement iscreated The same applies for the distal section of thehami including the hooks The extraordinary architectureof the hami suggests that the assembly process requiresdistinct coordination and control probably by a multicom-ponent system Another problem poses the stable anchor-ing of hami in the cytoplasmic membrane Besides thenecessary identification of genes and the isolation ofproteins involved the determination of the hamusrsquo basalstructure close to the membrane and inside thecytoplasm eg by cellular cryo-electron tomography(Baumeister 2002) would shed light on these intriguingquestions

The hami appear to consist of one major protein iethe 120 kDa component as revealed by gel electrophore-sis and immunological experiments This finding does notexclude the existence of protein species of the same sizeor of different proteins in low amounts And indeed thePAGE experiments showed further minor components

Fig 7 Immunogold labelling of hami extending from the surface of an SM1 euryarchaeal cell by the use of affinity-purified antibodies To avoid complete gold-covering of the hami a high dilution of the immunogold-labelled antibodies was used in this preparation Elec-tron micrograph negatively stained

Unique structure of archaeal lsquohamirsquo 367

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

However our data strongly suggest that the structuralcomponents of the hamus filament and the prickles areof the same size Whether they are structurally closelyrelated or even identical remains to be elucidated A pre-requisite for a more detailed investigation of this fascinat-ing microbial structure is knowledge of the genome andthe experimental availability of this currently uncultivatedeuryarchaeon as a laboratory culture

In a first glance the structure of the hami appears tobe distantly reminiscent of actin filaments (F-actin) dec-orated with myosin S1 heads (Milligan et al 1990Holmes et al 2003) The quantitative data howevershow the clear differences between both biopolymers (i)the diameter of the filament with 9ndash10 nm for actin and7 nm for the hami (ii) the number of strands two-strandedhelical for actin three-stranded for the hami (iii) the dis-tance of the sites where the arms are anchored to oremanate from the filament (about 55 nm for F-actin46 nm for the hami) and (iv) their molecular composition(two molecules actin plus myosin whereas only one sub-unit constitutes the hami presumably)

A further remarkable feature of the hami is the stabilityof the three-dimensional (3D) structure in a very broadpH and temperature range Amazingly they are even sta-ble at a temperature of 70infinC although they have beensynthesized at and may have been adapted to 10infinC thenatural growth temperature of the SM1 euryarchaeonThe discovery of the hami raises interesting questionsabout possible ecological functions Our attachmentexperiments showed that the hami mediate strong adhe-sion of single cells to each other and to surfaces of differ-ent chemical nature Therefore they are perfectly wellsuited for cell attachment to various organic and inor-ganic materials in nature They may also play a crucialrole in the formation of the microbial string-of-pearls com-munity by initial attachment to the specific bacterial part-ners of the SM1 euryarchaeon in its environment(Rudolph et al 2001 2004 Moissl et al 2003)Because of the highly specific archaealbacterial partner-ship in the pearls one could even suppose that in thefirst step of recognition the hami mediate cellcell com-munication between members of the two prokaryoticdomains

Nanostructured surfaces with dimensions of a fewnanometres exhibit unique physical and chemical proper-ties that can be utilized for many important technologicalapplications (Roukes 2002) The most complex functionalnanoscale structures are built efficiently from biomole-cules in biological systems especially from nucleic acidspolysaccharides and proteins Microorganisms in particu-lar have novel and interesting structures that could beexploited for example bacterial spore coats and the lat-tice-type crystalline arrays of bacterial S-layers (Sleytr andMessner 1983 Ricca and Cutting 2003) In this context

the discovery of the hami could open new windows in theemerging field of nanobiotechnology

Experimental procedures

In situ growth harvesting and specific separation of SM1 euryarchaeal cells

SM1 euryarchaeal cells were grown and harvested asdescribed (Moissl et al 2003) with the following modificationinstead of using Percoll (Moissl et al 2003) which is unsuit-able for further biochemical and electron microscopical stud-ies the SM1 euryarchaeal cells were specifically enriched bydifferential centrifugation at 20 000 g (10infinC 10 min) Thisstep resulted in a brownish cell pellet (consisting almostexclusively of bacteria) and a whitish cell cloud just aboveThis cloud was selectively removed by a pipette and con-sisted of up to 98 SM1 euryarchaeal cells the remainingorganisms were bacteria

Electron microscopy image processing and tomographical reconstruction

SM1 euryarchaeal cells or protein solutions were depositedon a carbon-coated copper grid and platinum shadowed asdescribed (Moissl et al 2003) or negatively stained with 2uranyl acetate pH 45 Samples were examined using aCM12 transmission electron microscope (Philips) operated at120 keV All images were digitally recorded using a slow-scanCCD camera that was connected to a PC running TVIPSsoftware (TVIPS GmbH) The pixel size on the specimen levelwas 056 nm

Noise reduction via Fourier filtering of images from nega-tively stained hami was performed in two variants For bothprocedures the hami were extracted from electron micro-graphs suitably boxed The images of the hami werestretched to give a straight longitudinal axis using the SEM-PER image processing system (Saxton 1996) Fourier trans-forms of hami images including the prickles were thresholdedfor the most prominent reflections according to the respectivepower spectrum The transforms of filaments without theprickles were filtered for the apparent layer lines indicated inthe power spectra in addition

For cryo-electron tomography a 5 ml droplet of an SM1euryarchaeal cell suspension in original marsh water wasapplied on a lacy carbon grid coated with 5 nm colloidal goldparticles After blotting the sample was vitrified by plungefreezing in liquid ethane (Dubochet et al 1988) Data collec-tion was performed at liquid nitrogen temperature using aPhilips CM300 transmission electron microscope equippedwith a field emission gun and a Gatan imaging filter operatedat 300 keV extraction voltage

The tilt series were recorded with a tilt range from -60infin toabout +67infin and an 15infin increment The images were aquiredwith a magnification of 43 975yen at -12 mm defocus under lowdose conditions The pixel size on the specimen level was136 nm in all the tomograms After alignment of the singleprojections using the nanogold particles as markers thereconstruction was performed by weighted backprojectionwith the EM software (Hegerl 1996) For 3D volume render-

368 C Moissl et al

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

ing the resulting tomograms of the hami were denoised bynon-linear anisotropic diffusion (Frangakis and Hegerl 2001)The final 3D visualization was performed by means of theAMIRA software (Visual concepts GmbH)

Isolation and purification of the SM1 hami

For hami preparations and washing steps a KPH buffer withan ionic composition similar to the original marsh water wasused 07 mM NaCl 01 mM MgCl2 16 mM CaSO4 10 mMHepesNaOH pH 65

For the isolation of the SM1 hami SM1 euryarchaeal cellswere either treated by an ultraturrax for 6 min at 4infinC (PolytronPT1200 Kinematica AG) by sonification for 35 min at 4infinCand 35 kHz (Sonorex super 10P Bandelin Electronic) or bythe use of glass beads (017ndash018 mm Braun Biotech Inter-national GmbH) For the glass bead extraction one volumeof glass beads was added to the cell suspension and stirredgently for 18 h at 4infinC The SM1 cells were removed bycentrifugation (34 000 g 30 min) and the hami were col-lected from the supernatant by ultracentrifugation (62 000 g1 h) After resuspension in KPH buffer the efficiency andquality of the different hami preparations were compared byelectron microscopy

SM1 cell extract and whole-cell protein preparation

SM1 euryarchaeal cells were washed twice with KPH After-wards the cells were disrupted by a French press (FrenchPressure Cell Press Aminco) Cells debris was removed bycentrifugation at 20 000 g (4infinC 15 min) To the supernatantthree volumes of ice cold acetone were added and the pro-teins precipitated for 2 h at -20infinC After centrifugation at20 000 g (4infinC 15 min) the resulting pellet was dried in anexsiccator and used for immunoblot analyses

Denaturing SDS-PAGE analysis

Denaturing SDS-PAGE was performed using the buffer sys-tem of Laemmli (1970) at a constant voltage (200 V) in avertical minigel apparatus (mini Protean III Bio-Rad) withgels containing 10 polyacrylamide in the separating gel and3 polyacrylamide in the stacking gel Samples were boiledin sample buffer for 8 min before loading on acrylamide gelsProtein bands were visualized by Coomassie blue staining

Western blot immunoblot and antibody affinity purification

After SDS-PAGE total SM1 cell extracts and hami prepara-tions were blotted onto PVDF membranes (Immobilon F Mil-lipore) using a Trans-Blot SD transfer cell (Bio-Rad) at 15 Vfor 45 min Transfer of proteins was verified by staining themembranes with 02 Ponceau S (wv) in 3 TCA (vv)

For immunoblots the membranes were blocked for 14 h at4infinC by soaking in 5 milk powder (wv) in TBST [0242TrisHCl (wv) 08 NaCl (wv) 01 Tween 20 (vv)pH 76] The membranes were washed three times in TBST(10 min) Afterwards they were transferred into 5 milk pow-

der in TBST containing the anti-SM1 serum and incubatedfor 3 h at 20infinC After three washing steps for 10 min in TBSTthe membranes were incubated for 2 h with a 110 000 dilu-tion of peroxidase-conjugated rabbit anti-chicken IgG (inTBST with 5 powdered milk Dianova GmbH) The mem-branes were washed in TBST and the protein-bound antibod-ies were detected by shaking the membranes in a 4-chlornaphtol solution for 20 min at 20infinC The 4-chlornaphtolsolution was composed of 10 ml of 50 mM TrisHCl pH 7510 ml of 30 H2O2 and 3 mg of 4-chlornaphtol

For affinity purification of the anti-SM1 serum hami protein(120 kDa) blotted onto PVDF membranes was used Themembrane pieces of excised protein bands were collected inan Eppendorf tube containing 5 (wv) powdered milk inTBST for the blocking step (14 h 4infinC) All membrane pieceswere treated with anti-SM1 serum (11000) in TBST (with 5powdered milk) for 3 h at 20infinC and then washed three timesin TBST buffer The pieces were transferred into an Eppen-dorf tube containing 1 ml of 100 mM triethylamine (pH 115)After 15 min the solution was neutralized by adding 01 mlof 1 M TrisHCl (pH 80) The affinity-purified antibodies wereconcentrated and washed by the use of Centricon YM-30centrifugal filter devices (Millipore) according to the sup-plierrsquos instructions

Immunogold labelling for electron microscopy

SM1 euryarchaeal cells in KPH were incubated with affinity-purified antibodies for 2 h at 20infinC The cells were washedand a 1500 solution of a donkey anti-chicken colloidal gold-labelled (12 nm) IgG (Dianova) was added After incubation(2 h 20infinC) the cells were washed twice gently resuspendedin KPH applied onto carbon-coated copper grids and nega-tively stained with uranyl acetate as described

PAS staining and PNGase F treatment of glass bead-separated hami

PAS staining was performed as described (Segrest and Jack-son 1972) using Avidin as a positive control PNGase F(New England BioLabs) studies were performed as describedin the manufacturerrsquos instruction The hami were treated with1 mg ml-1 PNGase F (1 h 37infinC) and analysed by SDS-PAGE Ovalbumin was used as a positive control

Cell adhesion studies and hami stability experiments

Adhesion forces of single cells were investigated by the useof a computer-controlled inverted microscope equipped witha continuously operating neodymium-doped yttrium alumi-num garnet laser (NdYAG laser) The emission wavelengthof the laser is in the near infrared at 1064 nm the maximumoutput power 25 W (Huber et al 1995 Huber and Stetter2001) The laser can be focused to a spot size of less than1 mm in diameter by the use of a high-numerical-aperture oilimmersion objective (100yen) As a consequence of the strongintensity of the laser light optical trapping and manipulationof single cells in mm size in three dimensions is possible(lsquooptical tweezers traprsquo lsquolaser traprsquo Ashkin and Dziedzic1987 Ashkin et al 1987)

Unique structure of archaeal lsquohamirsquo 369

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

For cell adhesion studies glass slides (76 yen 26 yen 1 mmMarienfeld) were coated with polylysine polyglutamategelatine bovine serum albumine laminin fibronectine (each01 wv in KPH final concentration) or with a bind-silanesolution The solution was prepared by adding 15 ml of bind-silane to a mixture of 12 ml of ethanol and 375 ml of aceticacid (10 wv) Ten microlitres from a cell suspension ofSM1 euryarchaeal cells were spotted onto each slide surfaceAfter sedimentation of most of the cells a cover slide wasplaced on top of each droplet The glass slides were placedon the stage of the laser microscope and single adhered cellswere trapped in the laser beam After adjustment of the laserto its maximum output power we attempted to detach thetrapped cells from the solid surfaces into the liquid phase

Next we investigated the stability of isolated hami In oneseries of experiments the hami were incubated for 1 h atvarious temperatures The influence of low and high pH val-ues was studied at 20infinC after 1 h of incubation the pH ofthe samples was re-adjusted to 70 with HCl or NaOH Theeffect of the different treatments was analysed by electronmicroscopy (presence or absence of hami) Enzymatic diges-tion of hami preparations was tested by adding proteinase K(Merck KG) protease (Subtilisin A Sigma-Aldrich ChemieGmbH) pronase or trypsin (Boehringer) The suspensionswere incubated for 1 h at 37infinC (proteinase K protease pro-nase) or 25infinC (trypsin) As a positive control bovine serumalbumine was used The effect of the enzymes was investi-gated by SDS-PAGE (presence or absence of the 120 kDaprotein band)

Acknowledgements

We are indebted to the Government of Bavaria Germany fora sampling permit Financial support from the DeutscheForschungsgemeinschaft (HU 7112) is gratefullyacknowledged

References

Adler J (1966) Chemotaxis in bacteria Science 153 706ndash716

Ashkin A and Dziedzic JM (1987) Optical trapping andmanipulation of viruses and bacteria Science 235 1517ndash1520

Ashkin A Dziedzic JM and Yamane T (1987) Opticaltrapping and manipulation of single cells using infraredlaser beams Nature 330 769ndash771

Ball P (2001) Lifersquos lessons in design Nature 409 413ndash416Bardy SL Ng SYM and Jarrell KF (2003) Prokaryotic

motility structures Microbiology 149 295ndash304Baumeister W (2002) Electron tomography towards visual-

izing the molecular organization of the cytoplasm CurrOpin Struct Biol 12 679ndash684

Bayley DP and Jarrell KF (1998) Further evidence tosuggest that archaeal flagella are related to bacteria typeIV pili J Mol Evol 46 370ndash373

Berry RM (2001) Bacterial flagella flagellar motor InNature Encyclopedia of Life Sciences London NaturePublishing Group [www document] URL httpwwwelsnet [doi 101038npgels0000744]

Cohen-Krausz S and Trachtenberg S (2002) The structureof the archeabacterial flagellar filament of the extremehalophile Halobacterium salinarium R1M1 and its relationto eubacterial flagellar filaments and type IV pili J Mol Biol321 383ndash395

DeLong EF (1992) Archaea in coastal marine environ-ments Proc Natl Acad Sci USA 89 5685ndash5689

Dickinson MH (1999) Bionics biological insight intomechanical design Proc Natl Acad Sci USA 96 14208ndash14209

Doddema HJ Derksen JWM and Vogels GD (1979)Fimbriae and flagella of methanogenic bacteria FEMSMicrobiol Lett 5 135ndash138

Dubochet J Adrian M Chang JJ Homo JC LepaultJ McDowall AW and Schultz P (1988) Cryo-electronmicroscopy of vitrified specimens Q Rev Biophys 21 129ndash228

Eichler J (2004) Facing extremes archaeal surface-layer(glyco) proteins Microbiology 149 3347ndash3351

Faguy DM Bayley DP Kostyukova AS Thomas NAand Jarrell KF (1996) Isolation and characterization offlagella and flagellin protein from the thermoacidophilicarchaea Thermoplasma volcanium and Sulfolobus shiba-tae J Bacteriol 178 902ndash905

Fernaacutendez LA and Berenguer J (2000) Secretion andassembly of regular surface structures in Gram-negativebacteria FEMS Microbiol Rev 24 21ndash44

Frangakis AS and Hegerl R (2001) Noise reductionin electron tomographic reconstructions using nonlinearanisotropic diffusion J Struct Biol 135 239ndash250

Fuhrman JA McCallum K and Davis AA (1992) Novelmajor archaebacterial group from marine plankton Nature356 148ndash149

Hahn E Wild P Hermanns U Sebbel P GlockshuberR Haumlner M et al (2002) Exploring the 3D moleculararchitecture of Escherichia coli type 1 pili J Mol Biol 323845ndash857

Hegerl R (1996) The EM program package a platformfor image processing in biological electron microscopy JStruct Biol 116 30ndash34

Holmes KC Angert I Kull FJ Jahn W and SchroumlderRR (2003) Electron cryo-microscopy shows how strongbinding of myosin to actin releases nucleotide Nature 425423ndash427

Huber R and Stetter KO (2001) Discovery of hyperther-mophilic microorganisms In Methods in EnzymologyAdams MWW and Kelly RM (eds) London AcademicPress pp 11ndash24

Huber R Burggraf S Mayer T Barns SM RossnagelP and Stetter KO (1995) Isolation of a hyperthermo-philic archaeum predicted by in situ RNA analysis Nature376 57ndash58

Jarrell KF Bayley DP and Kostyukova AS (1996) Thearchaeal flagellum a unique motility structure J Bacteriol178 5057ndash5064

Jarrell KF Bayley DP Correia JD and ThomasNA (2001) Archaeal flagella In Nature Encyclopediaof Life Sciences London Nature Publishing Group[www document] URL httpwwwelsnet [doi 101038npgels0000386]

Laemmli UK (1970) Cleavage of structural proteins during

Unique structure of archaeal lsquohamirsquo

363

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

finding that was not obvious from negatively stained prep-arations According to longitudinal and cross-sections ofthe reconstructed hamus (Fig 4A and E) the onsets ofthe prickles are not exactly aligned along the filament axisbut may be distant by

ordf

2ndash5 mn The prickles assume anangle of 45

infin-

60

infin

with respect of the filament axis pointtowards the hook and are distant by about 120

infin

from eachother (Fig 4E) The same angular arrangement applies tothe three arms of the hook suggesting that the centralfilament possesses a threefold regular basic structure Inaccordance to the data presented in Figs 2 and 3 thearms of the hook show the same curvature and the some-what thicker anchor-like tips (Fig 4)

The tomographic reconstructions already suggested ahelical architecture of the hamus filament which was cor-roborated by typical layer line reflections in power spectraof negatively stained preparations (Fig 5D) Because thecomplex hamus structure and the non-ideal arrangementof the prickles prevented straightforward interpretation ofthe layer line data and reliable application of helical recon-struction schemes we used Fourier filtering to suppressobliterating noise The filtered images in Fig 5 reveal thehelical twist of the filament and show that the helicalperiodicity (pitch) correlates with the regularly positionedattachment sites of the prickles The Fourier spectrumcontains particularly strong information at spatial frequen-cies between 146 nm

-

1

and 146 nm

-

1

(Fig 5D) corre-sponding to the distances of repetitive prickle sites

Fig 2

Electron micrograph of a negatively stained fibre (lsquohamusrsquo) in its total length from the SM1 euryarchaeon after cell lysis by addition of 001 SDS (final concentration) showing the prickle and hook region The three enlarged sections reveal the well-defined base-to-top architecture of the hamus

Fig 3

Ultrastructure of hami from the SM1 euryarchaeon negative staining Bar

=

100 nmA Electron micrographs of grappling hooks located at the distal ends of the hami Arrows indicate location of the barbs ( )B Electron micrograph of high level structured SM1 hami The hami show prickles ( ) and grappling hooks ( )

364

C Moissl

et al

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

(46 nm) and of the smallest structural units (46 nm)resolved in the images of hami (Fig 5B and C) The datasuggest that 10 of these units build a recurring segmentof one protofilament and that three protofilaments formthe filamentous core of the hamus Filtering of a distalprickle-free 120 nm long portion of the hamus corrobo-rated the helical arrangement of 46 nm large units in thefilament but did not reveal further structural detail (datanot shown)

The prickles are thinner than the helical filament Thewidth ranges from 35 to 45 nm whereas the central fila-ment is about 7ndash8 nm in diameter (Figs 4 and 5) In accor-dance with the latter the prickles appear to be built fromunits 46 nm in size along the prickle axis Thus it istempting to speculate that they are structurally closelyrelated to the hamus protofilaments The prickles appearto consist of six (five to seven) units each accounting fora length of

ordf

30 nmThe hami from many different cells observed over an

extended period of time and in numerous experimentsalways showed the same basic architecture the samedimensions and proportions The only variable detectedwas the total length of the hami ranging from about 1

m

mup to 3

m

m Taking all the data together we suggest amodel of the hamus structure as illustrated in Fig 4D

Purification of the SM1 hami and biochemical analyses

For biochemical characterization the hami were extracted

from batch cultures of SM1 euryarchaeal cells that hadbeen grown on polyethylene nets in their biotope (Moissl

et al

2003) To isolate the hami these cells were treatedwith different mechanical methods like ultraturrax sonifi-cation or glass beads Electron microscopy of the differenthami preparations revealed that superior results wereobtained by the use of glass beads We could extractsignificant amounts of hami (

ordf

40

m

g protein per hamipreparation) with minimal lysis of cells The preparationsobtained consisted almost exclusively of hami and weretherefore used for most experiments without furtherpurification Alternatively hami were obtained by gentlelysis of the cells by the addition of SDS (001 finalconcentration)

The hami were very resistant against physical andchemical treatment Their structure as judged by electronmicroscopy remained stable for at least 1 h at tempera-tures between 0

infin

C and 70

infin

C and pH values ranging from05 (adjusted with HCl) to 115 (adjusted with NaOH) At80

infin

C or at pH 125 the hami were destroyed and were nolonger detectable in the electron microscope

We found that proteinase K digests the hami of the SM1euryarchaeal cells (Moissl

et al

2003) In agreement withthe proteinaceous structure the hami were also highlysensitive against enzymatic treatment with protease(Subtilisin A) pronase and trypsin The protein composi-tion was analysed by SDS-PAGE experiments Underreducing conditions the hami dissociated to a major sub-unit with an apparent mass of 120 kDa (Fig 6)

Fig 4

Cryo-electron tomography of an SM1 hamusA and B Longitudinal section through the hookprickle region of the three-dimensionally recon-structed hamus (A) before and (B) after denois-ing of the tomographical data The widths of the sections are 110 nm eachC 3D model of the hamus structure as visual-ized by surface rendering of the denoised data setD Model of the hamus with the characteristic dimensions indicatedE Series of cross-sections through the original tomogram perpendicular to the hamus axis The sections are distant by 136 nm from each other and illustrate the onsets and the number of the prickles of the prickle site indicated in (A) The series is displayed in the order from the bottom close to the central filament towards the tips of the prickles oriented towards the hook regionF Projection through the last three sections in (E) illustrating the positions of the filament and the prickles more clearlyG Corresponding projection through four con-secutive sections in the hook region Image size of cross-sections 67 nm

Unique structure of archaeal lsquohamirsquo

365

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Assuming that the hami consist of three protofilamentsthe cumulative length of one repetitive segment is3

yen

46

=

138 nm (or 30 units) whereas the pricklesaccount for about 3

yen

30

=

90 nm (or 20 units) ie

ordf

65of the building blocks in the filament If the protein formingthe prickles were significantly different in size we wouldexpect a second prominent protein band in SDS gels

N-glycosylation of flagella and S-layer proteins is wide-spread within the archaea (Jarrell

et al

2001 Upreti

et al

2003 Eichler 2004) Therefore the 120 kDa proteinwas tested for possible glycosylation using PAS-stainingor digestion with PNGaseF however both assays werenegative

Immunological studies

For immunological experiments we used polyclonal anti-

bodies raised against about 2

yen

10

8

SM1 euryarchaealcells (Moissl

et al

2003) By immuno-FISH we found thatthese antibodies (anti-SM1 serum) target the SM1 hami(Moissl

et al

2003) In the next step we wanted to specifythe target molecules for these antibodies in more detailImmunoblot analysis of total cell extracts and hami prep-arations showed in both cases a strong antibody reactionwith the 120 kDa protein To verify that the identified120 kDa protein is the substantial part of the hami severalSDS-PAGE gels of hami fractions were blotted and the120 kDa protein bands excised collected and used as atemplate for antibody affinity purification The antibodiesobtained showed specific reaction against the 120 kDaprotein in immunoblot studies In the next step the affinity-purified antibodies were used for immunogold labellingElectron microscopy showed that these antibodiesreacted specifically against the hami of the SM1 eur-yarchaeal cells (Fig 7) This specific antibody binding wasconfirmed independently in immuno-FISH experimentsand we conclude that the 120 kDa protein is the majorconstituent of the hami

Fig 5

Fourier filtering of a hamus in the prickle regionA Original image extracted from a micrograph of a negatively stained preparation Bar indicates 20 nmB Nois- reduced filtered image of the same hamus regionC More rigorously filtered image of the central filament illustrating the helical arrangement of structural units D Central region of a power spectrum from the hamus partly shown in A The hamus was optically isolated by a box-like mask of 650 nm in length in the original image before Fourier transformation The arrows indicate particularly strong reflections (layer lines) and the positions of characteristic spatial frequencies (figures denote the real spacings given in nm) The circle indicates the position of data at 2 nm resolution

Fig 6

SDS-PAGE of a crude cell extract of SM1 euryarchaeal cells (B) and of purified SM1 hami after glass bead extraction and centrif-ugation (C) The arrow indicates the 120 kDa hami protein already visible in the crude extract (B) The molecular weight of the protein markers is given in kDa (A)

366

C Moissl

et al

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Cell adhesion studies and hami stability experiments

In order to address the function of the hami we designedadhesion experiments of single SM1 cells (Moissl

et al

2003) using a variety of surfaces with different chemicalproperties (polylysine polyglutamate gelatine bovineserum albumine laminin fibronectine bind-silane) Allcoating materials tested mediated the strong adherenceof these archaea They were no more detachable evenwhen the maximum output power (25 W) of the lsquoopticaltweezersrsquo laser was used (Huber

et al

1995 Huber andStetter 2001)

In further adhesion studies single SM1 euryarchaealcells in suspension were optically trapped in the laserbeam and placed upon single cells which were fixed to abovine serum albumine-coated glass slide All attempts toseparate the cell pairs by the lsquooptical tweezersrsquo failedindicating that strong adhesion forces also occur betweenSM1 euryarchaeal cells

Discussion

In this study we reported on the discovery and analysisof a unique prokaryotic cell surface structure formed bythe uncultivated cold-loving non-methanogenic SM1 eur-yarchaeon in nature (Rudolph

et al

2001 Moissl

et al

2003) These archaeal cocci were entirely covered bysurface appendages of unexpected high complexity witha well-defined base-to-top organization each filamentexhibited a barbwire-like morphology and carried a tripar-tite barbed grappling hook at its distal end Because of

its complex architecture and the obvious distinctivenessfrom all the bacterial and archaeal cell appendages wehave chosen the term lsquohamusrsquo (plural lsquohamirsquo) for this newclass of cell surface structures The hami closely resembleman-made fishhooks grapples and anchors and sponta-neously suggest a function for cell anchoring or adhesionIt appears that one of the most basic life forms of themicrobial world long ago developed a tool that mankindnow uses in technology worldwide a parallel of directinterest to the fields of bionics and biomimetrics(Dickinson 1999 Ball 2001 Sarikaya

et al

2003)In contrast to the hami architecture prokaryotic

appendages like pili or flagella appear more simply in theirfilament structure They form tubes or helical fibres withoutfurther morphological differentiation (Fernaacutendez andBerenguer 2000 Jarrell

et al 2001 Hahn et al 2002)From bacterial flagella the hami also differ significantly infilament diameter with 7ndash8 nm compared with 24 nm(Yonekura et al 2003) in this aspect they are more sim-ilar to bacterial pili and to archaeal pili-like fibres withdiameters of 5ndash6 nm and 3ndash5 nm respectively (Doddemaet al 1979 Leadbetter and Breznak 1996 Mirosh-nichenko et al 1998 Fernaacutendez and Berenguer 2000)Similar to archaeal flagella and bacterial pili (Cohen-Krausz and Trachtenberg 2002) no evidence for theexistence of a central channel was found in the hamifilaments as far as this can be concluded from the tomo-graphical reconstructions It appears unlikely that the hamigrow by the bacterial flagellum mode of assembly It ismore conceivable that new subunits are added to thebase similar as shown for bacterial pili and as postulatedfor archaeal flagella (Jarrell et al 2001 Li and Mobley2001Bardy et al 2003)

However it is currently beyond our imagination how theprickle structures are synthesized or lsquoattachedrsquo to the fila-ment and by what mechanism the regular arrangement iscreated The same applies for the distal section of thehami including the hooks The extraordinary architectureof the hami suggests that the assembly process requiresdistinct coordination and control probably by a multicom-ponent system Another problem poses the stable anchor-ing of hami in the cytoplasmic membrane Besides thenecessary identification of genes and the isolation ofproteins involved the determination of the hamusrsquo basalstructure close to the membrane and inside thecytoplasm eg by cellular cryo-electron tomography(Baumeister 2002) would shed light on these intriguingquestions

The hami appear to consist of one major protein iethe 120 kDa component as revealed by gel electrophore-sis and immunological experiments This finding does notexclude the existence of protein species of the same sizeor of different proteins in low amounts And indeed thePAGE experiments showed further minor components

Fig 7 Immunogold labelling of hami extending from the surface of an SM1 euryarchaeal cell by the use of affinity-purified antibodies To avoid complete gold-covering of the hami a high dilution of the immunogold-labelled antibodies was used in this preparation Elec-tron micrograph negatively stained

Unique structure of archaeal lsquohamirsquo 367

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

However our data strongly suggest that the structuralcomponents of the hamus filament and the prickles areof the same size Whether they are structurally closelyrelated or even identical remains to be elucidated A pre-requisite for a more detailed investigation of this fascinat-ing microbial structure is knowledge of the genome andthe experimental availability of this currently uncultivatedeuryarchaeon as a laboratory culture

In a first glance the structure of the hami appears tobe distantly reminiscent of actin filaments (F-actin) dec-orated with myosin S1 heads (Milligan et al 1990Holmes et al 2003) The quantitative data howevershow the clear differences between both biopolymers (i)the diameter of the filament with 9ndash10 nm for actin and7 nm for the hami (ii) the number of strands two-strandedhelical for actin three-stranded for the hami (iii) the dis-tance of the sites where the arms are anchored to oremanate from the filament (about 55 nm for F-actin46 nm for the hami) and (iv) their molecular composition(two molecules actin plus myosin whereas only one sub-unit constitutes the hami presumably)

A further remarkable feature of the hami is the stabilityof the three-dimensional (3D) structure in a very broadpH and temperature range Amazingly they are even sta-ble at a temperature of 70infinC although they have beensynthesized at and may have been adapted to 10infinC thenatural growth temperature of the SM1 euryarchaeonThe discovery of the hami raises interesting questionsabout possible ecological functions Our attachmentexperiments showed that the hami mediate strong adhe-sion of single cells to each other and to surfaces of differ-ent chemical nature Therefore they are perfectly wellsuited for cell attachment to various organic and inor-ganic materials in nature They may also play a crucialrole in the formation of the microbial string-of-pearls com-munity by initial attachment to the specific bacterial part-ners of the SM1 euryarchaeon in its environment(Rudolph et al 2001 2004 Moissl et al 2003)Because of the highly specific archaealbacterial partner-ship in the pearls one could even suppose that in thefirst step of recognition the hami mediate cellcell com-munication between members of the two prokaryoticdomains

Nanostructured surfaces with dimensions of a fewnanometres exhibit unique physical and chemical proper-ties that can be utilized for many important technologicalapplications (Roukes 2002) The most complex functionalnanoscale structures are built efficiently from biomole-cules in biological systems especially from nucleic acidspolysaccharides and proteins Microorganisms in particu-lar have novel and interesting structures that could beexploited for example bacterial spore coats and the lat-tice-type crystalline arrays of bacterial S-layers (Sleytr andMessner 1983 Ricca and Cutting 2003) In this context

the discovery of the hami could open new windows in theemerging field of nanobiotechnology

Experimental procedures

In situ growth harvesting and specific separation of SM1 euryarchaeal cells

SM1 euryarchaeal cells were grown and harvested asdescribed (Moissl et al 2003) with the following modificationinstead of using Percoll (Moissl et al 2003) which is unsuit-able for further biochemical and electron microscopical stud-ies the SM1 euryarchaeal cells were specifically enriched bydifferential centrifugation at 20 000 g (10infinC 10 min) Thisstep resulted in a brownish cell pellet (consisting almostexclusively of bacteria) and a whitish cell cloud just aboveThis cloud was selectively removed by a pipette and con-sisted of up to 98 SM1 euryarchaeal cells the remainingorganisms were bacteria

Electron microscopy image processing and tomographical reconstruction

SM1 euryarchaeal cells or protein solutions were depositedon a carbon-coated copper grid and platinum shadowed asdescribed (Moissl et al 2003) or negatively stained with 2uranyl acetate pH 45 Samples were examined using aCM12 transmission electron microscope (Philips) operated at120 keV All images were digitally recorded using a slow-scanCCD camera that was connected to a PC running TVIPSsoftware (TVIPS GmbH) The pixel size on the specimen levelwas 056 nm

Noise reduction via Fourier filtering of images from nega-tively stained hami was performed in two variants For bothprocedures the hami were extracted from electron micro-graphs suitably boxed The images of the hami werestretched to give a straight longitudinal axis using the SEM-PER image processing system (Saxton 1996) Fourier trans-forms of hami images including the prickles were thresholdedfor the most prominent reflections according to the respectivepower spectrum The transforms of filaments without theprickles were filtered for the apparent layer lines indicated inthe power spectra in addition

For cryo-electron tomography a 5 ml droplet of an SM1euryarchaeal cell suspension in original marsh water wasapplied on a lacy carbon grid coated with 5 nm colloidal goldparticles After blotting the sample was vitrified by plungefreezing in liquid ethane (Dubochet et al 1988) Data collec-tion was performed at liquid nitrogen temperature using aPhilips CM300 transmission electron microscope equippedwith a field emission gun and a Gatan imaging filter operatedat 300 keV extraction voltage

The tilt series were recorded with a tilt range from -60infin toabout +67infin and an 15infin increment The images were aquiredwith a magnification of 43 975yen at -12 mm defocus under lowdose conditions The pixel size on the specimen level was136 nm in all the tomograms After alignment of the singleprojections using the nanogold particles as markers thereconstruction was performed by weighted backprojectionwith the EM software (Hegerl 1996) For 3D volume render-

368 C Moissl et al

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

ing the resulting tomograms of the hami were denoised bynon-linear anisotropic diffusion (Frangakis and Hegerl 2001)The final 3D visualization was performed by means of theAMIRA software (Visual concepts GmbH)

Isolation and purification of the SM1 hami

For hami preparations and washing steps a KPH buffer withan ionic composition similar to the original marsh water wasused 07 mM NaCl 01 mM MgCl2 16 mM CaSO4 10 mMHepesNaOH pH 65

For the isolation of the SM1 hami SM1 euryarchaeal cellswere either treated by an ultraturrax for 6 min at 4infinC (PolytronPT1200 Kinematica AG) by sonification for 35 min at 4infinCand 35 kHz (Sonorex super 10P Bandelin Electronic) or bythe use of glass beads (017ndash018 mm Braun Biotech Inter-national GmbH) For the glass bead extraction one volumeof glass beads was added to the cell suspension and stirredgently for 18 h at 4infinC The SM1 cells were removed bycentrifugation (34 000 g 30 min) and the hami were col-lected from the supernatant by ultracentrifugation (62 000 g1 h) After resuspension in KPH buffer the efficiency andquality of the different hami preparations were compared byelectron microscopy

SM1 cell extract and whole-cell protein preparation

SM1 euryarchaeal cells were washed twice with KPH After-wards the cells were disrupted by a French press (FrenchPressure Cell Press Aminco) Cells debris was removed bycentrifugation at 20 000 g (4infinC 15 min) To the supernatantthree volumes of ice cold acetone were added and the pro-teins precipitated for 2 h at -20infinC After centrifugation at20 000 g (4infinC 15 min) the resulting pellet was dried in anexsiccator and used for immunoblot analyses

Denaturing SDS-PAGE analysis

Denaturing SDS-PAGE was performed using the buffer sys-tem of Laemmli (1970) at a constant voltage (200 V) in avertical minigel apparatus (mini Protean III Bio-Rad) withgels containing 10 polyacrylamide in the separating gel and3 polyacrylamide in the stacking gel Samples were boiledin sample buffer for 8 min before loading on acrylamide gelsProtein bands were visualized by Coomassie blue staining

Western blot immunoblot and antibody affinity purification

After SDS-PAGE total SM1 cell extracts and hami prepara-tions were blotted onto PVDF membranes (Immobilon F Mil-lipore) using a Trans-Blot SD transfer cell (Bio-Rad) at 15 Vfor 45 min Transfer of proteins was verified by staining themembranes with 02 Ponceau S (wv) in 3 TCA (vv)

For immunoblots the membranes were blocked for 14 h at4infinC by soaking in 5 milk powder (wv) in TBST [0242TrisHCl (wv) 08 NaCl (wv) 01 Tween 20 (vv)pH 76] The membranes were washed three times in TBST(10 min) Afterwards they were transferred into 5 milk pow-

der in TBST containing the anti-SM1 serum and incubatedfor 3 h at 20infinC After three washing steps for 10 min in TBSTthe membranes were incubated for 2 h with a 110 000 dilu-tion of peroxidase-conjugated rabbit anti-chicken IgG (inTBST with 5 powdered milk Dianova GmbH) The mem-branes were washed in TBST and the protein-bound antibod-ies were detected by shaking the membranes in a 4-chlornaphtol solution for 20 min at 20infinC The 4-chlornaphtolsolution was composed of 10 ml of 50 mM TrisHCl pH 7510 ml of 30 H2O2 and 3 mg of 4-chlornaphtol

For affinity purification of the anti-SM1 serum hami protein(120 kDa) blotted onto PVDF membranes was used Themembrane pieces of excised protein bands were collected inan Eppendorf tube containing 5 (wv) powdered milk inTBST for the blocking step (14 h 4infinC) All membrane pieceswere treated with anti-SM1 serum (11000) in TBST (with 5powdered milk) for 3 h at 20infinC and then washed three timesin TBST buffer The pieces were transferred into an Eppen-dorf tube containing 1 ml of 100 mM triethylamine (pH 115)After 15 min the solution was neutralized by adding 01 mlof 1 M TrisHCl (pH 80) The affinity-purified antibodies wereconcentrated and washed by the use of Centricon YM-30centrifugal filter devices (Millipore) according to the sup-plierrsquos instructions

Immunogold labelling for electron microscopy

SM1 euryarchaeal cells in KPH were incubated with affinity-purified antibodies for 2 h at 20infinC The cells were washedand a 1500 solution of a donkey anti-chicken colloidal gold-labelled (12 nm) IgG (Dianova) was added After incubation(2 h 20infinC) the cells were washed twice gently resuspendedin KPH applied onto carbon-coated copper grids and nega-tively stained with uranyl acetate as described

PAS staining and PNGase F treatment of glass bead-separated hami

PAS staining was performed as described (Segrest and Jack-son 1972) using Avidin as a positive control PNGase F(New England BioLabs) studies were performed as describedin the manufacturerrsquos instruction The hami were treated with1 mg ml-1 PNGase F (1 h 37infinC) and analysed by SDS-PAGE Ovalbumin was used as a positive control

Cell adhesion studies and hami stability experiments

Adhesion forces of single cells were investigated by the useof a computer-controlled inverted microscope equipped witha continuously operating neodymium-doped yttrium alumi-num garnet laser (NdYAG laser) The emission wavelengthof the laser is in the near infrared at 1064 nm the maximumoutput power 25 W (Huber et al 1995 Huber and Stetter2001) The laser can be focused to a spot size of less than1 mm in diameter by the use of a high-numerical-aperture oilimmersion objective (100yen) As a consequence of the strongintensity of the laser light optical trapping and manipulationof single cells in mm size in three dimensions is possible(lsquooptical tweezers traprsquo lsquolaser traprsquo Ashkin and Dziedzic1987 Ashkin et al 1987)

Unique structure of archaeal lsquohamirsquo 369

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

For cell adhesion studies glass slides (76 yen 26 yen 1 mmMarienfeld) were coated with polylysine polyglutamategelatine bovine serum albumine laminin fibronectine (each01 wv in KPH final concentration) or with a bind-silanesolution The solution was prepared by adding 15 ml of bind-silane to a mixture of 12 ml of ethanol and 375 ml of aceticacid (10 wv) Ten microlitres from a cell suspension ofSM1 euryarchaeal cells were spotted onto each slide surfaceAfter sedimentation of most of the cells a cover slide wasplaced on top of each droplet The glass slides were placedon the stage of the laser microscope and single adhered cellswere trapped in the laser beam After adjustment of the laserto its maximum output power we attempted to detach thetrapped cells from the solid surfaces into the liquid phase

Next we investigated the stability of isolated hami In oneseries of experiments the hami were incubated for 1 h atvarious temperatures The influence of low and high pH val-ues was studied at 20infinC after 1 h of incubation the pH ofthe samples was re-adjusted to 70 with HCl or NaOH Theeffect of the different treatments was analysed by electronmicroscopy (presence or absence of hami) Enzymatic diges-tion of hami preparations was tested by adding proteinase K(Merck KG) protease (Subtilisin A Sigma-Aldrich ChemieGmbH) pronase or trypsin (Boehringer) The suspensionswere incubated for 1 h at 37infinC (proteinase K protease pro-nase) or 25infinC (trypsin) As a positive control bovine serumalbumine was used The effect of the enzymes was investi-gated by SDS-PAGE (presence or absence of the 120 kDaprotein band)

Acknowledgements

We are indebted to the Government of Bavaria Germany fora sampling permit Financial support from the DeutscheForschungsgemeinschaft (HU 7112) is gratefullyacknowledged

References

Adler J (1966) Chemotaxis in bacteria Science 153 706ndash716

Ashkin A and Dziedzic JM (1987) Optical trapping andmanipulation of viruses and bacteria Science 235 1517ndash1520

Ashkin A Dziedzic JM and Yamane T (1987) Opticaltrapping and manipulation of single cells using infraredlaser beams Nature 330 769ndash771

Ball P (2001) Lifersquos lessons in design Nature 409 413ndash416Bardy SL Ng SYM and Jarrell KF (2003) Prokaryotic

motility structures Microbiology 149 295ndash304Baumeister W (2002) Electron tomography towards visual-

izing the molecular organization of the cytoplasm CurrOpin Struct Biol 12 679ndash684

Bayley DP and Jarrell KF (1998) Further evidence tosuggest that archaeal flagella are related to bacteria typeIV pili J Mol Evol 46 370ndash373

Berry RM (2001) Bacterial flagella flagellar motor InNature Encyclopedia of Life Sciences London NaturePublishing Group [www document] URL httpwwwelsnet [doi 101038npgels0000744]

Cohen-Krausz S and Trachtenberg S (2002) The structureof the archeabacterial flagellar filament of the extremehalophile Halobacterium salinarium R1M1 and its relationto eubacterial flagellar filaments and type IV pili J Mol Biol321 383ndash395

DeLong EF (1992) Archaea in coastal marine environ-ments Proc Natl Acad Sci USA 89 5685ndash5689

Dickinson MH (1999) Bionics biological insight intomechanical design Proc Natl Acad Sci USA 96 14208ndash14209

Doddema HJ Derksen JWM and Vogels GD (1979)Fimbriae and flagella of methanogenic bacteria FEMSMicrobiol Lett 5 135ndash138

Dubochet J Adrian M Chang JJ Homo JC LepaultJ McDowall AW and Schultz P (1988) Cryo-electronmicroscopy of vitrified specimens Q Rev Biophys 21 129ndash228

Eichler J (2004) Facing extremes archaeal surface-layer(glyco) proteins Microbiology 149 3347ndash3351

Faguy DM Bayley DP Kostyukova AS Thomas NAand Jarrell KF (1996) Isolation and characterization offlagella and flagellin protein from the thermoacidophilicarchaea Thermoplasma volcanium and Sulfolobus shiba-tae J Bacteriol 178 902ndash905

Fernaacutendez LA and Berenguer J (2000) Secretion andassembly of regular surface structures in Gram-negativebacteria FEMS Microbiol Rev 24 21ndash44

Frangakis AS and Hegerl R (2001) Noise reductionin electron tomographic reconstructions using nonlinearanisotropic diffusion J Struct Biol 135 239ndash250

Fuhrman JA McCallum K and Davis AA (1992) Novelmajor archaebacterial group from marine plankton Nature356 148ndash149

Hahn E Wild P Hermanns U Sebbel P GlockshuberR Haumlner M et al (2002) Exploring the 3D moleculararchitecture of Escherichia coli type 1 pili J Mol Biol 323845ndash857

Hegerl R (1996) The EM program package a platformfor image processing in biological electron microscopy JStruct Biol 116 30ndash34

Holmes KC Angert I Kull FJ Jahn W and SchroumlderRR (2003) Electron cryo-microscopy shows how strongbinding of myosin to actin releases nucleotide Nature 425423ndash427

Huber R and Stetter KO (2001) Discovery of hyperther-mophilic microorganisms In Methods in EnzymologyAdams MWW and Kelly RM (eds) London AcademicPress pp 11ndash24

Huber R Burggraf S Mayer T Barns SM RossnagelP and Stetter KO (1995) Isolation of a hyperthermo-philic archaeum predicted by in situ RNA analysis Nature376 57ndash58

Jarrell KF Bayley DP and Kostyukova AS (1996) Thearchaeal flagellum a unique motility structure J Bacteriol178 5057ndash5064

Jarrell KF Bayley DP Correia JD and ThomasNA (2001) Archaeal flagella In Nature Encyclopediaof Life Sciences London Nature Publishing Group[www document] URL httpwwwelsnet [doi 101038npgels0000386]

Laemmli UK (1970) Cleavage of structural proteins during

364

C Moissl

et al

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

(46 nm) and of the smallest structural units (46 nm)resolved in the images of hami (Fig 5B and C) The datasuggest that 10 of these units build a recurring segmentof one protofilament and that three protofilaments formthe filamentous core of the hamus Filtering of a distalprickle-free 120 nm long portion of the hamus corrobo-rated the helical arrangement of 46 nm large units in thefilament but did not reveal further structural detail (datanot shown)

The prickles are thinner than the helical filament Thewidth ranges from 35 to 45 nm whereas the central fila-ment is about 7ndash8 nm in diameter (Figs 4 and 5) In accor-dance with the latter the prickles appear to be built fromunits 46 nm in size along the prickle axis Thus it istempting to speculate that they are structurally closelyrelated to the hamus protofilaments The prickles appearto consist of six (five to seven) units each accounting fora length of

ordf

30 nmThe hami from many different cells observed over an

extended period of time and in numerous experimentsalways showed the same basic architecture the samedimensions and proportions The only variable detectedwas the total length of the hami ranging from about 1

m

mup to 3

m

m Taking all the data together we suggest amodel of the hamus structure as illustrated in Fig 4D

Purification of the SM1 hami and biochemical analyses

For biochemical characterization the hami were extracted

from batch cultures of SM1 euryarchaeal cells that hadbeen grown on polyethylene nets in their biotope (Moissl

et al

2003) To isolate the hami these cells were treatedwith different mechanical methods like ultraturrax sonifi-cation or glass beads Electron microscopy of the differenthami preparations revealed that superior results wereobtained by the use of glass beads We could extractsignificant amounts of hami (

ordf

40

m

g protein per hamipreparation) with minimal lysis of cells The preparationsobtained consisted almost exclusively of hami and weretherefore used for most experiments without furtherpurification Alternatively hami were obtained by gentlelysis of the cells by the addition of SDS (001 finalconcentration)

The hami were very resistant against physical andchemical treatment Their structure as judged by electronmicroscopy remained stable for at least 1 h at tempera-tures between 0

infin

C and 70

infin

C and pH values ranging from05 (adjusted with HCl) to 115 (adjusted with NaOH) At80

infin

C or at pH 125 the hami were destroyed and were nolonger detectable in the electron microscope

We found that proteinase K digests the hami of the SM1euryarchaeal cells (Moissl

et al

2003) In agreement withthe proteinaceous structure the hami were also highlysensitive against enzymatic treatment with protease(Subtilisin A) pronase and trypsin The protein composi-tion was analysed by SDS-PAGE experiments Underreducing conditions the hami dissociated to a major sub-unit with an apparent mass of 120 kDa (Fig 6)

Fig 4

Cryo-electron tomography of an SM1 hamusA and B Longitudinal section through the hookprickle region of the three-dimensionally recon-structed hamus (A) before and (B) after denois-ing of the tomographical data The widths of the sections are 110 nm eachC 3D model of the hamus structure as visual-ized by surface rendering of the denoised data setD Model of the hamus with the characteristic dimensions indicatedE Series of cross-sections through the original tomogram perpendicular to the hamus axis The sections are distant by 136 nm from each other and illustrate the onsets and the number of the prickles of the prickle site indicated in (A) The series is displayed in the order from the bottom close to the central filament towards the tips of the prickles oriented towards the hook regionF Projection through the last three sections in (E) illustrating the positions of the filament and the prickles more clearlyG Corresponding projection through four con-secutive sections in the hook region Image size of cross-sections 67 nm

Unique structure of archaeal lsquohamirsquo

365

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Assuming that the hami consist of three protofilamentsthe cumulative length of one repetitive segment is3

yen

46

=

138 nm (or 30 units) whereas the pricklesaccount for about 3

yen

30

=

90 nm (or 20 units) ie

ordf

65of the building blocks in the filament If the protein formingthe prickles were significantly different in size we wouldexpect a second prominent protein band in SDS gels

N-glycosylation of flagella and S-layer proteins is wide-spread within the archaea (Jarrell

et al

2001 Upreti

et al

2003 Eichler 2004) Therefore the 120 kDa proteinwas tested for possible glycosylation using PAS-stainingor digestion with PNGaseF however both assays werenegative

Immunological studies

For immunological experiments we used polyclonal anti-

bodies raised against about 2

yen

10

8

SM1 euryarchaealcells (Moissl

et al

2003) By immuno-FISH we found thatthese antibodies (anti-SM1 serum) target the SM1 hami(Moissl

et al

2003) In the next step we wanted to specifythe target molecules for these antibodies in more detailImmunoblot analysis of total cell extracts and hami prep-arations showed in both cases a strong antibody reactionwith the 120 kDa protein To verify that the identified120 kDa protein is the substantial part of the hami severalSDS-PAGE gels of hami fractions were blotted and the120 kDa protein bands excised collected and used as atemplate for antibody affinity purification The antibodiesobtained showed specific reaction against the 120 kDaprotein in immunoblot studies In the next step the affinity-purified antibodies were used for immunogold labellingElectron microscopy showed that these antibodiesreacted specifically against the hami of the SM1 eur-yarchaeal cells (Fig 7) This specific antibody binding wasconfirmed independently in immuno-FISH experimentsand we conclude that the 120 kDa protein is the majorconstituent of the hami

Fig 5

Fourier filtering of a hamus in the prickle regionA Original image extracted from a micrograph of a negatively stained preparation Bar indicates 20 nmB Nois- reduced filtered image of the same hamus regionC More rigorously filtered image of the central filament illustrating the helical arrangement of structural units D Central region of a power spectrum from the hamus partly shown in A The hamus was optically isolated by a box-like mask of 650 nm in length in the original image before Fourier transformation The arrows indicate particularly strong reflections (layer lines) and the positions of characteristic spatial frequencies (figures denote the real spacings given in nm) The circle indicates the position of data at 2 nm resolution

Fig 6

SDS-PAGE of a crude cell extract of SM1 euryarchaeal cells (B) and of purified SM1 hami after glass bead extraction and centrif-ugation (C) The arrow indicates the 120 kDa hami protein already visible in the crude extract (B) The molecular weight of the protein markers is given in kDa (A)

366

C Moissl

et al

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Cell adhesion studies and hami stability experiments

In order to address the function of the hami we designedadhesion experiments of single SM1 cells (Moissl

et al

2003) using a variety of surfaces with different chemicalproperties (polylysine polyglutamate gelatine bovineserum albumine laminin fibronectine bind-silane) Allcoating materials tested mediated the strong adherenceof these archaea They were no more detachable evenwhen the maximum output power (25 W) of the lsquoopticaltweezersrsquo laser was used (Huber

et al

1995 Huber andStetter 2001)

In further adhesion studies single SM1 euryarchaealcells in suspension were optically trapped in the laserbeam and placed upon single cells which were fixed to abovine serum albumine-coated glass slide All attempts toseparate the cell pairs by the lsquooptical tweezersrsquo failedindicating that strong adhesion forces also occur betweenSM1 euryarchaeal cells

Discussion

In this study we reported on the discovery and analysisof a unique prokaryotic cell surface structure formed bythe uncultivated cold-loving non-methanogenic SM1 eur-yarchaeon in nature (Rudolph

et al

2001 Moissl

et al

2003) These archaeal cocci were entirely covered bysurface appendages of unexpected high complexity witha well-defined base-to-top organization each filamentexhibited a barbwire-like morphology and carried a tripar-tite barbed grappling hook at its distal end Because of

its complex architecture and the obvious distinctivenessfrom all the bacterial and archaeal cell appendages wehave chosen the term lsquohamusrsquo (plural lsquohamirsquo) for this newclass of cell surface structures The hami closely resembleman-made fishhooks grapples and anchors and sponta-neously suggest a function for cell anchoring or adhesionIt appears that one of the most basic life forms of themicrobial world long ago developed a tool that mankindnow uses in technology worldwide a parallel of directinterest to the fields of bionics and biomimetrics(Dickinson 1999 Ball 2001 Sarikaya

et al

2003)In contrast to the hami architecture prokaryotic

appendages like pili or flagella appear more simply in theirfilament structure They form tubes or helical fibres withoutfurther morphological differentiation (Fernaacutendez andBerenguer 2000 Jarrell

et al 2001 Hahn et al 2002)From bacterial flagella the hami also differ significantly infilament diameter with 7ndash8 nm compared with 24 nm(Yonekura et al 2003) in this aspect they are more sim-ilar to bacterial pili and to archaeal pili-like fibres withdiameters of 5ndash6 nm and 3ndash5 nm respectively (Doddemaet al 1979 Leadbetter and Breznak 1996 Mirosh-nichenko et al 1998 Fernaacutendez and Berenguer 2000)Similar to archaeal flagella and bacterial pili (Cohen-Krausz and Trachtenberg 2002) no evidence for theexistence of a central channel was found in the hamifilaments as far as this can be concluded from the tomo-graphical reconstructions It appears unlikely that the hamigrow by the bacterial flagellum mode of assembly It ismore conceivable that new subunits are added to thebase similar as shown for bacterial pili and as postulatedfor archaeal flagella (Jarrell et al 2001 Li and Mobley2001Bardy et al 2003)

However it is currently beyond our imagination how theprickle structures are synthesized or lsquoattachedrsquo to the fila-ment and by what mechanism the regular arrangement iscreated The same applies for the distal section of thehami including the hooks The extraordinary architectureof the hami suggests that the assembly process requiresdistinct coordination and control probably by a multicom-ponent system Another problem poses the stable anchor-ing of hami in the cytoplasmic membrane Besides thenecessary identification of genes and the isolation ofproteins involved the determination of the hamusrsquo basalstructure close to the membrane and inside thecytoplasm eg by cellular cryo-electron tomography(Baumeister 2002) would shed light on these intriguingquestions

The hami appear to consist of one major protein iethe 120 kDa component as revealed by gel electrophore-sis and immunological experiments This finding does notexclude the existence of protein species of the same sizeor of different proteins in low amounts And indeed thePAGE experiments showed further minor components

Fig 7 Immunogold labelling of hami extending from the surface of an SM1 euryarchaeal cell by the use of affinity-purified antibodies To avoid complete gold-covering of the hami a high dilution of the immunogold-labelled antibodies was used in this preparation Elec-tron micrograph negatively stained

Unique structure of archaeal lsquohamirsquo 367

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

However our data strongly suggest that the structuralcomponents of the hamus filament and the prickles areof the same size Whether they are structurally closelyrelated or even identical remains to be elucidated A pre-requisite for a more detailed investigation of this fascinat-ing microbial structure is knowledge of the genome andthe experimental availability of this currently uncultivatedeuryarchaeon as a laboratory culture

In a first glance the structure of the hami appears tobe distantly reminiscent of actin filaments (F-actin) dec-orated with myosin S1 heads (Milligan et al 1990Holmes et al 2003) The quantitative data howevershow the clear differences between both biopolymers (i)the diameter of the filament with 9ndash10 nm for actin and7 nm for the hami (ii) the number of strands two-strandedhelical for actin three-stranded for the hami (iii) the dis-tance of the sites where the arms are anchored to oremanate from the filament (about 55 nm for F-actin46 nm for the hami) and (iv) their molecular composition(two molecules actin plus myosin whereas only one sub-unit constitutes the hami presumably)

A further remarkable feature of the hami is the stabilityof the three-dimensional (3D) structure in a very broadpH and temperature range Amazingly they are even sta-ble at a temperature of 70infinC although they have beensynthesized at and may have been adapted to 10infinC thenatural growth temperature of the SM1 euryarchaeonThe discovery of the hami raises interesting questionsabout possible ecological functions Our attachmentexperiments showed that the hami mediate strong adhe-sion of single cells to each other and to surfaces of differ-ent chemical nature Therefore they are perfectly wellsuited for cell attachment to various organic and inor-ganic materials in nature They may also play a crucialrole in the formation of the microbial string-of-pearls com-munity by initial attachment to the specific bacterial part-ners of the SM1 euryarchaeon in its environment(Rudolph et al 2001 2004 Moissl et al 2003)Because of the highly specific archaealbacterial partner-ship in the pearls one could even suppose that in thefirst step of recognition the hami mediate cellcell com-munication between members of the two prokaryoticdomains

Nanostructured surfaces with dimensions of a fewnanometres exhibit unique physical and chemical proper-ties that can be utilized for many important technologicalapplications (Roukes 2002) The most complex functionalnanoscale structures are built efficiently from biomole-cules in biological systems especially from nucleic acidspolysaccharides and proteins Microorganisms in particu-lar have novel and interesting structures that could beexploited for example bacterial spore coats and the lat-tice-type crystalline arrays of bacterial S-layers (Sleytr andMessner 1983 Ricca and Cutting 2003) In this context

the discovery of the hami could open new windows in theemerging field of nanobiotechnology

Experimental procedures

In situ growth harvesting and specific separation of SM1 euryarchaeal cells

SM1 euryarchaeal cells were grown and harvested asdescribed (Moissl et al 2003) with the following modificationinstead of using Percoll (Moissl et al 2003) which is unsuit-able for further biochemical and electron microscopical stud-ies the SM1 euryarchaeal cells were specifically enriched bydifferential centrifugation at 20 000 g (10infinC 10 min) Thisstep resulted in a brownish cell pellet (consisting almostexclusively of bacteria) and a whitish cell cloud just aboveThis cloud was selectively removed by a pipette and con-sisted of up to 98 SM1 euryarchaeal cells the remainingorganisms were bacteria

Electron microscopy image processing and tomographical reconstruction

SM1 euryarchaeal cells or protein solutions were depositedon a carbon-coated copper grid and platinum shadowed asdescribed (Moissl et al 2003) or negatively stained with 2uranyl acetate pH 45 Samples were examined using aCM12 transmission electron microscope (Philips) operated at120 keV All images were digitally recorded using a slow-scanCCD camera that was connected to a PC running TVIPSsoftware (TVIPS GmbH) The pixel size on the specimen levelwas 056 nm

Noise reduction via Fourier filtering of images from nega-tively stained hami was performed in two variants For bothprocedures the hami were extracted from electron micro-graphs suitably boxed The images of the hami werestretched to give a straight longitudinal axis using the SEM-PER image processing system (Saxton 1996) Fourier trans-forms of hami images including the prickles were thresholdedfor the most prominent reflections according to the respectivepower spectrum The transforms of filaments without theprickles were filtered for the apparent layer lines indicated inthe power spectra in addition

For cryo-electron tomography a 5 ml droplet of an SM1euryarchaeal cell suspension in original marsh water wasapplied on a lacy carbon grid coated with 5 nm colloidal goldparticles After blotting the sample was vitrified by plungefreezing in liquid ethane (Dubochet et al 1988) Data collec-tion was performed at liquid nitrogen temperature using aPhilips CM300 transmission electron microscope equippedwith a field emission gun and a Gatan imaging filter operatedat 300 keV extraction voltage

The tilt series were recorded with a tilt range from -60infin toabout +67infin and an 15infin increment The images were aquiredwith a magnification of 43 975yen at -12 mm defocus under lowdose conditions The pixel size on the specimen level was136 nm in all the tomograms After alignment of the singleprojections using the nanogold particles as markers thereconstruction was performed by weighted backprojectionwith the EM software (Hegerl 1996) For 3D volume render-

368 C Moissl et al

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

ing the resulting tomograms of the hami were denoised bynon-linear anisotropic diffusion (Frangakis and Hegerl 2001)The final 3D visualization was performed by means of theAMIRA software (Visual concepts GmbH)

Isolation and purification of the SM1 hami

For hami preparations and washing steps a KPH buffer withan ionic composition similar to the original marsh water wasused 07 mM NaCl 01 mM MgCl2 16 mM CaSO4 10 mMHepesNaOH pH 65

For the isolation of the SM1 hami SM1 euryarchaeal cellswere either treated by an ultraturrax for 6 min at 4infinC (PolytronPT1200 Kinematica AG) by sonification for 35 min at 4infinCand 35 kHz (Sonorex super 10P Bandelin Electronic) or bythe use of glass beads (017ndash018 mm Braun Biotech Inter-national GmbH) For the glass bead extraction one volumeof glass beads was added to the cell suspension and stirredgently for 18 h at 4infinC The SM1 cells were removed bycentrifugation (34 000 g 30 min) and the hami were col-lected from the supernatant by ultracentrifugation (62 000 g1 h) After resuspension in KPH buffer the efficiency andquality of the different hami preparations were compared byelectron microscopy

SM1 cell extract and whole-cell protein preparation

SM1 euryarchaeal cells were washed twice with KPH After-wards the cells were disrupted by a French press (FrenchPressure Cell Press Aminco) Cells debris was removed bycentrifugation at 20 000 g (4infinC 15 min) To the supernatantthree volumes of ice cold acetone were added and the pro-teins precipitated for 2 h at -20infinC After centrifugation at20 000 g (4infinC 15 min) the resulting pellet was dried in anexsiccator and used for immunoblot analyses

Denaturing SDS-PAGE analysis

Denaturing SDS-PAGE was performed using the buffer sys-tem of Laemmli (1970) at a constant voltage (200 V) in avertical minigel apparatus (mini Protean III Bio-Rad) withgels containing 10 polyacrylamide in the separating gel and3 polyacrylamide in the stacking gel Samples were boiledin sample buffer for 8 min before loading on acrylamide gelsProtein bands were visualized by Coomassie blue staining

Western blot immunoblot and antibody affinity purification

After SDS-PAGE total SM1 cell extracts and hami prepara-tions were blotted onto PVDF membranes (Immobilon F Mil-lipore) using a Trans-Blot SD transfer cell (Bio-Rad) at 15 Vfor 45 min Transfer of proteins was verified by staining themembranes with 02 Ponceau S (wv) in 3 TCA (vv)

For immunoblots the membranes were blocked for 14 h at4infinC by soaking in 5 milk powder (wv) in TBST [0242TrisHCl (wv) 08 NaCl (wv) 01 Tween 20 (vv)pH 76] The membranes were washed three times in TBST(10 min) Afterwards they were transferred into 5 milk pow-

der in TBST containing the anti-SM1 serum and incubatedfor 3 h at 20infinC After three washing steps for 10 min in TBSTthe membranes were incubated for 2 h with a 110 000 dilu-tion of peroxidase-conjugated rabbit anti-chicken IgG (inTBST with 5 powdered milk Dianova GmbH) The mem-branes were washed in TBST and the protein-bound antibod-ies were detected by shaking the membranes in a 4-chlornaphtol solution for 20 min at 20infinC The 4-chlornaphtolsolution was composed of 10 ml of 50 mM TrisHCl pH 7510 ml of 30 H2O2 and 3 mg of 4-chlornaphtol

For affinity purification of the anti-SM1 serum hami protein(120 kDa) blotted onto PVDF membranes was used Themembrane pieces of excised protein bands were collected inan Eppendorf tube containing 5 (wv) powdered milk inTBST for the blocking step (14 h 4infinC) All membrane pieceswere treated with anti-SM1 serum (11000) in TBST (with 5powdered milk) for 3 h at 20infinC and then washed three timesin TBST buffer The pieces were transferred into an Eppen-dorf tube containing 1 ml of 100 mM triethylamine (pH 115)After 15 min the solution was neutralized by adding 01 mlof 1 M TrisHCl (pH 80) The affinity-purified antibodies wereconcentrated and washed by the use of Centricon YM-30centrifugal filter devices (Millipore) according to the sup-plierrsquos instructions

Immunogold labelling for electron microscopy

SM1 euryarchaeal cells in KPH were incubated with affinity-purified antibodies for 2 h at 20infinC The cells were washedand a 1500 solution of a donkey anti-chicken colloidal gold-labelled (12 nm) IgG (Dianova) was added After incubation(2 h 20infinC) the cells were washed twice gently resuspendedin KPH applied onto carbon-coated copper grids and nega-tively stained with uranyl acetate as described

PAS staining and PNGase F treatment of glass bead-separated hami

PAS staining was performed as described (Segrest and Jack-son 1972) using Avidin as a positive control PNGase F(New England BioLabs) studies were performed as describedin the manufacturerrsquos instruction The hami were treated with1 mg ml-1 PNGase F (1 h 37infinC) and analysed by SDS-PAGE Ovalbumin was used as a positive control

Cell adhesion studies and hami stability experiments

Adhesion forces of single cells were investigated by the useof a computer-controlled inverted microscope equipped witha continuously operating neodymium-doped yttrium alumi-num garnet laser (NdYAG laser) The emission wavelengthof the laser is in the near infrared at 1064 nm the maximumoutput power 25 W (Huber et al 1995 Huber and Stetter2001) The laser can be focused to a spot size of less than1 mm in diameter by the use of a high-numerical-aperture oilimmersion objective (100yen) As a consequence of the strongintensity of the laser light optical trapping and manipulationof single cells in mm size in three dimensions is possible(lsquooptical tweezers traprsquo lsquolaser traprsquo Ashkin and Dziedzic1987 Ashkin et al 1987)

Unique structure of archaeal lsquohamirsquo 369

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

For cell adhesion studies glass slides (76 yen 26 yen 1 mmMarienfeld) were coated with polylysine polyglutamategelatine bovine serum albumine laminin fibronectine (each01 wv in KPH final concentration) or with a bind-silanesolution The solution was prepared by adding 15 ml of bind-silane to a mixture of 12 ml of ethanol and 375 ml of aceticacid (10 wv) Ten microlitres from a cell suspension ofSM1 euryarchaeal cells were spotted onto each slide surfaceAfter sedimentation of most of the cells a cover slide wasplaced on top of each droplet The glass slides were placedon the stage of the laser microscope and single adhered cellswere trapped in the laser beam After adjustment of the laserto its maximum output power we attempted to detach thetrapped cells from the solid surfaces into the liquid phase

Next we investigated the stability of isolated hami In oneseries of experiments the hami were incubated for 1 h atvarious temperatures The influence of low and high pH val-ues was studied at 20infinC after 1 h of incubation the pH ofthe samples was re-adjusted to 70 with HCl or NaOH Theeffect of the different treatments was analysed by electronmicroscopy (presence or absence of hami) Enzymatic diges-tion of hami preparations was tested by adding proteinase K(Merck KG) protease (Subtilisin A Sigma-Aldrich ChemieGmbH) pronase or trypsin (Boehringer) The suspensionswere incubated for 1 h at 37infinC (proteinase K protease pro-nase) or 25infinC (trypsin) As a positive control bovine serumalbumine was used The effect of the enzymes was investi-gated by SDS-PAGE (presence or absence of the 120 kDaprotein band)

Acknowledgements

We are indebted to the Government of Bavaria Germany fora sampling permit Financial support from the DeutscheForschungsgemeinschaft (HU 7112) is gratefullyacknowledged

References

Adler J (1966) Chemotaxis in bacteria Science 153 706ndash716

Ashkin A and Dziedzic JM (1987) Optical trapping andmanipulation of viruses and bacteria Science 235 1517ndash1520

Ashkin A Dziedzic JM and Yamane T (1987) Opticaltrapping and manipulation of single cells using infraredlaser beams Nature 330 769ndash771

Ball P (2001) Lifersquos lessons in design Nature 409 413ndash416Bardy SL Ng SYM and Jarrell KF (2003) Prokaryotic

motility structures Microbiology 149 295ndash304Baumeister W (2002) Electron tomography towards visual-

izing the molecular organization of the cytoplasm CurrOpin Struct Biol 12 679ndash684

Bayley DP and Jarrell KF (1998) Further evidence tosuggest that archaeal flagella are related to bacteria typeIV pili J Mol Evol 46 370ndash373

Berry RM (2001) Bacterial flagella flagellar motor InNature Encyclopedia of Life Sciences London NaturePublishing Group [www document] URL httpwwwelsnet [doi 101038npgels0000744]

Cohen-Krausz S and Trachtenberg S (2002) The structureof the archeabacterial flagellar filament of the extremehalophile Halobacterium salinarium R1M1 and its relationto eubacterial flagellar filaments and type IV pili J Mol Biol321 383ndash395

DeLong EF (1992) Archaea in coastal marine environ-ments Proc Natl Acad Sci USA 89 5685ndash5689

Dickinson MH (1999) Bionics biological insight intomechanical design Proc Natl Acad Sci USA 96 14208ndash14209

Doddema HJ Derksen JWM and Vogels GD (1979)Fimbriae and flagella of methanogenic bacteria FEMSMicrobiol Lett 5 135ndash138

Dubochet J Adrian M Chang JJ Homo JC LepaultJ McDowall AW and Schultz P (1988) Cryo-electronmicroscopy of vitrified specimens Q Rev Biophys 21 129ndash228

Eichler J (2004) Facing extremes archaeal surface-layer(glyco) proteins Microbiology 149 3347ndash3351

Faguy DM Bayley DP Kostyukova AS Thomas NAand Jarrell KF (1996) Isolation and characterization offlagella and flagellin protein from the thermoacidophilicarchaea Thermoplasma volcanium and Sulfolobus shiba-tae J Bacteriol 178 902ndash905

Fernaacutendez LA and Berenguer J (2000) Secretion andassembly of regular surface structures in Gram-negativebacteria FEMS Microbiol Rev 24 21ndash44

Frangakis AS and Hegerl R (2001) Noise reductionin electron tomographic reconstructions using nonlinearanisotropic diffusion J Struct Biol 135 239ndash250

Fuhrman JA McCallum K and Davis AA (1992) Novelmajor archaebacterial group from marine plankton Nature356 148ndash149

Hahn E Wild P Hermanns U Sebbel P GlockshuberR Haumlner M et al (2002) Exploring the 3D moleculararchitecture of Escherichia coli type 1 pili J Mol Biol 323845ndash857

Hegerl R (1996) The EM program package a platformfor image processing in biological electron microscopy JStruct Biol 116 30ndash34

Holmes KC Angert I Kull FJ Jahn W and SchroumlderRR (2003) Electron cryo-microscopy shows how strongbinding of myosin to actin releases nucleotide Nature 425423ndash427

Huber R and Stetter KO (2001) Discovery of hyperther-mophilic microorganisms In Methods in EnzymologyAdams MWW and Kelly RM (eds) London AcademicPress pp 11ndash24

Huber R Burggraf S Mayer T Barns SM RossnagelP and Stetter KO (1995) Isolation of a hyperthermo-philic archaeum predicted by in situ RNA analysis Nature376 57ndash58

Jarrell KF Bayley DP and Kostyukova AS (1996) Thearchaeal flagellum a unique motility structure J Bacteriol178 5057ndash5064

Jarrell KF Bayley DP Correia JD and ThomasNA (2001) Archaeal flagella In Nature Encyclopediaof Life Sciences London Nature Publishing Group[www document] URL httpwwwelsnet [doi 101038npgels0000386]

Laemmli UK (1970) Cleavage of structural proteins during

Unique structure of archaeal lsquohamirsquo

365

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Assuming that the hami consist of three protofilamentsthe cumulative length of one repetitive segment is3

yen

46

=

138 nm (or 30 units) whereas the pricklesaccount for about 3

yen

30

=

90 nm (or 20 units) ie

ordf

65of the building blocks in the filament If the protein formingthe prickles were significantly different in size we wouldexpect a second prominent protein band in SDS gels

N-glycosylation of flagella and S-layer proteins is wide-spread within the archaea (Jarrell

et al

2001 Upreti

et al

2003 Eichler 2004) Therefore the 120 kDa proteinwas tested for possible glycosylation using PAS-stainingor digestion with PNGaseF however both assays werenegative

Immunological studies

For immunological experiments we used polyclonal anti-

bodies raised against about 2

yen

10

8

SM1 euryarchaealcells (Moissl

et al

2003) By immuno-FISH we found thatthese antibodies (anti-SM1 serum) target the SM1 hami(Moissl

et al

2003) In the next step we wanted to specifythe target molecules for these antibodies in more detailImmunoblot analysis of total cell extracts and hami prep-arations showed in both cases a strong antibody reactionwith the 120 kDa protein To verify that the identified120 kDa protein is the substantial part of the hami severalSDS-PAGE gels of hami fractions were blotted and the120 kDa protein bands excised collected and used as atemplate for antibody affinity purification The antibodiesobtained showed specific reaction against the 120 kDaprotein in immunoblot studies In the next step the affinity-purified antibodies were used for immunogold labellingElectron microscopy showed that these antibodiesreacted specifically against the hami of the SM1 eur-yarchaeal cells (Fig 7) This specific antibody binding wasconfirmed independently in immuno-FISH experimentsand we conclude that the 120 kDa protein is the majorconstituent of the hami

Fig 5

Fourier filtering of a hamus in the prickle regionA Original image extracted from a micrograph of a negatively stained preparation Bar indicates 20 nmB Nois- reduced filtered image of the same hamus regionC More rigorously filtered image of the central filament illustrating the helical arrangement of structural units D Central region of a power spectrum from the hamus partly shown in A The hamus was optically isolated by a box-like mask of 650 nm in length in the original image before Fourier transformation The arrows indicate particularly strong reflections (layer lines) and the positions of characteristic spatial frequencies (figures denote the real spacings given in nm) The circle indicates the position of data at 2 nm resolution

Fig 6

SDS-PAGE of a crude cell extract of SM1 euryarchaeal cells (B) and of purified SM1 hami after glass bead extraction and centrif-ugation (C) The arrow indicates the 120 kDa hami protein already visible in the crude extract (B) The molecular weight of the protein markers is given in kDa (A)

366

C Moissl

et al

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Cell adhesion studies and hami stability experiments

In order to address the function of the hami we designedadhesion experiments of single SM1 cells (Moissl

et al

2003) using a variety of surfaces with different chemicalproperties (polylysine polyglutamate gelatine bovineserum albumine laminin fibronectine bind-silane) Allcoating materials tested mediated the strong adherenceof these archaea They were no more detachable evenwhen the maximum output power (25 W) of the lsquoopticaltweezersrsquo laser was used (Huber

et al

1995 Huber andStetter 2001)

In further adhesion studies single SM1 euryarchaealcells in suspension were optically trapped in the laserbeam and placed upon single cells which were fixed to abovine serum albumine-coated glass slide All attempts toseparate the cell pairs by the lsquooptical tweezersrsquo failedindicating that strong adhesion forces also occur betweenSM1 euryarchaeal cells

Discussion

In this study we reported on the discovery and analysisof a unique prokaryotic cell surface structure formed bythe uncultivated cold-loving non-methanogenic SM1 eur-yarchaeon in nature (Rudolph

et al

2001 Moissl

et al

2003) These archaeal cocci were entirely covered bysurface appendages of unexpected high complexity witha well-defined base-to-top organization each filamentexhibited a barbwire-like morphology and carried a tripar-tite barbed grappling hook at its distal end Because of

its complex architecture and the obvious distinctivenessfrom all the bacterial and archaeal cell appendages wehave chosen the term lsquohamusrsquo (plural lsquohamirsquo) for this newclass of cell surface structures The hami closely resembleman-made fishhooks grapples and anchors and sponta-neously suggest a function for cell anchoring or adhesionIt appears that one of the most basic life forms of themicrobial world long ago developed a tool that mankindnow uses in technology worldwide a parallel of directinterest to the fields of bionics and biomimetrics(Dickinson 1999 Ball 2001 Sarikaya

et al

2003)In contrast to the hami architecture prokaryotic

appendages like pili or flagella appear more simply in theirfilament structure They form tubes or helical fibres withoutfurther morphological differentiation (Fernaacutendez andBerenguer 2000 Jarrell

et al 2001 Hahn et al 2002)From bacterial flagella the hami also differ significantly infilament diameter with 7ndash8 nm compared with 24 nm(Yonekura et al 2003) in this aspect they are more sim-ilar to bacterial pili and to archaeal pili-like fibres withdiameters of 5ndash6 nm and 3ndash5 nm respectively (Doddemaet al 1979 Leadbetter and Breznak 1996 Mirosh-nichenko et al 1998 Fernaacutendez and Berenguer 2000)Similar to archaeal flagella and bacterial pili (Cohen-Krausz and Trachtenberg 2002) no evidence for theexistence of a central channel was found in the hamifilaments as far as this can be concluded from the tomo-graphical reconstructions It appears unlikely that the hamigrow by the bacterial flagellum mode of assembly It ismore conceivable that new subunits are added to thebase similar as shown for bacterial pili and as postulatedfor archaeal flagella (Jarrell et al 2001 Li and Mobley2001Bardy et al 2003)

However it is currently beyond our imagination how theprickle structures are synthesized or lsquoattachedrsquo to the fila-ment and by what mechanism the regular arrangement iscreated The same applies for the distal section of thehami including the hooks The extraordinary architectureof the hami suggests that the assembly process requiresdistinct coordination and control probably by a multicom-ponent system Another problem poses the stable anchor-ing of hami in the cytoplasmic membrane Besides thenecessary identification of genes and the isolation ofproteins involved the determination of the hamusrsquo basalstructure close to the membrane and inside thecytoplasm eg by cellular cryo-electron tomography(Baumeister 2002) would shed light on these intriguingquestions

The hami appear to consist of one major protein iethe 120 kDa component as revealed by gel electrophore-sis and immunological experiments This finding does notexclude the existence of protein species of the same sizeor of different proteins in low amounts And indeed thePAGE experiments showed further minor components

Fig 7 Immunogold labelling of hami extending from the surface of an SM1 euryarchaeal cell by the use of affinity-purified antibodies To avoid complete gold-covering of the hami a high dilution of the immunogold-labelled antibodies was used in this preparation Elec-tron micrograph negatively stained

Unique structure of archaeal lsquohamirsquo 367

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

However our data strongly suggest that the structuralcomponents of the hamus filament and the prickles areof the same size Whether they are structurally closelyrelated or even identical remains to be elucidated A pre-requisite for a more detailed investigation of this fascinat-ing microbial structure is knowledge of the genome andthe experimental availability of this currently uncultivatedeuryarchaeon as a laboratory culture

In a first glance the structure of the hami appears tobe distantly reminiscent of actin filaments (F-actin) dec-orated with myosin S1 heads (Milligan et al 1990Holmes et al 2003) The quantitative data howevershow the clear differences between both biopolymers (i)the diameter of the filament with 9ndash10 nm for actin and7 nm for the hami (ii) the number of strands two-strandedhelical for actin three-stranded for the hami (iii) the dis-tance of the sites where the arms are anchored to oremanate from the filament (about 55 nm for F-actin46 nm for the hami) and (iv) their molecular composition(two molecules actin plus myosin whereas only one sub-unit constitutes the hami presumably)

A further remarkable feature of the hami is the stabilityof the three-dimensional (3D) structure in a very broadpH and temperature range Amazingly they are even sta-ble at a temperature of 70infinC although they have beensynthesized at and may have been adapted to 10infinC thenatural growth temperature of the SM1 euryarchaeonThe discovery of the hami raises interesting questionsabout possible ecological functions Our attachmentexperiments showed that the hami mediate strong adhe-sion of single cells to each other and to surfaces of differ-ent chemical nature Therefore they are perfectly wellsuited for cell attachment to various organic and inor-ganic materials in nature They may also play a crucialrole in the formation of the microbial string-of-pearls com-munity by initial attachment to the specific bacterial part-ners of the SM1 euryarchaeon in its environment(Rudolph et al 2001 2004 Moissl et al 2003)Because of the highly specific archaealbacterial partner-ship in the pearls one could even suppose that in thefirst step of recognition the hami mediate cellcell com-munication between members of the two prokaryoticdomains

Nanostructured surfaces with dimensions of a fewnanometres exhibit unique physical and chemical proper-ties that can be utilized for many important technologicalapplications (Roukes 2002) The most complex functionalnanoscale structures are built efficiently from biomole-cules in biological systems especially from nucleic acidspolysaccharides and proteins Microorganisms in particu-lar have novel and interesting structures that could beexploited for example bacterial spore coats and the lat-tice-type crystalline arrays of bacterial S-layers (Sleytr andMessner 1983 Ricca and Cutting 2003) In this context

the discovery of the hami could open new windows in theemerging field of nanobiotechnology

Experimental procedures

In situ growth harvesting and specific separation of SM1 euryarchaeal cells

SM1 euryarchaeal cells were grown and harvested asdescribed (Moissl et al 2003) with the following modificationinstead of using Percoll (Moissl et al 2003) which is unsuit-able for further biochemical and electron microscopical stud-ies the SM1 euryarchaeal cells were specifically enriched bydifferential centrifugation at 20 000 g (10infinC 10 min) Thisstep resulted in a brownish cell pellet (consisting almostexclusively of bacteria) and a whitish cell cloud just aboveThis cloud was selectively removed by a pipette and con-sisted of up to 98 SM1 euryarchaeal cells the remainingorganisms were bacteria

Electron microscopy image processing and tomographical reconstruction

SM1 euryarchaeal cells or protein solutions were depositedon a carbon-coated copper grid and platinum shadowed asdescribed (Moissl et al 2003) or negatively stained with 2uranyl acetate pH 45 Samples were examined using aCM12 transmission electron microscope (Philips) operated at120 keV All images were digitally recorded using a slow-scanCCD camera that was connected to a PC running TVIPSsoftware (TVIPS GmbH) The pixel size on the specimen levelwas 056 nm

Noise reduction via Fourier filtering of images from nega-tively stained hami was performed in two variants For bothprocedures the hami were extracted from electron micro-graphs suitably boxed The images of the hami werestretched to give a straight longitudinal axis using the SEM-PER image processing system (Saxton 1996) Fourier trans-forms of hami images including the prickles were thresholdedfor the most prominent reflections according to the respectivepower spectrum The transforms of filaments without theprickles were filtered for the apparent layer lines indicated inthe power spectra in addition

For cryo-electron tomography a 5 ml droplet of an SM1euryarchaeal cell suspension in original marsh water wasapplied on a lacy carbon grid coated with 5 nm colloidal goldparticles After blotting the sample was vitrified by plungefreezing in liquid ethane (Dubochet et al 1988) Data collec-tion was performed at liquid nitrogen temperature using aPhilips CM300 transmission electron microscope equippedwith a field emission gun and a Gatan imaging filter operatedat 300 keV extraction voltage

The tilt series were recorded with a tilt range from -60infin toabout +67infin and an 15infin increment The images were aquiredwith a magnification of 43 975yen at -12 mm defocus under lowdose conditions The pixel size on the specimen level was136 nm in all the tomograms After alignment of the singleprojections using the nanogold particles as markers thereconstruction was performed by weighted backprojectionwith the EM software (Hegerl 1996) For 3D volume render-

368 C Moissl et al

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

ing the resulting tomograms of the hami were denoised bynon-linear anisotropic diffusion (Frangakis and Hegerl 2001)The final 3D visualization was performed by means of theAMIRA software (Visual concepts GmbH)

Isolation and purification of the SM1 hami

For hami preparations and washing steps a KPH buffer withan ionic composition similar to the original marsh water wasused 07 mM NaCl 01 mM MgCl2 16 mM CaSO4 10 mMHepesNaOH pH 65

For the isolation of the SM1 hami SM1 euryarchaeal cellswere either treated by an ultraturrax for 6 min at 4infinC (PolytronPT1200 Kinematica AG) by sonification for 35 min at 4infinCand 35 kHz (Sonorex super 10P Bandelin Electronic) or bythe use of glass beads (017ndash018 mm Braun Biotech Inter-national GmbH) For the glass bead extraction one volumeof glass beads was added to the cell suspension and stirredgently for 18 h at 4infinC The SM1 cells were removed bycentrifugation (34 000 g 30 min) and the hami were col-lected from the supernatant by ultracentrifugation (62 000 g1 h) After resuspension in KPH buffer the efficiency andquality of the different hami preparations were compared byelectron microscopy

SM1 cell extract and whole-cell protein preparation

SM1 euryarchaeal cells were washed twice with KPH After-wards the cells were disrupted by a French press (FrenchPressure Cell Press Aminco) Cells debris was removed bycentrifugation at 20 000 g (4infinC 15 min) To the supernatantthree volumes of ice cold acetone were added and the pro-teins precipitated for 2 h at -20infinC After centrifugation at20 000 g (4infinC 15 min) the resulting pellet was dried in anexsiccator and used for immunoblot analyses

Denaturing SDS-PAGE analysis

Denaturing SDS-PAGE was performed using the buffer sys-tem of Laemmli (1970) at a constant voltage (200 V) in avertical minigel apparatus (mini Protean III Bio-Rad) withgels containing 10 polyacrylamide in the separating gel and3 polyacrylamide in the stacking gel Samples were boiledin sample buffer for 8 min before loading on acrylamide gelsProtein bands were visualized by Coomassie blue staining

Western blot immunoblot and antibody affinity purification

After SDS-PAGE total SM1 cell extracts and hami prepara-tions were blotted onto PVDF membranes (Immobilon F Mil-lipore) using a Trans-Blot SD transfer cell (Bio-Rad) at 15 Vfor 45 min Transfer of proteins was verified by staining themembranes with 02 Ponceau S (wv) in 3 TCA (vv)

For immunoblots the membranes were blocked for 14 h at4infinC by soaking in 5 milk powder (wv) in TBST [0242TrisHCl (wv) 08 NaCl (wv) 01 Tween 20 (vv)pH 76] The membranes were washed three times in TBST(10 min) Afterwards they were transferred into 5 milk pow-

der in TBST containing the anti-SM1 serum and incubatedfor 3 h at 20infinC After three washing steps for 10 min in TBSTthe membranes were incubated for 2 h with a 110 000 dilu-tion of peroxidase-conjugated rabbit anti-chicken IgG (inTBST with 5 powdered milk Dianova GmbH) The mem-branes were washed in TBST and the protein-bound antibod-ies were detected by shaking the membranes in a 4-chlornaphtol solution for 20 min at 20infinC The 4-chlornaphtolsolution was composed of 10 ml of 50 mM TrisHCl pH 7510 ml of 30 H2O2 and 3 mg of 4-chlornaphtol

For affinity purification of the anti-SM1 serum hami protein(120 kDa) blotted onto PVDF membranes was used Themembrane pieces of excised protein bands were collected inan Eppendorf tube containing 5 (wv) powdered milk inTBST for the blocking step (14 h 4infinC) All membrane pieceswere treated with anti-SM1 serum (11000) in TBST (with 5powdered milk) for 3 h at 20infinC and then washed three timesin TBST buffer The pieces were transferred into an Eppen-dorf tube containing 1 ml of 100 mM triethylamine (pH 115)After 15 min the solution was neutralized by adding 01 mlof 1 M TrisHCl (pH 80) The affinity-purified antibodies wereconcentrated and washed by the use of Centricon YM-30centrifugal filter devices (Millipore) according to the sup-plierrsquos instructions

Immunogold labelling for electron microscopy

SM1 euryarchaeal cells in KPH were incubated with affinity-purified antibodies for 2 h at 20infinC The cells were washedand a 1500 solution of a donkey anti-chicken colloidal gold-labelled (12 nm) IgG (Dianova) was added After incubation(2 h 20infinC) the cells were washed twice gently resuspendedin KPH applied onto carbon-coated copper grids and nega-tively stained with uranyl acetate as described

PAS staining and PNGase F treatment of glass bead-separated hami

PAS staining was performed as described (Segrest and Jack-son 1972) using Avidin as a positive control PNGase F(New England BioLabs) studies were performed as describedin the manufacturerrsquos instruction The hami were treated with1 mg ml-1 PNGase F (1 h 37infinC) and analysed by SDS-PAGE Ovalbumin was used as a positive control

Cell adhesion studies and hami stability experiments

Adhesion forces of single cells were investigated by the useof a computer-controlled inverted microscope equipped witha continuously operating neodymium-doped yttrium alumi-num garnet laser (NdYAG laser) The emission wavelengthof the laser is in the near infrared at 1064 nm the maximumoutput power 25 W (Huber et al 1995 Huber and Stetter2001) The laser can be focused to a spot size of less than1 mm in diameter by the use of a high-numerical-aperture oilimmersion objective (100yen) As a consequence of the strongintensity of the laser light optical trapping and manipulationof single cells in mm size in three dimensions is possible(lsquooptical tweezers traprsquo lsquolaser traprsquo Ashkin and Dziedzic1987 Ashkin et al 1987)

Unique structure of archaeal lsquohamirsquo 369

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

For cell adhesion studies glass slides (76 yen 26 yen 1 mmMarienfeld) were coated with polylysine polyglutamategelatine bovine serum albumine laminin fibronectine (each01 wv in KPH final concentration) or with a bind-silanesolution The solution was prepared by adding 15 ml of bind-silane to a mixture of 12 ml of ethanol and 375 ml of aceticacid (10 wv) Ten microlitres from a cell suspension ofSM1 euryarchaeal cells were spotted onto each slide surfaceAfter sedimentation of most of the cells a cover slide wasplaced on top of each droplet The glass slides were placedon the stage of the laser microscope and single adhered cellswere trapped in the laser beam After adjustment of the laserto its maximum output power we attempted to detach thetrapped cells from the solid surfaces into the liquid phase

Next we investigated the stability of isolated hami In oneseries of experiments the hami were incubated for 1 h atvarious temperatures The influence of low and high pH val-ues was studied at 20infinC after 1 h of incubation the pH ofthe samples was re-adjusted to 70 with HCl or NaOH Theeffect of the different treatments was analysed by electronmicroscopy (presence or absence of hami) Enzymatic diges-tion of hami preparations was tested by adding proteinase K(Merck KG) protease (Subtilisin A Sigma-Aldrich ChemieGmbH) pronase or trypsin (Boehringer) The suspensionswere incubated for 1 h at 37infinC (proteinase K protease pro-nase) or 25infinC (trypsin) As a positive control bovine serumalbumine was used The effect of the enzymes was investi-gated by SDS-PAGE (presence or absence of the 120 kDaprotein band)

Acknowledgements

We are indebted to the Government of Bavaria Germany fora sampling permit Financial support from the DeutscheForschungsgemeinschaft (HU 7112) is gratefullyacknowledged

References

Adler J (1966) Chemotaxis in bacteria Science 153 706ndash716

Ashkin A and Dziedzic JM (1987) Optical trapping andmanipulation of viruses and bacteria Science 235 1517ndash1520

Ashkin A Dziedzic JM and Yamane T (1987) Opticaltrapping and manipulation of single cells using infraredlaser beams Nature 330 769ndash771

Ball P (2001) Lifersquos lessons in design Nature 409 413ndash416Bardy SL Ng SYM and Jarrell KF (2003) Prokaryotic

motility structures Microbiology 149 295ndash304Baumeister W (2002) Electron tomography towards visual-

izing the molecular organization of the cytoplasm CurrOpin Struct Biol 12 679ndash684

Bayley DP and Jarrell KF (1998) Further evidence tosuggest that archaeal flagella are related to bacteria typeIV pili J Mol Evol 46 370ndash373

Berry RM (2001) Bacterial flagella flagellar motor InNature Encyclopedia of Life Sciences London NaturePublishing Group [www document] URL httpwwwelsnet [doi 101038npgels0000744]

Cohen-Krausz S and Trachtenberg S (2002) The structureof the archeabacterial flagellar filament of the extremehalophile Halobacterium salinarium R1M1 and its relationto eubacterial flagellar filaments and type IV pili J Mol Biol321 383ndash395

DeLong EF (1992) Archaea in coastal marine environ-ments Proc Natl Acad Sci USA 89 5685ndash5689

Dickinson MH (1999) Bionics biological insight intomechanical design Proc Natl Acad Sci USA 96 14208ndash14209

Doddema HJ Derksen JWM and Vogels GD (1979)Fimbriae and flagella of methanogenic bacteria FEMSMicrobiol Lett 5 135ndash138

Dubochet J Adrian M Chang JJ Homo JC LepaultJ McDowall AW and Schultz P (1988) Cryo-electronmicroscopy of vitrified specimens Q Rev Biophys 21 129ndash228

Eichler J (2004) Facing extremes archaeal surface-layer(glyco) proteins Microbiology 149 3347ndash3351

Faguy DM Bayley DP Kostyukova AS Thomas NAand Jarrell KF (1996) Isolation and characterization offlagella and flagellin protein from the thermoacidophilicarchaea Thermoplasma volcanium and Sulfolobus shiba-tae J Bacteriol 178 902ndash905

Fernaacutendez LA and Berenguer J (2000) Secretion andassembly of regular surface structures in Gram-negativebacteria FEMS Microbiol Rev 24 21ndash44

Frangakis AS and Hegerl R (2001) Noise reductionin electron tomographic reconstructions using nonlinearanisotropic diffusion J Struct Biol 135 239ndash250

Fuhrman JA McCallum K and Davis AA (1992) Novelmajor archaebacterial group from marine plankton Nature356 148ndash149

Hahn E Wild P Hermanns U Sebbel P GlockshuberR Haumlner M et al (2002) Exploring the 3D moleculararchitecture of Escherichia coli type 1 pili J Mol Biol 323845ndash857

Hegerl R (1996) The EM program package a platformfor image processing in biological electron microscopy JStruct Biol 116 30ndash34

Holmes KC Angert I Kull FJ Jahn W and SchroumlderRR (2003) Electron cryo-microscopy shows how strongbinding of myosin to actin releases nucleotide Nature 425423ndash427

Huber R and Stetter KO (2001) Discovery of hyperther-mophilic microorganisms In Methods in EnzymologyAdams MWW and Kelly RM (eds) London AcademicPress pp 11ndash24

Huber R Burggraf S Mayer T Barns SM RossnagelP and Stetter KO (1995) Isolation of a hyperthermo-philic archaeum predicted by in situ RNA analysis Nature376 57ndash58

Jarrell KF Bayley DP and Kostyukova AS (1996) Thearchaeal flagellum a unique motility structure J Bacteriol178 5057ndash5064

Jarrell KF Bayley DP Correia JD and ThomasNA (2001) Archaeal flagella In Nature Encyclopediaof Life Sciences London Nature Publishing Group[www document] URL httpwwwelsnet [doi 101038npgels0000386]

Laemmli UK (1970) Cleavage of structural proteins during

366

C Moissl

et al

copy 2005 Blackwell Publishing Ltd

Molecular Microbiology

56

361ndash370

Cell adhesion studies and hami stability experiments

In order to address the function of the hami we designedadhesion experiments of single SM1 cells (Moissl

et al

2003) using a variety of surfaces with different chemicalproperties (polylysine polyglutamate gelatine bovineserum albumine laminin fibronectine bind-silane) Allcoating materials tested mediated the strong adherenceof these archaea They were no more detachable evenwhen the maximum output power (25 W) of the lsquoopticaltweezersrsquo laser was used (Huber

et al

1995 Huber andStetter 2001)

In further adhesion studies single SM1 euryarchaealcells in suspension were optically trapped in the laserbeam and placed upon single cells which were fixed to abovine serum albumine-coated glass slide All attempts toseparate the cell pairs by the lsquooptical tweezersrsquo failedindicating that strong adhesion forces also occur betweenSM1 euryarchaeal cells

Discussion

In this study we reported on the discovery and analysisof a unique prokaryotic cell surface structure formed bythe uncultivated cold-loving non-methanogenic SM1 eur-yarchaeon in nature (Rudolph

et al

2001 Moissl

et al

2003) These archaeal cocci were entirely covered bysurface appendages of unexpected high complexity witha well-defined base-to-top organization each filamentexhibited a barbwire-like morphology and carried a tripar-tite barbed grappling hook at its distal end Because of

its complex architecture and the obvious distinctivenessfrom all the bacterial and archaeal cell appendages wehave chosen the term lsquohamusrsquo (plural lsquohamirsquo) for this newclass of cell surface structures The hami closely resembleman-made fishhooks grapples and anchors and sponta-neously suggest a function for cell anchoring or adhesionIt appears that one of the most basic life forms of themicrobial world long ago developed a tool that mankindnow uses in technology worldwide a parallel of directinterest to the fields of bionics and biomimetrics(Dickinson 1999 Ball 2001 Sarikaya

et al

2003)In contrast to the hami architecture prokaryotic

appendages like pili or flagella appear more simply in theirfilament structure They form tubes or helical fibres withoutfurther morphological differentiation (Fernaacutendez andBerenguer 2000 Jarrell

et al 2001 Hahn et al 2002)From bacterial flagella the hami also differ significantly infilament diameter with 7ndash8 nm compared with 24 nm(Yonekura et al 2003) in this aspect they are more sim-ilar to bacterial pili and to archaeal pili-like fibres withdiameters of 5ndash6 nm and 3ndash5 nm respectively (Doddemaet al 1979 Leadbetter and Breznak 1996 Mirosh-nichenko et al 1998 Fernaacutendez and Berenguer 2000)Similar to archaeal flagella and bacterial pili (Cohen-Krausz and Trachtenberg 2002) no evidence for theexistence of a central channel was found in the hamifilaments as far as this can be concluded from the tomo-graphical reconstructions It appears unlikely that the hamigrow by the bacterial flagellum mode of assembly It ismore conceivable that new subunits are added to thebase similar as shown for bacterial pili and as postulatedfor archaeal flagella (Jarrell et al 2001 Li and Mobley2001Bardy et al 2003)

However it is currently beyond our imagination how theprickle structures are synthesized or lsquoattachedrsquo to the fila-ment and by what mechanism the regular arrangement iscreated The same applies for the distal section of thehami including the hooks The extraordinary architectureof the hami suggests that the assembly process requiresdistinct coordination and control probably by a multicom-ponent system Another problem poses the stable anchor-ing of hami in the cytoplasmic membrane Besides thenecessary identification of genes and the isolation ofproteins involved the determination of the hamusrsquo basalstructure close to the membrane and inside thecytoplasm eg by cellular cryo-electron tomography(Baumeister 2002) would shed light on these intriguingquestions

The hami appear to consist of one major protein iethe 120 kDa component as revealed by gel electrophore-sis and immunological experiments This finding does notexclude the existence of protein species of the same sizeor of different proteins in low amounts And indeed thePAGE experiments showed further minor components

Fig 7 Immunogold labelling of hami extending from the surface of an SM1 euryarchaeal cell by the use of affinity-purified antibodies To avoid complete gold-covering of the hami a high dilution of the immunogold-labelled antibodies was used in this preparation Elec-tron micrograph negatively stained

Unique structure of archaeal lsquohamirsquo 367

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

However our data strongly suggest that the structuralcomponents of the hamus filament and the prickles areof the same size Whether they are structurally closelyrelated or even identical remains to be elucidated A pre-requisite for a more detailed investigation of this fascinat-ing microbial structure is knowledge of the genome andthe experimental availability of this currently uncultivatedeuryarchaeon as a laboratory culture

In a first glance the structure of the hami appears tobe distantly reminiscent of actin filaments (F-actin) dec-orated with myosin S1 heads (Milligan et al 1990Holmes et al 2003) The quantitative data howevershow the clear differences between both biopolymers (i)the diameter of the filament with 9ndash10 nm for actin and7 nm for the hami (ii) the number of strands two-strandedhelical for actin three-stranded for the hami (iii) the dis-tance of the sites where the arms are anchored to oremanate from the filament (about 55 nm for F-actin46 nm for the hami) and (iv) their molecular composition(two molecules actin plus myosin whereas only one sub-unit constitutes the hami presumably)

A further remarkable feature of the hami is the stabilityof the three-dimensional (3D) structure in a very broadpH and temperature range Amazingly they are even sta-ble at a temperature of 70infinC although they have beensynthesized at and may have been adapted to 10infinC thenatural growth temperature of the SM1 euryarchaeonThe discovery of the hami raises interesting questionsabout possible ecological functions Our attachmentexperiments showed that the hami mediate strong adhe-sion of single cells to each other and to surfaces of differ-ent chemical nature Therefore they are perfectly wellsuited for cell attachment to various organic and inor-ganic materials in nature They may also play a crucialrole in the formation of the microbial string-of-pearls com-munity by initial attachment to the specific bacterial part-ners of the SM1 euryarchaeon in its environment(Rudolph et al 2001 2004 Moissl et al 2003)Because of the highly specific archaealbacterial partner-ship in the pearls one could even suppose that in thefirst step of recognition the hami mediate cellcell com-munication between members of the two prokaryoticdomains

Nanostructured surfaces with dimensions of a fewnanometres exhibit unique physical and chemical proper-ties that can be utilized for many important technologicalapplications (Roukes 2002) The most complex functionalnanoscale structures are built efficiently from biomole-cules in biological systems especially from nucleic acidspolysaccharides and proteins Microorganisms in particu-lar have novel and interesting structures that could beexploited for example bacterial spore coats and the lat-tice-type crystalline arrays of bacterial S-layers (Sleytr andMessner 1983 Ricca and Cutting 2003) In this context

the discovery of the hami could open new windows in theemerging field of nanobiotechnology

Experimental procedures

In situ growth harvesting and specific separation of SM1 euryarchaeal cells

SM1 euryarchaeal cells were grown and harvested asdescribed (Moissl et al 2003) with the following modificationinstead of using Percoll (Moissl et al 2003) which is unsuit-able for further biochemical and electron microscopical stud-ies the SM1 euryarchaeal cells were specifically enriched bydifferential centrifugation at 20 000 g (10infinC 10 min) Thisstep resulted in a brownish cell pellet (consisting almostexclusively of bacteria) and a whitish cell cloud just aboveThis cloud was selectively removed by a pipette and con-sisted of up to 98 SM1 euryarchaeal cells the remainingorganisms were bacteria

Electron microscopy image processing and tomographical reconstruction

SM1 euryarchaeal cells or protein solutions were depositedon a carbon-coated copper grid and platinum shadowed asdescribed (Moissl et al 2003) or negatively stained with 2uranyl acetate pH 45 Samples were examined using aCM12 transmission electron microscope (Philips) operated at120 keV All images were digitally recorded using a slow-scanCCD camera that was connected to a PC running TVIPSsoftware (TVIPS GmbH) The pixel size on the specimen levelwas 056 nm

Noise reduction via Fourier filtering of images from nega-tively stained hami was performed in two variants For bothprocedures the hami were extracted from electron micro-graphs suitably boxed The images of the hami werestretched to give a straight longitudinal axis using the SEM-PER image processing system (Saxton 1996) Fourier trans-forms of hami images including the prickles were thresholdedfor the most prominent reflections according to the respectivepower spectrum The transforms of filaments without theprickles were filtered for the apparent layer lines indicated inthe power spectra in addition

For cryo-electron tomography a 5 ml droplet of an SM1euryarchaeal cell suspension in original marsh water wasapplied on a lacy carbon grid coated with 5 nm colloidal goldparticles After blotting the sample was vitrified by plungefreezing in liquid ethane (Dubochet et al 1988) Data collec-tion was performed at liquid nitrogen temperature using aPhilips CM300 transmission electron microscope equippedwith a field emission gun and a Gatan imaging filter operatedat 300 keV extraction voltage

The tilt series were recorded with a tilt range from -60infin toabout +67infin and an 15infin increment The images were aquiredwith a magnification of 43 975yen at -12 mm defocus under lowdose conditions The pixel size on the specimen level was136 nm in all the tomograms After alignment of the singleprojections using the nanogold particles as markers thereconstruction was performed by weighted backprojectionwith the EM software (Hegerl 1996) For 3D volume render-

368 C Moissl et al

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

ing the resulting tomograms of the hami were denoised bynon-linear anisotropic diffusion (Frangakis and Hegerl 2001)The final 3D visualization was performed by means of theAMIRA software (Visual concepts GmbH)

Isolation and purification of the SM1 hami

For hami preparations and washing steps a KPH buffer withan ionic composition similar to the original marsh water wasused 07 mM NaCl 01 mM MgCl2 16 mM CaSO4 10 mMHepesNaOH pH 65

For the isolation of the SM1 hami SM1 euryarchaeal cellswere either treated by an ultraturrax for 6 min at 4infinC (PolytronPT1200 Kinematica AG) by sonification for 35 min at 4infinCand 35 kHz (Sonorex super 10P Bandelin Electronic) or bythe use of glass beads (017ndash018 mm Braun Biotech Inter-national GmbH) For the glass bead extraction one volumeof glass beads was added to the cell suspension and stirredgently for 18 h at 4infinC The SM1 cells were removed bycentrifugation (34 000 g 30 min) and the hami were col-lected from the supernatant by ultracentrifugation (62 000 g1 h) After resuspension in KPH buffer the efficiency andquality of the different hami preparations were compared byelectron microscopy

SM1 cell extract and whole-cell protein preparation

SM1 euryarchaeal cells were washed twice with KPH After-wards the cells were disrupted by a French press (FrenchPressure Cell Press Aminco) Cells debris was removed bycentrifugation at 20 000 g (4infinC 15 min) To the supernatantthree volumes of ice cold acetone were added and the pro-teins precipitated for 2 h at -20infinC After centrifugation at20 000 g (4infinC 15 min) the resulting pellet was dried in anexsiccator and used for immunoblot analyses

Denaturing SDS-PAGE analysis

Denaturing SDS-PAGE was performed using the buffer sys-tem of Laemmli (1970) at a constant voltage (200 V) in avertical minigel apparatus (mini Protean III Bio-Rad) withgels containing 10 polyacrylamide in the separating gel and3 polyacrylamide in the stacking gel Samples were boiledin sample buffer for 8 min before loading on acrylamide gelsProtein bands were visualized by Coomassie blue staining

Western blot immunoblot and antibody affinity purification

After SDS-PAGE total SM1 cell extracts and hami prepara-tions were blotted onto PVDF membranes (Immobilon F Mil-lipore) using a Trans-Blot SD transfer cell (Bio-Rad) at 15 Vfor 45 min Transfer of proteins was verified by staining themembranes with 02 Ponceau S (wv) in 3 TCA (vv)

For immunoblots the membranes were blocked for 14 h at4infinC by soaking in 5 milk powder (wv) in TBST [0242TrisHCl (wv) 08 NaCl (wv) 01 Tween 20 (vv)pH 76] The membranes were washed three times in TBST(10 min) Afterwards they were transferred into 5 milk pow-

der in TBST containing the anti-SM1 serum and incubatedfor 3 h at 20infinC After three washing steps for 10 min in TBSTthe membranes were incubated for 2 h with a 110 000 dilu-tion of peroxidase-conjugated rabbit anti-chicken IgG (inTBST with 5 powdered milk Dianova GmbH) The mem-branes were washed in TBST and the protein-bound antibod-ies were detected by shaking the membranes in a 4-chlornaphtol solution for 20 min at 20infinC The 4-chlornaphtolsolution was composed of 10 ml of 50 mM TrisHCl pH 7510 ml of 30 H2O2 and 3 mg of 4-chlornaphtol

For affinity purification of the anti-SM1 serum hami protein(120 kDa) blotted onto PVDF membranes was used Themembrane pieces of excised protein bands were collected inan Eppendorf tube containing 5 (wv) powdered milk inTBST for the blocking step (14 h 4infinC) All membrane pieceswere treated with anti-SM1 serum (11000) in TBST (with 5powdered milk) for 3 h at 20infinC and then washed three timesin TBST buffer The pieces were transferred into an Eppen-dorf tube containing 1 ml of 100 mM triethylamine (pH 115)After 15 min the solution was neutralized by adding 01 mlof 1 M TrisHCl (pH 80) The affinity-purified antibodies wereconcentrated and washed by the use of Centricon YM-30centrifugal filter devices (Millipore) according to the sup-plierrsquos instructions

Immunogold labelling for electron microscopy

SM1 euryarchaeal cells in KPH were incubated with affinity-purified antibodies for 2 h at 20infinC The cells were washedand a 1500 solution of a donkey anti-chicken colloidal gold-labelled (12 nm) IgG (Dianova) was added After incubation(2 h 20infinC) the cells were washed twice gently resuspendedin KPH applied onto carbon-coated copper grids and nega-tively stained with uranyl acetate as described

PAS staining and PNGase F treatment of glass bead-separated hami

PAS staining was performed as described (Segrest and Jack-son 1972) using Avidin as a positive control PNGase F(New England BioLabs) studies were performed as describedin the manufacturerrsquos instruction The hami were treated with1 mg ml-1 PNGase F (1 h 37infinC) and analysed by SDS-PAGE Ovalbumin was used as a positive control

Cell adhesion studies and hami stability experiments

Adhesion forces of single cells were investigated by the useof a computer-controlled inverted microscope equipped witha continuously operating neodymium-doped yttrium alumi-num garnet laser (NdYAG laser) The emission wavelengthof the laser is in the near infrared at 1064 nm the maximumoutput power 25 W (Huber et al 1995 Huber and Stetter2001) The laser can be focused to a spot size of less than1 mm in diameter by the use of a high-numerical-aperture oilimmersion objective (100yen) As a consequence of the strongintensity of the laser light optical trapping and manipulationof single cells in mm size in three dimensions is possible(lsquooptical tweezers traprsquo lsquolaser traprsquo Ashkin and Dziedzic1987 Ashkin et al 1987)

Unique structure of archaeal lsquohamirsquo 369

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

For cell adhesion studies glass slides (76 yen 26 yen 1 mmMarienfeld) were coated with polylysine polyglutamategelatine bovine serum albumine laminin fibronectine (each01 wv in KPH final concentration) or with a bind-silanesolution The solution was prepared by adding 15 ml of bind-silane to a mixture of 12 ml of ethanol and 375 ml of aceticacid (10 wv) Ten microlitres from a cell suspension ofSM1 euryarchaeal cells were spotted onto each slide surfaceAfter sedimentation of most of the cells a cover slide wasplaced on top of each droplet The glass slides were placedon the stage of the laser microscope and single adhered cellswere trapped in the laser beam After adjustment of the laserto its maximum output power we attempted to detach thetrapped cells from the solid surfaces into the liquid phase

Next we investigated the stability of isolated hami In oneseries of experiments the hami were incubated for 1 h atvarious temperatures The influence of low and high pH val-ues was studied at 20infinC after 1 h of incubation the pH ofthe samples was re-adjusted to 70 with HCl or NaOH Theeffect of the different treatments was analysed by electronmicroscopy (presence or absence of hami) Enzymatic diges-tion of hami preparations was tested by adding proteinase K(Merck KG) protease (Subtilisin A Sigma-Aldrich ChemieGmbH) pronase or trypsin (Boehringer) The suspensionswere incubated for 1 h at 37infinC (proteinase K protease pro-nase) or 25infinC (trypsin) As a positive control bovine serumalbumine was used The effect of the enzymes was investi-gated by SDS-PAGE (presence or absence of the 120 kDaprotein band)

Acknowledgements

We are indebted to the Government of Bavaria Germany fora sampling permit Financial support from the DeutscheForschungsgemeinschaft (HU 7112) is gratefullyacknowledged

References

Adler J (1966) Chemotaxis in bacteria Science 153 706ndash716

Ashkin A and Dziedzic JM (1987) Optical trapping andmanipulation of viruses and bacteria Science 235 1517ndash1520

Ashkin A Dziedzic JM and Yamane T (1987) Opticaltrapping and manipulation of single cells using infraredlaser beams Nature 330 769ndash771

Ball P (2001) Lifersquos lessons in design Nature 409 413ndash416Bardy SL Ng SYM and Jarrell KF (2003) Prokaryotic

motility structures Microbiology 149 295ndash304Baumeister W (2002) Electron tomography towards visual-

izing the molecular organization of the cytoplasm CurrOpin Struct Biol 12 679ndash684

Bayley DP and Jarrell KF (1998) Further evidence tosuggest that archaeal flagella are related to bacteria typeIV pili J Mol Evol 46 370ndash373

Berry RM (2001) Bacterial flagella flagellar motor InNature Encyclopedia of Life Sciences London NaturePublishing Group [www document] URL httpwwwelsnet [doi 101038npgels0000744]

Cohen-Krausz S and Trachtenberg S (2002) The structureof the archeabacterial flagellar filament of the extremehalophile Halobacterium salinarium R1M1 and its relationto eubacterial flagellar filaments and type IV pili J Mol Biol321 383ndash395

DeLong EF (1992) Archaea in coastal marine environ-ments Proc Natl Acad Sci USA 89 5685ndash5689

Dickinson MH (1999) Bionics biological insight intomechanical design Proc Natl Acad Sci USA 96 14208ndash14209

Doddema HJ Derksen JWM and Vogels GD (1979)Fimbriae and flagella of methanogenic bacteria FEMSMicrobiol Lett 5 135ndash138

Dubochet J Adrian M Chang JJ Homo JC LepaultJ McDowall AW and Schultz P (1988) Cryo-electronmicroscopy of vitrified specimens Q Rev Biophys 21 129ndash228

Eichler J (2004) Facing extremes archaeal surface-layer(glyco) proteins Microbiology 149 3347ndash3351

Faguy DM Bayley DP Kostyukova AS Thomas NAand Jarrell KF (1996) Isolation and characterization offlagella and flagellin protein from the thermoacidophilicarchaea Thermoplasma volcanium and Sulfolobus shiba-tae J Bacteriol 178 902ndash905

Fernaacutendez LA and Berenguer J (2000) Secretion andassembly of regular surface structures in Gram-negativebacteria FEMS Microbiol Rev 24 21ndash44

Frangakis AS and Hegerl R (2001) Noise reductionin electron tomographic reconstructions using nonlinearanisotropic diffusion J Struct Biol 135 239ndash250

Fuhrman JA McCallum K and Davis AA (1992) Novelmajor archaebacterial group from marine plankton Nature356 148ndash149

Hahn E Wild P Hermanns U Sebbel P GlockshuberR Haumlner M et al (2002) Exploring the 3D moleculararchitecture of Escherichia coli type 1 pili J Mol Biol 323845ndash857

Hegerl R (1996) The EM program package a platformfor image processing in biological electron microscopy JStruct Biol 116 30ndash34

Holmes KC Angert I Kull FJ Jahn W and SchroumlderRR (2003) Electron cryo-microscopy shows how strongbinding of myosin to actin releases nucleotide Nature 425423ndash427

Huber R and Stetter KO (2001) Discovery of hyperther-mophilic microorganisms In Methods in EnzymologyAdams MWW and Kelly RM (eds) London AcademicPress pp 11ndash24

Huber R Burggraf S Mayer T Barns SM RossnagelP and Stetter KO (1995) Isolation of a hyperthermo-philic archaeum predicted by in situ RNA analysis Nature376 57ndash58

Jarrell KF Bayley DP and Kostyukova AS (1996) Thearchaeal flagellum a unique motility structure J Bacteriol178 5057ndash5064

Jarrell KF Bayley DP Correia JD and ThomasNA (2001) Archaeal flagella In Nature Encyclopediaof Life Sciences London Nature Publishing Group[www document] URL httpwwwelsnet [doi 101038npgels0000386]

Laemmli UK (1970) Cleavage of structural proteins during

Unique structure of archaeal lsquohamirsquo 367

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

However our data strongly suggest that the structuralcomponents of the hamus filament and the prickles areof the same size Whether they are structurally closelyrelated or even identical remains to be elucidated A pre-requisite for a more detailed investigation of this fascinat-ing microbial structure is knowledge of the genome andthe experimental availability of this currently uncultivatedeuryarchaeon as a laboratory culture

In a first glance the structure of the hami appears tobe distantly reminiscent of actin filaments (F-actin) dec-orated with myosin S1 heads (Milligan et al 1990Holmes et al 2003) The quantitative data howevershow the clear differences between both biopolymers (i)the diameter of the filament with 9ndash10 nm for actin and7 nm for the hami (ii) the number of strands two-strandedhelical for actin three-stranded for the hami (iii) the dis-tance of the sites where the arms are anchored to oremanate from the filament (about 55 nm for F-actin46 nm for the hami) and (iv) their molecular composition(two molecules actin plus myosin whereas only one sub-unit constitutes the hami presumably)

A further remarkable feature of the hami is the stabilityof the three-dimensional (3D) structure in a very broadpH and temperature range Amazingly they are even sta-ble at a temperature of 70infinC although they have beensynthesized at and may have been adapted to 10infinC thenatural growth temperature of the SM1 euryarchaeonThe discovery of the hami raises interesting questionsabout possible ecological functions Our attachmentexperiments showed that the hami mediate strong adhe-sion of single cells to each other and to surfaces of differ-ent chemical nature Therefore they are perfectly wellsuited for cell attachment to various organic and inor-ganic materials in nature They may also play a crucialrole in the formation of the microbial string-of-pearls com-munity by initial attachment to the specific bacterial part-ners of the SM1 euryarchaeon in its environment(Rudolph et al 2001 2004 Moissl et al 2003)Because of the highly specific archaealbacterial partner-ship in the pearls one could even suppose that in thefirst step of recognition the hami mediate cellcell com-munication between members of the two prokaryoticdomains

Nanostructured surfaces with dimensions of a fewnanometres exhibit unique physical and chemical proper-ties that can be utilized for many important technologicalapplications (Roukes 2002) The most complex functionalnanoscale structures are built efficiently from biomole-cules in biological systems especially from nucleic acidspolysaccharides and proteins Microorganisms in particu-lar have novel and interesting structures that could beexploited for example bacterial spore coats and the lat-tice-type crystalline arrays of bacterial S-layers (Sleytr andMessner 1983 Ricca and Cutting 2003) In this context

the discovery of the hami could open new windows in theemerging field of nanobiotechnology

Experimental procedures

In situ growth harvesting and specific separation of SM1 euryarchaeal cells

SM1 euryarchaeal cells were grown and harvested asdescribed (Moissl et al 2003) with the following modificationinstead of using Percoll (Moissl et al 2003) which is unsuit-able for further biochemical and electron microscopical stud-ies the SM1 euryarchaeal cells were specifically enriched bydifferential centrifugation at 20 000 g (10infinC 10 min) Thisstep resulted in a brownish cell pellet (consisting almostexclusively of bacteria) and a whitish cell cloud just aboveThis cloud was selectively removed by a pipette and con-sisted of up to 98 SM1 euryarchaeal cells the remainingorganisms were bacteria

Electron microscopy image processing and tomographical reconstruction

SM1 euryarchaeal cells or protein solutions were depositedon a carbon-coated copper grid and platinum shadowed asdescribed (Moissl et al 2003) or negatively stained with 2uranyl acetate pH 45 Samples were examined using aCM12 transmission electron microscope (Philips) operated at120 keV All images were digitally recorded using a slow-scanCCD camera that was connected to a PC running TVIPSsoftware (TVIPS GmbH) The pixel size on the specimen levelwas 056 nm

Noise reduction via Fourier filtering of images from nega-tively stained hami was performed in two variants For bothprocedures the hami were extracted from electron micro-graphs suitably boxed The images of the hami werestretched to give a straight longitudinal axis using the SEM-PER image processing system (Saxton 1996) Fourier trans-forms of hami images including the prickles were thresholdedfor the most prominent reflections according to the respectivepower spectrum The transforms of filaments without theprickles were filtered for the apparent layer lines indicated inthe power spectra in addition

For cryo-electron tomography a 5 ml droplet of an SM1euryarchaeal cell suspension in original marsh water wasapplied on a lacy carbon grid coated with 5 nm colloidal goldparticles After blotting the sample was vitrified by plungefreezing in liquid ethane (Dubochet et al 1988) Data collec-tion was performed at liquid nitrogen temperature using aPhilips CM300 transmission electron microscope equippedwith a field emission gun and a Gatan imaging filter operatedat 300 keV extraction voltage

The tilt series were recorded with a tilt range from -60infin toabout +67infin and an 15infin increment The images were aquiredwith a magnification of 43 975yen at -12 mm defocus under lowdose conditions The pixel size on the specimen level was136 nm in all the tomograms After alignment of the singleprojections using the nanogold particles as markers thereconstruction was performed by weighted backprojectionwith the EM software (Hegerl 1996) For 3D volume render-

368 C Moissl et al

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

ing the resulting tomograms of the hami were denoised bynon-linear anisotropic diffusion (Frangakis and Hegerl 2001)The final 3D visualization was performed by means of theAMIRA software (Visual concepts GmbH)

Isolation and purification of the SM1 hami

For hami preparations and washing steps a KPH buffer withan ionic composition similar to the original marsh water wasused 07 mM NaCl 01 mM MgCl2 16 mM CaSO4 10 mMHepesNaOH pH 65

For the isolation of the SM1 hami SM1 euryarchaeal cellswere either treated by an ultraturrax for 6 min at 4infinC (PolytronPT1200 Kinematica AG) by sonification for 35 min at 4infinCand 35 kHz (Sonorex super 10P Bandelin Electronic) or bythe use of glass beads (017ndash018 mm Braun Biotech Inter-national GmbH) For the glass bead extraction one volumeof glass beads was added to the cell suspension and stirredgently for 18 h at 4infinC The SM1 cells were removed bycentrifugation (34 000 g 30 min) and the hami were col-lected from the supernatant by ultracentrifugation (62 000 g1 h) After resuspension in KPH buffer the efficiency andquality of the different hami preparations were compared byelectron microscopy

SM1 cell extract and whole-cell protein preparation

SM1 euryarchaeal cells were washed twice with KPH After-wards the cells were disrupted by a French press (FrenchPressure Cell Press Aminco) Cells debris was removed bycentrifugation at 20 000 g (4infinC 15 min) To the supernatantthree volumes of ice cold acetone were added and the pro-teins precipitated for 2 h at -20infinC After centrifugation at20 000 g (4infinC 15 min) the resulting pellet was dried in anexsiccator and used for immunoblot analyses

Denaturing SDS-PAGE analysis

Denaturing SDS-PAGE was performed using the buffer sys-tem of Laemmli (1970) at a constant voltage (200 V) in avertical minigel apparatus (mini Protean III Bio-Rad) withgels containing 10 polyacrylamide in the separating gel and3 polyacrylamide in the stacking gel Samples were boiledin sample buffer for 8 min before loading on acrylamide gelsProtein bands were visualized by Coomassie blue staining

Western blot immunoblot and antibody affinity purification

After SDS-PAGE total SM1 cell extracts and hami prepara-tions were blotted onto PVDF membranes (Immobilon F Mil-lipore) using a Trans-Blot SD transfer cell (Bio-Rad) at 15 Vfor 45 min Transfer of proteins was verified by staining themembranes with 02 Ponceau S (wv) in 3 TCA (vv)

For immunoblots the membranes were blocked for 14 h at4infinC by soaking in 5 milk powder (wv) in TBST [0242TrisHCl (wv) 08 NaCl (wv) 01 Tween 20 (vv)pH 76] The membranes were washed three times in TBST(10 min) Afterwards they were transferred into 5 milk pow-

der in TBST containing the anti-SM1 serum and incubatedfor 3 h at 20infinC After three washing steps for 10 min in TBSTthe membranes were incubated for 2 h with a 110 000 dilu-tion of peroxidase-conjugated rabbit anti-chicken IgG (inTBST with 5 powdered milk Dianova GmbH) The mem-branes were washed in TBST and the protein-bound antibod-ies were detected by shaking the membranes in a 4-chlornaphtol solution for 20 min at 20infinC The 4-chlornaphtolsolution was composed of 10 ml of 50 mM TrisHCl pH 7510 ml of 30 H2O2 and 3 mg of 4-chlornaphtol

For affinity purification of the anti-SM1 serum hami protein(120 kDa) blotted onto PVDF membranes was used Themembrane pieces of excised protein bands were collected inan Eppendorf tube containing 5 (wv) powdered milk inTBST for the blocking step (14 h 4infinC) All membrane pieceswere treated with anti-SM1 serum (11000) in TBST (with 5powdered milk) for 3 h at 20infinC and then washed three timesin TBST buffer The pieces were transferred into an Eppen-dorf tube containing 1 ml of 100 mM triethylamine (pH 115)After 15 min the solution was neutralized by adding 01 mlof 1 M TrisHCl (pH 80) The affinity-purified antibodies wereconcentrated and washed by the use of Centricon YM-30centrifugal filter devices (Millipore) according to the sup-plierrsquos instructions

Immunogold labelling for electron microscopy

SM1 euryarchaeal cells in KPH were incubated with affinity-purified antibodies for 2 h at 20infinC The cells were washedand a 1500 solution of a donkey anti-chicken colloidal gold-labelled (12 nm) IgG (Dianova) was added After incubation(2 h 20infinC) the cells were washed twice gently resuspendedin KPH applied onto carbon-coated copper grids and nega-tively stained with uranyl acetate as described

PAS staining and PNGase F treatment of glass bead-separated hami

PAS staining was performed as described (Segrest and Jack-son 1972) using Avidin as a positive control PNGase F(New England BioLabs) studies were performed as describedin the manufacturerrsquos instruction The hami were treated with1 mg ml-1 PNGase F (1 h 37infinC) and analysed by SDS-PAGE Ovalbumin was used as a positive control

Cell adhesion studies and hami stability experiments

Adhesion forces of single cells were investigated by the useof a computer-controlled inverted microscope equipped witha continuously operating neodymium-doped yttrium alumi-num garnet laser (NdYAG laser) The emission wavelengthof the laser is in the near infrared at 1064 nm the maximumoutput power 25 W (Huber et al 1995 Huber and Stetter2001) The laser can be focused to a spot size of less than1 mm in diameter by the use of a high-numerical-aperture oilimmersion objective (100yen) As a consequence of the strongintensity of the laser light optical trapping and manipulationof single cells in mm size in three dimensions is possible(lsquooptical tweezers traprsquo lsquolaser traprsquo Ashkin and Dziedzic1987 Ashkin et al 1987)

Unique structure of archaeal lsquohamirsquo 369

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

For cell adhesion studies glass slides (76 yen 26 yen 1 mmMarienfeld) were coated with polylysine polyglutamategelatine bovine serum albumine laminin fibronectine (each01 wv in KPH final concentration) or with a bind-silanesolution The solution was prepared by adding 15 ml of bind-silane to a mixture of 12 ml of ethanol and 375 ml of aceticacid (10 wv) Ten microlitres from a cell suspension ofSM1 euryarchaeal cells were spotted onto each slide surfaceAfter sedimentation of most of the cells a cover slide wasplaced on top of each droplet The glass slides were placedon the stage of the laser microscope and single adhered cellswere trapped in the laser beam After adjustment of the laserto its maximum output power we attempted to detach thetrapped cells from the solid surfaces into the liquid phase

Next we investigated the stability of isolated hami In oneseries of experiments the hami were incubated for 1 h atvarious temperatures The influence of low and high pH val-ues was studied at 20infinC after 1 h of incubation the pH ofthe samples was re-adjusted to 70 with HCl or NaOH Theeffect of the different treatments was analysed by electronmicroscopy (presence or absence of hami) Enzymatic diges-tion of hami preparations was tested by adding proteinase K(Merck KG) protease (Subtilisin A Sigma-Aldrich ChemieGmbH) pronase or trypsin (Boehringer) The suspensionswere incubated for 1 h at 37infinC (proteinase K protease pro-nase) or 25infinC (trypsin) As a positive control bovine serumalbumine was used The effect of the enzymes was investi-gated by SDS-PAGE (presence or absence of the 120 kDaprotein band)

Acknowledgements

We are indebted to the Government of Bavaria Germany fora sampling permit Financial support from the DeutscheForschungsgemeinschaft (HU 7112) is gratefullyacknowledged

References

Adler J (1966) Chemotaxis in bacteria Science 153 706ndash716

Ashkin A and Dziedzic JM (1987) Optical trapping andmanipulation of viruses and bacteria Science 235 1517ndash1520

Ashkin A Dziedzic JM and Yamane T (1987) Opticaltrapping and manipulation of single cells using infraredlaser beams Nature 330 769ndash771

Ball P (2001) Lifersquos lessons in design Nature 409 413ndash416Bardy SL Ng SYM and Jarrell KF (2003) Prokaryotic

motility structures Microbiology 149 295ndash304Baumeister W (2002) Electron tomography towards visual-

izing the molecular organization of the cytoplasm CurrOpin Struct Biol 12 679ndash684

Bayley DP and Jarrell KF (1998) Further evidence tosuggest that archaeal flagella are related to bacteria typeIV pili J Mol Evol 46 370ndash373

Berry RM (2001) Bacterial flagella flagellar motor InNature Encyclopedia of Life Sciences London NaturePublishing Group [www document] URL httpwwwelsnet [doi 101038npgels0000744]

Cohen-Krausz S and Trachtenberg S (2002) The structureof the archeabacterial flagellar filament of the extremehalophile Halobacterium salinarium R1M1 and its relationto eubacterial flagellar filaments and type IV pili J Mol Biol321 383ndash395

DeLong EF (1992) Archaea in coastal marine environ-ments Proc Natl Acad Sci USA 89 5685ndash5689

Dickinson MH (1999) Bionics biological insight intomechanical design Proc Natl Acad Sci USA 96 14208ndash14209

Doddema HJ Derksen JWM and Vogels GD (1979)Fimbriae and flagella of methanogenic bacteria FEMSMicrobiol Lett 5 135ndash138

Dubochet J Adrian M Chang JJ Homo JC LepaultJ McDowall AW and Schultz P (1988) Cryo-electronmicroscopy of vitrified specimens Q Rev Biophys 21 129ndash228

Eichler J (2004) Facing extremes archaeal surface-layer(glyco) proteins Microbiology 149 3347ndash3351

Faguy DM Bayley DP Kostyukova AS Thomas NAand Jarrell KF (1996) Isolation and characterization offlagella and flagellin protein from the thermoacidophilicarchaea Thermoplasma volcanium and Sulfolobus shiba-tae J Bacteriol 178 902ndash905

Fernaacutendez LA and Berenguer J (2000) Secretion andassembly of regular surface structures in Gram-negativebacteria FEMS Microbiol Rev 24 21ndash44

Frangakis AS and Hegerl R (2001) Noise reductionin electron tomographic reconstructions using nonlinearanisotropic diffusion J Struct Biol 135 239ndash250

Fuhrman JA McCallum K and Davis AA (1992) Novelmajor archaebacterial group from marine plankton Nature356 148ndash149

Hahn E Wild P Hermanns U Sebbel P GlockshuberR Haumlner M et al (2002) Exploring the 3D moleculararchitecture of Escherichia coli type 1 pili J Mol Biol 323845ndash857

Hegerl R (1996) The EM program package a platformfor image processing in biological electron microscopy JStruct Biol 116 30ndash34

Holmes KC Angert I Kull FJ Jahn W and SchroumlderRR (2003) Electron cryo-microscopy shows how strongbinding of myosin to actin releases nucleotide Nature 425423ndash427

Huber R and Stetter KO (2001) Discovery of hyperther-mophilic microorganisms In Methods in EnzymologyAdams MWW and Kelly RM (eds) London AcademicPress pp 11ndash24

Huber R Burggraf S Mayer T Barns SM RossnagelP and Stetter KO (1995) Isolation of a hyperthermo-philic archaeum predicted by in situ RNA analysis Nature376 57ndash58

Jarrell KF Bayley DP and Kostyukova AS (1996) Thearchaeal flagellum a unique motility structure J Bacteriol178 5057ndash5064

Jarrell KF Bayley DP Correia JD and ThomasNA (2001) Archaeal flagella In Nature Encyclopediaof Life Sciences London Nature Publishing Group[www document] URL httpwwwelsnet [doi 101038npgels0000386]

Laemmli UK (1970) Cleavage of structural proteins during

368 C Moissl et al

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

ing the resulting tomograms of the hami were denoised bynon-linear anisotropic diffusion (Frangakis and Hegerl 2001)The final 3D visualization was performed by means of theAMIRA software (Visual concepts GmbH)

Isolation and purification of the SM1 hami

For hami preparations and washing steps a KPH buffer withan ionic composition similar to the original marsh water wasused 07 mM NaCl 01 mM MgCl2 16 mM CaSO4 10 mMHepesNaOH pH 65

For the isolation of the SM1 hami SM1 euryarchaeal cellswere either treated by an ultraturrax for 6 min at 4infinC (PolytronPT1200 Kinematica AG) by sonification for 35 min at 4infinCand 35 kHz (Sonorex super 10P Bandelin Electronic) or bythe use of glass beads (017ndash018 mm Braun Biotech Inter-national GmbH) For the glass bead extraction one volumeof glass beads was added to the cell suspension and stirredgently for 18 h at 4infinC The SM1 cells were removed bycentrifugation (34 000 g 30 min) and the hami were col-lected from the supernatant by ultracentrifugation (62 000 g1 h) After resuspension in KPH buffer the efficiency andquality of the different hami preparations were compared byelectron microscopy

SM1 cell extract and whole-cell protein preparation

SM1 euryarchaeal cells were washed twice with KPH After-wards the cells were disrupted by a French press (FrenchPressure Cell Press Aminco) Cells debris was removed bycentrifugation at 20 000 g (4infinC 15 min) To the supernatantthree volumes of ice cold acetone were added and the pro-teins precipitated for 2 h at -20infinC After centrifugation at20 000 g (4infinC 15 min) the resulting pellet was dried in anexsiccator and used for immunoblot analyses

Denaturing SDS-PAGE analysis

Denaturing SDS-PAGE was performed using the buffer sys-tem of Laemmli (1970) at a constant voltage (200 V) in avertical minigel apparatus (mini Protean III Bio-Rad) withgels containing 10 polyacrylamide in the separating gel and3 polyacrylamide in the stacking gel Samples were boiledin sample buffer for 8 min before loading on acrylamide gelsProtein bands were visualized by Coomassie blue staining

Western blot immunoblot and antibody affinity purification

After SDS-PAGE total SM1 cell extracts and hami prepara-tions were blotted onto PVDF membranes (Immobilon F Mil-lipore) using a Trans-Blot SD transfer cell (Bio-Rad) at 15 Vfor 45 min Transfer of proteins was verified by staining themembranes with 02 Ponceau S (wv) in 3 TCA (vv)

For immunoblots the membranes were blocked for 14 h at4infinC by soaking in 5 milk powder (wv) in TBST [0242TrisHCl (wv) 08 NaCl (wv) 01 Tween 20 (vv)pH 76] The membranes were washed three times in TBST(10 min) Afterwards they were transferred into 5 milk pow-

der in TBST containing the anti-SM1 serum and incubatedfor 3 h at 20infinC After three washing steps for 10 min in TBSTthe membranes were incubated for 2 h with a 110 000 dilu-tion of peroxidase-conjugated rabbit anti-chicken IgG (inTBST with 5 powdered milk Dianova GmbH) The mem-branes were washed in TBST and the protein-bound antibod-ies were detected by shaking the membranes in a 4-chlornaphtol solution for 20 min at 20infinC The 4-chlornaphtolsolution was composed of 10 ml of 50 mM TrisHCl pH 7510 ml of 30 H2O2 and 3 mg of 4-chlornaphtol

For affinity purification of the anti-SM1 serum hami protein(120 kDa) blotted onto PVDF membranes was used Themembrane pieces of excised protein bands were collected inan Eppendorf tube containing 5 (wv) powdered milk inTBST for the blocking step (14 h 4infinC) All membrane pieceswere treated with anti-SM1 serum (11000) in TBST (with 5powdered milk) for 3 h at 20infinC and then washed three timesin TBST buffer The pieces were transferred into an Eppen-dorf tube containing 1 ml of 100 mM triethylamine (pH 115)After 15 min the solution was neutralized by adding 01 mlof 1 M TrisHCl (pH 80) The affinity-purified antibodies wereconcentrated and washed by the use of Centricon YM-30centrifugal filter devices (Millipore) according to the sup-plierrsquos instructions

Immunogold labelling for electron microscopy

SM1 euryarchaeal cells in KPH were incubated with affinity-purified antibodies for 2 h at 20infinC The cells were washedand a 1500 solution of a donkey anti-chicken colloidal gold-labelled (12 nm) IgG (Dianova) was added After incubation(2 h 20infinC) the cells were washed twice gently resuspendedin KPH applied onto carbon-coated copper grids and nega-tively stained with uranyl acetate as described

PAS staining and PNGase F treatment of glass bead-separated hami

PAS staining was performed as described (Segrest and Jack-son 1972) using Avidin as a positive control PNGase F(New England BioLabs) studies were performed as describedin the manufacturerrsquos instruction The hami were treated with1 mg ml-1 PNGase F (1 h 37infinC) and analysed by SDS-PAGE Ovalbumin was used as a positive control

Cell adhesion studies and hami stability experiments

Adhesion forces of single cells were investigated by the useof a computer-controlled inverted microscope equipped witha continuously operating neodymium-doped yttrium alumi-num garnet laser (NdYAG laser) The emission wavelengthof the laser is in the near infrared at 1064 nm the maximumoutput power 25 W (Huber et al 1995 Huber and Stetter2001) The laser can be focused to a spot size of less than1 mm in diameter by the use of a high-numerical-aperture oilimmersion objective (100yen) As a consequence of the strongintensity of the laser light optical trapping and manipulationof single cells in mm size in three dimensions is possible(lsquooptical tweezers traprsquo lsquolaser traprsquo Ashkin and Dziedzic1987 Ashkin et al 1987)

Unique structure of archaeal lsquohamirsquo 369

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

For cell adhesion studies glass slides (76 yen 26 yen 1 mmMarienfeld) were coated with polylysine polyglutamategelatine bovine serum albumine laminin fibronectine (each01 wv in KPH final concentration) or with a bind-silanesolution The solution was prepared by adding 15 ml of bind-silane to a mixture of 12 ml of ethanol and 375 ml of aceticacid (10 wv) Ten microlitres from a cell suspension ofSM1 euryarchaeal cells were spotted onto each slide surfaceAfter sedimentation of most of the cells a cover slide wasplaced on top of each droplet The glass slides were placedon the stage of the laser microscope and single adhered cellswere trapped in the laser beam After adjustment of the laserto its maximum output power we attempted to detach thetrapped cells from the solid surfaces into the liquid phase

Next we investigated the stability of isolated hami In oneseries of experiments the hami were incubated for 1 h atvarious temperatures The influence of low and high pH val-ues was studied at 20infinC after 1 h of incubation the pH ofthe samples was re-adjusted to 70 with HCl or NaOH Theeffect of the different treatments was analysed by electronmicroscopy (presence or absence of hami) Enzymatic diges-tion of hami preparations was tested by adding proteinase K(Merck KG) protease (Subtilisin A Sigma-Aldrich ChemieGmbH) pronase or trypsin (Boehringer) The suspensionswere incubated for 1 h at 37infinC (proteinase K protease pro-nase) or 25infinC (trypsin) As a positive control bovine serumalbumine was used The effect of the enzymes was investi-gated by SDS-PAGE (presence or absence of the 120 kDaprotein band)

Acknowledgements

We are indebted to the Government of Bavaria Germany fora sampling permit Financial support from the DeutscheForschungsgemeinschaft (HU 7112) is gratefullyacknowledged

References

Adler J (1966) Chemotaxis in bacteria Science 153 706ndash716

Ashkin A and Dziedzic JM (1987) Optical trapping andmanipulation of viruses and bacteria Science 235 1517ndash1520

Ashkin A Dziedzic JM and Yamane T (1987) Opticaltrapping and manipulation of single cells using infraredlaser beams Nature 330 769ndash771

Ball P (2001) Lifersquos lessons in design Nature 409 413ndash416Bardy SL Ng SYM and Jarrell KF (2003) Prokaryotic

motility structures Microbiology 149 295ndash304Baumeister W (2002) Electron tomography towards visual-

izing the molecular organization of the cytoplasm CurrOpin Struct Biol 12 679ndash684

Bayley DP and Jarrell KF (1998) Further evidence tosuggest that archaeal flagella are related to bacteria typeIV pili J Mol Evol 46 370ndash373

Berry RM (2001) Bacterial flagella flagellar motor InNature Encyclopedia of Life Sciences London NaturePublishing Group [www document] URL httpwwwelsnet [doi 101038npgels0000744]

Cohen-Krausz S and Trachtenberg S (2002) The structureof the archeabacterial flagellar filament of the extremehalophile Halobacterium salinarium R1M1 and its relationto eubacterial flagellar filaments and type IV pili J Mol Biol321 383ndash395

DeLong EF (1992) Archaea in coastal marine environ-ments Proc Natl Acad Sci USA 89 5685ndash5689

Dickinson MH (1999) Bionics biological insight intomechanical design Proc Natl Acad Sci USA 96 14208ndash14209

Doddema HJ Derksen JWM and Vogels GD (1979)Fimbriae and flagella of methanogenic bacteria FEMSMicrobiol Lett 5 135ndash138

Dubochet J Adrian M Chang JJ Homo JC LepaultJ McDowall AW and Schultz P (1988) Cryo-electronmicroscopy of vitrified specimens Q Rev Biophys 21 129ndash228

Eichler J (2004) Facing extremes archaeal surface-layer(glyco) proteins Microbiology 149 3347ndash3351

Faguy DM Bayley DP Kostyukova AS Thomas NAand Jarrell KF (1996) Isolation and characterization offlagella and flagellin protein from the thermoacidophilicarchaea Thermoplasma volcanium and Sulfolobus shiba-tae J Bacteriol 178 902ndash905

Fernaacutendez LA and Berenguer J (2000) Secretion andassembly of regular surface structures in Gram-negativebacteria FEMS Microbiol Rev 24 21ndash44

Frangakis AS and Hegerl R (2001) Noise reductionin electron tomographic reconstructions using nonlinearanisotropic diffusion J Struct Biol 135 239ndash250

Fuhrman JA McCallum K and Davis AA (1992) Novelmajor archaebacterial group from marine plankton Nature356 148ndash149

Hahn E Wild P Hermanns U Sebbel P GlockshuberR Haumlner M et al (2002) Exploring the 3D moleculararchitecture of Escherichia coli type 1 pili J Mol Biol 323845ndash857

Hegerl R (1996) The EM program package a platformfor image processing in biological electron microscopy JStruct Biol 116 30ndash34

Holmes KC Angert I Kull FJ Jahn W and SchroumlderRR (2003) Electron cryo-microscopy shows how strongbinding of myosin to actin releases nucleotide Nature 425423ndash427

Huber R and Stetter KO (2001) Discovery of hyperther-mophilic microorganisms In Methods in EnzymologyAdams MWW and Kelly RM (eds) London AcademicPress pp 11ndash24

Huber R Burggraf S Mayer T Barns SM RossnagelP and Stetter KO (1995) Isolation of a hyperthermo-philic archaeum predicted by in situ RNA analysis Nature376 57ndash58

Jarrell KF Bayley DP and Kostyukova AS (1996) Thearchaeal flagellum a unique motility structure J Bacteriol178 5057ndash5064

Jarrell KF Bayley DP Correia JD and ThomasNA (2001) Archaeal flagella In Nature Encyclopediaof Life Sciences London Nature Publishing Group[www document] URL httpwwwelsnet [doi 101038npgels0000386]

Laemmli UK (1970) Cleavage of structural proteins during

Unique structure of archaeal lsquohamirsquo 369

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

For cell adhesion studies glass slides (76 yen 26 yen 1 mmMarienfeld) were coated with polylysine polyglutamategelatine bovine serum albumine laminin fibronectine (each01 wv in KPH final concentration) or with a bind-silanesolution The solution was prepared by adding 15 ml of bind-silane to a mixture of 12 ml of ethanol and 375 ml of aceticacid (10 wv) Ten microlitres from a cell suspension ofSM1 euryarchaeal cells were spotted onto each slide surfaceAfter sedimentation of most of the cells a cover slide wasplaced on top of each droplet The glass slides were placedon the stage of the laser microscope and single adhered cellswere trapped in the laser beam After adjustment of the laserto its maximum output power we attempted to detach thetrapped cells from the solid surfaces into the liquid phase

Next we investigated the stability of isolated hami In oneseries of experiments the hami were incubated for 1 h atvarious temperatures The influence of low and high pH val-ues was studied at 20infinC after 1 h of incubation the pH ofthe samples was re-adjusted to 70 with HCl or NaOH Theeffect of the different treatments was analysed by electronmicroscopy (presence or absence of hami) Enzymatic diges-tion of hami preparations was tested by adding proteinase K(Merck KG) protease (Subtilisin A Sigma-Aldrich ChemieGmbH) pronase or trypsin (Boehringer) The suspensionswere incubated for 1 h at 37infinC (proteinase K protease pro-nase) or 25infinC (trypsin) As a positive control bovine serumalbumine was used The effect of the enzymes was investi-gated by SDS-PAGE (presence or absence of the 120 kDaprotein band)

Acknowledgements

We are indebted to the Government of Bavaria Germany fora sampling permit Financial support from the DeutscheForschungsgemeinschaft (HU 7112) is gratefullyacknowledged

References

Adler J (1966) Chemotaxis in bacteria Science 153 706ndash716

Ashkin A and Dziedzic JM (1987) Optical trapping andmanipulation of viruses and bacteria Science 235 1517ndash1520

Ashkin A Dziedzic JM and Yamane T (1987) Opticaltrapping and manipulation of single cells using infraredlaser beams Nature 330 769ndash771

Ball P (2001) Lifersquos lessons in design Nature 409 413ndash416Bardy SL Ng SYM and Jarrell KF (2003) Prokaryotic

motility structures Microbiology 149 295ndash304Baumeister W (2002) Electron tomography towards visual-

izing the molecular organization of the cytoplasm CurrOpin Struct Biol 12 679ndash684

Bayley DP and Jarrell KF (1998) Further evidence tosuggest that archaeal flagella are related to bacteria typeIV pili J Mol Evol 46 370ndash373

Berry RM (2001) Bacterial flagella flagellar motor InNature Encyclopedia of Life Sciences London NaturePublishing Group [www document] URL httpwwwelsnet [doi 101038npgels0000744]

Cohen-Krausz S and Trachtenberg S (2002) The structureof the archeabacterial flagellar filament of the extremehalophile Halobacterium salinarium R1M1 and its relationto eubacterial flagellar filaments and type IV pili J Mol Biol321 383ndash395

DeLong EF (1992) Archaea in coastal marine environ-ments Proc Natl Acad Sci USA 89 5685ndash5689

Dickinson MH (1999) Bionics biological insight intomechanical design Proc Natl Acad Sci USA 96 14208ndash14209

Doddema HJ Derksen JWM and Vogels GD (1979)Fimbriae and flagella of methanogenic bacteria FEMSMicrobiol Lett 5 135ndash138

Dubochet J Adrian M Chang JJ Homo JC LepaultJ McDowall AW and Schultz P (1988) Cryo-electronmicroscopy of vitrified specimens Q Rev Biophys 21 129ndash228

Eichler J (2004) Facing extremes archaeal surface-layer(glyco) proteins Microbiology 149 3347ndash3351

Faguy DM Bayley DP Kostyukova AS Thomas NAand Jarrell KF (1996) Isolation and characterization offlagella and flagellin protein from the thermoacidophilicarchaea Thermoplasma volcanium and Sulfolobus shiba-tae J Bacteriol 178 902ndash905

Fernaacutendez LA and Berenguer J (2000) Secretion andassembly of regular surface structures in Gram-negativebacteria FEMS Microbiol Rev 24 21ndash44

Frangakis AS and Hegerl R (2001) Noise reductionin electron tomographic reconstructions using nonlinearanisotropic diffusion J Struct Biol 135 239ndash250

Fuhrman JA McCallum K and Davis AA (1992) Novelmajor archaebacterial group from marine plankton Nature356 148ndash149

Hahn E Wild P Hermanns U Sebbel P GlockshuberR Haumlner M et al (2002) Exploring the 3D moleculararchitecture of Escherichia coli type 1 pili J Mol Biol 323845ndash857

Hegerl R (1996) The EM program package a platformfor image processing in biological electron microscopy JStruct Biol 116 30ndash34

Holmes KC Angert I Kull FJ Jahn W and SchroumlderRR (2003) Electron cryo-microscopy shows how strongbinding of myosin to actin releases nucleotide Nature 425423ndash427

Huber R and Stetter KO (2001) Discovery of hyperther-mophilic microorganisms In Methods in EnzymologyAdams MWW and Kelly RM (eds) London AcademicPress pp 11ndash24

Huber R Burggraf S Mayer T Barns SM RossnagelP and Stetter KO (1995) Isolation of a hyperthermo-philic archaeum predicted by in situ RNA analysis Nature376 57ndash58

Jarrell KF Bayley DP and Kostyukova AS (1996) Thearchaeal flagellum a unique motility structure J Bacteriol178 5057ndash5064

Jarrell KF Bayley DP Correia JD and ThomasNA (2001) Archaeal flagella In Nature Encyclopediaof Life Sciences London Nature Publishing Group[www document] URL httpwwwelsnet [doi 101038npgels0000386]

Laemmli UK (1970) Cleavage of structural proteins during

370 C Moissl et al

copy 2005 Blackwell Publishing Ltd Molecular Microbiology 56 361ndash370

the assembly of the head of bacteriophage T4 Nature 227680ndash685

Leadbetter JR and Breznak JA (1996) Physiologicalecology of Methanobrevibacter cuticularis sp nov andMethanobrevibacter curvatus sp nov isolated from thehindgut of the termite Reticulitermes flavipes Appl EnvironMicrobiol 62 3620ndash3631

Leifson E (1960) Atlas of Bacterial Flagellation LondonAcademic Press

Li X and Mobley HLT (2001) Bacterial pili and fimbriaeIn Nature Encyclopedia of Life Sciences London NaturePublishing Group [www document] URL httpwwwelsnet [doi 101038npgels0000304]

Milligan RA Whittaker M and Safer D (1990) Molecularstructure of F-actin and location of surface binding sitesNature 348 217ndash221

Miroshnichenko ML Gongadze GM Rainey FAKostyukova AS Lysenko AM Chernyh NA andBonch-Osmolovskaya EA (1998) Thermococcus gorgo-narius sp nov and Thermococcus pacificus sp novheterotrophic extremely thermophilic archaea from NewZealand submarine hot vents Int J Syst Bacteriol 48 23ndash29

Moissl C Rudolph C and Huber R (2002) Naturalcommunities of novel archaea and bacteria with astring-of-pearls-like morphology molecular analysis ofthe bacterial partners Appl Environ Microbiol 68 933ndash937

Moissl C Rudolph C Rachel R Koch M and Huber R(2003) In situ growth of the novel SM1 euryarchaeon froma string-of-pearls-like microbial community in its coldbiotope its physical separation and insights into its struc-ture and physiology Arch Microbiol 180 211ndash217

Morgan DG and Khan S (2001) Bacterial flagella InNature Encyclopedia of Life Sciences London NaturePublishing Group [www document] URL httpwwwelsnet [doi 101038npgels0000301]

Olsen GJ Lane DJ Giovannoni SJ Pace NR andStahl DA (1986) Microbial ecology and evolution aribosomal RNA approach Ann Rev Microbiol 40 337ndash365

Pace NR Stahl DA Lane DJ and Olsen GJ (1986)The analysis of natural microbial populations by ribosomalRNA sequences Adv Microbiol Ecol 9 1ndash55

Ricca E and Cutting SM (2003) Emerging applications ofbacterial spores in nanobiotechnology J Nanobiotechnol1 6

Roukes MLF (2002) Foreword understanding nanotech-

nology In Understanding Nanotechnology ScientificAmerican (ed) New York Warner Books pp VIndashX

Rudolph C Wanner G and Huber R (2001) Natural com-munities of novel archaea and bacteria growing in coldsulfurous springs with a string-of-pearls-like morphologyAppl Environ Microbiol 67 2336ndash2344

Rudolph C Moissl C Henneberger R and Huber R(2004) Ecology and microbial structures of archaealbac-terial strings-of-pearls communities and archaeal relativesthriving in cold sulfidic springs FEMS Microbiol Ecoldoi101016jfemsec2004051006

Sarikaya M Tamerler C Jen AKJ Schulten K andBaneyx F (2003) Molecular biomimetics nanotechnologythrough biology Nat Mater 2 577ndash585

Saxton WO (1996) Distortion compensation selective aver-aging 3-D reconstruction and transfer function correctionin a highly programmable system J Struct Biol 116 230ndash236

Segrest JR and Jackson RC (1972) Molecular weightdetermination of glycoproteins by polyacrylamide gel elec-trophoresis in sodium dodecyl sulfate In Methods in Enzy-mology Vol XXVIII Ginsburg V (ed) New York andLondon Academic Press pp 54ndash63

Skerker JM and Shapiro L (2000) Identification and cellcycle control of a novel pilus system in Caulobacter cres-centus EMBO J 19 3223ndash3234

Sleytr UB and Messner P (1983) Crystalline surface lay-ers on bacteria Ann Rev Microbiol 37 311ndash339

Soto GE and Hultgren SJ (1999) Bacterial adhesinscommon themes and variations in architecture and assem-bly J Bacteriol 181 1059ndash1071

Stetter KO (1999) Extremophiles and their adaption to hotenvironments FEBS Lett 452 22ndash25

Upreti RK Kumar M and Shankar V (2003) Bacterialglycoproteins functions biosynthesis and applicationsProteomics 3 363ndash379

Woese CR Kandler O and Wheelis ML (1990) Towardsa natural system of organisms proposal for the domainsArchaea Bacteria and Eucarya Proc Natl Acad Sci USA87 4576ndash4579

Yonekura K Maki-Yonekura S and Namba K (2003)Complete atomic model of the bacterial flagellar filamentby electron cryomiscroscopy Nature 424 643ndash650

Zillig W Gierl A Schreiber G Wunderl S Janekovic DStetter KO and Klenk HP (1983) The archaebacteriumThermofilum pendens represents a novel genus of thethermophilic anaerobic sulfur respiring ThermoprotealesSyst Appl Microbiol 4 79ndash87