The Gram-Positive Bacterial Cell Wall

G am Posi i e Pa hogens 3 d Edi ion Chap e : The G am-posi i e bac e ial cell wall By Man ed Rohde P o . D . Man ed Rohde Helmhol z Cen e o In ec ion Resea ch, HZI Cen al Facili y o Mic oscopy, ZEIM Inho ens asse 7 D-38124 B aunschweig, Ge many e-mail: [email p o ec ed] Tel ++49 (0)531-61814413 THE GRAM-POSITIVE BACTERIAL CELL WALL 1. His o ical backg ound 2. The bac e ial cell wall 2.1. Chemis y o he cell wall backbone 2.2. Biochemical syn hesis o he pep idoglycan laye 2.3. Tu no e o pep idoglycan 3. Lipo eichoic acids and wall eichoic acids as majo cons i uen s o he cell wall 4. Capsule 5. Ex acellula esicles o G am-posi i e bac e ia 6. A specialized cell wall in Mycobac e ia 7. Elec on mic oscopic echniques applied o s udy mo phology o G am- posi i e bac e ia cell en elopes 7.1. Hea y me al coa ing and shadowing 7.2. Nega i e-s aining 7.3. Con en ional embedding 7.4. C yo-me hods 7.4.1. F eeze- ac u ing and eeze-e ching 7.4.2. High p essu e eezing and eeze-subs i u ion (HPF-FS) 7.4.3. C yo-sec ions o hyd a ed i i ied bac e ia (CEMOVIS) and C yo- FIB-SEM 7.4.4. C yo-elec on omog aphy 8. Ou look The G am-posi i e bac e ial cell wall 1. His o ical backg ound Back in 1884, he Danish bac e iologis Hans Ch is ian G am had de eloped a s aining p ocedu e o iew s ained bac e ia unde he ligh mic oscope (47). His s aining me hod, nowadays simply called G am-s aining, disc imina ed be ween a G am-posi i e and G am- nega i e bac e ial cell wall. He in oduced a dye, gen ian iole , which pene a es o e he cell wall and cy oplasmic memb ane, hus, s aining he cy oplasm o he hea ixed bac e ia. A e addi ion o iodine an insoluble complex is o med which is e ained by he G am-posi i e bac e ial cell wall upon addi ion o a decolo ize such as e hanol. The e o e, G am-posi i e bac e ia appea almos pu ple while G am-nega i e bac e ia e ain he dye o a lesse ex en o no a all and ha e o be coun e s ained wi h a second dye, sa anin o uchsine, appea ing pink o eddish. No ewo hy, some mycobac e ia showed an indi e en s aining beha io when applied o G am-s aining sugges ing ha he cell wall o mycobac e ia migh be somehow di e en om he o he wo ypes. In he ollowing decades, i became ob ious ha cell walls/cell en elopes u ned ou o be mo e di e se and G am-s aining alone o en could lead o misin e p e a ions o he cell wall composi ion. Un il he ea ly 1950s, when he chemical composi ion o bac e ial cell walls was no known, i was specula ed i chi in o cellulose, polyme s ecognized as p o iding igid s uc u es o o he o ganisms, ep esen ed also he building ma e ial o he bac e ial cell wall. In 1951, expe imen s wi h phenol-insoluble ma e ial om Co ynebac e ium diph he ia (57) e ealed glucosamine and diaminopimelic acid as componen s o he bac e ial cell wall which a e associa ed wi h polysaccha ides. Chemical examina ion o s ep ococci cell wall laye s highligh ed he p esence o amino acids and hexosamines in he cell wall ex ac as well as hamnose as a main componen in G am-posi i e bac e ia (83, 109). Sys ema ic analyses o a numbe o G am- posi i e bac e ia iden i ied he wo hexosamines glucosamine and mu amic acid as majo componen s oge he wi h h ee p e alen amino acids, namely D-alanine, lysine o diaminopimelic acid and glu amic acid. By hen also a ypical basic basal uni in G am-posi i e cell walls was ecognized in which glucosamine and mu amic acid a e linked wi h h ee amino acids ia a pep ide bond (27, 129). G am-nega i e bac e ia exp ess he iden ical basal uni . Nume ous analysis o o he bac e ia e ealed ha each bac e ial genus o e en species a e o en cha ac e ized by a dis inc i e pa e n o amino acids, amino suga s and suga s connec ed o he basic basal uni . I was discussed ha hese di e ences should gi e a aluable pa e n o disc imina e be ween bac e ial genus/species (28, 130). O e he ollowing yea s o he compounds o he G am-posi i e cell wall we e ecognized as eichoic acids, which a e poly ibi yl phospha es (8), and lipo eichoic acid. Fu he mo e, nume ous p o eins had been ound linked o he cell wall. S aining me hods o bac e ia o ligh mic oscopic examina ions ha e limi a ions since he esolu ion is no high enough o e eal s uc u al de ails. Wi h he ad en o ansmission elec on mic oscopes (TEMs) in he 1930s and he pa allel de elopmen o p epa a ion me hods o biological samples, elec on mic oscopy imaging o ul a hin sec ions o embedded bac e ia became he me hod o choice o s udy bac e ial cell walls in de ail a high esolu ions (25, 65, 66, 67). Wi h his me hodology, i was possible o he i s ime o disc imina e be ween he s uc u es o G am-posi i e and G am-nega i e bac e ia based on mo phological di e ences in an image. Fi s ly, elec on mic oscopic p epa a ion p o ocols de eloped o euka yo ic cells o issues we e also applied o bac e ia. The mos ui ul e a s a ed when embedding p o ocols we e cus omized o bac e ia and new kinds o embedding esins became a ailable; o example he Lowic yl esins o low empe a u e embedding which allowed in oducing he p og essi e lowe ing o empe a u e (PLT) me hod (1, 5, 22,). This de elopmen was pa alleled by echnical in en ions, especially c yo-me hods in which bac e ia a e physically ixed ins ead o chemically, and opened up a new ho izon in unde s anding bac e ial cell walls. I should be men ioned ha e en oday new me hodologies a e a ising and push mo phological s udies owa ds i i ied and uns ained bac e ia in a ully hyd a ed s a us and he e o e in a close- o- na u e condi ion. No ewo hy, majo de elopmen s in elec on mic oscopic me hodology equi ed a long ime pe iod o in en ion and es ing be o e he echnique was in oduced o he ma ke . Fo example, h ee-dimensional (3D) elec on mic oscopy was de eloped app . 30 yea s a e in en ion o he TEM. In en ion and p e-comme cial de elopmen o c yo-elec on omog aphy (CET) was b ough o he ma ke ano he 30 yea s la e . Due o he apid de elopmen o compu e pe o mance and p og ess in specialized so wa e, nowadays one can es ima e ha new imaging echniques a e in oduced as e . Fo example, he in oduc ion o c yo-FIB ( ocused ion-beam) combined wi h a scanning elec on mic oscope (c yo-FIB-SEM) as a new close- o-na u e app oach was sold a ew yea s a e he i s ad en o FIB-SEM mic oscopes o con en ional esin embedded biological samples. 2. The bac e ial cell wall Bac e ia a e mos ly unicellula o ganisms, which can be ound in a e y wide a ie y o di e en en i onmen s. The e o e, bac e ial cell walls dese e special a en ion because hey a) a e he essen ial s uc u e o bac e ial iabili y by p o ec ing agains he o en hos ile en i onmen , b) a e composed o unique componen s ound nowhe e else in na u e, c) a e esponsible o he shape o he bac e ia, d) p o ide hal o ligands and p o eins o adhe ence o hos cells, e) expose ecep o si es o d ugs o i uses, ) ep esen he mos impo an si es o a ack o an ibio ics, g) p o ide s uc u es o immunological dis inc ion and a ia ion and h) can cause symp oms o disease in animals and humans. 2.1. Chemis y o he bac e ial cell wall backbone The majo backbone o he bac e ial cell wall is he pep idoglycan, also called mu ein, which consis s o epea ing linea uni s o he disaccha ide N-ace yl glucosamine (NAG) linked o N- ace yl mu amic acid (NAM). The disaccha ides a e c oss-linked ia o en lexible pen apep ide amino acid chains o ming a mesh like amewo k (123). Chemically, he pep idoglycan consis s o al e na ing β-1,4-linked N-ace ylglucosamine (GlcNAc; NAG) and N-ace ylmu amic acid (Mu NAc, NAM, a a ian o GlcNAc wi h a D-lac a e a ached o he C-3 by an e he bond). Te mina ion o a pep idoglycan s and is achie ed a he educing end by a 1,6-anhyd oMu NAc esidue, in which he C-1 and C-6 o he suga backbone a e bound h ough an e he linkage. The appea ance o he unusual 1,6-anhyd oMu Nac is used o de e mine he end o he s ands. The pep ide s ems a e co alen ly linked o he glycan s ands wi h an amide bond o he ca boxyl ca bon o he D-lac yl g oup o Mu NAc. One hallma k o he pep idoglycan is ha he glycans a e conse ed ac oss bac e ial species whe eas he pep ide s em is o en modi ied and di e se con aining D- amino acids. An L-alanine (L-Ala) is usually ound in he i s posi ion o he pen apep ide s em om he lac yl g oup o Mu NAc, which can be eplaced by glycine o L- se ine in some a e excep ions. The second amino acid is mos ly occupied by a D-isoglu amic acid (D-iGlu). In S ep ococcus pneumoniae his D-iGlu is amida ed o yield a D-isoglu amine (D-iGln) (134). The γ-ca bon o D-iGlu is bound o he hi d amino acid. This amino acid in he hi d posi ion o he pep ide s em has he highes di e si y among bac e ia. Gene ally one can summa ize ha in mos G am-nega i e bac e ia and some G am-posi i e bac e ia, like in Bacilli and Mycobac e ia, his hi d posi ion is occupied by he unusual amino acid meso- diaminopimelic acid (m-Dap). In con as , in mos o he G am-posi i e bac e ia i is usually a L- lysine (L-Lys) (see Fig. 1). The pep ide s em is inally e mina ed by wo D-alanines (D-Ala), al hough di e en D-amino acids can be ound in his place, oo (123). In summa y, one hallma k o G am-posi i e bac e ia is he obse ed di e ences in he ypes o c osslinks in which he pep ides a e connec ed o he pep idoglycan. Today mo e han 100 chemo ypes can be dis inguished and hei di e ences a e based on di e en linking uni s and subs i uen s in he pep ide chain (114). Due o i s unique chemical s uc u e, he pep idoglycan sacculus o ms a e y la ge polyme ha can be isola ed and iewed e en in he ligh mic oscope (see Fig. 2). The di e ence be ween G am-nega i e and G am-posi i e bac e ia is gi en by he hickness o he pep idoglycan laye su ounding he cy oplasmic memb ane. G am-posi i e bac e ia exhibi a laye o pep idoglycan s ands which can each a size be ween 30-100 nm o e en hicke , whe eas G am-nega i e bac e ia show a laye o only a ew nanome e s (see Fig. 3). While he chemical composi ion o pep idoglycan and he amily o p o eins o assembling is known o a numbe o di e en bac e ia, he o e all a angemen o hese componen s in G am-posi i e cell walls is no ully sol ed. Fo G am-nega i e bac e ia i has been shown wi h CET (c yo elec on omog aphy) ha indi idual e y hin densi ies, mos p obably ep esen ing glycan s ands, un ci cum e en ially a ound he long axis o he bac e ial cell (42). In con as , he 3-dimen ional a angemen o pep idoglycan in G am-posi i e bac e ia is s ill unde discussion (124). Mo e o less h ee di e en models ha e been p oposed o e he yea s. The i s model sugges s ha glycan s ands un ci cum e en ially a ound he long axis as in G am-nega i e bac e ia. This model was called he “ci cum e en ial” o “laye ed” model (43). In he second model he glycan s ands a e supposed o un pe pendicula o he bac e ial cell wall in a hexagonal la ice, and is called he e o e “pe pendicula ” o “sca old” model (31, 85). This model was p oposed on he basis o NMR s udies applying a syn he ic 2 kDa agmen o he pep idoglycan o med o NAG-NAM (pen apep ide)-NAG-NAM(pen apep ide). NMR e ealed ha his agmen o ms a igh handed helix wi h a pe iodici y o h ee NAG-NAM pe helix u n. The i s wo amino acids can adop a limi ed numbe o di e en con o ma ions (85). A omic o ce s udies (AFM) wi h gen ly dis up ed sacculi o Bacillus sub ilis es ablished he so-called “coiled cable” model. He ein, bundles o glycan s ands o m hicke moie ies o a ound 50 nm which un a ound he cell (53). I should be no ed ha a model wi h glycan s ands unning pa allel o he long axis has ne e been conside ed because i was unclea how such a sacculus could elonga e. In addi ion, he “coiled cable” model is no conside ed nowadays since CET did no show any cable-like s uc u es in he hick pep idoglycan laye . These ea lie obse a ions migh ha e been based on he ac ha isola ed pep idoglycan was ha es ed by boiling o bac e ia, opened up by a F ench P ess cell, dilu ed in wa e and ai d ied on mica be o e AFM imaging was pe o med. 2.2. Biochemical syn hesis o he pep idoglycan laye Syn hesis o he pep idoglycan is a h ee-s ep mechanism, which is localized a h ee di e en loca ions wi hin a bac e ium. The sequen ial Mu ligase pa hway is in ol ed in biosyn hesis o he pep idoglycan. The ea ly s eps o syn hesis s a in he bac e ial cy oplasm whe e p ecu so s linked o undecap enylpy ophospha e (UDP), like UDP-N-ace ylmu amyl- pen apep ide (UDP-NAM) and UDP-N-ace ylglucosamine (UDP-NAG), a e o med. In a second s ep UDP-NAM is bound o ano he cy oplasmic memb ane-bound UDP unc ioning as a anspo lipid. This complex is called lipid I and is loca ed a he inne cy oplasmic ace o he memb ane. Co alen a achmen o he second p ecu so UDP-NAG o ms he anspo lipid complex lipid II. Then, o example in case o S. au eus, a pep ide c oss-b idge is a ached o he hi d amino acid in he pen apep ide consis ing o 5 glycine esidues. Nex , he en i e lipid II complex is lipped o e he cy oplasmic memb ane o he ex acellula side by a lippase. The p ecise biochemical p ocess o he lipping mechanism is no ye ully unde s ood. Lipid II is inco po a ed in o nascen g owing pep idoglycan by penicillin binding p o eins (PBPs) on he ex acellula side o he memb ane. This hi d s ep in ol es i s ly a ansglycosyla ion and secondly a anspep ida ion eac ion pe o med by PBPs o inco po a ing new glycans wi h lexible pep ides in o he exis ing pep idoglycan laye (10, 16, 110, 111, 121, 122, 134). Fo de ailed eading o chemical eac ions and enzymes in ol ed in his p ocess, please e e o he e iew o Teo and Rope (134). 2.3. Tu no e o pep idoglycan The i s epo desc ibing he bac e ial cell wall u no e was published o he G am-posi i e bac e ium Bacillus mega e ium mo e han 50 yea s ago (23). La e on, pulse-chased expe imen s demons a ed wi h adioac i ely labeled cell wall p ecu so s ha all s udied G am- posi i e bac e ia ca y ou a cell wall u no e as well as G am-nega i e bac e ia (14, 34, 81, 107). The model o pep idoglycan g ow h in G am-posi i e bac e ia implies an inside- o-ou side g ow h in which newly syn hesized pep idoglycan is deli e ed o he cy oplasmic memb ane ace o he pep idoglycan laye in a elaxed o m. Wi h polyme iza ion and c oss-linking, pep idoglycan is mo ing o he ou side o he cell wall and ge s s e ched due o he high u go p essu e wi hin he bac e ial cell (68). Once he maximally s e ched pep idoglycans in he ou e laye s s a o age, hey a e subsequen ly hyd olyzed by au olysins (117). I was es ima ed ha a ound 50% o he o al cell wall mass is u ned o e wi hin one gene a ion. This would ha e been a massi e loss o esou ces o he bac e ia and i was specula ed ha hyd olyzed cons i uen s o he cell wall migh be ecycled by bac e ia. Indeed, his was ound o be he case in G am-nega i e bac e ia like E. coli and he biochemical pa hways a e well unde s ood (64, 94). I G am-posi i e bac e ia would also ecycle cell wall ma e ial emained unclea . I was ound ha high amoun s o cell wall agmen s could be de ec ed in g ow h medium o exponen ially g own G am-posi i e bac e ia like in Bacillus, Lac obacillus, Lis e ia and S aphylococcus s ains (64). Thus, a obus u no e o cell wall componen s was es ablished G am-posi i e pep idoglycan laye s. CET could se e as he me hod o choice o cla i y he ansmig a ion h ough he pep idoglycan laye since bac e ia a e snap- ozen and e en as e en s can be ixed. Howe e , he sea ch o such e en s in omog ams can be e y ime consuming. Ne e heless, mo e and mo e e idence is eme ging poin ing ou ha MVs o G am-posi i e bac e ia play an impo an ole in pa hogenesis since o S. au eus i is epo ed ha MVs con ain penicillin-binding p o eins, which can block ac i i y o ß-lac am an ibio ics. Fu he mo e, he global egula o Ms R, which is in ol ed in me hillicin esis ance, was also de ec ed in S. au eus MVs. MVs can be conside ed as T ojan ho ses being in ol ed in passing on esis ance genes among G am-posi i e bac e ia (108). In addi ion, se e al oxins ha e been ound in MVs like lis e olysin O (LLO) in L. monocy ogenes and pneumolysin (Ply) in S. pneumoniae. Bo h oxins induce po e o ma ion in he hos cells and a e he e o e impo an i ulence ac o s o coloniza ion and in asion (72, 93). In G oup A s ep ococci, MVs ha e also been desc ibed and i s con en cha ac e ized in de ail (see Fig. 6). In summa y, no only i ulence associa ed p o eins like M1 p o ein, s ep olysin O (SLO) and se ine p o ease H A we e de ec ed, bu also nume ous me abolic p o eins esiding in he s ep ococcal cy oplasm we e iden i ied as well as su ace exposed p o eins including ancho less su ace p o eins, lipids and RNA. Fu he mo e, he in ol emen o he i ulence associa ed wo-componen egula o Co RS was demons a ed and loss o Co RS esul ed in inc eased esicle o ma ion (105). 6. A specialized cell wall in Mycobac e ia Mycobac e ia a e classi ied as G am-posi i e bac e ia, al hough a e also e e ed o as acid- as bac e ia due o he high densi y o lipids in he cell wall, which p e en s an accu a e G am- s aining. Thus, he s aining is pe o med wi h Ziehl-Neelsen s ain. The complexi y o Mycobac e ia cell walls is a dis inc ea u e ha is no ound in o he bac e ia. Th ee majo mac omolecules, pep idoglycan, a abinogalac an and mycolic acids a e he building blocks o he mycobac e ial cell wall. S uc u al desc ip ion o he mycobac e ial cell wall was conduc ed in he 1960s and 1970s and elec on mic oscopy played an impo an ole in desc ibing he unusual mo phological s uc u es o he cell wall. The cu en accep ed model o he cell wall is based on s udies which iden i ied he mycolyl-a abino-galac an-pep idoglycan complex as he co e s uc u e o Mycobac e ia (86). This unique a angemen wi h lipids and p o eins being included is esponsible o he cha ac e is ically impo an and e y e icien pe meabili y ba ie o he mycobac e ial cell wall, pa icula ly agains d ugs and p o ide he basis o he e y po en pa hogenici y o mycobac e ia (see Fig. 7). Due o he p esence o a high amoun o lipids in he cell wall, ea lie s udies we e con on ed wi h he di icul y o ex ac ing he lipids du ing he dehyd a ion p o ocol. The e o e, o a long ime i was discussed whe he he lipids o med a lipid bilaye in he cell wall, as sugges ed by Minnikin in 1982 (86). He sugges ed an asymme ical memb ane o which he mycolic acids a e co alen ly a ached as an inne lea le . The p esence o such a lipid bilaye was con i med by eeze- ac u e s udies, which clea ly de ined a second ac u e plane, ypical o a lipid bilaye . These indings suppo ed he hypo hesis o a second bilaye ou side he cy oplasmic memb ane, e en hough hese s udies we e pe o med wi h Co ynebac e ia (101, 136). Howe e , he exis ence o a bilaye ou side o he cy oplasmic memb ane was s ill hea ily c i icized because he p oposed bilaye has ne e been clea ly iden i ied in ul a hin sec ions due o a e ac -p oducing embedding p epa a ions like chemical ixa ion and dehyd a ion wi h ace one. Ins ead, a mo e o less lucen zone, ou e laye , mos p ope ly ep esen ing lipids and mycolic acids, was de ec ed di iding he mycobac e ial cell wall in o a iple laye s uc u e composed o he cy oplasmic memb ane, cell wall and ou e laye (see Fig. 8). This anslucen zone is co e ed wi h a e y hin s ainable laye consis ing o capsule and a ached p o eins. F eeze-subs i u ion e ealed mo e o less simila images e en hough he cell wall appea ed hinne (95, 96, 97). A majo s ep o wa d in elucida ing he mycobac e ial ou e cell wall s uc u e was pe o med wi h close- o-na u e imaging applying c yo ul a hin sec ions and CET o ully hyd a ed and i i ied samples (55). These s udies e ealed impo an changes o he cu en model. Fi s ly, a lipid bilaye was de ec ed co e ing he ou side o he cell wall and he e o e, indica ing ha mycobac e ia exp ess a simila ou e memb ane esembling G am- nega i e bac e ia. Secondly, no e idence was ound o an asymme ical memb ane, ins ead, a symme ic memb ane and an addi ional pe iplasmic space was pos ula ed (136). In addi ion, i is clea ha ex ac able lipids play a dominan ole o mycobac e ial memb ane in eg i y and p ope ies. The mycobac e ial pep idoglycan is syn hesized as obse ed in o he bac e ia, in he cy oplasm, using UDP and is hen lipped o e he cy oplasmic memb ane and inse ed in he g owing pep idoglycan ne wo k by he ac ion o hyd olases and PBPs. Ne e heless, Mycobac e ia exhibi a numbe o di e ences when compa ed o model bac e ia. Fi s ly, mycobac e ial pep idoglycan is ex emely c oss-linked, secondly he c oss-links a e based on up o 80% o he o al pep idoglycan on 3-3 pep ide c oss-links ins ead o he 4-3 pep ide c oss-links ound in o he bac e ia, and hi dly he pep idoglycan backbone shows modi ica ions such as glycolyla ion’s o NAM and amida ion o D-Glu and m-DAP (62, 70, 74, 75, 102). Fu he mo e, he mycobac e ial pep idoglycan is su ounded by a laye o a abinogalac an, a disaccha ide, which has long a abinan polyme s a ached. No ewo hy, some galac ans emain ee o a abinan polyme s and mos impo an , he a abinan chain ends a e b anched. These b anched ends a e he binding pa ne s o he long ca bon chains o mycolic acid. These inco po a ed a y acids a e esponsible o he ex emely hick waxy coa o mycobac e ia and make he mycobac e ial cell wall mos ly impe meable, con ibu ing o pa hogenici y. Fo de ailed eac ion and enzymes in ol ed in he p ocess please e e o he e iew o Janku e e al. (62). 7. Elec on mic oscopic echniques applied o s udy mo phology o G am-posi i e bac e ia cell en elopes Since he ea ly 1950s ansmission elec on mic oscopy has been applied o s udying he mo phology and ul as uc u e o G am-posi i e bac e ial cell walls. Su p isingly, e en oday a single unique me hod, ha would allow s udying he ul as uc u al de ails o all he di e en G am-posi i e bac e ia unde close- o-na u e condi ions in an elec on mic oscope, is s ill needed. The newly de eloped CET migh be he cu en me hod o choice, hough CET bea s some d awbacks and es ic ions especially when bac e ia, wi h a wid h o mo e han 0.4 o 0.5 µm, ha e o be imaged. The new supe esolu ion ligh mic oscopy me hods like STED (s imula ed emission deple ion), PALM (pho oac i a ed localiza ion mic oscopy), SIM (s uc u ed illumina ion mic oscopy) and TIRF ( o al in e nal e lec ion luo escence) we e all e y p omising, bu a b eak h ough was hinde ed by he limi a ions when imaging immune luo escen labeled s uc u es. The exp ession o luo escence ags o luo escence p o eins, like GFP p o eins, migh al e he in i o biological ac i i ies o a ce ain ex en , he e o e gi ing ise o non-accu a e localiza ions wi hin he bac e ial cell. I he eade is in e es ed in ollowing his in de ail, i is ecommended o ollow he li e a u e ega ding he M eB p o ein (in ol ed in he bac e ial di ision p ocess) o e he las yea s. Depending on he high esolu ion imaging me hod applied, di e en assump ions abou i s dis ibu ion, a angemen and localiza ion in Bacillus sub ilis we e made, i.e., looking i M eB o ms helices in he bac e ial cell o no (39). Se e al a emp s ha e been unde aken o elucida e he ul as uc u e o G am-posi i e cell walls. In ea ly TEM imaging o embedded and ul a hin cu bac e ia he p epa a ion scheme included chemical ixa ion wi h aldehydes, in oduc ion o hea y me als, dehyd a ion wi h ace one/e hanol and embedding in sui able esins. All hese p epa a ions s eps had o be done o cope wi h he “hos ile” en i onmen c ea ed by he elec on mic oscope, namely high acuum and bomba dmen wi h high-ene gy elec ons esul ing in hea ing up he sec ion. Thus, i is ob ious ha hese ea men s migh no esul in a p ope p ese a ion o he na i e cell wall. To o e come some o hese de imen al e ec s in he p epa a ion o bac e ia, c yo-me hods ha e been in oduced like eeze-subs i u ion o hyd a ed c yo-ul a hin sec ions. Wi h he ad en o high p essu e- eezing echniques p ese a ion o bac e ial cell wall s uc u es was pushed u he in he di ec ion o close- o-na u e condi ions. Nowadays, CET is he bes me hod o pe o m imaging in a ozen i i ied hyd a ed s a e o he bac e ia (91). Howe e , d awbacks o his echnique a e ha i is only a ailable in ce ain ins i u es and i needs a sophis ica ed in as uc u e and ime o pe o ming in dep h analysis. The u u e will show i he newly de eloping c yo- ocused-ion-beam scanning elec on mic oscopy mic omachining (C yo-FIB- SEM) will ad ance he deciphe ing o he ul as uc u al de ails o he bac e ial cell wall since he examined bac e ia a e in hei ully hyd a ed condi ion and physically ozen o e coming he p oblem o chemical ixa ion wi h aldehydes. This echnique allows obse ing lamellas (app . 10-20 nm in hickness) cu ou o he bac e ium; hus, gaining access o small ul as uc u al de ails. I should also be men ioned ha o he echniques like x- ay di ac ion and X- ay li hog aphy ha e been unsuccess ul because he bac e ial cell wall is no c ys alline. A omic o ce mic oscopy (AFM) has also been implemen ed, bu wi h AFM only he su ace o a sample can be imaged and he e o e, only limi ed ul as uc u al in o ma ion was ob ainable. The same es ic ion holds ue o scanning elec on mic oscopy. Wi h he ad en o ield emission scanning elec on mic oscopes (FESEM) i was possible o s udy bac e ial s uc u es a e y high magni ica ion (up o 400,000- old) and esolu ion. Ne e heless, FESEM has ne e been able o p o ide he amoun o ul as uc u al de ails obse ed wi h TEM on ul a hin sec ions. This is simply due o he ac ha FESEM e eals only he su ace opog aphy o a bac e ial cell. No ewo hy, FESEM does no allow disc imina ing be ween G am-posi i e and G am-nega i e bac e ia (see Fig. 9). In addi ion, FESEM samples need o be coa ed o be conduc i e. This so-called spu e -coa ing is o en he las s ep in a scanning EM p epa a ion p o ocol. Samples a e usually spu e coa ed wi h a hin 5-8 nm ilm o ei he gold, gold palladium o pla inum. E en hough hese laye s a e e y hin, i migh co e some ine ul as uc u al de ails o in e es when obse ed a high magni ica ions. Su p isingly images a high esolu ion o he G am-posi i e cell wall do mo e o less no exis . On he o he side, FESEM has been e y use ul in s udying pa hogenic bac e ia in e ac ions wi h hos cells. In he ollowing a gene al desc ip ion o mos o he elec on mic oscopic me hods applied o s udying he bac e ial su ace s uc u es will be gi en. I he eade is in e es ed in ully de ailed p o ocols please e e o specialized ex books o elec on mic oscopic me hods. 7.1. Hea y me al coa ing o shadowing When biological samples we e examined o he i s ime unde a TEM i became ob ious ha he con as o he biological ma e ial is undamen al and ha me hods had o be de eloped o inc ease he con as o TEM images. One o he i s app oaches applied was me al coa ing wi h hea y me als (87, 128). A shadow line behind he exposed s uc u es appea ed when he me al coa ing was pe o med unde a ce ain angle. Knowing he coa ing angle and he measu ed leng h o he esul ing shadow, he heigh o he s uc u e could be de e mined. In ea lie yea s, me al coa ing had become he me hod o choice o he ul as uc u al desc ip ion o egula ly pa e ned cell wall s uc u es, named S-laye s, a ached o he cell wall o bac e ia (6, 60, 61, 116). 7.2. Nega i e-s aining The me al coa ing app oach has i s es ic ions when mac omolecules o p o ein complexes ha e o be imaged. Fo such pu poses he idea o embedding mac omolecules in o hea y me al sal s like Na-K-phospho ungs a e, and la e on u anyl ace a e o o he s, we e conside ed. Ad an ages o nega i e-s aining a e mani old: a) eliable and epea able, b) as , c) a oids la ening o mac omolecules on he suppo ilm when ai -d ying, e) s abilizes he p o ein in he elec on beam, ) allows o de e mine he shape and qua e na y s uc u e o an enzyme complex a a ound 1.3 nm esolu ion and, g) usage o di e en hea y me al sal s esul s in highe o lowe con as s (4, 17, 18). No ewo hy, nega i e s ained i uses and la ge enzymes opened he doo o 3D mic oscopy and image p ocessing beginning in he 1970s (41, 59). Nega i e s aining was he me hod o choice when isola ed pep idoglycan sacculi o pole caps we e analyzed (13). Ne e heless, nega i e-s aining is no sui able o di e en ia e be ween G am- nega i e and G am-posi i e bac e ia (see Fig. 10). 7.3. Con en ional embeddings Since he 1950s, he embedding echnique was in oduced o analyze he ul as uc u al de ails o bac e ia (25) because in ac bac e ia we e unsui able o hese s udies. The e o e, ul a hin sec ioning o bac e ia was needed o gain access o in e nal mo phological s uc u es. Wi h he in en ion o ul amic o omes, i was possible o ob ain ul a hin sec ions o biological samples, hus acili a ing de ailed s udies o bac e ial cell walls (see Fig. 11). F om hen on, mo phological s udies o bac e ial cell walls s a ed o blossom. Ul a hin sec ions ha e a hickness o a ound 50-80 nm, meaning ha a single bac e ium measu ing 1 µm in leng h can be cu in o nea ly 15 sec ions. Again, one was di ec ly con on ed wi h he p oblem o low con as o biological samples. Thus, he ea ly embedding p o ocols usually included ixa ion wi h aldehydes, con as ing wi h hea y me als like osmium e oxide, u henium ed and u anyl ace a e, dehyd a ion wi h ace one/e hanol depending o he esin used o embedding. A ha ime mos ly epoxy o me hac yla e esins we e used and polyme iza ion was ca ied ou a 60- 70°C. Nowadays, many di e en esins a e a ailable and e e y class o esin o e s a sligh ly di e en image o he embedded bac e ial ul as uc u e, depending on he embedding p o ocol (see Fig. 12). In addi ion, he coun e -s aining o ul a hin sec ions be o e TEM examina ion in luences he appea ance o ul as uc u al de ails in he sec ions. These p o ocols e ealed unequi ocally he isible di e ences be ween G am-posi i e and G am-nega i e bac e ial cell walls. In mos o he ul a hin sec ions, he G am-posi i e cell wall appea s as an amo phous s uc u e and, depending on he esin and applied embedding p o ocol; some s uc u al de ails could be de ec ed like he discussed pe iplasmic space in G am-posi i e cell walls (12, 46). I should be clea ly s a ed he e ha hese embedding p o ocols a e p one o induce a i ac s in he samples and he e o e in luence he in e p e a ion o he obse ed ul as uc u al de ails (44). Ne e heless, hese me hods se ed as a basis o mos o he desc ip ion o bac e ial ul as uc u e and hey a e widely a ailable in nea ly all li e science elec on mic oscopy uni s. 7.4. C yo-me hods Con en ional embedding app oaches lack he accu acy o in es iga e iny ul as uc u al de ails conside ing po en ial ad e se e ec s o chemical ixa i es, in oduc ion o hea y me als and dehyd a ion du ing p epa a ions. C yo-me hods began o de elop om he beginning o he 1980s onwa ds, when i i ica ion o wa e in biological samples o elec on mic oscopic s udies was applied o he i s ime by Maye and B üggele (82) and Duboche e al. (35, 36, 37). Ea lie on eeze- ac u ing was in oduced in he 1960s (88). 7.4.1. F eeze- ac u ing and eeze e ching One o he ea lies c yo-me hod applied was eeze- ac u ing and eeze-e ching. Samples a e ozen in ni ogen slush, he eby wa e in he samples is b ough in o i s i i ied s a e. Then samples a e ac u ed, some imes e ched and subsequen ly coa ed wi h me al o ca bon o bo h. F om his sample a eplica is p oduced which exhibi s he su ace opog aphy. The dep h o he opog aphical s uc u es depends on he e ching ime (58, 103). Usually, du ing eeze- ac u e, he ac u e line is in he hyd ophobic egion o a memb ane, i.e., in he bac e ial cy oplasmic memb ane. Thus, exposing ansmemb ane o memb ane bound p o eins. Only a ely does a ac u e line un ac oss he cell wall o inside a cell wall. A eas which a e exposed gi e a mo e o less ea u eless ma ix o , as in c oss ac u es, show a polyme ic ne wo k which could no be u he esol ed. These indings did no succeed in gaining conside able new unde s andings o he G am-posi i e cell wall (11). 7.4.2. High-p essu e eezing and eeze-subs i u ion (HPF-FS) The de elopmen o eeze-subs i u ion o quickly ozen samples, which was pa alleled by he in en ion o low empe a u e embedding esins as he Lowic yl se ies o me hac yla e esins, was s a ed in he 1980s (1, 22). Bac e ia a e snap- ozen in liquid p opane o e hane and hen apidly ans e ed in o a subs i u ion medium con aining osmium and/o u anyl ace a e in ace one. Rema kably, i was demons a ed ha a ce ain wa e con en in ace one (up o 4%) esul ed in a much be e isibili y o memb anes (125). Samples a e hen kep o 2 days a - 80°C, wa med up o -50°C and -20°C and le o 1 day a each s ep. The ollowing embedding can be pe o med wi h low empe a u e esins, Lowic yl esins, o samples a e b ough o ambien empe a u e and embedded wi h con en ional esins (44, 45, 96). Du ing subs i u ion, bac e ia a e s ained and dehyd a ed esul ing in a isibly be e p ese a ion o ul as uc u al de ails. Mos o eeze-subs i u ed bac e ia in ul a hin sec ions a e ecognizable by he ac ha no dis inc DNA egion can be obse ed, whe eas, in con en ional embedding, DNA mos ly agg ega es and o ms he ypical lucen DNA egion in he middle o he bac e ial cell (see Fig. 13). Cu en ly, hund eds o subs i u ions p o ocols do exis which a e cus omized o ul il he needs o he examined biological samples and o add ess he s udy pu pose, e.g., o ul as uc u al s udies o immune cy ochemical localiza ion s udies. F eeze-subs i u ion was pushed e en mo e o wa d by high-p essu e eezing o bac e ia. This me hod was de eloped in he 1960s (88). A ambien empe a u es adequa e eezing o bac e ia is eached wi h cooling a es o mo e han 10.000 K/s o i i y he wa e con en in he sample. High p essu e is a po en physical c yo-p o ec an because i lowe s he eezing poin o wa e conside ably and hicke samples can be i i ied. The cu en ly a ailable equipmen eezes samples a app . 2000 ba . A his p essu e, samples o up o 200 µm can be ozen wi hou o ma ion o ice c ys als (56, 88, 118, 119). The combina ion o hese wo me hods is nowadays conside ed o be he bes app oach o ul as uc u al s udies on bac e ia when no access o CET is possible. One esul o such s udies is he discussed appea ance o a pe iplasmic space also in G am-posi i e bac e ia (79, 80, 136). Ne e heless, CET obse a ions ha e pu hese assump ions in o ques ion. 7.4.3. 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Figu e legends Fig. 1 The bac e ial cell wall backbone, pep idoglycan; shown a e he wo glycan s ands (in black) and pep ide s ems a e depic ed in black (le side) and he second pep ide s em in blue, no e he c oss-linking NH (in ed) ia he wo unusual amino acids m-diaminopimelic acid (m- Dap in ed) and he p esence o D-alanine in he pep ide s ems; wo mo e pep ide s ems (g een and pink) a e depic ed which can in e ac o build he nex c oss-linking be ween glycan s ands. Fig. 2 T ansmission elec on mic oscopic image aken a an accele a ion ol age o 80 kV o a pep idoglycan sacculus o E. coli a e boiling o 3 h in 10% SDS. The mesh like sacculus was nega i ely s ained wi h 1% aqueous u anyl ace a e, ai -d ied and obse ed in a no mal TEM. Fig. 3 Schema ic d awing o G am-nega i e and G am-posi i e cell walls; a cha ac e is ic o G am-nega i e cell walls is he p esence o wo memb anes i) he cy oplasmic memb ane and ii) he ou e memb ane, be ween bo h memb anes he pe iplasmic space is ound in which a e y hin laye o pep idoglycan is ound; a ached o he ou e memb ane a e lipopolysaccha ides and in he ou e memb ane po ins a e inse ed. A hick laye o pep idoglycan and he lack o an ou e memb ane a e he main cha ac e is ics o G am-posi i e cell walls; ins ead o lipopolysaccha ides G am-posi i e bac e ia ha e lipo eichoic acid and eichoic acid localized in he cell wall. The discussed pe iplasmic space is no d awn since he exis ence o such a pe iplasm in G am-posi i e bac e ia is s ill ongoing. Fig. 4 Visualiza ion o G am-posi i e bac e ial capsules. A) ca ionic gold nanopa icles (lysine coa ed 15 nm gold nanopa icles) label he hick capsule o S ep ococcus pneumoniae a e ixa ion wi h 1% o maldehyde a low pH (s a s), B) c yo-FESEM a close- o-na u e condi ions e eals he hick capsule laye o S. pneumoniae ma ked wi h whi e s a s, he hickness is compa able o he labeled capsule in A; samples we e ni ogen slush ozen, eeze- ac u ed a -105°C, eeze-e ched a -105°C o 30 sec and spu e coa ed wi h gold/palladium, C) o ul a hin sec ions capsules can be p ese ed wi h lysine- u henium- ed osmium embedding p o ocol (see 52) ollowed by embedding in LRWhi e esin, S ep ococcus suis is su ounded by a dense capsule laye (whi e s a s). Fig. 5 Good p ese a ion o s ep ococcal capsules unde in i o condi ions. A) S ep ococcal capsules (S ep ococcus pyogenes adminis e ed i. .) a e well p ese ed (black s a s) in spleen unde in i o condi ions in he mouse model e en a e ixa ion wi h glu a aldehyde and o maldehyde, dehyd a ion wi h ace one and embedding in epoxy esin and ul a hin sec ioning, B) enla gemen o ano he bac e ium depic ing nicely p ese ed capsule (black s a s). Mos likely p o eins in he blood ha e co e ed and p ese ed he capsule and p e en loss o capsule du ing aldehyde ixa ion. Fig. 6 Fo ma ion o memb ane esicles (MVs) on he su ace o S ep ococcus pyogenes M1 se o ype imaged wi h FESEM a e chemical ixa ion wi h aldehydes, dehyd a ion wi h ace one, c i ical-poin d ying, and spu e coa ing wi h gold/palladium. Fig. 7 Schema ical d awing o a mycobac e ial cell wall; cha ac e is ic is a hin laye o pep idoglycan and a abinogalac an o which high amoun s o mycolic acids a e a ached; ano he unusual compound is lipoa abinomannan which is a ached o he cy oplasmic memb ane, on he ou e mos ou side glycolipids a e a ached o he mycolic acids, anspo is acili a ed by inse ed po ins. The “mycobac e ial ou e memb ane” is no d awn in he scheme since he p esence o such an ou e memb ane is s ill unde discussion. Fig. 8 Typical appea ance o a iple laye s uc u e o he mycobac e ial cell wall o Mycobac e ium a ium ssp. pa a ube culosis a e special embedding applying he OTO me hod (osmium- hioca bohyd azide(TCH)-osmium); his me hod especially p ese es lipids much be e because a e he i s osmium e oxide s ep TCH binds o he sample bound osmium and in he second osmium s ep mo e osmium is bound o TCH, he e o e s abilizing lipids; in addi ion bac e ia we e embedded applying he PLT me hod (p og essi e lowe ing o empe a u e) down o -50°C and bac e ia a e hen embedded in he hyd ophobic Lowic yl esin HM20; his p o ocol allows o clea ly de ine he iple laye s uc u e o he mycobac e ial cell