JOURNAL OF BACTERIOLOGY,0021-9193/98/$04.0010

Aug. 1998, p. 3923–3932 Vol. 180, No. 15

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Structural and Biochemical Analysis of the Sheathof Phormidium uncinatum

EGBERT HOICZYK*

Max-Planck-Institut fur Biochemie, D-82152 Martinsried, Germany

Received 6 March 1998/Accepted 26 May 1998

The sheath of the filamentous, gliding cyanobacterium Phormidium uncinatum was studied by using light andelectron microscopy. In thin sections and freeze fractures the sheath was found to be composed of helically ar-ranged carbohydrate fibrils, 4 to 7 nm in diameter, which showed a substantial degree of crystallinity. As in allother examined motile cyanobacteria, the arrangement of the sheath fibrils correlates with the motion of the fil-aments during gliding motility; i.e., the fibrils formed a right-handed helix in clockwise-rotating species and aleft-handed helix in counterclockwise-rotating species and were radially arranged in nonrotating cyanobacte-ria. Since sheaths could only be found in old immotile cultures, the arrangement seems to depend on the pro-cess of formation and attachment of sheath fibrils to the cell surface rather than on shear forces created by thelocomotion of the filaments. As the sheath in P. uncinatum directly contacts the cell surface via the previouslyidentified surface fibril forming glycoprotein oscillin (E. Hoiczyk and W. Baumeister, Mol. Microbiol. 26:699–708, 1997), it seems reasonable that similar surface glycoproteins act as platforms for the assembly and at-tachment of the sheaths in cyanobacteria. In P. uncinatum the sheath makes up approximately 21% of the totaldry weight of old cultures and consists only of neutral sugars. Staining reactions and X-ray diffraction analysissuggested that the fibrillar component is a homoglucan that is very similar but not identical to cellulose whichis cross-linked by the other detected monosaccharides. Both the chemical composition and the rigid highlyordered structure clearly distinguish the sheaths from the slime secreted by the filaments during glidingmotility.

Electron microscopic studies have shown that cyanobacteriapossess complex gram-negative cell walls. Proceeding from theinside, the cytoplasmic membrane is covered by a peptidogly-can layer and an outer membrane. Also, some filamentousgliding cyanobacteria have additional extracellular cell walllayers. These layers are formed by an S layer attached to theouter membrane and an array of parallel, helically arrangedsurface fibrils on top of the S layer. In all species studied thusfar, the helical arrangement of these surface fibrils correspondsto the sense of rotation during gliding motility (11).

While moving, the filaments secrete slime that seems to be anecessary prerequisite for this specific type of motility. Theslime is composed of carbohydrate fibrils which are orientedduring the translational motion along the helically arrangedsurface fibrils before they are shed off and left behind as acollapsed slime tube (11). However, many cyanobacteria areable to form sheaths, which are another distinct type of extra-cellular carbohydrate (2, 4, 19). The rigid sheath in Phormi-dium spp. is closely associated with the extracellular cell walllayer on top of the cell surface and impairs the gliding motilityof the filaments in aged cultures (11).

Previous studies have demonstrated that various cyanobac-terial sheaths consist of a meshwork of polysaccharide fibrilswhich are variably oriented with respect to the cell surface. Insome motile cyanobacteria the fibrils extend radially from thecell surface (13, 15), whereas in other species a helical orien-tation has been demonstrated (14). However, until now, noreports have been published concerning the chemistry of thesefibrils, which Frey-Wyssling and Stecher (7) thought to be

cellulose in the genus Nostoc. How these sheaths are anchoredon the cell surfaces also has not been studied.

In the present study, the structural and biochemical charac-terization of the sheath of the gliding filamentous cyanobacte-rium Phormidium uncinatum is described. It is demonstratedthat the fibrils of the sheath have properties very similar butnot identical to those of cellulose. It is also shown that theorganization of the fibrils in the sheaths of various glidingcyanobacteria always correlate with the motion of the species.Finally, it is suggested that specific surface glycoproteins suchas oscillin (12) act as platforms for the assembly and attach-ment of these extracellular carbohydrate structures.

MATERIALS AND METHODS

Cyanobacterial strains and cultivation. P. uncinatum Baikal was a gift fromD.-P. Hader, University of Erlangen, Erlangen, Germany. Other strains exam-ined and listed in Table 2 included Anabaena variabilis (B1403-4b), Lyngbyaaeruginosa (B47.79), Oscillatoria amoena (B1459-7), Oscillatoria chalybea(B1459-2), Oscillatoria geminata (B1459-8), Oscillatoria sancta (B74.79), Oscilla-toria tenuis (B75.79), Phormidium autumnale (B78.79), and Phormidium foveo-larum (B1462-1) from the Gottingen algal culture collection. Anabaena sp. strainC12, Lyngbya sp. strain C36, and Phormidium ambiguum were kindly provided byW. Nultsch, University of Marburg, Marburg, Germany. Oscillatoria princeps andOscillatoria limosa were both isolated from a canal at Schloss Nymphenburg,Munich, Germany. For the studies described here, all species were grown pho-toautotrophically on a mineral medium as previously described (11).

Light and electron microscopy. A Zeiss Axiovert 10 microscope equipped withbright-field, phase-contrast, and differential-interference contrast optics was usedto examine living trichomes and to determine the presence of sheaths on thefilaments of the different cyanobacteria. To visualize the arrangement of thefibrils within the sheaths, ensheathed filaments were either broken by ultrasoni-cation or viewed with polarized light by using crossed Nicolprisms and a l/2 filter.For fluorescence microscopy, the samples were observed after staining withvarious fluorochromes by using epifluorescence illumination and blue-light ex-citation (filter combination, FT 510 and LP 520).

Isolated sheaths were adsorbed on glow-discharged carbon-coated grids andnegatively stained with 2% (wt/vol) unbuffered uranyl acetate or 1.5% (wt/vol)sodium phosphotungstate at pH 6.8, containing 0.015% glucose to promotehomogeneous staining. Alternatively, the structures of the sheaths were visual-

* Present address: Laboratory of Cell Biology, The Rockefeller Uni-versity, 1230 York Ave., New York, NY 10021. Phone (212) 327-8181.Fax: (212) 327-7880. E-mail: Hoiczye@rockvax.rockefeller.edu.

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ized after air drying by unidirectional shadowing with 1-nm platinum-carbon(Pt/C) under a nominal angle of 45°.

To visualize the deposited slime tubes, carbon-reinforced 50-mesh nickel grids(covered with Pioloform plastic were glow discharged, immersed in medium, andkept in a Petri dish with Phormidium filaments. After 1 or 2 h under appropriatelight conditions, rapidly gliding filaments moved over the grid surface, leavingtheir slime trails behind. The samples were then either negatively stained asdescribed above or rapidly frozen, freeze-dried, and shadowed with Pt/C undera nominal angle of 45°. Electron micrographs were obtained with a Philips CM12 at an operating voltage of 100 kV.

Freeze fracturing. Filaments of P. uncinatum used for freeze fracturing wereharvested by centrifugation and immediately frozen by plunging or slammingwithout fixation or cryoprotection. Alternatively, the cells were stepwise infil-trated with glycerol (25%) and frozen in a cryojet (Balzers Process Systems,

Balzers, Liechtenstein). Frozen samples were fractured and replicated in a Bal-zers BA 360 freeze-etching device at 2115°C by the protocol of Moor andMuhlethaler (17). The carbon reinforced Pt/C replicas were cleaned with 70%sulfuric acid, rinsed several times with distilled water, and picked up on un-coated, 400-mesh copper grids.

Freeze substitution. For freeze substitution, ensheathed filaments or formedhormogonia-like cell chains were immediately cryofixed by plunging them intoliquid ethane. Substitution was performed in diethyl ether containing 2% (wt/vol)osmium tetroxide by the protocol described earlier (11).

Isolation of the sheath. The protocol used for isolating cell-free sheaths wasbased on procedures described by Weckesser and Jurgens (24). Sheathed fila-ments of P. uncinatum and the other cyanobacterial species were harvested bycentrifugation and washed twice in 20 mM ammonium acetate buffer (pH 7.0).The filaments were then resuspended in the buffer and broken by ultrasonication.

FIG. 1. (A) Electron micrograph of negatively stained slime tubes of P. uncinatum. The uranyl acetate stain used not only contrasts with the highly hydrated slimetubes negatively but sometimes infiltrates the tubes (arrowheads). Bar, 30 mm. (B) Electron micrograph of a frozen and partially freeze-dried, collapsed slime tube whichwas left behind a rapidly gliding P. uncinatum filament. The surface of the elastic tube shows characteristic folds. Bar, 10 mm.

TABLE 1. Characteristic features of the different exopolysaccharides produced by P. uncinatum

PropertyCharacteristic features of P. uncinatuma:

Sheath Slime

Microscopic visibility Clearly visible with phase and differential-interference contrast Visible only after stainingAnisotropy Strong Barely detectableCrystallinity Yields sharp X-ray diffraction pattern No diffraction detectableAutofluorescence Yellow-green under blue-light excitation Not detectablePhysical properties A rigid structure formed by parallel helically arranged fibrils An elastic tube formed by fibrilsCompositional analysis Complex heteropolysaccharide containing homoglucan fibrils Complex heteropolysaccharideChemical stability Stable in 10 N KOH for 1 h Completely dissolved in 1 N KOH after 5 minProduction according to

age of culturesFound only in aged cultures Found in young, highly motile cultures

a The terms “rigid” and “elastic” are used descriptively to emphasize the different appearances of the slime tube (see Fig. 1) and the sheath (see Fig. 2, 3, and 7).

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Crude sheath preparations were obtained by centrifugation (15 min at 750 3 g)and further purified from residual cell wall material by treatment with lysozyme.The enzyme was added to a protein/sheath ratio of 1:5 (dry weight/wet weight),and the digest was performed at 37°C for 24 h. After being washed, the sheathswere extracted with Triton X-100 (2% [wt/vol] in 10 mM disodium EDTA atroom temperature for 20 min) or sodium dodecyl sulfate (SDS; 4% [wt/vol] inacetate buffer at 60°C for 15 min). Finally, the purified sheath material wasrepeatedly washed with distilled water prior to lyophilization.

Isolation of slime. Young, motile cultures of P. uncinatum were grown on agarplates covered with a nitrocellulose filter. These culture conditions were selectedto optimize gliding motility and, therefore, to favor the production of slime (11).After the filaments had spread over the filter surface, the formed “pellicle” wascarefully scraped off, washed several times with water, and mildly homogenizedby ultrasonication. The suspension was diluted with water to decrease the vis-cosity and centrifuged (15 min at 5,000 3 g) to remove the bacterial cells. Todissolve the polysaccharide completely, the supernatant was heated for 30 min at80°C. After centrifugation (30 min at 100,000 3 g), this crude preparation waseither used directly for precipitation or purified from contaminating proteins bytreatment with trichloroacetic acid (12%). Finally, the slime was precipitated bythe dropwise addition of 2 volumes of 95% (wt/vol) ethanol and then centrifugedand lyophilized (9).

FTIR spectroscopy. Spectroscopic analysis of the purified sheaths was per-formed by means of Fourier transform infrared (FTIR) spectroscopy. The spec-tra were recorded with a Nicolet 740 FTIR spectrometer. About 50 mg of thesheath material, suspended in water, was dried on the Ge crystal designed formultiple internal reflections. A total of 512 scans were taken for each spectrumat a resolution of better than 2 cm21.

X-ray diffraction analysis and powder diffraction. For X-ray diffraction anal-ysis, purified lyophilized sheath samples were placed in thin-walled X-ray capil-lary tubes, mounted in an Iso-Debeyeflex 100 flat-film vacuum camera, andsubjected to CuKa radiation. The resulting X-ray patterns were recorded onKodak direct-exposure film DEF-5 at a specimen-film distance of 7.5 cm.

To improve the detection of weak diffraction signals, the purified sheathmaterial was also measured in an X-ray powder diffractometer (Stoe, Darmstadt,Germany) by using monochromatic MoKa irradiation. The counted impulse ratewas recorded online and analyzed with a software package of the manufacturer.

Carbohydrate analysis procedure. The monosaccharide compositions of theisolated sheaths and the slime were determined by anion-exchange chromatog-raphy (HPAE system; Dionex) with appropriate sugar mixtures used as a cali-bration standard. The relative amounts of the single sugars were calculated fromthe areas under the peaks. For the chosen concentrations the relative amountsand the detector signal intensities were directly proportional. For the analysis, 5mg of lyophilized sheath and slime material was hydrolyzed in 4 M trifluoroaceticacid (TFA) for 3 h at 100°C. The hydrolysates were dried, dissolved in bidistilledwater, and applied to the column equilibrated with 15 mM NaOH at a flow rateof 1 ml/min (6). Amino sugars were assayed as ninhydrin derivatives on an aminoacid analyzer (Beckman). The uronic acid content was determined by the methodof Blumenkrantz and Asboe-Hansen (3) with glucuronic acid as a standard.

Further analytical-chemical methods. Organic phosphate analysis was doneaccording to the procedure of Lowry et al. (16). Sulfate was quantified by thecolorimetric rhodizonic acid assay described by Terho and Hartiala (23). Forprotein and amino acid detections the isolated sheath material was hydrolyzedwith 6 N HCl for 1 h at 150°C and analyzed on an amino acid analyzer (Beck-man). Analysis of 2-keto-3-deoxyoctonic acid, an indicator of outer membranecontamination in sheaths, was done by the method of Skoza and Mohos (21) withlipopolysaccharide purified from Escherichia coli (Sigma) as a standard.

SDS-PAGE and amino acid sequencing. For SDS-polyacrylamide gel electro-phoresis (PAGE), the tricine system of Schagger and von Jagow was used (20),and the 5 to 13% minigels were routinely stained with colloidal Coomassie blue(18). After being blotted on siliconized glass fibers, the extracted protein wascleaved with endoproteinase Lys-C (Boehringer), and the resulting peptideswere separated as described previously (reference 12 and references therein).Finally, the amino acid sequence was determined by using a gas phase sequencer(type 470A; Applied Biosystems).

RESULTS

Occurrence of the different exopolysaccharides. As summa-rized in Table 1, the production of exopolysaccharides in P. un-cinatum differs according to the age of the culture. A highlyhydrated slime is produced during the gliding motility periodand is left behind as a collapsed thin tube (Fig. 1). By lightmicroscopy it could only be visualized by staining with Indiaink particles that adhere to the mucus. Although sheaths formedafter prolonged cultivation (6 to 7 weeks) were easily visiblewhen using phase contrast or differential interference contrast(Fig. 2A and Fig. 3), these old cultures showed little or no mo-tility. Particularly, ensheathed filaments were never observed

to move, and so it seems that the rigid sheaths inhibit glidingmotility in Phormidium spp. and in the other cyanobacterialspecies. After the formation of hormogonium-like cell chainswithin these ensheathed filaments, the cell chains lost contactwith the tight-fitting sheath and started to move again. Occa-sionally, these short “filaments” left the sheath, and it could beshown by India ink staining that the cells secrete slime duringtheir movements. Therefore, the slime and the sheath seem toplay different roles in gliding motility.

Light microscopic observations. As can be seen from Fig.2A, the protocol used for the isolation of the sheaths of P. un-cinatum resulted in preparations that were devoid of cells. Themechanical stress necessary to break up the filaments oftentears the tube-like sheaths, resulting in the curled structures

FIG. 2. (A) Differential interference contrast micrograph of the isolatedsheaths of P. uncinatum (large arrowheads). Some of the tube-like structureswere disrupted by the ultrasonication used during preparation. The curly ap-pearance of disrupted sheaths (small arrowheads) is the consequence of thehelical arrangement of the fibrils within the sheaths. Bar, 50 mm. (B) Electronmicrograph of the thin section of an isolated sheath. Note the compact fabric ofthe sheath fibrils after cryosubstitution and the absence of remnants of the outermembrane. Bar, 2 mm.

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visible on the micrograph (see also Fig. 3B). After furtherpurification the sheaths were considered to be homogeneousand of fairly high purity (Fig. 2B) based upon the absence ofphosphorus and 2-keto-3-deoxyoctonic acid, suggesting negli-gible membrane contamination. The isolated sheaths showed aweak yellow-green autofluorescence and an intense stainingwith the cellulose-specific fluorochrome Calcofluor White, withaniline blue, and with sulfuric acid-iodine. None of these stain-ing reactions could be observed for the isolated slime secretedby the filaments during gliding motility.

Furthermore, the zinc chloride-iodine reaction of the sheathfor cellulose was negative, as was the phloroglucinol reactionfor lignins and the I2KI staining for amyloids. However, themost striking difference between both exopolysaccharides wasthe strong positive anisotropy of the sheaths. This anisotropywas weak or barely detectable for the slime. The characteristicbirefringence indicated that the sheaths are composed offibrillar elements arranged helically with respect to the fila-ment long axis, which possesses a substantial degree of crys-tallinity.

FIG. 3. Differential-interference contrast micrographs illustrating the correlation between the structure of the sheaths and the motion of various filamentouscyanobacteria. (A) The counterclockwise-rotating species O. princeps, in which the counterclockwise-arranged sheath fibrils can be directly observed (arrowheads). (B)The sheath of the clockwise-rotating species O. tenuis has been disrupted by ultrasonication and shows a curly appearance caused by the clockwise helical arrangementof the fibrils within the sheath (the appearance is nearly identical to that of the sheath of P. uncinatum in Fig. 7). (C and D) P. foveolarum and O. geminata, respectively,are nonrotating cyanobacteria that possess sheaths formed by radially arranged fibrils (see also Fig. 5B), as indicated by the straight ends of the sheaths after disruption(arrowheads) and the characteristic anisotropy observed when using polarized light (pictures not shown). All bars, 10 mm.

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Fine structure of the sheath. Ultrathin sections of cryosub-stituted old-aged filaments of P. uncinatum showed that thesheath consists of a single layer of fine fibrillar material with atotal thickness of up to 0.3 mm (Fig. 2B and 4A). The sheath isdirectly attached to the complex extracellular cell wall layerformed by an S layer and the oscillin surface fibrils (Fig. 4). Inaccordance with studies on Leptothrix discophora and Sphaero-tilus natans (5, 8, 10), the structural appearance of the Phor-midium sheath revealed a more compact fabric after cryosub-stitution than conventionally processed samples (compare Fig.4A to Fig. 5A). Nevertheless, as expected from light micros-copy, both preparations clearly show that the individual sheathfibrils form a right-handed helix around the cylindrical tri-chome of P. uncinatum (Fig. 6 and 7; compare also with Fig.3B). Therefore, the orientation of the sheath fibrils corre-sponds with the sense of rotation during the screw-like motionof the filaments (11). This correlation between the sheath fibrilarrangement and the motion of different cyanobacteria was acharacteristic feature found in all gliding filamentous cyano-bacteria used in this study and is presented in Table 2 and Fig.3 and 5. Interestingly, species like Anabaena variabilis or Phor-midium foveolarum (Fig. 5B and Fig. 3C), which do not rotate,developed in aged cultures sheaths in which the sheath fibrilswere radially arranged with respect to the cell surfaces. It

would be difficult to explain this arrangement by the proposedshear-oriented polymerization of the fibrils during gliding mo-tility (14).

Freeze fracture was used to study the carbohydrate networkof the sheath of P. uncinatum without fixation or dehydration,confirming the helical arrangement of the sheath fibrils (Fig.6). Individual fibrils measured 4 to 7 nm in diameter and lay inparallel with a pitch of about 65 6 3°. The preparations alsoallowed visualization of the contact zone between the sheathand the underlying cell wall surface, which could not be ob-served in thin sections. As seen in the inset of Fig. 6, the sheath

FIG. 4. (A) Cross-section of a cryosubstituted ensheathed filament of P. un-cinatum showing the close contact between the sheath (S) and the underlyingcomplex external layer of the cell wall (EL). (B) Cross-section of a cryosubsti-tuted newly formed hormogonium-like cell chain. After the contact with thetight-fitting sheath is loosened, the cells still possess the complex external cellwall layer with its serrated substructure formed by the oscillin fibrils. EL, externallayer; OM, outer membrane; P, peptidoglycan. All bars, 100 nm.

FIG. 5. P. uncinatum. Tangential section of an old ensheathed filament show-ing the helical, clockwise arrangement of the individual sheath fibrils (SF). Thefibrillar network is more visible in these conventionally processed filaments thanin the cryosubstituted samples shown in Fig. 4A. (B) Structure of the sheath ofA. variabilis. As in all other nonrotating cyanobacteria, the individual sheathfibrils are radially arranged with respect to the filament surface. All bars, 1 mm.

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fibril orientation corresponds to the orientation of the oscillinglycoprotein surface fibrils previously described (11, 12).

Electron microscopy of metal-shadowed, air-dried prepara-tions showed the fibrillar texture of the sheaths (Fig. 7). Similarto the appearance of a plant cell wall, adjacent sheath fibrilsare connected by anastomoses forming domains similar to thecrystallites of the cellulose microfibrils. Additionally, individualfibrils may be cross-linked through short pin-like connections

(arrowheads in Fig. 7). The observed anisotropic properties ofthe sheath seemed to be determined by these highly orderedfibril aggregates, which are composed of carbohydrate chainswith their axes parallel to the axes of the fibrils.

Compositional analysis of isolated sheaths. Comparison ofthe dry weight of the isolated sheath material with intact cellfilaments demonstrated that the sheath accounted for approx-imately 21% of the biomass in old Phormidium cultures; this

FIG. 6. Freeze fracture of an ensheathed filament of P. uncinatum showing the helical, clockwise arrangement of the sheath fibrils (small arrowheads). The closecontact between the sheath and the complex external layer of the cell wall is clearly visible at the lower right edge of the broken cell, where the serrated surface fibrils,formed by the glycoprotein oscillin (OF), directly interact with the overlying sheath (compare with Fig. 4). The double-headed arrow indicates the long axis of thefilament. CW, cross wall; OF, oscillin fibrils; P, peptidoglycan; S, sheath. Scale bar, 1 mm. The inset shows the enlarged view of the contact zone between the sheathand the cell surface. Note the correspondence of the arrangement of the sheath fibrils (SF) and the oscillin fibrils. Bar, 100 nm.

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was a much higher value than the amount of extruded slimematerial, which was estimated to be only about 5% of the bio-mass (11).

The identification of the component monosaccharides of theisolated sheaths was done by high-pressure liquid chromatog-raphy of acid-hydrolyzed samples. To ensure detection of allmonosaccharides, the polysaccharide material was subjected to

hydrolysis under several different conditions. By these proto-cols, five different neutral sugars were identified as major con-stituents of the sheath: glucose (ca. 60%), galactose (18%),xylose (12%), arabinose (,5%), and rhamnose (,5%). Thesame neutral sugars were also found to compose the slime andthe carbohydrate moiety of the surface glycoprotein oscillin(Table 3). The predominance of glucose in the sheath was even

FIG. 7. Electron micrograph of a metal-shadowed, air-dried sheath of P. uncinatum. The tube-like structure is disrupted by ultrasonication and clearly shows thehelical arrangement of the sheath fibrils. Bar, 2 mm. The parallel bundles of fibrils connected by anastomoses and cross-linked through short pin-like elements(arrowheads) are enlarged in the inset. Bar, 200 nm.

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more pronounced (ca. 80%) after a pretreatment with 1 MTFA for 1 h at 100°C, which dissolved the pin-like connectionsand loosened the fibrillar meshwork (Fig. 8). The intact ap-pearance of the remaining fibrils upon electron microscopystrongly suggests that the fibrillar elements of the sheaths arecomposed of a homoglucan. Neither amino sugars nor uronicacids, phosphate, or sulfate groups could be detected, suggest-ing that the content of these components was below the de-tection limits for the assays employed.

SDS-PAGE of isolated sheaths revealed only a weaklystained band with a molecular mass of 130 kDa (Fig. 9), sug-gesting that this sheath-associated protein is the surface fibril-forming protein oscillin (12). Oscillin is a 66-kDa protein whichnot only forms the contact zone between the filament surfaceand the sheath but is arranged helically just as the sheath fibrilsare (see also Fig. 5A and Fig. 6). This interpretation was fur-ther confirmed by sequencing of a Lys-C-derived peptide of thesheath-associated protein. The revealed amino acid sequence,GxDxIGFFTGAGDGSNNLGLNLSG, is identical to the os-cillin sequence between amino acid positions 136 and 159 (Gen-Bank accession number AF002131). Generally, there were nodifferences in the chemical compositions of the sheaths iso-lated with or without detergents. However, in the SDS-treatedfractions no association between the sheath and oscillin couldbe detected.

Biophysical characterization of the sheath. The results ofthe compositional analysis of isolated sheath and slime mate-rial could be further confirmed by FTIR spectroscopy. Bothrecorded spectra showed a characteristic triple peak centeredat 1,030 cm21 that is typical for polysaccharides but which

clearly differed within this region, reflecting the molecular dif-ferences of the two polymers.

In order to obtain more information about the bonding ofthe carbohydrate backbone in the crystalline part of thesheaths, X-ray crystallography was used. As shown in Fig. 10A,the isolated and purified sheaths gave a diagram with fourdiffraction lines: a set of three weak rings at 0.541, 0.333, and0.253 nm and a stronger sharp ring at 0.221 nm. This diffractionpattern was clearly different from the characteristic lines ofcellulose (Fig. 10B), which displayed reflections at 0.605, 0.541,0.391, and 0.263 nm. To detect even weaker diffraction signals,the sheath material was also measured in an X-ray powderdiffractometer (Fig. 11). The counted impulse rate was re-corded, and four additional diffraction signals could be iden-tified corresponding to 0.170, 0.146, 0.132, and 0.124 nm.Therefore, the fibrillar component of the sheath of P. uncina-tum has a substantial degree of crystallinity, a finding in agree-ment with the strong anisotropy observed by light microscopy.

DISCUSSIONDifferent exopolysaccharides for different purposes. P. un-

cinatum is able to produce different exopolysaccharides whichseem to serve different functions. The slime is only producedby actively moving filaments and consists of highly hydratedheteropolysaccharide fibrils which show, especially at the inner

FIG. 8. Electron micrograph of Pt/C shadowed sheath fibrils after treatmentwith TFA for 1 h at 100°C. The pin-like connections between neighboring sheathfibrils are dissolved, and the fibrillar meshwork disintegrates into single homo-glucan fibrils. Bar, 50 nm.

TABLE 2. Correlation between the arrangement of the sheathfibrils and the motion of different filamentous cyanobacteria

during gliding motility as examined in this study

Cyanobacterial groupings based on rotation and arrangement

Clockwiserotation, clockwise

arrangement

Counterclockwiserotation, counterclock-

wise arrangement

Nonrotating species,radial arrangement

O. chalybea O. limosa O. rubescensa

O. tenuis O. princeps O. geminataO. sancta L. aeruginosa P. foveolarumO. amoena A. variabilisP. uncinatum Anabaena sp. strain C12P. autumnaleP. ambiguumLynbgya sp. strain C36

a The observation reported for O. rubescens is according to the work of Jost(13).

TABLE 3. Comparison of the monosaccharide compositionsof sheath, slime, and surface glycoprotein oscillin

SugarMonosaccharide composition (%)a of:

Sheath Slime Oscillin

Arabinose ,5 11 ,5Fucose 9 ,5Galactose 18 10 3Glucose 60 34 18Rhamnose ,5 3 9Xylose 12 33 60

a All determinations reported here are not quantitative and indicate only therelative amounts of the individual sugar components.

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contact zone, an orientational arrangement identical to that ofthe oscillin fibrils on the filament surface as previously shown(11). These slime fibrils form a flexible tube which is onlytemporarily attached to the filament before it is shed off andleft behind. Unlike slime, the sheath is only produced in oldcultures and may protect the filaments from unfavorable con-ditions such as desiccation. It forms a rigid tube-like structurecomposed of helically arranged fibrils which are permanentlyattached to the cell surface and thereby inhibit gliding motility.Various nutritional and environmental factors seem to controlwhich type of exopolysaccharide is formed by the Phormidiumfilaments. As in Leptothrix discophora strain SS-1 (1) or Gloeo-thece sp. (22), the sheath of Phormidium appears not to be astable cell structure, and the ability to form a sheath was fre-quently lost during repeated subculture, whereas the ability tosecrete slime was invariably found as long as the filamentsdisplayed gliding motility.

Structure and formation of the sheaths. A striking feature ofall investigated sheaths is that the orientation of the sheathfibrils is always correlated with the motion of the filaments dur-ing gliding; i.e., the fibrils form a right-handed helix in theclockwise-rotating species and a left-handed helix in the coun-terclockwise-rotating species and are radially arranged in thenonrotating cyanobacteria (Table 2; Fig. 3 and Fig. 5). The cor-respondence of fibril arrangements with the paths of the fila-ments was thought to be the result of a shear-oriented poly-merization of the sheath monomers (14). Since ensheathedfilaments were never observed to move, it is unlikely that thearrangement of the fibrils could be the consequence of shearforces during motility. Instead, it now seems likely that theassembly and attachment of the sheaths via helically or radiallyarranged surface glycoproteins are the reasons for the ob-served identical orientational arrangement, an interpretationstrongly suggested by the following circumstantial evidence.First, in thin sections the oscillin fibrils directly contact thesheath. Second, both structures are arranged in an identicalhelical fashion around the filament, which can even be directly

visualized in freeze fractures. Finally, the only protein found tobe associated with the sheaths after isolation is the surfaceglycoprotein oscillin.

These observations further indicate that the direct bindingof the sheath and the surface proteins can be disrupted by thefilament to form short hormogonium-like parts of the filament.These hormogonium-like cell chains are able to move againand have all of the features suggested to be required for glidingmotility; i.e., they secrete slime and possess a surface topogra-phy created by specific glycoproteins such as oscillin (12).

Fibrillar component of the sheath. According to the light-microscopic observations and the X-ray diffraction pattern, thesheath of P. uncinatum consists mainly of a fibrillar componentwith a substantial degree of crystallinity. Furthermore, theseresults, together with the positive staining reactions, the com-positional analysis, and the relatively high resistance of thesheath to chemical degradation, indicate that the microfibrilsare formed by a homoglucan with properties very similar butnot identical to those of cellulose. The FTIR spectroscopy gaveweak indications that the sheath fibrils might consist of a b-1,3-homoglucan; however, the actual type of bonding between theglucose monomers has not been assigned.

From the comparison of the composition of the sheath be-fore and after acid pretreatment, the other four monosaccha-rides found in the sheath seem to be the components of thepin-like structures. These complex heteropolysaccharide ele-ments might stabilize the fabric of the sheath by cross-linking,giving the structure its characteristic rigidity.

Although it now seems reasonable to explain how complexcarbohydrate structures such as sheaths are attached to cya-nobacterial cell surfaces, it is still difficult to understand theactual processes of their secretion and assembly. Onemight speculate that the single fibrils are synthesized by thejunctional pore complexes. These pore complexes also seem tobe involved in the process of slime secretion (11), and so it maybe that the cells are able to switch their polysaccharide pro-duction in response to different environmental stimuli.

FIG. 9. SDS-PAGE of the proteins associated with the isolated sheath ofP. uncinatum. The only protein found in larger quantities is the surface glyco-protein oscillin, which seems to act as a platform for the assembly and attach-ment of the sheath to the cell surface (compare with Fig. 4). Oscillin could notbe detected in the SDS-isolated sheaths.

FIG. 10. X-ray diagrams of the isolated and purified sheaths of P. uncinatum(A) and of a test specimen of cellulose powder from cotton linters (B). Bothmaterials gave sharp diffraction lines representing different types of bonding andlattice orders of the homoglucans.

VOL. 180, 1998 ANALYSIS OF SHEATH OF P. UNCINATUM 3931

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ACKNOWLEDGMENTS

I thank Wolfgang Baumeister for continuous generous support andencouragement, Julius Schneider for expert assistance in powder dif-fractometry, Margit Bauer for her help in X-ray crystallography, andMary Kania for critical reading of the manuscript.

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FIG. 11. X-ray powder diffraction pattern of the isolated and purified sheaths of P. uncinatum. The recorded sharp signals indicate a substantial degree ofcrystallinity of the sheath material. The four prominent peaks correspond to the four diffraction lines in the X-ray diagram (Fig. 10A). The additional diffraction signalsindicate further characteristic small spacings within the lattice of the crystalline sheath material.

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