Glucan Common to the Microcyst Walls of Cyst-Forming Bacteria IAN W

Home , Glucan, Stigmatella aurantiaca

JOURNAL OF BACTRIOLOGY, Feb. 1977, p. 599-605 Vol. 129, No. 2 Copyright C 1977 American Society for Microbiology Printed in U.S.A. Glucan Common to the Microcyst Walls of Cyst-Forming Bacteria IAN W. SUTHERLAND* AND C. LINDSEY MACKENZIE Department ofMicrobiology, University ofEdinburgh, Edinburgh, EH9 3JG Scotland Received for publication 21 September 1976 Chemical analysis indicated that 1)-glucose is the major neutral monosacchar- ide present in the microcysts of a range of gram-negative bacteria. Varying amounts ofother neutral sugars were found. The glucose was mainly present as a glucan that could be extracted from microcysts of representative strains with alkali or mild acid treatment. The glucan could be identified as an a-1,3-linked polymer on the basis of (i) periodate resistance ofthe extracted polymer and the material present in microcysts; (ii) lectin agglutination of the microcysts; (iii) lectin precipitation ofthe extracted glucans; and (iv) susceptibility ofthe glucan either in the walls or after extraction to a specific a-1,3-glucanase from Asper- gillus nidulans, yielding glucose as the sole hydrolysis product. The galactosamine found in microcysts of Myxococcus xanthus by other workers is clearly a component of another polymer, distinct from the glucan. The presence of an a- 1,3-linked glucan, common to microcyst walls ofvarious bacterial genera, probably contributes to the rigidity ofthe walls ofthese forms and, inter alia, to their resistance to ultrasonic treatment. Preliminary experiments indicate that the glucan is discarded on germination of the microcysts rather than being broken down by specific enzymes. Gram-negative bacteria are unable to form ance were associated with the synthesis of an endospores, but a limited number ofspecies can additional cell layer. The presence of glucose, form a resting phase resistant to ultrasonic ir- associated with peptidoglycan in both vegeta- radiation but not to heat. The resting form is tive bacteria and microcysts ofM. xanthus, was generally termed a microcyst (or, in myxobac- reported (8). The amount of glucose present in teria, a myxospore). Such microcysts are con- the lipopolysaccharide of bacilli is much fined in their occurrence to four groups ofbacte- smaller than that found in cysts, and the proba- ria: Azotobacteriaceae, Methylomonadaceae, ble loss oflipopolysaccharide during cyst forma- Sporocytophaga species, and Myxobacterales tion suggests that the sugar is present in differ- (2). In myxobacteria the microcysts are gener- ent polymers (17). The glucose is probably not ally found within fruiting bodies of varying part ofthe same polymer as the galactosamine, complexity, but in the other three bacterial since the neutral sugar content of myxospores groups they are found free. The morphological was variable but increased after galactosamine changes occurring during microcyst formation synthesis had apparently ceased, the total car- and germination in Azotobacter vinelandii and bohydrate continuing to increase over a 24-h in Myxococcus xanthus are well documented period (1, 10). and have been recently reviewed by Sadoff (15) The walls of A. vinelandii microcysts also and Sudo and Dworkin (16), respectively. Use differ in their carbohydrate composition from of these two species has the advantage that, the walls of the vegetative cells (15). 1-Glucose through the use of appropriate inducers, syn- is again present, although mannose, xylose, chronous conversion ofbacillary forms to micro- and rhamnose were also reported. Associated cysts can be achieved and relatively large with the neutral sugars are -mannuronic acid amounts of material can be prepared. and L-guluronic acid from bacterial alginate, Microcysts of M. xanthus contain relatively the proportions of the two uronic acids, not - large amounts of n-glucose and r.-galactosa- and L-isomers of the same acid (12), varying in mine, and the increase in amino sugar content the intine and exine layers of the microcysts after microcyst induction coincides with the in- (15). creased resistance to ultrasonic treatment (21). It seemed possible that similarities existed Dworkin (6) suggested that optical refractility, between the composition of the walls of all mi- increased galactosamine, and ultrasonic resist- crocyst-forming bacteria, and this paper reports 599 600 SUTHERLAND AND MACKENZIE J. BACTERIOL. a comparison of the chemical composition of Polysaccharide extracts. Microcysts (myxo- microcysts from different genera and species spores) were extracted with 1 N NaOH at 50°C for 30 representing three of the four microcyst-form- min (21) or with 5% (wt/vol) KOH for 16 h at 37°C ing bacterial groups. Attempts to obtain ade- (9). After neutralization, the extracts were dialyzed exhaustively against running tap water and dis- quate quantities of Sporocytophaga myxococ- tilled water and lyophilized. Acetic acid extracts coides microcysts, free from medium-derived were prepared as described earlier (17). cellulose, were unsuccessful. Chemical analysis. Polysaccharide preparations were hydrolyzed with 1 N H2SO4 for 12 h in sealed MATERIALS AND METHODS tubes, neutralized with saturated Ba(OH)2, and sep- Bacterial strains and methods of culture. Azoto- arated into neutral and basic fractions by prepara- bacter species were grown routinely in Burks nitro- tive paper electrophoresis. Acidic material was gen-free medium with glucose as carbon source. found in Azotobacter preparations, and its identity After 36 h at 30°C in 250-ml flasks on a reciprocal as a mixture of D-mannuronic acid and i-glucuronic shaker, yielding mid- to late-log-phase cells (E530 = acid was confirmed by paper chromatography. 0.7 to 0.8), vegetative cells were centrifuged asepti- Paper chromatography and microanalysis. These cally and microcysts were induced by resuspension assays were done by methods previously described in Burks medium containing 0.2% (wt/vol) 6-hy- (16, 17). Glucose was determined by the oxidase droxybutyric acid and incubated for a further 48 h at reagent; galactosamine was assayed with the galac- 30°C. Cysts were then recovered by centrifugation tose oxidase procedure. Xylose was used as an inter- and washed in saline and water before lyophiliza- nal standard. to correct for losses of carbohydrates tion. Bacilli for lectin studies were taken from the during separation procedures. All chromatograms initial cultures. M. xanthus strain FB was grown in were run in descending solvent systems. These Casitone broth (medium C10 of Reichenbach and were: butan-1-ol-pyridine-water (6:4:3), solvent A; Dworkin) (14) for 4 h at 30°C in 1-liter amounts in 2- ethyl acetate-pyridine-acetic acid-water (5:5:1:3), liter Erlenmeyer flasks on a shaker, to give early- solvent B; and butan-1-ol-acetic acid-water log-phase cells (E530 = ca. 0.4). Microcysts were (50:12:25), solvent C. Irrigation times were 24, 24, induced by the addition of 0.5 M glycerol directly to and 60 h, respectively. the cultures and harvested after a further 4 or 16 h Enzymes. Cellulase from Trichomonas viride and (7). To obtain fruiting bodies of other myxobacterial a Cytophaga 8-glucanase preparation were pur- strains, cultures were grown on cell agar (0.1% [wt/ chased from British Drug Houses, Poole, England; vol] Enterobacter aerogenes cells in tap water agar) a- and 8-amylases were from the Boehringer Corp., for 6 to 14 days at 30°C. Fruiting bodies were re- Lewes, England. The specific exo a-1,3-glucanase covered as described by Sutherland and Thomson fromAspergillus nidulans (25) was the generous gift (18). The single strain of Cystobacter velatus was of B. J. M. Zonneveld. grown similarly on yeast cell agar. Vegetative cells Lectin tests. Slide agglutination tests were per- were isolated from cultures on 2% (wt/vol) Casitone formed according to Cruickshank (5), using lectin agar. All myxobacterial cultures were incubated in solutions (5 mg/ml) and vegetative cell or microcyst sealed polythene bags to maintain the humidity of suspensions containing 109 and 108/ml, respectively. the cultures. Results were viewed after 2 min at 20°C and reexam- The culture of "Methylomonas albus" was grown ined at 5 and 10 min. in a salts medium gassed with methane for 4 to 5 Capillary precipitation tests were done by using days at 30°C as described by Whittenbury et al. (23). soluble extracts (10 mg/ml) added to equal volumes Mycrocyst isolation. Microcysts from Azotobacter of aqueous lectin solution (60 or 120 ,ug/ml) in fine species were the only form ofcells present in induced capillary tubes. The tubes were examined under a cultures and were recovered directly by centrifuga- hand lens after 16 h at 20°C. tion. The other bacteria investigated contained a mixture of vegetative forms and myxospores in both RESULTS induced cultures and fruiting bodies. In addition, Composition of microcysts. The properties most produced relatively large amounts of exopoly- common to microcysts or myxospores from dif- saccharides. Myxospores were separated from con- taminating material by centrifugation after de- ferent bacterial species and genera indicated struction of vegetative bacteria by ultrasonic treat- the possibility that similar or identical chemi- ment: 15-s. exposure to a nominal 20 kc/s in an MSE cal components were present to confer these (London) sonic oscillator. The myxospores were properties. Only Azotobacter vinelandii micro- thoroughly washed in distilled water. For lectin ag- cysts are readily lysed to yield good wall prepa- glutination, some of the myxospores from fruiting rations. Alternatively, acetic acid extracts can bodies or induced with glycerol were also suspended be obtained from most microcysts, yielding directly in distilled water without ultrasonic treat- much ofthe cell wall carbohydrate. Analyses of ment to obviate the possible removal of surface lay- these preparations revealed that all contained ers. Methylobacterial microcysts were prepared in the same way as those from myxobacteria. 1-glucose as the major identifiable carbohy- Lectins. Lectins were obtained from Pharmacia drate component (Table 1). Most myxospores Ltd. (London) and used in slide agglutination tests also contained D-galactosamine, whereas Azo- or capillary precipitation tests by standard immuno- tobacter and related species contained glucosa- logical procedures (5). mine. To confirm that no other polysaccharides VOL. 129, 1977 MICROCYST WALL GLUCAN 601 TABLz 1. Composition of bacterial microcyst polysaccharides Sugars present (as % of total) Microorganism Strain Source of cysts Prepn Glucosa- Galactosa- Glucose Galactose mine mine Azotobacter vinelandii ATCC 12837 Induced Cysts 73.8 0 20.1 5.9 A. vinelandii Av Induced Cysts 60.5 4.6 34.8 0 A. vinelandii Az Induced Cysts 73.7 0 20.2 5.8 A. chroococcum NC1B 8002 Induced Cysts 46.2 8.5 52.3 0 Beverinckia indica NCIB 8005 Induced Cysts 71.4 1.8 25.2 0 "Methylomonas albus" BG8 5-Day culture Cysts 60.9 6.6 15a 4.5 Myxococcus xanthus FB FBb Cysts 70.1 0.5 4.5 23.9 Induced Cysts 63.1 0.2 6.8 26.7 Myxococcus sp. E FB Cysts 51.5 15.6 9.9 23.5 F2 FB Cysts 68.3 1.4 0 30.3 F3 FB Cysts 49.7 0.7 0.7 48.7 Cystobacter velatus Plv9 FB Cysts 53.8 19.2 3.4 20.6 Archangium sp. W FB Cysts 68.7 11.2 5.0 15.1 P FB Cysts 67.2 4.0 0 28.0 A FB Cysts 60.2 1.5 1.9 36.4 W3 FB Cysts 69.4 4.6 0 25.9 a Various unidentified amino sugars - total amino sugar. b FB, Fruiting bodies. remained unextracted, the microcyst residues TABLz 2. Recovery ofglucose after periodate were also hydrolyzed and the monosaccharides oxidation of microcysts and extracts were separated by paper electrophoresis and Microorga-Microorga* Strain Prepn ery% Recov-of glu- paper chromatography (solvent A). The sugars nism present were the same as those in the acetic cosea acid extracts, albeit in very small amounts, Mxyococcus FB Microcysts 85-91 confirming that almost all the polymers had xanthus Acetic acid 94 NaOH extract 93 been extracted from each microcyst prepara- KOH extract 91 tion. The A. vinelandii preparations contained varying amounts of two uronic acids, which Cystobacter Plv9 Microcysts 87 velatus NaOH extract 91 were chromatographically identical with n- mannuronic acid and i-glucuronic acid stan- Azotobacter ATCC 12837 Microcysts 70-73 dards (solvents B and C). Since these are com- vinelandii NaOH extract 83 ponents ofbacterial alginate, present as an exo- KOH extract 90 polysaccharide closely associated with the wall a Expressed as (glucose content after oxidation)/(glucose in this species, they were not quantified fur- content before oxidation). ther. Nature of the polysaccharide present in microcysts. The glucose present in the microcysts Preparation and properties of glucans. Glu- may form part of a heteropolysaccharide or be cans can be extracted from eukaryotic micro- derived from a glucan. The observation that in organisms by treatment with alkali, and a sim- M. xanthus strain FB the glucose and galactos- ilar technique was used to prepare glucose-con- amine contents of microcysts increased inde- taining material from myxospores of M. xan- pendently (10) indicated that two distinct poly- thus strain FB (20). Both alkaline extraction mers were probably present. Neither the intact procedures were used to obtain glucans from cysts nor extracts were susceptible to digestion three representative microcyst (myxospore) with cellulase preparations either alone or in preparations. These preparations were essen- the presence of peptidoglycan-degrading en- tially free of amino sugars when aliquots were zymes. Helix pomatia juice, used to digest yeast hydrolyzed and examined chromatographically; cell walls, was also inactive. In an attempt to neutral sugars other than glucose were also deternine the position ofthe interglucose link- present in only trace amounts. The residual ages, microcysts and extracts were treated with microcyst material contained the same amino periodate. This did not lead to any appreciable sugars as were originally present, and there diminution in the glucose content of either the appeared to be little diminution in their cysts or the extracted material (Table 2). The amounts. Small amounts of glucose were also resistance to periodate indicates that glucose is present in the residual myxobacterial material, present predominantly in n-glucopyranosyl 1 and rather larger amounts were present in the 3-D-glucopyranose links in the polymer. Azotobacter microcyst residues. 602 SUTHERLAND AND MACKENZIE J. BACTERIOL. The highest yields of glucan were obtained saccharides as the dominant components,indi- from M. xanthus myxospores using the 5% cated the possible application of lectins. Al- KOH procedure; the yield by this method though concanavalin A (ConA) reacts with a- amounted to 25 to 30% of the dry weight. glucosyl, a-mannosyl, or fructosyl residues, Slightly lower yields were obtained using the mannose and fructose are absent from the mi- alternative (NaOH) alkaline extraction proce- crocysts. Any interaction can thus be ascribed dure. The amount ofmaterial extracted fromA. to a-glucosyl residues. Wheat germ agglutinin vinelandii and from C. velatus by KOH extrac- is specific for 43-linked N-acetyl-D-glucosamine, tion was approximately 20% of the dry weight. whereas soyabean agglutinin binds toN-acetyl- Partial acid hydrolysis of the glucan prepara- n-galactosamine (11). Accordingly, these three tions from all three sources, using the proce- Iectins were used in slide agglutination tests at dure adopted by Johnston (9) for Aspergillus a final concentration of 5 mg/ml to test the niger glucan, yielded glucose and an oligosac- agglutinability of bacilli and the corresponding charide that, from its chromatographic proper- microcysts from a range ofdifferent strains and ties (solvents A and B), was probably nigerose species (Table 3). Although autoagglutination and differed from all other available glucose- of the lectin-free controls was a problem in containing disaccharide standards. The glucans Azotobacter preparations, in general it is clear were periodate resistant (Table 2), providing that ConA agglutinated the microcysts but not further indication that they are 1 -*3 linked. the corresponding vegetative bacilli. An excep- Cellulase preparations, a- and 8-amylases, tion was the M. albus strain, which is known to and a ,3-glucanase preparation from Cytophaga contain a lipopolysaccharide with high 1)-glu- species all failed to hydrolyze the polymers, but cose content (I. W. Sutherland, unpublished glucose was released by the action of a specific data). However, in this strain the microcysts exo a-1,3-glucanase from Aspergillus nidulans were very much more strongly agglutinated (25). Typical results for release of glucose from than were the bacilli. Despite the high galac- M. xanthus glucan yielded over 90%o of the tosamine content ofmyxospores, only one prep- available glucose in a 20-h period under the aration was agglutinated by the soyabean lec- conditions used; glucose release from the A. tin; no increased agglutination ofthe Azotobac- vinelandii glucan and from that of C. velatus ter microcysts were observed with the wheat was somewhat slower and amounted to 70 to germ agglutinin. 80%o of the total glucose after 36 h. Glucose Slide agglutination has the advantage of release in all three systems was linear with no being a simple technique that uses small lag. This, taken with the small amount of glu- amounts ofmaterials, but has the disadvantage cose in these preparations that was destroyed that results are difficult to quantify. Capillary by periodate, indicates the possible contamina- precipitation tests were therefore performed on tion ofthe a-1,3-glucan with other glucose-con- the extracted glucans that were available and taining material. on a preparation ofAzotobacter vinelandii "ex- Use of the a-1,3-glucanase to digest intact ine," which was also known to contain rela- microcysts also led to some glucose release. tively large amounts of n-glucose. In all cases Even after prolonged incubation, this was precipitations occurred with ConA, the mini- never more than 15 to 20% of the available mum concentrations being ofthe order of6 to 12 glucose. To identify the products of enzyme ,g of ConA to precipitate 1 mg of extract dis- treatment, portions of all digests, i.e., polymer solved in 100 ,ul (Table 4). The Azotobacter and microcyst, were applied to chromatograms exine preparation also precipitated with the along with glucose and various disaccharides as other lectins, and one M. xanthus preparation standards. After development in solvent A and showed slight precipitation with soyabean ag- treatment with alkaline silver nitrate, glucose glutinin. This particular material contained was the only product identified. more galactosamine than did the KOH extract Lectin studies. Although sufficient material of microcysts from the same strain. was available from some of the strains to per- Attempts to isolate an a-1,3-glucanase from mit analysis, periodate oxidation, etc., of var- bacilli or from genninating microcysts of sev- ious preparations, relatively small amounts of eral of the species used in this study have been fruiting-body-derived myxospores were ob- unsuccessful. Probably the replacement of the tained. Yields of Methylomonas albus micro- glucan in the microcyst wall by the characteris- cysts were also low. Since some material was tic lipopolysaccharide of the bacilli involves needed to perform the analyses presented in complete rejection of the polymer rather than Table 1, an alternative method of examining enzymatic degradation (17). This agrees with the nature of the polymer in these microcysts cytological evidence indicating loss of large was required. The identification ofthree mono- amounts ofthe microcyst wall material (19, 20). TABLE 3. Lectin agglutinability of some bacteria and microcysts Microorganism Strain Prepn ConA WGAa SYAb Azotobacter vinelandiii ATCC 12837 Bacilli *C Microcysts * Myxococcus xanthus FB Bacilli Myxospores +_ Myxosporesd +_ Myxosporese +_ Myxococcus E Bacilli Myxospores +_

Myxococcus Fl Bacilli Myxospores +_ Myxococcus F2 Bacilli Myxospores +_ Myxococcus F3 Bacilli Myxospores +_ Cystobacter velatus Plv9 Bacilli Myxospores +_ Archangium W3 Bacilli

Myxospores +_

Archangium W Bacilli Myxospores Archangium A Bacilli Myxospores +_ Archangium p Bacilli Myxospores +_

"Methylomonas albus" BG8 Bacilli +_

Microcysts +_ aWGA, Wheat germ agglutinin. b SYA, Soyabean agglutinin. *, Control autoagglutinates. Other symbols: +, Maximal agglutination in 2 min at 200C (slide test); , most preparations failed to agglutinate, one batch of vegetative cells agglutinated weakly after 5 min at 200C; -, no agglutination (slide or tube tests). d Induced; other myxospores are derived from fruiting bodies. eNot ultrasonically treated. TABLE 4. Lectin precipitation of microcyst material Capillary precipitationa Microorganism Strain Prepn Extract ConA WGA SYA A. vinelandii ATCC 12837 Microcyst NaOH + - - KOH + - - Exineb + + + C. velatus Plv9 Myxospores NaOH + M. xanthus FB Myxospores NaOH + - + KOH + - - a Abbreviations: WGA, Wheat germ agglutinin; SYA, soyabean agglutinin. Symbols: +, Strong precipitation at liquid interface (no visible result with lower lectin concentrations); +, slight precipitate with 120 ,ug oflectin/ml, stronger precipitate with 250 jig/ml; -, no visible reaction with lectin concentrations of60 to 500 jig/ml. b Exine is the outer layer of the Azotobacter microcyst coat. 603 604 SUTHERLAND AND MACKENZIE J. BACTERIOL. DISCUSSION have not previously been identified as bacterial The common properties of gram-negative wall components. On the other hand, a-1,3- bacterial microcysts could be expected to in- linked glucans have been found in eukaryotic clude some form of wall polymer, and although walls (3, 24). The disaccharide D-glucopyrano- the peptidoglycan of M. xanthus is known to syl-1 -* 3-n-glucopyranose was obtained from differ in the vegetative and myxospore forms of nigeran, a wall glucan containing both a-1,3 this organism (22), no evidence appears to have and a-1,4 linkages (9). An alkali-soluble glucan been presented for a polymer common to all containing only a-1,3 linkages was later identi- microcysts. On the basis of the high glucose fied in the wall ofAspergillus nidulans (24); to content of all the microcysts examined in the date no similar polymer appears to have been current study, this polymer can be postulated to found in the walls of vegetative bacteria. A. be a glucan. This confinns the results of White nidulans is also the source of a constitutive and his colleagues (1, 21, 22) for M. xanthus. enzyme specifically hydrolyzing a-1,3-glucans Surprisingly, studies on Azotobacter microcysts to glucose; it has no action on nigeran or other had not revealed a similar polymer although polymers with mixed linkage types (25). The glucose had been identified as a component of relatively inflexible 1,3-linked type of polymer the exine preparations from A. vinelandii (15). presumably confers a strong and rigid wall for The neutral sugars in such preparations are, both eukaryotic species and bacterial micro- however, accompanied by large amounts of cysts. Although the evidence obtained for mi- uronic acids derived from the bacterial algin- crocysts from methylobacteria was partially ate, which is closely associated with both exine presumptive, it appears that this type of poly- and intine layers (15). The present work has mer may be common to many bacterial micro- demonstrated that a glucan can be isolated cysts. It may be of interest to determine from representative microcyst preparations. On whether its occurrence extends to all microcysts the basis of its periodate resistance, precipita- and to other bacterial species in which some bility with ConA, and susceptibility to a spe- form of differentiation if found. cific a-1,3-glucanase, there seems little doubt ACKNOWLEDGMENTS that it is a homogeneous polymer of the type The gift of strains by H. Reichenbach, Stockheim, West -(a-Glc,p-1 --3 3-Glcp)n,- Germany; M. Dworkin, University of Minnesota; and E. The lectin agglutinability of the microcysts Williams of this Department is gratefully acknowledged. indicates that the glucan is a surface compo- We thank E. Chalmers for her technical assistance and nent that is readily accessible to the lectin. In E. Hogg for growing the methylobacteria. A. vinelandii, it may well form part of the "barklike" exine layer observed by Pope and LITERATURE CITED Wyss (13) and known to contain glucose and 1. Bacon, K., D. Clutter, R. H. Kottel, M. Orlowski, and other sugars In M. the D. White. 1975. Carbohydrate accumulation during (15). xanthus, glucan myxospore formation in Myxococcus xanthus. J. Bac- was postulated to be bound to peptidoglycan (8) teriol. 124:1635-1636. and could thus be expected to exist at or near 2. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Ber- the myxospore surface. It would appear that gey's manual of determinative bacteriology, 8th ed. the glucan forms part of the surface that The Williams & Wilkins Co., Baltimore. layer 3. Bull, A. T. 1970. Chemical composition ofwild-type and White (21) and others have termed capsules. By mutant Aspergillus nidulans cell walls. The nature of analogy with A. vinelandii, the term exine polysaccharide and melanin constituents. J. Gen. Mi- might be more apposite, since myxospores crobiol. 63:75-94. found within fruiting bodies are surrounded by 4. Ceska, M., K. Granath, B. Norrman, and B. Guggen- heim. 1972. Structural and enzymic studies on glu- capsular material that is chemically distinct cans synthesized with glucosyltransferases of some from the glucan and similar if not identical to strains of oral streptococci. Acta Chem. Scand. that surrounding vegetative cells (18). In con- 26:2223-2230. trast, the galactosamine present in M. xanthus 5. Cruickshank, R. 1960. Handbook of bacteriology, 10th ed. Livingstone, Edinburgh. myxospores does not lead to lectin agglutinabil- 6. Dworkin, M. 1972. The Myxobacteria: new directions in ity. This could be due to the amino sugar not studies of procaryotic development. Crit. Rev. Micro- being acetylated or to its being located below biol. 1:435-452. the microcyst surface. The first possibility can 7. Dworkin, M., and S. M. Gibson. 1964. A system for studying microbial morphogenesis. Rapid formation be excluded (21), and it must be assumed that of microcysts in Myxococcus xanthus. Science the glucan, and perhaps other material, oc- 146:243-244. cludes the galactosamine-contaiming polymer. 8. Johnson, R. Y., and D. White. 1972. Myxospore forma- An a-1,3-linked glucan produced as an exopo- tion in Myxococcus xanthus: chemical changes in the cell wall during cellular morphogenesis. J. Bacteriol. lysaccharide by a strain of Streptococcus mu- 112:849-855. tans has been reported (4), but such polymers 9. Johnston, I. R. 1965. The partial acid hydrolysis of a VOL. 129, 1977 MICROCYST WALL GLUCAN 605 highly dextrorotary fragment of the cell wall of As- of polysaccharides produced by Myxococcus strains. pergillus niger. Biochem. J. 96:659-664. J. Gen. Microbiol. 89:124-132. 10. Kottel, R. H., K. Bacon, D. Clutter, and D. White. 19. Voelz, H. 1966. The fate of the cell envelopes of Myxo- 1975. Coats from Myxococcus xanthus: characteriza- coccus xanthus during microcyst germination. Arch. tion and synthesis during myxospore differentiation. Mikrobiol. 55:110-115. J. Bacteriol. 124:550-557. 20. Voelz, H., and M. Dworkin. 1962. Fine structure of 11. Nicolson, G. L. 1974. The interactions of lectins with Myxococcus xanthus during morphogenesis. J. Bacte- animal cell surfaces. Int. Rev. Cytol. 39:89-180. riol. 84:943-952. 12. Page, W. J., and H. L. Sadoff. 1975. Relationship be- 21. White, D. 1975. Myxospores of Myxococcus xanthus, tween calcium and uronic acids in the encystment of p. 44-51. In P. Gerhardt, R. N. Costilow, and H. L. Azotobacter vinelandii. J. Bacteriol. 122:145-151. Sadoff (ed.), Spores VI. American Society for Micro- 13. Pope, L. M., and 0. Wyss. 1970. Outer layers of the biology, Washington, D.C. Azotobacter vinelandii cyst. J. Bacteriol. 102:234-239. 22. White, D., M. Dworkin, and D. J. Tipper. 1968. Peptido- 14. Reichenbach, H., and M. Dworkin. 1969. Studies on glycan ofMyxococcus xanthus: structure and relation Stigmatella aurantiaca (Myxobacterales). J. Gen. Mi- to morphogenesis. J. Bacteriol. 95:2186-2197. crobiol. 58:3-14. 23. Whittenbury, R., S. L. Davies, and J. F. Davey. 1970. 15. Sadoff, H. L. 1975. Encystment and germination in Exospores and cysts formed by methane-utilizing bac- Azotobacter vinelandii. Bacteriol. Rev. 39:516-539. teria. J. Gen. Microbiol. 61:219-226. 16. Sudo, S., and M. Dworkin. 1973. The comparitive biol- 24. Zonneveld, B. J. M. 1971. Biochemical analysis of the ogy ofprokaryotic resting cells. Adv. Microb. Physiol. cell wall of Aspergillus nidulans. Biochim. Biophys. 9:153-224. Acta 249:506-511. 17. Sutherland, I. W. 1976. Novel surface polymer changes 25. Zonneveld, B. J. M. 1972. A new type ofenzyme, an exo- in development of Myxococcus spp. Nature (London) splitting a-1,3 glucanase from non-induced cultures 259:46-47. of Aspergillus nidulans. Biochim. Biophys. Acta 18. Sutherland, I. W., and S. Thomson. 1975. Comparison 258:541-547.