Chemistry and Uhrastructure of Surface Layers in Prim- itive Myxobacteria: Cytophaga hutchinsonii and Sporo- cytophaga myxococcoides g. P. VERMA 1) and H. 14. MARTIN ~)

Institut fftr Angewandte Botanik, Tcchnische Hochschule, Mtinehen, Germany

In the past research on bacterial surface structures has been most actively carried on in organisms belonging to the eubacteria. Similar studies on other bacterial groups are still rare, but un- doubtedly many important surface phe- nomena remain to be discovered among the more exotic taxa. The subjects of our present studies are Cytophaga hutchin- sonii and Sporocytophaga myxococcoides, two primitive species of myxobacteria.

Among many other remarkable pro- perties, the myxobacter ia have two distinctive features which are closely related to the structure and the bio- synthetic activities of their surface layers:

First ly: In the so-called vegetative state, myxobacter ial cells are slender, rod-shaped or spindle-shaped organisms which are capable of very rapid flexing or twisting movements. No similar flexi- bil i ty is known in any of the eubacteria which surround their cell contents with compact, rigid cell walls. This functional difference has prompted several authors to conclude that vegetat ive myxobaete- ria, in contrast to the eubacteria, do not have proper cell walls at all (Stanier, 1942, 1947; Thimann, 1963).

1) Postdoctoral fellow of the Deutsche Akade- mische Austauschdicnst, Pe rmanen t address: Dept. of Botany, Universi ty of Kalyani , Kalyani , West Bengal, India.

2) Present address: Ins t i tu t ftir Mikrobiologie, Tcchnischc Hochschule, Darmstad t , Germany.

A second peculiarity is the complex life cycle of these organisms. The flexible and allegedly wall-less vegetat ive cells cooperate in a multicellular effort to form fruiting bodies for the production of microcysts or myxospores. These are spherical cells with rigid cell walls. In the genus Sporocytophaga this meta- morphosis is reduced to the simplest state. The entire spindle-shaped cells are converted into the spherical microcysts wi thout the intervention of fruiting bodies.

Cytophaga exists only in the vegetat ive form. No microcysts are formed here.

Our own work on Cytophagaand Sporo- cytophaga was under taken to answer the following questions: Do the flexible, vegetat ive cells of myxobacter ia possess a cell wall? Is murein, the specific, s tructural polymer of bacterial cell walls, also present in the non-rigid myxobaeter ial cell walls, and what are the chemical properties of the flexible murein ? Finally, what kind of chemical and morphological changes in surface structure can be observed in Sporocyto- phaga during the conversion of the flexible vegetative cells into the rigid- walled mierocysts ?

As a first lead it is useful to recall the earlier work of Mason and Powelson (1958) and Adye and Powelson (1961) on Myxococcus xanthus. From both vegetat ive cells and microcysts of this myxobacter ium Powelson and eoworkers

1967 SURFACE LAYERS IN PRIMITIVE I~IYXOBACTERIA 249

obtained cell fractions which appeared to be cell walls. As chemical components t h e y identified a set of no less than 17 different amino acids, including the murein component diaminopimelic acid. Several neutral sugars and amino sugars were also found. One may infer from these studies tha t the Myxococcus ma- terials represented complex wall prepara- tions containing perhaps some sort of murein as a minor component.

Fig. 1 presents an electron micrograph of the starting material for our cell wall work, Cytophaga hutchinsonii, grown in aerated glucose-mineral salts medium.

The bacteria were mounted on carbon- coated grids and negatively stained with 1 per cent sodium phosphotungstate in distilled water at p H 7.2. The picture immediately demonstrates the m a j o r source of difficulties which we encoun- tered in our a t tempts to isolate myxo- bacterial cell walls. The bacteria are embedded in large quantities of extra- cellular slime. Even fairly dilute bacterial suspensions have the consistency of a sticky jelly, which makes every kind of manipulation and extraction very dif- ficult.

The slime originates as a multitude of individual filaments from the cell surface and permeates the entire substrate. The average diameter of the individual fila- mentsis in the neighbourhood of ]30/~.

Vegetative cells of Sporocytophaga myxococcoides gave essentially the same picture.

We found eventual ly that diethylene glycol is a good solvent for the myxo- bacterial slimes. The dissolved slimes from both Cytophaga and Sporocytophaga were precipitated by cetylpyridinium chloride. Data on the chemical composi- t ion of the mucoid mat te r are given in Table 1. We know nothing yet about the chemical homogenei ty of the slimes. From the preliminary results we conclude ten ta t ive ly that t hey are acidic hetero- polysaccharides, and that glucuronic acid

Table 1. Chemical composition of extracellular myxobacterial slimes

Cytophaga hutchinsonii Sporocytophaga , myxococcoides

XFlose Xylose Arabinose Arabinose l~r Mannose Glucose Glucose Glucuronic acid Galaetose

Glucuronic acid

is the acidic component in both cases. Fig. 2. shows a negatively stained

preparation of Sporocytophaga microcysts preparation of Sporocytophaga micro- cysts. Under our growth conditions, Sporocytophaga cultures reach the sta- t ionary phase of vegetat ive growth after about 24 hours and then undergo large- scale conversion into microeysts within another 24--28 hours (52 hours alto- gether). The microcysts are densely packed, their cell borders are sharply defined. There is no evidence of extra- cellular slime or of a capsule. Thus, extensive degradation of the superficial slime must take place during conversion of vegetat ive Sporocytophaga into micro- cysts. The large, electron-dense bodies which are present inside almost every microcyst, were found to be meta- chromatic particles (polymetaphosphate).

Table 2 presents an outline of the pro- cedure which we applied in our search for mvxobacterial murein. Stat ionary phase }0aeteria were first given a rapid heat t rea tment to prevent the loss of murein by the action of autolytic en- zymes during the subsequent manipula- tion. The bacteria were then separated from their extracellular slime by dis- solving the slime in aqueous diethylene glycol (1 vol. d ie thyleneglyeol : 1 vol. water).

The cells were t rea ted with proteolytic enzymes and nucleases in order to solubilize and remove the heat-denatured cell contents. After this t rea tment the insoluble bacterial residue already re-

250 J . P. VERMA AND H. H. MARTIN

Table 2. Isolation of myxobaeterial cell walls

Vol. 12

Vegetative 1Vlyxobaeteria

Harvested at early stationary phase

Flash heat t reatment (96 ~ C) (Inactivation of autolytic enzymes)

1 Diethylene 91yeal/water 11 : 1)

Two extractions, eentrifugation at 10,000 rpm for 45 minutes I

Supernatant

I Extracellular slime - - ] acidic hoteropolysaecharide I

Supernatant (dissolved cell contents)

Supernatant Carotenoid pigments

Sediment

Bacteria I

Proteases and nucleases (Trypsin, DNase, RNase)

I

Insoluble residue

4 % Sodium dodeeylsulphate or 90% Phenol

Two hot extractions

Differential eentrlfugatlon

Sedimentation at 100,000 • g, 1--2 hourB

Sediment

IKUREIN SACCULI

sembled more or less empty cell walls under the phase-contrast microscope. However, the material still retained the intense yellow colour which is a typical

feature of Cytophaga and vegetat ive Sporocytophaga. The coloured material was dissolved by hot 2 per cent sodium dodecylsulphate solution. 90 percent phe-

1967 SITRFACE L A Y E R S I N P R I M I T I V E M Y X O B A C T E R I A 25I

nol acted in a similar way. The adsorption spectrum of the pigment solution showed a maximum at 450 nm. This resembled the spectrum of a carotenoid pigment, found by Anderson and Ordal (1961) in Cytophaga succinicans.

As a final product of cell fractionation we obtained an insoluble fraction of very light particles which could be sedimented only by prolonged centrifugation at 100,000 g.

Fig. 3 shows an electronmicrograph of a metal-shadowed preparation of this final sediment fraction from Cytophaga. We find very delicate, empty, tubular structures of the size and shape of the vegetative myxobacter ial cells. In their morphology the particles fit the definition which Weidel has given for the macromo]ecular, shape-conferring com- ponent of the bacterial cell wall, and which he proposed to call "Sacculus".

Fig. 4. shows a similar preparation of vegetative Sporocytophaga, revealing the entire length of a sacculus from a typical, long, slender and spindle-shaped cell.

In microcysts of Sporocytophaga the preparation of cell walls is much less of

a problem. Since there is no slime, no dicthylene glycol extraction is necessary. The carotenoid pigment of the vegetative cells is also absent. Digestion of heat- t reated microcysts with proteolytic en- zymes and nucleases, followed by sodium dodeeylsulphate extraction immediately yields empty, balloon-shaped particles, which are shown in Fig. 5. These struc- tures appear much more solid than the vegetat ive sacculi. They sediment from aqueous suspension in the centrifuge at low speed or even spontaneously upon standing.

The following tables present quantita- tive information on the chemical com- position of the cell wall preparations.

Table 3 shows data of Cytophaga walls at different stages of purification. The major wall components are the typical amino sugars and amino acids of murein: muramie acid, glucosamine, glutamic acid, alanine and diaminopimelic acid. Other amino acids are also present in smaller amounts, but repeated t rea tment with proteolytic enzymes (II) reduces their amounts still further without chang- ing the quantities of murein components~

Table 3. Molar ratio of a m m o ac ids a nd amino sugars of Cytophaga cell wa l l p r epa ra t i ons

Sam- M U R ple No.

GLU A L A DAP GIcN A SP THR S E R G L Y VAL I L E U LEU

Murein compo - nen t s ;

t o t a l ~/o- of dry weigh t

I 0.66 1.25 1.89 1.0 0.74 0.17 0.11 0.14 0.19 0.14 0.15 0.24 60.6 I I 0.69 1.16 1.85 1.0 0.61 0.07 0.04 0.06 0.08 0.05 0.05 0.07 71.1 I I I 0.70 1.05 1.90 1.0 0.75 . . . . . . . 73.6 I V 0.61 1.19 1.80 1.0 0.51 0.35 0.15 0.21 0.27 0.16 0.17 0.21 40.0

Treat, merit8:

I - - Cell wa l l s : d i e t h y l e n e glycol , t r y p s i n a nd sod ium dodecy l su lpha t e ; I I - - Cell wal l s : s a m e as I p lus p a n c r e a t i n ; I I I - - Lysozym,J sp l i t p r o d u c t s ( to t a l m urope p t i de s ) ; I V - - Cell wads : (non-heated) , d i e t h y l e n e glycol a n d sod ium dodecy l su l pha t e .

A b b r e v i a t i o n s : M U l = t - m u r a m i c ac id ; G L U - - g l u t a m i c ac id ; ALA - - a l an ine ; D A P - - d i a m i n o p i m e l i c ac id : GlcN - - g luc os a m ine ; A S P - - a spa r t i c acid; T H R - - t h r e o m i n e ; S E R - - ser ine; VAL - - va l ine ; I L E U - - i soleucine;

G L Y - - g lyc ine ; LEIY - - leucine .

252 J . P . V E R M A A N D H. H . M A R T I N Vol. 12

Tab le 4. Molar ra t io o f a m i n o ac ids a n d a m i n o sugars of Sporocytophaga cell wall p r e p a r a t i o n s

M U R G L U A L A D A P ASP G l e n T H R ~am-

ple No.

S E R G L Y V A L I L E U L E U

Mure i~ compo- n e n t s ;

t o t a l ~o of dry weight

]: 0.68 0.99 1.87 1.0 0.69 0.04 0.01 0.07 0.01 0.01 0.01 0.02 48.3 I I 0.68 0.99 1.86 1.0 0.86 . . . . . . . 73.0 I I I 0.71 1.15 2.20 1,0 0,56 0.28 0.11 0.21 0.32 0.18 0.17 0.23 22.1 IV 0.68 1.01 1.96 1,0 0.72 . . . . . . . 83.9 V 0.89 1,14 2.40 1,0 0.80 0,37 0.19 0.21 0.34 0.22 0.15 0,31 58.9

T r e a t m e n t s :

I - - Vege t a t i ve cell walls: d i e t hy l ene glycol, t r yps in , s o d i u m dodecy l su lpha te ; I I - - Vege t a t i ve cell walls: l y s o z y m e spl i t p roduc t s ( to ta l muropep t i de s ) ; I I I - - V e g e t a t i v e cell walls: (non-hea ted) , d i e thy l ene glycol , s o d i u m dodecy l su lpha te ; IV - - Mieroeys t cell walls: t r yps i n , s o d i u m dodecy l - su lpha te ; V - - Microcys t cell walls: (non-hea ted) , s o d i u m dodecy l su lpha to .

which at this stage account for more than 70 per cent of the to ta l sacculus dry weight.

The Cytophaga murein is dissolved by lysozyme. The analysis of the soluble muropeptides (III) stresses the fact tha t the quali tat ive and quant i ta t ive com- position of the Cytophaga murein is rather conventional and probably iden- tical with the composition of the mureins of such eubacteria as Escherichia coli and Proteus mirabilis, if the inevitable de- struction of the amino sugars during hydrolysis is taken into account.

Sample IV is a crude and certainly impure wall preparation which was ob- ta ined by diethylene glycol- and hot sodium dodecylsulphate extract ion alone. No proteolyt ic enzymes were used. Even here murein is the prominent cell wall component.

Table 4 shows vir tual ly the same results for Sporocytophaga. Lower "mu- rein yields" were obtained in walls of vegetat ive cells because slime extraction with diethylene glycol was less efficient than in Cytophaga.

The microcyst cell walls contain mu- rein with practically the same composi- tion as the vegetative murein. To all appearances microcyst walls are virtu- ally pure murein sacculi.

Returning to our initial questions we come to the following conclusions: Veg- etat ive myxobacter ia certainly have a cell wall. In this cell wall a murein sacculus of conventional chemical com- position is present as a major structural component. In Sporocytophaga the transi- tion from vegetat ive murein into micro- cyst murein involves no drastic changes in gross chemistry. Combining chemical and electron microscopical data, cell wall- metamorphosis during microcyst forma- tion can be understood as transformation of the very delicate murein sheet of the vegetat ive cell (which may perhaps be a monolayer), into the solid murein wall of the microcysts, where several layers of murein sheeting are probably stacked upon each other.

Information on the thickness of murein sacculi in vegetative Sporocytophaga and mieroeysts was obtained from electron- micrographs of thin-sectioned sacculi; (this work was done in collaboration with Dr. Hermann Frank, Max-Planck- Ins t i tu t ftir Virusforschung, Ttibingen).

Cross sections of vegetat ive saceuli (Fig. 6) are about 20--25 A thick. They are thus in the same range as murein sacculi of Escherichia coli and Proteus mirabilis (Frank & Dekegel, 1965; Mar- tin, Preuss & Hofsehneider, in press).

1967 S U R F A C E L A Y E R S IN P R I M I T I V E M Y X O B A C T E R I A 253

The profile of microcyst sacculi has a width of about 90--100 A (Fig. 7). I t thus has about four times the thickness of vegetat ive murein sheets.

A final word should be said on the flexibility of the vegetat ive myxobacteria. In the beginning we entcrtained the idea tha t vegetative mureins might be flcxible because they have a low degree of peptide crosslinkage, in analogy to the non-rigid mureins which are synthesized in the presence of penicillin in some L-forms of Proteus mirabilis (]V[artin, 1964; 1967). The rigidity of the microcyst murein might then have been due to an increase in peptide crosslinkage. However, chem- ical end-group determinations and mu- ropeptide chromatography of ]ysozyme split-products have provided no evidence for such a difference. Mureins from vegetative Sporocytophaga and from mi- erocysts seem, in fact, to be crosslinked to a similar extent and to a fairly high degree. On the other hand, it must be recalled that the delicate murein sacculus of Escherichia cell, for instance, is, str ictly speaking, not at all a "rigid" (i.e. inflexible) structure, once it has been

R e f e r e n c e s

Adye , J . C., Powelson , D. M.: ~licrocyste of Myxo- coccus xanthus. Chemical composition of the cell wall. J. Bacter iol . 81 : 780, 1961.

Ande r son , R. L. , Ordal , E. J . : Cytophaga succinlcan~ sp. N., a facultatively anaerobic aquatic 2tlyxo. bacterium. J . Bacter iol . 81 : 130, 1961.

P r a n k , H . , Dekegel , D.: Zur Interpretation yon Zellwandstrukturen in Di~nnachnitten gram-negati- ver Bakterien. Zbl. Bakter io l . Ab t . I , 198 : 81, 1965.

Mar t in , H . H . : Chemical composition of cell wall mucopolymers from penicillin-induced spheroplusts and normal eella of Proteus mirabilia. (Abstract ) . In : Prec . S ix th I n t e r n . Congr. B iochemis t ry , Now York, p. 518, 1964.

Mar t in , H . H . : Surface structure of normal bacteria and L-lotto* of Proteus mlrabilia and the site of action ofpeniciUln. Fol. microbiol . 12 : 234, 1967.

Mason , D. J . , Powelson , J . : The cell wall of ,~1yxo. coccus xanthus. Biochem. b iophys . A c t a 2 9 : 1 , 1958.

Startler, R . Y . : The Cytophaga group. Bacter iol . Rev . 6 : 143, 1942.

Stanier , R. Y.: Studies on nonfruitlng myxobacteria. I . Cytophaga johnsonae n. sp., a chain decomposinq myxobacteri~um. J. Bacter lo l . 5 3 : 2 9 7 , 1947.

separated from the large amounts of plastic cell wall polymers, with which it is associated in the complex cell wall (Weidel, Frank & Martin, 19601.

Much of the "r igidi ty" of the entero- bacterial cell wall probably comes from the plastic wall polymers (lipoprotein and lipopolysaccharide), which are ce- mented onto the murein basal structure.

As a working hypothesis we picture the vegetative myxobacter ial cell wall as a flexible murein monolayer tube with few, if any, accessory wall components. Additional protection for the cell contents is probably provided by the extracellular slime in which the bacteria are firmly embedded.

In its ultrastructure, the rigid sacculus of the microcyst resembles the cell wall of Gram - positive bacteria. I t may be envisaged as a complex of several super- posed and covalently interlinked murein networks.

W e are g r e a t l y i n d e b t e d to Professor O. K a n d l o r for n ~ k i n g t h e faci l i t ies a n d resources o f h is labor- s t o r y ava i lab le to us.

Our work was s u p p o r t e d b y a g r a n t f rom t h e I ) e~ t sche Forschhuagsgemeinschaf t to H . i t . M.

T h i m a n n , K . V . : The Life of Bacteria. 2rid. ed., T h e Macmi l l a~ Co., New York , N.Y. , p. 909, 1963.

Weide l , W. , F r a n k , H . , Mar t in , H . H . : The r /g /d layer of the celt wall of Escherichia col~ 8train B. J . gen. Microbiol. 22 : 158, 1960.

Diseussion

Rogers: I n cons ider ing t h e a p p a r e n t r ig id i ty o f t h e wal l of a m ic roo rgan i sm p e r h a p s one shou ld Mso con- s ider t h e in t e rna l pressure . Fo r e x a m p l e an inf la ted ba l loon appea r s r igid, t h e col lapsed care does no t . I t m a y be t h e pr inciple cha rac te r i s t i c o f t he t h i n layers of mucopep t i do in s o m e m i c r o o r g a n i s m s in tens i le s t r e n g t h r a t h e r t h a n r igidi ty . Rig id i ty , on t h e o t h e r h a n d , m a y come w h e n t h e m u c o p e p t i d e l aye r is v e r y t h i c k as in some Gram-pos i t i ve o rgan i sms . I f t he r e are seve ra l i n t e r c o n n e c t e d l ayers of cha in ma i l t he se m i g h t be r igid whe rea s one or two l ayers wou ld no t .

Verma: There is no f u r t h e r c o m m e n t f rom our side. I n fac t , t h e essence o f dr. Roge r ' s r e m a r k comes ve ry close to one o f t h e m a i n conc lus ions w h i c h we h a v e c o m m u n i c a t e d in our pape r .

254 J. P. VERMA AND H. H. MARTIN Vol. 12

Nermut: I t seems to me that the mucopeptide layer ensures the strength of the cell wall, not the rigidity in the proper sense of the word, because the ceils are always flexible. I did not observe any collapse of isolated mucopeptide membranes pre- pared by hot formamide extraction of cells.

Work: I am going to show you some E.M. pictures illustrating an interesting relationship between structure and composition in walls of Mi, crococcu.s rad4odurans. This organism, although a Gram-positive coccus, contains lipoprotein in its walls which are very complex. The organism is extremely resistant to ionlsing and other radiations, being able to s tand 1,000 times the usually lethal dose of X-rays.

In the electron microscope, the wall preparations show at least 3 layers: the outer one is protein in nature and does not appear in trypsinised prepara- tions. After t rypsin there remain 2 main structural entities, an outer layer which on negative staining is shown to consist of hexagonalty-packed spheres, each joined to its neighbours by pegs; and an inner layer of relatively structureless material penetrated by holes. The hexagonal layer is plastic and fragile and easily stripped from the inner layer, it then appears as fiat plates still maintaining the hexagonal packing; sometimes moir6 patterns are seen as well as the basic patterns. These moir6 pat terns could be artifacts resulting from slight displacement of 2 identical superimposed layers.

The holey layer is rigid and has the shape of the bacterial cell. I t consists largely of a muco- peptide, which is unusual since it contains L-orni- thine as its diamino acid (Work, 1964, Nature, 201 : 1107). In view of the large amount of protein in the whole walls, it was at first only possible to determine mucopeptide composition by examining the soluble lysozyme digest after removal of protein with TCA. (Table 1 shows the composition of this fraction.) However, subsequently in certain pre- parations it proved possible to separate the lipo- protein and the mucopeptide layers.

The wild-type organism is pink and sometimes during wall preparation something happened which removed all the pink material from the bulk of the walls. ~ ' h e n these preparations were centrifuged at 2,500 rpm in a sucrose gradient, a pink band remained on top, while a white band separated about half way down the gradient. Fur ther gra- dient3 were run on tile white band which was finally found to consist of the "holey" layer com- pletely freed of hexagonally packed material and to analyse as mucopeptide (Table 1).

Table 1. Composition of mucopeptides of Micro- coccus radlodurans

Molar ratios of constituents

Sol. lysozyme White band digest of whole from graktient

walls

Glutamic acid 1.0 1.0 Glycine 2.5 1.8 Alanine 1.9 1.7 ~.Ornlthine 1.4 1.2 Glucosamine 0.93 0.97 ]Yluramic acid 1.1 1.1

The pink layer, which was soft and gelatinous, contained only masses of hexagonally-packed ma- terial and was lipeprotein and polysaecharide in nature. Table 2 shows some of the components of

Table 2. Distribution of constituents in trypsinised walls of Alicrococcus radiodurans

Unfrac. t ionated

I walls

Pink band from

gradient

White band from

gradient

Protein + + - - Mucopeptide + - - -{- Extractable

Lipid + d- Galactose -4- not examined Glucose + + + Marmose + + - - Rhanmose + +

the whole walls and the separated layers. Apart from the amino acid differentiation into protein and mucopeptide, different sugars were also found in the 2 layers; also extractable lipid was only present in the pink layer where it made up 25% of the weight

I t is evident tha t a rigid mucopeptide.containing layer, penetrated by holes, is overlaid by a lipo- glycoprotein layer containing also carotinoids. I t is not thought tha t these specific structures play any part in determining radiation resistance, since wall structure and composition in various mutants do not line up with radiation resistance.