VOL. 2 9 (1958) BIOCHIMICA ET BIOPHYSICA ACTA I

T H E CELL W A L L OF MYXOCOCCUS X A N T H U S

D. J. MASON* AND D O R O T H Y P O W E L S O N

Department o/Biological Sciences, Purdue University, La/ayette, Ind. (U.S.A .)

INTRODUCTION

The microorganisms classified as Myxobacteria exhibit traits which suggest that these bacteria are transitional organisms between the Eubacteria and more organized forms. Myxobacteria are characterized by a peculiar gliding motility on solid surfaces, but the cells have no flagella. The long vegetative cells, which are flexible, will aggregate into complex fruiting structures and form microcysts. These microcysts may be bacillary, oval or spherical.

Because of their unique behavior, one might suspect that the Myxobacteria have cell walls different from those of the Eubacteria. We are becoming more familiar with the chemistry of cell walls of Eubacteria 1, 3, 3. Practically all that we know about cell walls of Myxobacteria is that they are susceptible to penetration and lysis by phagO and that the microcyst wall looks very much like walls of some Eubacteria 5. This paper describes research on the isolation and characterization of the cell wall of Myxobacteria.

METHODS

Myxococcus xanthus was used for all of the experiments. The culture was isolated and identified by HOLT e. All cell crops were grown in a modified NOREN'S broth ~ containing o. 5 % trypticase (BBL), 0.2 % K~HPO 4, o.i % NaC1, o.oi % MgSO,- 7HzO, traces of Ca(NO3)2, FeSO 4 and MnSO 4. The p H of the autoclaved broth was about 7.3-

The cells were grown in i-1 flasks containing 15o ml of the broth. Inoculated flasks were shaken at 27 ° (lOO-2 ½ inch strokes]min). The culture originally isolated grew slowly and clumped in the broth, bu t s t ra ins were isolated which grew dispersed in the broth. These strains did not spread on agar, bu t they did form fruiting bodies and microcysts 8. A dispersed growing culture designated s t ra in ANS was used. Growth curves were made f rom optical density changes at 61o m/~ (Coleman model 14 spectrophotometer) . The strain was subcultured in bro th every 48 h at least three t imes before inoculation into flasks to get experimental batches of ceils; the inoculum was 5 ml. The ceils were harvested f rom cultures jus t entering the s ta t ionary phase of growth and washed at least three t imes by centrifugation. All water used for washing and resuspending cells was de-ionized.

A t t empt s were made to isolate cell walls by a var ie ty of techniques. No "clean" walls could be isolated f rom cells which had been broken wi th glass beads or by sonic oscillation. A slight modification of techniques used by •OMURA AND HOSADA 9 and SALTON 10 permit ted the isolation of cell-wall material. Washed myxobacter ia l cells from 300 ml of culture were resuspended in IO ml water. The cells were squir ted into ~r4o nil water previously heated to 75 °, and the suspension was mainta ined at this t empera ture for 15 min. The heated cells were washed one t ime in water and resuspended in 5 ° m l water . 25 ml of o.o 5 M Na veronal buffer (pH 8.o) were added along wi th 5 ml of salt-free crystalline t ryps in (i mg/ml) ; the mixture was incubated at 45 ° for 4 to 6 h.

A variat ion of this procedure was also used. Washed cells from 3o0 ml culture were suspended in ioo ml of 7 ° % ethanol for 45 rain. The cells were washed two t imes with water and resuspended

* Predoctoral fellow of the National Ins t i tu tes of Health. Present address: Depar tment of Microbiology, The Upjohn Company, Kalamazoo, Mich. U.S.A.

Re/erences p. 7.

2 D . J . MASON, D. POWELSON VOL. 29 (x958)

in 5 ° ml water. Twenty-five ml 0.05 M Na veronal (pH 8.0) buffer and 5 ml crystalline salt4ree chymostrypsin (i mg/ml) were added. The mixture was incubated at 45 ° for 4 to 6 h.

In both procedures, the particulate material resistant to enzyme digestion was washed once with water and then once with i M NaC1. The cell walls were separated from any whole cells and large debris by centrifugation at 4o00 g for ie lain. The supernatant material was washed eight times with water (centrifugation at io,ooo g for 15 min). Most data reported were obtained by analysis of material from heat-killed, trypsin-treated cells, though some analyses were also made on ethanol-fixed, chymotrypsin-treated cells,

Analyses Cell-wall yields were determined by dry-weight determinations. Cells and cell walls were

dried at 8o ° and weighed until three consecutive weighings 24 h apart, were within i mg of each QLher. All quantitative measurements were run in duplicate.

"Lipid" content was determined by extracting the wall material with ethanol-ether (3/1). The walls were suspended in the extracting solvent for 12 h at 25 °. Aliquots were dried and weighed before and after extraction. The pigments tha t were extracted were chromatographed on celite or silicic acid columns using petroleum ether (b.p. 6o-11o °) as a developing agent.

Total carbohydrate was determined with a quantitative Molisch reaction (DlscltE) 11 and measured in terms of glucose. Total nitrogen was determined by the Kjeldaht method described by WILl.ITS AND OGG 12.

Determination o/amino acids To determine amino acid content, cell walls were refluxed in HCI for 24 h, about 5O-lOO mg

(dry weight) of walls and 2o ml 6 N HC1. The hydrolysate was dried and freed from HC1. The hydrolysate was spotted as bands on Whatman No. i chromatograph paper and separated into individual bands of amino acids. N-butanol-acetic acid-water (4 : i : 5 and 49-5 : I : 49.5), phenol- HsO, pH 12.o buffered phenol, pH 8. 4 buffered phenol and pH 8. 4 buffered n-butanol-benzyl alcohol x3 were used as developers. Separated bands were detected by spraying narrow strips of the chromatograms with ninhydrin. The amino acids were eluted from the bands and cochromato- graphed with known amino acids.

Special color sprays for sulfur amino acids and the prolines were used for detecting these materials li. Diaminopimelic acid was detected as follows. An acid hydrolysate (about I nag cell wall material) was applied as a 4-inch band on Whatman No. 3 MM paper and the cystine converted to cysteic acid with H,Oz 14. The paper was saturated with pH 5.4 Na acetate buffer (o.o5 M). A potential of 8 V/cm was applied to the paper for 6 h with Reco-E-8oo-2 paper electrophoresis equipment. The neutral amino acid band and the bands corresponding to the basic amino acids were eluted and cochromatographed for diaminopimelic acid with methanol-pyridine-HC1-H,O TM

and also to detect lysine, arginine and histidine.

Determination o] sugars Cell walls (50-1oo mg dry wt.) were hydrolyzed in 20 ml of 2 N HC1 at IOO ° for 4 h in sealed

tubes. After centrifugation at 2o,ooo g for 15 re.in, the supernatant was dried to remove HC1. The hydrolysates were chromatographed one-dimensionally and two-dimensionally on Whatman No. I paper. One-dimensional chromatograms were run with n-butanol-acetic acid-water (4: I : 5) and n-butanol-ethanol-water (5:1:4). Two dimensional chromatograms were developed in one direction in the butanol--ethanol-water solvent and in the other direction with phenol-water. Aniline acid phthalate, p-anisidine phosphate and aramoniacal AgNO3 x4 were used as detecting and identifying sprays. Amino sugars were detected by the Elson-Morgan spray 14.

Cytology The degree of isolation of the cell walls was determined by electron microscopy. Only prep-

arations which were completely free of cytoplasmic debris and granules were used in the analyses. Cells and isolated cell walls were fixed in OsO 4 and embedded in methyl-methacrylate for thin sectioning.

RESULTS

Fig. I s h o w s a t y p i c a l g r o w t h c u r v e of s t r a i n A N S of M. xanthus. Cells h a r v e s t e d for

ce l l -wal l i so la t ions were f r o m cu l t u r e s j u s t e n t e r i n g t h e s t a t i o n a r y g r o w t h p h a s e

(O.D. = ca. 1.4o ). T h i n sec t ions of t h e s e cells s h o w t h a t each cell is s u r r o u n d e d b y t w o

e l e c t r o n - d e n s e l aye r s s e p a r a t e d b y a n o n - e l e c t r o n - d e n s e l a y e r (Figs. 2, 3, a n d 4). T h e t w o

e l e c t r o n - d e n s e l aye r s are each a b o u t 75 ]k a n d t h e n o n - d e n s e l a y e r a b o u t IOO A th ick .

Re#fences p. 7.

VOL. 29 (1958) CELL WALL OF Myxococcus xanthus 3

O.D. 610 m~ 1.50

1.40

1.30

1.20

1.10

1.00

0.90

0.80

0.70

0.60

0.50

0.40

0 .30

0.20

0.10,

I I I I ! I I I I I i I

0 4 8 12 16 20 24 26 32 315 40 44 4B H o u r s

Fig. I. G rowth curve of M. xanthus (disperse g rowing s t ra in) a t 27 ° in t ryp t i case sa l t s bro th .

Sections of the resting cells or microcysts (of M. stipitatus) show that the walls are similar in morphology to vegetative cell walls but are probably more rigid; the photo- graphs are not clear enough to be included here.

Walls isolated from heat-killed M. xctnthus are shaped as though removed from spherical cells (Fig. 5). Packed pellets of the walls are orange and non-turbid. Thin sections of these wails show that the two electron-dense layers are still present, but they are separated by a wider middle layer than that found in whole cells (Fig. 7). Walls isolated from ethanol-treated cells retained the rod shape of the vegetative cells (Fig. 6) ; these walls were not pigmented.

Table I summarizes quanti tat ive data on walls from heated and from ethanol- treated cells. The pigments present in the walls from heated cells were extracted by ethanol-ether. This fraction was separated into one orange and two yellow pigments.

During the refluxing of the walls from heated cells in 6 N HC1 a white material collected on the cooling coils of the reflux condenser. This material was not liberated from the walls of ethanol-extracted cells or from walls extracted with ethanol-ether (3 : I). Samples removed from the condenser were insoluble in water and IO % KOH. The material was soluble in ether, ethanol, acetone, chloroform, methanol and petroleum ether (b.p. 6O-lOO°). Refluxing whole cells of Escherichia coli in 6 N HC] yielded a substance with similar properties. This peculiar lipid has not been identified.

Table I I lists the amino acids and sugars detected by paper chromatography of hydrolyzed walls from the heated cells. Amino acids and sugars in the walls from ethanol-treated cells were not identified. Preliminary paper chromatography of

References p. 7,

4 D . J . MASON, D. POWELSON VOL. 29 (1958)

Figs. 2 and 4- Electron micrographs of thin sections of M. xanthus. Fig. 3. Higher magnification of area outlined in Fig. 2.

VOL. 29 (1958) CELL WALL OF Myxococcus xanthus 5

Fig. 5. Elec t ron m; c rog raph of air-dried cell-wall p repa ra t ion f rom cells by hea t and t r yps in t r e a t m e n t . C h r o m i u m shadow.

Fig. 6. Elec t ron mic rograph of air-dried cell-wall p repa ra t ion f rom cells by e thano l and c h y m o - t r yps i n t r e a t m e n t . C h r o m i u m shadow.

Fig. 7. E lec t ron mic rograph of a th in sec t ion of cell walls p repared f rom cells by hea t a n d t ryps in t r e a t m e n t . Wal ls no t washed in I M ~NaCI.

D. J. MASON, D. POWELSON VOL. 2 9 (I958)

TABLE I

GROSS C O M P O S I T I O N OF C E L L W A L L S I S O L A T E D F R O M M . x a ~ t h u s

Wall~ /tom Walls/rora ethanol-Itched

heated ceils cells

% of cell dry weight recovered as cell walls

% of cell wall dry weight extracted with e thanol -e ther (3 : i)

% cell wall dry weight as Kjeldahl nitrogen

% of cell wall dry weight as carbohydrate (as glucose)

7.4-8.4 4.4-4.8

49-54 23-25

5.o-5.4 8.4-9, 3

4-7-5.I 7.2-7,5

TABLE II

A M I N O A C I D S A N D S U G A R S D E T E C T E D B Y P A P E R C H R O M A T O G R A P H Y OF H Y D R O L Y Z E D

C E L L W A L L S F R O M H E A T E D M. xamhus

Amiz~o acids Sugars

Alanine Leucine Arginine Lysine Aspartic acid Phenylalanine Cystine Proline Diaminopimelic acid Serine Glutamic acid Threonine Glycine Tyrosine Hydroxyproline Valine Isoleucine

Galactose Glucose Hexosamine Rhamnose

hydrolysates from these walls indicated that they probably had the same amino acids and sugars as the walls from heated cells.

DISCUSSION

The walls isolated from M. xanthus are similar in composition to the walls isolated from other gram-negative bacteria 3. Because of the higher lipid content of the Myxobacterial walls, however, the total carbohydrate and total nitrogen are relatively low. At the present time, we have no way of knowing how the natural distribution of materials has been altered by the isolation procedures. The use of proteolytic enzymes has been said to strip the wall to its "basal" constituents in the case of gram-positive bacteria but not gram-negatives 2.

The discovery of carotinoid pigments of M. xanthus in the walls of the cells was unexpected. Except for the walls of Rhodospirillum rubrum, the walls of pigmented bacteria seem to be colorless ~.

An interesting comparison can be made between walls isolated from heated cells and from ethanol-treated cells of M. xanthus. The ethanol (7 ° %) apparently extracts considerable lipid (including the pigments) without removing very much carbohydrate or protein. The total carbohydrate and nitrogen then constitute a greater proportion of the walls isolated from ethanol-treated cells than from heated cells.

Re/erences p. 7.

VOL. 29 (1958) CELL WALL OF Myxococcus xanthus 7

Some observations related to the round shape of walls from heated cells should be briefly presented here. The heated cells remained rod-shaped until they were treated with trypsin. When they were also treated with 7 ° % ethanol before digestion with trypsin the walls were rod-shaped. Likewise, when the cells were treated first with ethanol and then with heat (75 ° for 15 min) before digestion with trypsin the walls remained rod-shaped. Several possible explanations for the effect of ethanol treatment can be considered. The ethanol may denature and coagulate the proteins in the wall, so preserving the shape of the cells, as by fixation. Or the enzymic digestion of struc- tural protein complexes may be prevented because of the alteration of the molecules by the ethanol; however, proteins fixed by ethanol are usually susceptible to trypsin or pepsin. On the other hand, the extraction of lipid by the ethanol may reduce the flexibility of the walls.

Nothing was observed that would give a clue about the creeping movement of Myxobacteria. No evidence was found for pores through the cell wall or for contractile fibers in the wall. The high lipid content characteristic of the isolated walls, from cells not treated with ethanol, might explain the flexibility of the living cells.

S U M M A R Y

T h e v e g e t a t i v e cell of Myxococcus xanthus h a s a wall a b o u t 25o J~ thick. Wal ls can be i so la ted f rom t h e s e cells by d iges t ing h e a t e d or e t hano l - t r e a t ed cells w i th t r yps in or c h y m o t r y p s i n . Wal ls in t he s h a p e of sphe res are ob ta ined f rom h e a t e d cells while e t hano l - t r e a t ed cells yield rod - shaped walls. Wal l s isolated f rom h e a t e d cells a re p i g m e n t e d and are a b o u t 5o % lipid, 5 % n i t rogen a n d 5 % c a r b o h y d r a t e . Wal l s f r om e t h a n o l - e x t r a c t e d cells are colorless a n d are lower in lipid and h igher in n i t r ogen a n d c a r b o h y d r a t e c o m p o n e n t s . Seven teen amino acids were identif ied in isolated walls of M. xanthus. C o n s t i t u e n t suga r s in t h e walls were: glucose, galactose, r h a m n o s e a n d a hexosamine .

R E F E R E N C E S

1 C. S. CVMMINS, Intern. Rev. Cytol., 5 (1956) 25. E. WORK, Nature, 179 (1957) 841.

3 M. 1~. J. SALTON, in Bacterial Anatomy, Cambr idge Un ive r s i t y Press , Cambridge, 1956, p. 81. 4 R. L. ANACKXR AND E. J. ORDAL, J. Bacteriol, 7 ° (1955) 738. 5 M. E. LOEBECK AND E. J. ORDAL, J. Gen. Microbiol., 16 (1957) 76. e j . HOLT, Ph .D . thesis in p repara t ion , P u r d u e Unive r s i ty , La faye t t e , 1958. 7 B. INIOREN, Svensk Botan. Tidshr., 46 (1952) 324 . 8 j . ADYE, P h . D . thesis in p repa ra t ion , P u r d u e Unive r s i ty , La faye t t e , 1958. 9 M. NOMURA AND J. HOSODA, J. Bacteriol., 72 (1956) 573.

10 M. R. J. SALTON, Biochim. Biophys. Acta, io (1953) 512. 11 Z. DISCHE, in D. CLICK, Methods o/Biochemical Analysis, Vol. II, In te rsc ience Pub l i she rs Inc. ,

New York, 1955, p. 313 • is C. O. WILLITS AND C. L. OGG, J. Assoc. Off. Agr. Chemists, 33 (195 o) 179. la E. F. MCFARREN, Anal. Chem., 23 (I951) 168. 14 R. J. BLOCK, R. L~STRANGE AND G. ZWEIG, Paper Chromatography, A Laboratory Manual,

Academic Press Inc. , New York, 1952. t5 L. E. RHULAND, E. WORK, R. F. DENMAN AND D. S. HOARE, J. Am. Chem. Soc., 77 (1955) 4844 •

Received January I l th , 1958