A study of the microbial lysis of the cell walls of soil yeasts (Cryptococcus spp.)

Soil Bid. Biochem. Vol. 1, pp. 145-151. Pergamon Press 1969. Printed in Great Britain

A STUDY OF THE MICROBIAL LYSIS OF THE CELL WALLS OF SOIL YEASTS (CR YPTOCOCCUS SPP.) D. JONES,J. S. D. BACON,V. C. FARMERand D. M. WEBLEY The Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen Stmumuy-The di!Terencein the ability of lytic microorganisms to bring about dissolution of the cell walls of the soil yeasts Cryprocoec~ albidus and C. &rem has been exam&d. It was found that the composition of the cell walls, which varied according to the cultural conditions employed, de&mined the extent to which the walls were lysed. Thus walls from cells of C. ulbidus grown under conditions favourable for growth contained O- and ~glucans and chltin as major components and were lysed by two Drsptomyces spp. but not by a nonfruiting myxobacteriumcyfoplragujohnsonii.Sign&ant lysis of C. ulbidm walls by the myxobacterium as well as by the Streptomyces sp. occunzd, however, when the a-glucan component was considerably reduced by growing the yeast under unfavourable conditions. Ultra&u&& studies showed the absence of deflrdtelayers in the wall. The chitinous residueaftachemicalextractionofthewallsretainedthe~~shapeofthecellandwascome&f;gz&ifi&, in contrast to the granular chitinous residues from other yeasts e.g. .

INTRODUCTION IT IS IMPORTANT that the nature of the cell walls of soil microorganisms and the manner in which they are &graded be elucidated in view of their contribution to the soil organic matter complex on the death of the cell. An understanding of the microbial population responsible for the decay of this material is also required since there is a possibility of using lytic microorganisms as agents for controlling certain fungal plant pathogens. The present paper reports observations on the lytic activity of two species of Streptomyces and of Cyrophaga johnsonii, a non-fruiting myxobacterium, on the cell walls of two soil yeasts, Cryptococclrs &z&s and C. termus. Some aspects of the chemistry and ultrastructure of the cell walls of these yeasts have also been investigated. MATERTALS AND

METHODS

Growthof yeasts andpreparation of cell walls The yeasts were isolated during studies on soil aggregating agents (Jones, 1964) and identified by Dr. L. J. Wickerham, Fermentation Laboratory, Agricultural Research Service, Peoria, Illinois, U.S.A. One of the yeasts examined was ‘near, but possibly not identical with Cryptococcuscrlbidus’which has been previously isolated in Britain from building materials in 1936, but there is no record of C. terreus having been found in Britain. The yeasts were cultured in flasks at 27°C in a New Brunswick incubator shaker using the following media: (1) Oxoid mycological peptone, 10 g; dextrose, 40 g; water, 1000 ml; (2) Ammonium sulphate (O-1 “/,), RH2P04 (O-1 %), MgSO, (O-05 %), glucose (lx), thiamine, O-2 mg/l (Aschner and Cury, 1951). The cells were harvested by high-speed centrifugation, washed, made into a thick paste with ballotini glass beads (No. 9) and treated alternately (15 min) on a M.S.E. ultrasonic disintegrator and Mickle cell disintegrator until practically all had 145

146

D. JONES, J. S. D. BACON, V. C. FARMER AND D. M. WEBLEY

broken and were devoid of contents. Clean cell wall preparations were obtained by successive washing with water at the centrifuge until the supernatants were free of debris. Lytic organisms The Streptomyces spp. Nos 5 and 6 (see Jones and Webley, 1968) were cultured at 27°C in a basie mineral salts solution (SkujiqS, Potgieter and Alexander, 1965) containing 1 mg/ml cell walls or whole cells of the yeasts. For comparison some experiments were performed with the non-fruiting myxobacterium Cytophagajohnsonii (Follett and Webley, 1965) which has been shown to lyse the walls of Saccharomyces cerevisiae actively (Bacon, Mime, Taylor and Webley, 1965). It was grown on the mineral salts medium of Starrier (1947) to which were added 0 * 1 ‘A (w/v) Difco peptone and walls of either C. albidus or C. terreus. Culture tIltrates were obtained by centrifugation at high speed and examined for laminarinase and lytic activity towards cell walls of the two yeasts (see Jones and Webley, 1967). Unless otherwise stated, a typical incubation mixture was: culture fluid, 0.8 ml; preheated (30 min at 8O’C) yeast cell-wall suspension(l6 mg dry wt/ml), 0.05ml; 5Om~ tris-HCI buffer (pH 7.5) or citrate phosphate buffer (pH 5 .O), 0.15 ml. In some incubations 2mercaptoethanol was added to give a final concentration of 20 mu. Chemical and physical examination

Chemical and infrared analyses of the yeast cell walls were made by methods described elsewhere (Jones, Bacon, Farmer and Webley, 1968). Specimens for electron microscopy were prepared by methods also described previously (Jones and Webley, 1967; Jones and McHardy, 1967).

Good growth of C. albidus and C. terreus was obtained in the glucose-peptone medium (6 - 8 mg and 5 mg dry wt/mI respectively) after 6 days. The corresponding weights in the Aschner and Cury’s medium were 3 mg and 1.7 mg after the same period. During the preparation of walls from cells harvested from the peptone-dextrose medium the strands of capsular material characteristic of this genus (see Figs l-3; cf. also C. neoformans, Edwards, Gordon, Lapa and Ghiorse, 1967) appeared to have been removed (see Figs 4 and 5). The test (Wickerham, 1951) for starch-like material (cf. Kooiman, 1963) in these walls was negative. A positive result was obtained, however, with walls of C. terreus prepared from Aschner and Cury’s medium; autoclaving removed some of this starch-like material from the walls. Very little starch-like material was produced by C. albidus under these conditions. In order to obtain the widest difference in composition (see later) cells from cultures of C. albidus grown for 2 days in glucose-peptone and 15 days in Aschner and Cury’s medium were used to prepare walls for use in the growth and incubation experiments described below. Growth experiments

Good growth of the Streptomyces spp. occurred when walls of the yeasts (prepared from cells harvested from either medium) were supplied as sole carbon source in the mineral salts solution; the walls remaining after 3-5 days were extremely thin and sometimes disintegrated. Almost complete dissolution of whole cells of C. albidus took place in flasks shaken for 4 days at 25°C and only small particles resembling contracted protoplasts remained. With whole cells of C. terreus as growth substrate a large number of cell walls in various

4

5

Electron micrographs of Cryptococcus ferrcw (Fig. 1) end Cryprococcus abidus (Figs Z-9). The spccimms examined were cultured in pcptone-dextrose medium with the exception of that shown in Fig. 8 (Aschner and Cury’s medium). FIGS 1, 2. Ultra-thin sections of cells illustrating strands of capsular material (arrowed). Fro. 3. Shadowed preparation of whole cdl illustrating capsular material (arrowed). For clarity this photograph was printed from a nepativc plate and not from a positive plate as in Fips 4, 7 and 9. FIGS4, 5. Isolated cell walls illustratmg absence of capsular material in shadowed specimens (Fig. 4) add ulra-thin section (Fig. 5). Magnifications: Fig. I-x

SBB f.p. 1461

22,400: Fig. 2-x

59,500: Fig. 3-x

21,000: Fig. 4-x

7700; Fig. 5-x

70,OlKl.

6

8

FIGS 6, 7. Isolated cell walls after lysis by S~eptomyres culture fluid; note eroded areas in thin section (Fig. 6) and shadowed specimen (Fig. 7). FIG. 8. Ultra-thin section of isolated cell wall; compare with Fig. 5. FIG. 9. Shadowed residue of walls after extraction with HCI followed by NaOH; note microtibrillar appearance. Magntfications: Fig. bx21,ooO: Fig. 6 (inset)--_9100; Fig. 7--..‘3850; Fig. 8-x70,000; Fig. 9--x16,800.

LYSIS OF

CryptOCOCCUS

CELL WALLS

147

stages of lysis persisted. In comparison, Cytophagajohnsonii was unable to lyse the walls of C. terreus or C. albidus prepared from cells grown in peptone dextrose; significant lysis occurred, however, with walls of C!.albidusprepared from cells harvested from Aschner and Guy’s medium. As the chitin residue obtained after acid followed by alkali extraction (Houwink and Rreger, 1953) of the yeasts was utilixed by the myxobacterium, it appears that this component is not responsible for the resistance of the cell walls to lysis. Changes in components and ultrastructureof cell walls of C. albidus and C. terreus during lysis Culture filtrates from the growth experiments were tested for lytic activity on yeast walls. Mercaptoethanol, which enhances the lysis of walls of Saccharomyces cereoisiae by other lytic organisms (Bacon et al., 1965; Jones and Webley, 1967), had no effect on the lysis of the walls of Cryptococcus. Consequently in the experiments described below 2-mercaptoethanol was included in the incubation mixtures with walls of S. cerevisiaebut not with Cryptococcus. Cells harvestedfrom peptone-dextrose medium The infrared spectra of the cell walls prepared, from cells harvested after 2 days indicated no signifkant difference between the species. Their spectra (Fig. 1OA) suggested the presence of both a and /Lghrcans and chitin. These were later confirmed by examination of various fractions from the cell walls (Fig. 10). The presence of a(1 +3) glucan was indicated by absorption bands 11.75 p and 12.15 w. Rather less a-glucan was indicated in spectra of walls prepared from C. aZbiduscells harvested after 15 days. A detailed account of this component, which is concentrated in the alkali-soluble fraction (Fig. lOB, compare Fig. lOC), has been given by Bacon, Jones, Farmer and Webley (1968). The infrared spectrum of the residue (Fig. 1OD) remainin g after extraction of cells of C. albidus with alkali, which removed the a-glucan, indicated the presence of chitin and a /3-glucan. Although the pattern of absorption approximated to that of laminarin, a /I(1 +3) glucan (Fig. lOE), the material was clearly not a pure specimen of this fy$e. Chitin in almost pure form was identified by infrared analysis (Fig. 1OF) of a microfibrillar residue (Fig. 9) which remained after acid/alkali extraction of cells of C. albidus; determination by the anthrone method showed that the glucan content of this residue did not exceed 2 per cent. The presence of a fi(1+3) glucan in the walls was confirmed by the TABLE 1. LAIblINARlNAssAcTNlTY IN FILlluTEs PROM CULTURES OF StreptOmycCs OF Cryptococcus terreus AND C. albiahs SHAKENFOR 3 DAYS Test organism

Growth experiment substrate (cell walls) in growth medium PH

Streptomyces Streptomyces Streptomyces Streptomyces

No. No. No. No.

6 6 6 6

c~ptowccus Cryptococcus Cryptococcus Cryptococcus

albidus albiaks teweus terreus

Streptomyces Streptomyces Streptomyces Streptomyces

No. No. No. No.

5 5 5 5

Cryptococcus Cryptowccus Cryptococcus Cryptococcus

afbiahs albidus terreus terreus

SpP.

GROWN

ON CELL

WALLS

AT 25°C

Incubation experiment Reducing sugar (me) released per 100 ml culture fluid per day Calculated from CMculated from 1 day’s incubation* 3 days incubation

;:; 5.0 7.5 5-o ::; 7-5

+ No reducing sugar detected in culture fluid incubated alone.

540 182

540 180

30 12

::

312 141 134 70

364 112 ii

148

D. JONES, J. S. D. BACON, V. C, FARMER AND D. M. WBLEY

I

3

I

4

I

5

I

6

I

7

I

Fl

,

0

WAVELENGTH

Fm. 10. A. B. C. D. EL F.

,

lo

,

11

(

12

,

,

,

13

14

yj

$tm,

I&iared spectra of: Celi walls of Cryptocoecus&i&s. Alkali-soluble&can from C. u&&h ~(14 3) glucan from Aspergilhun&w (FractionIVR of Johnston, 1965). Alkali-insolublerusidue from C. Irltilrius. L4xmhah, a Ip(1-b3) gluean. Chitin residue &om acid and alkaline digestion of C. u&i& ceils.

development of laminarinase in cultures of the Strepromyces spp. grown on C. uuiidur walk (Table 1); this enzyme was not induced with either $(1+6) or a(1 -+3) glucan. In this connexion it is of interest to note that culture filtrates from Sfreptomyces sp. No. 5 grown on cell walls of C. albidus and C. rerreus almost completely lysed cell walls of bakers’ yeast in which a laminarin+ke glucan is a major component. Although Cytophaga jo~o~i was unable to lyse the walls of the two yeasts in growth experiments, as judged by optical observations, the culture fluid was found to possess an active /3(1+3) glucanase. In a further experiment a 16 - Efold concentrated culture fluid of the myxobacterium, after growth on walls of S. cerevisiue, was incubated with walls of C. albidus at pH 7 - 5 for 6 days. This culture fluid possessed a very high &glucanase activity but produced no marked visual lysis of

LYSIS OF CryptOCOCCw

CELL WALLS

149

the walls. Nevertheless, infrared analysis of the wall residue showed a marked increase in the a-glum content, indicating preferential attack on the /3-glucan. Paper chromatography of the concentrated de-salted supernatant from this experiment showed glucose and other oligosac&arides. No N-ace@ glucosamine or glucosamine was detected. Filtrates from a culture of Strqfomyces sp. No. 5, grown on Cryptowccw aibidus walls in shake flasks for 3 days at WC, were tested for lytic activity towards preheated walls of C. ulbidw After 24 hr a considerable number of walls had thinned and disintegrated at pH 5 -0 (few at pH 7 - 5) as judged by phase-contrast microscopy. At both pH values, after 5 days a~ro~~t~ly 50 per cent of the walls were extremely thin and many ~~nte~t~. In another experiment with walls, a weight loss of 20 per cent and 50 per cent at pH 7 - 5 and 5.0, respectively, was recorded due to lysis. Infrared examination of the residue remaining after lysis indicated no preferential removal of wall components. Paper chromatography showed that the products of lysis included glucose and nigerose. The cell walls of C. terreus when incubated with the filtrate from a culture of Streptumyces sp. No. 5, grown on cell walls of C. terreus, did not lyse as rapidly as those of C. ~~. Electron micrographs bang the lysed walls of C. albidus are given in Figs 6, 7. Uneven attack generally oumrred and this gave a pitted appearance to the walls in thin section. Occasionally sections of wall residues showed electron dense ‘shells’with no pits, (see inset, Fig. 6), which could be the inner layer sometimes observed in unlysed walls. Culture f&rates from the myxobacterium grown in the presence of C. terreus or C. aim w4s did not lyse these walls in incubation experiments at pH 7-5 as judged by the optical microscope. Cells harvested from Aschner and Gay’s medium In thin section (compare Figs 5, g) the walls of C. a~‘&& prepared from cells harvested after 15 days were much thicker on average (0.3~) than those prepared from cells harveaM after 2 days from the peptone-dextrose medim (0.17~). No detite layers in the walls were observed. Little a-glucan was detectable in the infrared spec&um of C. albti walls prepared from cells harvested after 2 days, less after 6 days and none after 15 days growth, but lytic experiments (see below) confirmed that a small amount was present. The walls of C. rrlbidrtswere partially lysed in growth experiments by the rn~o~~~~; in incubation experiments at pH 5-O these walls were also partially lysed by culture fluids of C. johnsonti after its growth on these walls. Infrared examination of the wall residues then showed a high content of u(l+3) glucan, indicating that the myxobacterium had lysed only the &glucan. The same culture fluid partially lysed Succhuromyces cerevisiae cell walls at pH 7 -5 and paper chromatography showed the presence of glucose and oligosaaharide. Infrared examina tion of the wall residue of C. aim renkning afk in&&ion with a culture fhrid from C. johnsonii grown on S. cerevisiae further confirmed the presence of a-glucan. Paper chromatography of the supernatant after de-salting and reducing in volume gave glucose and other oligosaccharides but no N-acetyl glucosamine or glucosamine. The culture fluid from Streptomyces sp. No. 5, grown on the Cryptococcus walls, possessed a very active laminarinase and almost completely lysed the corresponding walls in incubation experiments at pH 5 -0. It did not lyse to any marked extent, as judged by optical floppy, cell walls of C. a&&s from the peptone-dextrose medium; paper chromatography of the concentrated supernatants indicated that some attack on the chitin and protein had taken place. The infrared spectrum of the wall residue showed increased absorption in the a-glucan region at 12pm indicating a preferential removal of the other. components. The culture fluid

150

D. JONES,

J. S. D. BACON,

V. C. FARMER

AND D. M. WEBLEY

brought about hydrolysis of a pure a(1 +3) glucan substrate (Duff, 1952) with the production of oligosaccharide, appreciable amounts of n&rose and glucose. The infrared spectra of walls prepared from 6 and 15day-old cells of C. terreus showed the presence of sign&ant amounts of a(1 +3) glucan. Culture fluidsof Streptomyces sp. No. 5 grown on C. terreus cell walls also partially lysed these walls in incubation experiments at pH 5 -0. The starch-like material present in the walls was degraded, as the iodine reagent test proved negative. DISCUSSION

If walls of the two yeasts investigated are to be lysed to a significant extent in soil then it is necessary to have microorganisms present which produce an a-glucanase. If /3-glucanase and chitinase are also present, as in the streptomycete No. 5, then almost complete lysis should occur. In the myxobacterium the a-glucanase appeared to be absent but some lysis must have occurred possibly because of the laminarinase activity of this organism. One interesting aspect of the present work is the change in the composition of the walls that occurs on altering the cultural methods (cf. Wilson and Niederpruem, 1967). As a result the activities of the lytic organisms were affected. Walls in which a(1 +3) glucan was a minor component could be seen to undergo lysis by a myxobacterial culture filtrate and suflicient &glucan was removed to make the a-glucan detectable by infrared. This tiding illustrates the value of using various microorganisms to elucidate the wall composition. It is also worth mentioning that the wall composition of these soil yeasts growing on ‘natural’ substrates in soil might differ from that produced under laboratory conditions. The results presented also give an insight into the ultrastructure of the walls of the yeasts. The microfibrils in the chitinous residue of C. albidus are not unlike those observed in !Yamentous fungi (e.g. Jones et al., 1968). Aronson and Preston (1960) have also demonstrated the occurrence of microfibrils in walls of certain lower Phycomycetes and consider them to be composed of chitin. The microfibrils of C. albidusare composed of chitin and in this respect contrast with the chitin granules that occur in other yeasts e.g. Saccharomyces cerevisiae, (Houwink and Kreger, 1953). Walls of C. albid& and C. terreus also differ from those of S. cereuisiue in the apparent absence of well-defined bud scars in chemically extracted and enzymically degraded preparations (Houwink and Kreger, 1953; Bacon, Davidson, Jones and Taylor, 1966; Jones and Webley, 1967). The present work has illustrated how a combination of techniques which included electron microscopy, infrared spectrometry, biochemical and microbiological methods, has helped to unravel some of the complexities of one component of the soil organic matter. Ac&nowfe&emenfs-We wish to thank Miss J. NORMINOT~N,Miss B. R. ALLUHAN and Mrs E. CRUICKTHANKfor valuable tech&al assistance. RBmRJxNcEs ARONSONJ. M. and pREsr0~ R. D. (1960) Au electron microscopic and X-ray analysis of the wails of selected lower Phycomycetes. Proc. R. Sot. B 152,346-352. ASCHNERM. aud CURYA. (1951) Starch production in the genus Trichosporon. J. Bad. 62,350-352. BACONJ. S. D., DAVILWNE. D., Jam D. and TA~R I. F. (1966) The location of chitin in the yeast cell wall. Biochem. J. 101, 36c-38~. BACONJ. S. D., JONES D., FARMER V. C. aud Waaw D. M. (1968) The occurrence of a(1 + 3) glucan in Cryptococcus. Schizosaccharomyces and Polyporus species, and its hydrolysis by a Streptomyces culture liltrate lysiug cell walls of Cryptococcus. Biochim. biophys. Acta 158,313-315. BACONJ. S. D., MILNEB. D., TAYLORI. F. and WEBLEYD. M. (1965) Features of the cell-wall structure of yeast revealed by the action of euzymes from a non-fruiting myxobacterium (Cytophaga johnsonii). Biochem. J. 95,2803Oc.

LYSIS OF CryptOCOCCUSCELL WALLS

151

DUFF R. B. (1952) The constitution of a glucosan from the fungus Polyporus betubnus. J. them. Sot. 25922594. EDWARDS M. R, GORDONM. A., LAFAE. W. and Gmoxs~ W. C. (1967) Micromorphology of Cryptowccus neoform.

J. Bact. Q4,766-777.

For~fir

E. A. C. and Wnxnxv D. M. (1965) An electron microscope study of the cell surface of Cytopiwgu johnsonii and some observations on related organisms. Antom vun Leeuwenhoek 31,361382. HO~~INK A. L. and KRUGER D. R. (1953) Observations on the cell wall of yeasts; an electron microscope and X-ray diffraction study. Anfonie vun &euwenkoe& 19, l-24. JO~INSTON I. R. (1965) The partial acid hydrolysis of a highly dextrorotary fragment of the cell wall of Aspergilfus nigcr. Isolation of the a(1 + 3)-linked dextrin series. Biochem. J. %, 65P-664. Jones D. (1964) Studies on Soil h4icroorganisms with Particukzr Reference to Soil Structure, Ph.D. The&l, University of Wales. JONESD. and MCHARDYW. (1967) Location of specimens in Araldite blocks for ultra-thin seotioning. Bull: Br. mycol. Sot. 1,39.

Jomrs D. and WESLEYD. M. (1967) Lysis of the cell walls of yeast (Succharomyces ceredae)

by soil fungi.

Trans. Br. mycol. Sot. 60,14P-154. JONESD. and WEBLPY D. M. (1968) A new enrichment technique for studying lysis of fungal cell walls in soil. PI. Soil 23,147-157. JONESI),, BACONJ. S. I)., Fw V. C. and W~~LZ!Y D. M. (1968) Lysls of cell walls of Mucor ramanniatm,r Mallet by a St~ptomy~s sp. Ar+onie vats Leeuwenboek 34,173-l 82. KOOIMANP. (1963) The chemical structure of the extracellular ‘starch’ produced by Cryptococcxs &&us and C. Iaurentii var. j&zvescens. Antonie van Leeuwedwek 29, 169-176. SKUJ@ J. J., Poronrraa H. J. and mna M. (1965) Dissolution of fimgal cell walls by a streptomycete chitinase and fl(1--c3) glucanase. Arch. Biochem. Biophys. 111,35&364.

STANIER R. Y. (1947) Studies on nonfruiting myxobacteria. I. Cytop&afohnsoMe,n. sp., a chitin-decomposins myxobacterium. J. Buck 53,2Q7-315. Wrm L. J. (1951) Taxonomy of Yeasts* Tech&al Bulletin of the U.S. wt of Agriculture No. 1029. %LSON R. W. and N IEDERPRUEM D. J. (1967) Cellobiose as a paramorphogen in Schizophylfum commune. Can. J. Microbioi. l3, 16634670.