Phosphatases and the utilisation of inositol hexaphosphate by soil yeasts of the genus Cryptococcus

PHOSPHATASES AND THE UTILISATION OF TNOSITOL HEXAPHOSPHATE BY SOIL YEASTS OF THE GENUS CRYPTOCOCCUS. A. J. Department

GREENWOOD*

of Botany,

and D. H. LEWIS

The University.

(Accepted

Sheffield

14 September

SlO ZTN, England

1976)

Summary-~Six strains of 5 Cryptococcus spp. were tested for growth on inositol hexaphosphate (NaIHP) as sole P source and for production of acid and alkaline phosphatases when grown in the presence or absence of orthophosphate. The enzymes of the three species which did grow on Na-IHP hydrolysed p-nitrophenylphosphate, fl-glycerophosphate, pyrophosphate and Na-IHP, but not insoluble salts of IHP. These results are discussed with respect to the utilisation of soil IHP and organic P.

INTRODUCTION In most soils, the concentration of inorganic phosphate in the soil solution is extremely low (lnM-1pM) (Bieleski, 1973; Tinker, 1975). This, combined with the relative immobility of the ion in the soil solution (Bhat and Nye. 1973 and 1974) can cause P supply to be the limiting factor for plant growth. Organic P can constitute 4-90”; of the total soil P (Cosgrove, 1967) and, of this. inositol hexaphosphate (IHP), probably adsorbed on to clay or as insoluble aluminium, ferric and calcium phytates (Martin and Cartwright. 1971; Anderson et al., 1974). can form a substantial component, sometimes more than 50”,b (Anderson, 1967; Martin. 1970; Halstead and McKercher. 1975). Many soil micro-organisms can hydrolyse Na-IHP (Greaves and Webley, 1969) and even aseptic plant roots have phytase activity and can utilise soluble NaIHP as a P source (Saxena. 1964; Ridge and Rovira, 1971; Martin, 1973). Detailed studies have been made of the phytases of some bacteria and mycelial fungi (Greaves et ul., 1967; Powar and Jagannathan, 1967; Yamada rt ul.. 1968; Theodorou, 1971; Irving and Cosgrove, 1971; 1974). Soil yeasts have been almost completely ignored although. in one survey of a range of micro-organisms for production of extracellular phytase. none of the yeasts examined (species not named) was found to produce any (Shieh and Ware, 196X). Relatively little is known about the ecology and possible biochemical activities of yeasts in the soil (Casas-Campillo, 1967). They are widespread (Lund, 1954: di Menna, 1965; do Carmo-Sousa, 1969) and, since their populations are distinct from foliage yeasts (di Menna, 1959). are not just casual contaminants. It is possible that numbers of yeasts in the soil have been underestimated because several species of Cryptococcus, at least, are psychrophilic, failing to grow at temperatures commonly used in isolation procedures, as shown by Buhagair and Barnett (1971). * Present address: Department of Botany and Microbiology, The University College of Wales. Penglais. Aberystwyth, SY23 2DA.

Yeasts of the genus Cryptococcus were chosen for this study because they arc the dominant yeast species in several soils (di Menna, 1965) and because one of their most important generic characteristics is the ability to utilise inositol as a C source (Pfaff and Fell, 1970). Soil IHP and lower esters of inositol are therefore potential sources of both C and P to these yeasts. MATERIALS AND METHODS

Cultures of the following species were obtained from Dr. J. A. Barnett of the University of East Anglia: Cryptococcus alhidus (Saito) Skinner (L49), C. clif,puens (Zach) Lodder et Kreger-van Rij (NCYC460) (=ahfus (Saito) Skinner var. d@urns (Zach) Pfaff et Fell), C. injirmo-miniatus (Okunuki) Pfaff et Fell (L115), C. macerans (Frederiksen) Pfaff et Fell (NCYC586), C. hrentii (Kufferath) Skinner var. laurentii (NCYCl39), C. laurentii (Kufferath) Skinner var. Pavescens (Saito) Lodder et Kreger-van Rij (H343). They were maintained on slopes of malt agar (3g malt extract, log glucose. 5g peptone and 3g yeast extract.l- ‘). Utilisation of Na-IHP was tested using a defined liquid culture medium similar to that used by Wickerham (1946) but with KH,PO, replaced by K,S04 and KCI. Solutions of pure Na-IHP (Sigma), either autoclaved separately or sterilised using a millipore filter. were added to the autoclaved medium. Standard 0.5 ml inocula from 2-3 day old cultures grown on KH2P0, or -P media were used in IOOml of medium and the cultures were incubated on a rotary shaker at 15-20°C. Growth was routinely measured by the absorbance of suitable dilutions of the cultures at 600 nm using a Unicam SP600 spectrophotometer. Values presented in tables and figures have been corrected for dilution and converted to dry weight from standard curves relating absorbance to dry weight for each strain of yeast. p-Nitrophenylphosphatase (PNPP-ase) of whole cells and filtrates from cultures grown on different P sources were determined as follows. Cells were harvested by centrifugation and washed twice with distilled water. They were resuspended in various buffers prepared according to Gomori (1955) and at concen161

162

A. J.

GRtENWOOl>

and D. H. Lt,w~s

Table I. Yields of Cr~~procwcu.s spp. grown on various yield in less than

Species

KH,PO,

sources of phosphorus at 23/i& P. mlIO days (Values as mg dry ut. ml-‘) Phosphorus Na-IHP (autoclaved)

4.87 4.6X 5.90 5.7x 4.72

5.41

trations of cells which gave readily measurable rates of hydrolysis of PNPP. Samples of culture filtrate were passed through a millipore filter to remove all cells. Substrate (in the form of 40 mg pre-weighed capsules from Sigma) was dissolved in 30 ml distilled water and 1.5ml was added to 1.O ml of yeast suspension in buffer. At the end of the incubation at 20°C (usually 30 min or less), the reaction was stopped by adding 5 ml of 0.1 M NaOH, which also raised the pH, ionising the p-nitrophenol (PNP) produced by hydrolysis to give a bright yellow solution. After centrifuging to remove yeast cells, absorbance at 410 nm was measured and results expressed as pmoles PNP.mg dry wt. -’ h _’ from a calibration curve of PNP. IHP-ase. pyrophosphatase (PP-asc) and fi-glycerophosphatase (b-GP-asc) were measured by incubating solutions of their Na-salts at 150pg.m1- ‘P in acetate buffer. pH5.6, with a suspension of the pellet fraction of cells disrupted by freezing (using ethanol cooled with dry ice) and thawing 4 times. Orthophosphate produced was measured by the vanadomolybdate method of Bartlett and Lewis (1970). Intact yeast cells were not used in these assays to avoid the problem of phosphate uptake. K, Ca and Mg salts were added to the enzyme assay media at the same concentrations as present in the growth medium (0.1 g CaClJ ‘. 0.5g MgSOJ’. 0.5g K,SOJ’, 0.5g KCI.1~‘). As these salts had no effect on PNPP-ase, they were not included in this assay mixture. Controls without substratc and without yeast were always included to cheek for any endogenous hydrolysis of organic P in the broken cells and chemical hydrolysis of substrate. Utilisation of insoluble forms of IHP (Ca, Al and Fe salts) prepared as described by Greaves it trl., 1969) was tested by incubating them with resuspended pellets of disrupted cells and measuring phosphate produced. Growth on insoluble forms of IHP was determined by measuring the diameters of colonies grown on agar plates of -P medium with autoclaved, washed suspensions of the salts spread over the surface at concentrations equivalent to 0. I, 0.2 and 0.5 g KH2P0,.I’ of medium.

4.26* I .47* 5.83 5.X6 0.25* 0.?6*

’ for IO d* or to maximum

Sources

Na-IHP (filter-sterilised) 0.09* 0.03* 5.90 5.70 0.04* 0.04*

Only C. 177m~~u17.s and C. with filter-sterilised Na-IHP

grew well as sole P source (Table

ir!fir,,lo-rllirliaflr,s

0.04* 0.02* 0.01* 0.01 * 0.02* 0.01*

I) but C. alhidus grew slowly with autoclaved Na-IHP (Fig. 1) to give a final yield equal to that on KH,PO,. This phenomenon. which may be due to slight decomposition of the Na-IHP during autoclaving to produce orthophosphate and more readily utilized lower esters of inositol, is being further investigated. The autoclaved Na-IHP medium contained 0.27 pg P.ml- ’ as orthophosphate compared with 0.09 in the filter sterilised Na-IHP medium and 23 in the KH2POI medium. PNPP-L1.W

All species showed higher activities of PNPP-ase when grown on Na-IHP or -P media than when grown on orthophosphate (Table 2), although absolute activities varied according to the stage of growth. Repressed PNPP-ase activities during growth on KH,PO, ranged from 1690 nmoles PNP.mg dry wt ‘. h- ’ for all strains except C’. hurentii var. JuIW~(IIZ.~which had values an order of magnitude higher. For each species. the pH activity curve of PNPP-ase throughout growth was similar when grown on Na-IHP or -P media but differed from that of KHzPO,-grown cultures (Figs 2-7). The pH optima of PNPP-ases of cultures grown in the absence of orthophosphate varied greatly between species (even between the two varieties of C. laurentii) and were not related to ability to utilise Na-IHP. C. wucerut~s, C. tlijjhxs and C. hurentii var. laurentii had derepressed PNPP-ases with alkaline pH optima; C. trlhitlus and C. ir$irmo-miniatus had derepressed PNPP-ascs with slightly acid pH optima and C. hrcv~ii var. ,&W.S~LWS(which. as mentioned above, had a high activity of non-rcprcssible PNPP-ase) differed

h RESULTS

-P

h

Fig. I. Growth of Cr~~ptococcus spp.with potassium orthophosphate (----. open symbols) or sodium inositol hexaphosphate (autoclavcd) ( -. closed symbols) as sole source of P C. III(I~TUII,S0. n : C. i,?fi,,,io-mirfiutlrs A, A; C‘.N/hidW\0. 0.

Phosphatases

163

of soil yeasts

Table 2. Activities and pH optima of PNPP-ases produced during growth of Cr~ptococcus spp. (KH2P04 as P source) or derepressing (autoclaved Na-IHP as P source or no P source) conditions substrate hydrolysed mg-’ dry wt. h-‘) Repressing Conditions PNPP-ase activity at pH optimum pH optimum

Species C.

alhidus

C. dijluens C. in$rmominiutus C. macerans C. laurentii var. luurrntii C. laurentii

0.01~0.02 0.02 0.05 0.040.09

<3.0 <4.0 4. I

0.02-0.04 0.03-~0.05 0.5&0.75

Derepressing PNPP-ase activity at pH optimum

10.0LO.0 0.55I .o

under

repressing

( values as ALmoles

Conditions pH optimum

5.8 8.0

0.4- 1.o

6.1

4.3 5.2

I sk2.0 0.5%1.0

>9.0 8.1

4.5

2.5-5.0

4.6

var. jaucscens

the others in showing no change in pH optimum in derepressed as opposed to repressed conditions. The PNPP-ase activities of culture filtrates of NaIHP cultures of the three species which grew on Na-

from

IHP were measured at a range C. alhidus (tested over a pH range

of pH values. For of 3.64.6), an aver-

age of 2.1% of the PNPP-ase activity was in the culture filtrate (values varied from 1.0&3.263,,); for C.

mucrrans (pH range of 3.0-9.0). the average value was 9.37’); (varying from 5.86-l 1.743;,) and, for C. infirmorniniutus (pH range of 3X%6.6), it was 21.2”,, (varying from 17.5S23.7y/,). This last species, with the highest extracellular enzymic activity. was chosen for experiments to test growth on insoluble salts of IHP since it also had IHP-ase activity in the culture filtrate (Table 3).

3 PH

(3)

PH

Colbidus

/

C.laurentli

flovescens

var.

20 t

03L

4

I

I

‘,\

5

6

7

1

_

8

PH

(6)

curves of p-nitrophenylphosphatase of intact cells of Cryptococcus spp. (Fig. Fig. 3: C. macrrans; Fig. 4: C. alhidus; Fig. 5: C. d(fluen.s; Fig. 6: C. laurerltii 7: C. kaurentii var. laurerltii.) grown with potassium orthophosphate (----) and with either no P (-. closed symbols) or sodium inositol hexaphosphate (---, open symbols). For each pair of graphs on each fig., values are expressed as “;, maximum on that source of P. These maximum values, as {lmoles substrate hydrolysed rng-’ dry wt. h-’ arc inserted on each fig. Key to buffers A, a: 0.05M citrate, +. 0: 0.1~ acetate. 0, 0: 0.05~ Tris-maleate. W. 0: 0.05~ Tris-HCl. Figs 2-7.

2: C.

pH activity

infirmo-mi,liatus; var. JIawscens: Fig.

164

A.

J.

GREENWCODand

D. H. LEWIS

Table 3. Relative values of /&GP-ase, PP-ase and IHP-ase of resuspended pellet fractions of frozcll and thawed, log phase, Na-IHP-grown cells in 0.1M acetate bulfer, pH 5.6. (Values as pmolcs substrate hydroiyscd (/?-GP.PP) or phosphate produced (IHP) rng-’ dry wt. h-’ and shown as percentages of PNPP-ase in brackets) Enzyme activity

C. ir!firttict-rairliurlrs

C. ulhidu.s

PNPP-ase /I-GP-ase PP-ase IHP-ase IHP-ase in culture l&ate

14.36(100”,,) I h.53( I 1S’;,) l7.42(121”,,) 0.02(0.1”,,) nd.

c. ,lit,(‘~‘MllS

O.ZhO(I W,,) 0.066(X”,,) 0.064(25”,) 0.1.3”(60” ,>) n.d.

0.670(lOOI’,,) 0.309(45”,,) (X354(52”,,) 0.066( Io”,,) 0.027 (39”;, of total IHP-ase)

Table 4. Growth of C. ir?firr,lo-/nirtiutus on agar plates with KH,PO, at 0.1. 0.2 or 0.5g.l ’ or equivalent amounts of P as Al-, Fe- or C’a-IHP (Average colony diameters in mm after growth for IOd. 10 colonies measured per plate; 2 plates per treatment) Concentration (-g.l-’ KH2P0,)

*Colony diameters (mm) KH2P0, 0.87( 1-0.14) 0.90(~0.07) 0.90( & 0.13)

0.1 0.2 0.5

* Average colony diameters on -P Othrr

phospharusr

AI-IHP 0.41(&0,08) 0.44( f 0.04) 0.43( kO.07) _. plates were 0.33 ( t0.04) mm.

activities

IHP-ase, /SGP-asc and PP-ase activities were measured for Na-IHP grown cultures of three species at pH 5.6, the pH of the growth medium (Table 3). f. u~~~j~~s had barely detectable IHP-ase but extremely high PNPP-ase, fl-GP-ase and PP-ase. C. it?firm-miniatus had much higher IHP-ase than C. ulhidus but p-GP-ase, PP-ase and PNPP-ase were higher than IHP-ase. C. ~m~~‘ru~z.~ (which showed the best growth on Na-IHP) had the highest IHP-ase, higher for this species than ii-GP-ase and PP-ase but not as high as PNPP-ase. For this species, IHP-ase had a pH optimum at 5.6 but was undetectable at pH 7.4 and above whereas the pH optimum of PNPP-ase was >9 (Fig. 3). /SGP-asc had a similar pH activity curve to PNPP-ase but the optimum of PP-ase was lower (pH 8.0). For all three species, analysis of the culture filtrates during growth showed the disappearance of Na-IHP but no appearance of orthophosphate.

Ca-IHP 0.67(+0.16) n.d. n.d.

Fe-IH P (X9( f 0.02) 0.37( *0.05) 0.41( +0.05)

these species during the 24 h incubation, so that rates could not be determined. However, as the same anion is present in both Na-IHP and Ca-IHP assay mixtures. rates of hydrolysis of the two forms are likely to be the same for these species also. The experiment to test the growth of t. ir!fi)7110-l)lil2i(ltll.~ on agar plates with insoluble IHP salts (Table 4) also suggested that Ca-IHP but not Al- or Fc-IHP could be utilised. Despite release of phosphatasc and Na-IHPasc by this species during growth and microscopical observation of direct physical contact between particles of Al- and Fc-IHP and yeast colonies. it appeared that IHP was not utiliscd in these forms. DISCC'SSION

In all the species examined, phosphatases were derepressed in the absence of orthophosphate. The pH optima of these enzymes with PNPP as substrate were very different for pairs of closely related species. e.g. C. dhidus and C. rl$j%~ns (varieties of the same species according to Pfaff and Fell, 1970) and the two Utilisatim of’ Ca-, Fe- and Al-IHP varieties of C. lanrrrxtii. In C. IIIUW~YE~IS, the fact that the IHP-ase and PNPP-asc (with /i-GP-ase) of dcIncubation of the resuspended pellet fractions of cells of C. ~~~~i~~~.s, C. if?~ntlo-fnirliarfts and C. ~~~~~~~~~1,srcprcssed cells had different pH optima suggests that several phosphatascs are present. The kinetic characfor 24h with the insoluble calcium. ferric and aluteristics and responses to inhibitors. temperature etc. minium salts of IHP (img.ml- ‘) showed no othoof these enzymes and those of repressed cells remain phosphate release from Al- or Fe-IHP. but considerable hydrolysis of Ca-IHP. Vanadomolybdate analyto be studied. ses of organic phosphate in the incubation mixtures Seller and Swatek (1974), in a survey of the distribefore addition of the yeast showed that there was bution of Cryptococors species in Californian soils, no soluble IHP present in the Al- or Fe-IHP mixtures found that different species were associated with definite temperature and pH ranges. C. ~~~bi~~~~s (9 isobut that a considerable amount of Ca-IHP was soluble at the pH used (5.6). C. ulhidus hydrolysed Calates) was found in soils at 15 21 ‘C and pH 5.45.6, c’. ri@~t~s (7 isolates) in soils at 21.- 26°C and pH IHP at the same rate as Na-IHP (I? nmoles phos6.9 7.9 and C. luuwr~tii (2 isolates, varieties unspeciphate produced’mg dry wt.- ’ h-l). As C. infirnzofied) in soils at 27 and 29 C and pH 8.1 8.3. Thcsc miniulzns and C. InucerNns hydrolysed Na-IHP at faspH ranges correlate well with the pH optima of the ter rates (values of 48 and 157 respectively), all Caderepressed PNPP-ases: pH 5.8 for C. ~~~~i~~i~.s, pH IHP supplied in this experiment was hydrotysed by

Phosphatases 8.0 for C. difiuens and pH 8.3 for C. luurentii var. laurentii, suggesting that these enzymes may have some ecological function in the soil. In this study, tem~rature optima for growth were not measured but it was noticed that only C. ~~~t~~n.~ and C. /mumtii var. laurmfii grew rehably at 25&C, whereas all the species grew at 15’ C. C. mucmms and C. ir!firr)lo-rlliniutus, the two species which grew well on soluble Na-IHP, differ from most Cryptococ~~s spp. in having bright red carotenoid p~gnle~lts in their young colonies. The two species are probably closely related (Pfaff and Fell. 1970) and both have been observed to produce pseudomycelium and thick-walled, chlamydospores resembling teliospores (Ahearn and Roth, 1966). which supports a wide variety of other evidence, both biochemical and morphological (Fell. 1970: Kreger-van Rij and Veehius. 1971: Bastide, Travc and Bastide. 1975). that all Clp tococ’msspp. arc imperfect basidiomycetes. It is interesting that C. IVWCVX(~~~.S is often the main organism involved in the field retting of flax (Frederiksen, 1956), which contains relatively large amounts of free WJVinositol and other cyclitols relative to other soluble carbohydrates such as sucrose (L. J. Littlefield and D. H. Lewis. llnpublished). Even these two species seemed unable to utilise IHP in insoluble forms, so it appears unlikely that they would have access to it in the soil. How insoluble salts of IHP and lower esters of inositol are degraded in soils remains an enigma (Halstead and McKerchcr, 1975: Tinker and Sanders. 1975). Ackrzowlt,~~en,rtt.s---This work was supported by a Natural Environmental Research Council studentship awarded to A. J. Greenwood. We are grateful to Dr. J. A. Barnctt for supplying the yeast cultures.

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A. J. GKEENWGOUand D. H. Lt:wts

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