Mycol. Res. 95 (6), 712-714 (1991) Printed in Great Britain

Uptake of zinc by Penicillium notatum

A. P. STARLING AND I. S. ROSS

Deparlmenl of Biological Sciences, Universily of Keele, Keele, Siaffordshire, STS SBG

712

Zinc uptake by Penicillum nolalum was energy-dependent and occurred via a high affinity (Km = 6'66 nM) transport system whenexternal zinc concentrations were low (5-100 nM). This system was inhibited competitively by Cdz+ and non-competitively by Cu2

+,

other cations having no effect. Uptake was enhanced during growth of mycelium in medium low in zinc, and was reduced in zinc­rich medium. At higher external zinc concentrations (10-100 11M), uptake occurred by a low-affinity transport system (Km = 5'90 11M),which was inhibited competitively by all ions tested. Uptake of zinc by this system was stimulated by growth in medium with alow magnesium concentration. It is therefore proposed that zinc uptake in P. nolalum takes place by a high-affinity specifictransporter at low external concentrations and by a low-affinity non-specific system, probably a magnesium transporter, at higherconcentrations.

Zinc is essential for growth of fungi (Perlman, 1949; Thind &

Rawla, 1967; McHan & Johnson, 1970), becoming toxic onlyat high concentrations. Zinc is also required for the productionof various secondary metabolites including aflatoxins, ergotalkaloids and penicillin (Weinberg, 1977; Failla & Niehaus,1986).

The uptake of zinc by fungi includes a rapid and reversiblephase, generally considered to be binding to the cell surface,which mayor may not be followed by a slower, apparentlyirreversible phase considered to represent transport into thecells (Fuhrman & Rothstein, 1968; Paton & Budd, 1972; Faillaet ai., 1976; Mowll & Gadd, 1983). In yeasts, zinc transport isdependent on metabolic energy and appears to fall into twocategories: high-affinity specific transport (Failla et ai. 1976;Lawford et ai., 1980; White & Gadd, 1987); or low-affinity,non-specific transport (Fuhrman & Rothstein, 1968; Ponta &

Broda, 1970; Mowll & Gadd, 1983). In the filamentous fungi,energy-dependent uptake of zinc has only been observed inNeocosmospora vasinfecta (Paton & Budd, 1972; Budd, 1988)and Aspergillus parasiticus (FaIila & Niehaus, 1986). In thiswork we investigated the uptake of zinc by the filamentousfungus Penicillium notatum.

MATERIALS AND METHODS

Organism and culture methods

Penicillium notatum obtained from Griffin and George (Lough­borough, UK) was maintained on malt extract agar. Liquidculture studies were carried out in a medium containing(gl-I): citric acid, 2'8; K2HP04, 5'5; MgS04.7H20, I;CaCl2 ·6H20, 0'25; (NH4)zS04' 2; FeS04.7H20, 0'005;ZnS04· 7H20, 0'00175; CuS04.5H20, 0'0001;

MnS04 .4H20, 0'0001; glucose 20; yeast extract 0'1 anddistilled water. The pH was adjusted to 5'5 with KOH. Wheremedium of low Zn content was used, ZnS04 • 7HzO wasomitted and yeast extract replaced with D-biotin at a con­centration of 10 llg ml-1

. Where medium of low magnesiumcontent was required, only 0'005 g 1-1 MgS04 • 7H20 wasused. Flasks containing 400 ml medium were inoculated with106 spores ml-1 and incubated at 25°C on an orbital shaker(200 rev. min-I). Cultures were harvested in early exponentialphase (20 h incubation) by centrifugation, washed once indistilled water, once in 50 mM-2-(N-morpholino)ethane­sulphonic acid (MES) buffer adjusted to pH 5'5 with KOH,and resuspended to a density of 0'05 mg ml-1 in MES buffer,pH 5-5.

Uptake of zinc

Uptake experiments were carried out using 20 ml volumes ofa mycelial suspension prepared as described above andincubated at 30° for 30 min. Zn2+ uptake was initiated by theaddition of a solution of 65Zn2+ (Amersham International: finalactivity 0'4 llCi ml-1

). When required, glucose was added at afinal concentration of 50 mM at the start of the incubationperiod. The metabolic inhibitor carbonyl cyanide p-(tri­fluoromethoxy)-phenylhydrazone (FCCP) was added to a finalconcentration of 100 11M 5 min before the addition of metalions. Samples (I ml) were removed at intervals and filteredthrough 0-45 11m membrane filters and washed with 10 ml50 mM-MES buffer containing I mM non-radioactive Zn2+, foruptake experiments using up to 100 nM zinc, and 10 mM-Zn2+,for experiments using up to 100 !-AM zinc. Competition byother divalent metal cations was studied by addition of theirchlorides I min prior to the addition of 65Zn2+.

A. P. Starling and 1. S. Ross 713

RESULTS

Energy dependence of uptake

Zinc uptake was relatively low in mycelium grown in mediumwith a normal (26'8 j..lM-ZnS04 • 7H20) zinc concentration,was enhanced when mycelium was grown in the absence ofadded zinc, and was completely suppressed in mediumcontaining 1 mM zinc (Fig. 1). Zinc was therefore omitted fromgrowth medium unless otherwise stated. The uptake of zincwas readily demonstrated in experiments with 10 nM externalzinc (Fig. 2) and was abolished by the metabolic inhibitorFCCP and was unaffected by the presence of glucose. Therelatively small uptake evident in the presence of FCCP wasassumed to be zinc bound to cell surfaces. When myceliumwas starved by incubation in 50 mM-MES buffer for 18 h,uptake was glucose-dependent with 10 nM external zinc (Fig.3) and 20 j..lM external zinc. These results confirm the energydependence of zinc uptake.

50

Kinetics of uptake

The kinetics of ZnH uptake were determined from the initialrate of uptake, which was taken to be linear over the first 10min, using a range of Zn2+ concentrations from 5-100 nM and1O-100j..lM. Data from five experiments were analysed bylinear regression of the Lineweaver-Burk plots. An apparenthalf-saturation constant, Km , of 6'66 nM (range 5'54-7'78) anda maximal rate of transport, Vmax of 4'01 nM r.1in- 1 g-l (range3'23-4'79) for uptake in the concentration range 5-100 nM Znwas obtained. For the range 10-100 j..lm Zn2+, Km was5'90 j..lM (range 4'99-6'81) and Vmax was 4'54 j..lM min-1 g-l(range 3'64-6'44).

Specificity of uptake

The effect on Zn2+ uptake of various metal ions wasdetermined at metal ion concentrations given in Table 1. OnlyCu2+ and Cd2+ had any significant effect on uptake from

50

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S Of)

'" -0..I< S 25'"i5. S:::l

'"u ..I<

" '"N i5. ! l:::l

U

"~N ~

o 10 20Time (min)

Fig. 1. Uptake from 10 nM zinc by P. notatum grown in mediumcontaining 26'8 ~M zinc (e), no zinc (0) and I mM zinc (0). Errorbars represent S.E.M. values from 5 replicate experiments.

Time (min)Fig. 2. Zinc uptake from 10 nM zinc in the presence of 50 mMglucose (e), in the absence of glucose (0) and in the presence ofglucose and 100 ~M-FCCP (0). Error bars represent S.E.M. valuesfrom 5 replicate experiments.

Table 1. Effect of competing metal ions on uptake of zinc. Data representthe uptake of zinc in the presence of a competing cation expressed as apercentage of uptake of zinc alone after 20 min incubation at 300

calculated from mean data from 5 replicate experiments

lIO9291

8949

2

2664

40

6141

39

Zn2+ uptake(percentage of

control)

Mg,+}Mn 2+

N°2+

C~'+ 10 ~MCu2 -+-

Cd'+

Mg,-}Mn'+N"+C~'+ 100 ~MCu 2-t-

Cd'+

Competing metalion and concn

Zn'+conen

20 ~M

10 mM

o 10 20Time (min)

Fig. 3. Zinc uptake from 10 nM zinc by starved mycelium in thepresence of 50 mM glucose (e), in the absence of glucose (0) andin the presence of glucose and 100 ~M-FCCP (0). Error barsrepresent the S.E.M. values from 5 replicate experiments.

2010o

50

,.~C"0Of)

-0S 25S'"..I<

'"i5.:::l

U

"N

Uptake of zinc by Penicillium notatum 714

2

-0·2 -0·1 0 0·1 0·21S

Substrate conc (5) (nM)

Fig. 4. Lineweaver-Burk plot of zinc uptake in the range 5-100 nMin the absence of added cations (.) and in presence of 500 nMcopper (6) and 500 nM cadmium (0). Data are means from 3replicate experiments.

to those obtained for the manganese transport system of C.ufilis (Parkin & Ross, 1986), P. notafum (Starling & Ross, 1990)and bacteria (Silver & Lusk, 1987). The non-competitiveinhibition seen with Cu2+ was probably due to toxicity of thision. Uptake was sensitive to the zinc content of the mycelium,being enhanced by growth in medium low in Zn2+ butabolished by growth in medium with a high Zn2 + content.

At an external zinc concentration of 20 I-lm, uptake wascompetitively inhibited by all the ions tested, suggestinguptake by a system with a broad specificity, probably themagnesium transporter, as growth of mycelium in mediumwith a low magnesium content stimulated zinc uptake from20 I-lM-Zn 2

+ The much higher Km obtained with higherconcentrations of zinc indicates a much lower affinity of thenon-specific transporter for zinc. These results are in broadagreement with those obtained with relatively high concen­trations of zinc by Budd (1988) - though in that work zincuptake was stimulated by growth in medium low in magnesiumat all external zinc concentrations tested - and are comparablewith data on manganese uptake by C. uti/is (Parkin & Ross,1985), P. notatum (Starling & Ross, unpublished data) andbacteria (Silver & Lusk, 1987).

/I

I0//

/I

I/

1V

Ic'6

10 nM-Zn2+. However, all ions tested inhibited zinc uptakefrom 20 I-lm-Zn2+. A Lineweaver-Burk plot (Fig. 4) of zincuptake in the range 5-100 nM showed that Zn2+ transport wascompetitively inhibited by Cd2+ and non-competitivelyinhibited by Cu2+. Zinc uptake in the range 10-100 I-lM wascompetitively inhibited by all the ions tested. Further, it wasnoted that uptake of Zn2+ from 20 I-lM was markedly stimulatedby growth of mycelium in medium with a low magnesiumcontent (data not shown), suggesting that at this concentrationzinc uptake may have involved the magnesium transportsystem.

DISCUSSION

The results presented above show that uptake of zinc byPenicillium notatum from concentrations of 10 nM and 20 I-lM isenergy-dependent and shows saturation kinetics as reportedin yeasts (Fuhrman & Rothstein, 1968; Failla et a/., 1976;Mowll & Cadd, 1983; White & Cadd, 1987) and otherfilamentous fungi (paton & Budd, 1972; Budd, 1988). Theability of FCCP to abolish uptake indicates the dependence oftransport on metabolic activity, though dependence On glucosecould only be demonstrated in mycelium which had beenstarved to deplete internal energy reserves. Failure of glucoseto stimulate Zn2+ uptake was observed in Neocosmosporavasinfecta, though no data on starved mycelium were reported(Budd, 1988). It was not possible from these experiments todetermine the nature of energy coupling, as the functions ofFCCP as an uncoupler or as a proton conductor could not bedistinguished.

The kinetic data obtained indicate the existence of twodistinct transport systems, with Km and Vmax values differingby three orders of magnitude. At an external zinc concentrationof 10 nM, uptake was highly specific, only Cd2 + offeringcompetitive inhibition. These results are similar to thoseobtained by Failla et al. (1976) for Candida uti/is and analogous(Received for publication 22 June 1990 and in revised form 5 October 1990)

The authors wish to thank the S.E.R.C. for a researchstudentship for A. P. S.

REFERENCES

Budd, K. (1988). A high affinity system for the transport of zinc inNeocosmospora vasinfecta. Experimental Mycology 12, 195-202.

Failla, M. L., Benedict, C. D. & Weinberg, E. D. (1976). Accumulation andstorage of zinc by Candida utilis. Journal of General Microbiology 94, 23-26.

Failla, L. j. & Niehaus, W. G. (1986). Regulation of zinc uptake andversicolorin A synthesis in a mutant strain of Aspergillus parasiticus.

Experimental Mycology 10, 35-41.

Fuhrman, G. F. & Rothstein, A. (1968). The transport of Zn2-, Co2- and Ni 2+

into yeast cells. Biochimica et Biophysica Acta 163, 325-330.

Lawford, H. G., Pik, j. R., Lawford, G. R., Williams, T. & Kligerman. A. (1980).

Hyperaccumulation of zinc by Zinc-depleted Candida utilis grown inchemostat culture. Canadian Journal of Microbiology 26, 71-76.

McHan, F. & johnson, G. T. (1970). Zinc and amino acids: importantcomponents of a medium promoting growth of Monascus purpureus.

mycologia 62, 1018-1031.

Mowll, j. L. & Gadd, G. M. (1983). Zinc uptake and toxicity in the yeastsSporobolomyces roseus and Saccharomyces cerevisiae. Journal of General

Microbiology 130, 279-284.

Parkin, M. j. & Ross, I. S. (1985). Uptake of copper and manganese by theyeast, Candida utilis. Microbios Leliers 29, 115-120.

Parkin, M. j. & Ross, I. S. (1986). The speCific uptake of manganese in theyeast Candida utilis. Journal of General Microbiology 132, 2155-2160.

Paton, W. H., N. & Budd, K. (1972). Zinc uptake in Neocosmospora vasinfecta.

Journal of General Microbiology 72, 173-184.

Perlman, D. (1949). Effects of minor elements on the physiology of fungi.Botanical Reviews 15, 195-220.

Ponta, H. & Broda, E. (1970). Mechanisrnen der Aufnahme von Zink durchBackerhefe. Planta 95, 18-26.

Silver, S. & Lusk, j. E. (1987). Bacterial magnesium, manganese and zinctransport In Ion Transport in Prokaryotes (ed. B. P. Rosen & S. Silver), pp.165-201. New York, U.s.A.: Academic Press.

Thind, K. S. & Rawla, G. S. (1967). Trace element studies on six species ofHelminthosporium. Proceedings of the Indian Academy of Sciences 66, 250-272.

Weinberg, E. D. (1977). Mineral element control of microbial secondarymetabolism. In Microorganisms and Minerals (ed. E. D. Weinberg), pp.298-316. New York: Dekker.

White, C. & Gadd, G. M. (1987). The uptake and cellular distribution of zincin Saccharomyces cerevisiae. Journal of General Microbiology 133, 727-737.