The Effect of Cycloheximide on Nitrogen and Carbohydrate Metabolism of Cladosporium cladosporioides and Cunninghamella echinulata grown on different nitrogen sources

Zbl. Mikrobiol. 137 (1982), 125-132 [Fac. of Science, Botany Dept.; Zagazig University, Zagazig, Egypt)

The Effect of Cycloheximide on Nitrogen and Carbohydrate Metabolism of Cladosporium cladosporioides and Cunninghamella echinulata Grown on Different Nitrogen Sources A. A. ELESSAWY, S. M. GHAZI, M. E. YOUNIS and A. A. ABD EL RAZAK

Summary Cycloheximide induced a reduction in the uptake of nitrates, sugars and amino acids by the growing fungal mycelia of Cladosporium cladosporioides and Cunninghamella echinulata. As indicated in the experimental results, reducing sugars and total soluble-N including amino-N were partly recovered in the metabolism solutions of the differently treated samples in presence of cycloheximide. This was considered as evidence of leakage of metabolites from the growing mycelia and of inversion of sucrose in the respective media, prior to its uptake.

Zusammenfassung Cycloheximid fuhrte zu einer Hemmung der Aufnahme von Nitraten, Zucker und Aminosiiuren beim Wachstum des Mycels von Cladosporium cladosporioides und Cuninghamella echinulata. Wie aus den Versuchsergebnissen ersichtlich, wurden reduzierende Zucker und der gesamte Iosliche Stickstoff einschlieElich der Aminosauren zum Teil in den Niihrliisungen der verschieden behandelten Proben in Gegenwart von Cycloheximid wiedergefunden. Das liiBt die Abgabe von Stoffwechselprodukten von den wachsenden Mycelien an die jeweiligen Nahrlosungen vermuten und die Urnwandlung der Sucrose vor der Aufnahme.

The effect of cycloheximide on carbohydrate and nitrogen-metabolism has received much attention. Thus, WHIFFEN (1950), BENNETT-CLARK et al. (1964), DEKLOET (1966), RAO and GROLLMAN (1967), SISLER and SIEGEL (1967), MAYO et al. (1968), MACDONALD and ELLIS (1969), have dealt with this subject in their investigations. The present study is an attempt to investigate further the effect of cycloheximide on nitrogen and carbohydrate metabolism of the two isolated fungi, Cladosporium cladosporioides and Cunninghamella echinulata, grown on different nitrogen sources, namely: sodium nitrate, Dl-serine, and L( -)tryptophan in the case of Cl. cladosporioides and sodium nitrate and DL-serine for C. echinulata.

Material and Methods Organisms The two experimental fungi, Cladosporium cladoeporioides (Fres) and Cunninghamella echinulata (Thaxter) were isolated from Egyptian soil, collected from Zagazig area. They were identified according to GILMAN (1957) and DE VRIES (1952). The identification was kindly confirmed by Prof. Dr. A. H. MOBASHER, Dean of Faculty of Science, Assiut University, Egypt, who is also in charge of the culture of fungi there, and to whom the authors are greatly obliged. Cultural conditions and composition of media For this experiment, the two fungi were separately inoculated in 50 ml sterile liquid media in 250 ml Erlenmeyer flasks and incubated at 27 DC for 10 days. The media used were N-free basal medium containing 3 g% sucrose (replaced by an equivalent weight of glucose in case of C. echinu-

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laltt) 0.1 g% potassium dihydrogen phosphate; 0.05% magnesium sulphate; 0.05 g% potassium chloride and one ml% of microelements solution (YOUSEF and ALLAM 1967). The various nitrogen sources (sodium nitrate, Dl-serine or L(-)tryptophan) were separately added in equivalent weights to the basal medium each being used as the sole nitrogen source that was added as cold sterile stock solution to the sterile basal medium. Cold sterile cycloheximide was prepared in such concentrations that not more then one ml had to be added to the culture medium to maintain 100 mg/l for 01. clodosporioidee and 30 mg/l for O. ecliinulata, The media were inoculated under septic conditions with the standard inoculum of one ml of the spore suspension from 7-day old cultures. At the end of incubation period, the fungal mycelia were filtered off, thoroughly washed several times with warm sterile distilled water and then dried in an oven at 60°C to constant weight. The dried mycelia were used for extraction and analysis of carbohydrate and nitrogen fractions. Also the cell-free culture filtrates were subjected to estimation of sugars and nitrogen fractions. Method of analysis Estimation of direct reducing value (D.R.V.): This was estimated by the method of SOMOGYI (1937) as modified by HANES (unpublished) and described by YOUNIS (1963). The direct reducing value of the sample that includes hexoses and non fermentable reducing substances will be considered in this study as equivalent to reducing sugars and is expressed as glucose equivalent. Estimation of total reducing value after sucrose hydrolysis (T.R.V.): To a known volume the mycelial extract and adequate amount of B.D.H. invertase preparation was added and enough time for complete hydrolysis of sucrose was allowed. The reducing value was then determined as mentioned above. Estimation of polysaccharides: According to NAGUIB (1963). Estimation of nitrogenous constituents: Various techniques have been developed for extraction of nitrogenous fractions from plant tissues with different degrees of specificity. The method used in this investigation was essentially that of YEMM and WILLIS (1956). Estimation of total soluble nitrogen: The total soluble N was determined by the conventional micro-Kjeldahl method (PIRIE 1955). Estimation of amino-N: The technique used was that of MUTING and KAISER (1963). Estimation of total nitrogen: Total nitrogen was determined by the conventional micro-Kjeldahl method.

Results A. Analysis of the culture media Nitrogen and sugar content The culture media were analysed for their total soluble N, amino-N, and reducing sugars, according to the nitrogen source present in presence and absence of cycloheximide. Results are presented in Table 1 and 2. Comparing the amount of nitrogen absorbed from the different nitrogen-containing medium, it appears that the highest amount of nitrogen absorbed was from the sodium nitrate media and the lowest was from L(-)tryptophan in case of Cladosporium dadosporioides. In case of Cunninghamella echinulata, the disappearance of nitrogen was highest from sodium nitrate and lowest from the Dl-serine nitrogen media. The presence of cycloheximide in the media apparently retarded the rate of nitrogen absorption by the two fungi. Moreover, the presence of arnino-N in the NaN0 3 media may point to leakage of nitrogen from the mycelia of the two fungi. Also from Tables 1 and 2 it is evident that the two fungi absorbed sugars at variable rates from the different media. The following sequence of media: NaN0 3 > DLserine> L(-)tryptophan and NaN0 3 > DL-serine was displayed with respect to absorption of sugars by Cl. cladosporioides and C. echinulate, respectively.

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Table 1. Analysis of nitrogen fractions and 1 educing sugars in the differently treated culture medium on which Cladosporium cladosporioides was grown for 10 days. The average values listed are expressed as mg Nand mg glucose equivalent/l00 g mycelial dry weight Nitrogen or Carbohydrate fraction

Sodium nitrate

159.7 Amino-N 2,211.1 Total Soluble N Reducing sugars 54,143.7

Dl-Serine

Dl-Serine Cyelo-

+

L(.)Tryp. tophan Oycloheximide

L(-) Tryptophan

Sodium nitrate Oycloheximide

177.8 2,379.3

186.2 2,846.4

265.5 4,741. 7

275.6 4,922.5

309.8 5,379.0

62,425.7

89,615.9

281,990.5

245,387.5

268,145.2

Moreover, the inclusion of cycloheximide absorption of sugars by the two fungi.

+

III

heximide

+

the media markedly retarded the

B. Analysis of the fungal mats 1. Nitrogen content of Cladosporium cladosporioides

The amounts of the nitrogen constituents of Ol. cladosporioides depend from the nitrogen sources and from the presence or absence of cycloheximide as it is shown in Table 3. It is evident that more or less comparable amounts of nitrogen absorbed from the different media appeared in part as amino and total soluble N and in part accumulated in the mycelial mats as protein nitrogen. Due to low absorption of nitrogen in presence of cycloheximide, markedly lower amounts of protein-N were detected in the respective mycelial mats. A corresponding increase in amino-N and in total soluble N was detected in media with cycloheximide compared to media free from fungicide. It should be mentioned also t hat in presence of cycloheximide, less protein was formed from sodium nitrate than from amino acids media. 2. Carbohydrate content of Oladosporium. cladoeporioides The full data of the carbohydrate content of the differently treated samples of Ol, cladosporioides are presented in Table 4. Sucrose was either completely absent or

was present in negligible amounts and hence it was not presented in the Table. As apparent from the Table, comparable amounts of reducing sugars, polysaccharides and total carbohydrates were detected in the different nitrogen media. After treatment with cycloheximide a marked increase of carbohydrate content was observed above that present in mycelial mats grown on nitrogen sources alone. 3. Nitrogen content of Ounninghamella echinulata Table 5 shows the amount of fractions influenced by different sources in presence or absence of cycloheximide. In NaN0 3 media, lower amount of amino-N than that present in Dl-serine media was detected. Addition of cycloheximide in the media induced accumulation of different amounts of amino-N in the mycelial mats. Total soluble N was markedly increased by cycloheximide and those amounts detected in the nitrate media were comparatively higher than those detected in mycelia grown on DL-serine media.

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In regard to protein-N content, comparable amounts accumulated in mycelia grown on both nitrogen media. In presence of cycloheximide markedly lower amounts of protein nitrogen were determined in the respective mycelia. Moreover, building up of protein was lower in the case of nitrate than in the case of DL-serine used as a nitrogen source. The behaviour of total N followed a similar pattern to that of protein nitrogen which is the predominant fraction in the nitrogen pool of O. echinulata. 4. Carbohydrate content of Ounninghamella echinulata The effects of various nitrogen sources in presence or absence of cycloheximide on the carbohydrate content of O. echinulata are presented in Table 6. Table 2. Analysis of nitrogen fractions and reducing sugars in the differently treated culture media on which Ounninghamclla cchinulata was grown for 10 days. The average value listed are expressed as mg Nand mg glucose equivalent/100 g mycelial dry weight Nitrogen or Carbohydrate fraction

Sodium nitrate

DI-Serine

Sodium nitrate Cycloheximide

+

DI-Serine + Cycloheximide

114.6

153.3

193.1

Total soluble N

2,340.3

2,739.2

4,631.9

5,652.5

Reducing sugars

98,245.6

126,488.7

855,555.5

696,045.2

Amino-N

211.0

Table 3. Analysis of nitrogen content of Cladosporium cladosporioides mycelia grown for 10 days on media containing different nitrogen sources in presence or absence of cycloheximide. The average values listed are expressed as mg N/100 g dry weight of mycelium Nitrogen fraction

Sodium nitrate

DI-Serine

L( -)Tryptophan

Sodium nitrate + Cycloheximide

D1-Serine Cycloheximide

+

L(-)Tryptophan + Cycloheximide

40.8

43.7

46.6

69.9

58.3

61.2

499.9

416.7

458.3

666.7

541.7

583.3

Protein-N

3,780.1

3,663.3

3,701.7

1,113.3

1,438.3

1,336.7

Total N

4,280.0

4,080.0

4,160.0

1,780.0

1,980.0

1,920.0

Amino-N Total soluble N

Table 4. Analysis of the carbohydrate content of Oladosporium cladosporioides mycelia grown for 10 days on media containing different nitrogen sources in presence or absence of cycloheximide. The average values listed are expressed as mg glucose equivalent/IOO g dry weight of mycelium Carbohydrate fraction

Sodium nitrate

Dl-Serine

L( -)Tryptophan

Sodium nitrate + Cycloheximide

Dl-Serine Cyoloheximide

+

L(-)Tryptophan + Cycleheximide

479.3

426.0

452.6

585.8

532.5

559.1

Polysaccharides

26,525.0

25,293.8

25,959.4

29,287.5

27.956.3

28,621.9

Total carbohydrate

27,104.3

25,719.8

26,412.0

29,873.3

28,488.8

29,181.0

Reducing sugars

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The Effect of Cycloheximide

Table 5. Analysis of the nitrogen content of Cunninghamella echinulata mycelia grown for 10 days on media containing different nitrogen sources in presence or absence of cycloheximide. The average values are expressed as mg N/100 g dry weight of mycelium Nitrogen fraction Amino-N

Total soluble N Protein-N Total N

Sodium nitrate 37.40 333.5 3,410.5 3,744.0

Dl-Serine

46.75 266.8 3,433.2 3,700.0

Sodium nitrate Cycloheximide

+

+

Dl-Serine Cycloheximide

68.63 667.00

65.45 566.95

993.00 1,660.00

1,377.05 1,944.00

Table 6. Analysis of the carbohydrate content of Cunninghamella echinulata mycelia grown for 10 days on media containing different nitrogen sources in presence or absence of cycloheximide. The average values listed are expressed as mg glucose equivalent/1 00 g dry weight of mycelium Carbohydrate fraction

Sodium nitrate

Reducing sugars 159.8 Polysaccharides 5,591.3 5,751.0 Total sugars

+

Dl-Serine

Sodium nitrate Cycloheximide

DI-Serine Cycloheximide

133.1 5,325.0 5,458.1

213.0 6,390.0 6,603.0

186.4 6,123.0 6,309.4

+

As in the case with Cl. cladosporioides, estimation of sucrose let to negligible amounts and hence it was not presented in the Table. Reducing sugars were present in low amounts in mats grown on nitrogen media alone and, when supplemented with cycloheximide, higher amounts of reducing sugars were observed. Marked amounts of polysaccharides were determined in mats grown on nitrogen media. In presence of cycloheximide more accumulation of polysaccharides was evident. The behaviour of total sugars followed a pattern similar to that of polysaccharides which appeared to be the predominant fraction in the carbohydrate pool of C. echinulata.

Discussion Of the well known antifungal antibiotics, cycloheximide has been reported by FORD et al. (1958). It is produced by Streptomyces griseus and has been shown to be very active against a wide range of fungi (PETERSEN and CATION 1960; MCOLURE and CATION 1951; HAMILTON and SZKOLNIK 1953 and 1958; KLOS and FRONEK 1964; LIVINGSTON 1953; SILVERMAN and HART 1954 ; WALLEN 1955 and 1958; HOCKING and WHITE 1965; Moss et al. 1960; Moss 1961; HELTON and ROHRBACH 1967; PHELPS et al. 1957 and 1966). Cladosporium cladosporioides and Cunninghamella echinulata have been chosen to study the effect of cycloheximide on their nitrogen and carbohydrate metabolism in presence of different nitrogen sources. The Tables 1-6 indicate the influence of cycloheximide on the metabolism of Cl. cladosporioides and C. echinulata in presence of different types of nitrogen sources.

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Table I showed that Ol. cladosporioides absorbed highest amount of nitrogen from the sodium nitrate medium and the lowest amount L(-)tryptophan medium. In case of O. echinulata (Table 2) the nitrogen disappeared from sodium nitrate medium at the highest rate whereas lowest rate of absorption was from the Dl-serine medium. In general, as apparent from Table 1 and 2, the use of amino acids as nitrogen sources increased the amount of reducing sugars, amino nitrogen and total soluble nitrogen in the metabolism solutions compared with media containing sodium nitrate as nitrogen source. That increase may be due to the decrease in the assimilation of sugars and nitrogen from the medium or to the increased leakage of nitrogenous metabolites from the fungal mycelia into the culture media. In case of Ol. cladosporioides, sucrose in the culture media appeared to be converted into reducing sugars due to surface invertase activity and this might have been a prerequisite for its uptake (PUTMAN and HASSID 1954; HARLEY and SMITH 1950; HATCH and GLAZIOU 1963; YOUNIS et aI. 1969b, 1970b). However the present results do not allow us to exclude the possibility that sucrose may penetrate intact as suggested by PORTER and MAY (1955) and KRIEDEMANN and BEEVERS (1967). It is also evident, from the data in the Tables 1 and 2, that treatment with cycloheximide increased the amount of the reducing sugars, amino-N and total soluble N irrespective of the nitrogen source used. This may be due, in part, to inhibition of sugar and nitrogen uptake in presence of cycloheximide as earlier reported by Ross (1974), JUAN et aI. (1973) and Lux and PETZOLD (1976) and, in part, to excessive leakage of sugars and nitrogen compounds from the fungal mycelia into the culture media. In this connection the observations of HEINICH (1976) that cycloheximide stimulates the intracellular protein degradation and those of BERTHE et al. (1977) that cycloheximide may affect the chemical composition of cell wall which may facilitate the leakage of metabolites, are of interest. Also, leakage of nitrogen has been shown to be operative from plant tissues under many physiological situation (YOUNIS et aI. 1969a, b; YOUNIS et al. 1970a, b; YOUNIS and EL-SHAFEY 1973). Most of the absorbed nitrate rapidly disappeared in the plant tissues with a correspending increase in total soluble nitrogen, protein nitrogen and total nitrogen content of the mycelium of the two tested fungi. This might indicate that nitrate was reduced and in presence of carbon skeleton, from the carbohydrate, transformed into amino acids; the latter being subsequently synthesised into proteins. The rate of protein synthesis appeared to be faster than the rate of reduction and hence the observed reduction in the amino nitrogen content of the plant tissues. The possible pathway for nitrate utilization is in accordance with the suggestion of BURSTROM (1945) that, in a group of plants represented by fungi and perhaps roots of higher plants, nitrate is reduced inorganically down to ammonia before its utilization. Other investigators, using various plant tissues, forwarded evidence that nitrate utilization takes place through the classical reduction pathway (MYERS and CRAMER 1948; MENDEL and VISSER 1951; TOLBA and SALAMA 1957; FEWSON and NICHOLAS 1961).

In a preliminary experiment (EL-RAZAK 1979) O. echinulata was shown unable to utilize L(-)tryptophan as a sole nitrogen sources whereas Ol. cladosporioides utilized L(-)tryptophan as sole nitrogen source but at a lower rate than DL-serine utilization. The absorbed amino acid nitrogen was partly accumulated in the fungal tissues and some was detected as protein nitrogen. Thus, the absorbed amino nitrogen was utilized in building up proteins. Several workers reported utilization of amino acids by fungi and higher plant tissues either directly or through transamination (LEONIAN and LILLY 1938; STEINBERG 1942; SAID and YOUNIS 1952 and 1953; STEINER 1959; JOY and FOLKES 1965).

The Effect of Cycloheximide

131

Furthermore, ROBERTS et al. (1955) have demonstrated, in microorganisms, that externally supplied amino acids may be assimilated and incorporated into protein. The presence of cycloheximide with sodium nitrate, DL-serine or L(-)tryptophan (in case of OZ. cladosporioides) and with sodium nitrate or DL-serine (in case of C. echinulata) suppressed the rate of nitrogen uptake (Table 3 and 5) and vigorously retarded protein synthesis and hence there was greater accumulation of total soluble nitrogen. These results are in agreement with those achieved by many investigators (SISLER and SIEGEL 1967; RAO and GROLLMAN 1967; BALIGOR et al. 1969 ; VANCE and SHERWOOD 1976; HEINICH 1976). Carbonate analysis in the mycelia of the two tested fungi (Table 4 and 6) revealed that higher amounts of carbohydrates were accumulated in the fungal mats grown on media containing sodium nitrate than in those grown on DL-serine or L(-)tryptophan. The reduction in the carbohydrate content of the mycelia grown on media containing amino acids (Dl-serine or L(-)tryptophan) may be due to the role of amino nitrogen in the activation of respiration as suggested by SCHWABE (1932), SAID (1934), SAID and YOUNIS (1952 and 1953) and YOUNIS (1960). Presence of cycloheximide - with any nitrogen source used - increased the carbohydrate content of the mycelia of the two tested fungi. The accumulation of carbohydrate in the presence of cycloheximide may be due to the inhibitory effect of cycloheximide on respiration as reported by WHIFFEN (1950), MACDONALD and ELLIS (1969) and GARBER et al. (1973).

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