Polyphosphate in Zygomycetes: a cytochemical study

Polifosfato em Zygomycetes: um estudo citoquímico

POLYPHOSPHATE IN ZYGOMYCETES: A CYTOCHEMICAL STUDY

Allana Elesbão do Nascimento Shari'a^1,2; Aline Elesbão do Nascimento^1,2; Marcos Antonio Barbosa de Lima²; Galba Maria de Campos-Takaki^1,2*; Wanderley de Souza³

¹ Núcleo de Pesquisas em Ciências Ambientais, Departamentos de Biologia e Química, Universidade Católica de Pernambuco, Boa Vista, PE, Brasil. ² Laboratório de Imunopatologia Keizo Asami (LIKA), Microscopia Eletrônica, Universidade Federal de Pernambuco (UFPE), Boa Vista, PE, Brasil. ³ Laboratório de Ultraestrutura Celular, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil

Submitted: August 08, 2001; Retusrned to authors for corrections: November 22, 2001; Approved: May 13, 2002

ABSTRACT

The inorganic phosphorus content, and distribution, structure and localization of polyphosphate in mycelia of zygomycetous fungi was evaluated. Ultrastructural cytochemistry was successfully used to identifying the localization and distribution of polyphosphate in Absidia cylindrospora, Gongronella butleri and Mucor javanicus. The results revealed differences in the cytochemical staining pattern in all species studied and a uniform labeling on the cellular surface (cell wall and citoplasmic membrane). Reaction products were observed in intracellular structures and cellular membrane. Intracellular staining was observed in trabecullar, vacuolar and vesicular structures, in dense bodies and in the cytoplasm. However, the cytochemical staining intensity varied during the cellular growth. Analytical procedure revealed the phosphorus content during cell growth. The results demonstrated that phosphorus content varied during cultivation time and could be related to the polyphosphate cytochemical staining. Polyhosphate functions such as storage and utilization are discussed.

Key words: Ultrastructural cytochemistry, polyphosphate, Zygomycetes.

INTRODUCTION

Members of Zygomycetes occur on or within a variety of substrata. Some species cause plant or animal diseases. They are often notable in culture because of their extremely fast growth rate, a characteristic important in their ability to compete in the soil. Some members not only are common soil saprophytes but also are important or are involved in technical aspects of industrial procedures causing spoilage of stored grains and diseases in humans, animals and plants. Some are used to produce commercially important products, such as organic acids, pigments, fermented oriental foods, alcohols and modified steroids (3,5,9,25,31,32). Some members have been called "oportunistic fungi" to emphasize the aspect of a normally saprophitic fungus which can suddenly become parasitic and pathogenic when introduced in animal body, particularly in humans (1,14,20,21,23,24,26,34,35).

In microbial cells, inorganic polyphosphate (polyP) plays a significant role in increasing cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. PolyP is a chain of tens or many hundreds of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. It has long been hypothesized that polyphosphate performed numerous and varied biological functions depending on the need and its location in a specie, cell or subcellular compartment. Among these are phosphate and energy reserve, chelator of metals, buffer against alkali, involvement in cell envelope formation and function, gene activity control, bacterial transformation, disposal of pollulant phosphate, substrate for glucose phosphorylation, minimization of toxic effects of heavy metals, nucleic acid and phospholipid metabolism. The most valuable biological effect of polyphosphate is in physiological adjustment to growth, development, stress response and nutrient deprivation (10,11,16,17,19,27,36).

The subcellular localization of the polyphosphate in different organisms is still controversial. Microscopists are familiar with the conspicous so-called volutin or metachromatic granules occurring in many microorganisms and it is generally agreed that these granules contain polyphosphates. A great difference is observed between prokaryotes and eukaryotes in their polyP-metabolizing enzymes. The synthesis and degradation of polyP in each specialized organelle or compartment are mediated by different sets of enzymes (2,6,11,12,16,17,18,19,36).

Polyphosphates have been known to occur in numerous filamentous fungi and in the yeasts. The ability to acumulate polyP as a response of physiological adaptation during growth and development has been observed in some fungi. In these organisms polyphosphate cover a wide range of molecular sizes, particularly those with low molecular weight and are found in the form of dense metachromatic granules (7,22,29,30,33).

The present paper reports the quantification of inorganic phosphorus, localization, structure and distribution of polyphosphate in the mycelium of Absidia cylindrospora, Gongronella butleri and Mucor javanicus.

MATERIALS AND METHODS

Microorganisms and culture conditions -Absidia cylindrospora (URM 3351) HAGEN, Gongronella butleri (URM 2151) (LENDNER) PEYRONEL and DAL VASCO, and Mucor javanicus (URM 1989) WHEMER (Mucorales - Zygomycetes) were kindly supplied by the Culture Collection of the Universidade Federal de Pernambuco. The cultures of Zygomycetes were mantained on Difco PDA (Potato Dextrose Agar) slants, incubated at 26ºC. The liquid medium used for large scale cultures was SMM (Synthetic Medium for Mucoralean) modified (4) without agar. Actively growing mycelia, grown on PDA, was transferred to Erlenmeyer flasks containing 40 ml of SMM and incubated during 15 days, at 26ºC, at 250 hz. All samples were prepared in five replicas.

Growth curves - Samples collected at 3, 6, 9, 12 and 15 days of culture were submitted to liophylization and mantained in a vacuum dissecator until constant weight. The final value corresponded to arithmetic media of five replicas of each sample.

Analytical procedure - Liophylized samples (2mg) corresponding to 3, 6, 9, 12 and 15 days of culture were mixed in distilled water, and frozen during 24 hours. After this period, samples were thawed, and then disrupted with glass stick and centrifuged at 3000 rpm, during 15 minutes. The supernatant was used to determine polyphosphate content as inorganic phosphorus form. Total inorganic phosphorus content was determined by using the Sigma Kit ¾ Fiske and Subbarow method. To each sample of 1 ml of supernatant, 1 ml of distilled water, ammonium molibdate 2.5% and ascorbic acid 5.0% were added, and the samples were boiled for 5 minutes. Samples were analysed through spectrophometry at 660 nm.

Electron microscopy

Routine studies - Samples of Absidia cylindrospora, Gongronella butleri and Mucor javanicus, grown in liquid SMM, were collected after 3, 6, 9, 12 and 15 days of culture. Samples were washed twice in PBS (phosphate-buffered saline) pH 7.2 for 10 minutes and fixed in 2.5% glutaraldehyde, 0.1M cacodylate buffer pH 7.4, during 2 hours, at room temperature, post-fixed in 1.0% osmium tetroxide for 1 hour at room temperature. Post-fixation was followed by washing in 0.1M cacodylate buffer, pH 7.4. Samples were dehydrated in acetone and embedded in Epon. Samples were cut in a REICHERT JUNG ultramicrotome, collected on copper grids and contrasted with uranyl acetate and lead citrate. Eletronmicrographs were obtained in a JEOL 100-CX (80 Kv) transmission electron microscope.

Polyphosphate cytochemistry ¾ The cytochemical detection of polyphosphates was performed according to Ebbel et al. 1958 (8). Absidia cylindrospora, Gongronella butleri and Mucor javanicus samples, grown in liquid SMM, were collected after 3, 6, 9, 12 and 15 days of culture. Samples were washed twice in PBS, pH 7.2 for 10 minutes, fixed in 2.5% glutaraldehyde, 0.1M cacodylate buffer pH 6.5 during 1 hour at 4ºC and washed in distilled water. Samples were incubated in 2% lead nitrate solution, pH 3.4 during 4 hours at 4ºC, and in 1% ammonium sulphite, during 30 minutes at 4ºC. After this period, samples were washed in distilled water, post-fixed in 1.0% osmium tetroxide for 1 hour at room temperature. Post-fixation was followed by washing in 0.1M cacodylate buffer, pH 7.4. Samples were dehydrated in acetone and embedded in Epon. Thin sections and photographs were obtained as described previously. Control samples were incubated in trichloroacetic acid after the fixation during 6 hours at 4ºC. After this period, samples were treated as cited before.

RESULTS

Figs. 1, 2 and 3 exhibit Absidia cylindrospora, Gongronella butleri and Mucor javanicus growth curves and the inorganic polyphosphate content, respectively. It was possible to observe an increase in dry weight in the first three days of culture. After this, a progressive decrease in the dry weight was observed. A similar pattern of cell growth was observed among the species studied.

Absidia cylindrospora exhibited the lowest phosphorus content on the third day of growth compared to other species. A visible phosphorus content decrease was observed on the sixth day of growth. In the ninth day the phosphorus content increased and reached the maximum value on the twelfth day of culture. After this period, there was a new decrease which was accentuated after fifteen days of growth. During the initial period of growth, cells of Gongronella butleri and Mucor javanicus had substantially higher inorganic phosphorus levels than cells on the final period of cultivation. A pattern similar to that exhibited by Absidia cylindrospora was observed for Gongronella butleri and Mucor javanicus.

Figs. 4, 5 and 6 show the ultrastructure of Absidia cylindrospora, Gongronella butleri and Mucor javanicus cells, respectively. Cells presented homogeneous cytoplasm, thin cell wall and well defined organelles, such as mitochondria, lomassomes, endoplasmic reticulum, electron-dense bodies and vesicles at the initial period of growth. Some hyphae presented septa, inumerous vacuoles and multivesicular bodies (Fig. 7). The cellular wall became thicker after six and twelve days of cultivation (Fig. 8). The cytoplasm became heterogeneous after fifteen days. The cell wall presented different layers and became thicker compared to those observed in cells with twelve days. A intense process of vacuolization was observed through the cell growth (Figs. 9 and 10).

Gongronellabutleri presented the highest surface polyphosphate labeling and the presence of electron-dense bodies in the cytoplasm (Fig. 11). Mucor javanicus exhibited the highest polyphosphate labeling in the area correspondent to the contact between cell wall and cytoplasmatic membrane (Fig. 12). Reaction products were observed in different intracellular structures. All species presented polyphosphate labeling in vacuoles in the form of internal granular precipitates, reaction products in their membranes or in the form of electron-dense bodies or deposits associated to membranes (Figs. 13 and 14). The highest content of polyphosphate deposits were observed in Gongronella butleri and Absidia cylindrospora compared to Mucor javanicus (Figs. 11, 13 and 14). An intense polyphosphate labeling was higher in cells with three days of cultivation. Indeed, Absidia cylindrospora cells with nine days of cultivation presented the highest intense labeling in the cell surface (Fig. 15).

After nine days of cultivation Mucor javanicus cells, exhibited a low intensity of polyphosphate labeling in cell surface. However, reaction products were observed in cell membrane, cytoplasm and electron dense precipitates. Cells with twelve days exhibited an intense labeling in the cell membrane, vacuoles and inclusions. On the fifteenth day of cultivation cells exhibited the highest labeling intensity in vacuoles, cytoplasm and cell surface compared to Absidia cylindrospora and Gongronella butleri (Figs. 16 A-C). Electron-dense bodies and inclusions were not observed in samples on the fifteenth day of cultivation.

Samples with twelve days of cultivation showed a decrease of the polyphosphate reaction products at the cell surface of all species studied and the lowest intensity labeling was observed in Absidia cylindrospora. The cells presented an intense vacuolization process and an increase of the cell wall thickness (Fig. 17).

DISCUSSION

The occurrence of polyphosphate has been reported for various organisms, both eukaryotes and prokaryotes. The cytochemical deteccion of polyphosphate shows very distinct features among these organisms. The cytochemical staining of cellular polyphosphate by lead salts permit the observation of polyphosphate precipitated in situ with lead as metachromatic granules after exposure to the electron beam (8,11). However, the polyphosphate distribution in Zygomycetes has not previously been reported. Localization of polyphosphate within a cell also depends on the chain lenght. Low molecular weight polyphosphate are located in the cytoplasm, in granules, free in the cytosol or bound to cytoplasm compounds such as ribonucleic acids. More highly polymerized polyphosphates are found in the plasma membrane and in the cell wall (18).

Electron microscopic investigations published in recent years have shown that polyphosphate appears as electron-dense granules in association with a number of subcellular entities. Associations among polyphosphate granules and cell wall, cell membrane, vacuoles and in cytoplasm were reported (2,32,35).

The results presented in this work revealed the polyphosphate location, structure and distribution in Mucor javanicus, Gongronella butleri and Absidia cylindrospora. The present findings showed that the Zygomycetes studied exhibited a cytochemical polyphosphate labeling in different structures as well different amounts according to the specie. These results reflect the different ways in which the species store polyphosphate and may represent different functions related to its location. Also, a uniform polyphosphate labeling on the cellular surface, cell wall and cell membrane of all species studied was visualized. The presence of electron-dense bodies, trabecullar structures and vacuoles with reaction products was observed. In the cytoplasm the polyphosphate labeling was observed as dispersed patches or linear arrangements in the membrane of vacuoles. Electron-dense bodies presenting core with labeling intensity lower than the peripheric area were visualized. These results suggest that the Zygomycetes studied present polyphosphate molecules with different chain lenghts.

The cellular location, structure and distribution of polyphosphate are related to the cell growth phase. This suggests that the polyphosphate is developped in distinct phases of the growth cycle and is accumulated under certain growth conditions. Bacteria accumulate polyphosphates only under adverse growth conditions, others appear to accumulate during exponential growth phase in spite of the synthesis of nucleic acids, which depends of polyphosphate degradation (2,19,21).

In fungi, metabolic changes associated to pH of the culture medium, vacuolar pH, phosphorus content and polyphosphate chain size in cells on stationary and logarithmic growth phases were demonstrated in Saccharomyces cerevisiae, Gigaspora margarita and Candida humicola (12,24,31).

Polyphosphates have been implicated in energy storage due to the energy liberated in hydrolysis of their phosphoanhydride bonds, their associations with energy producing cellular structures and their enzymatic synthesis from cellular energy carriers (12,18,19,21).

It is very apparent that there were changes in the phosphorus content that take place during the growth cycle. The analytical study performed in this work revealed that the cells in exponential growth phase exhibited the highest inorganic phosphorus content compared to cells in other growth phases, indicating the necessity to evolve metabolic activities in this period of cellular growth. The inorganic phosphorus content decrease during the cellular growth cycle suggesting its utilization for molecular synthesis of cellular compounds.

The increase in the phosphorus content on the twelfth day of growth observed in this work presumably resulted from the transport of newly formed inorganic phosphorus out of intracellular structures from polyphosphate degradation, since newly phosphorus content was not added in the medium.

Some authors have suggested a mural localization of polyphosphate. The presence of polyphosphate on cellular surface of whole cells was observed in Neisseria gonorrheae, Propionibacterium acnes andHelicobacter pilori (2,17).

In fungi, the presence of polyphosphate was detected in isolated cell walls and mycelia of Zygomycetes and in Neurospora crassa and Kluyveromyces marxianus by analytical methods (4,7,35). The cytochemical localization of polyphosphate in the yeast Saccharomyces cereviseae was observed outside of plasma membrane (32).

The results obtained in this work revealed an intense polyphosphate labeling in cell wall and cell membrane of all species studied, similar to what have been described in bacteria.

The polyphosphate aggregation with the cell surface is involved in the maintenance of cytoplasmic membrane integrity and in the transport-associated phosphorylation of glucose and glucose derivatives (30). The occurrence of amorphous polyphosphate cytoplasmic precipitates was detected in Helicobacter pilori (2).

Mucor javanicus was the only Zygomycetes to present amorphous polyphosphate precipitated in cytoplasm.

The existence of vacuolar polyphosphate is a well known phenomenom in yeasts and bacteria. In these organisms the vacuolar polyphosphate is associated to storage material and functions as biological cation exchanger. Electron dense bodies containing polyphosphate were observed in Nostoc punctiforme, Helicobacter pilori and Propionibacterium acnes. These results were confirmed by rays X- microanalysis (2,15,17,18,32,35).

Despite of central roles of phosphate and ATP in metabolism and regulation, scant attention has been given to ubiquitous, abundant and conserved polyphosphate in fungi. The physiological roles of polyphosphate could be determined through studies on its distribution and structure at cellular level in order to identify and characterize the enzymes involves in polyP metabolism.

ACKNOWLEDGEMENTS

The authors are greateful to PRONEX, PADCT and CNPq for the financial support.

RESUMO

Polifosfato em Zygomycetes: um estudo citoquímico

O conteúdo de fósforo e a distribuição, estrutura e localização de polifosfato em micélios de Zygomycetes foram avaliados. A citoquímica ultrastrutural foi utilizada com sucesso para identificar a localização e a distribuição de polifosfato em Absidia cylindrospora, Gongronella butleri e Mucor javanicus. Os resultados obtidos revelaram diferenças no padrão de marcação citoquímica em todas as espécies estudadas, bem como, uma marcação uniforme sobre a superfície celular, correspondendo à parede celular e à membrana citoplasmática. Produtos de reação, resultantes da marcação citoquímica, foram também visualizados em estruturas intracelulares e na membrana celular. Estruturas trabeculares, vacuolares, vesiculares, sob a forma de corpos eletron densos e grânulos dispersos no citoplasma foram visualizadas. Adicionalmente, a marcação citoquímica variou durante as diferentes fases do crescimento celular. O conteúdo de fósforo variou ao longo do crescimento celular e do tempo de cultivo, possivelmente devido aos padrões da marcação citoquímica observados por meio da microscopia eletrônica de transmissão. São discutidas as funções do polifosfato como material de reserva e sua utilização.

Palavras-chave: citoquímica ultraestrutural, polifosfato, Zygomycetes.

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*

Corresponding Author. Mailing Address: Rua Nunes Machado, 42, Bloco J. UNICAP. 50050-900 - Boa Vista, PE. Brasil. Fax: [phone].

Publication Dates

• Publication in this collection
  27 Jan 2003
• Date of issue
  June 2002

History

• Accepted
  13 May 2001
• Reviewed
  22 Nov 2001
• Received
  08 Aug 2001