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Revista de Microbiologia

Print version ISSN 0001-3714

Rev. Microbiol. vol. 29 n. 4 São Paulo Oct./Dec. 1998

http://dx.doi.org/10.1590/S0001-37141998000400009 

INTRA AND EXTRACELLULAR NUCLEASE PRODUCTION BY ASPERGILLUS NIGER AND ASPERGILLUS NIDULANS1 

 

Adlane V. B. Ferreira*
Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

Submitted: September 30, 1997; Returned to authors for corrections: February 17, 1998;
Approved: July 23, 1998

 

SHORT COMMUNICATION

 

 


ABSTRACT

Intra and extracellular nuclease production by strains of Aspergillus niger and Aspergillus nidulans was estimated using a modified DNAse test agar and cell-free extract assays. Differences in the production of nucleases by A. niger and A. nidulans were observed. These observations suggest that the DNAse test agar can be helpful for a quick screening for some types of nucleases in filamentous fungi. The assays using cell-free extracts can also be useful for initial characterization of other types of nucleases.

Key words: Nucleases, Aspergillus niger, Aspergillus nidulans, filamentous fungi


 

 

Nucleolytic enzymes have several important biological roles, such as nutrition (pancreatic enzymes), protection (restriction enzymes), repair and recombination mechanisms or DNA transport. Nucleases can be inconvenient when genetic manipulation techniques are used. The frequency of genetic transformation can decrease dramatically if the vector is degraded by nucleases. Mink et al. (7) observed an increase in transformation efficiency of Saccharomyces cerevisiae and Aspergillus nidulans when protoplasts were obtained with an endonuclease-free enzyme preparation. Transient gene expression in rice tissues was also increased when plantules were exhaustively washed before the vector was added (2). Many nuclease inhibitors such as heparin (11) and spermidine (2) have been used in genetic transformation of fungi and other organisms.

Aspergillus niger and A. nidulans, despite being considered taxonomically closely related, show very different transformation frequencies in heterologous and in homologous systems. As an example, transformation of A. nidulans with the homologous oliC 31 gene resulted in frequencies of 10 to 70 transformants/µg DNA (12). In the transformation of A. niger with the correspondent oliC 3 gene (13), frequencies were lower than 2 transformants/µg DNA. In order to investigate the basis for this difference the levels of nuclease production was compared in a few strains of both A. nidulans and A. niger. A modified DNAse test agar originally described by Jeffries et al. (5) mainly for assaying bacterial nucleases was used to assay extracellular nucleases. Two other methods were used to assay nucleases in cell-free extracts.

Strains of A. niger used were: lgp 73 (pabA1, nicA1, olvA3, lgpB73 – Laboratório de Genética de Microrganismos, UNICAMP, Brazil) and wild type 350 (provided by M. Devchand, Allelix Inc., Canada). A. nidulans strains were biA1 methG1 (biaA1, methG1 - provided by I. Baracho, UNICAMP, Brazil) and T580 (pyrG) (provided by M. Devchand, Allelix Inc., Canada). Aspergillus Minimal Media - MM (9) and Complete Media - CM were used. CM is MM supplemented with 2 g peptone, 1.5 g hydrolysed casein and 0.5 g yeast extract per litre; vitamins are added as required. Incubation temperatures for A. niger and A. nidulans were 30º and 37ºC, respectively.

For the DNAse test agar, strains were inoculated in CM or MM agar plates, supplemented with 2 g/l of herring sperm DNA (HS DNA-Sigma). Strains were also grown in MM without inorganic phosphate. After 3 days of incubation, precipitation of nucleic acids was performed by adding 1N HCl to the plates for 5 minutes. Formation of hyaline halos around the colonies indicated DNAse activity, in contrast to an opaque background for unhydrolysed DNA. Nuclease S1 from Aspergillus oryzae and an endonuclease from Escherichia coli (Sigma) were spotted on the plates prior to addition of HCl as controls for the DNA degradation and precipitation.

A modification of a microtiter dish assay (1) was performed using a cell-free extract. About 106 protoplasts, obtained as described by Hamlym et al. (4), were resuspended in 500µl reaction buffer (TM - Tris-Cl 50 mM pH8, MgCl2 10 mM) and ultrasonicated by inserting a small tip into the suspension (3 cycles of 45 W for 10 sec.). After a 2 min. centrifugation at 12,000 g, the supernatant was collected. The wells of a microtiter dish were filled with 100 µl of TM containing 100 mg HS DNA/ml. Fifty microliters of the cell-free extract was applied to the first well and 1:2 dilutions were done for the next 10 wells. After overnight incubation at 37ºC one microgram of ethidium bromide was added to each well. The plate was placed on a UV light box and photographed through a red filter. Degradation of DNA was followed by the reduction in fluorescence intensity. Electrophoretic analysis of DNA exposed to extracts (from A. niger lgp73 and A. nidulans T580) was performed using three micrograms of a 5.8 kb plasmid or five micrograms of chromosomal DNA (from A. niger lgp73 strain). Incubation was done with 50 µl of the cell-free extract in the presence of TM at 37oC. Aliquots (10 µl) were taken from the reaction mixture at different intervals (from 5 to 45 minutes), electrophoresed in a 0.8 % agarose gel which was photographed on a UV light box, through a red filter. Degradation of DNA was detected by the formation of smaller fragments, as compared with high molecular weight chromosomal or plasmid DNA.

The possibility that nucleases could be interfering with transformation efficiency of strain lgp 73 was first suggested because of unsuccessful attempts to isolate intact DNA from mycelia using 25mM EDTA in the extraction buffer. When the concentration of EDTA was increased 4-fold, intact chromosomal DNA was obtained, suggesting an inhibition of enzyme activity on DNA.

The DNAse test agar was used to detect extracellular nucleases. Hydrolysis halos were not detected when the strains were grown on Complete Media or Minimal Media supplemented with HS DNA, possibly due to excess of phosphate sources in those media. Phosphate-repressible nucleases have been isolated from A. nidulans (6) and Neurospora crassa (3). For this reason the composition of the Minimal Medium was modified so that the main source of phosphate was derived from HS DNA (no inorganic phosphate added).

Table 1 shows the sizes of halos around colonies grown on modified MM. Growth of all colonies was normal in this medium but halos were formed only around A. niger colonies. A.niger wt350 produced more nucleases than lgp 73 as shown by the ratio halo/colony (Table 1). To evaluate if diffusion was the reason for the difference observed, the plates were kept at 4ºC for 4 days after the regular incubation times. The resulting hydrolysis halos were larger around A. niger colonies but no halos were observed around A. nidulans colonies (not shown). These results indicate that extracellular nucleases were not produced or secreted in the A. nidulans strains in the conditions tested.

 

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If during the process of protoplast production some cells are disrupted nucleases may be released that could influence transformation efficiency. The microtiter dish assay was performed to detect differences in the amount of other nucleases in A. niger and A. nidulans. Protoplasts were washed twice before sonication, so most of the extracellular nucleases of the protoplast forming mixture were eliminated at this point. Both A. nidulans and A.niger cell-free extracts had high activity on HS DNA in the microtiter dish assay and differences in the amount of enzyme were not detected (not shown). The ability of the enzymes contained in the cell-free extracts to degrade intact double stranded DNA was also evaluated by electrophoresis. Fig. 1 shows the digestion of fungal chromosomal DNA by nucleases contained in the extracts of A. niger and A. nidulans at different time intervals. Degradation of DNA was observed in samples from both species within the first 5 minutes of incubation and was not significantly increased after 45 minutes. The specificity of the nucleolytic enzymes contained in the extracts was different between the two species: the DNA fragments resulting from A. nidulans digestion were very large, ranging from about 20 Kb to less than 1 kb and A. niger enzymes produced fragments around 1 kb in the first 5 minutes of reaction. When plasmid DNA (5.8kb) was used fragments were very small and were not detected in a 0.8% agarose gel (not shown).

 

0009i02.jpg (17830 bytes)

Figure 1 - Electrophoresis of chromosomal DNA after exposure to cell-free extracts of A. niger (NG) and A. nidulans (ND). lgp 73 DNA was exposed to NG and ND cell-free extracts at diferent time intervals (5 to 45 min. as shown above the lanes); Int DNA: intact lgp 73 DNA; l HIII: lambda DNA digested with Hind III.

 

These results show that there is a difference in the production and/or secretion of extracellular nucleases between the A. niger and A. nidulans strains tested and that the specificity of other nucleases is also different between the two species. As some of A. nidulans (6) and N. crassa (3) nucleases the extracellular nucleases detected by the modified DNAse test agar in A. niger seem to be repressed by high phosphate levels. In addition, the A. niger nucleases detected here do not act as a modification/restriction system since degradation was observed with DNA from the A. niger lgp73 strain. Even though A. niger is a very important fungus used for production of several enzymes (8), nuclease production has not been analysed intensely in this species. The Aspergillus oryzae S1 nuclease is widely used in molecular biology protocols and recently, the gene for the nuclease O of A. oryzae was cloned (10). The characterization of the nucleases detected in this work could be of interest for biotechnology.

It can also be suggested from this study that the higher production of nucleases by A. niger strains can be one of the causes for the lower transformation efficiencies generally observed for this species. Several parameters have been considered for improving transformation efficiency in filamentous fungi. However, the production of nucleases by the manipulated fungus has not been given much consideration. To confirm the implication of intrinsic nucleases on transformation efficiency in A. niger however a more comprehensive analysis including strains of different backgrounds will be necessary. The DNAse test agar can be helpful for a quick screening of strains that produce large amounts of extracellular nucleases. This screening would avoid the use of such strains in transformation or point out to precautions such as handling of protoplasts at lower temperatures, additional washes of protoplasting cells or use of nuclease inhibitors.

 

 


RESUMO

Produção de nucleases intra e extracelulares por Aspergillus niger e Aspergillus nidulans

A produção de nucleases intra e extracelulares foi estimada em algumas linhagens de Aspergillus niger e Aspergillus nidulans usando um teste para DNAse em placa e um ensaio com extratos livres de células. Pôde-se observar diferenças na produção de nucleases entre A. niger e A. nidulans. Estas observações sugerem que o teste de DNAse em ágar pode ajudar na detecção rápida de nucleases extracelulares em fungos filamentosos. Os ensaios usando extratos livres de células podem também servir na caracterização inicial de outros tipos de nucleases.

Palavras-chave: Nucleases, Aspergillus niger, Aspergillus nidulans, fungos filamentosos.


 

 

REFERENCES

1. Ball, T.K.; Saurugger, P.N.; Benedik, M.J. The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli. Gene, 57:183-192, 1987.        [ Links ]

2. Dekeiser R.A.; Claes, B.; De Ricke, R.M.U.; Habets M.E.; van Montagu,M.C.; Caplan,A.B. Transient expression in intact and organized rice tissues. The plant cell 2:591-602, 1990.        [ Links ]

3. Fraser, M.J. Alkaline deoxyribonucleases realeased from Neurospora crassa mycelia: two activities by mutants with multiple sensitivities to mutagens. Nucl. Acid Res. 6:231-246, 1979.        [ Links ]

4. Hamlym, P.F.; Bradshaw, R.E.; Mellon, G.M.; Santiago. C.M.; Wilson, J.M.; Peberdy, F. Efficient protoplast isolation from fungi using commercial enzymes. Enzyme Microb. Tech. 3:321-325, 1981.        [ Links ]

5. Jeffries, C. D.; Holtman, D. F.; Gusee, D. G. Rapid method for determining the activity of microorganisms on nucleic acids. J. Bacteriol. 73:590-591, 1957.         [ Links ]

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7. Mink, M.; Hôltke, H.-J.; Kessler, C.; Ferenczy, L. Endonuclease-free, protoplast-forming enzyme preparation and its application in fungal tranformation. Enzyme. Microb. Technol. 12:612-615, 1990.        [ Links ]

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10. Sano, M.; Nakamura,A.; Masaki H.; Uozomi, T. Isolation and characterization of the nuclease O gene (nucO) from Aspergillus oryzae. Curr. Genet. 30:312-317, 1996.        [ Links ]

11. Schweizer, M.; Case, M.E.; Dykstra, C.C.; Giles, N.H.; Kushner, S.R. Identification and characterization of recombinant plasmids carrying the complete qa gene cluster from Neursopora crassa including the qa-1+ regulatory gene. Proc. Natl. Acad. Sci. USA 78:5086-5091, 1981.        [ Links ]

12. Ward, M. Production of calf chimosin by Aspergillus awamori. In: Herhberger, C.L.; Queener, S.N.; Hegeman, G. Genetics and Molecular Biology of Industrial Organisms. Washington, 1986.        [ Links ]

13. Ward, M. Wilkinson, B.; Turner, G. Transformation of Aspergillus nidulans with a cloned olygomycin-resistant ATP-synthase subunit 9 gene. Mol. Gen. Genet. 202: 265-270, 1988.        [ Links ]

 

 

1 This paper is dedicated to the memory of Dr. Renato Bonatelli Jr. who was the supervisor of the author at the Departamento de Genética e Evolução, Universidade de Campinas, SP, Brasil.

* Corresponding author. Mailing Address: Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, CEP 31270-910, Belo Horizonte, MG, Brazil. E-mail: lane@icb.ufmg.br   - FAX: (+5531) 499 2567

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