The extracellular proteases of

Aspergillus nidulans:

a study of structural genes and their regulation.

Patricia Ann vanKuykBSc(Hons) The University of Melbourne

A thesis submitted for the degree of Doctor of Philosophy of the University of NewEngland.

October, 1997

Patricia Ann v Kuyk.

I certify that the substance of this thesis has not already been submitted for anydegree and is not currently being submitted for any other degree or qualification.

I certify that any help received in preparing this thesis, and all sources used, havebeen acknowledged in this thesis.

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With the exception of the plasmid pAlk/Arg2 (section 5.3.1), which was constructed

by M. Katz, the entire contents of this thesis are the sole work of Patricia Ann

vanKuyk.

Abstract.

Polyacrylamide gel electrophoresis and specific protease inhibitors were used to

identify three serine proteases and one metallo-protease in the culture filtrate of A.

nidulans cultures subject to nutrient-limiting conditions.

A gene encoding a putative acid protease was isolated from an A. nidulans gene library

using a heterologous probe. Sequence analysis showed that the A. nidulans

aspergillopepsin gene, designated prtB, shows similarity to the A. fumigatus and A.

niger aspergillopepsin genes, with nucleotide identities of 67.7% and 54.8%

respectively. When the putative PrtB protein sequence was compared to the protein

sequences of other aspergillopepsin genes, it was found that although the active sites

were conserved, there was an 8 amino acid deletion prior to the second active site. It

is unknown what effect this deletion has on the function of the PrtB protein. No prtB

transcript was detected, using Northern analysis or RT-PCR, in RNA obtained from

cultures subjected to 4 hours nitrogen limitation at pH 3 and pH 8. It is possible that

the A. nidulans prtB gene is not expressed, or prtB may be expressed under conditions

other than those tested.

Transformation was used to generate strains in which the prtA gene, encoding an

extracellular serine protease, was disrupted. Data from protease assays showed that

the prtAAstrains produced significantly lower levels of protease activity. Analysis of

culture filtrate using native polyacrylamide gel electrophoresis determined that the

band of protease activity absent in the prtAA strains corresponded to a band which

had been identified as a serine protease. The serine protease gene, prtA, was mapped

to the region between the hxA and riboD genes on chromosome V. Examination of

the promoter region of prtA showed that there was a region of approximately 100 bp

which was highly conserved in a similar position in the promoters of other Aspergillus

extracellular serine proteases. Preliminary experiments using gel mobility shift assays

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identified proteins in nuclear protein extracts which bound in a specific manner to

DNA fragments which contained this highly conserved region.

Two mutants which did not produce extracellular protease in response to sulphur

limitation, were isolated in a mutagenesis screen. Haploidisation analysis showed that

one mutation, designated xprl I, was linked to a translocation event involving

chromosomes II and VII. Further genetic characterisation of the second mutant,

suggested that the phenotype of interest was the result of two mutations.

Characterisation of strains carrying the xprl mutation have shown that it did not

produce extracellular protease under sulphur-, nitrogen-, or carbon-limiting conditions.

It was also shown, that the secretory process in strains carrying the xprl mutation was

not impaired. The ability of strains carrying the xprl I mutation to utilise a variety of

carbon, nitrogen, and sulphur sources was examined. No pleiotrophic effects were

observed, suggesting that xprl may play a role in the regulation of the extracellular

proteases of A. nidulans.

Acknowledgments.

There are many people who I wish to thank:

Firstly, Dr Margaret Katz, who has been a great supervisor, for her guidance and

encouragement.

Meryl for her good advice, Sarah, Julie, Richard, and Rachael for their friendship and

encouraging me to leave Melbourne.

The "fly lab" -especially Netty, Shari, Ronnie, and Kate for making me feel welcome in

Armidale, Greg, Mick, and Lynette for their friendship, and Carol and Ruth for

always being friendly and helpful.

Alex Adrianopolous showing me how to do gel mobility shift assays, Charles

Claudianous for advice on phylogenetic analysis, and Brian Cheetham, Keith Greg, and

Brenton Hamdorf for their advice.

The various people who have been members of the lab while I've been there, especially

Amir, for the many interesting and enlightening conversations, and Pam, who has been

a truly great friend. The pseudo-lab members, especially Gab for her friendship and

for making weekends in the lab much more fun.

The Flynns, for making me feel like part of the family.

My parents, Ben and Ilka, for their support and encouragement.

Stephen for his limitless patience, kindness, and understanding.

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Contents.

Chapter 1. Literature Review. 1

1.1. The extracellular proteases of Aspergillus nidulans. 2

1.1.1. Proteases as pathogenicity factors. 2

1.1.2. Extracellular proteases and their effect on the use of filamentous

fungi in industry. 4

1.1.3. A model system for the study of secretion in eukaryotes. 5

1.1.4. A model system for the study of multifactorial gene regulation 8

1.2. Regulation of the extracellular proteases of A. nidulans. 10

1.2.1. Carbon catabolite repression. 10

1.2.2. Nitrogen metabolite repression. 13

1.2.3. Sulphur metabolite repression. 17

1.2.3.1. Neurospora crassa: A model system for the study of sulphur

metabolite repression. 17

1.2.3.2. Sulphur metabolite repression in A. nidulans. 21

1.2.4. The affect of environmental pH on extracellular protease expression. .24

1.2.5. Induction of extracellular proteases by exogenous protein. 27

1.2.6. Mutations in A. nidulans which affect the expression of the

extracellular proteases. 28

1.3. Protease structural genes. 29

1.3.1. Proteolytic enzyme classification. 29

1.3.2. Proteases known in the Aspergilli. 30

1.4. Prelude to this study. 31

Chapter 2. Materials and methods. 33

2.1. Strains. 34

2.1.1. Bacterial strains. 34

2.1.2. Aspergillus strains. 34

2.2. Chemical reagents. 34

2.3. Bacterial media. 34

2.4. Aspergillus media and genetic techniques. 36

2.5. Oligonucleotides. 36

2.6. Preparation of Aspergillus genomic DNA. 38

2.7. Preparation of Aspergillus RNA. 38

2.8. X, library screens. 38

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2.9. Preparation of protein samples. 39

2.9.1. Preparation of culture filtrate (extracellular protein) samples. 39

2.9.2. Preparation of culture filtrate (intracellular protein) samples. 39

2.10. Gel electrophoresis. 40

2.11. Recombinant DNA methods. 41

2.11.1. Restriction endonuclease digests. 41

2.11.2. Subcloning. 41

2.11.3. Gel purification. 42

2.11.4. Plasmid DNA preparations. 42

2.11.4.1. Small-scale alkaline lysis plasmid preparations. 42

2.11.4.2. Lightning mega-preps. 42

2.11.4.3. Cracking preps. 42

2.11.4.4. Rapid boil preps. 43

2.11.5. X, DNA preparations. 43

2.11.6. PCR. 44

2.11.6.1. PCR. 44

2.11.6.2. Crude cell extracts for PCR. 44

2.11.7. RT-PCR. 45

2.11.8. Miscellaneous molecular methods. 45

2.12. Preparation of labelled DNA probes. 45

2.12.1. DIG-labelled DNA probes. 45

2.12.2. 32-P-labelled DNA probes. 46

2.13. Hybridisation analysis. 47

2.13.1. Southern blotting. 47

2.13.2. Northern blotting. 47

2.13.3. DIG-detection. 47

2.13.4. Autoradiography. 47

2.14. DNA sequencing. 48

2.15. Transformation. 48

2.15.1. E. coli transformation. 48

2.15.2. A. nidulans transformation. 48

2.16. Enzyme assays. 49

2.16.1. Detection of protease activity in culture filtrate. 49

2.16.2. Zymograms and protease inhibition study. 49

2.16.3. Invertase activity. 50

2.16.4. Phosphatase activity. 50

2.17. Preparation of Aspergillus nuclear extract. 51

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2.18. Gel mobility shift assays. 51

2.19. Computer analysis. 51

Chapter 3. The number and types of proteases found in the culturefiltrate of A. nidulans. 52

3.1. Background. 53

3.2. Rationale and aims. 54

3.3. Results. 55

3.3.1. Empirical determination of suitable methodology. .553.3.1.1. SDS PAGE to detect extracellular protease activity. 55

3.3.1.2. Native PAGE to detect extracellular proteases. 58

3.3.2. The effect of different nutrient-limiting conditions. 59

3.3.3. Protease inhibition study. .62

3.3.4. Detection of acid protease activity in the culture filtrate

of derepressed A. nidulans cultures. 653.3.5. A comparison of proteases present in culture filtrate and

mycelial extracts. 67

3.4. Discussion. 69

Chapter 4. Isolation and analysis of prtB, a putative A. nidulans

aspartic protease gene. 72

4.1. Background ...74

4.2. Rationale and aims. .74

4.3. Results. .75

4.3.1. Heterologous probing of A. nidulans genomic DNA. 75

4.3.2. Screening of the A. nidulans genomic DNA X, library. 76

4.3.3. Restriction mapping of XPVK2. 76

4.3.4. Copy number analysis. 81

4.3.5. DNA sequence analysis of prtB. 81

4.3.6. Putative protein sequence of prtB. 88

4.3.6.1. Comparison to other fungal aspartic proteases. 88

4.3.6.2. Phylogenetic analysis. 94

4.3.7. Expression of prtB. 98

4.3.7.1. Northern analysis. 98

4.3.7.2. RT-PCR analysis. 98

4.4. Discussion. .103

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Chapter 5. Analysis of the extracellular serine protease gene, prtA. 108

5.1. Background. 109

5.2. Rationale and aims. 110

5.3. Results 110

5.3.1. Strategy for generation of prtA gene disruption mutants. 113

5.3.2. The screen for prtAA transformants. 113

5.3.2. Southern blot analysis of the prtAA transformants. 113

5.3.3. Assays for extracellular protease production 117

5.3.3.1. Plate tests. 117

5.3.3.2. Protease assays -extracellular protease levels under

sulphur-limiting conditions. .119

5.3.3.3. Protease assays -extracellular protease levels under

nitrogen-limiting conditions. 119

5.3.3.4. Protease assays -extracellular protease levels under

carbon-limiting conditions. .122

5.3.3.5. Protease assays -induction versus time experiment. 122

5.3.3.6. Protein gels. 124

5.3.4. Genetic analysis of the prtAA transformant. 127

5.3.4.1. Haploidisation analysis. 127

5.3.4.2. Further genetic mapping of prtA. 127

5.3.5. Analysis of the promoter region of prtA. 130

5.3.5.1. Comparison of promoter regions of Aspergillusextracellular proteases. 130

5.3.5.2. Gel mobility shift assays. 130

5.4. Discussion. 137

Chapter 6. Isolation of mutations which affect the regulationof the extracellular proteases of A. nidulans. 143

6.1. Background. 144

6.2. Rationale and aims. 144

6.3. Results. 145

6.3.1. The effects of the sB I mutation on the production of

extracellular proteases. 145

6.3.2. The mutant screen. 145

6.3.2.1. Selection strategy. 145

6.3.2.2. Mutagenesis and selection. 147

6.3.3. Analysis of the xprl j mutation. 149

6.3.3.1. Haploidisation analysis. 149

6.3.3.2. Utilisation of alternative carbon, nitrogen, and sulphur

sources by xpri 1 strains. 149

6.3.3.3. Biochemical analysis of xprI i mutants. 152

6.3.3.4. Further genetic analysis of xprI 1 . 156

6.3.3.4.1. Determination of the dominance relationship. 156

6.3.3.4.2. Crosses to other mutations effecting extracellular

protease production. 156

6.3.4. Analysis of xprJ1 . 161

6.3.4.1. Genetic analysis of xpr, I 1 . 161

6.3.5. Do any of the mutations which result in a reduction in

protease production effect secretion? 163

6.4. Discussion. .165

Chapter 7. General discussion. 169

7.1. Summary. 170

7.2. Future direction for the study of the extracellular

proteases of A. nidulans. ...174

References. 178

Appendices. 194

1 Raw data and statistical analysis of the levels of

protease produced by the prtAA strains, MK189

and MK190, and the wildtype strains, MH2 and

MK191, under sulphur-nutrient-limiting conditions. 195

2. Raw data and statistical analysis of the levels of

protease produced by the prtAA strains, MK189

and MK190, and the wildtype strains, MH2 and

MK191, under nitrogen-limiting conditions. .198

3. Raw data and statistical analysis of the levels

of protease produced by the prtAA strains,

MK189 and MK190, and the wildtype strains,

MH2 and MK191, under carbon-nutrient-limiting

conditions. 202

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4. Raw data and statistical analysis of the levels

of protease produced by the prtAA strains,

MK189 and MK190, and the wildtype strains,

MH2 and MK191, comparing the effects of

exogenous protein and time. 205

5. Analysis of the haploids derived from MK189/J3. 227

6. Mapping of prtA to a region of chromosome V

using a marker strain. Analysis of the cross

between MK189 x A613. ...228

7. Mapping prtA to lysE .233

8. Comparison of the levels of proteolytic activity

produced by the wildtype strain MH97, sB 1 strain

MK130, the xprl i strain MK169, and the sB i xprl Istrain, MK170, under sulphur-repressing and

sulphur-derepressing conditions 238

9. Comparison of the levels of proteolytic activity

produced by the wildtype strain MH97, skistrain MK130, the xprl .1 strain MK169, and the

sB I xprl .1 strain, MK170, under nitrogen-repressing

and derepressing conditions 246

10. Comparison of the levels of proteolytic activity

produced by the wildtype strain MH97, sB 1strain MK130, the xprl 1 strain MK169, and the

sB 1 xprI i' strain, MK170, under carbon-repressing

and derepressing conditions ...250

11. Assays examining phosphatase activity in the culture

filtrate of strains grown in phosphate limiting media. 254

12. Results of the cross between strains carrying the

xprl i and xprG2 mutations. 262

13. Crosses to strains carrying the xpr. 1 1 mutation. 264

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List of Figures.

1.1. A hypothetical secretory pathway for filamentous

fungi. 7

1.2. A molecular model of the regulatory circuit involved

in sulphur metabolite repression in Neurospora crassa. 19

1.3 Sulphur metabolite repression in Aspergillus nidulans. 22

1.4. A model for gene regulation in response to environmental

pH 26

3.1. SDS-PAGE of culture filtrate from the wildtype strain

MH2 after transfer to protease-repressing and

protease-derepressing conditions for 4 hours. 56

3.2. Milk overlay used to detect protease activity in a protein

gel which had been renatured after SDS-PAGE. 57

3.3. The spectrum of proteases observed in the culture filtrate

of wildtype strains of A. nidulans under nutrient-limiting

conditions. 60

3.4. Effects of a variety of protease inhibitors on the proteases

observed in the culture filtrate of A. nidulans. 64

3.5. Zymogram comparing the proteases present in intracellular

and extracellular samples obtained from wildtype strain

MH2 .68

4.1. Partial restriction map of the 17 kb A. nidulans genomic

DNA insert of XPVK2 and the 2.75 kb BglIUXbalfragment containing the aspartic protease gene, prtB. 77

4.2. Southern blot showing that the A. niger pepA probe

hybridises, under low stringency conditions, to single

fragments of the A.PVK2 clone. 78

4.3. High stringency Southern blot of XPVK2 and A. nidulansgenomic DNA 79

4.4. Southern blot demonstrating that the size difference

between hybridising fragments from XPVK2 and

A. nidulans genomic DNA is not specific to a single

fragment, but is an artefact of the genomic DNA

preparation. 80

4.5. Southern blot showing that the XPVK2 fragment is

identical in size to the A. nidulans genomic DNA

fragment. 82

4.6. Low stringency Southern blot to determine if there

are multiple copies of prtB. 83

4.7. Comparison of the DNA sequence of the A. nidulansprtB gene and the A. fumigatus gene, pepF. .84

4.8. Comparison of the deduced amino acid sequences

of Aspergillus aspartic proteases. 89

4.9. Comparison of the deduced amino acid sequences

of fungal aspartic proteases. 90

4.10. 3-dimensional structure of the aspartic protease

of Penicillium janthinellum. 92

4.11. 3-dimensional structure of the aspartic protease of

Rhizopus chinnensis. 93

4.12. PCR products from the prtB gene amplified from DNA

from an A. nidulans laboratory strain (MH2), and a strain

of A. nidulans recently isolated from the environment. 95

4.13. Phylogenetic tree of the fungal aspartic proteases

generated by unrooted parsimony analysis. 96

4.14. Phylogenetic tree of the fungal aspartic proteases

generated by distance neighbour joining analysis. 97

4.15. Northern blot probed with the 7-actin gene. 99

4.16. RT-PCR of the 7-actin gene of A. nidulans. 101

4.17 RT-PCR of the aspergillopepsin genes of A. nidulansand A. niger...102

4.18 The "18-mer" -a DNA sequence motif common to a

number of Aspergillus extracellular acid protease

promoters .105

5.1. The DNA sequence of the 1312 nucleotides 5' of the ,prtA

start of translation. 111

5.2 Schematic diagram showing the strategy for disrupting the

A. nidulans prtA gene 112

5.3. PCR screen for prtAA mutants. 114

5.4. Southern blot analysis of two putative prtAA mutants. 115

5.5. Southern blot to determine the number of integration

events that occurred in generating the prtAA mutants. 116

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5.6. Growth and protease production on solid media,

where milk is the sole nitrogen source, at pH 4.5. 118

5.7. Graph showing the effect of sulphur-limitation on

the prtAA transformants. 120

5.8. Graph showing the effect of nitrogen-limitation on

the prtAA transformants. 121

5.9. Graph showing the effect of carbon-limitation on

the prtAA.transformants 123

5.10. Graphs comparing the effects of induction and time

on protease production by the wildtype strains, MH2

and MK191, and the prtAA strains, MK189 and MK] 90. 125

5.11. Comparison of proteases present in intracellular and

extracellular samples obtained from strains grown

under carbon repressed and carbon derepressed

conditions. 126

5.12. Graph showing the number of prtAA haploids carrying

chromosome markers from the J3 and MK189 strains. 128

5.13. Chromosome location of the prtA gene, as determined

by the cross between MK189 and A613. 129

5.14. Chromosomal location of the prtA gene with respect

to the lysE gene, both of which are located in the region

between the hxA and riboD genes. 129

5.15. The highly conserved region (HCR) -a DNA sequence

common to the promoter regions of Aspergillusextracellular serine protease genes. .131

5.16. Example of a gel mobility shift assay used to optimise

components in the binding reaction. 133

5.17. Schematic diagram showing the probes used in the gel

mobility shift assays, for the study of the prtA promoter ...134

5.18. Gel mobility shift assay in which the XhollSacI fragment,

which contains the HCR, was used in an experiment in

which electrophoresis occurred over a period of 5.5 hours. 136

5.19. Gel mobility shift assay using a 110 bp PCR fragment

containing the HCR. 138

6.1. Results from the initial assay examining the effects of

the mutations on the production of extracellular protease. 148

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6.2. Protease assay comparing the response of the wildtype

strain and mutant strains to sulphur-limiting conditions. 153

6.3. Protease assay comparing the response of the wildtype

strain and mutant strains to nitrogen-limiting conditions. 154

6.4. Protease assay comparing the response of the wildtype

strain and mutant strains to carbon-limiting conditions 155

6.5. Phosphatase activity of mutant and wildtype strains at

pH 6.0 and pH 10.0. 157

6.6. Growth on solid media containing milk as the sole

nitrogen source. 158

6.7. Schematic diagram showing how segregants with a

fluffy phenotype may have arisen from a cross where one

strain is carrying a balanced translocation 160

6.8. Comparison of proteases present in intracellular and

extracellular samples obtained from strains grown under

carbon repressed and carbon derepressed conditions. 165

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List of Tables.

1.1. Protease genes which have been isolated from Aspergilli. 32

2.1. Bacterial strains used in this study. 34

2.2. Aspergillus strains used in this study. 35

2.3. Details of all custom oligonucleotides used. 37

2.4. Protease inhibitors used in the inhibition study. 50

3.1. The effect of different protease inhibitors on the activity

of the proteases of A. nidulans 65

3.2. pH of unbuffered media (pH 6.5) after incubation

with A. nidulans mycelia 62

6.1. Growth and extracellular protease production of

segregants from MH205 x MSF. 146

6.2. Haploidisation analysis to determine the chromosomal

location of the xprl gene. 150

6.3. Growth of wildtype, xprll and heterozygous diploid

strains on a variety of carbon, nitrogen, and sulphur.

sources 151

6.4. Haploidisation analysis to determine the chromosomal

location of the xprJ gene. 162

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