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THE FUNCTION, ACCUMULATION AND LOCALIZATION OF THE NOR-l
PROTEIN INVOLVED IN AFLATOXIN BIOSYNTHESIS; THE FUNCTION OF
THE I'IUP GENE ASSOCIATED WITH SPORULATION IN
ASPERGHL US PARASITICUS

BY

RENQIN G ZIIOU

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition
and
Multidisciplinary Graduate Program in Environmental Toxicology

1997

ABSTRACT

THE FUNCTION, ACCUMULATION AND LOCALIZATION OF THE NOR-l
PROTEIN INVOLVED IN AFLATOXIN BIOSYNTHESIS; THE FUNCTION OF
THE HUP GENE ASSOCIATED WTTH SPORULATION IN
ASPER GILL US PARASI 77 C US

BY

Renqing Zhou

Aflatoxin contamination can cause public health and economic problems in many areas
of the world. A study of the mechanisms regulating aflatoxin biosynthesis and the biology
of the aflatoxigenic fungus Aspergillus parasiticus may play a role in controlling aflatoxin
contamination. '

To study the function of the Nor-l protein, which is directly involved in aflatoxin Br
biosynthesis in A. parasitic-us, a nor-1 cDNA was expressed in Escherichia coli. The
resulting Nor-1c protein was evaluated in an enzyme activity assay.

To study the acammlation of the Nor-l protein in A. parasiticus, polyclonal antibodies
were raised against the Nor-1c protein and were used to monitor the accumulation of the
Nor-1 protein. A nor-llGUS reporter construct was also used to monitor the activity of
the nor-l promoter.

Cell fi’actionationN/estem blot analysis and in situ immunolocalization experiments were
used to study the localization of the Nor-l protein. A nor-I/GU S reporter construct was used

to localize the activity of the nor-1 promoter.

Gene disruption was used to study the function of the fluP gene which encodes a
putative polyketide synthase in A. parasiticus.

The data presented in this dissertation suggest that: The Nor-l protein is a ketoreductase
which converts the aflatoxin B1 pathway intermediate norsolorinic acid to averantin. The
accumulation of the Nor-1 protein is regulated temporally in liquid media and
temporally/spatially on solid media. The Nor-1 protein is mainly localized in the cytosol of
the vegetative cell. The Nor-l protein is localized in developing conidial head, conidial stalks,
vegetative hyphal cells, and mature conidia in the order fi'om the highest to the nondetectable
level, respectively. The localization of the Nor-l protein and the localization of nor-1
promoter activity follow a temporal and spatial pattern and are correlated to the process of
spomlation in A. pausiticms grown on solid media. Disruption of fluP resulted in slow
growth with cotton-like hyphae, no sporulation, and reduced aflatoxin accumulation
suggesting that fluP is associated with the vegetative cell growth, sporulation, and in turn

indirectly influences the accumulation of aflatoxin in A. parasiticus.

This work is dedicated to my aunt (Zhou Chunlan), my uncle (Ma Zhichun), my grand
mother (Cong Guicang) and my sister (Ma Lijun) for their emotional and financial support
during the most difficult time in my life; and to my daughter (Zhou Ymgchiao) for her
understanding and self sacrifice during my seven-year absence, working and studying in

the United States.

iv

ACKNOWLEDGMENTS

I would like to deeply acknowledge the academic and technical guidance and advice of
Dr. John E. Linz throughout my graduate study at Michigan State University. It was his
continuous encouragement and inspiration that helped me succeed in these studies.

I am very grateful to receive the advice and help from Dr. James J. Pestka, Dr. William
G. Heiferich, and Dr. L. Patrick Hart who have graciously served on my guidance
committee.

Special thanks are also expressed to Matt Rarick for his assistance in sequence analysis,
and last but not least to Dr. Tzong-Shoon Wu, Dr. Ludmila Rose, Dr. N‘mee Mahanti and
all of my colleagues in the Food Microbiology Laboratory, the Department of Food

Science and Human Nutrition (Michigan State University) for their technical expertise.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. x

LIST OF FIGURES ..................................................................................

CHAPTERI

.xi

1

LITERATURE REVIEW ............................................................................. 5
PART 1.MYCOTOXIN BIOSYNTHESIS IN ASPERGEL US ................ 5

SECONDARY METABOLISM...

...5

FUNCTIONS or SECONDARY METABOLI‘I'ES......................................' 1...... .' .' .....7

BIOSYNTHESIS OF MYCOTOXINS BY ASPERGILLUS

Biosynthesis Of Isoprenoid Mycotoxrns
Isopentenyl Pyrophosphate... .
Hexahydropolyprenols...
Biosynthesis Of Amino Acrd-Isoprenord Mycotoxms
Funntremorgrn
Biosynthesis Of Polyketrde Mycotoxms ...............................................
Pamlm...
Stengmatocystm, Stengmatrn, ”and Autocystrn .................................
Biosynthesis Of Amino Acid-Polyketide Mycotoxins”
Ochratoxin...
Biosynthesis Of Polypeptide Mycotoxms ............................................
Dicoumarol... .
Gliotoxin... ....
TryptoqurvalrneM
Biosynthesis Of Isoprenord-Polyketide Mycotoxrns

Fumagillin

8
ll

.....11
.....13
......13
.....13
...16

16

......18
......18
...19

21

22
......22
...24

24

......25
......25
......27
.....29

......32
.....32
...32

TerritremB... . ............34
Biosynthesis Of Amino Ac1d-Isopreno1d-Polyket1de Mycotoxms ................. 36
Cyclopiazonic Acid... . . .. 36
Epilog.... 37

TOXICITY OF AFLATOXIN S ................................................................ 37
BIOSYNTHESIS OF AFLATOXIN B1 .................................................... 42
Biosynthesis Of Aflatoxin B1.. 42
Genes Involved In The Aflatoxin B1 Pathway ................................... 51
Purification OfEnzymes Involved In The AFBr Pathwa... 56
Regulation Of Aflatoxin Biosynthesis. . 56
BIOCONTROL OF AFLATOXIN CONTAMINATION .............58
Possible Function Of Aflatoxins ......................................................... 58
Species OfTheAqoergilIusflavus Group... 58
BiocontrolOfAflatoxin Contamination... ... 60
Perspectives On The Use Of Fungal Biocontrol Agents .................................... 64

CHAPTER2
ENZYMATIC FUNCTION OF THE NOR-l PROTEIN INVOLVED IN
AFLATOXIN B1 BIOSYNTHESIS IN ASPERGHLUS PARASIHCUS.... . . .........67
ABSTRACT ................................................................................... 67
MATERIALS AND METHODS .. 71
BacterialStrain, Fungal Strams,andCultureCond1trons .........71
Nucleotide Sequence Analysis... ........71
Construction of pMN1,a pMAL-c2-nor-l-cDNA Expression Vector ......... 73
Preparation of the Nor-ICIMBP Fusion Protein and the Nor-lo Protein ...... 73
Fungal Culture and Crude Protein Extract Preparation.....................74
lmmunoafinity Ptnification of the Native Nor-1 Protein ........................... 75
Western Blot Analysis... 75
NorsolorinicAcid (NA)Preparatron 76
Averantin (AVN) Preparation... .7.......6
Enzyme Activity Assay of the Nor-lolMBP Fusion Protein and
the Nor-1c protein .................................................................. 77
UV/VIS, Mass, and NMR Spectroscopy of NA and AVN... ............. 77
Aflatoxin Analysis by TLC and ELISA78
RESULTS ........................................................................................... 78
Sequence Analysis ofthe nor-l cDNA78
Preparation of the Nor-ldBMP Fusion Protein and
the Nor-1c Protein ................................................................................... 82
Immunoafinity Purification ofthe Native Nor-1 Protein 82
Enzyme Activity of the Nor-le/BMP Fusion Protein and
the Nor-1c Protein .................................................................................... 82
UV/VIS, Mass, and NMR Spectra ofNA85

UV/VISMass,andN1VlRSpectraofAVN.......89
AFBr Accumulation m A. parasiticus SU-l and A. parasztzcus ANor-1.90

CHAPTER 3
THE ACCUMULATION OF THE NOR-l PROTEIN AND THE ACTIVITY
OF THE NOR-l PROMOTER DURING AFLATOXIN Br BIOSYNTHESIS

IN ASPERGEL US PARASITICUS .......................................................................... 95
ABSTRACT ........................................................................................................ 95
INTRODUCTION ............................................................................ 95
MATERIALS AND METHODS ............................................................. 98

Bacterial Strain, Fungal Strains, and Growth Media ........................... 98
Fungal Culture, Dry Weight Determination, and Analysis of Fungal

Morphology .................................................................................... 99
Temporal and Regional Collection of Samples from

Solid Growth Medium ............................................................................ 99
Crude Protein Extract Preparation... .100
Preparation and Purification of Polyclonal Antrbodres 101
Western BlotAnalysisoftheNative Nor-1 Protein" .......102
Aflatoxin Analysis By TLC and ELISA.102
Quantitative Assay of B-Glucuronidase Activity ................................... 103

RESULTS ...................................................................................... 103

Titer and Specificity of Polyclonal Antibodies... ... ..103
The Accumulation of the Nor-l Protein and the Accumulation of AFB1

in A. parasiticus SU-l Grown in YES Liquid Medium .............. 105
The Accumulation of the Nor-l Protein and the accumulation of AFBr

in A. parasiticus SU-l in Nutritional Shift Assay ....................... 107
Morphology of A. pm'asiticus SU-l grown on YES Agar .................. 109
Morphology of Development and the Accumulation of AFBr at

theColonervelinA.parasiticus Grown onYESAgar ................ 111

The Accumulation of the Nor-1 Protein, the Activity of
the nor-l Promoter, and the Accumulation of Aflatoxin in
A. parasiticus SU-l Grown on YES Agar ...................................... 112
DISCUSSION ................................................................................ l 16

CHAPTER4
LOCALIZATION OF NOR-l PROTEIN ACCUMULATION AND
LOCALIZATION OF nor-l PROMOTER ACTIVITY DURING
AFLATOXIN Bl BIOSYNTHESIS IN ASPERGHLUS PARASHTCUS..........124
ABSTRACT ...................................................................................................... 124
Fungal Strains and Culture Media... 127
Cell Fractronatron128

viii

Western Blot Analysis of the Nor-1 Protein in Cell Fractions .................... 130

Analysis of Marker Enzyme Activities ........................................................ 131
Nor-1 Protein Localization by Immunofluorescence Microscopy ............. 133
Semiquantitative Petri Dish Assay for nor-l/GUS Activity
(Overlay Procedure) ................................................................................. 134
RESULTS... .. ...134
Distributions of Marker Enzymes and the Nor-1 Protein 111 "Cell Fractrons 147
Subcellular Immunolocalization of the Nor-1 Protein... ...147
In Situ Localization of nor-1 Promoter Activity in a Colony of"
A. parasiticus ........................................................................................... 148
DISCUSSION ............................................................................................................ 152
CHAPTERS
ISOLATION AND ANALYSIS OF HUP, A GENE ASSOCIATED WITH HYPHAL
GROWTH AND SPORULATION IN ASPERGEL US PARASIHCUS ............... 159
ABSTRACT ..................................................................................................... 159
INTRODUCTION... .. 159
MATERIALS AND METHODS .161
Bacterial Strain, Fungal Strains and Culture Condrtrons ..........161
Fungal Culture, Dry Weight Determination, Growth Rate
Measurement, and Aflatoxin Analysis ............................................. 161
Temporal and Regional Collection of Samples fi'om Solid Medium ..... 163
Plasmid Preparation and Protoplast Transformation ................................. 163
Nucleotide Sequence Analysis and Amino Acid Sequence Comparison....164
Isolation and Analysis of Genomic DNA and Total RNA164
Analysis of the Codrng Region offluP 165
Construction of a Disruption Vector pPRS ...............168
Transformation of A. parasiticus NR-3 and Disruption of fluP ................. 173
fluP Transcript Accumulation 1n the erd-Type Strain and
thefluP-Disnrpted Transformant... 181
Aflatoxin B1 Accumulation 1n the erd-Type Strain and
thefluP-Disrupted Transformant... 182

CONCLUSIONS 192

APPENDICES
APPENDIX A. CLONING OF THREE EcoRI DNA FRAGMENT S FROM
COSMID Nor-A AND ANALYSIS OF RELATED TRANSCRIPTS ............ 194
APPENDIX B. IDENTIFICATION OF VERSICONAL CYCLASE GENE ......... 195

APPENDIX C. A MODIFIED PROCEDURE FOR CRUDE EXTRACT
PREPARATION FROM FUNGAL CELLSl96

ix

LIST OF TABLES

Table 3-1. Titers and specificities of anti Nor-lc/MBP PAb ..................................... 105

Table 3-2. The accumulation of aflatoxin Bl in A. parasiticus SU-l in nutritional shifi

Table 3-3. The morphological characteristics and the acmnmlation of AFB: in
A. parasitic-w SU-l gown on YES agar112

Table 3-4. The accumulation of the Nor-l protein, the activity of nor-l/GUS
(GUS activity), and the accumulation of AFB] in A. parasiticus SU-l
gown on YES agar ............................................................................... 115

Table 4-1. Distributions of marker enzymes and the Nor-l protein in cell fi'actions
obtained by Potter homogenization and difi‘erential centrifitgation..........l38

Table 4-2. Distributions of marker enzymes and the Nor-l protein in total
organelle and the cytosol fractions obtained by protoplast

pelmeabilimion and ceuuifitgation. ............................................................ 139
Table 5-1. Mycelial dry weight and aflatoxin Bl accumulation in A. pwasitz’cus NR-3
and the fluP-disrupted transformant gown on YES agar ................ 183

LIST OF FIGURES

Figure 1-1. Representatives of mycotoxins produced by species of Aspergillus........ . . . ..
Figure 1-2. Biosynthesis of isopentenyl pyrophosphate from acetyl CoA...
Figure 1-3. Biosynthesis of hexahydropolyprenols in A3pergill113fi1miga1113... . . . . . . . . .
Figure 1-4. Biosynthesis ofaflatrem inAspergiIIusflavus................

Figure 1-5. Biosynthesis offumitremorgin A inA.spergiIlu3fi1migalu3.........

Figure 1-6. Biosynthesis of sulochrin in A3pergill1131erre113.................
Figure l-7.Biosynthesis ofpatulininPeniciIliumpa111h1m......................................

Figure 1-8. Biosynthesis of citrinin in Aspergillus candzdus

Figure 1-9. Biosynthesis of sterigmatocystin, sterignatin, and austocystin in

Avergillus ver31'color ..........................................................
Figure 1-10. Biosynthesis of ochratoxin in Aspergillus ochraceu3
Figure 1-11. Biosynthesis of cytochalasin B in Aspergillus clava1113 ....................
Figure 1-12. Biosynthesis ofdicoumarol inA.spergiIlu3fi1miga1113......

Figure 1-13. Biosynthesis ofgliotoxin in Trichodema vmdz

Figure 1-14. Biosynthesis of tryptoquivaline M in Aspergillusfianigatus..................
Figure 1-15. Biosynthesis ofpenicillin in Aspergillus nidulam......... 28
Figure l-16. Biosynthesis ofaustin inAspergiIlus 1131113...

Figure 1-17. Biosynthesis offumagillin in Aspergillusfimigatus.............

10

12

12

14

..15

15

.20

.20

20

23

23

.26

.26

28

.33

.33

Figure 1-18. Biosynthesis ofterritrem B inAspergillus 1erre113............ 35

Figure 1-19. Biosynthesis of cyclopiazonic acid in some species of Aspergillus. . . ........35
Figure 1-20. Structures ofAflatoxin B1, Bz, Gr, and 62 41
Figure 1-21. Origin of the 16 skeletal carbon atoms of aflatoxin Br ........................ 41
Figure 1-22. Aflatoxin B1 biosynthetic pathway in Aspergillus parasmcus43
Figure 1-23. Restriction endonuclease and transcript map of cosmid Nor-A.................53
Figure 2-1. Proposed multiple alternative pathways which convert NA to AVF. ............ 69
Figure 2-2. Construction of the expression vector pMN 1 ......................................... 72

Figure 2-3. Nucleotide and predicted amino acid sequence of the S’and 3’-ends of
nor-1 cDNA in expression vector pMN179

Figure 2-4. SDS-PAGE detection of the Nor- 1 c/MBP fusion protein and
the Nor-1c protein ................................................................... 80

Figure 2-5. Western blot analysis of the native Nor-l protein in A. pw'asiricus SU-l ......... 81
Figure 2-6. Enzyme activity of the Nor-l/MBP filSlOll protein and the Nor-1c protein ......... 83
Figure 2-7. UV/VIS spectroscopy of norsolorinic acid (NA) and averantin (AVN) ........... 86
Figure 2-8. Mass spectroscopy ofnorsolorinic acid (NA) and averantin (AVN)...............87
Figure 2-9. NMR spectroscopy of norsolorinic acid (NA) and averantin (AVN). . . . .........88

Figure 3-1. Western blot analysis demonstrating the specrficrty of PAb from different
levels ofpurification.... ......104

Figure 3-2. Western blot analysis of the accumulation of the Nor-1 protein in
A. para31‘11‘c113 SU-l gown in YES liquid medium106

Figure 3-3. The accumulation of AFBi and mycelial dry weight of A pw'asiticus SU-l
gowninYES liquid medium... .. ... 108

Figure 3-4. Western blot analysis of the accumulation of the Nor-1 protein in
A. parasiticus SU-l in PMS-GMS nutritional shifi assay... 110

xii

Figure 3-5. Western blot analysis of the accumulation of the Nor-1 protein in

A. pamsitr’cus SU-l gown on YES agar...

Figure 3-6. TLC analysis of the accumulation of aflatoxins in A.para31’11'cus SU-l

gown on YES agar.

Figure 4-1. Western blot analysis of the Nor-1 protein in cell fractions of

A. parasitic“ ....................................................................................

Figure 4-2.1mnnmolocalization of the Nor-1 protein in hyphalm cells of A. pm'asm'cus

SU-l gown in YES liquid medium...

Figure 4-3. Inummolocalization of the Nor-l protein in A. paras’ticus SU-l gown

onYESagar... ......................................................................................

Figure 4-4. Imrmmolocalization of the Nor-l protein in A. parasificus SU-l by laser

scanning microscopy (LSM) ....................................................................
Figure4-5. Schematicofa conidiophore ofAmergiIlusparasiticus.....

FigunMInsimanalysisofnw-l promoteractrvrtywrthmacolonyof

A. pry-1131110113 gown on solid medium...

Figure$LAminoacid sequencecomparisonofflquithtwoPKS genesandoneFAS
gene ............................................................................................................

Figure 5-2. A schematic ofthe 9.1-kb SacI genomic DNA fragnent containing

the fl11P gene in A3pergr’ll113 par-03mm ...................................................

Figure 5-3. The fluP-disruption vector pPKS and a schematic description of the expected
restriction map ofgenomic DNA in thefluP-disrupted transformant........

Figure 5-4. Southern blot analysis of genomic DNA in A. parasitic-113 NR-3 and

the fluP-disrupted transfonnant ..................................................................

113

...114

136

...140

142

144

146

...149

166

168

169

....171

Figure 5-5. Morphology of A. parasitic-113 NR—3 and the fluP-disrupted transformant gown
on coconut agar medium (CAM) or on potato dextrose agar (PDA) ..... 174

Figure 5-6. Transcript accumulations of fluP, the B-tubulin gene, and the ver-rA gene
in A. parasiticus. .. ...................................................................................

Figure 5-7. Transcript accumulations of fluP in A. parantrcus NR-3 and

the fluP-drsrupted transformant...

xiii

....177

...179

INTRODUCTION

Aflatoxins are extremely toxic mycotoxins produced by certain strains of Aspergillus
parasitic-113, A. flaws, and A. 1101111113. These ubiquitous fungi gow on a variety of food
and feed crops and may cause serious aflatoxin contamination. Aflatoxin contamination
poses a threat to public health and in turn causes economic problems in many areas of the
world. Therefore, reducing or eliminating aflatoxin contamination is an urgent need.

Myresearchisonepartofalongtermresearchprojectaimedattheeliminationof
prebarvest contamination of crops by aflatoxin Bl (AFBl). The work is designed to provide
an understanding of the mechanisms which regulate aflatoxin biosynthesis at the molecular
level and an understanding of the general biology of the toxigenic firngi (e.g. regulatory
mechanisms involved in hyphal gowth, hyphal differentiation, and sporulation). This
information may lead to the development of new fimgicides, the improvement of
agonomic practices, the development of resistant crops (by genetic engineering or
classical breeding), and the development of biocontrol agents (such as nontoxigenic
competitive fungi) for control of aflatoxin contamination.

In the currently accepted general pathway of aflatoxin biosynthesis (Figure 1-22) (Bhatnagar
e1 at, 1992; Dutton, 1988; Yabe etal, 1993), the decaketide—derived norsolorinic acid (NA) is
the earliest stable pathway intermediate isolated (Dutton, 1988). NA is converted to averufin

(AVF) via one or more alternative pathways by converting NA to averantin (AVN) first or to

averufanin (AVNN) first (Figure 2-1) (Bennett and Cluistensen, 1983; Button, 1988;
McCormick e1 11]., 1987; Yabe et al., 1991b) Disruption of the nor-1 gene resulted in NA
accumulation and a significant reduction of aflatoxin in an aflatoxigenic strain of A. parasitim
(Trail et at, 1994). Therefore the nor-1 gene may function in one of these alternative
pathways. Analysis of the predicted amino acid sequence of nor-1 provided evidence that nor-
1 encodes a ketorcductase (Trail e1al., 1994). Comparison ofthe chemical structure ofNA
and AVN (Figure 2-1) demonstrated that the 5’-keto goup of NA is converted to the 5’-
hydroxyl goup of AVN suggesting that this reaction is catalyzed by a ketoreductase. The
accunmlation of nor-l transcript is subjected to temporal regulation in liquid arlmre (Skory et
al., 1993; Trail e1 01., 1994). Aflatoxins have been found to be concentrated in spores and
sclerotia (Wicldow and Cole, 1982; Cotty, 1988). These data mggest that the accumulation
of the Nor-1 protein is also subjected to temporal regulation in liquid culture and the
localization of the Nor-1 protein is correlated to the process of sporulation.

Based on thebackgound data, three hypotheses related to theNor-l protein are proposed.
(1) The Nor-l protein encoded by nor-l is a ketoreductase which converts NA to AVN in the
AFBr pathway. (2) The accumulation of the Nor-l protein is subjected to temporal regulation
in liquid media and to temporal and spatial regulation on solid media. (3) The Nor-l protein
resides either in the cytosol or in organelles. The localization of the Nor-1 protein is correlated
to the process of sporulation in A. marine-113 gown on solid media.

Previous data showed that a 680-bp genomic DNA fiagnent fiom A. par-1131110113 was able
to hybridize to a cDNA fi'agnent of a polyketide synthase (PKS) gene which encodes the B-

ketoacyl synthase functional domain of the 6-methylsalicylic acid synthase (MSAS) complex in

Penicifliwnpatuhan (Becketal, 1990) As expected, the deduced amino acid sequence fi'om
the 680-bp DNA fi'agnent was similar (72% identity) to that of the B-ketoacyl synthase
functional domain of MSAS. A search of the EMBL and GenBank data base h‘braries showed
that the 680-bp DNA fi'agnent is also very similar to a variety of other PKS genes (35.3% to
70. 8% identity in 216 amino acid overiap compared with 6 PKS genes). Because aflatoxins
are derived from a polyketide pathway (Kurtzman e101. , 1987), the producing fungi are
predicted to have a polyketide synthase (PKS) related to aflatoxin biosynthesis.

ItmshufiaflyhypothenzedflntflqumdbcmvohedmaflMmdnbiosymhedsbecause
there had been no published report on identifying patulin and/or its precursor 6-methylsalicylic
acid produced by A. parasiticus.

Polykides are secondary metabolites and are often involved in regulation of development or
difi‘erentiation (Summers et al., 1995; Revill et al., 1995; Keller and Adams, 1995).
Therefore an alternative hypothesis was that this PKS-hke gene (fluP) containing the 680-bp
DNA fiagment might be related to some kind of secondary metabolism (such as pignent
synthesis, hyphal gowth and/or sporulation).

In order to test these hypotheses, four goals were proposed: (1) to determine the enzymatic
function of the Nor-1 protein; (2) to determine the accmnulation pattern of the Nor-1 protein;
(3) to localize the accumulation of the Nor-1 protein; and (4) to test the firnction of the fluP
gene.

Fourexperimentsweredesigredtofiilfillthefourgoalsandintumtoaddressthefour
hypotheses. (1) The Nor-1c protein was produced in a host bacterium transformed with a nor-

1 cDNA expression vector. The Nor-1c protein was used to test the enzymatic function of the

native Nor-1 protein in the AFB! pathway. (2) Polyclonal antibodies (PAb) were raised
against the Nor-1c protein and used to monitor the accumulation of the native Nor-1 protein in
A. pausitiws gown in liquid media and on solid media A nor-l/GUS reporter construct was
used to monitor the activity of the nor-1 promoter (which could indirectly indicate the
accunmlation of the Nor-1 protein during aflatoxin biosynthesis) (3) Cell fiactionation plus
Western blot analysis and in situ irnmunolocalization were used to localize the Nor-1 protein at
thecellularlevel andthebyphal level. Anor-l/GUS reporter constructwasusedto localize
the activity of the nor-l promoter (which could indirectly indicate the localintion of the Nor-1
protein during aflatoxin biosynthesis) at the hyphal level and at the colony level (4) Southern
blot and Northern blot analyses were used to clone the fluP gene. Gene disruption was used to

arnlyzethefimctionoftheflngene.

CHAPTER]

LITERATURE REVIEW

My dissertation focuses attention on two genes. The first is the nor-l gene which is
involved in afiatoxin Br (AFBr) biosynthesis. The second is a suspected polyketide
synthase (PKS) gene, the fluP gene, which is associated with gowth and sporulation and
indirectly influences AFBr biosynthesis in the filamentous firngus Aspergillus para31‘tic113.
AFBr is just one of the numerous mycotoxins produced by Aspergillus. The first part of
this review is a general introduction to mycotoxin biosynthesis in Aspergillus, which
includes a brief discussion of secondary metabolism and its possible functions and several
biosynthetic pathways for representative mycotoxins produced by species of Aspergillus.
This dissertation is a part of a long term research project desigied to provide an
understanding of the regulation of aflatoxin biosynthesis which may lead to the
development of biocontrol agents for control of aflatoxin contamination. The second part
of this review includes a discussion of aflatoxin toxicity and biosynthesis, and biocontrol of

aflatoxin contamination.

PART 1. MY COTOXIN BIOSYNTHESIS IN ASPERGHL US

SECONDARY METABOLISM

Mycotoxins are a large and highly varied goup of natural products referred to as

secondary metabolites. Chemical compounds are synthesized and degaded by means of a
series of chemical reactions mediated by enzymes in the living organism. These processes
are known as metabolism which is comprised of catabolism (degadation) and anabolism
(synthesis). All organisms possess very similar primary metabolic pathways by which they
synthesize and degade certain biochemicals such as sugars, amino acids, common fatty
acids and nucleotides to generate energy and to build the polymers derived from them
(polysaccharides, proteins, lipids, RNA, and DNA). Compounds derived fiom primary
metabolic pathways are essential for the survival and well-being of the organism and are
called primary metabolites. Most organisms also utilize other metabolic pathways to
produce compounds which appear to have no apparent utility. These compounds are
called secondary metabolites. The pathways of synthesis and degadation of secondary
metabolites constitute secondary metabolism. These pathways are perhaps only activated
during particular stages of gowth and development or during periods of stress caused by
factors such as nutritional limitation or microbial attack. Most secondary metabolites are
produced only by a specific species or a small number of closely related species. For
example, aflatoxins have been found to be produced only by certain strains of three
Aspergillus species.

Primary metabolites have the same or similar structures such as the 20 common anrino
acids, RNA and DNA, while secondary metabolites have a wide diversity of structures.
Their unusual chemical structures include B-lactam rings, cyclic peptides containing
‘unnatural’ and non-protein amino acids, unusual sugars and nucleosides, unsaturated

bonds of polyacetylenes and polyenes and large macrolide rings. Examples of some of the

well known secondary metabolites with unusual structures include ephedrine (for
respiratory ailments), ricinine (a purgative), and salicin (aspirin is a synthetic analogue)
which are medicines; strychnine, coniline, and rotenone which are poisons; morphine
(fiom opium), tetrahydrocanabinol (from marijuana), and cocaine which are narcotics and
hallucinogens; caffeine which is a stimulant; geraniol (from rose oil), linalol (fiom
lavender), cinamaldehyde (fi'om cinamon), eugenol (fi'om cloves) and diallyl disuphide
(fi'om garlic) which are perfumes and spices.

The organisms which can produce secondary metabolites are usually considered to be
limited to bacteria, firngi, and plants. But some scientists believe that all organisms
including humans can produce secondary metabolites. For example, defensins, which are
endogenous antibiotic peptides produced by human leukocytes may be considered as
secondary metabolites (Ganz etal., 1985).

There is no unanimous definition of secondary metabolism. However, one proposed
definition may provide some hints about the nature of secondary metabolism. Davies
(1992) proposed that primary metabolism enables an organism to produce the next
generation. Secondary metabolism is responsible for interactions between the organism
and its environment and is concerned with what is going on outside the producing

organism, rather than events going on inside.

FUNCTIONS OF SECONDARY METABOLITES
The role of secondary metabolites for the producing organisms is still an area of

vigorous debate, but there is a gowing consensus that secondary metabolism benefits the

organism and this benefit usually reflects the nature of the metabolites themselves (Davies,
1992). Davies has proposed ten potential biological firnctions of secondary metabolites:
(l) competitive weapons against other organisms including bacteria, firngi, plants,
amoebae, and insects; (2) metal-transporting agents; (3) plant-microbe symbiosis; (4)
nematode-microbe symbiosis; (5) insect-microbe symbiosis; (6) sexual hormones
(pheromones); (7) difi'erentiation efl‘ectors between and within cells; (8) excretion of
unwanted products; (9) products of ‘selfish’ DNA; (10) reserve pool of new biochemical

pathways.

BIOSYNTHESIS OF MY COTOXIN S BY ASPERGEL US
Fungal secondary metabolites are relatively small molecules characterized not only by
their structural diversity (Turner and Aldridge, 1983), but also by their diversity of
biological activity which includes antibiotic activity, phytotoxicity, animal toxicity and a
very large array of physiological and pharmacological activities in a mammalian system.
The toxic secondary metabolites produced by firngi are called mycotoxins. The
diversity of mycotorn'ns produced by Aspergillus was reviewed by Moss (1977, 1994).
The precursors of mycotoxins produced by Aspergr'llus species are certain amino acids,
acetyl CoA, and isopentenyl pyrophosphate. The polymers of the three basic building
units are polypeptides (fi'om amino acids), polyketides (from acetyl CoA), and isoprenoids
(fi‘om isopentenyl pyrophosphate). Acetyl CoA is also the precursors of some amino acids
and isopentenyl pyrophosphate. Not only can the mycotoxins from Asvergillus be derived

fi'om the three kinds of precursors respectively, but also fi'om all the possible combinations

of the three kinds of precursors. The broad range of mycotoxins produced by Aspergillus
are contained in seven goups: (l) isoprenoids; (2) amino acid-isoprenoids; (3)
polyketides; (4) amino acid-polyketides; (5) polypeptides; (6) is0prenoid-polyketides; (7)
amino acid-isoprenoid-polyketides.

Some of the well known mycotoxins produced by Aspergillus are listed in Figure 1-1
along with their likely producers (may not be the major producers). In addition to the
mycotoxins listed in Figure 1-1, many other Aspergillus mycotoxins (each with one
producer) have been identified such as sulochrin (A. 1erre113), dicoumarol (A. fi1migatr13),
aflatrem (A. flaws), fumitremorgin (A. frmrr’gatus), emodin (A. alutaceus), kojic acid (A.
a1111ace113), neoaspergillic acid (A. alutaceus), penicillic acid (A. 111111042113), secalonic acid
A (A. alutaceus), gandidufin (A. candidus), terphenyllin (A. candidu3), xanthoascin (A.
cardidres), ascladiol (A. clavatu3), clavatol (A. clava1113), kotanine (A. clava1113), aflavinin
(A. flaws), aspergillic acids (A. flavr13), paspalinin (A. flaws), firmigaclavines (A.
fi1miga1113), firmigatin (A. fi1m1'ga1113), fumitoxins (A. fimrr'gatus), spinulosin (A.
flanigatus), verruculogen (A. fimrr’gatus), naphthoquinones (A. oryzae), nigagillin (A.
niger), aspergillomarasmin (A. oryzae), 3-nitropropionic acid (A. oryzae), oryzacidin (A.
oryzae), nidulatoxin (A. sydawii ), giseofirlvin (A. sydowii), terrein (A. terreus), terreic
acid (A. 1erre113), terretonin (A. 1erre113), terredionol (A. terre113), austarnid (A. 1131113),
wentilacton (A. wentir), and physicon (A. wenriz) (Frisvad, 1988).

With some exceptions such as 3-nitropropionic acid fi'om A. oryzae, the biosynthetic

pathways of most mycotoxins (as for many other secondary metabolites) in A3perg1'll113

10

.......................................................................................

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

gacid

 

 

 

 

 

 

 

 

 

 

 

 

 

anary metabohsm
Isopentenyl Acetyl CoA Amino acids
pyrophOSPhate
(Malonyl CoA)
. 1
Isoprenoids _, Polyketides Polypeptides
(1)1
:Hexahydro-E
polyprenols
--------------- (6) (3)" "(4) (5)
EFumagillin i Patulin Ochratoxin iGliotoxin 5
(A. fi1miga1113), (A. sclerofiorum),§ (A. alliaceus), :(A. famigatus),
5Austin 5 Austocystins 5 5 Aspochalasin 5Malforrnins '
5(A. 1131113), 5 (A. 1131113), 5 E A. microcysticus),§ (A. niger),
iTerritrems Citreoviridin Cytochalasin E, Xanthocillin
5 (A. 1erre113), 5 (A. terreus), 5 5 (A. clavatus), 5 A. chevalieri), 5
{Andibenins E Citrinin 1:; Sphingofrmgins 5Trytoquivaline 5
5(A. wm’ecolor); (A. 1erre113), :_.(A fumigatus) i ( A c lava1113), .
r, """""""""""" , 5 Sterigmatocysting' """""""""""""" :Penicillin
E (A nidulam), """""" (A nidulans)

E Asteltoxin (A. 31ella1113),
§ Aflatoxins (A. flaws),

5 Mevinolin (A. terrens),

5 Maltoryzine (A. niger),
Monacolin (A. terreus).

 

Figure 1-1. Representatives of mycotoxins produced by species of Aspergillus.

 

ll

involve two distinct phases. The first phase is the polymerization phase during which the
precursor molecules are linked together by enzyme complexes such as polyketide
synthases. The second phase is the modification phase during which the products of the
polymerization phase are modified by enzymes which carry out hydroxylations, oxidative
cleavages and rearrangements leading to the remarkable diversity of mycotoxins produced
by Aspergillus.

Although hexahydropolyprenol and penicillin may not be considered as mycotoxins,
their biosynthetic pathways have been well studied (Manitto, 1981; Baldwin and Abraham,
1988), and these two secondary metabolites are typically used as examples to discuss
isoprenoid mycotoxin biosynthesis and polypeptide mycotoxin biosynthesis. Biosynthetic
pathways of each of the seven goups of Aspergillus mycotoxins are described with one or
more examples. The best studied biosynthetic pathway is that utilized for AFBr

biosynthesis which is discussed separately.

Biosynthesis Of Isoprenoid Mycotoxins

Secondary metabolites containing a carbon backbone comprised of five-carbon isoprene
[CH2C(CH3)CHCH2), 2-methylbutadiene] units are called terpenes. If the carbon
backbone of a terpene is altered by addition or loss of carbon atoms, or by other
modifications, such a modified compound is called an isoprenoid.

Isopentenyl pyrophosphate. All isoprenoids originate from isopentenyl pyrophosphate

which is formed fiom acetyl CoA through an important intermediate mevalonic acid

(Figure 1-2).

12

cm H OH

HJC —C0A _, mow 2 / O—P—O—P—O
OH II ll
0 O
3 Acetyl CoA Mevalonic acid Isopentenyl pyrophosphate

Figure 1-2. Biosynthesis of isopentenyl pyrophosphate fi'om acetyl CoA.

>5 0 o
n o-r'i-o-i'i-ori —. \
O" n-3

Isopentenyl pyrophosphate Hexahydropolyprenols

Figure 1-3. Biosynthesis of hexahydropolyprenols in Aspergillusfimigatus.

13

Hexahydropolyprenols. A large goup of isoprenoids are the polyisoprenes which are
made up of more than 10 isoprene units, bound head to tail. Polyisoprenes which exist in
the alcohol form are called polyprenols. Among the polyprenols, hexahydropolyprenols
were isolated fi'om Aspergillus fumigams and their synthetic pathway (Figure 1-3) was

described by Manitto (1981).

Biosynthesis Of Amino Acid-Isoprenoid Mycotoxins

Most isoprenoids produced by Aspergr'llus species are not mycotoxins, but many amino
acid-isoprenoids are mycotoxins. A large goup of tremorgenic mycotoxins, such as
aflatrem and firmitremorgin, are isoprenoids with an irrdole nucleus provided by
tryptophan.

Aflatrem. Afiatrem was the first mycotoxin with tremorgenic activity isolated from the
mycelium and the sclerotium of a strain of A. W by Wilson and Wilson (1964) and its
structure was then elucidated by Gallagher and Wilson (1978). Later, other aflatrem-
related tremorgenic compounds from Aspergillus were isolated, such as paspalinine
(Gallagher et al., 1980), aflavarin, B-aflatrem (T ePaske et a1. , 1992), sulpinine,
secopenitrern B, and aflatrem B (Laakso e1al., 1992). Indeed, paspalinine, which may be
a precursor of aflatrem, was isolated fiom an aflatrem-producing isolate of A. flaws along
with the very unusual indole-isopentenyl pyrophosphate metabolites aflavinine and
dihydroxyaflavinine (Cole e1 111., 1981). The biosynthetic pathway of aflatrem in A. flaws

was proposed by Steyn and Vleggaar (1985). Aflatrem is formed fi'om tryptophan,

l4

Tryptophan
Paspalinine
o o
Ho-i'i-o-ij-o 5
on on
W my.
BAcetyl 00A H
O O
W-fiQ-g-Gfi \ o o Paspaline
0H 0H
0 o I yl
Mega ”WWW“
on or
om momma/l
o o
u 1 0-1
Naif-oi- Mrrr ..
°" °" 2 Isopenten pyrophosm
2 Dimethylally pyrophosphate Y' o
L 12 Acetyl CoA J Aflatrem

Figure. 1-4. Biosynthesis of aflatrem in Aspergillusflaws

15

aegis

Funltrernorg'm C o

 

3 Dimsthylally pyrophosphate Fmitramorgin A

Tryptophan+Prolina M-A—o-B-ou .— SAeetleoA
3Wmflmm

Figure 1-5. Biosynthesis of firmitremorgin A in Aspergillusfimigatus.

7 Malonyl-COA + Acetyl-00A

/ cu
m- @2233 ”a

o o c on c on
Polyketide (01s) Emodin-S-anthrone
on m o 0
WI?“ ”W“ ”etc
"’c'° ° on "ac-O 0 0H on 0 0H

Sulochrin Questin Emodin

Figure 1-6. Biosynthesis of sulochrin in quergillus 1erre113.

l6

geranylgeranyl pyrophosphate, and isopentenyl perphosphate with the loss of a methyl
goup during the formation of the polycyclic structure (Figure 1-4).

Fumitremorgin. During a survey of toxigenic food-borne firngi in Japan, Yamazaki et
a1. (1971) found metabolites fi'om certain strains of Aspergr'llus fiam‘gatus which caused
vigorous tremor and convulsions in experimental animals. Two indole-containing
metabolites, desigrated firmitremorgin A and B, were isolated fiom extracts of this firngus.
These tremorgenic metabolites are a family of compounds derived fi'om diketopiperazine.
Based on feeding experiments in A. fianigatus with [“Cl-labeled tryptophan, proline and
mevalonic acid, Yamazaki e1 11!. (1980) proposed that fumitremorgin C is synthesized
from two amino acids, tryptophan and proline, to which a C5 unit from dimethylally
pyrophosphate is added. Funritremorgin A is synthesized by adding two more C, units
fi'om dimethylallyl pyrophosphate (Figure 1-5). Their discovery and structural
characterization have been concisely reviewed by Yamazaki (1980). A derivative of

fumitremorgin C was also found in A. fimrigatus (Abraham and Arfinann, 1990).

Biosynthesis Of Polyketide Mycotoxins

Polyketides may represent the largest goup of secondary metabolites (Weiss and
Edward, 1980). These structurally diverse compounds typically contain a carbon
backbone with oxygen atoms at alternate positions. The name ‘polyketide’ was coined
more than 100 years ago by Collie and Myers (1893). They imagined that poly-B-keto
compounds could be produced by treatment of polyacetyl compounds with weak alkali.

Biochemical support for this idea was provided by Birch and Donovan (1953). According

17

to the isotopic labeling pattern of several fungal metabolites, they proposed that
polyketides must be formed fiom acetic and malonic acids by a process similar to the
biosynthesis of fatty acids. Polyketide chain gowth, however, is different fiom fatty acid
biosynthesis, because it lacks the faithfirl removal of each B-keto goup by a process

necessary in fatty acid biosynthesis. This hypothesis to explain the mechanism of
polyketide synthesis was further supported by the application of the isotopic labeling
method, and later by the development of sophisticated nuclear magretic resonance
spectroscopic techniques (Simpson, 1987; Vederas, 1987).

Knowledge of the enzyrnology of the polyketide synthase (PKS) enzymes was obtained
very “slowly prior to 1985 because of the dificulty encountered in enzyme assay and
purification. Only three PKS’s were purified by 1985: 6-methylsalicylic acid synthase (6-
MSAS) from the firngus Penicilliun patulum (Dimroth et at, 1970), naringenin chalcone
synthase (NCS) fi'om the parsley plant Petroselinum horren3e (Kreuzaler et al. , 1979), and
resveratrol (stilbene) synthase from the peanut plant Arachis hjpohaea (Schoppner and
Kindl, 1984). Studies on these three enzymes established the basic characteristics of PKS,
even though they displayed distinctly difl‘erent properties. The synthase of 6-MSA is a
large (800 kDa), tetrarneric multifirnctional protein to which all the substrates are
covalently attached, whereas NCS is a homodirner composed of 42 kDa subunits that acts
on the CoA esters of the substrates and lacks a firnctional equivalent to an acyl carrier
protein (ACP). From 1986 until late 1996, several other PKS’s or their components have
been identified and/or purified. The multienzyme of the erythromycin-producing

polyketide synthase, DEBSl, DEBS2 and DEB S3 (6-deoxyerythronolide B synthase)

18

were identified and purified from Saccharopolyspora erythraea (Cafl‘rey et aI. , 1992). A
proposed component of the dimeric polyketide synthase (actinorhodin acyl carrier protein-
dependent malonyltransferase) for the antibiotic actinorhodin was purified from
Streptomyces coelicolor A3(2) (Revill et al., 1995). With advanced molecular biology
techniques, other PKS’s or their components were expressed fi'om cDNAs, and then
purified. Acyl carrier proteins (ACPs) of the polyketide synthases for the aromatic
antibiotics actinorhodin, ganaticin, frenolicin and oxytetracycline were expressed in and
purified fi’om Escherichia coli (Crosby et a1. , 1995).

The biosyntheses of sulchrin, patulin, citrinin, sterignatocystin, sterignatin, and
autocystin in some species of Aspergillus have been well studied and are taken as
examples to discuss the charateristics of polyketide biosynthesis.

Sulochrin. Sulochrin is a derivative of a goup of mycotoxins called ergochromes
which were first isolated fi'om ergot, a name for the sclerotium of Claviceps purpurea.
Ergochrome BB was isolated from Aspergillus ochraceus, and ergochrome AC and
ergochrome BC fi'om Amergr‘llus aculea1113. All of the known ergochromes are dimers of
the monoxanthones which are synthesized fi'om polyketide precursors. The structure and
the possible biosynthetic pathway of sulochrin (Figure 1-6) were described by Franck
(1980).

Patulin. Patulin and penicillic acid are synthesized primarily by Penicillium and
Amergillus species. The biosyntheses of these two secondary metabolites are very similar.
Only the biosynthesis of patulin is discussed in this section. Patulin was first discovered by

Birkinshaw et a1. (1943) and its chemical structure was identified by Woodward and Singh

19

(1949). The toxicity of patulin has led to an increased awareness of its occurrence in fresh
apple juice which originates from brown rot of apples contaminated with Penicillium
mm Link. The production of patulin by Aspergillus clavatus gowing on spent
malted barley was implicated in the poisoning of cattle when this material was used as a
feed additive. Birch e1 11!. (1955) demonstrated the biosynthetic pathway of 6-
methylsalicylic acid (6-MSA) in experiments with [1-“C]-labeled acetate fed to
Penicilliran pa1ulr1m. 6-MSA is converted to patulin by a complex sequence of oxidation,
cleavage and rearrangements which form the second stage in the biosynthesis of patulin.
Zamir (1980) confirmed that 6-MSA is converted to gentisaldehyde, then phyllostine
which is the substrate for the oxidative cleavage reaction and the rearrangement which
results in the formation of patulin. The major steps are outlined in Figure 1-7. The
biosynthesis of patulin is a clear example of the two stages in the production of a
secondary metabolite. The polymerization of three molecules of malonyl CoA with a
molecule of acetyl CoA, during which a reduction and dehydration occur, leads to a
tetraketide derivative which readily cyclizes to form 6-methylsalicylic acid (6-MSA).
Although a relatively small molecule, 6-MSA has a special place in the history of our
understanding of polyketide biosynthesis.

Citrinin. Citrinin, first isolated fi'om Penicillin»: citrinum by Hetherington and
Raistrick (1931), is produced by numerous Penicilliwn and Aspergillus species (Rodig er
a]. , 1966). Citrinin exhibited marked antibiotic activity in vitro, but its nephrotoxigenic
properties prevented therapeutic application (Krogh et al. , 1973 ). The biosynthesis of

citrinin was determined initially by Birch et al. (1958). Addition of sodium [1-“C]-labeled

20

0'1 0
3MalonyCoA Ho 20-1 0“ °°=” °
: . -—> .__..o MW -—-
CPD
0'1

on 0 H0420
Acetyl
G-MSA Gentisaldehyde Phylloetlne Patulln

Figure 1-7. Biosynthesis of patulin in Penicilliwn patr1111m.

Pentaketlde
‘ W COA 3 Methionlne Cltrlnln

o on 0 on
meteoA+4MannvICOA-~ dim mam “mm
o C 0
o o 3 ea;
J4 arouncoorr
Figure 1-8. Biosynthesis of citrinin in Aspergillus candidus.

Starlgmatocyatln 0

H:
a
o coca /
AcatleoA 0 HO OH
+ —.—D —->—. b
OMannyCoA c
on c on
\b
4
I
on
on 0 on on 0 0H
AustocystinF SWM"

Figure 1-9. Biosynthesis of sterignatocystin, sterignatin, and austocystin in Aspergillus
versicolor.

21

acetate to cultures of Aspergillus candidus gave ["C]-citrinin. Gererally, during the
building of a polyketide chain, a number of additional goups can be added, especially to
the reactive mythylene goups, providing another source of diversity in structure. In the
biosynthesis of citrinin, three additional C1 goups are added to the pentaketide chain from
the biochemically active one carbon pool involving methionine (Figure 1-8). The common
occurrence of C-methyl goups in polyketide-derived metabolites produced by firngi is
often provided by malonyl CoA. These additional C1 goups are frequently further
oxidized. Thus, one of the three additional branched carbons in citrinin is oxidized to a
carboxylic acid goup.

Sterigmatocystin, sterigmatin, and autocystin. Sterignatocystin (Figure 1-9), a
xanthone which contains an angularly firsed bisdihydrofuran system, was originally isolated
from the nrycelium of Aspergillus versicolor Tiraboschi and proved to be
hepatocarcinogenic in rats (Hatsuda and Kuyama, 1954). Sterignatocystin is an
intermediate in the biosynthetic pathway leading to the aflatoxins. Sterignatocystin is
synthesized from a decaketide, which is probably derived from hexanoyl CoA with the
subsequent addition of seven more malonyl CoA units (Townsend et aL, 1984). With the
development of an understanding of the molecular biology of aflatoxin biosynthesis,‘
several genes and enzymes involved in sterignatosystin synthesis have been identified
(Skory e1 11]., 1992; Keller e1 11]., 1994; Yu and Leonard, 1995; Keller et 01., 1995).

Sterignatin (Figure 1-9), a metabolite isolated from A. versicolor, was the first
metabolite found with a linearly fused xanthone and bisdihydrofirran moiety (Harnasaki et

at, 1973). Steyn and Vleggaar (197 5) obtained other linear xanthones from corn cultures

22

of A. 1131113. The linear xanthones, called austocystins, fi'equently contain a chlorine atom,
or an isopentyl side chain. They are essentially substituted derivatives of sterignatin
produced (along with sterignatocystin) by A3perg1'II113 versicolor. They are believed to
share the early pathway of aflatoxin biosynthesis (Harnasaki e1 (11., 1973). A key
intermediate in this pathway is the postulated benzophyenone carboxylic acid (BCA)
(Figure 1-9). Rotation about bond (11) would lead to the sterignatocystins, whereas no

rotation or rotation about bond (b) would lead to sterignatin and the austocystins.

Biosynthesis Of Amino Acid-Polyketide Mycotoxins

This goup of mycotoxins is synthesized first by forming a polyketide backbone to
which amino acids are added. Ochratoxin and cytochalasin B are two examples of amino
acid-polyketide mycotoxins produced by Aspergillus.

Ochratoxin. Ochratoxin is a nephrotoxic metabolite of some species of Aspergillus.
Ochratoxin A is also a weak antibiotic (Heller e1 111., 1975). The discovery of toxigenic
strains of Aspergillus ochraceus (Scott, 1965) led to the isolation and structural
elucidation of ochratoxin A and B. The biosynthesis of ochratoxin A in Aspergillus
ochraceus was studied in feeding experiments by Seary e1 al. (1969) with ["C]-labeled
phenylalanine, by Steyn et a1. (1970) with [“C]-labeled acetate and methionine, by Wei et
a]. (1971) with [“Cl]-labeled sodium chloride, and by Maebayashi et a1. (1972) with
[13C]-labeled forrnate. These studies made it clear that the biosynthesis of ochratoxin A

(Figure 1-10) consists of the generation of a pentaketide which is modified by the addition

23

Methionine

muonyICoA Mofiggflo mmTwmn me

Ochratoxin A

 

Figure 1-10. Biosynthesis of ochratoxin in Aspergillus ochraceus.

Phenyl- \
alanine o 3 Methionine 5:0

0
O
O o ! €0.13 0 o
—. ——.——.

Oetakatide
k Cytochalasin E
Acetyl CoA 7 Malonyl CoA «j- 7 Acetyl CoA

7 Biotin-COOH

Figure 1-11. Biosynthesis of cytochalasin E in Aspergillus clava1113.

24

of a single methyl goup fi'om the C1 pooL This additional methyl goup is oxidized to a
carboxyl goup through which L-phenylalanine is linked by an amide bond.

Cytochalasin E. The discovery of cytochalasans was the result of the observation that
the culture filtrate of certain microorganisms produced morphological changes in hyphal
cells of test firngi and unusual efi‘ects in mammalian cells in tissue culture (Rothweiler and
Tamm, 1966). Cytochalasin B, one of more than 20 cytochalasans, was found in certain
species of Asperillus (Buchi et a1., 1973). Cytochalasans are characterized by a highly
substituted perhydroisoindolone goup, to which is fused a macrocyclic ring (which is
either a carbocyclic, a lactone, or a cyclic carbonate). The structure of cytochalasin B was
determined by Buchi et al. (1973). A detailed account of the biosynthetic pathway leading
to cytochalasans was provided by Tarnm (1980). Here, cytochalasin E produced by A.
clava1113 (Buchi e1 01., 1973) is utilized as an example to describe a possible biosynthetic
pathway for cytochalasans. The proposed pathway of cytochalasin B synthesis starts with
the formation of an octaketide which is produced fi'om one molecule of acetyl CoA and 7
molecules of malonyl CoA, followed by addition of a phenylalanine to form an amino acid-
polyketide complex to which three C1 goup fiom methionine are added. The synthesis is

terminated by formation of an epoxide (Figure 1-1 1).

Biosynthesis Of Polypeptide Mycotoxins
Anrino acids are the precursors of several distinct families of secondary metabolites
including polypeptides, cyclic polypeptides, amino acid-polyketides, and amino acid-

isoprerroids. The last two goups have already been described. Some mycotoxins

25

produced by Aspergillus are synthesized using one or two amino acids as building blocks.
Coumarin and related compounds are the derivatives of one moleclue of L-phenylalanine
or one molecule of L-tyrosine in several species of Aspergillus. Aspergillic acid is
synthesized fi'om one molecule of leucine and one molecule of isoleucine in Aspergillus
flaws. Many amino acid-derived mycotoxins or antibiotics are synthesized from more
than two anrino acids such as dicoumarol, gliotoxin, tryptoquivaline M, and penicillin.
Dicoumarol. During fermentation of hay, Aspergillus fianigatms transforms coumarin
into dicoumarol which is a powerful blood anticoagulant and can cause fatal hemorrhage
in cattle eating the spoiled bay. The immediate precursor of dicoumarol is 4-
hydroxycoumarin which is derived from either L-phenylalanine or 1..-tyrosine. The
methylene goup which connects the two 4-hydroxycoumarins is derived fiom
formaldehyde which is also produced during fermentation. The possible biosynthetic
pathway of dicoumarol in Aspergillusfimigatus (Luckner, 1990) is shown in Figure 1-12.
Gliotoxin. Gliotoxin was originally described as a metabolite with antifirngal activity.
The producing organism was subsequently identified as Gliocladium fimbriatwn Gilman
& Abbott (Webster and Lomas, 1964). Gliotoxin is also produced by several species of
Penicillium and Aspergillus such as A. chevalieri (Wilkinson and Spilsbury, 1965).
Gliotoxin belongs to a large goup of toxins called epipolythiodioxopeperazines with
phenylalanine, tryptophan, or tyrosine and sometimes alanine as their precursors, and with
methionine or cysteine as their sulfur suppliers. Gliotoxin causes genomic DNA

degadation preferentially in certain blood cell types including T lymphocytes and

26

KW“ m.“

L-Tyrosine L-Phenyla\lanine
on
can
-— \ng
p-Hydroxymelilotic acid acoumaric acid Melilotic acid

OH Formaldehyde

1°

B-Ketomelilotic acid 4—Hydroxycoumarin Dicoumarol

Figure 1-12. Biosynthesis of dicourrrarol in Aspergillusflmigatus.

H o
C1420"! °Hs H2014 CHzG'I
HOOCY‘JH;
CHzO-l Glrotoxrn
Sarita

Figure 1-13. Biosynthesis of gliotoxin in T richodemra viridi.

27

macrophages. Gliotoxin has previously been used to treat murine allogeneic bone marrow
prior to transplantation into irradiated recipients, and in this situation the drug prevents
development of gaft-versus-host disease. This goup of toxins includes hyalodendrin,
aranotin, epicorazine, sporidesnrin, chactocin, verticillin, chetomin, sirodesrnin, and a large
number of their derivatives.

An understanding of the gliotoxin biosynthetic pathway has been partially achieved
(Figure 1-13). Suhadolnik and Chenoweth (1958) were the first to demonstrate that
phenylalanine provides the reduced indole nucleus of gliotoxin in T richodenna viridi.
Almost 20 years later, Kirby et a]. (197 8) confirmed that cyclo-(Irphenylalanyl-L-seryl) is
an intermediate in gliotoxin biosynthesis in T richoderma viridi. For the biosynthetic
mechanism for introduction of sulfirr into gliotoxin, no firm conclusions have been
reached. Cysteine ( possibly in combination with pyridoxal) is suggested for the sulfirr
donor, and a dehydrodioxopiperazine is suggested for the sulfur acceptor. Consecutive
introduction of two thiol goups would be suficient for oxidative cyclization to an
epidisulfide compound (Kirby and Robins, 1980). The biosynthesis of gliotoxin is dealt
with in some detail by Kirby and Robins (1980). Gliotoxin was the first mold metabolite
to be recognized as belonging to the diketopiperazines with polysulfirr bridges.

Tryptoquivaline M. Two tremor-causing metabolites, tryptoquivaline and
tryptoquivalone, were isolated from a sample of rice implicated in the death of a child in
Thailand, and then demonstrated to be produced in a toxigenic strain of Aspergillus
clavatus by Clardy e1 11]. (1975). Later other tryptoquivaline-related metabolites were

isolated from Aspergillus fi1m1'gatr13 (Buchi e1 11!. , 1977; Yamamki et a1. , 1979).

28

"2N
*1 U
“ac-CHOW! HOW

Vuln- Maw “'6 OIK. 03:5,. cacao
. Mr 0 p o p o p
(1?” ”firm" 0&1; 0%; 012:?“

O

7W Alanhe Deoxynortryptovalne Tryptoqulvalone TWIN!“ M

Figure 1-14. Biosynthesis of tryptoquivaline M in A3perg1‘llusfi1m1’ga1113.

“WW
L-arphuminoadiprc acid L-valine We“
ach L-cystelne Phenylacetyl CoA
Mmfig” IpnA an’sf $0500Yfi9<
LLD-ACV lsopenicillin N Penicillin 6

Figure 1-15. Biosynthesis of penicillin in Aspergillus nidulans.

29

The basic precursors involved in biosynthesis of these complex molecules are probably
the amino acids tryptophan, valine, alanine and anthranilic acid, the latter being also an
intermediate in the shikimate pathway to tryptophan. In this section tryptoquivaline M is
utilized as an example to describe the possible pathway of tryptoquivaline biosynthesis in
A3pergillr13fi1m1‘ga1113 (Y amazaki et al. , 1979). Synthesis appears to be initiated to form a
tripeptide composed of anthranilic acid, tryptophan, and valine. Deoxynortryptoquivalone
may be the first metabolite formed in the pathway of tryptoquivaline by addition of
another amino acid alanine. The oxidation of dexoynortryptoquivalone results in the
formation of tryptoquivalone which is converted to nortryptoquivaline.
Nortryptoquivaline is then converted to tryptoquivaline M by simple epirnerization (Figure
1-14).

Penicillin. The discovery of penicillin has a special place in the history of human
medicine. In September 1928, Alexander Fleming, a Scottish bacteriologist working in
Almroth Wright’s Inoculation Department at St. Mary’s Hospital, London, returned from
holiday to his laboratory and found a contamination of mold (Penicillium n01a111m) in a
plate culture of staphylococci. The gowth of the bacterial colony was inhibited around
the mold. The inhibition was later shown to be caused by the production of a substance by
the mold to which Fleming gave the name “penicillin”. His discovery of penicillin was
published in 1929. Penicillin belongs to a goup of antibiotics with a common B—lactam
ring. Today, 69 years later, the understanding of penicillin (and other B-lactam
compounds) biosynthesis has been reached not only at the chemical and biochemical level,
but also at the genetic and molecular levels. The main interest is with Penicillium

chrysogeman and Acremonium chrysogenum which have been the commercial sources of

30

penicillin and other B-lactams. But in the late 1940s, screening of a wide range of fungi
for new antibiotics led to the detection of penicillin produced by Aspergillus nidulans
(Dulaney, 1947). Although this has never been of commercial sigrificance, A. nidulam
has been utilized as a model organism for genetic and molecular studies of penicillin
biosynthesis.

The biosynthetic pathway for penicillin G in A. nidulans fi'om amino acid precursors is
shown in Figure 1-15. The primary metabolites which are the starting point for penicillin
biosynthesis are the three amino acids L-or-aminoadipic acid, L-cysteine, and 1..-valine.
The first step is the formation of the tripeptide 5-(L-a-aminoadipyl)-L-cysteinyl-D—valine
(ACV) catalyzed by the single large enzyme ACV synthetase (ACVS). The tripeptide is
cyclized by is0penicillin N synthetase (IPNS), also called ACV cyclase, in an oxidative
reaction in which four hydrogen atoms are removed fi'om ACV, and one molecule of
oxygen is consumed (Baldwin and Abraham, 1988). Isopenicillin N is modified by the
enzyme acyltransferase (acyl CoA:6-amino-penicillanic acid acyltransferase, ACT). This
enzyme removes the aminoadipyl side chain, and replaces it by phenoxyacetyl to form
penicillin G.

Streptonwces clabvuligerus contains isopenicillin epirnerase which converts isopenicillin
to penicillin N. Certain fungi and many bacterial species further modify the lactam ring by 5
the sequential action of expandase and hydroxylase activities to form
decacetylcephalosporin C. This is converted into either cephalosporins or cephamycins,

depending on the produing organism.

31

Delta-Ha-aminoadipylylfcysteinyl-D-valine synthetase (ACVS) was first isolated
fi'om Aspergillus nidulans and studied by Van Liempt et al. (1989), and further analyzed
by MacCabe et al. (1991). Isopenicillin synthetase (IPNS) was first isolated and studied
by Baldwin and Abraham (1988) and was further studied by Roach et a1. (1995) and
Blackburn et al. (1995). Acyl CoAz6-amino-penicillanic acid acyltransferase (ACT) was
purified fi'om A. nidulans (Whiteman e1 111., 1990; Montenego e1 11]., 1990).

The first B-lactam biosynthetic gene to be cloned was the isopenicillin synthetase (IPNS)
gene of Amergillus chorsogenum. This gene was identified using oligonucleotides based
on a partial sequence of the purified enzyme (Samson e1 111., 1985). Availability of this
gene quickly led to the isolation of the IPNS gene, 1'pnA, of A. nidulans (Ramon er al. ,
1987) by heterologous hybridization. The discovery that a cephamycin biosynthetic gene
cluster from the Gram-negative bacterium, F lavobacterium spp. SCI and 154, hybridized
to the IPNS gene of P. chrysogenum, enabled the identification of the region containing
the 11ch gene encoding the 5-(L-or-arninoadipyl)—L-cysteinyl-D-valine synthetase (ACVS)
inA. 1111111111113 (Bumham e1 111., 1989). Later the ach gene was positively identified in A.
nidulans by MacCabe et a1. (1991). The isolation of the penDE gene encoding acyl
CoAz6-amino-penicillanic acid acyltransferase (ACT) was initially achieved in P.
chrysogenum (Barredo et aL, 1989), using oligonucleotides desigred fiom the N-terminal
amino acid sequence of the purified enzyme. The equivalent gene, acyA, of A. nidulans
was isolated using the P. chrysogenum penDE gene as a probe (Montenego et al. , 1990).
The Aspergillu3 nidulans rrpeA locus was identified by MacCabe et a1. (1990). The locus

consists of three contiguous genes in the order of ach-ipnA-acyA and is required for

32

penicillin biosynthesis. These three genes, which are clustered in chromosome V1 (3.0

Mb), were cloned fi'om A. nidulans. Beach gene is expressed as a single transcript from a

separate promoter (Martin and Gutierrez, 1995).

Biosynthesis Of Isoprenoid-Polyketide Mycotoxins

Some mycotoxins isolated fiom Aspergillus contain both isoprenoid and polyketide
moieties. Turner and Aldridge (1983) listed a number of isoprenoid-polyketide
mycotoxins produced by Aspergillus. Austin, firmagillin, and territrem B belong to this
goup of mycotoxins.

Austin. Austin, a polyketide-isoprenoid mycotoxin, was first isolated fi'om Aspergillus
1131113 by Cherral et a]. (1976). Austin was later found in A. 1erre113 by Springer et a1.
(1979). The biosynthetic pathway of austin in Aspergillus 1131113 was initially studied by
Wicnienski (1979) and proposed by Simpson er al. (1982). The major step in the
biosynthesis is the preliminary coupling of the sesquiterpene precursor farnesyl
pyrophosphate and the methylated polyketide. The three methyl goups come from
methionine. Rearrangement of this hypothetical intermediate leads to the formation of
austin (Figure 1-16).

. Fumagilliu. Furnagillin, first isolated from Aspergillus fi1m1'gatr13, was recogrized as
an antibiotic agent (Hoza, 1966). At high doses firmagillin was lethal and caused toxic
alteration in the liver, the kidney (Lauren et a1. , 1989), and the respiratory epithelium
(Anritani e1 11]., 1995). Fumagillin is a potent anti-angiogenic compound (I-Iorsman at al,

1995; Nishimura e1 111., 1996). Fumagillin is another example of a secondary metabolite

33

MGM 3M8bnleoAc—3Bloth-COOH'I'3AOCMCOA

 

Figure 1-17. Biosynthesis of fumagillin in Aspergillusfimigams.

34

assembled fiom two distinct pools of precursor intermediates, one is polyketide and the
other is isoprenoid. Birch and Hussain (1969) showed that in A. firmigatus the
decantetraendioic acid moiety, which is one of the two precursors of firmagillin, has a
polyketide origin and that the terminal carboxyl goup represents the original carboxyl end
of the polyketide. They proposed an hypothesis for the origin of the unusual nucleus of
fumagillin from farnesyl pyrophosphate via a'bergamotene intermediate. This hypothesis
was later supported by the detailed studies of Cane and Levin (1976) on the biosynthesis
of ovalicin of Pseudeurotium ovalis Stolk. Fumagillin is an intermediate in the ovalicin
biosynthetic pathway. The proposed biosynthetic pathway of firmagillin is shown in
Figure 1-17.

Territrem B. Territrem A and territrem B were first identified in stored unhulled rice
contaminated with Aspergillus ten-e113 in Taiwan by Chung et a1. (1971). Ling (1976)
demonstrated that both toxins induce tremor and convulsions in mice. Territrems do not
contain nitrogen. This is the most distinctive feature of territrems which sets them apart
fi'om the known N-containing tremorgenic mycotoxins. Other members of this family of
compounds have been identified and characterized by Ling er a]. (1984), Peng et a].
(1985), and Ling (1994). For example, territrem C was isolated fi'om the chloroform
extract of rice cultures of Aspergillus terreus 23-1, which also produces territrems A and
B. Ling et a1. (1984) also studied acute toxicity and some physicochernical properties of
territrems. Territrems contain isoprenoid and polyketide moieties. The biosynthetic

pathways of both territrem A and territrem B have been studied in Aspergillus terreus

35

OO

 

Fameeyl pyrophosphate

o
45-0-1"; -oa 0
on on
Isopentenyl pyrophosphate
W0 0' 9 o
41-04-011
5" 5H Hexaketide
Gennyl pyrophosphate
9 9
> , 95, 9 -P-O-P-O-1
-o-r.>-ou
6" on Isopentenyl pyrophosphate
Dlmathylally pyrophosphate

L W9

 

Figure 1-18. Biosynthesis of territrem B in Aspergillu3 1erre113.

 

 

O
I HN o
; 0'1
cc?“ ° \ °°
49-011
a.
Dimethylallypyrophosphate
"""°""" - /
9 9 H
7 f-o-g-ou
amateur _.

0401-1

Cycloplazonic acid
Isopentenyl pyrophosphate '

Figure 1-19. Biosynthesis of cyclopiazonic acid in some species of Aspergillus.

36

(Kuo and Yun, 1988; Lee et aL, 1992). The major step in the biosynthesis of territrem B
is the combination of one molecule of hexaketide and one molecule of farnesyl
perphosphate. Famesyl pyrophosphate is formed from one molecule of isopentenyl

pyrophosphate and one molecule of dimethylally pyrophosphate (Figure 1-18).

Biosynthesis Of Amino Acid-Isoprenoid-Polyketide Mycotoxins

This goup of mycotoxins contains all the three basic precursors: amino acid,
polyketide, and isoprenoid components. Cyclopiazonic acid belongs to this goup of
mycotoxins.

Cyclopiazonic acid. Cyclopiazonic acid is produced by strains of Aspergillus flaws,
A. oryzae and A. versicolor, as well as several species of Penicillium (Luk et a]. , 1977;
Gallagher e1 (11., 1978; Richard and Gallagher, 1979; Dorner, 1983; Domer et al., 1984;
Trucksess e1 01., 1987; Lee and Hagler, 1991). The biosynthesis of cyclopiazonic acid
was reviewed by Holzapfel (1980). The molecule is synthesized in some species of
Aspergillu3 (Figure 1-19) possibly from tryptophan and an acetyl CoA-derived diketide to
yield or-acetyl y—(B-indolyl) methyltetramine acid to which is added a C5 dimethylallyl
goup with subsequent cyclization to yield cyclopiazonic acid. Because the C5
dimethyallyl goup is derived fi'om isopentenyl pyrophosphate, cyclopiazonic acid is
composed of the three basic precursors: tryptophan an amino acid, a deketide, and an
isopentenyl pyrophosphate. Two of the enzymes involved in the later steps of this
pathway were isolated and partially characterized (Steenkamp et a1, 1973) and the

stereochenristry of the cyclization was thoroughly studied (Steyn e1 111., 197 5a).

37

Epilos

The biosynthesis of mycotoxins in Aspergillus introduced in this review represents a
small piece of the whole picture of the enormous chemical diversity and the complex
biosyntheses of mycotoxins of this genus. In addition to the challenges still ofi‘ered by the
mycotoxins of Aqrergillus to chemists and biochernists, their potential biological activity
including mammalian toxicity also challenge the medical and toxicological fields. One

important challenge now is to understand the molecular biology of the biosyntheses of this

goup of mycotoxins.

PART 2. AFLATOXINS
TOXICITYOFAFLATOXINS
AmmamsewndmynwtabohtessynfledmdbycatmnMOqurergIhufhmA
paun'ticus, andA. 1101111113(Gormanetal., 1992;Kurtzmanetal., 1987). Thesefirngiare
ubiqrutwsandfiequanlygowonavafiayoffoodmdfeedaopsandcauseaflatordn
contaminatoninmanyareasintheworld (Blourrt, 1961;]elineke1al, 1989;Ellise1 111., 1991).
Peands,cottonseed,comandteermtsarethemajorcropsafi‘ectedinfl1eUSA.
AfiatordnsarebasicaflyclassifiedhrtotheBoereriesbasedontheircoloroffiuorescence
under Iongwave (360 nm) UV light (B for bhre, and G for geen). Afiatoxin Br (AFBr),
afiatoxin B2 (AFBz), aflatoxin Gr (AF Gr), and afiatordn G2 (AFGz) are the four major
afiatordnsproducedbyfirngiinnatm'e.
ItislikclythataflatordcosiserdstedformanyyearspriortotheepizooficoutbreakinBritain

inl960knownasTurkedeisease(Blount,1961)inwhichmorethan100,000turkeyswere

38

destroyed with acute hepatotoxicity caused by afiatoxin-contaminated feed. However, that
dramaficornbreakdemonsu'atedtheseriousnessofafiatordncomanfinafion, andledtothe
recogrition that afiatoxin contamination is both an economic and a public health problem in
my areas of the world.

Asobsavedinfieldoutbreaks,acutestructuralandfirnctional damagetotheliver(suchas
fatty infiltration, necrosis, and extensive bile duct proliferation), the major target organ for
afiatothhavebewreponedmmoahbomtorymfimalsmdmsevaaldomesficanhnal
species(BusbyandWogan, 1984). Humanexposuretoaflatordnoccurreddirectlythrough
ingestion ofcontarninated crops and resulted in fatal aflatoxicosis in India (Krishnamachari et
at, 1975) and in West Afiica (Ngindu er al., 1982).

Inaddifionmaanehepatotorddty,afiatordmmesuongnnnagmsmdcarcmogeus
Aflatoxins have been demonstrated to be potent mutagens in a variety of test systems
causing difi‘erent mutations. For example, aflatoxins have been demonstrated to cause
mutations in: bacteriophage (Goze et al., 1975) and plasmids (Wood et al., 1987;
Courtemanche and Anderson, 1994); bacteria (Raina e1 (11., 1980; Lowery et at, 1983;
Refolo e1 01., 1987; Foster et at, 1988); Drosophila melanogaster (Lamb and Lilly,
1971; Graf e1 (11., 1984); cotton leaf-worm (Abdou e1 111., 1984); the chick embryo
(Bloom, 1984); monkeys (Adgigitov e1 11]., 1984); mice (Umeda et at, 1977;.Macgegor,
1979); and human cells (Kaden et al., 1987). The types of mutations linked to aflatoxin
exposure include substitutions, fi'arneshifts, sister chromatid exchanges, chromosome

aberrations, DNA single-strand breaks, nricronucleus formation, and lethal mutations.

39

The experimmtal data, which have been obtained from different animals ingesting AFBl such
as rat, rainbowtrout, duck, rhesus monkey and tree shrew, demonstrate that AFBl is the most
potent naturally occurring carcinogen (Adarnson e1 11]., 1973; Carnaghan, 1965; Gopalan et at,
1972; Reddy e1 11]., 1976; Sinnhuber e1 01., 1968; Tilak, 1975) in these experimental systems.
For example, early experiments with laboratory rats demonstrated that AFBl is a renal
carcinogen (Epstein et al., 1969). A small incidence of colon carcinomas resulted when AFBr
wasadmhusuatedtochha'mtseitheromflyorviaflredietMogmmdNewbene, 1967).
Administration of AFB1 elicited tumors in the central and peripheral nervous systems of
pregnant rats (Goerttler et al., 1980). Sieber et at (1979) found that long term exposure of
AFBr to monkeys induced hepatocelluar carcinomas, osteogerric sarcomas, bladder papillary
carcinoma, gall bladder adeno-carcinomas, and pancreatic carcinomas.

Emerimental evidence has linked AFBl to human lung cancer (Hayes etal, 1984; Mace e1
111., 1994). Epiderniologically, AFBl also has been suggested as a synergistic factor along with
hepafifisthusOiBWinthegenerafionofhumanpfimaryhvercancerQLC),andhuman
hepatocellular carcinoma (HCC) (Groopman et at, 1988). Very recently it was demonstrated
tint HBV and AFBl played a synergistic role in the development of hepatocellular
carcinoma in tree shrews (Yan e1al., 1996).

Afiatoxinscancauseavariety ofothertoxic efi‘ects. AFBl experirnentallyhasbeen shown
to be a potent teratogen in rat, hamster, and chick embryo (Butler et at, 1966; Elis and
DiPaolo, 1967; Venett e1 at, 1973). Afiatoxicosis altered the reproductive efliciency of both
male and female domestic animals, particularly poultry (Ottinger and Doerr, 1980). Afiatoxins

havepdmfialtordcefi‘easonmehmnmesystanmmhnalsremlfingmsrscepubflitym

40

diseases caused by bacteria, fungi, and viruses (Edds et a1, 1973; Richard et al., 1973;
Giambrone et at, 1985). Herrold (1969) reported that AFBl caused megalocytosis in the
prordmaltubulesinthekidney. Hemorrhagickidney syndromethatcomrnonlyoccursin
chickensinAfiicahasbeeulinkedtoAFBl (Datiillaetal, 1987). Inwlturedlddneycelllines,
AFBr induced mitochondrial degeneration and loss of microvilli (Y oneyama et at, 1987). The
possible efi‘ects of AFBl on nervous system function were proposed by finding this toxin in
tissue samples fi'om patients with Reye’s syndrome, a pediatric disease characterized by
cerebral edema and neuronal degeneration (Chaves-Carballo er al., 1976). In rats, AFBl
cmsedadeaeaseofsamomnmdhsmetabohtembrainmdalsomhibhedbrainstem
tryptophan metabolism (Coulombe e1 111., 1985).

The generally accepted biochemical mechanism of AFBl toxicity is its biotransformation to
the AFBl-8,9 epoxide by cytochrome Pea-dependent epoxidation of the terminal firran ring
double bond (Essignann e1 111., 1982; Stark, 1980; Soman and Wogan, 1993). AFBl-8,9
epoxide is a potent electrophilic molecule which can form adducts with protein and DNA. The
protein adducts may result in abnormal fimction and cause acute toxicity, and DNA adducts
may cause mutagenicity and carcinogenicity. In addition, formation of fi'ee radicals from
AFBl suggests a possible role of fi'ee radical metabolites in the carcinogenicity (Kodarna et
at, 1990).

As early as the late 19705, Autrup et a]. (1979) reported that colon tissues from rat and
harm were capable of activating AFBl to form DNA adducts. later it was demonstrated that

AFBl-8,9 epoxide combines mainly to the N-7 of guanine to form DNA adducts (Essignarm,

41

00
on

Aflatoxin 32

O O OCH3

 

O O OCH;
Aflatoxin G2

 

Figure 1-20. Structures of Aflatoxin B1, B2, Gr, an G2.

 

CHBCOZH CHsSCHzCI-IZOH(NHZ)C02H
ACETATE METI-IIONINE

Figure 1-21. The origin of the 16 skeletal carbon atoms of aflatoxin B1.

42

er al, 1982). These DNA adducts have been proven to be mutagenic in many in vitro
mutagen-detecting systems such as bacteria, firngi, drosophila, and mammalian cells. For
example, mutations in codon 61 of the Ha-ras proto-oncogene have been shown to occur
with a high fi'equency in AFBr-induced liver tumors in the CF] mouse (Bauer-Hofinan e1
111., 1990). Another major mutational hot spot for DNA adduct formation by AFBl in human
primary liver cancer (PLC) and human hepatocellular carcinoma (HCC) was demonstrated to
be codon 249 in the p53 tumor suppresser gene (Puisieux e1 01,1991). Experimental data
demonstrated that rmrtations in p53 occur later in tumor development, and therefore suggested
that these mutations may act as tumor promoters not as tumor initiators (Nose et al., 1993).
AFBl can also induce specific mutations which activate another proto-oncogene c-ki-ras
(Soman and Wogan, 1993).

Because of the threat of aflatoxin contamination to public health the US Food and Drug
Administration set action levels of 20 ppb for human foods (except for milk which is 0.5 ppb)

and 20to 300 ppbinmostanirnal feeds(Labuza, 1983).

BIOSYNTHESIS OF AFLATOXIN Bl
Biosynthesis Of Allatoxin B1

Afiatoxins are polycyclic, unsaturated compounds consisting of a coumarin mrcleus flanked
byabisfirran system containing eitherafilranring(Bl, G1 series) oradihydrofirranring (82, G2

series) on one side, and either a cyclopentanone (B series), or a six-member lactone (G-series)

43

mortar! CO MabnleoA o
,5“,me ,_j_ ,...,wvm °L4__ Eagle“,

TMalonleoA 7c02 o o o o

. we» . \_ a we
Ema/‘chs EmSJk/V‘CBJ o o o o 8.5
HexanoleoA Decaltetide

ouoouou err,
on 0110011011 01100110

"misfit. are... ‘3. WTW

\ on c on Avarantin (AVN) Norsolorinic acid (NA)

° 0110011
no oucu

/ Avezufanln $1.3] , on ‘ 016".

(MIN) / "° 0
on c on on c on Veniconal W1
acetate

...-(3:100... ,cec ,.

Averufin (AVF) °
1'4-lydroxyveraicolorone (HVA)

0110011 01100" ouoonv

WU.— ._

"o 0° "0 0° HO oouorr
° 0

Versioolorin A (VA) Versioolorin 3 (VB) Venisonal (VAL)

o c
on c on 0 can. 0
o o _, o _.
o o o ecu. 0 ° OCH: ° °°"'

Demetlrylaterig- Sterigmatocystin O-methylsterigma- Aflatox in 81 (AF 31)
matocyatin (ST) tocystrn
(DMST) (OM37)

 

Figure 1-22. The aflatoxin B1 biosynthetic pathway in Aspergillus parasiticus.

on the other side (Figure 1-20).

Most researchers in the field of aflatoxin biosynthesis have focused on aflatoxin Bl
(AFBr) because it is the most toxic and abundant aflatoxin produced by fungi. The
, currently accepted AFBl biosynthetic pathway begins with acetyl CoA and malonyl CoA
to form a proposed polyketide precursor, which is then converted sequentially to several
pathway intermediates in the order of norsolorinic acid (NA), averantin (AVN), averufanin
(AVNN), averufin (AVF), versiconal hemiacetal acetate (VHA), versiconal (VAL),
versicolorin B (VB), versicolorin A (VA), demethylsterignatocystin (DMST),
sterignatocystin (ST), O-methyl sterignatocystin (OMT), and finally aflatoxin B1 (AFBr)
(Figure 1-22) (Dutton 1988; Bhatnager e1 111., 1992; Yabe and Hamasaki, 1993). The
established pathway is the result of a combination of the use of Aspergillus mutants
blocked in the AFBl pathway in certain steps, radiolabeled-precursor feeding experiments,
enzyme isolation (purification) and activity determination, and chemical structure
determination by nuclear magnetic resonance (NMR) spectroscopy and mass
spectroscopy. Determination of the structure (especially the radiolabeled atom
distribution and the stereochemical configuration) of any potential precursor and the final
product is a very important requirement. Without accurate structure determination, all the
5 other experimental results are not fillly explainable. Therefore, any comprehensive review
on AFBl biosynthesis should include structure analysis. However, in this brief review,
only the most important experiments related to structure determination will be mentioned.
Steyn e1 al.(l980) presented an excellent review on structure determination related to

AFBl biosynthesis.

45

According to the chemical structure of the starter units, the intermediates and the final
product, and the demonstrated or speculated nature of the biochemical reactions involved,
the biosynthesis of AFB1 can be divided into seven distinct stages: (1) the fatty acid stage
in which it is proposed that a hexanoyl CoA is synthesized with one acetyl CoA and two
malonyl CoA as the building units; (2) the polyketide stage in which a proposed
decaketide is formed (by adding another seven malonyl CoA to the hexanoyl CoA in a
manner similar to fatty acid synthesis but without the complete reduction, dehydration,
and reduction cycles) and finished by formation of a stable anthraquinone norsolorinic
acid; (3) the internal ketal stage in which the six-carbon side chain of norsolorinic acid is
modified (through averantin, and averufanin) to form the internal ketal of averufin; (4) the
dihydrobisfuran stage in which the ketal of averufin is firrther modified by oxidative
reorganization (in the order of versiconal hemiacetal acetate, versiconal, versicolorin B) to
form versicolorin A which contains an internal ketal structure; (5) the anthraquinone-
xanthone conversion stage in which the anthraquinone versicolorin A undergoes a
complex set of oxidative cleavage and deoxygenation reactions 5 to form the xanthone
demethylsterignatocystin; (6) the methylation stage in which demethylsterignatocystin
is modified by methylation to form sterignatocystin and in turn O-methylsterignatocystin.
(7) the xanthone-coumarin conversion stage in which the xanthone structure of O-
methylsterignatocystin undergoes oxidative cleavage, subsequent cyclization and loss of
one carbon atom to form the coumarin nucleus of afiatoxin Br.

Acetyl CoA, malonyl CoA, and C-1 of methionine are the carbon sources of AFB1.

The study of the sources of the carbons in aflatoxin Br was started in the' late 1960’s.

Among many studies, the experiments conducted by Biollaz et a1. (1970) established
conclusively that AFBl is synthesized with acetate units as the starter. AFBl, prepared by
separately feeding of [l-"C]- and [2-"C]-acetate to an A. flaws culture, was analyzed by
step degadation. The origin of the 16 skeletal carbon atoms was determined (Figure 1-
21). The nearly equal distribution of the labeled carbon atoms at all the centers suggested
that AFBl is derived from a single, highly rearranged polyketide chain. This hypothesis
was firrther supported (Hsieh and Mateles, 1970; Hsieh and Mateles, 1971). In the study
by Biollaz et al. (1970), [methyl-“C]-methionine was found to specifically label the
methoxy carbon atom of AFBl. Later, the development of [13C] NMR spectroscopy,
which reduced the time used in these studies, confirmed that acetate is the source of
carbon for AFBl (Steyn et al., 1975b; Pachler et al.,1976; Gorst-Allman et al., 1978).

The first stage was proposed to occur via a fatty acid biosynthetic pathway to form a
hexanoate (Townsend et al., 1984; Brown et al., 1996).

The second stage was proposed to occur through a polyketide synthetic process to form

a decaketide (Lee et al., 1971; Peng and Leonard, 1995; Chang etal., 1995).

The third stage is the internal ketal stage in which a proposed decaketide goes through
norsolorinic acid, averantin, and averufanin to form averufin (an internal ketal). The
anthraquinones norsolorinic acid (NA) (Conrsoi) (Lee et al., 1971), averantin (AVN)
(Conzoo‘I) (Birkinshaw etal., 1966), and averufin (AVF) (canrror) (Donkersloot er al.,
1972), are each produced by blocked mutant strains derived fiom wild-type A. parasiticus.
Each compound is derived from a Car-decaketide, and was demonstrated to be an

intermediate in the AFBl pathway by feeding experiments. Bennett et a1. (1980)

47

demonstrated the transformation of [“C]-AVN into AVF, but not into NA in A.
parasiticr13. Therefore AVN was placed after NA and before AVF in the AFBl pathway.
McCormick et a]. (1987) found AVF in wild-type A. para31'11'c113. In studies with blocked
mutants they demonstrated ["Cj-radiolabeled averufanin (AVNN) accumulated in AVF
but not AVN. The results demonstrated that AVNN is a precursor in the AFBl pathway
afier AVN and before AVF. With improved I-IPLC, Yabe et al. (1991a) suggested that
5’-hydroxyaverantin may be an intermediate between AVN and AVNN.

The fourth stage is the dihydrobisfirran stage in which averufin goes through versiconal
hemiacetal acetate, versiconal, and versicolorin B (C) to form versicolorin (a
dihydrobisfirran). Lin et a1. (1973) first demonstrated that averufin (AVF) was converted
into AFBl by A. pwasiticus. Yao and Hsieh ( 1974) treated wild-type A. pamsiticr13 with
the insecticide dichlorvos (dimethyl 2,2-dichlorovinyl phosphate) which presumably acts as
a specific enzyme inhibitor, and found AFBl production was inhibited but versiconal
hemiacetal acetate (VHA) accumulated. In the presence of dichlorvos, AVF was
converted into VHA. Therefore AVF lies before VHA in the AFBl pathway. Several
mechanisms have been proposed for the rearrangement of averufin (AVF) to versiconal
hemiacetal acetate (VHA). Among them an epoxide rearrangement is consistent with
experimental evidence. Nidurufin (2’-hydroxyaverufin) is a natural metabolite of
Amergillus nidulans (Auch and Holzapfel, 1970). Hydroxylation at C-2 of averufin
could convert averufin into nidurufin. A rearrangement of nidurufin followed by a Baeyer-
Villiger oxidation (Townsend at al., 1982) was proposed to convert nidurufin into

versiconal hemiacetal acetate.

48

In feeding studies without dichlorvos treatment, VHA was converted to AFBl in wild-
type A. parasiticu3. In feeding studies with dichlorvos, versiconal (VAL), versicolorin B
(VB), and versicolorin A (VA), (but not VHA) were converted into AFBl (Y ac and
Hsieh, 1974; Fitzell et al., 1977; Hsieh et al., 1978). Therefore VHA was before VAL, '
VB and VA in the AFBl pathway. Hsieh et a1. (1989) and Anderson and Chung (1990)
found that VHA was converted to versiconal (VAL) which was further converted to
versicolorin C (VC) by A. parasiticus. Lin and Anderson (1992) and Silva et al. (1996)
demonstrated that a versiconal cyclase purified from A. parasiticus catalyzes the
dehydration of VAL to VB or versicolorin C (V C). Indeed versicolorin C (V C) is the.
racemate of VB. Yabe .et al. (1991b) found that VB was converted into VA in A.
pm'asiticus by a desaturase activity.

Townsend et at (1988) suggested that 1’-hydroxy-versicolorone (HVN) may be an
intermediate between AVF and VHA. Yabe and Hamasaki (1993) suggested that, in the
AFBl pathway, the conversion of AVF to VA goes through a metabolic gid involving
versiconal hemiacetal acetate, versiconol acetate, versiconol, and versiconal.

In the fifih stage, versicolorin A (an anthraquinone) is converted to
demethylsterignatocystin (a xanthone). Hsieh et al. (197 8) found that ["C]-labeled
versicolorin A (VA) was eficiently converted to sterigmatocystin (ST) by A. versicolor.
Thomas (1965) proposed that the conversion of versicolorin A to sterignatocystin could
occur by oxidative decarboxylation and elimination of an acetate-derived methyl carbon
atom. This hypothesis was further supported by the [13C]-NMR spectrum data of

sterignatocystin and aflatoxin Bl (Pachler et al., 1976). Jeenah and Dutton (1983) found

49

that. a VA-accumulating mutant of A. parasiticus was able to convert sterignatocystin to
both O-methylsterignatocystin (OMST) and AFBl suggesting that VA is an earlier
precursor than ST in the AFBl pathway. Yabe et a1. (1989) found that
demethylsterigmatocystin (DMST) was converted to AFBl; and in the presence of S-
adenosyl-[methyl-‘lflmethionine, DMST was first converted into ST, and then to OMST
in A. parasi11'c113. According to recent data, VA is the last anthroquinone and DMST is
the first-xanthOne in the AFBl pathway. DMST is clearly an intermediate between VA
and ST. At this stage the anthraquinone VA is converted into the xanthone DMST
through a possible oxidative cleavage and deoxygenation (Y abe et al. , 1989).

In the sixth stage, demethylsterignatocystin is methylated to form sterignatocystin
which in turn is methylated to form O-methylsterigmatocystin. Singh and Hsieh (1976)
demonstrated that A. pam3111c113 was capable of converting (“cl-sterigmatocystin (31)
into AFBl. Bhatnagar er a1. (1987) found an isolate of A. parasiticus which accumulated
O-methylsterignatocystin (OMST) without AFBl. When ["Cj-OMST was fed to the
mycelia of amutant of A. parasiticus, ["C]-AFB1 was produced and when ["C]-ST was
fed to the same mutant, [“C]-OMST was produced. Cleveland and Bhatnager (1987),
and Bhatnagar and Cleveland (1988) demonstrated that, in A. parasiticu3, a
postrnicrosomal activity (PMA) catalyzed the conversion of ST to OMST, and a
microsomal activity (MA) catalyzed the conversion of OMST to AFBl. These results
suggest that OMST is an intermediate in the AFBl pathway between ST and AFBl.

In the seventh stage, O-methylsterignatocystin is converted to aflatoxin B1. In this

stage, the xanthone structure of O-methylsterignatocystin undergoes oxidative cleavage,

50

subsequent cyclization and loss of one carbon atom to form the coumarin nucleus of
aflatoxin Bl. This is the complete current picture of AFB1 biosynthesis which is generally
accepted.

Based on the experimental data that vesicolorin A (VA) was a precursor of AFB1 (not
of AFB:) and versicolorin B (VB) was a precursor of both AFBl and AFB2, combined
with the fact that VB was converted to VA, McGuire et al. (1989) and Yabe et al.
(1991b) proposed that VB was the branch point leading both to AFBl and AFB2 in the
aflatoxin biosynthesis pathway.

Lee et a]. (1975) found two distinct O-methyltransferase activities in aflatoxin
biosynthesis. Based on their experiments, Cleveland e1 al.(l987) and Cleveland (1989)
suggested that AFBl/AFGl and AFB2/AFG2 are synthesized separately. This idea was
filrther confirmed. Yabe et a1. (1989) demonstrated that A. parasiticus NIAH-26, which
does not normally produce aflatoxins, was able to convert demethylsterignatocystin
(DMST) to aflatoxins Br and G1 (not to B2 and G2); and to convert dihydrodemethyl-
sterignatocystin (DHDMST) to aflatoxins B2 and G2 (not Br and G1). They also
demonstrated that O-methyltransferase I converts DMST and DHDMST into
sterigmatocystin and dihydrosterignatocystin, respectively; and O-methyltransferase II
converts sterignatocystin and dihydrosterignatocystin into O-methylsterignatocystin
(OMST) and dihydro-O-methylsterignatocystin (DHOMST), respectively. These data
support the idea that AFBl/AFGl and AFBz/AFG2 are synthesized separately.

Henderberg et a]. (1988) demonstrated that [um-sterignatocystin was converted to

AFGl in wild-type A. flaws and A. parasiticus but ["C]-AFBl was not. These data

51

suggested that AFGl may not be derived from AFBl; while AFBl and AFGl may share a
common pathway at least until sterignatocystin formation.

The relationship between of AFBl/AFGl and AFB2/AF G2 in aflatoxin biosynthesis then
may be summarized as follows: the four aflatoxins share a common pathway in their
biosynthesis up to versicolorin B. After versicolorin B, one branch leads to AFBl/AFGl,
the other to AFBz/AFG2. AFBl and AFGl share a common pathway up to

sterignatocystin; while AFB2 and AF G2 share a common pathway up to

dihydrosterignatocystin.

Genes Involved In The Allatoxin Br Pathway

In order to understand the regulatory mechanisms involved in aflatoxin biosynthesis, a
very important approach is to clone and sequence the relevant pathway genes. Woloshuk
et a1. (1989), Horng er a1. (1990), and Skory et a1. (1990) developed transformation
systems for A. flaws and A. parasiticus, respectively, which have been demonstrated to be
useful tools to introduce exogenous DNA into these fungi to identify, isolate, and
characterize the genes related to aflatoxin biosynthesis.

Genes can be isolated by the use of heterologous sequences to screen DNA libraries, by
the use of PCR primers to amplify desired genes from genomic DNA, by the use of
antibodies raised against purified proteins to probe DNA expression libraries, or by the use
of complementation tests with relevant mutants and appropriate selectable markers.

The first aflatoxin pathway gene isolated, named nor-1, is involved in the conversion of

norsolorinic acid (NA) to averantin (AVN) in the aflatoxin pathway, and was cloned by

52

genetic complementation of the aflatoxin blocked mutant strain, A. parasiticus B62 (nor-
1, niaD, brn-l), with a cosmid library which was constructed by inserting wild type A.
parasiticus genomic DNA into a vector containing a nitrate reductase gene (niaD) as a
selectable marker (Chang et al., 1992). Another gene, named vet-1 which encodes an
activity associated with the conversion of versicolorin A to sterignatocystin, was cloned
similarly by genetic complementation of another aflatoxin blocked mutant, A. parasiticus
CS 10 (ver-l, pyrG, wh-l), with a cost genomic DNA library and the homologous gene
(pyrG) encoding orotidine monophosphate decarboxylase for the selection of
transformants (Skory e1 al., 1992). Payne et a1. (1993) cloned a gene called 1117-2 from A.
flaws by using an aflatoxin defective strain and a genomic DNA hbrary fiom aflatoxigenic
A. flaws in a cosmid vector containing the pyr-4 gene of Neurosprora crassa.
Complernentation of the mutated afl-2 gene in a double mutant containing 1177-2 and a
lesion resulting in accumulation of norsolorinic acid suggested that the product of the gene
has a regulatory firnction (Payne et a1. , 1993) Another regulatory gene (apa-Z, which is
very similar to 11/7-2) has been cloned fiom A. parasiticus by transformation of strains with
a cosmid containing both nor-1 and ver-l (Chang et al., 1993) The two regulatory genes,
apa-2 and 011-2, are both called aflR Yu er al. (1993) isolated a methyltransferase-
related gene (omt-l) by the use of a polyclonal antibodies raised against a 40-kDa
methyltransferase to screen a cDNA library from wild type aflatoxigenic A. parasiticus
Even though there are no data showing accumulation of a stable intermediate (e. g a
polyketide between acetate and norsolorinic acid), recent molecular data support the

hypothesis that AFBl originates from a polyketide.

53

Figure 1-23. Restriction endonuclease and transcript map of cost Nor-A. Cosmid
Nor-A was cloned from genomic DNA of the wild-type Aspergillus par-1131110113.
Restriction enzymes: E, EcoRI; X, XbaI; H, HindIII; B, BamHI; Genes: pyrG (selective
marker in the cosmid), encoding orotidine monophosphate decarboxylase; pksA, encoding
a polyketide synthase (including acyl carrier protein, B-ketoacyl carrier protein synthase,
acyltransferase, and thioesterase firnctional domains); nor-l, encoding norsolorinic acid
reductase; fa3-2A, encoding a fatty acid synthetase or-subunit (including B-ketoreductase,
enoylreductase, and acyl carrier protein functional domains); fa3-1A(11vm8), encoding a
fatty acid synthetase B-subunit (including enoyl reductase and malonyl/palmityl transferase
functional domains); qflk involved in regulation of AFBl biosynthesis; ver-lA. encoding
an activity associated with the conversion of versicolorin A to sterignatocystin; Amp
(selective marker), encoding B-lactamase; nor-A, encoding a reductase activity; 31cV,
encoding a cytochrome P-450 protein. Symbols: narrow straight line, detected transcript
with unknown firnction; narrow straight line with abbreviation below, deduced functional
domain fi'om DNA sequence; narrow straight line with number below and gene name
above, detected transcript with possible known firnction; Abbreviations: ACP, acyl
carrier protein; KS, B-ketoacyl ACP synthase; KR B-ketoreductase; DH, dehydratase;
ER, enoylreductase; MP, malonyl-palmityl transferase.

54

sameness e. 3 Pizza e. on fleecim

 

 

6.2 can: an... a..: . ea...
and (Hum. I It . I
are. so <75. E». 3.3 5.3 To: <31 use

 

 

m ..m :2
new
in.” find 8:..— efid 6.3. ...—ad.
cacao $56 $55 .236 2.5 oz . «186
in... a..." en... es.. ea... can
.25 3236 moses «2.5 $56
«...an 2:55

Figure 1-23.

55

Disruption of the pics-A gene in A. pamsiticus resulted in a strain which lost both the
ability to produce aflatoxins and the ability to accumulate norsolorinic acid and all other
intermediates in the aflatoxin pathway (Feng and Leonard, 1995; Chang etal, 1995; Trail
et al., 1995). Sequence analysis showed four firnctional domains in this gene, acy carrier
protein, B-ketoacyl carrier protein synthase, acyltransferase, and thioesterase, all of which
are usually present in polyketide synthases and fatty acid synthetases Disruption of a
gene (fas-IA) physically located between nor-l and ver-l in A. parasiticus B62 (nor-l ,
niaD, bmA), which normally accumulates norsolorinic acid, resulted in a new set of
mutants which were unable to accumulate norsolorinic acid and AFBl (Mahanti et
al., 1996). The predicted amino acid sequence of this gene showed a high level of identity
with extensive regions in the enoyl reductase and malonyl/palmityl transferase functional
domains in the B-subunit of yeast fatty acid synthetase suggesting that fas-lA encodes a
novel fatty acid synthetase which synthesizes part of the polyketide backbone of AFB1.
Therefore, fas-lA and pksA are clearly involved in AFBl biosynthesis, function before
nor-1 in the AFBl pathway, and possibly work together at the fatty acid stage and
polyketide stage in the AFBl pathway.

A gene named nor-A was identified when an Aspergillus parasiticus cDNA library was
screened with monoclonal antibody raised against a purified 43-kDa protein demonstrating
norsolorinic acid reductase activity (Cary et al., 1996). Northern blot and Western blot
analyses showed that norA transcript and protein are present only when the firngus was
gown in medium conducive to aflatoxin biosynthesis norA is located between ver-l and

nor-l in the aflatoxin gene cluster, a region which is in part located in a cosrrrid named

Plenty l.v2_....e$ ...2<..n-
..Fxmv-z<n— fl-.-<ZRVC
——.f.— «.5! ar—’,

1? (.....t.

rat J...
—..a.— 31,. 7—9.

hr _ r... 2:21:12 1......—

 

\

1

TI
71:15:...1?) ..u...z f5?
x:v ..cor-.v.r.—:.—:..—. =v=>..

.».——...L::..I.._ ...... ......—
_.....: .—J ...... .....r <

.A— .... .2) v. .>. .ru

56

Nor-A (Figure 1-23 ), isolated fi'om A. parasiticus. The deduced amino acid sequence of
norA had 49% anrino acid identity with an aryl-alcohol dehydrogerrase gene fi'om

Phanerochaete chysosparium. The function of norA in the AFBl pathway is not clear.

Purification Of Enzymes Involved In The Allatoxin Br Pathway
PudfyhgmzymesflmwalyzeaflatordnsymhensfiomAflamorpranfiwsaude
mycdidMactsisndeasybecausethemzymesmpresahhmlafivdylowwnMafiom
and are extremely short lived (Dutton, 1988). Mashaly et a1. (1988) purified an enzyme (70
kDa) showing activity in converting sterignatocystin to aflatoxin B1. Bhatnagar et at
(1988) and Keller etal (1993) have purified two methyltransferases (168 kDa and 40 kDa) to
homogeneity, both enzymes catalyze conversion of sterignatocystin (ST) to O-
methylsterigmatocystin (OMST). A 38 kDa reductase that catalyzes the reduction of NA to
AVN was purified by Bhatnagar and Cleveland (1990); an isozyme (43 kDa) ofthe reductase
also has been purified to homogeneity (Bhatnagar, unpublished observation). Another NA
reductase (140 kDa) was also isolated (Chuturgoon and Dutton, 1991). Lin and Anderson
(1992) purified versiconal cyclase (72 kDa) catalyzing the dehydration of versiconal to
versicolorin B or versicolorin C Studies on protein isolation, purification, and enzymatic
reacfionmggeuflmdifi‘aunmzymesudmmemmecatdyficfimcfionmybeisolaedfiom

pertinent cells (Bhatnagar etal, 1991).

Regulation 01' Allatoxin Biosynthesis
The regulation of aflatoxin biosynthesis has been studied at difl‘erent levels by many

researchers. Many environmental factors can influence aflatoxin biosynthesis. The three

57

most important factors controlling aflatoxin formation in field crops are relative humidity,
moisture, and temperature (Russell, 1979; Wicklow, 1990; Gorman et al., 1992). At the
biochemical level, studies on the regulation of aflatoxin biosynthesis started in the 1970’s.
One of the early important studies was conducted by Shih and Marth (1974). They
investigated the influence of sodium azide on AFBl biosynthesis and found that, at low
concentration, sodium azide selectively inhibits the respiratory system of the mold,
enhances production of AFBl and total lipid, and promotes [ 1-"C]-glucose incorporated
into aflatoxin. Shih and Marth concluded that the decrease in oxidative respiration during
late logarithmic gowth favored an accumulation of acetate (acetyl CoA) and NADPH via
the Embden-Meyerhof pathway. Later, aflatoxin biosynthesis was found to be influenced
by many other biochemical factors, such as the cellular energy status (Buchanan e1 al.,
1987), the intracellular level of cyclic AMP (Khan and Venkitasubramanian, 1986), the
cellular redox state (Bhatnagar et al., 1986), and glycolytic activity (Gupta e1al., 1977).
Aflatoxin biosynthesis was also influenced by the level of glucose (Buchanan and Lewis,
1984), the levels of other carbohydrates (Abdollahi and Buchanan, 1981), and the pH
value of the gowth medium (Cotty, 1988).

In the 1990’s, aflatoxin biosynthesis was found to be regulated at the transcriptional
level (Skory et al., 1993; Trail et al., 1995). A regulatory gene, aflR, for aflatoxin
biosynthesis was characterized (Woloshuk e1_al., 1994). DNA sequence analysis showed
that the aim gene possesses a binuclear zinc finger DNA-binding domain suggesting that
the product of aflR is a regulatory protein (Chang e1 al. , 1995). Overexpression of qflR

relieved nitrate inhibition of aflatoxin biosynthesis (Chang et al., 1995).

58

BIOCONTROL OF AFLATOJGN CONTAMINATION
Possible Function Of Aflatoxins

Aflatoxins may provide a mechanism for a toxigenic firngus to survive in the
environment. Aflatoxins were found to be concentrated in both the conidium and the
sclerotium of aflatoxigenic strains (\Vrcklow and Cole, 1982; Cotty, 1988). The presence
of aflatoxins in sclerotia and conidia may have a long term survival value, because
aflatoxins are toxic to a variety of predators of firngi, especially insects (erletts and
Bullock, 1992).

Aflatoxins may have survival value only for the producing firngus in the field. There is
not a clear cut relationship between sclerotia and aflatoxin production. Some fungal
strains produce aflatoxins but not sclerotia and vice versa (Bennett et al. , 1979).
Aflatoxin production is highly variable within the overall A. flaws goup. Aflatoxin
production can vary by as much as a million-fold among isolates fiom the same species
(Clevstrom and Ljunggen, 1985). A. parasiticus and A. 1101111113 produce both the B-
series and G-series of aflatoxins (Kurtzrnan et al., 1987). A. flaws is observed to produce
only the B-series of aflatoxins (Samson and Frisvad, 1991). The ability to produce
aflatoxin is highly conserved among most wild type strains of A. parasiticus and A. flaws
(Domer et al., 1984). This ability is easily lost in culture (Cotty, 1989). Therefore,

aflatoxin may only have survival value for the aflatoxigenic fungi in the field.

Species Of TheAspagiHus Flaws Group

One primary requirement for studies on the field populations of members of Aspergillus

59

flavlls goup is the ability to classify species ofthe A. flaws goup. There are many
taxonomic schemes used to classify species in the A. flavus goup (Klich and Pitt, 1988;
Samson and Frisvad, 1991). The standard for classifying a species proposed by Papa
(1986) may be the best one. He proposed that a species represents a collection of strains
which behave as clonal organisms with the exception of occasional parasexuality between
members of the same vegetative compatibility goup (V CG). Parasexuality can be
observed in the laboratory only in the same VCG. According to King and Stransfield
(1985), clonal organisms are a goup of identical organisms descended from a single
common ancestor by mitosis. How to determine if a goup of organisms is originated
fi'om a single common ancestor? One standard is based on DNA polymorphism.

Analyzing DNA polymorphism, Bayman and Cotty (1993) proposed that the A. flaws
goup consists of four species which are Aspergillus flaws, A. parasitic-113, A. 1101111113, and
A. tamarii. Among the four, no aflatoxigenic isolates have been obtained fi'om A. tamarii.
Within each of the three aflatoxin producing species, there is geat variability. Bayman
and Cotty (1991) further classified each species into Vegetative Compatibility Groups
(V CGs). Each species is composed of several Vegetative Compatibility Groups (V CGs)
(Papa, 1986). Physiological and morphological traits such as enzyme production, plant
virulence and sclerotial morphology, are much more consistent within a VCG (Bayman
and Cotty, 1993).

By using this classification scheme as a tool, scientists now know the vast diversity of
strains within the Aspergillus flavus goup (Papa, 1986; Bayman and Cotty, 1991), the

vast diversity in ecological niches (Brown et al., 1991), the vast diversity of difl‘erent

5.0

regional population structures (Bayman and Cotty, 1991), and the vast diversity of
changing populations caused by the agricultural practices (Shearer e1 01. , 1992). The
distributions of the A. flaws goup fungi are very complex. Almost all species from the A.
flaws goup are better adapted to a soil environment, but certain species are better
adapted to niches above the soil. For example, A. parasiticus occurs more fiequently on
peanuts than on corn (Hill et al., 1985). Sometimes all species within the goup may

occur on the same crop or in the same field (Cotty, 1992b).

Biocontrol Of Aflatoxin Contamination
Eventhough aflatoxirrsareproduced onlybycertainspecies ofAmergiIIusflavus, A.
paran’ticus, andA. 1101111113, these firngi are ubiquitous and fiequently gow on a variety of food
and feed crops and cause aflatoxin contamination in many areas in the world (Blourrt, 1961;
Ellis etal, 1991; Jelinek et al, 1989).
AflmordnwmmmmonisdifiimhtodimefiommefoodchainwithomWfomsof
treatment. These compounds are resistant to normal food processing such as millingand
cooking, and some of their bio-metabolites are also very toxic and mutagenic such as AFMl in
cow milk. Therefore aflatoxin contamination causes serious Nth problems, and in turn
resultsineconomicproblerns. Therehavebeenseveralstrategiescurrentlyinuscorproposed
for reducing or eliminating aflatoxin fiom contaminated food and feed such as
screening/detection, removal/decontamination, or altered AFBl metabolism/DNA adduct
formation (Clru, 1991; Ellis et al., 1991; Park and Liang, 1993). On the other hand, strategies

formdudng/dinfinafingaflflordnmnmnfinafionattheprehmvestlevdmybebata

61

approaches. These strategies include improving agononric practices (such as irrigation and
fimgicide application) and classical breeding progarns (such as selecting aflatoxin-resistant
strains of crops). Thwe strategies of aflatoxin decontamination and contamination prevention
arestillveryusefirlandarewidelyused. Withthedevelopment ofmodernmolecular
techniques, studies on the mechanism ofaflatoxin biosynthesis at the molecular level has been
an active fours area. Such studies not only can provide information for improving agonomic
practices and breeding progams, for developing safe and specific ‘aspergicides’, for
developing genetically engineered crops, but also can provide new biocontrol agents by
knocking out genes involved specifically in aflatoxin biosynthesis to generate genetically stable
nontordgeuicstrainsofAspergillus. Thesestrainsirrtheorymaybesafelyutilizedintlrefieldto
mducemdhfinfleaflatordnwmmmmonoffoodorfeedaopsbybiologcalmrdusion
(biocontrol). Therefore the following brief review will focus on the possrbility of developing

such biocontrol agents.

, The idea of using biocontrol agents to control aflatoxin contamination is that
nonaflatoxigenic strains or mutants of the A. flaws goup can compete with the toxigenic
strains in the field for gowing space and nutrition, and/or by the production of interfering

compounds to reduce or eliminate aflatoxin contamination.

It is possible to obtain nonaflatoxigenic strains for use as biocontrol agents. Aflatoxin
production is highly variable (up to a million-fold) within the overall A. flaws goup.
Many strains cannot produce detectable aflatoxins (Clevstrom and Ljunggen, 1985).
Over the centuries, industrial strain selection has reduced fungal toxicity and increased

fungal traits associated with both product quality and emcient fermentation in products

62

using Amergilli. Therefore, it is also possible to identify nonaflatoxigenic isotates in the
field. Alternatively, nonaflatoxigenic strains can be created by the use of molecular
techniques to genetically disrupt specific genes only in aflatoxin biosynthesis. However,
not all nonaflatoxigenic strains are capable of reducing aflatoxin contamination when
inoculated in the field (Cotty, 1992a). Following selection, the co-inoculation of both the
producing and nonproducing strains should be tested both in the geenhouse and in the
field.

It is safe to use nonaflatoxigenic strains as biocontrol agents. The inoculation of
nonaflatoxigenic strains or mutants in the field may cause some concern about the danger
of those potential pathogens to humans. Indeed, in many areas people respire high
concentrations ofAspergiII113 spores but without noticeable disease. For example, in the
koji, baking, and brewing industries workers over several generations have been exposed
to very high concentrations of spores throughout their working years with a very low
incidence of disease (Barbesgaard et al. , 1992). It is important to note that inoculation of
nonaflatoxigenic strains in the field can result in A. flaws population with altered
composition, but without increased population size (Cotty, 1992c). Indeed, inoculation of
nonaflatoxigenic strains in the field may provide the opportunity to improve the overall
safety of fungal populations by reducing human exposure to aflatoxin through both dietary
and respiratory routes. Aflatoxins can cause several physiological efl‘ects on the host
crops and on domestic animals (Mclean e1 01., 1992; Robens and Richard, 1992).
Reducing the aflatoxigenic population of fungi in the field can also reduce the

physiological or toxic efi‘ects on aflatoxins of crops and domestic animals.

63

Normal agricultural practices generate a very large quantity of organic materials
contaminated with A. flaws goup fungi in the field. Because those materials consist of
crop remnants, corn cobs, gin trash, inoculation of the A. flaws goup filngi occurs
naturally without our notice. The main difference between natural inoculation and
artificial inoculation is that in the latter only nonaflatoxigenic strains are inoculated.

Inoculation of nonaflatoxigenic strains in the field generally will not increase the
infection level of crops Crop infection by the A. flavus goup ofien happens when crops
are wounded or under the stress of nutrition limitation. Aflatoxins are not required for the
producing fungi to infect the host crops (Cotty, 1989). Infection is more heavily
dependent on host predisposition and the environment. than on the number of A. flaws.
Therefore nonaflatoxigenic strains can compete with the aflatoxigenic strains for areas
which are infection-predisposed in the crops. In three-year tests on cotton, inoculation of
nonaflatoxigenic strains in the field has not resulted in an increase of infection rates (Cotty,
1992c).

It is efi‘ective to use nonaflatoxigenic strains as biocontrol agents. The advantage of
inoculation of nonaflatoxigenic strains of A. flavus over other types of microbial
biocontrol agents is that nonaflatoxigenic strains are adapted to similar environmental
conditions as aflatoxigenic strains. Other potential agents, such as bacteria, may be
inactive under the hot, dry conditions associated with aflatoxin contamination (Brown e1
al., 1992). The A. flaws goup fungi are associated with crops in the field during crop

development and remain associated with the crop during harvest, storage and processing.

64

Inoculation of nonaflatoxigenic strains in the field both before and after harvest has been
shown to provide protection fiom aflatoxin contamination of corn (Brown et aI. , 1991).
Nonaflatoxigenic strains or mutants of the A. flaws goup have been proven to reduce
aflatoxin production and crop contamination by competition for gowing space and
nutrition, and/or by the production of interfering compounds (Cotty, 1990; Shantha e101. ,

l 990).

Perspectives On The Use Of Fungal Biocontrol Agents

Based on the above discussion, the possibility of using nontoxigenic firngi as biocontrol
agents to reduce or eliminate aflatoxin contamination is promising. However there are still
some questions which need to be answered. One is the biology of a firngal population in
the field and the other is the firnction of aflatoxin to the producing organism.

Because of the tremendous reproductive and dispersal abilities of the A. flaws goup
and the influence of different cr0ps and difi‘erent soils on the population structure of the
firngus, scientists still do not know the change in population structure over time, or the
mechanism of natural selection in the firngal population. Therefore studies on one goup
of firngi have often turned out to have limited application to other goups (Cotty and
Bayman, 1993 ).

Even though there is much research left to be done, the preliminary results of using the
nonaflatoxigenic strains to control the aflatoxigenic strains are very positive. For example,
in geenhouse and field experiments, in which cotton bolls and corn ears were inoculated

with combinations of different strains of the A. flavus goup, nonaflatoxigenic strains

65

reduced aflatoxin contamination by 80 to 90% (Cotty, 1990; Brown et al., 1991). Domer
e1 01. (1992) demonstrated that nonaflatoxigenic A. parasiticus strains reduced aflatoxin
contamination when concentrated mycelia/spore suspensions were applied to developing
peanuts. Those procedures are both reliable and economic, and are active in the field
under hot, dry conditions that are near optimal for the aflatoxin production by the A.
flaws group.

As discussed above, it is possible to identify nontoxigenic isolates in the field as
biocontrol agents. Alternatively, nontoxigenic biocontrol agents (strains) can be created
by the use of molecular techniques to genetically disrupt specific genes (gene-knock-out
strategy) involved in aflatoxin biosynthesis. The gene-knock-out biocontrol agents may
share the same niche with toxigenic strains in the ecosystem, and therefore, may be more
effective in the field than the natural nontoxigenic isolates.

An understanding of the regulation of aflatoxin biosynthesis and the general biology of
the toxigenic fungus (e.g. regulatory mechanisms involved in hyphal gowth, hyphal
difl‘erentiation, and sponrlation) may also lead to the development of new fungicides, the
improvement of agononric practices, the development of resistant crops (by genetic
engineering or classical breeding), and the development of biocontrol agents (such as
nontoxigenic competitive fungi) for control of aflatoxin contamination.

In the following chapters, the enzymatic filnction of the Nor-l protein, the pattern of
Nor-1 protein accumulation in A. parasiticus gown in liquid media and on solid media,
the localization of Nor-l protein accumulation and the localization of nor-1 promoter

activity during aflatoxin biosynthesis, and the firnction of the flap gene which is associated

66

with hyphal development and the process of sporulation and indirectly influences the

accumulation of aflatoxin in A. parasiticus are presented.

CHAPTERZ

ENZYMATIC FUNCTION OF THE NOR-l PROTEIN INVOLVED IN
AFLATOXIN Br BIOSYNTHESIS IN ASPERGEL US PARASHYCUS

ABSTRACT: To study the function of the nor-l gene, which is directly involved in
aflatoxin B1 (AFBl) biosynthesis Aspergillus pw'asiticus, a nor-1 cDNA was expressed in
Weinbcoli. TheresultingNor—lcproteinwasusedinanenzymeactivity assaytotestthe
function ofthe nor-1 gene in the AFB! pathway. The results confirmed that the Nor-1c protein
is a ketoreductase which converts the AFBl pathway intermediate norsolorinic acid (NA) to
averantin (AVN) in the presence of NADPH and a supernatant fiaction (105,000 x g) obtained
fiom E. coli DHSor. The results suggest that an unidentified cofactor(s) may be necessary for
the function of the native Nor-l protein and that the nor-l gene is involved in only one of
multiple proposed pathways for the conversion of NA to averufin (AVF) in the AFB: pathway

in A. parasiziaw.

INTRODUCTION
The anthraquinones norsolorinic acid (NA) (CmHlsoy), averantin (AVN) (ConzoO'l),
and averufin (AVF) (CmeO-I) (Figure 2-1) are each produced by blocked mutant strains

of the wild-type A. parasiticus, and were demonstrated to be intermediates in the aflatoxin

67

68

Br (AFBl) pathway by feeding experiments (Lee et al., 1971; Birkinshaw et al., 1966;
Donkersloot et al., 1972; Dutton, 1988). NA is converted to AVF via one or more proposed
alternative pathways by converting NA to either AVN or to averufanin (AVNN) initially
(Frgm'e 2—1) (Bennett and Christensen, 1983; Button, 1988; McCormick et al., 1987; Yabe er
al., 1991b).

The nor-l gene is physically located in the AFB] pathway gene cluster (Figure 1-23) in the
genome of A. parasiticus suggesting that nor-l is involved in AFBl biosynthesis. Many genes
physically arranged in a cluster in the genome of fungi are found to be involved in the
same biosynthetic pathway (Timberlake and Barnard, 1981; Feitelson er a1. , 1985; Chater,
1992; Kimura and Tsuge, 1993; Hohn et al., 1993; Rainey et al., 1993; Ueda et al., 1993;
Keller and Adams, 1995) for eficient expression (F eitelson et al., 1985). Disruption of the
nor-1 gene in a toxigenic strain of A. parasiticus (SU-l) resulted in significant NA
accmrmlation and significant reduction of aflatoxin accumulation (Trail et al., 1994) suggesting
that nor-l is directly involved in AFBl biosynthesis inA. parasitic-tar.

Analysis of the proposed amino acid sequence of nor-l provided evidence that this gene
encodes a ketoreductase (Trail et al., 1994). Comparison of the chemical structure of NA and
AVN (Figure 2-1) suggests that the 5’-keto group of NA can be converted to the S’-hydroxyl
group of AVN by a ketoreductase. Therefore it is reasonable to hypothesize that the Nor-l
protein encoded by nor-l can convert NA to AVN in the AFB: pathway and that the nor-1
gene is involved in one branch of proposed alternative patlmays for conversion of NA to AVF.

To prove this hypothesis requires isolation of the native Nor-l protein.

69

 

 

 

 

O
5'-Hydroxyaverantin (HAVN)

v v v
H H \ H H
H3 __
Ho H Ho CH’
0 o

Averufanin (AVNN) Averufin (AVF)

 

 

Figure 2-1. Proposed multiple alternative pathways which convert NA to AVF (Bennett
and Christensen, 1983; Button, 1988; McCormick er al., 1987; and Yabe etal, 1991).

70

Several NA reductase (38, 140, and 43 kDa) activities have been purified to homogeneity
fiom toxigenicA. parasiticus (Bhatnagar and Cleveland, 1990; Chuturgoon and Dutton, 1991;
Bhatnagar, unpublished data) which can convert NA to AVN. But these three NA reductases
donothavethesamemolecularmassastheNor-l protein(3l kDadeduced fromtheDNA
sequence of nor-1). These data suggest that: (l) the Nor-l protein is extremely unstable; (2)
the fungal cell contains ememdy low levels of the Nor-1 protein; and/or (3) the Nor-l protein
needs a cofactor(s) (in addition to NADPH) to perform the reductase activity and the
cofactor(s)waslostduringpurification. Thesedataalsosuggestthatanaltemativestrategyis
required to determine the fimction of the Nor-1 protein. A nor-l cDNA cloning and
expression strategy was chosen in this study.

Thegoal ofthisstudywasto determinetheenzymaticfimction oftheNor-lcprotein. The
Nor-1c protein was produced in a host bacterium transformed with a nor-l cDNA expression
vector. The enzymatic function ofthe Nor-1c protein was tested to analyze the fimction ofthe
nor-1 gene in the AFB: pathway.

The results confirmed that the Nor-1c protein is a ketoreductase which converts NA to
AVN in the presence ofNADPH and a supernatant fiaction (105,000 x g) obtained fiom
Escherichia coli DHSa. The data also suggest that an unidentified cofactor(s) may be
necessary for the native Nor-l protein fiinction and that the nor-1 gene is involved in only one

of multiple proposed pathways of NA conversion to AVF.

71

MATERIALS AND METHODS

Bacterial Strain, lfimgal strains, and Culture Conditions. Plasmids were amplified in
EWI'I'Chid coli DHSa F' °[PendA1 hem 7(rx" mid) upE44 thi-I recAI gyrA (Nal')
relA IA(IacZYA-argF)mgg (m801acZAM15)] using standard methods (Ausubel et at, 1987).
Amergillus parasiticus NRRL 5862 (ATCC 56775, SU-l) (American Type Culture
Collections) was used as the aflatoxin-producing wild-type strain. AWIIus parasiticus
ATCC 56774, which accumulates averantin, was used as a source of averantin. A nor-l
disrupted strain of A.‘ parasiticus (Trail er al., 1994) (designated ANor-l) was used for NA
preparation. An aflatoxin—inducing liquid growth medium consisting of 2% (w/v) yeast extract
and 6% (w/v) sucrose (pH 5.8) (YES broth) was used to grow mycelia at 29°C in the dark
with constant shaking (150 rpm) for protein preparation and aflatoxin extraction. YES solid
medium [YES broth plus 1.5% (w/v) Bacto-agar] was used for spore preparation. Fungal
strains were maintained as fiozen spore stock suspensions at -80°C. Spore stock wspensions
were prepared by suspending spores in 20% (v/v) glycerol.

Nucleotide Sequence Analysis. A nor-1 cDNA (with an EcoRI adapter at the 5'-end and
an Mrol adapter at the 3'-end) was cloned into the EcoRI/Mrol sites of pBluescriptSKII'
(Stratagene Cloning Systems, La Jolla, CA) resulting in pNOR which was kindly provided by
Dr. Perng-Kuang Chang (USDA. ARS. SRRC). Nucleotide sequence analysis of the nor-l
cDNAwasperfonnedbythedideoxychaintemfinafionmethodwithSequenaseH
(Biochemical Corp., Cleveland, Ohio) as described in the manufacturer's instructions. The

clonedinsertwassequencedonbothstrandswiththefiandTSprimers

72

  

fl

ColEl origin

  
  

pMNl
, 7731hps ' maIE

  
   
  
   
 

I malE-nor-l cDNA
fusion

anI

nor-l cDNA

Figure 2-2. Construction of expression vector pMN 1. An EcoRl/Sall fi‘agment (1040 bp)
containing a nor-l cDNA was cloned into expression vector pMAL-c2. The nor-l cDNA
in the resulting expression vector pMNl was fiised with the MIPS gene encoding maltose
biding protein (MBP) in the same reading flame to form a maIE/nor-l-cDNA firsion
which was expressed as a Nor-l c/MBP fiision protein.

73

supplied in the kit. In order to resolve the ambiguities in the initial sequence data, one
internal l7-nucleotide primer (5’GTA'I'I‘TGGTCACCGGGG3’) was also used for
sequwcing.

Construction of pMNl, a pMAL—cz-nor-l-cDNA Expression Vector. The nor-l
cDNA fi'agment was removed from pNOR using the restriction enzymes BamHI and mi and
cloned into the BamI-II/Kanl sites of the expression vector pQE31 (QIAexpress, QIAGEN Inc.
Chatsworth, CA). The nor-l cDNA was removed again fi'om pQE31 with the restriction
enzymes EcoRI and SalI and cloned into the EcoRI/Sall sites of the expression vector pMAL-
c2 (New England Biolabs Inc., Beverly, MA). The resulting vector was cut with EcoRI, and
the protruding ends were filled with dN'I'Ps by the Klenow fiagment of -E. coli DNA
polymerase I to create blunt ends. Blunt-end ligation created a new expression vector called
pMNl (Figure 2-2) in which themr-l cDNAwas fused in framewiththe 3’-end ofthe maIE
gene (encoding the maltose-binding protein) in pMAL-c2.

Preparation of the Nor-lc/MBP Fusion Protein and the Nor-1c Protein. The
expression vector pMNl was transformed into E. coli DHSa using standard methods (Ausubel
et at, 1987). The Nor-lclMBP firsion protein and the Nor-1c protein were prepared following
the instructions of the Protein Fusion & Pmification System (New England Biolabs Inc.
Beverly, MA). The resulting proteins were concentrated to approximately 1 mg/ml in an
Amicon Centriprep concentrator (Amicon Inc., Beverly, MA). Cleavage of the fiision protein
with protease factor Xa and separation of the Nor-1c protein fiom MBP by afinity
chromatography were carried out according to the manufacturer’s instructions (New England

Biolabs Inc., Beverly, MA).

74

. Fungal Culture and Crude Protein Extract Preparation. For culture of the
fungus in liquid media, a stock spore suspension was inoculated into liquid media with a
final spore concentration of 1.5x105 spores/ml, and gown at 29°C in the dark with
constant shaking (150 rpm). Mycelia were collected by filtration, and were either used
immediately, or flow under liquid nitrogen and stored at -80°C until use. The dry weight
of mycelia gowing in liquid media was determined after drying the mycelium in a
preweighed flask overnight at 80°C. To generate a crude cell extract, mycelia were
gound under liquid nitrogen with a mortar and pestle to produce a fine powder. The
flow mycelial powder was quickly transferred to a 50-ml centrifuge tube containing
precooled extraction bufi‘er (PEB; a volume approximately 5 times the mass of cell
material) consisting of 0.01 M Tris-HCl (pH 8.0), 0.14 M NaCl, and 0.025% (w/v) NaNs.
Just before mixing with mycelia, the following chemicals were added to PEB: 20 mM
phenylrnethylsulfonyl fluoride (PMSF), 0.4 TIU (trypsin inhibition unit)/ml aprotinin, 10
mM iodoacetarnide, 4 mM 2-mercaptoethanol, 10 mM EDTA, and 5% (v/v) glycerol.
Cell debris was removed by centrifugation two times at 1,000 x g for 10 min. The
resulting supernatant fiaction was further centrifuged at 10,000 x g for 1 h to generate a
crude extract (10,000 x g supernatant). For a protein concentration assay, sodium
dodecyl sulfate (SDS) was added to the crude extract [to a 2% (w/v) final concentration]
. and was boiled at 100°C for 5 min. The protein concentration was then determined with
the Bio-Rad Protein Assay reagent (Bio-Rad Lab., Hercules, CA) according to the

manufacturer’s instructions.

75

Immunoafl'mity Purification of the Native Nor-l Protein. Afinity column preparation
using CNBr-activated Sepharose 4B (Sigma, St. Louis, MO) and polyclonal antibody (PAb)
raised against the Nor-ldMBP fusion protein (Chapter 3. Preparation and Purification of
Polyclonal Antibodies) was carried out according to published standard methods (Ausubel et
al., 1995c). The basic procedure for aflinity purification of the Nor-1 protein was established
by Dr. R. Mehigh in our laboratory. Briefly, an afinity coltunn (1.2 x 5.5 cm) containing 4.5
mlSepharose-4Bresinconjugatedwith l mgPAbwasequilibratedwithhighsaltphosplmte-
bufl‘ered saline (HS-PBS) consisting of 390 rnM NaCl, 10 mM NazI-IPO4, and 10 rnM
NaI-IzPO4 (pH 7.2). Twenty mg of crude extract (5 mg/ml) fi'om A. parasitic” SU-l or
ANor-l (nor-l disrupted strain) cultured in YES liquid medium for 60 h was loaded onto the
column. ThecohnnnwaswashedwithHS—PBSunfiltheODnat-mretumedtothebaseline
ThenativeNor-l proteinwaselutedwithO.1 MglycineHCl (pH2.5)andcollectedwitha
fiaction collector. Bach fiction (2 ml) was neutralized immediately with 0.3 ml 1 M Tris-HCl
(pH 8.0). Fractions were pooled and dialyzed against three changes of low salt phosphate-
bufi‘ered saline (LS-PBS) consisting of 130 mM NaCl, 10 rnM NW, and 10 mM
NaHzP04(pH7.2)at4°Covera36hperiod.

Western Blot Analysis. Proteins were resolved by SDS«PAGE (polyacrylamide gel
electrophoresis) and blotted to a polyvinylidene difluoride (PVDF) membrane (Du Porrt Co.,
Boston, MA) using published standard methods (Ausubel et al., 1995b). Irrmmnodetection
wascarriedoerithachemiluminescerrtdetectionldt(Schleicher& Schuell, Keene,NH)

according to the manufacturer‘s instructions.

76

Nomlorinic Acid (NA) Preparation. Conidia (about 1x10° ) from a nor-1 disrupted
snairLpr'asificusANor-l, wereinoculatedintoZLonES liquidmedimnandculturedfor
2daysat29°Cwithconstantshaldng(150rpm)inthedark. Theculturewasthenincubated
under stationary conditions at 30°C in the dark for an additional 13-day period of time. The
mycelia (red color) were collected by filtration and a portion (250 g wet weight) was extracted
with300mlacetonethreetimes. ‘I‘heredpurpleextractsohrtionwasdriedwithaRotovapor
R110 (Brinlonarmlnstrumentslnc. Westbury,NY) at 65°C. Theresulting solid materialswere
extracted with 100 ml chloroform. The chloroform extract was concentrated with a Rotovapor
R110at72°C(5-6mlfinalvolumelefi)andextractedagainwith100mlacetone. TheNAin
theacaone-amwmnplered)wasfiuflrapmifiedbynuunngprepmafivethinhya
clnomatogaphyCI'LCMPIG’ SILICAGEL60A, 20x20cm,WhatmanInc.Clifton,NJ)with
chlorofomr-acetone (9:1) as the developing system. The NA spot (brown color) on the TLC
platewasscraped ofi‘witharazorbladeandtheNAwasextractedwithchloroform.

Averantin (AVN) Preparation Averantin was prepared following the method developed
by Bennett et al. (1980) with modifications. A freeze-dried hyphal culture of A. parasitic-us
ATCC 56774, which accumulates averantin, was reconstituted according to ATCC's
instructions. Corridia (about 1x10”) ofstrain ATCC 56774 were inoculated into 2 L of YES
liquidmediurn,andculhuedwithconstarfishaldng(150rpm)for2daysat29°Chrthedark
followed by incubation without shaking for an additional l3-day period of time at 29°C in the
dark. Approximately 210 g (wet weight) of orange mycelia were collected by filtration and
extracted with acetone until colorless. Water was added to make a 30% (v/v) acetone

solution. The acetone solutionwaswashed with hexane (1:1 ratio), and the orange pignents

77

were then partitioned fiom the acetone into chloroform (l :1 ratio). The chloroform extract
was concentrated to 5 ml at room temperature, and then further purified by preparative TLC
(HPK SILICA GEL 60 A 20 x 20 cm, Whatrnam Inc. Clifton, NJ) with chloroform-acetone
(9: 1) as the developing system. The AVN spot (yellow-brown color) on the TLC plate was
scrapedofi‘witharazorbladeandtheAVNwasextractedwithchlorofonn.

Enzyme Activity Assay of the Nor-lc/MBP Fusion Protein and the Nor-1c Protein.
Theenzymaticassaywas conducted accordingto apublished method (Yabeetal,1991b)with
modifications. The reaction mixture included an appropriate quantity of protein (70 to 105
pg), 90 M NA, 0.23 rnM NADPH, 90 rnM KHzP04 (pH 7.5), and 10% (v/v) glycerol in a
totalvohrmeoflOOuL Reactionswereconductedat37°C inthedarkforappropriateperiods
oftime(30to90min),andthenstoppedbyadding900ulethylacetate. Theethylacetate
extract was air dried and the residue was extracted by adding 400 ul chloroform. The reaction
products in chloroform extract were resolved by TLC (HPK SILICA GEL 60 A, 10 x 10 cm,
Whatmam Inc. Clifton, NJ) with benzene-ethyl acetate (7:3) as the developing system.

UV/VIS, Mass, and NMR Spectroscopy of NA and AVN. UV/VIS spectroscopy was
conducted using a UV/Visible light spectrophotometer (CARY 3E, Varian, Australia Pty. Ltd.,
Australia). Mass spectrometry was carried out on a double-focusing mass spectrometer (IEOL
AX-SOSH, Varian, Australia Pty. Ltd., Australia) with the help of Dr. B. Charnberlin
(Department of Biochemistry, Michigan State University, East Lansing MI) under the
following conditions: electron energy, 70 eV; scan range, m/z 45-600. The 1H proton nuclear
magnetic resonance (NMR) spectroscopy was performed with a NMR spectrometer (VX12-
500, Varian, Australia Pty. Ltd., Australia) with the help of Dr. K. Johnson (Department of

78

Chemistry, Michigan State University, East Lansing, MI) under the following conditions:
proton probe at 80 mHz; range, 0 to 12 ppm; solvent, deuterated dimethyl sulfoxide
(d6DMSO).

AflatoxinAnalysisbyTLCand ELISA. Aflatoxinswereextractedwith lOrnlof
chloroform fi'om mycelial liquid culture medium (not including mycelium) (1 ml) or from
mycelial solid culture medium (including mycelium) (1 mg). Chloroform extracts were tested
either semiquantitatively by TLC (for total aflatoxins) with chloroforrn—acetone (95:5) as the
developing system, or quantitatively by enzyme-linked irnrnunosorbent assay (ELISA) (for
AFBr). The direct competitive ELISA was carried out according to the method ofPestka et
at (1980) using anti-AFBr antibodies kindly provided by Dr. J. Pestka (Department of Food

Science and Human Nutrition, Michigan State University, East Lansing, MI).

RESULTS

Sequence Analysis of the nor-1 cDNA. Nucleotide sequence analysis (Figure 2-3)
showed that the nor-1 cDNA was missing 6 nucleotides (ATGAAC) fiom the protein-coding
region at 5'-end of the proposed transcript beginning with the translation initiation codon
(ATG) based on a comparison with the nor-1 genomic DNA sequence (T rail et al., 1994). It
msthoughtthatthisshort deletionrnightnothaveasignificantefl‘ectontheNor—lcprotein
activity. Oneofthethreereadingfi'amesofthemr-l cDNAencoded acomplete openreading
fiameand had three in-fi'ame stop codons at the 3'-end which in theory could geneate proteins
withpredicted molecularrnassof31 kDa, 34 kDa, or38 kDaifreadthrough oftheprimary

translation termination codon occurred.

79

Factor Xa cutting sit: ( nor-1 cDNA 5'... ATG AAC)

l
DNA 5':de....§ FAQ fig AQG ATT TOA GAA TTA ATTOGG OAO GAGGGATOA CTI' AGO CAG CAO GATCM
N-AA 001 E G R l S E L I R H E G S L S O H D Q

DNAS': GAGAGGCTCTOTAOGCOATAOCGGGATGGACOGOOTGAGGAGAOGGTGTAT'ITGGTOAOC
MMERLSTPYRDGPPEFTVYLVT

 

N—AA: 041— 230

DNAS': TGG TAG GAO GOT AGT GAO GACGMGOG OAG TOA TATGTT TOT GAG ACGAGCAGA MOGTG
N-AA2281WS1DASDDEAQSYVSETSRNV

DNAS’: OTG GOG TGG AGO TGG TOO AGA MG GOG COG TGA GGT COT GGG TTC GTG TOG COG COG CTT
N—MI301LAWSWSRKARSZGPGFVSRRL

DNAS': TOG OTC CAT CAG TTO GTG TAT GAO CTT TIC GGT OTT TTC OGT TCO GTC TGT 'l'l'A GGO TOT
NM321$LDQFVYDLFGLFRSVCLGS

DNAS': OACMGATAMACCAMTTGAMCOTAAOGTI'OGTTTTOAMAMMAAMMAMAAAC
3411-! K I K P N #3 N L T F V F K K K K K K T

DNAS‘: TOG A66 666 GGC COG GTA COO CGG GTC DC T A
N-AA: 318 R G G P V P R

Figure 2-3. Nucleotide and predicted amino acid sequence of the 5’ and 3’-ends of nor-1
cDNA in expression vector pMNl. The underlined nucleotides represent the carboxyl
terminus of the maltose binding protein encoded by maIE (in the expression vector pMAL-c2)
which is fused in-fi'ame with the nor-1 cDNA. The nucleotides in italics were introduced
during vector construction. The nucleotides in bold represent the nor-1 cDNA. The
nucleotides in parentheses (ATGAAC) were missing fiom the protein-coding region at 5'-end
of the proposed transcript beginning with the translation initiation codon (ATG) based on a
comparison with the nor-1 genomic DNA sequence. The factor Xa cutting site is indicated by
an arrow. The two Nor-1c proteins (starting fiom the factor Xa cutting site, 32 and 36 kDa)
are predicted to be derived fiom translation termination at the first two stop codons (marked
#1 at residue 282, and #2 at residue 311), respectively. The two Nor-1c proteins are therefore
predictedtolacktwoarnino acids(M, andT)which are replacedby8 amino acid(I, S, E, L, 1,
IL H, and E) at the amino terminus. N-AA indicates the N-terrninus of the amino acid
sequence.

80

97*
m a“ «78‘
_ to .....—
66‘ ~ 9* 74
., ...,
45-- Q. """’ EL? .- «42
...... "-3
r..- *36
31—b - ‘c
22—. -

Figure 2-4. SDS-PAGE detection of the Nor-IcIMBP firsion protein and the Nor-1c
protein. Expression vector pMN 1 containing the nor-l cDNA fused with maIE encoding
maltose binding protein (MBP) was transformed into Escherichia coli DHSor. Under
iSOphenylthiogalactoside (IPTG) induction, two Nor-lclMBP fusion proteins were
produced in the bacterial host. Lanes: 1, molecular mass standard (kDa); 2, total crude
extract (10,000 x g supernatant) from E. coli DHSor; 3, total crude extract from E. coli
DHSor transformed with expression vector pMNl; 4, two Nor-lc/MBP fusion proteins
(74 kDa, and 78 kDa) purified by affinity chromatogaphy; and 5, two Nor-1c proteins (32
kDa, and 36 kDa) and MBP (42 kDa) obtained by factor Xa cutting of the two Nor-

1c/MBP fusion proteins.

81

¢- Nor-1

 

Figure 2-5. Western blot analysis of the native Nor-l protein in A. parasiticus SU- 1. Each
lane contained 10 pg protein. The primary antibody was the IgG fraction of the antiserum
raised against the Nor-ldMBP fusion protein (Chapter 3) (10 ug/ml). Lanes: 1, total crude
extract (10,000 x g supernatant) fiom the nor-l-disrupted strain A. pw'asiticus ANor-l
cultured in YES liquid medium for 60 h; 2 and 3, total crude extract fiom the wild-type A.
parasiticus SU—l cultured in YES liquid medium for 48 h and 60 h, respectively; 4, the native
Nor-1 protein (31 kDa) purified with anti-Nor-lc/MBP firsion protein PAb aflinity column
from the total crude extract of A. parasiticus SU-l cultured in YES medium for 60 h.

82

. Preparation of the Nor-ldMBP Fusion Protein and the Nor-1c Protein. The
express'onvectorpMNl wastransformedintoE coliDHSor. Afier2hinductionwith
isophenylthiogalactoside (IPT G), two Nor-ldMBP fusion proteins (approximately 74, and 78
kDa)wereproducedinthehostcdlandwaedetectedbySDS-PAGE(Figne24). The
firsimpmtdnswaepnifiedbynnhoserednammychmmatogaphyandanwhhpmtease
factorXamrltinghrtheappearanceofthemaltosebhrdingproteinMZ kDa)andtwoNor-1c
proteins(approximately32kDa, and36kDa)(Figure2-4).

Immunoaflinity Purification of the Native Nor-l Protein. Twenty mg of crude extract
obtained fi‘om the aflatoxin-producing strainA. pen-asia’als SU-l, or fi'om the nor-l disrupted
strainpr'asiticw; ANor-l, was used to purify the native Nor-1 protein by imrnunoaflinity
chromatogaphy. The concentrated Nor-1 protein, resolved by SDS-PAGE, was detected by
Westernblotanalysis. Onlyonemajorproteinbandcorrespondingtothe sizepredictedforthe
Nor-l protein was detected by the IgG fiaction of the anti-Nor-lc/MBP firsion protein serum
(Chapter 3) in the wild-typeA. parasiticus SU-l (Figure 2-5, lane 4). This protein band was
notdetectedusingthe sameantibodiesinthenor-l disrupted strainprusiticusANor-l
(Figure 2-5, lane 1). Using the same antibodies, in addition to the expected Nor-1 protein,
severalotherproteinsweredetected inthecr'udeextract ofA. pm'asr'tr'cus SU-l (Figure 2-5,
lanes 2, 3) suggesting that cross-reactivity occurred.

Enzyme Activity of the Nor-ldMBP Fusion Protein and the Nor-1c Protein. The
imnnmoafinity purified Nor-ldMBP fusion protein, the Nor-ICIMBP fusion protein in the
crude extract (10,000 x g supernatant) ofE coli cells containing expression vector pMNl, and

thehmumaflimtypmifiedNor-lcpmtdnwereassayedfortheuabflhymwnvatNAm

83

Figure 2-6. Enzyme activity assay of the Nor-lc/MBP fusion protein and the Nor-1c protein.
All reactions were conducted at 37°C in the dark. The reaction products were resolved by
TLC with benzene-ethyl acetate (7:3) as the developing system. The photo of reactions 1
through 9 was taken under white light. The photo of reactions 10 through 18 was taken
under UV light. Reactions: 1, NA standard; 2, AVN standard; 3, NADPH plus NA
control. *, crude extract (10,000 x g supernatant) of E. coli DHSot; v, pellet (105,000 x
g) of crude extract from E. coli DHSor; 4, supernatant (105,000 x g) of crude extract
fiom E. coli DHSa; o, 35 ug anti-Nor-lc/MBP firsion protein PAb; 0, Nor-1dMBP
fusion protein was stored for 4 months at -80°C; + or -, containing or not containing the
component in the same row in the “Reaction components” column.

team
:eom

:8 mm m n ma):
:cuum . .0 mg 2.32
=8 ..m mg
zoomsw n n ma):
Am :06 ..N mg
:8 ..w o _ LoZ

:8 m
=8 .m

 

85

AVN (Figure 2-6). In the presence of NADPH, only the crude extract fi'om E coli DH5a
containing the Nor-lc/MBP fusion protein (reaction 6), the mixture of the afinity purified Nor-
lc proteinandtheE colicell crudeextract (reaction 8), andthe mixture ofthe afinitypurified
Nor-1c/MBP firsion protein and the E coli cell crude extract (reaction 10) completely
convertedNA(90uM)toacompoundthatco—migatedwithAVNuponTLCanalysis.
Neither the afinity purified Nor-1c protein (not shown) nor the control E coli extract (reaction
4) converted NA to AVN in the presence of NADPH. These data suggested that E coli
provided some essential cofactor(s) required for NA reductase activity. The cofactor(s) was
foundinthe 105,000 xgsuper'natant (reaction 12)andnotinthe 105,000 xgpellet oftheE
coliextract (reaction 11). NADPH, E coli cell extract, andE coli cell extract containing
MBP were unable to convert NA to AVN (reactions 3, 4, and 5). Anti-Nor-lc/MBP fusion
protein PAb inhibited the Nor-ldMBP activity (reaction 13) suggesting that the enzymatic
activity originated fiorn the Nor-1c protein. The freshly prepared Nor-lclMBP firsion protein
finished the NA-AVN conversion in 30 min and the stored (at —80°C for 4 months)
Nor-lc/MBP fusion protein finished the same conversion in 50 min (reactions 14 to 18)
suggesting that storage reduced the Nor-1c protein activity.
UVIVIS,Mass,andNMRSpectra ofNA. To confinntheidentityofthecompomrd
usedasthesrbsuatemthemzymeacfivityassay,thecompoundwaspmifiedmtwo
consecutiveTLCsteps. Thepurified compound, whichhadthesameRfvahre(0.45)with
benzene-ethyl acetate (7:3) as the developing system and the same color (brown under white

light)asthestandardNA,wasscrapedfromtheTLCplateandthenanalyzedbyUV/VISCUV

A 4 .9990:I
3 .0999J
2 .9099-

1.8880-

 

 

9.009 , ' . .
209.99 399.99 499.99 599.99

 

B 3 .8880-

 

 

 

Fignre 2-7. UV/VIS spectroscopy of norsolorinic acid (NA) and averantin (AVN). A:
UV/VIS spectrum (200-600 nm) of NA (rn ethanol). 3. max (EtOHXe): 235nm(24,500),
269nm(16,900), 284mn(18,600), 297nm(19,900), 314nm(22,900), and 465nm(7,760). B:
UV/VIS spectrum (200-600 nm) of AVN (in ethanol). 9e mx(EtOH)(a): 222nm(26,784),
262nm(15,810), 298nm(20,386), 315nm(22,022), 453 nm(6,658).

87

 

 

 

 

 

 

 

 

 

 

 

* A 2..
J
88-1
J
t
...: l
‘ L
P
48: 3?? 31,8
; 3 4
‘ Z
. 3 1 l
O
50 100 150 200 250 300 358
"/2
100
83‘
i r
en 1
. . l
49- 395 .
20‘ Z 7 '
, 3 1 r
‘ l' 373.,“ is ‘T’ ’
62:?3.‘ laLE?§1 l'lfignll IIlaLnxgrnna'1..-11-Aaa?3:aaa gilllllll [In-galalfixlsi Ian'.-a.'xx-3;?l6n-;-.x .1 an. i
300 328 349 360 383 403

"/2

Figure 2-8. Mass spectroscopy of norsolorinic acid (NA) and averantin (AVN). A: The
massspectrumofthepurifiedNA [relativeintensity(%): molewlarionatm/2370;fiagments
at m/z 327, 314, 299, 272]. B: The mass spectrum ofthe enzymatic end product AVN
[relative intensity (“/o): molecular ion at m/z 371; fragments at m/z 354, 325, 311, 297, and
285].

88

DMSO

 

 

cam. 5.15
conm, 3.53

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2-9. NMR spectroscopy of norsolorinic acid (NA) and averantin (AVN). A:
NMR spectrum of NA (in d6DMSO) (6pprn): CH3(6'), 0.88(3H); CH2(4'5'), 130(2H);
CHZC“). 1-60(2H); 012(2). 281(2H); CH(7). 6-61(1H); CH(5). 7-l3(1H); CH(4). 7220K)-
B: NMR spectrum of the end product AVN (in d6DMSO) (fippm): CH3(6’), 0.85(3H);
CH2(3',4',5'), l.30(6I-I); CI-I2(2'), 1.84(2H); COH(1'), 3.53(1H); CH(1'), 5.15(1H); H(7),
653(111);H(5). 7-19(1H); 11(4). 7220“).

89

and visrhle light), mass, and NMR spectroscopy. The six maximum absorbence values of the
WMS spectrum (200-600 nm) of the purified compound in ethanol (Figure 2-7A) were
consistent with the data for NA reported by Cole and Cox (1981) [A max (EtOHXa):
235nm(24,500), 269mn(l6,900), 284nm(18,600), 297nm(19,900), 314nm(22,900), and
465nrn(7,760)]. The mass spectrum (Figure 2-8A) and the NMR spectrum (Figure 2-9A) of
thepurified compoundwerealso consistentwiththepublisheddataforNA(ColeandCox
1981’) [mass spectrum (relative intensity (%): molecular ion at m/z 370; fiagments at m/z 327,
314, 299, 272; NMR spectrum (in d6DMSO, 8pprn): CH3(6'), 0.88(3H); CH2(4',5'),
130(21‘1); CH2(3'). 1-60(2H); CH2(2'). 2-81(2H); CH(7). 6-6l(1H); (311(5). 7-13(1H); CH(4).
7.22(1I-I)].

UV/VIS,Mass,andNMRspectraofAVN. Toidentifytheendproductinthe
conversion of NA by the Nor-lc/MBP fusion protein, the enzymatic reaction was scaled up 10
fold. The enzymatic end product, which had the same Rf value (0.37) with benzene-ethyl
acetate (7:3) as the developing system and the same color (yellow under white light ) as the
standardAVNon'I'LC,werescrapedfi'omthe'ILCplateandthenanalyzedbyUV/VISCUV
and visible light), mass, and NMR spectroscopy. The five maximum absorbence values of the
WMS spectrum (200-600 nm) of the end product in ethanol (Figure 2-7B) were consistent
with AVN data reported by Bennett et at (1980) [A mx(EtOI-I) (a): 222nm(26,784),
262nm(15,810), 298nm(20,386), 315nm(22,022) 453 nm(6,658)]. The mass spectrum (Figure
2-8B) of the end product was consistent with AVN data published by Bennett et al. (1980)
[relative intensity (%): molecular ion at m/z 372; fiagments at m/z 354, 325, 311, 309, 297, and

285] with one exception (molecular ion at m/z 371, not at m/z 372 as expected). The NMR

90

spectrum of the end product in d6DMSO (Figure 2-9B) was consistent with the data for AVN
published by Bennett et at (1980) [(5ppm): CHB(6'), 0.85(3H); CHZ(3’,4',5'), 1.30(2H);
CH2(2'), 1.74(2H); COH(1’), 3.53(1H); CH(l’), 5.15(lI-I); CH(7), 6.S3(1H); CH(S), 7.02
(1H); CH(4), 7.02(1H)]. Two “new peaks” appeared in the NMR spectrum of AVN. One
was COH(1'), 3.53(8ppm); another CH(l'), 5.15(6ppm). These two new peaks resulted fi'orn
keto-reduction by the addition of two more hydrogen atoms. The standard AVN purified fiom
thefimgal snainAptrr'asitiatsATCC 56774was shownto luvethe sameUV/VIS, mass, and
NMRspectraasAVNgeneratedintheNAreductasereaction.

AFBr Accumulation in A. pansiticus SU-I and A. panniticus ANor-I. Direct
competitive ELISA analysis was utilized to quantify AFBr accumulation in the wild-type A.
pen-annals SU-l and the nor-1 disrupted transformarrt, A. pen-asiticus ANor-l, arltured in
YESliquidmedium. After2,and5daysofculture, approximately25 foldmoreAFBr was
produced by the wild-type A. pw'asr'ticus SU-l (4.55 i- 0.02 mg, and 6.8 :t: 0.03 mg of AFBl
per garn of dry mycelial mass, respectively) when compared with the nor-l disrupted

transforrrrant A. pw'asr'ricus ANor-l (0.18 :t 0.02 mg, and 0.27 :l: 0.03 mg, respectively).

DISCUSSION

The data presented in this study demonstrated that the Nor-1c protein (or the Nor-lclMBP
firsion protein) is able to convert the aflatoxin Br pathway intermediate NA to AVN but only in
the presence of NADPH and a cofactor(s) obtained fi'om E coli DHSot. This suggests that the

rmtive Nor-1 protein is an NADPH dependent NA ketoreductase requiring an unidentified

91

cofactor(s) and that nor-1 encodes an activity involved in one of multiple proposed pathways
for the conversion ofNA to AVF in the AFBl pathway inA. par-annals SU-l.

The Nor-1c protein has a ketoreductase activity capable ofconverting NA to AVN in vitro.
TheUVNIS, mass, anthrIRspectrmndataconfirmedthatthesubstratewasNAandtheend
product was AVN. The conversion of the 1'-keto goup of NA to the l’-hydroxyl goup of
AVNandtherequirementofNADPHintheenzymaticconversion confirmedthattheactivity
involved in this conversion is a NADPH dependent NA ketoreductase activity. The conclusion
thumlytheNor-lcpmtdnmmiskaoreduaaseacfivuymmemzynwacfivhyassaywas
deduced fi'om the following three observations: (1) the conversion of NA to AVN was
inhibited by the addition of the anti-Nor-lc/MBP firsion protein PAb when the Nor-1c protein
was involved in the enzyme activity assay, (2) NADPH, the E coli protein plus NADPH, and
the MBP plus the E coli protein and NADPH were unable to convert NA to AVN; and. (3)
analysis of the proposed amino acid sequence of nor-l provided evidence that the Nor-1c
proteinwasaketoreductase (Trail eral, 1994).

Bhatnagar and Cleveland (1990) proved that the conversion of NA to AVN is reversible in
the presence of a 38-kDa reductase plus NADP. This may also be true for the Nor-1c protein,
butthereversereactionwasnot conductedinthecurrent study.

The Nor-1c protein showed NA ketoreductase activity only in the presence of a cofactor(s)
fi'omE coli DHSa suggesting that a cofactor(s) may be also necessary for the native Nor-1
protein to show the NA ketoreductase activity. A 38-kDa reductase (Bhatnagar and
Cleveland, 1990), a 43-kDa isozyme of the reductase (Bhatnagar, unpublished observation),

and a 140-kDa reductase (also called dehydrogenase) (Chuturgoon and Dutton, 1991) have

92

been purified to homogeneity, and are reported to convert NA to AVN. Surprisingly the
purification of the 31-kDa native Nor-l protein encoded by nor-1 has not been reported. This
obsavafionmaybeetplaimblewiththedatapresentedmthisstudfiseebdow).

Western blot analysis using PAb raised against the Nor-le/MBP firsion protein showed
that:flreacarmrflafionofa3l-kDapmtdnhadapattanconsistentwiflrAFBr accumulationin
A. parturition: SU-l (Chapter 3); the 31-kDa protein purified fiom a crude extract ofA.
parasitic” SU-l byafinitychrormtographywasdetectedasasingleband; andthe31-kDa
protein was not detected in a crude extract fi'om the nor-l disrupted strain ANor-l. These
observations suggest that the 31-kDa protein is the native Nor-1 protein encoded by the nor-1
gene. But, the nor-1c protein alone does not show the reductase activity. Therefore, it is
possible that a cofactor(s) is needed for the activity of the native Nor-1 protein. The
cofactor(s)mayhavebeenlostdmingpurificationstepinprevious studiesresultingininability
to purify Nor-1. Such a cofactor(s) was possibly provided by E coli DHSa in this study.

Alternatively, because polyketide synthase (PKS) and fatty acid synthetase (FAS) enzymes
often firnction in multisubunit complexes, it is possible that the native Nor-1 protein is a part of
a multiple polypeptide complex required for AFBl biosynthesis. Therefore, only when it is
mchtdedmdheldinafirncfionalconfigmafiondhecflyassodatedwhhthecomplag doesthe
native Nor-l protein have the reductase activity. It is hypothesized that certain low mass
element(s) in an E coli supematant (105,000 x g ) substitute for a required cofactor or
eliminate the requirement for protein/protein interaction to help the Nor-1 protein to show

reductaseactivity. Fmtherresearchiswarranted.

93

Thenar-l geneiscorrfirmed tobedirecdyinvolvedhrAFBrbiosynthesisinAparan'fic-ras,
an observation which was reported previously (Skory et al., 1993; Trail et at, 1994). ELISA
datainthisstudyshowedthatthenw-ldisruptedstrainANor-l reducedtheaccunmlationof
AFBrbyapproxirnatelyZSfold. TheNor-lcproteinencodedbythenar-l cDNAwasableto
convertNAtoAVN,andbothNAandAVNhavebeenproventobetheintermediatesin
AFBl pathway(Bennett etaL, 1980;Yabe etaL, 1993).

ThemassandNMRspectroscopicdatastronglysuggestthatNA(370Da)isconvertedto
AVN(372Da)(the l'oketogoupisreducedtohydroxylgoup), eventhoughthetheoretical
molewlarmassofAVNis372Da,buttheobservedmassspectrumofAVNhadabasepeak
atm/z371,notatm/z3729sexpected. OfienionsCM—l)*arldsonretimes(1\d-2)*,(M-3)*are
observedintheprocessofmassspectroscopyduetolossesofhydrogenatom(s)fi'omthe
molecular ion M. Sometimestheion (M-1)*ismuch moreabundantthanthemolecular ion
M(RoseandJohnstone, 1982)asoccurredherewithAVN. TheNMRspectrumdataofNA
wasalsoconsistentwiththeliteratme(ColeandCox,1981),withoneexception.Norsolorinic
addmflnsresmrchwasprepmedudmomaaymflimfionaepfliueforeflnNMRspewtm
ofNAinthisstudynotonlyhadamajorpeakatZS5ppmwhichoriginatedfiomDMSO,but
alsohadapeakat3.46ppmwhichoriginatedfiomwatdzTheNMRdataofAVNweremore
complicated. TheerrzyrnaficelidproducLAVN,andtheAVNpreparedfiomA.pa-asificus
ATCC56774mdnsaudyhad-flxsmneNMRspchumThisNMRspem‘mnwasmnfigau
withthedatareportedbyBennettetal. (1980),butwaslessconsistentwiththedatareported

by Birkinshaw et al (1966) [(8ppm): CHB(6'), 1.00(3H); CHZ(3’,4',5'), l.40(21-I);CH2(2'),

94

1.90(2H); CH(7), 6.15(1H); CH(S), 6.80(1H); CH(4), 6.82(1H)]. These observed difl‘erences
couldrenrltfiomdifl‘eremquafityofsolvemsanddifi‘ereminsumnm

Insurnmary, thedatapreserrtedinthisstudyconfirmedthattheNor—lcproteinisa
ketoreductase which converts the AFBl pathway intermediate norsolorinic acid (NA) to
averatin (AVN) in the presence ofNADPH and a supernatant fraction (105,000 x g) obtained
fi'omE coliDI-ISG. Thissuggeststhatanuniderrtified cofactor(s)maybenecessaryforthe
nativeNor-l proteinfirnctionandthatthem—l geneisirrvolvedinonly one ofrmrltiple
proposedpathwaysfortheNAconversiontoaverufin(AVF)intheAFBr pathwayinA.

pw'asiticus.

CHAPTER3

THE ACCUMULATION OF THE NOR-l PROTEIN AND THE ACITVITY OF
THE NOR-l PROMOTER DURING AFLATOXIN B1 BIOSYNTHESIS IN
ASPERGEL US PARASIHCUS

ABSTRACT: Two difi‘erent procedures were utilized to study the expression of the nor-l
gene during aflatoxin Br biosynthesis in Aspergillus parasiticus. First, polyclonal
antibodies, which were raised against the Nor-1c protein (expressed in Escherichia coli
fiom a nor-1 cDNA), were used to monitor the accumulation of the native Nor-1 protein.
Second, a nor-l/GUS reporter construct was used to monitor the activity of the nor-1
promoter. The results showed that the accumulation of the Nor-1 protein (in liquid media
and on solid media) and the activity of the nor-1 promoter (on solid media) are regulated
in the same pattern as the accumulation of afiatoxin and are also closely correlated to the
development of sporulation in A. pw'asiticus. The results also suggested that the
expression of nor-1 is regulated in a temporal and spatial pattern in A. parasiticus gown

on sold media.

INTRODUCTION

A major focus of current studies on aflatoxin biosynthesis in Aspergillus parasiticus is

the regulation of the expression of the genes involved in this complex pathway. Northern

95

96

blot. analysis demonstrated that the expression of nor-1, which is directly involved in
aflatoxin B1 biosynthesis, is regulated at the level of transcript accumulation in the
toxigenic strain A. parasitism SU-l gown in liquid media (Skory et al., 1993; Trail et al. ,
1994; Trail 01 al., 1995). However, the expression of nor-1 can also be studied at the
level of the accumulation of the Nor-l protein and at the level of the activity of the nor-l
promoter in A. parasiticus.

Condifimsofmrdgancfimggnwingmfiquidmediamevaydifi‘erentfiomthenatural
environmentalconditionsofthe sarnefungigowinginsoil oronhost plantsinthefield. For
example, sporulation usually cannot be observed in liquid media. Therefore, studies conducted
infiqlfidmediamaynotcompletdyrefiecttheregflafionofthegenesinvolvedinafiatoxin
biosynthesis in toxigenic fungi gown on solid media.

Aflatoxins have been found in high concentrations in spores (\Vrcklow and Cole, 1982;
Wrcklow and Shotwell, 1983; Cotty, 1988; Bayman and Cotty, 1990). Mycelial gowth rate
wasndrcedunsponranonandadmdnaecrnudanonweetotnlyinnbnednammmd
A. parasiticus by addition of 2.0% (v/w) phosphates to Sabouraud dextrose agar (Lebron et
al., 1989). These data suggest that the regulation of aflatoxin biosynthesis may be correlated
to sponrlation.

Basedonthesedataandobservations, themajorhypothesisinthisstudyisthatthe
acclmmlationoftheNor-l protein(in liquidmediaandon solid media) andtheactivityofthe
nor-l promoteractivity(on solid media) areregulatedinthesamepatternastheaccunmlation

of aflatoxin and are correlated to the deveIOpment of sporulation in A. pw'asificus.

97

The data presented in Chapter 2 demonstrated that the Nor-1c protein was expressed in
E coli fiom a nor-1 cDNA, the Nor-1 protein had the same molecular mass .as that
predicted fi'om the nor-l gene sequence, and the Nor-1 protein had the enzymatic activity .
necessary to convert norsolorinic acid (NA) to averantin (AVN), two intermediates in the
AFBl pathway. These data suggested that the Nor-1c protein and the Nor-1 protein may
have very similar, if not identical, amino acid sequences, and therefore, have the same
inununogenicity and the same anitgenicity. Hence, a subsidiary hypothesis is that
polyclonal antibodies (PAb) raised against the Nor-1c protein would be useful to monitor
the accumulation of the Nor-l protein.

The Escherichia coli uidA gene encoding B—glucuronidase (GUS) has been used in fungi
as a reliable reporter for gene expression at the level of promoter activity (Monke and
Schafer, 1993). Therefore, the activity of nor-1 promoter can be monitored by a nor-1
promoter/GUS construct (nor-l/GUS) providing another tool to address the major
hypothesis.

Accordingly, two experimental procedures are proposed to address the major hypothesis
in this study. First, polyclonal antibodies (PAb) will be raised against the fusion of the
Nor-1c protein and the maltose binding protein (MBP) (N or-lc/MBP). The PAb will be
used to detect the accumulation of the Nor-1 protein. Second, a GUS reporter construct
will be used to monitor the activity of the nor-l promoter. The accumulation of the Nor-1
protein and the activity of the nor-1 promoter will be compared with the pattern of

afiatoxin accumulation and the development of sponrlation during aflatoxin biosynthesis.

98

The data presented in this study showed that the accumulation of the Nor-1 protein (in
liquid media and on solid media) and the activity of the nor-1 promoter (on solid media)
are regulated in the same pattern as the accumulation of aflatoxin and are closely
correlated to the development of sporulation in A. parasiticus. The data also suggested
that the expression of nor-1 is regulated in a temporal and spatial pattern in A. parasiticus

gown on sold media.

MATERIALS AND METHODS

Bacterial Strain, Fungal Strains, and Growth Media. Plasmids were amplified in
Escherichia coli DHSa F' °[F'endAI hstI 7(rK-rnK+)supE44 thi-l recAI gyrA (Nal‘)
relAI (IacZYA-argF) 0169 (m801acZ AM15)] using standard methods (Ausubel et al. ,
1987). Aspergillus parasiticrts NRRL 5862 (ATCC 56775, SU-l) was used as the
aflatoxin-producing wild-type strain. An A. parasiticus transformant (designated nor-
1/GUS) containing pAPGUSNN, a reporter construct containing the nor-1 promoter
fused with the E coli uidA gene encoding B-glucuronidase (kindly provided by David
Wilson in our laboratory) was used to monitor the activity of nor-l promoter. In this
transformant, pAPGUSNN was inserted at the 5’-end of the chromosomal nor-1 locus.
Fungal strains were maintained as frozen spore stock suspensions [in 20% (v/v) glycerol]
at -80°C. Aflatoxin-inducing media, YES broth [2% (w/v) yeast extract, 6% (w/v)
sucrose, pH 5.8] and YES agar [YES broth containing 1.5% (w/v) Bacto-agar] were used

to gow mycelia for protein preparation and aflatoxin extraction.

99

Fungal Culture, Dry Weight Determination, and Analysis of Fungal Morphology.
For gowth of fungi on solid medium, 5 ul of a spore stock suspension (1.5 x 10°
spores/ml) was inoculated onto the center of YES agar media and gown at 29°C in the
dark. Mycelia were collected using a spatula to scrape the mycelia from the surface of
agar. The collected mycelia were either used immediately or flow under liquid nitrogen
and stored at -80°C until use. The dry weight of mycelia gowing on solid media was
determined by a filtration/drying method described by Olsson and Jennings (1991).

For gowth of firngi in liquid media, a stock spore suspension was inoculated into liquid
media with a final spore concentration of 1.5 x 105 spores/ml and gown at 29°C with
constant shaking (150 rpm) in the dark. Mycelia were collected by filtration and were
either used immediately or fiozen under liquid nitrogen and stored at -80°C until use. The
dry weight of mycelia gown in liquid media was determined by drying the mycelial pad in
a preweighed flask overnight at 80°C before weighing. A nutritional shift culture assay
using nonaflatoxin-inducing peptone-rnineral salts (PMS) medium and aflatoxin-inducing
glucose—mineral salts (GMS) medium was performed according to Wiseman and Buchanan
(1987). Briefly, spores were inoculated into PMS medium (1.5 x 10’ spores/ml) and
incubated for 60 h and then shifted into GMS medium for a 60-h period of incubation.

The morphological characteristics of a colony gown on YES agar were examined
under a dissection microscope.

Temporal and Regional Collection of Samples From Solid Growth Medium. Corridia
(7.5x 10’)wereinoculatedatthecenteronESagarinPeuidishes. Colonieswiththe same

gowthratewereselected. Forcoflecfingsmnplesfiomthecmtalregionatleast4cultures

100

wereneeded. Twodaysaiterinocrrlafiontheareacoveredbythemycelimn (infourPetri
dishes)wasmarkedwithacircleanddesignatedthe‘centralregion’. Two days(4days, 5
days, or6days)afierinoarlafion,themycelimnwhichcoveredthecenfialregion ofthefirst
(second, third, or fourth) Petri dish was collected as the ‘2-day’ (‘4—day’, ‘S-day’, or ‘6-day’)
‘central-region’ sample C2 (C4, C5, or C6). For collecting samples fiom the middle region, at
least3 cultureswereneeded. TwodaysaflerinoarlafiontheareacoveredbythemyceliumCm
threePetridishes)wasmarkedwithadrcle (the2-daymark). Fourdaysafierinoculation, the
areacoveredbythemyceliurn(rnthreePetridishes)wasmarkedwithasecondcircle(the4-
daymark). TheareabetweentheZ-daymarkandthe4—daymarkwasdesignatedthe ‘middle
region. Fourdays(5 days, or6days)afierinoculation,themyceliawhichcoveredthemiddle
region of the first (second, or third) Petri dish was collected as the ‘4-day’ (‘5-day’, or ‘6-day’)
‘middle-region’ sample M4 (M5, or M6). For collecting samples from the peripheral region, at
least2arltureswereneeded. Fourdaysafierinoarlafiontheareacoveredbythemyceliumfin
twoPetridishes)wasmarkedwithacircle(the4-daymark). Theareabetweenthe4-daymark
andthedrcularedgeoftheagarwasdesignatedthe ‘peripheralregion’. Five 5 days(or6
days)aflerinoculation, themyceliawhichcoveredtheperipheral regionofthefirst (or second)
Petri dish was collected as the ‘5-day’ (or ‘6-day’) ‘peripheral region’ sample P5 (or P6). The
sampling procedure is shown schematically in Figure 3-5B.

Crude Protein Extract Preparation. Crude protein extract preparation fiom fungal
cells was conducted as described in Chapter 2.

Preparation and Purification of Polyclonal Antibodies. A nor-1 cDNA was cloned

into the expression vector pMAL-c2 resulting in pMN 1 . The Nor-lc/MBP (fusion

101

protein) was produced in E coli transformed with pMNl. The Nor-lclMBP was cut with
factor Xa to yield the Nor-1c protein and the MBP. The Nor-lclMBP and the Nor-1c
protein were purified by afinity chromatogaphy as described in Chapter 2.

The antigen-adjuvant mixture (100 pl of Hunter ’3 TiterMaxTM #R-l adjuvant plus 120
ug of the Nor-lclMBP in 50 ul of 10 mM phosphate-bufl‘ered saline) was prepared
according to the manufacturer’s instructions (Cthx Corporation, Atlanta, GA) and was
used for primary injection and boosts. The anti-Nor-lc/MBP serum of the rabbit was
prepared according to Ausubel et al.(1995a). Briefly, the antigen-adjuvant emulsion was
injected into the rabbit subcutaneously at four difi‘erent sites on the rabbit's back. The
rabbit was boosted subcutaneously 4 weeks later after the primary injection and reboosted
2 weeks after the initial boost. The titer ofthe antiseum was estimated by indirect ELISA
according to Ausubel et al.(l995a) using the Nor-lolMBP and the Nor-1c protein as
coating antigens respectively. Briefly, a microtiter plate was coated with 10 ug/ml antigen
and blocked with 0.25% (w/v) bovine serum albumin (BSA). The antigen-coated and
BSA-blocked wells of the microtiter plate were incubated with serial dilutions of the
antiserum (10 to 10° dilution). The wells were then incubated with goat anti-rabbit-IgG
antibodies conjugated with alkaline phosphatase (Signa, St. Louis, MO). The activity of
the captured alkaline phosphatase was determined according to the manufacturer's
instructions. The dilution of the antiserum which showed 2 times the alkaline phosphatase
activity of the preimmune serum in ELISA assay was defined as the titer.

The IgG fiaction of the antiserum was precipitated with 33% (v/v) saturated ammonium

sulfate (SAS). The double-purified polyclonal antibodies (PAb) were obtained by passing

102

a portion of the IgG fiaction twice through an afinity column containing MBP plus crude
protein (10,000 x g supernatant) of E coli DHSa as ligands. The triple-purified PAb
were obtained by passing a portion of the double—purified PAb through a second amnity
column containing the Nor-1c protein as the ligand. Aflinity column preparation using
CNBr-activated Sepharose 4B (Signa, St. Louis, MO) and ligand proteins (either a mixture of
MBP and E coli DHSa protein in a 1:1 ratio, or Nor-1c protein alone) was carried out
accordingtoapublishedstandardmethod(Ausubeletal, 1995c). Theprocedureforafinity
pmificationofPAbwascaniedoutaccordingtothemethod established byDr. R. Mehighin
our lab with modifications (Chapter 2, Immunoafiinity purification of the Nor-l protein). Five
mg of the IgG fiaction (or the double purified PAb) (5 mg/ml) was loaded onto the aflinity
column for purification of the double purified PAb (or the triple purified PAb).

Western Blot Analysis of the Native Nor-1 Protein. Protein, separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was blotted to a
polyvinylidene difluoride (PVDF) membrane (Du Pont Co., Boston, MA) using a standard
publishedmethod(Ausubeletal, 1995b). Irmmmodctectionwascaniedoutwitha
dramhumnescandetecfionldt(Schleidrer&SdmdLKeeneNFDaccordingtoflle
marmfactm’er’s instructions.

Afiatoxin Analysis by TLC and ELISA. Aflatoxins were extracted for 1- h at room
temperature with chloroform (10 ml) either fiom mycelial liquid gowth media (not
including mycelia) (1 ml) or fiom mycelial solid gowth media (including mycelia) (1 g).
Chloroforrn-extracted aflatoxins were tested either semiquantitatively by thin layer

chromatogaphy (TLC) with chloroforrn-acetone (95:5) as the developing system, or

103

quantitatively by enzyme-linked irnmunosorbent assay (ELISA). For ELISA, only
aflatoxin B1 (AFBl) was tested according to the method of Pestka et al. (1980) using anti-
AFBl antibodies kindly provided by Dr. J. Pestka (Department of Food Science and Human
Nutrition, Michigan State University, East Lansing , Nfl).

Quantitative Assay of B-Glucuronidase Activity. Quantitative analysis of B-
glucuronidase activity was performed by spectrophotometry as described by Jefi‘erson et
al. (1986), and Tada et al. (1991) using p-nitrophenylglucuronide as the substrate. One
unit was defined as the amount (nanomole) of p-nitrOphenol produced in one min at 37°C

by 1 mg protein (nmol/min per mg protein).

RESULTS

Titer and Specificity of Polyclonal Antibodies. The titer of the anti-Nor-lc/MBP
serum was determined by ELISA. The titers of antisera from the first and second boost
were similar (450,000 and 500,000). The irnmunogen used for antibody production in this
study was the Nor-lclMBP composed of the MBP (42 kDa) and the Nor-1c protein (31
kDa). Because the serum contained antibodies against both portions of the fusion protein,
the titer which was determined using the Nor-lclMBP as the antigen was higher than the
titer which was determined using the Nor-1c protein as the antigen.

In Western blot analysis of crude fungal cell extracts, the IgG fiaction detected at least
7 protein bands (Figure 3-1). The double-purified PAb detected two protein bands (Figure

3-2). The triple-purified PAb detected only one major protein band (Figure 3-1) with a

104

‘— Nor-l

 

Figure 3-1. Western blot analysis demonstrating the specificity of PAb fiom different
levels of purification. All crude extracts (except lane 5) were obtained from A. parasiticus
SU-l grown in YES liquid medium. Each lane contained 10 [lg crude protein. Lanes: 1,
crude extract (from the 60-h sample) was probed by the triple-purified PAb (10 [lg/ml); 2,
3, and 4, crude extracts from the 60-h, 54-h, and 48-h samples, respectively, were probed
with the IgG fiaction (10 ug/ml); 5, crude extract from nor-l disrupted strain of A.
parasiticus gown in YES liquid medium (for 60 h) was probed with the IgG fraction (10
uglml). The molecular mass of the Nor-l protein (top band) was 31 kDa.

105

Table 3-1. Titers and specificities of anti Nor-lc/MBP PAb.

 

 

 

 

 

 

Samples Titer against the Nor- Titer against the Specificity
lclMBP Nor-1c protein (cross reaction)
Antiserum from primary injection 200,000 50,000 > 7 bands
Antiserum from first boost 450,000 110,000 > 7 bands
Antiserum fiom second boost 500,000 120,000 > 7 bands
Double-purified PAb 2 bands
Triple-purified PAb 1 band

 

 

 

 

 

 

Notes:

(I). The antiserum fiom primary injection was obtained 4 weeks afier the primary
immunization. The antiserum fiom the first boost and the second boost were obtained 2
weeks after the first boost and the second boost respectively.

(2). The titers of antisera were estimated using either the Nor-lclMBP, or the Nor-1c
protein as the antigen.

(3). The relative specificity was estimated by Western blot analysis.
(4)ThespedfidtyoftheanfisamnwaseflnfledusingtheIgGfiacfioaneuanblot
analysis.

size of 31 kDa which was the expected size of the Nor-1 protein. The 31-kDa protein
band was also detected by both the IgG fiaction and the double-purified PAb. The
relative specificities of the IgG fiaction, the double-purified PAb, and the triple-purified
PAb (as measured by the degee of cross reactivity) are summarized in Table 3-1.

The Accumulation of the Nor-l Protein and the Accumulation of AFB1 in A.
pm'asiticus SU-l Grown in YES Liquid Medium. The pattern of the accurmrlation of the
Nor-1 protein was analyzed in A. parasiticus SU-l (an aflatoxin-producing strain) gown in
YESliquidmedium(anaflatoxin-inducingmedimn). Westernblotanalysisofcrudeextracts

fiom cells harvested at the appropriate time points showed that the Nor-1 protein was not

106

<— Nor-l

 

24h 36h 48h 60h 72h 84h 96h 108h

Figure 3-2. Western blot analysis of the accumulation of the Nor-l protein in A.
parasiticus SU—l gown in YES liquid medium. Each lane contained 10 ug crude protein.
Lanes: 1 through 8, crude extract from firngal cells gown for 24 h, 36 h, 60 h, 72 h, 84 h,

96 h, and 108 h, respectively, and probed by the double-purified PAb (10 rig/ml). The
molecular mass of the Nor-1 protein (top band) was 31 kDa.

107

detectedat24h ofincubation, butwasdetected from 36 hto 108 hofincubation (Figure 3-2).
The maxirmrm level ofthe Nor-1 protein appeared between 36 h and 72 h ofincubation and
decreased fi'om 72 h to 108 h ofincubation. ELISA analysis ofthe growth medium showed
thatAFBr beganto accmnulateafier24hofinaibation(Figure3-3). Themaximurnrateof
AFBraccunmlationoccurredbetween36hto60hofincubation (fiom 19to 121 mg/L).
AFBl accmmrlationremainedatalmostthesamelevel fiom60hto 108 hofincubation. The
maxirmrmrateofaccunmlation oftheNor-l proteinoccurredatthesametimeasthemaxinnnn
rate ofAFBr accumulation.

The Accumulation of the Nor-l Protein and the Accumulation of AFB1 in A.
parasiticus SU-l in Nutritional Shift Assay. A nutritional shift assay was conducted to
menarre the accumulation ofthe Nor-1 protein inA. pwasiticras SU-l gown in a nonaflatoxin-
inducingPMSmediumandthenshifiedinto aflatoxin-inducingGMSmedium. Afier60hof
incubation in PMS no Nor-l protein was detected by Western blot analysis (Figure 3-4). The
Nor-1proteinwasdetectedasearlyas6hafierashififi’omPMStoGMS. Thecorresponding
accumulationofAFBr inthegowthmedimnwasdeterminedbyEIJSACl‘able 3-2). Afierthe
mrtritional shifi, AFBl increased more slowly inthe fresh PMS (fiom 0.13 to 2.42 mg/L) than
inthefi'eshGMS (fiorn 0.13 to 129.80 mg/L). Inthenutritional shift assay, thepattern ofthe
accumulation of the Nor-1 protein was consistent with the pattern of the accrmrulation of

AFB1.

108

 

i

.s ..a

G O N

O 90
N

 

u-l

 

3

[iill[lilllliillllll|lllilllTTlllil

 
   

Time vs AF 81
Time vs dry weight

 

 

L l

 

N
0

AF B1 production (mg/L)
8
Dry mycelial weight (glflask)

 

 

 

 

I I I I I I

0 12 24 36 48 60 72 84 96108
Culture time (hour)

Figure 3-3. The accumulation of AFB1 and mycelial dry weight in A. parasiticus SU-l
gown in YES liquid medium. The concentration of AFB1 was assayed by ELISA in
samples taken 12 h, 24 h, 36 h, 60 h, 72 h, 84 h, 96 h, and 108 h after inoculation. The
mycelial dry weight was estimated at the same time points.

109

Table 3-2. The accumulation of aflatoxin Br in A. parasiticus SU-l in nutritional shifi

 

 

 

 

 

 

 

assay.
Incubationtimeaftera AFByinPMSafiershifi: AFBrinGMSaftershifl:
shifl fi'om PMS (h) mean t SE (mg/L) mean :1: SE (mg)

0 0.13 10.09 0.13 $0.09

6 0.16 :t: 0.07 7.89 :t 0.16

12 0.59 :l: 0.06 25.36 i 0.25
24 1.06 :i: 0.07 45.47 :1: 0.28
48 2.12 :l: 0.08 115.36 :1: 1.23
60 2.42 :t 0.11 129.80 :t 1.26

 

 

 

 

 

Note: A. parasiticus SU-l was gown in a nonaflatoxin-inducing PMS medium for 60 h and
then shifted into aflatoxin-inducing GMS medium. Each value represents the mean i
standard error (SE) of triple tests.

Morphology of A. parasiticus SU-l Grown on YES Agar. The morphology of A.
musicals SU-l gown on YES agar was observed under a dissection microscope.
Irmnediatelyafierinoculation, theareaoccupiedbytheinoculated sporeswasirregular. After
one day of incubation, the germination of individual spores was randomly oriented. Afier two
daysofincubation, awhiteroundcolony(0.5-l cmindiameter)appearedwithawhitecircular
bandofsubstratehyphae(0.1- 0.2cminwidth)atthemarginandawhiteromldareaofaerial
hyphae(0.3 -0.6cmindiameter)atthecentralregion. Afierthreedaysofincubation, the
white circular band of substrate hyphae (at the margin of the colony) moved outwards followed
byanadjacentwhitecircularbandofaerialhyphae(0.2-0.4cminwidth). Ayellow-geen
roundareaofimmaturespores(l.4- 1.6 crnindiarneter) appeared atthecentralregion. After
faudaysofhmbafionmewlfitedrmhrbarflofmbmhyphaeandflnadjacauwlnte

circular band of aerial hyphae continued to move outwards followed by a yellow-green circular

110

‘— Nor-l

 

G6 G12 G24 G48 G60 P60

Figure 3-4. Western blot analysis of the accumulation of the Nor-1 protein in A.
parasiticus SU—l in PMS-GMS nutritional shift assay. Each lane contained 10 pg crude
protein and was probed by the double-purified PAb (10 rig/ml). Lanes: 1, 2, 3, 4, and 5,
crude extract from cells collected alter incubation in GMS for 6 h, 12 h, 24 11, 4811, and 60
h (G6, GIZ, 624, G48, and G60), respectively, alter a shifi from a 60-h incubation in
PMS (P60); 6, crude extract from cells collected afier a 60-h period of incubation in PMS
(0 h in GMS). The molecular mass of the Nor-l protein (top band) was 31 kDa.

111

band ofinrrnature spores (1.0 - 2.0 cm in width). Abrown-geen round area ofmature spores
(1.0-2.0cmindiameter) appearedatthecentralregionofthePetridish. Afierfivedaysof
incubation, the white circular band of substrate hyphae reached the edge of the agar, the
adjacanwhhedmdmbandofaaidhyphaeandtheydlow-geendraflmbmdofhmnamm
sporesconfirmedtomoveoutwardsfollowedbyabrown-geencirmlarband ofmature spores
(1.5-2.5 crninwidth). Thehyphaeinthecentralregionstartedtodegenerate. Aflersixdays
ofharbafionthebmdofwbsfifiehyphaemdthebandofaaialhyphaedisappearedThe
bandofimmaturesporesandthebandofmature sporesmoved outwardsandabrownround
areaofdegenelatedhyphae(0.2- 0.5 crnindiarneter) appearedatthecenter.

Morphological Development and the Accumulation of AFB1 at the Colony Level in A.
paradtiaasGrownonYESAgar. SporeswereinoculatedontothecenteronESagarina
Petri dish and samples (C2, C4, C5, C6, M4, M5, M6, P5, and P6) were collected as described
(Materials and Methods) and shown schematically in Figure 3-5B. The accunmlation of AFBl
in each sample was determined by ELISA. The morphological characteristics and the
accurmllation of AFB1 in each samples were summarized in Table 3-3. By comparing the
morphological characteristics with the accumulation of AFBl, a pattern was found. In the
gowing colony, the concentration of AFBl increased from the region with substrate hyphae
plus aerial hyphae (3.2 mg/g), to the region with substrate hyphae plus aerial hyphae plus
irmnature spores (4.3-4.7 mg/g), to the region with irmnature spores plus mature spores (5.8-
7.4 mg/g), to the region with mature spores (7.4—9.0 mg/g), and to the region with mature

spores plus degenerated hyphae (7.9 mg/g) in a colony. The pattern of the accumulation

112

Table 3-3. The morphological characteristics and the accumulation of AFB1 in A. pm'as'iticus
SU-l gown on YES agar.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Samples Collecting Incubation Morphological characteristics AFBl
region time(day) (mm
CZ Central 2 Substrateandaerialhyphae 3.2:t0.8
C4 Central 4 Immatureandmaturespores 5.8:0.4
C5 Central 5 Mathew 7.4:03
C6 Central 6 Maturespores, anddegemratedhyphae 1910.7
M4 Middle 4 Wandaerialhyphae,andimmatmegxxes 4.31:0.5
M5 Middle 5 Inunatureandmaturespores 7.43:0.6
M6 Middle 6 Maulregtores 9.01: 1.2
P5 Peripheral 5 swarmeandauialhyphmandimmatmeqrores 4.7:tO.4
P6 Peripheral 6 Imrmtureandmatmespores 6.91:0.3
Notes:

(1) Conidia were inoculated at the center of YES agar in a Petri dish. Samples: Samples C2,
C4, C5, and C6 were collected fi'om the central region 2 days, 4 days, 5 days, and 6
days after inoculation; samples M4, M5, and M6 were collected from the middle region 4
days, 5 days, and 6 days afier inoculation; samples P5, and P6 were collected from the
peripheral region 5 days, and 6 days alter inoculation. A schematic of the sample
collection procedure is shown in Figure 3-5B.

(2) The accumulation of AFB1 (mg/g dry filngal mass) was determined by ELISA
(Materials and Methods).

(3) Each value represents the mean i standard error (SE) of three independent tests.

of AFB1 in the gowing colony was correlated to the morphological development of
sponrlation in A. parasiticus SU-l.

The Accumulation of the Nor-l Protein, the Activity of the nor-1 Promoter, and
the Accumulation of Aflatoxin in A. parasiticus SU-l Grown on YES Agar. Fungal
spores were inoculated onto the center of YES agar in a Petri dish. Samples (C2, C4, C5,

C6, M4, M5, M6, P5, and P6) were collected as described (Materials and Methods) and

shown schematically in Figure 3-5B. Western blot analysis of crude mycelial extracts

 

113

Nor-l

 

P6 P5 M6 M5 M4 CG C5 C4 C2

P5, P6

C4, C5, C6
C2, C4, C5, C6 I a

Figure 3-5. Westem blot analysis of the accumulation of the Nor-l protein in A.
parasiticus SU-l gown on YES agar. Each lane contained 10 ug crude protein and was
probed with the double-purified PAb (10 rig/ml). A: Lanes: C2, C4, C5, and C6,
samples collected from the central region 2 days, 4 days , 5 days , and 6 days afier
inoculation; M4, M5, and M6, samples collected from the middle region 4 days, 5 days,
and 6 days after inoculation; P5, and P6, samples collected from the peripheral region 5
days , and 6 days afler inoculation. B: A schematic of the sample collection procedure.

114

«AFB1
e—AFG1

 

C2 C4 CS C6 M4 M5 M6 P6 P5

Figure 3-6. TLC analysis of the accumulation of aflatoxins in A. parasiticus SU-l gown
on YES agar. Aflatoxins from each sample (mycelia and agar) were extracted with
chloroform and assayed by TLC with chloroform-acetone (95:5) as the developing system.
Fluorescence was detected with long wavelength UV light. A. Lanes: C2, C4, C5, C6,
M4, M5, M6, P5, and P6 are samples collected as explained in the legend of Figure 3-5.

115

Table 3-4.

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116

showed that the Nor-1 protein (31 kDa) was present in all the samples (Figure 3-5A). In
the central region, the Nor-1 protein appeared 2 days after inoculation (C2), reached the
highest level 4 days after inoculation (C4), and declined 5 days and 6 days after
inoculation (C5 and C6). In the middle region, the Nor-l protein appeared 4 days alter
inoculation (M4), and increased 5 days and 6 days after inoculation (MS and M6). In the
peripheral region, the Nor-1 protein appeared 5 days after inoculation (PS), and increased
6 days after inoculation (PG). For each sample, the GUS activity was measured
spectrophotometrically. The accumulation of aflatoxins was determined by TLC (Figure
3-6). The relative intensities of the Nor-l protein in Western blot and AFB1 on TLC were
determined with an EPSON ES-IOOOC scanner (EPSON Accessory, Singapore) and
quantitated with Sigma Gel software. These results (along with the accumulation of AFB1
measured by ELISA) are listed in Table 3-4 to facilitate comparison. The quantity of
AFB: in ELISA analysis and the relative quantity of AFB1 in TLC analysis in each sample
was consistent with the relative quantity of Nor-l protein in Western blot analysis (Figure

3-5), and was consistent with the level of nor-l promoter activity in the GUS assay.

DISCUSSION

The data presented in this study demonstrated that the PAb raised against the Nor-
lc/MBP were suficiently specific for detection of the (native) Nor-1 protein by Western
blot analysis. The data showed that the accumulation of the Nor-1 protein (in liquid media
and on solid media) and the activity of the nor-l promoter (on solid media) are consistent
with the pattern of the accumulation of aflatoxin and are closely correlated to the

morphological development of sporulation in A. parasiticus. The data also suggested that

117

the expression of nor-l is regulated in a temporal and spatial pattern in A. pm'asiticus
grown on solid media.

The first step in this study was to prove that anti-Nor-lc/MBP PAb were suficiently
specific to detect the Nor-1 protein in Western blot analysis. This step was necessary not
only because the imrnunogen used in this study was a fission protein composed of the Nor-
lc protein (instead of the Nor-l protein) and MBP, but also because the fusion protein
was likely contaminated with the bacterial protein. It was not surprising that the antiserum
specificity was low (indicated by the cross reactivity in Western blot analysis). Therefore
the antiserum could not be used in Western blot analysis of the Nor-1 protein without
validation. The only major protein band detected by the triple-purified PAb not only had
the expected size of the Nor-l protein (31 kDa) but also was detected by the IgG fi'action
and the double-purified PAb in crude mycelial extracts fi'om the wild-type A. parasiticus
SU—l. The same protein band was not detected in crude mycelial extracts from the nor-l
disrupted strain. Therefore the double-purified PAb (or even the IgG fiaction) could be
used to monitor the accumulation of the Nor-l protein in Western blot analysis.

A 28-kDa protein band was also detected by the IgG fraction and the double-purified
PAb, but was not detected by the triple-purified PAb. One possible explanation is that the
28-kDa protein and the Nor-l protein have similar antigenicity. One fraction of the total
PAb (raised against the Nor-lc/MBP) may have a higher afinity to the 3 l-kDa Nor-1
protein and a lower afinity to the 28-kDa protein. Another fraction may have a lower
amnity to the 3 l-kDa Nor-1 protein but a higher amnity to the 28-kDa protein.

Therefore, if an excess of the double-purified PAb was used for further purification using

118

amnity chromatography, the binding sites for the Nor-1c protein on the afinity column
could be saturated by the PAb fiaction which had the higher afinity to the 31-kDa Nor-l
protein. This fi'action was then purified (triple-purified PAb). This could be one possible
reason that the double-purified PAb detected both the Nor-1 protein and the 28-kDa
protein, but the triple-purified PAb detected only the Nor-1 protein in Western blot
analysis.

The pattern of the 28-kDa protein accumulation was consistent with the pattern of the
Nor-l protein accumulation. It is possible that the 28-kDa protein is also involved in the
AFB1 pathway. The 28-kDa protein and the Nor-l protein may be synthesized at the
same time and combine with each other in order to increase the eficiency of aflatoxin
synthesis. The combination of the two proteins may stabilize the 28-kDa protein.
Therefore, when the nor—1 gene is disrupted, there is no Nor-l protein existing and the 28-
kDa protein is rapidly degraded.

The temporal pattern of the accumulation of the Nor-1 protein and the accumulation of
AFB: are closely correlated in A. parasiticus grown in liquid medium. In batch
fermentation (YES liquid medium), the accumulation of the Nor-1 protein and the
accumulation of AFB1 increased simultaneously and quickly during late log-phase of the
fiingal growth and reached a maximum level at stationary phase. The level of the
accumulation of the Nor-1 protein declined at approximately the same time that the
accumulation of AFB1 reached its peak level.

In the nutritional shift assay, the accumulation of the Nor-l protein and the

accumulation of AFB1 began simultaneously and increased quickly a short time after the

ll9

shift fi'om the nonafiatoxin-inducing PMS medium to the aflatoxin-inducing GMS medium.
The Nor-l protein accumulation data were consistent with the nor-1 transcript
accumulation data previously reported (Skory et al., 1993; Trail et al., 1994; Trail at al.,
1995).

In PMS medium, no Nor-1 protein was detected, but AFB: was detected. This
apparently inconsistency may be explained in two possible ways. In the nutritional shift
culture, the AFB: concentration in PMS medium was much lower than in GMS medium
after the shift. Therefore, the corresponding Nor-l protein in PMS could be too low to be
detectable in Western blot analysis in this study. The second possible explanation may
come fi'om the result of nor-l disruption. The disruption of the nor-l gene resulted in an
accumulation of norsolorinic acid (NA) and a significant decrease in aflatoxin
accumulation (Trail etal, 1994). Thisresult suggeststhatasmall quantityofAFB: couldbe
synthesizedandacctmmlatedwithoutthefimction oftheNor-l proteininPMS medium.

The data from liquid batch fermentation and the nutritional shift assay confirmed the
observed expression pattern of the nor-l gene (at the level of transcript accumulation) in
A. pamsiticus grown in liquid media previously reported (Skory et al., 1993; Trail at al.,
1994; Trail at al., 1995) and demonstrated that the accumulation of the Nor-l protein can
provide an alternative measurement for the expression of nor-l. Therefore, it is also
reasonable to predict that the accumulation of the Nor-l protein can be used as a
measurement for the expression of nor-l in A. parasiticus grown on solid media.

The accumulation of the Nor-l protein was higher at 72 h than at 36 h in YES liquid

medium, but the rate of AFB1 accumulation between 60 h and 72 h was much lower than

120

between 36 h and 48 h. One possible explanation is that at 36 h most of the Nor-1 protein
was active, while at 72 h most of the Nor-l protein was inactive possibly because of the
absence of NADPH (Chapter 1, Regulation of aflatoxin biosynthesis) or other unknown
factors (Chapter 2, Discussion).

An alternative explanation comes fi'om the observation that the A. flaws group is not
very eficient at degrading aflatoxins under normal conditions (Doyle and Marth, 1978). It
is possible that when aflatoxin concentrations reach high levels in the medium, fungal cells
are forced to eficiently degrade aflatoxins to survive. AFB: could be synthesized at the
same rate in the period fi'om 60 h to 70 h and inthe period from 36 h to 48 h because the
similar accumulation of the Nor-1 protein was observed in the two periods of time. But in
the period fi'om 60 h to 70 h, a certain amount of AFB: was possibly degraded. This
degradation could result in the much lower rate of AFB: accumulation.

The morphological development at the colony level of A. parasiticus grown on solid
media in a Petri dish appears to be controlled by genetics and environmental influences.
Here, morphological development at the colony level is defined as the development of the
shape and color of a colony and distributions and colors of substrate hyphae, aerial hyphae,
immaturespores, maturespores, anddegenerated hyphalcellsinthecolony. Regardlessofthe
irregulargeometricalshapeoftheareaocwpiedbytheinoculated sporesandtherandom
orientation of the germination of individual spores, the resulting colony and the concentric
regions of distinct morphology in the colony are almost always nearly circular. Nutrients in
YESagarareevenlydistributed. Nutrientsincoconutagararenotalwaysevenlydismbuted

becauseitmaycontainlargepiecesofcocomrtflakeswithdifi‘erentsizes(Chapter4).The

121

nearly circular shape of the colony and the concentric regions of distinct morphology in the
colonyappearedonbothmedia. Butthesetwomediadohavedifi‘erentinfluencesonthe
colors and widths of concentric circular regions of distinct morphology in the colony. These
observations suggested that the morphological development of A. parasiticrrs is controlled by
genetic and environmental influence at the colony level. In another words, morphological
developmentappearstoberegulatedatthecolonylevel.

Sporulation is one example of morphological development. The Aspergillic m‘dulansfluG
geneisnecessaryforthesynthesisofasmalldifirsiblefactorwhichmaybeanextracellular
sigrfldirecfingasemdsponflafionmdpehapsomeraspecmofmlonygrowmaeeand
Adams, 1994a, 1994b, and 1996). This observation suggests that sporulation is regulated at
the colony level by cell-cell communication.

The accumulation of the Nor-1 protein, the activity of the nor-l promoter, and the
accumulation of aflatoxin are consistent with each other and are possibly regulated at the
colony level through an unknown mechanism which is closely related to the morphological
development of sporulation in A. parasitic-us grown on YES agar. Because of the inherent
dificulties encountered in the separation of substrate hyphae, aerial hyphae, immature
spores, and mature spores, the procedure of collecting samples from YES agar used in this
study was a crude approach to achieve this separation. Nevertheless, the accumulation of
the Nor-l protein and the activity of the nor-1 promoter (indicated by GUS activity)
increased and decreased at the same time (temporal level) in A. pw'asiticus SU-l grown
on YES agar. Higher levels of the Nor-l protein accumulation and the nor-l promoter

activity were detected in regions containing conidiophores and immature conidia than in

122

regions containing only substrate hyphae and aerial hyphae (spatial regulation). Decreased
levels of the Nor-l protein accumulation and the nor-l promoter activity were observed in
regions containing only mature spores and degraded hyphal cells. In the center of the
colony, the accumulation of AFB: increased (per unit of dry weight) at the same time that
the accumulation of the Nor-l protein (or GUS protein) decreased. This phenomenon
may result fi'om the fact that AFB: is much more stable than the Nor-l protein. The value
for AFB: accumulation likely represents a true measurement of accumulative. The value
for Nor-1 protein accumulation represents synthesis and degradation. In the center of the
colony, when degenerated hyphae were not able to synthesize new Nor-l protein, the
accumulation of the Nor-1 protein decreased but the accumulation of AFB: still increased.
These patterns were consistent with the pattern of the relative intensity and distribution of
blue color (indicating the activity of nor-l/GUS) shown on a colony using the same nor-
l/(RIS reporter construct with S-bromo-4~chloro-3 -indolyl glucuronide as the substrate
(Chapter 4). Also consistent with these observations, immunolocalization data presented
later in this dissertation (Chapter 4) showed that more Nor-1 protein accumulated in
conidiophores and conidia than in vegetative cells. Further, when a sporulation related
gene (fluP) was disrupted in an aflatoxin-producing strain of A. parasiticus, fungal hyphae
gown on solid media did not sporulate and AFB: remained at the same low level during a
6—day period of incubation (Chapter 5).

Because sporulation appeared to be regulated at the colony level and a close correlation
between sporulation and the accumulation of aflatoxin (as well as the accumulation of the

Nor-1 protein and the activity of the nor-1 promoter) was observed, the accumulation of

123

the .Nor-l protein and the activity of the nor-l promoter may also be regulated at the
colony level.

The correlation between aflatoxin biosynthesis and sporulation in toxigenic A.
pausitr’cus needs further study. Certain sponrlation-related pignents with the basic structure
of norsolorinic acid were detected in the Awergillus genus (Steyn et at, 1980). Spore
pigments from A. nidulans and A. parasiticus also appear to be produced via
polymerization of polyketide precursors (Brown et al., 1993; Brown and Salvo, 1994). A
development-related gene wA in A. nidulans possibly encodes a polyketide (Mayorga and
Timberlake, 1992). The A. parasiticus polyketide synthase gene pksA is required for
AFB: biosynthesis (Chang et al., 1995). Therefore, it is possible that a common signal(s)
at an early stage of development triggers both aflatoxin synthesis and sporulation. When
sporulation is inhibited in liquid media, only aflatoxin synthesis occurs in A. parasiticus.
When the mechanism of aflatoxin synthesis is absent in nontoxigenic fungi or mutation(s)
of the genes involved in aflatoxin synthesis occurs in toxigenic fungi, only sporulation
happens-

In summary, the data presented in this study showed that the accumulation of the Nor-
] protein (in liquid media and on solid media) and the activity of the nor-1 promoter (on
solid media) are regulated in the same pattern as the accumulation of aflatoxin and are
closely correlated to the morphological development) of sponrlation in A. parasiticus.
These results also suggested that the expression of nor-1 is regulated in a temporal and

spatial pattern in A. parasiticus gown on solid media.

CHAPTER4

LOCALIZATION OF NOR-l PROTEIN ACCUMULATION AND
LOCALIZATION OF nor-1 PROMOTER ACTIVITY DURING AFLATOXIN Bl
BIOSYNTHESIS IN ASPERGILL US PARASITICUS

ABSTRACT: Anti-Nor-lc protein polyclonal antibodies were used in immunolocalimtion
experiments to localize the accumulation of the Nor-l protein (at the cellular and 11pr
level) and a nor-l promoter/GU S (nor-l/GUS) reporter construct was used to localize the
activity of the nor-1 promoter (at the hyphal and colony level) during aflatoxin B1
biosynthesis in Aspergillus pamsitr'cus gown in liquid and/or on solid gowth media.

The Nor-l protein was mainly localized in the cytosol of vegetative hyphae (although a
sigrificant quantity appeared to be associated with particles of unknown composition) either in
liquid gowth medium using cell fiactionation/Western blot procedure and in situ
innmmolocalintionprocedmesorfiomsolidgowthmedimnusinganinsim
imrmrnolocalization procedure. On solid gowth media, the highest level of the Nor-1 protein
wasassociatedwithimmatureconidia higherlevelswereassociatedwithconidialstalkslower
levelswereassociatedwithvegetativehyphae, andnonewasassociatedwithmatureconidia(at
thehyphallevel). Onsolidgrowthmediimthehighestlevelofflreactivityofthemr-l
promoter was detected in conidial heads, higher levels in conidial stalks, and lower levels in

vegetative hyphae (at the hyphal level). At the colony level, the activity of the nor-1 promoter

124

125

wasmtdetededhregonswmaMngomymbsuatehyphaeormatumconidiaMW
detected in regions containing aerial hyplne (higher levels) and/or irmnature conidia (the
highest level). These data mggest that localization of Nor-l protein accumulation and
localization of nor-l promoter activity are closely correlated to the process of sporulation in a
colony. Therefore the regulation of nor-l gene expression may be regulated at the colony level

dmingaflatoxinbiosymlresisirrApar-asiticusonsolidmedia.

INTRODUCTION

Manystudieshavebeenconductedwithagoal ofunderstandingthegenesandtheproteins
involved in afiatoxin biosynthesis (Chapter 1). Little effort, however, has bear directed toward
definingwhereaflatordnbiosymhesisoccursatthecelhrlarlevel, atthehyphal level (vegetative
hyphae, conidial heads, conidial stalks, and conidia), and at the colony level (regions in a colony
corrtairringsubstatehyphaeaerialhyphaeimmamreconidia, and/ormatureconidiaina
colony).

SeveralenzymeactivitiesinvolvedinaflatordnB: (AFB1) syntlresishavebeendetectedin
the cytosol while others are associated with organelles (Bhatnagar et at, 1989; Yabe et at,
1989; Yabe et at, 1993; Yabe and Hamasaki, 1993; Matsushima et al., 1994). These data
wggwmmmfiomhedsomedniscaniedommdifimwmrhrwmpamm
Therefore, aflatoxin pathway enzymes or intermediates may be transferred fi'orn one cellular
compartment to another during aflatoxin biosynthesis.

Westernblotanalysesandnor—l/GUS assayspresentedinChapterB suggestthatthe

regulation of Nor-1 protein accumulation and the regulation of nor-1 promoter activity are

126

closelycon‘elatedtotbeprowssofspomlationatthecolonylevel. Anunderstandingofthe
localintion ofNor-lproteinacarmulationandthelocalimtion ofmr-l promoteractivitymay
remhmabeaamdastmdingofflrewndafionbaweardresemolocdimfionsandthe
processofspomlationduringaflatordnbiosynthesis. T'hisinformationmaydirectlyorindirectly
leadtothedevelopmentofsafe, specificfirngicidesornaturalplantproductswhichcanblock
flnmuacdhdunansfaofAFBrpathwayenzymesorhnamedimesfiomomwnmamnmtto
another,md/orhnafaewiflrflnregdafionofaflatordnbiosyndrefisatthewlonylwdmdin
truninhibitAFBrbiosynthesisinpr'asr'tims.

Twohypothesesareproposedinthisstudy:(l)theNor-lproteinresideslocallyincertain
organelles or in the cytosol; (2) localization of Nor-l protein accumulation and localimtion of
nor-l promoteractivityareclosely correlated to theprocessofsponrlationduringaflatordn
biosyrrthesisinpr'au'ticw.

ThedfiapresemdmChapta-BflsodanonsuatedthmmepolydonflmmbodiesGAb)
raisedagainsttheNor—lc proteinweresuficiently specifictodetectthenativeNor—l protein
and that regulation of Nor-l protein accumulation and regulation of nor-1 promoter activity
wereconsistentwiththepattemofAFBraccunmlation. Thereforetwogoalsareproposedin
thisstudy:(l)localizetheaccurmrlationoftheNor-l proteinattlrecelhrlarlevelandatthe
hyphallevelusingthePAbraisedagainsttheNor-lcprotein;(2)10calizetheactivityofthe
nor-1 promoter at the hyphal level and at the colony level using a nor-UGUS reporter
construct.

Thelocalizationdatapresented inthis study demonstrated that the Nor-l proteinwas mainly

localized in the cytosol of vegetative hyphae. The highest level of the Nor-l protein was

127

associated with immature conidia, higher levels were associated with conidial stalks, lower
levdswaeassodaedwithvegaafivehyphaeandmnewasassodaedwnhmaunewmdia
The highest level ofnor-l promoter activity was detected in conidial heads, higher levels in
conidial stalks, and lower levels in vegetative hyphae. At the colony level, the activity of the
nor-l promoter was not detected in regions of a colony containing only substrate hyphae or
matumconidiahnwasdeteaedmregonscomairfingaaialhyphaeafighalevds)or
immature conidia (highest level). These data suggest that localization of Nor-l protein
accumulation and localimtion of nor-1 promote activity are closely conelated to the process of
sponflafionandhmcefliesepmcessmaybemgdatedamewlonylwddufingaflatordn

biosynthesis in A. pw-asiticus on solid media.

MATERIALS AND METHODS

Fungal Strains and Culture Media. Awergilhzspamsificus NRRL 5862 (ATCC 56775,
SU-l) was used as the aflatoxin-producing wild-type strain. An A. manuals strain, nor-
1/GUS, which contains the nor-llGUS reporter construct (Chapter 3, Materials and
Methods), was used to monitor nor-l promoter activity. Fungal strains were maintained as
fiozen spore stock suspensions in 20% (v/v) glycerol at -80°C (Chapter 2, Materials and
Methods). Aflatoxin-inducing liquid gowth medium consisting of 2% (w/v) yeast extract, and
6% (w/v) sucrose (pH 5.8) (YES broth), and solid gowth medium YES agar [YES broth plus
1.5% (w/v) Bacto—agar] were used to gow fungal cells for localization of Nor-l protein

accurmrlation and for localintion of nor-l promoter activity.

128

Fungal Culture. For culture offirngal colonies on solid medium, 5 ul ofa stock spore
suspension (1.5 x106 spores/ml) was inoculated onto the center onES agar and incubated at
29°Cinthedarkforappropriateperiodsoftime. Forfirngalgowthinliquidmediurrtaspore
stockwspmsimwasmoadatedhnoYESbrothwafimlspomconcanrafionoflfixw’
spores/ml. Thewlunewashrarbatedat29°Cwithconstamflnldng(150rpm)mflredarkfor
appropriateperiodsoftime. Myceliawerecollectedbyfiltration. Forirnrrarnolocalization,the
collectedmyceliawereusedinrrnediately. ForWestanblotarmlysisthecollectedmycelia
wacdfluusedhmnedimdyorfiomundafiqifidmmgmmdstoredat-SWCunfiluse For
fimgalshdeufluneflremethodaccordhigtoflanis(l986)wasmodifiedasfoflows A
mia'oscopeslidewasplacedonflrecerna'ofaPenidishcomairnngYESagar. Twoblocksof
YESagar(appmrdnmdy200nnn3)waephcedattwosepmatelocafionsontheslide A
vohrmeof2ulofsporestocksuspension(l.5xlO‘spores/ml)wasinoarlatedoneachsideof
theYESagarblockexceptthebottomandthetopsidesfollowedbyplacemartofacover
glassontopoftheYESagarblock. 'I‘lreslideswereincrrbatedat29’Cforappropriateperiods
oftinreinthedark. Theslidesandthecoverglasseswithhyphaeattachedwereused
immediatelyforirmmmolocalimtionstudies.

Cell Eactionation. Two protocols were used for cell fractionation. Protocol 1. Cell
breakage by Potter homogenization of protoplasts followed by differential
centrifugation. Thisprotocolwascaniedoutat4°C according toMulleretal(1991)with
certainmodifications. Briefly,mycelia,gowninYESliquidmedimn,wereharvestedby
Monmfifiereaflfighyphflmflwaswadwdmdcewimmfingmfl‘awndsfingofom

M Tris-Cl (pH 8.0), 0.14 M NaCl, and 0.025% (w/v) NaN3 (T SA). Portions (20 g wet weight)

129

ofthewashed hyphalmatwereresuspended in 100 nrl ofcell-wall-digestion bufl‘er consisting
of 1.5% (w/v) Novozyme 234, 0.1 M citric acid (pH 6.0), and 0.6 MKCl for 4 h at 30°C with
gentleshaking. Theprotoplastswerecollectedbypassagethrougha29nnnnylonmeshand
washed three times with protoplast-washing bufl‘a' consisting of 2-(N-morpholino) ethane
sulphorric acid (pH 7.5) and 0.6 M KCl (MES). The resulting protoplasts were resuspended
with modified MES supplemented with 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 0.4
nypsininln‘bitionmrit/mlaprofininSdeithiotlneitoLandO.6MKCL Theresuspended
protoplastsweredisruptedbyaPoflerhomogerfizedAuflrerHThomasCo.) Celldebriswas
removedbycentrifirgationtwiceatl,000ngor 10min. Theresultingcelllysatewas
desigrated the PH attract (obtained by Potter homogenization). The PH extract was
fiacfionfledflodifi‘aanpmddefiacfiomandafinalwpanmamfiacfionbydifi‘amfial
centrifirgation. The1,000xgpelletfraction(PL)wasobtainedbycentrifirgationofthePH
extractatl,000ngor20minfollowedbythreewasheswithl\4ES. Tasmanian
(NU)wasobmmedfiomthepoa-l,000xgmpanmmfiacfimbycamifirgafimm6,000xg
for20minfollowedbythreewasheswithMES. ThenntochondnalfiacfionMDwas
obtainedfi'omthepostnuclear supernatant fiactionbycentrifiigationfor 30minat20,000xg
followedbythreewasheswithMES. Thelysosomalfiaction(LS)wasobtainedfiomthe
postmitochondrial supernatant fiactionbycentrifirgation for 30 min at 50,000 ngollowedby
threewasheswithMES. ThemicrosomalfiactionMC)waspreparedfi'omthepostlysosomal
supernatantfiactionbycentriflrgationforlhat100,000ngollowedbythreewasheswith
MES. The rrbosomal fiaction (RS) was prepared fiom the postmicrosomal supernatant

fi'action by centrifugation at 140,000 x g for l h followed by three washes with MES. The

130

postribosomalsupernatamfiomthisstepwassavedasthe 140,000 x gcytosol fiaction (CS).
Forproteinconcentrationdetermination,aknownvolumeofeachfiactionwasmixedwithan
equal vohrrne of 4% (w/v) sodium dodecyl sulfate (SDS) bufl‘er and boiled for 5 min. Then
drewmemafimwasdeenfinedwithmeBieRadpmtenassaykiflBieRadIabomofies,
Hecules, CA) according to the mamfacmrer’s instructions.
PmtoeolLSeparationoforganeflesandtbecytosolfractionbypmtoplast
permeabil'nation. Protoplasts were prepared as described above. Permeabilization of
protoplasts and separation of the organelle and cytosol fractions separation wee conducted at
4°C accordingtothemethodsodea etaL (1993). Briefly, protoplastswerewashedusing
thefollowingschenezaSminwashwithbufl’el [250mMsorbitoL20mMHEPES-KOH
(pH6.8), 150mMpotassirmracetatemrd5mMmagresiumacetate];a3nfinwashwnhbufi'e
2(50mMsorbitoLand5anagresiumacetate);a5minwashwithbufi‘e3(2.0M
pomsimnaceateZSOmMsorbnoLandSangredmnaceate);andaSnnnwashwith
bufl‘e4(250'mMsorbitol,and5mMmagnesiumacetate). Afieeachwashthecellswere
harvestedbycentrifirgationfor45 secat13,000xg. Thecombinedsupematantsfiomthefour
washesweethecytosolfiacfionmdtherenflfingpdlesweetheorgmeflefiacdon The
proteinconcentrationofeachfiactionwasdeterminedasdescribedabove. Thesetwofiactions
werecalledthePPextracts(obtainedfiompemeabilizedprotoplasts). ’
WesternBlotAnalysisoftheNor-l ProteininCellFractions. Proteinsineachcell
fi'action wee separated by SDS-polyacrylamide gel electrophoresis (PA(E) and blotted to a
polyvirrylidene difluoride (PVDF) membrane (Du Pont Co., Boston, MA) using standard

published methods (Ausubel er al., 1995b). The double-purified anti-Nor—lc protein polyclonal

131

mfibodies(PAb)(Chapte3)weeusedasthepfinmryanfibodymWeuenblmanalysis
hummodeecfionofthepfimmyarmbodywascmfiedomthachenfihmnnescendeecfion
kit (Schleiche & Schuell, Keene, NH) according to the mamrfacturer‘s instructions. The
rdafivethsityofNor-lpmtdnheachhnehWeflenbManalysiswasdderfinedby
scanning the X-ray autoradiograph with an EPSON ES-lOOOC scanner (EPSON Accessory,
Singapore)andquantitatedwithSignagelsoftware.
AnalysisofMarkerEnzymeActivities. Themeasm'emertoftheactivityofsuccinate
dehydrogenasewascarried out according to Singe eta]. (1965) withmodifications. This
mahodwasongrrallyusedwmethewtdmnmmwmchebytheuseofmercess
unknownamomrtofsuccinatedehydrogenase. Inthecurrentstudy,thereactionwasusedto
deennnemcdnatedehydrogenaseacfivitybyflreuseofanercessknownmnwmof
succinate. Thereactionwasoptimizedandcarriedoutfor30and60minatroomternperature
inatotalvolumeof3mlconsistingof0.5mlof0.3MTrisbufi‘e(pH7.6),0.1mlof0.lM
potassiumfenicyanide, 0.3 nil of 0.01M succinate, and 0.05 mg of protein. The optical density
ofthereactionmixturewasmeasuredat450nm. Oneunitofsuccinatedehydrogenasewas
definedasflreamoumOfenzymecausingadeeeaseoflnnnolewcdnatemlmhr.
Themeasurement ofcatalase activitywasconducted asdescribedbyCohen etal (1970).
Thereactionwasoptimizedandcaniedoutat4°Cfor1,5,and20mininatotalvolumeof9
ml consisting of] nrl of 2% (v/v) H202 solution in 0.01M phosphate bufi‘e (pH 7.0), and 0.02
mgofproteirr. Thereactionwasstoppedbyadditionoflmlof6NH2804followedby

additionof7mlof0.01NKMnO.. Theresidualeozwasmeasuredbymeamring

132

absorbanceat480nm. Oneunitofcatalasewasdefinedasthearnountofproteinrequiredto
liberate halfthe peroxide oxygen from a hydrogen peoxide solution in 100 s (Luck, 1965).

The activity of acid phosphatase was measured with a commercial assay kit (Signa
Chenical Co., St. Louis, MO) according to the manufacture’s instructions. The reaction was
carried out for30minat37°C inatotalvolumeofS ml consistingof0.5 mlof4mg/mlp-
nitrophenyl phosphate, 5.0 ml of 0.1 N NaOI-I, and 0.02 mg of protein. The measurement is
based on the hydrolysis of p-nitropherryl phosphate by the acid phosphatase yielding p-
nitrophenol. Under alkaline conditions, p-nitrophenol is converted to a yellow complex readily
nreasured by absorbance at 410 nm. The activity of acid phosphatase (Signa units/ml) was
calculated according to the manufacturer’s instructions.

The activity of glucose—6—phosphatase was measured according to the method of Harpe
(1965) with modifications to optimize the reaction conditions. The reaction was canied out at
37°C for 30 min in a total volume of0.3 ml consisting of0.1 ml ofO.l M citrate buffer (pH
6.5), 0.1 ml of 0.1 M glucose-6-phosphate, and 0.03 mg of protein. The reaction was stopped
by addition of 1 ml of 10% (w/v) trichloroacetic acid. After centrifugation, 1 ml of supernatant
was mixed with 5 ml of2 rnM molybdate solution and 1 ml ofreducing agent (40 mM 1-
amino-Z-naphthol-4-sulphonic acid) for 15 to 30 nrin at room temperature. The optical density
was measured at 660 nm. One unit of glucose-6-phosphatase was defined as the quantity of
enzyme required to liberate 1 mole phosphate fiom glucose-6-phosphate in l min.

The activity of glucose-6—phosphate dehydrogerrase was carried out according to Niehaus
and Dilts (1984). The reaction was canied out at 30°C from 1 to 30 nrin in a total volume of

2.5 ml consisting of 2.2 ml of 0.5 mM glucose-6-phosphate, 25 u] of 25 rnM NADP, 25 pl of

133

25 .mM phenazine methosulfate, 200 pl of 5 mM 3-(4,5-dimethylthiazol-2-y|)-2,5-
diphenylteflamlhnnbronfideando.5mgofprotein. Thereactionwasmonitoredby
measuringabsorbanceat578nm. Oneunitwasdefinedastheamomrtofenzymethat
catalyzes the NADP-dependent oxidation of 1 mrnole of glucose-6-phosphate in 1 nrin.

Nor-1 Protein Localization by Immunofluorescence Microscopy. Hyphae gown in
YEShqrfidmediumweewfleaedbyfilmfionmdrewspmdedmfieshYESfiqrMmedimn
(1 mgwethyphaeinSmlliquidmedium). AvolumeofSOulofthehyphal arspensionwas
droppedontoaslidepreneatedwith2°/o(w/v)glycinesolutionandairdried. Thefirngal cells
onflreslideeithefiomliqiriderlnneorfiomslideadmrewerefixed for4hatroom
ternpeature or 16 h at 4°C in I-listochoice Tissue Fixative (Amresco, Solon, OH). For partial
digesfimofflwcdlwalfimgflcdlsweewbjeaedtoacdlwafldigesfimmzymesohfion
consisting of 1.5% (w/v) Novozyme 234 in wate for 1 h at 30°C. The cell membrane ofthe
hyphalcellwaspermeabilizedwitho.l% (w/v) saponininTris-bufl‘eed saline (TBS) consisting
of 100 rnM Tris-Cl (pH 7.5) and 0.9% (w/v) NaCl for 30 rrrin at room temperature. The
hyphae were the: exposed to the triple-purified anti-Nor-lc protein polyclonal antibodies
(PAb) (see Chapter 3) [10 ug /ml in modified TBS supplenented with 0.1% (w/v) saponin
and 1% (w/v) bovine serumalbumin(BSA)] at room temperature for 1 h. This incubationwas
followedbythreerinseswithTBS for 10mineach. The slide preparationwasthenincubated
at room temperature with labeled secondary antibodies [a 100-fold dilution of fluorescein
isothiocyanate (FITC)-conjugated anti-rabbit IgG (Signa Chemical Co., St. Louis, MO)] for 1
h. Afieafinalwashwithwater,thepreparationwasairdried,mountedon slideswith Signa

Diagrostics mounting medium (Signa Chemical Co., St. Louis, MO), and examined under a

134

Nikon Labophot fluorescent microscope or a Zeiss 10 lase scanning microscope (LSM) to
obseve FIT C fluorescence. LSM micrographs wee taken with the help of Dr. J. H. Whallon
(Lase Scanning Microscope Laboratory, Michigan State Univesity, East Lansing, MI).
Semiquantitative Petri Dish Assay for ml/GUS Activity (Overlay Procedure). The
seniquantitativeinsitumeasurenert ofGUSactivitywasconducted accordingtoMbnkeand
Schafer(1993) with certainmodifrcations. Briefly, melted agar [12% (w/v) inwater] was kept
at 45°C in a wate bath and used as the overlay agar. X-glucuronide (5-bromo-4-chloro-3-
indolyl glucuronide, X-gluc, Sigma Chemical Co., St. Louis, MO) was dissolved in
dimethylsulfoxide (DMSO) (0.5 mg/ul), and added to 5 ml of the overlay agar at a final
concentration of 0.3 mg/ml (substrate/agar) and irnrrrediately poured onto a Petri dish
containingamycelial colonywhichhadbeerrinarbated fortheappropriateperiods oftime.
ThePetridishwasgenlyshakenundlflreerfirecolonywascoveedwiththeovelayagar.
Thedishwithovelaywasincubatedatroomtenperaurreuntilthebhre colorflillydeveloped

(W3 h)

RESULTS

Distributions of Marker Enzymes and the Nor-l Protein in Cell Fractions. Cell
fiactionation of mycelial cells was conducted using two methods: (1) disruption of fungal
protoplasts using a Potter homogenizer followed by difi‘eential certrifirgation (the PH method)
and (2) separation of the cytosol fi'om organdles by protoplast permeabilization followed by
centrifugation (the PP method). The resulting cell fractions wee subjected to marke erzyme

assay, to Western blot analysis for the Nor-l protein, and to protein concentration

135

determination. The results of Western blot analysis for the Nor-1 protein are shown in Figure
4-1. Distributions of marke enzymes, the pecentage of the Nor-1 protein (measured by the
relative intensity of the Nor-1 protein band in the Western blot autoradiogaph), and the
pecerrtage of the total protein in each fraction obtained by the PH method and the PP method
are summarized in Table 4-1 and Table 4-2, respectively.

Succinate dehydrogenase (DH) was used as a mitochondrion marke, glucose-6-phosphatase
(G-6-phosphatase) as a errdoplasrrric reticulum (ER) marker, catalase as a peoxisome marke,
and acid phosphatase as a lysosome marke (Muller et at, 1991). Glucose-6-phosphate
dehydrogenase (G6PDH) was used as a cytosol marke (Vida et at, 1993).

High pecentage ofeach organelle marker erzyme was detected in the cytosol fiaction using
the PH method (Table 4-1) (succinate DH, 16.4%; G-6-phosphatase 50.6%; catalase, 39.0%;
and acid phosphatase, 64.0%). Thee data suggest that the PH method disrupts a certain
fiaction of each organelle. Succinate DH (mitocondrion marker) activity was also detected in
the mitochondrial fiaction (32.5%) and the lysosomal fraction (20.1%). Some overlap in
localization of the two markers was expected because mitochondia and lysosomes have similar
sedirnertation coeficients (10‘ to 105 5).

Analyzing the data for the organelle fiaction only, the highest level of G-6—phosphatase (ER
marke) was detected in the microsomal fraction (24.5%). The highest level of catalase
(peoxisome marke) was detected in the lysosomal fraction (20.6%) and the mitochondrial

fiaction (20.5%) because the sedimentation coeficient of peroxisomes (5 x 10‘ s) falls within

136

Figure 4-1. Western blot analysis of the Nor-1 protein in cell fiactions of A. pm'asitr'cus.
Mycelial cells were gown in YES liquid medium for 60 h with constant shaking (150
rpm) at 29°C in the dark. Cell fractions were obtained from the wild-type strain SU-l
(except sample NA‘). The proteins in each fiaction were resolved by SDS-PAGE and
then transferred to a PVDF membrane. Each lane was loaded with 10 ug protein. The
membrane was probed with the double-purified PAb (10 pig/ml). A: Cell fiactions were
obtained using a Potter homogenizer followed by differential centrifugation (the PH
method). Lanes: 1 (CT), cytosol fiaction; 2 (RB), free ribosomal fraction; 3 (MC),
microsomal fraction; 4 (LY), lysosomal fiaction; 5 (MT), mitochondrial fiaction; 6 (NU)
nuclear fiaction; 7 (1K), 1,000 x g pellet from crude extract; 8 (NA‘), crude extract from
a norsolorinic acid (NA)-accumulating strain in which nor-1 was disrupted; 9 (WL), crude
extract from the wild-type strain SU-l. B: Cell fractions were obtained by protoplast
permeabilization followed by centrifugation (the PP method). Lanes: 1 (CT), cytosol
fiaction; 2 (OG), total organelle fiaction; 3 (NA‘), crude extract from an NA-
accumulating strain in which nor-1 was disrupted.

137

Figure 4-1.

‘- Nor-1»

   

CT RB MC LY MT NU 1K NA+ WL CT 06 NA+

138

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Table 4-1.

139

Table 4-2. Distributions of marker enzymes and the Nor-l protein in total organelle and the
cytosol fiactions obtained by protoplast permeabilization and centrifugation.

 

 

 

 

 

 

 

Total organelles Cytosol
Glucose-6—phosphatedehydrogenase (%) 3.0: 0.8 97.0:t2.6
Succinate dehydrogenase (%) 89. 4 i 5. 4 10. 6 :I: 4. 2
Catalase (%) 85. 7 :1: 4. 6 14. 3 t 10. 5
Totalprotein(%) 13.0117 87.0:t11.7
Nor-l relative intensity 15. 0 :t 3. 9 100
Nor-l(%) 2.2:t0.7 91812.3

 

 

 

 

 

Notes:

(1) Total protein (%): (the total protein in total organelles or in the cytosol) + (the sum ofthe
total protein in total organelles and the cytosol).

(2) Nor-l (relative) intensity: 100 x [the intensity of Nor-l protein in one lane (from total
organelles or the cytosol fi'action) on the Western blot autoradiograph] + (the intensity of the
Nor-1 protein in the cytosol on the same Western blot autoradiograph) (Figure 4-lB).

(3) Nor-l (%): {[the intensity of Nor-1 protein in one lane (from total organelles or the cytosol
fi'action)] x [the total protein (%) of total organelles or the cytosol fi'actionI} + { the sum of
[theintensityofNor-l proteinineachlanex thetotal protein (%) ofthe corresponding
fi'action1}.

(4) Each value was the mean :t standard error (SE) from tests performed in triplicate.

the range of sedimentation coeficients of lysosomes and mitochondria (104 to 105 s). The
highest level of acid phosphatase (lysosome rmrker) was detected in the mitochondrial fi'action
(12.4%) and the lysosomal fiaction (11.7%) because of the same reason as mentionw above.
These data suggest that difl‘erential cerm'ifirgation method can successfully fi'actionate
organeflesaccordingmthdrsedhnauafioncoefidanseventhoughahrgemmtbaof
organelles were destroyed by the PH method.

By the PP method, 97.0% of GGPDH (cytosol marker) was detected in the cytosol fi'action
while 89.4% of succinate DH (mitochondrion marker) and 85.7% of catalase (peroxisome

marker) were detected in the organelle fraction (total organelles). In comparison, 83.6% of

140

Figure 4-2. Irmnunolocalizafion of the Nor-l protein in hyphal cells of A. musiticw SU-l
grown in YES liquid medium. Mycelial cells were incubated in YES liquid medium for 60 h
with constant shaking (150 rpm) at 29°C in the dark. The triple-purified PAb was used as
the primary antibodies (10 113/ml). The secondary antibodies (FIT C-coniugated anti-rabbit
IgG) were diluted loo-fold. Photographs (magnification, 200 x): Al, fluorescence
micrograph of SU-l (the wild-type) grown for 48 h; A2, sample Al under white light; Bl,
fluorescence micrograph ofa nor-l-dismpted strain grown for 48 h; 32, sample Bl under
white light; Cl, fluorescence micrograph of SU-l (the wild-type) grown for 18 h; C2, sample
Cl under white light.

14]

Figure 4-2.

 

142

Figure 4-3. Immlmolocelimtion ofthe Nor-l protein inA. pa'an'ticw SU-l gown on YES
agar. IheflmgtswasinwbatedonYESagarat29°C for60hinthedarlaThe triple-purified
PAb was used as the primary antibodies (10 ug/ml). The secondary antibodies (FITC-
conjugated anti-rabbit IgG) were diluted loo-fold. Pbotogaphs (magnification, 280 x): Al,
fluorescence nncrogaph of SU-l (the wild-type); A2, sample A1 under white light; Bl,
fluorescence microgaph ofa typical hyphal cell SU-l (the wild-type); B2, sample B1 under
whitelight;Cl,fluorescencemicrogaphofanor-l disruptedstrain;C2,sampleCl under

143

Figure 4-3.

 

144

Figure“. Inmmolocalization oftheNor-l protein inA. pmuw'ficus SU-l bylaserscanning
microscopy(LSM). MycelialcellsweregrowninYES liquidmediumat29°Cinthedarkfor
48 h with constant shaking (150 rpm). The triple-purified PAb was used as the primary
antibodies (10 ug/ml). The secondary antibodies (FIT C-conjugated anti-rabbit IgG) were
diluted loo-fold. Pbotogaphs (magnification, 350 x): A, hyphal cells under white light; B,
fluorescence micrograph of sample A; C, fluorescence micrograph (the lower part) taken in the
X-Yplaneandthephi—zsectionmicmgaph(theupperpart)takenalongtheZaxisand
throughasflaigmwhitefinesdeaedmthelowerpan(seedetailsinDiswssion)

145

Figure 4.4.

C=689 I=218

200l=70 (2:435 I=Q44 F0 L488

C=425 I=454 F0 L488

 

146

  

Vesicle

Vegetative cell

Foot cell

/

  

Figure 4-5. Schematic of a conidiophore of Aspergillus maritime The process of
sporulation in A. parasiticus starts with a substrate somic hyphal cell which is called the .
foot cell. The foot cell branches to give rise the conidiophore which is a long, erect. hyphal
cell terminating in a bulbous vesicle. As the multinucleate vesicle develops, a large
number of conidiogenous cells, which are called phialides (or sterignata), are produced
over the vesicle. One or two layers of phialides (the first layer of phialides is sometimes
called metula) may be produced. As phialides reach maturity, they begin to form conidia
at their tips one below the other in chains (Alexopoulos and Mims, 1979; Kale et al.,
1994). The conidiophore does not include the foot cell and conidia. To facilitate
description of the localization of Nor-l protein accumulation and the localization of Nor-1
promoter activity, the vesicle with phialides attached is called “the conidial head”; the long
and erect structure of the conidiophore is called the conidial stalk in this study. The
schematic figure of a conidiophore was drawn according to previous descriptions
(Alexopoulos and Mims, 1979; Kale et al., 1994) and scanning electron microgaphs
(Tsuneda, 1983).

147

succinate DH and 61.0% ofcatalase were detected in the organelle Won by the PH method.
msmggestflmfewaorganefleswaedesuoyedbythePPmahodthanthePHmdhod

The majority of Nor-l protein was detected in the cytosol fraction (95.4%) and a trace
Wwasddeaedmthemdear,numchondfiaLlymsomaLnfiaosomaLandnbosomal
fi'actions (0.8%, 2.4%, 0.6%, 0.2%, and 0.6%, respectively) by the PH method. The majority
of the Nor-l protein was detected in the cytosol fiaction (97.8%) and a trace amount was
detectedinthe(total)organellefiaclion(2.2°/o)bythePPmethod. Thesedatasuggestthatthe
Nor-l proteinisnottightlyassodatedwithcellularorganelles.

Subcellular Immunolocalization of the Nor-l Protein. The Nor-l protein was localized
insiminAparasiticusgowninliquidmedimnandon solidmedimnusingafluorescent
antibodyprobe. TheappearanceofgeenfluorescencewresenceofFHC-labeledsecondary
antrbody) was used as an indicator of the level of Nor-1 protein acammlation (Figure 4-2).
Usingthisinurnmolocalimtiontechnique, mostoftheNor-l proteinappearedtobelocalizedin
thecytosol andasigrificantquantityofNor—l proteinwasfoundto associatewith“particles”
of uncharacterized compositions in hyphal cells florn the wild-type strain A. parasitic“ SU-l
gowninYES liquidmediumfor48h(Frgure4—2A). Incontrast,thefluoresccncewasnot
detectedintwonegativecontrols, anor-l mutant (genedisruption) ofiApaasificusgownin
YES liquidmediumfor48 h(Frgure4-ZB) arrdflrewfld-typeApmariticras SU-l gownin
YES liquid medium for 18 h (Chapter 2) (Figure 4~2C). More information was obtained fl'om
thefungusgownon solid media. Theimmatm‘econidium showedthemostintensivegneen
fluorescence (during photogaphy, the most intensive geen color artifactually appeared
yellow); while the corresponding conidial stalk showed stronger fluorescence than that

148

observed in the vegetative hyphal cell (Figure 4-3A). In ageernent with the data flom liquid
culture, the majority of Nor-1 protein was localized in the cytosol. However, a significant
quantitywasalsofoundtoassociatewith‘particles”inhyphalcellsofthewild-typeA.
parasiticus SU-l gown on YES solid medium for 60 h (slide culture) (Figure 4-3B). The nor-
1 disrupted rrrutant (negative control) gown on YES solid medium (slide culture) for 60 h did
not show detectable fluorescence (Figure 4-3C). Laser seaming microscopy (LSM) was used
to collect more information about the subcellular location of the Nor-1 protein. Micrographs
ofhyphalcellsfi‘ompr'asiticusSU-l gowninYES liquidnwdiumfor48 hweregenerated
by LSM using white light (figure 4—4A), fluorescence (Figure 4-43), and fluorescence in phi-z
section (Figure 4-4C). The phi-z section represents the optical section taken in the vertical Z
plane(theupperpart ofFrgure4-4C) whichpassesthrough a straight line (thewhitelinewhich
passes through a selected fluorescent “particle” shown in the lower part of Figure 4-4C) in the
micrograph taken in the X-Y plane (the lower part of Figure 4-4C). The position and the
shapeofthe fluorescent“particle”inthe phi-z section indicatedthatitwaslocatedwithinthe
cellandnot stucktotheoutsidesurface. Aschematic ofthe strucmreandthedevelopment of
a conidiophore are shown in Figure 4-5 for reference.

In Situ Localization of nor-l Promoter Activity in a Colony of A. Wars. The
close correlation between nor-1 promoter activity and the process of sporulation in A.
parasiticwgownon solidmediawasinitiallyobservedusinginvitroanalysis(Chapter3). In
orderto confirmthe invitro data, in situ localization ofnor-l promoteractivitywasconducted

in a fungal colony. An A. pw-asificus strain, nor-llGUS (the same strain as used in

149

Figure4-6. Insituanalysisofnor-l promoteractivitywithinacolonyoprmasiacusgown
onsolidmedium.Thefimgusinallthecolonies(exceptcolonyB)wasaA.pausificus
transformantcontainingamr-l/GUSreporterconstruct. Thefimgusincolonwaasthe
wild-type SU-l which did not contain the nor-l/GUS reporter construct. All colonies were
hmbatedonYESagarmPeuidislresmdthmovaiaidwimmovaiayagarwmmfinga
chrornogenic GUS substrate X-glucuronide. Photogaphs: A and B (magnification, 0.7 x),
coloniesgownfor3days(colonyAanchadthesamemorphologybeforebeingoverlaid);
C (magrification, 5 x), part of a colony gown for3 days; D (magnification, 30x), vegetative
hyphalcells,andhyphalcellmasses(bhre)ofacolonygownfor5days;E(magnification,25
x),conidialheadsandconidiaofacolonygownfor4days.

150

Figure 4-6.

 

151

Chapter 3) which contains a nor-l/GUS reporter construct integated at the nor-1 locus
(Chapter3),wasincubatedon YES solidmedimnforappropriateperiods oftimeandthen
overlaid with melted agar (45°C) containing a chromogenic B-glucuronidase substrate (5-
bromo-4-chloro-3-indolyl glucuronide). Accumulation of a blue colored end product
generated by B-glucuronidase indicates B-glucuronidase activity. The blue color was observed
inallareasofthecolonyexceptattheoutermarginmgrre4-6A). Nobluecolorwas
observed in the control colony of the wild—type SU-l which did not contain the nor-l/GUS
reporter construct (figure 4-6Bl

The following describes GUS activity in a section (Figure 4—6C with a higha magnification
than Figure 4—6A) of a colony of the nor-l/GUS strain (gown for 3 days) proceeding from the
outer margin gadually toward the center. No blue color was detectable in the peripheral
region (0.1-0.4 cm in width) in which only substrate hyphae were observed. An blue color was
observedintheadjacent region(0.l-0.4cminwidth)inwhichaerial hyphaeweredominant. A
more intense blue color was observed in the next adjacent region (0.8-1.0 cm in width) in
which immature conidia were dominant. A less intense blue (or no blue) color was observed
atthecenter(0.04-0.06cmindiameter)inwhichonlymatureconidiawerefound.Theinsitu
datawaeconsistauwiththemvimdata(Chapta3)wlflchwggestedthatmr-l promoter
acfivitywasdoselycorrelatedtoflrepmcessofsponflafioninpr'asifials. At certain stages
of gowth on solid media (e.g. afler 5 days of incubation), some vegetative hyphae formed
tightly interwoven round structures which showed an intense blue color (Figure 4-6D). When
a colony was overlaid with the overlayer agar (containing X-glucuronide) and was observed

underadissectionmicroscopeatlS-nrinfinreirrtervalsthebhrecolorappearedflrstinthe

152
conidialheathhconidiaaflachedsecondintheconidialstallgandlastinthevegetative

hyphal cell (Figure 4-6E).

DISCUSSION

Threetentafivecondusionscanbedmwnfi‘omflredatapresentedinflfissmdy: (l)the
Nor-l proteinwaslocalizedmainlyinflrecytosolofthehyphalcefl;(2)theNor-l proteinwas
foundminmrauuewmdhandwmdidheadswmdidaalksvegaafivehyphaeandm
conidia in the order flom the highest level to the lowest level (undetectable); and (3) the
localizationofnor-l promoteractivityiscloselyconelatedtotheprocessofspomlationinA.
parasitic-us SU—l gownon solidmedia.

Theprimarypurposeofcellfi'actionationinthissmdywastocheckiftheNor-l proteinis
mdosedmormongyaswdaedwnhcatdnmbcdmmpmfidesororgandlesfihemahods
used for sample preparation for cell fractionation was a major factor which influenced the
reliabilityofsubcellularlocalizationoftheNor-l proteininthisstudy. Oneofthe most widely
used methods for determimtion ofthe effectiveness ofcell fractionation is the bioassay for
specific marker enzymes. The marker enzyme assay suggested that Potter homogenization (the
PH method) destroyedalargemrmberoforganelles. However,themarkerenzymeassayalso
suggested flmdifl‘aarfialcamifirgafionatecluflqueMcedbytheNobdlaureaterat
Claude, efl‘ecfivdysepmateddifl‘aemmbceflrflarparfidesaccordingtothdrdifl‘aem
sedimentation coeficients when used afier sample preparation by the PH method.

The protoplast permeabilization (PP) method was shown to be more efl‘ective in preserving

theintegrityofcellularorganelles. 'I'hismethodcanremovethecytosolfl‘omthepermeabilized

153

cell and maintain organelle associations with the permeabilized cell (Vida et al, 1993). The
bufl‘ers used to remove cytosol contain physiological levels of magnesium and potassium ions,
whichamreqtfiredtomahnainorganefleassodafionsudththepermeabifizedcdl Wrthshort
fimeandlowspwdcaflifirgafionfiecytowlmflpmneabflimdcdbueeflecfivdysepmfled
(Vida etal, 1993).

For example, glucose-6-phosphate dehydrogenase (G6PDH) has been established as a usefirl
cytosolmarkerinAmergiIIus (archasA. niger,A. mduIans, A. apergillus)byseveral
researchers (Carson and Cooney, 1990; Broek et al., 1995; Vida et al., 1993). The high
percentage(97%)ofG6PDH detectedinthecytosolfiactionbythePPmethodinthisstudy
was similar to the data (98%) of Vida er al (1993) and Broek et a1. (1995). Higher
percentages of succinate DH (mitochondrion marker) and of catalase (peroxisome marker)
weredetectedintheparficlefiactionbythePPmethodthanbythePHmethod.

Similar percentages ofthe Nor-1 protein were detected in the cytosol fiction by either the
PH method (96%) orthe PP method (98%) suggesting the Nor-1 protein is not enclosed in or
strongly associated with a particle(s) or an organelle(s). Ifthe Nor-1 protein were enclosed in
or strongly associated with a particle(s) or an organelle(s), it might be predicted that a higher
percentage of the Nor-l protein would have been detected in the cytosol by the PH method
thanthePPmethodbecausemoreorganellesweredesfloyedbythePHmethodthanthePP
mahod. The data did not support this hypothesis.

Even thoughthedata suggestthattheNor-l protein is not enclosed in or strongly associated
with a particle(s) or an organelle(s), the highest concentration of Nor-1 protein was detected in

the free ribosomal flaction by the PH method suggesting that a significant quantity of the Nor-1

154

protein is associated with flee ribosomes or with particles (possible the complex consisting of
proteins involved in the AFB1 pathway) with significant mass which comigrated with free
n‘bosomesdmingdifl‘erentialcentrifirgation.
'I'heimrmmolocalimtiondatasupportedanassociation of the Nor-1 proteinwithaparticle(s)
orsomesort. Forexample,inmycelialcellsgowninYESliquidmediumorgownonYES
solidmedium,mostoftheNor-l proteinappearedtobelocalizedinthecytosolbuta
significant quantity appeared to be associated with particles by imnnmolocalization. This
observation was confirmed by Laser Scanning Microscopy (LSM). Comparing the micrograph
takeninflreX-Yplanewiththernicrogaphtakenhrtheverficallplane,afluorescentparticle
waslocalizedinsidethecell. ThererrmhfingNor-lproteinwlrichwasnotassociatedwith
particles, could in theory spread throughout the cytosol Whar the phi-z section was observed
inareaswheretheNor-l proteinwasnotattachedtoanyparticles,thereshouldbea“srnear”
withweakfluorescmcemdflmhdgnofme“mreaf’flmgmez-ardsduecfionshmfldbe
approxirmtelyequaltothecrosssectioml diarneterofthehyphalcell. IfalloftheNor-l
proteinweresimplyattachedtothesurfaceofthecell,thephi-zsectionwouldhaveshowna
circle offluorescence with no “smear”. Inthe actual experiment, the phi-z section showed that
the Nor-l protein, when it was not associated with any particle(s) or organelle(s), was
extensivelydism'butedthroughoutthecytosol.
TheobsavafimflratasigfificmflqumfityoftheNor-lpmtdnappearedwbeassodaed
whh“parfldes”ofhrgemassbymsimmmmolocdinfionmflrstappeammbemmnsistmt
withthedataobtainedbythePHmethodandthePPmethodbywhichonlyalittlearnountof

the Nor-l protein was localized in particles or organelles. One possible explanation which fits

155

the experimental data is that the Nor-1 protein is associated with “particles” of small mass.
'l'hesesmallparticlesareintrnnlooselyassociatedwithparticlesororganellesofrmrchlarger
mass which are detected in situ immunolocalimtion. Such an association could be destroyed
during protoplast homogenization and protoplast permeabilization. Because of limitations of
resolution and magnification, fluorescence microscopy and laser scanning microscopy cannot
distinguishsmallorganelles suchasn'bosomesandperoxisomes. EveniftheNor-l proteinwas
assodatedwithsmaflparfideswhichdism‘hnedeaensivdymthechtheNm-l protein
would still seem to be localized in the cytosol. However, a larger fiaction ofthe small particles
nfighranainassodmeduimmehrgepmfideswhmcdbwaefixedunmedimdyafiahmvefl
forinsituimnurnolocalimtion. Thishypothesiscanbeaddressedmoreeasilybyelectron
microscopy.

TheNor-l protein maybe associated with small particles which may still reside primarily in
thecytosolforanimportantreason. AsanearlyintermediateintheAFBrpathway,
norsolorinic acid could be a precursor for other secondary metabolites. A goup of pignents
withthebasic structure ofnorsolorinic acid andagoup ofxanthoneswiththebasicstmcture
of sterigmatocystin were detected in the germsAmergillus in the 1960’s and 1970’s (Steyn et
al., 1980). Norsolorinic acid, which does not possess a bisfuran ring, does not exhibit
genotoxicity. Versicolorin A (VA) and other later intermediates (after VA) in the AFBr
pathway, which possess a bisfuran ring, are mutagenic and genotoxic (Mori er al., 1985).
Therefore the earlier nontoxic or less toxic intermediates (like norsolorinic acid) in the AFBl

pathwaymaymtbemmpamnanalizedandnmymkepmtmothabiosynflwficpmhways The

156

more toxic intermediates may be compartmentalized in order to protect the producing cell fiom
toxicity and to increase the eficiency of aflatoxin biosynthesis.

The localization of Nor-1 protein accumulation is closely correlated to the process of
sporulation in A. pamiticus SU-l gown on YES agar. Immunolocalimtion data showed that
the conidial head, on which immature conidia were dominant, showed the highest level of the
Nor-1 protein, the conidial stalk showed a higher level of the Nor-l protein than the vegetative
hyphalcdkarrdmatureconidia, whichdidnotattachtotheconidialhead, showedundetectable
Nor-1 protein. Thesedatawereconsistemwithflieinviuodataforflreacamndafionofthe
Nor-1 protein, the accumulation of aflatoxin, and the activity of the nor-1 promoter presented
previously in this dissertation (Chapter 3).

The localimtion of nor-l promoter activity was also closely correlated to the process of
sporulation in A. par-milieu: SU-l gown on YES agar. The in vivo localization of nor-l
promoter activity in a colony was monitored by the nor-l/GU S reporter construct (blue color).
Areas of the colony containing substrate hyphae showed no detectable blue color, areas
containing aerial hyphae showed an intense blue color, areas containing immature conidia
showed a more intense blue color, and areas containing mature conidia showed dark-green
color which was the color of wild-type conidia (during photogaphy the dark-geen color
artifactually turned to dark-brown). The pattern of appearance of blue color was consistent
with the process of sporulation. Sporulation initiates with a foot cell (at the substrate level)
whichgeneratesanaerialhyphalcell. Thisaerialhyphalcellthengoestln'oughaseriesof

morphological steps including a conidial stalk, a conidial head with immature conidia, mauu'e

157

conidia attached to the conidial head, to mature conidia falling off the conidial head. Therefore
the localization of the nor-1 promoter activity was consistent with the process of sporulation.

The correlation between the localization of nor-1 promoter activity and the process of
sporulation was firrther supported by observation of the blue color development in individual
conidiophores under a dissection microscope at 15-min time intervals. The conidial head with
immature conidia was first to show the blue color, the conidial stalk second, and the vegetative
hyphalcelllast. 'I'hisdifl‘erenceintheappearanceofthemr—l promoteractivityindifl'erent
location could be due to two possible reasons: (1) real difl‘erences of the nor-l promoter
activityand GUS activityin the conidial head, the conidial stalk, andthevegetative hyphal cell;
or (2) difi‘erences in substrate permeability. A demonstration of the second hypothesis is
difiarhbecmrsembsfiatepermeabflhyisdeterfinedbyatleasttwofactors:thestructureof
ceflwaflandceflmanbmneofflrefimgusandthechenficalandphysicalnatumofthe
substrate. Howeverthefirsthypothesisissupponedbyinsimimnumoloealizafiondata
Further research is required to confirm this observation.

Sclerotia (surviving structures) are relatively massive structures composed entirely of
vegaafivecdlswhichafisebympeatedhyphalbmdfingmdhnenselyunawomAt
maturity, sclerotia contain a mixture of darkly pignented and hyaline (coloriess) hyphae
(Deacon, 1980). Accumulation of aflatoxins in sclerotia was observed in some strains of A.
flaws and A. parasiticus (Wreklow and Shotwell, 1983). In this study, relatively massive
structures composed entirely of vegetative cells, which showed an intense blue color, were
observed wggesting that these intensely stained blue colored masses may be developing or

irmnature sclerotia. A wild range of environmental and internal factors influence the initiation

158

and subsequent development of sclerotia. For example, internal morphological factors have
been suggested to be involved in the initiation of sclerotia (Chet and Herris, 1975). Therefore,
it is possrble that aflatoxin gene expression is corregulated with two difl‘erent developmental
processes (sporulation and scelrotial development) in A. parasitic-us.

In summary, the localization data suggest that the Nor-l protein resides mainly in the
cytosol of vegetative hyphae and that localintion of Nor-1 protein accumulation and the
localimtion ofnar-l promote activity are closely correlated to the process ofsporulation inA.
parasiticras. ThesnidiesinflfischapterpresanprdMnarydataonthembcdhflmlocalimfion
ofNor-l protein. Theinsitu subcellularimmunolocalimtiondataneedtobeconfirmedbythe

use of electron microscopy.

CHAPTERS

ISOLATION AND ANALYSIS OF HUP, A GENE ASSOCIATED WITH HYPHAL
GROWTH, HYPHAL DEVELOPMENT AND
SPORULATION IN ASPERGEL US PARASIUCUS

ABSTRACT: To study a gene (fluP) encoding a putative polyketide synthase, a
disruption vector was constructed and transformed into Amergillus parasiticus.
Disruption of fluP resulted in slow gowth with cotton-like hyphae, no sporulation, and
reduced aflatoxin accumulation. The data suggest that fluP is associated with vegetative
cell gowth, sporulation, and in turn indirectly influences aflatoxin accumulation in A.

parasiticus.

INTRODUCTION

Research on the molecular biology of aflatoxin biosynthesis may lead to development
of novel methods to reduce or eliminate aflatoxin contamination. However research on
the general biology of the toxigenic fungi encompassing areas such as the, regulatory
mechanisms involved in hyphal gowth, hyphal difi‘erentiation, and sporulation is also
usefirl. Blocking or interfering in any of these processes would help to eliminate or reduce
aflatoxin accumulation. Studies on the genes involved in these basic cell fimctions may
also provide useful information for the development of new fungicides, improvement of

agonomic practices, development of resistant crops (by genetic engineering or classical

159

160

breeding), and development of biocontrol agents (such as nontoxigenic Competitive firngi)
for control of aflatoxin contamination. Unfortunately, relatively little research data have
been published related to this area.

Because aflatoxins are derived from a polyketide pathway (Kurtzman er al., 1987), the
producing fungi are predicted to have a polyketide synthase (PKS) related to aflatoxin
biosynthesis. Previous studies showed that a 680-bp CIaI/HindIII DNA fiagment from an
aflatoxin-producing strain, A. parasitials SU-l, hybridized to a 1.5-kb cDNA fiagment
containing the B—ketoacyl synthase functional domain of 6-methylsalicylic acid synthase
(MSAS) complex fiom Penicilliwn patulwn (Beck et at, 1990). As expected, the deduced
ammaddsequencefiomthe680-prlaUHbrdeNAfingnanwasvaysinflhr(72%
identity)tothatofthe B-ketoacyl synthasefimctional domain ofMSAS ofP.patqun. Itwas
originally hypothesizedthatthegene containingthe680~bp DNAfiagnrerrtfromAparasiticus
amdesaPKSwfichishvoNedhaflflordnbioWefisbecmmemwbfisheddflamggefled
thatpamfinmd/mhsprewmor6-methylsahcyficaddisproducedmprasificus Onthe
other hand, it is also known that certain polyketides are closely related to cell
difl‘erentiation, development, and sporulation (Summers er al., 1995; Revill et al., 1995;
Keller and Adams, 1995). Therefore a reasonable alternative hypothesis is that the gene
containing the 680-bp DNA fiagment is a PKS gene involved in either aflatoxin
biosynthesis, cell gowth, cell difl‘erentiation, or sporulation in A. parasiticus. The data
presented in this chapter demonstrated that disruption of the suspected PKS gene (fluP)
resulted in slow gowth with cotton-like hyphae, no sporulation, and reduced aflatoxin

accumulation. This suggests that fluP is associated with hyphal cell gowth, hyphal cell

161

development, and sponrlation, and in turn indirectly influences aflatoxin synthesis in A.

parasiticus.

MATERIALS AND METHODS

Bacterial Strain, Fungal strains, and Incubation Conditions. Plasmids were
amplified in Escherichia coli DHer Fem/emu] W1 7(rK' mK+)supE44 thi—I recAI

gyrA (Nair) relAI A(lacZYA-argF) Ul69 (m8OIacZAM15)] using standard methods
(Ausubel et al., 1987). Aspergillus parasiticus NRRL 5862 (ATCC 567755, SU-l) was
used as the aflatoxin-producing wild-type strain. A. pw'asr'ticus NR-3, a nitrate reductase
(encoded by the m'aD gene) deficient strain derived fi'om A. parasiticus SU-l (Horng er
al., 1990), was used as the host stain for fluP disruption. Fungal strains were maintained
as stock spore suspension at -80°C in 20% (v/v) glycerol. Czapek-Dox (Difco
laboratories, Detroit, MI) agar (CDA) medium containing sodium nitrate as the sole
nitrogen source was used for selection of niaD” transformants. Aflatoxin-inducing media,
coconut agar medium (CAM) (Arseculeratne et al. , 1969), potato dextrose agar (PDA)
medium (Difco Laboratories, Detroit, MI), and yeast extract-sucrose (YES) liquid
medium and YES agar (Chapter 4) were used to gow fungal mycelia for genomic DNA
and total RNA preparation, for aflatoxin extraction, and for observation of morphology.
Reddy’s medium (Reddy er a1. , 1971), a chemically defined and aflatoxin-inducing
medium, was also used to gow mycelia for RNA preparation.

Fungal Culture, Dry Weight Determination, Growth Rate Measurement, and

Aflatoxin Analysis. For gowth of fungi on solid medium, 5 ul of a stock spore

162

suspension (1.5 x 10‘ spores/ml) were inoculated onto the center of agar media and
incubated at 29°C in the dark. For the non-sporulating fluP-disrupted strain, hyphae were
removed fi'om a flufl'y colony with a toothpick and transferred onto the center of agar
media and incubated at 29°C in the dark. The quantity of hyphae transferred was
determined by scraping the hyphae fi'om the toothpick, resuspending in 1 ml of YES liquid
medium, and spreading the hyphal suspension on CAM Petri dishes. The number of
colonies which gew on each Petri dish was between 50 and 200. The differences in
inoculum level did not appear to influence the gowth rate of the colonies. The majority
of the colonies (95%) initiated fiom toothpick-transfer had the same gowth rate.
Colonies initiated either from spore-suspension (more than 95% showed the same gowth
rate) or from hyphal-transfer with difl‘erent gowth rates (compared with the majority of
the colonies prepared by the same procedure) were eliminated from the analysis. Mycelial
samples were collected by using a spatula to scrape mycelia fi'om the surface of agar. The
collected mycelia were either used immediately, or fi'ozen under liquid nitrogen and stored
at -80°C until use. The dry weight of mycelia gown on solid medium was determined by
the filtration-drying method of Olsson and Jennings (1991). For gowth of fungi in liquid
medium, a stock spore suspension was inoculated into liquid medium to a final spore
concentration of 1.5x10s spores/ml, and gown at 29°C in the dark with constant shaking
(150 rpm). Mycelia in liquid media were collected by filtration through Miracloth
(Calbiochem-Novabiochern Corporation, La Jolla, CA), and were either used immediately, or

frozen under liquid nitrogen and stored at -80°C until use.

163

Colony gowth rate was measured by the time (in days) which the colony took to cover
the surface of agar medium in a Petri dish or by the mycelial dry weight (mg) per Petri
dish.

AflatoxinBr wasanalyzedbyusingdirectcompetifiveenzyme—linkednmmrneabsorbem
assay (ELISA) according to the method of Pestka (1980). Aflatoxin Br monoclonal antibodies
usedinthisstudywereldndlyprovidedbyDr. J. Pestka(DepartmentofFood Scienceand
Human Nutrition, Michigan State University, East Lansing, MI ).

Temporal and Regional Collection of Samples from Solid Medium. To measure
aflatoxin acctmrulation in wild-type A. pm'asiticus NR-3 gown on YES agar in a Petri dish,
mycdialsampleswaecoflectedaccordhrgtoaspecifictemporalandregional scheduleas
described previously (Chapter 3, Materials and Methods). Briefly, samples were collected
fromthecentralregiononESagarZ, 4, 5,and6daysafierinoculation; fi'omthemiddle
region4, 5,and6daysafierinoculation;andfi'omtheperipheral region 5,and6daysafler
inoculation. For collecting samples of the fluP-disrupted transformant gown on YES agar, the
sameregional schedulewasusedbutthetimepointswerechangedto 6,12,15, and 18 days
alter inoculation, because the colony of wild-type NR-3 took 6 days to cover the YES agar in a
Petri dish, while the fluP-disrupted transfonnant took 18 days to cover the same area.

Plasmid Preparation and Protoplast Transformation. Plasmid minipreparation was
performedbytheboilingmethod, andlarge-scalepreparationwasperfonnedbythealkaline
lysis procedure (Ausubel et al., 1987). Transformation of protoplasts of A. pausitr‘cus NR-3
with the disruption vector was conducted using minor modifications of the polyethylene glycol

method (Oakley et at, 1987) as previously described (Skory et at, 1990).

164

Nucleotide Sequence Analysis and Amino Acid Sequence Comparison. Nucleotide
sequawedatawereanalyzedwiththeWuconsinGeneficsComprneerupPadmge
Comparisons of the predicted amino acid sequence to EMBL and GenBank databases hbraries
wereconductedwith’I'FastAandGapandalignedwithPileup fromtheWisconsin Genetics
Computer Group Package.

Isolation and Analysis of Genomic DNA and Total RNA. Fungi mycelia collected fiom
eitherliquidmediaorsolid samplewaeusedforDNAandRNAisolationandpmification.
Genomic DNA was isolated and purified by a published modification (Skory et al., 1990) of a
phenol-chloroform protocol developed for mammalian DNA isolation and purification
(Ausubel et a1, 1995d). Total RNA was isolated and purified using the hot-phenol method as
previously described (Ausubel etal,1995e). Restriction endonucleases utilized in analysis of
DNAwerepm‘chasedfiomBoehringerMannheirnBiochemicals (Indianapolis, IN) orNew
England Biolabs (Beverly, MA) and were used according to the marnrfircturer’s instructions.
Northern (RNA) and Southern (DNA) hybridization analyses were conducted using published
procedures (Ausubel et al, 1995c, 1995f) with the modified hybridization bufl'er and
conditiomrecommended by Stratagene Cloning Systems (LaJolla, CA). Approximatelle pg
ofgenomicDNAor30ugoftotalRNApersamplewereseparatedbyagarosegel
dchophorefisandflwnMnsfenedbycapflhwacfiontoanylonmanbmnemym‘
manbrane, SchleicherandSclurellInc,Keene,NI-I)which servedasasolid support. Radio-
labeledDNAprobesweregeneratedwith[32P]-dCTP(DuPontCo.,Boston,MA)andthe
Random Primed DNA labeling Kit (Boehring Mannheim Biochemical, Indianapolis, IN)
accordingtothemamrfactrua’sinstructions. Afterthefinalwashthemembraneswere

165

exposed to X-ray film (Dodak-XARS, Eastman Kodok Co., Rochester, NY) at -80°C. DNA

probes(specifiedinthefigurelegends)wereusedinbothSouthernandNorthernanalysis.

RESULTS

AnalysisoftheCodingRegionoffluP. InaprevimrssuidyconrhactedbyRRasoolyin
our laboratory, a 680-bp CIaI/HindlII DNA fi'agnent from A. parasiticras was shown to
hybridizetoal.5-kb cDNAfi'agmentcontainingtheB-ketoacylsynthasefirnctional domain of
6-methylsalicylic acid synthase (MSAS) complex fiom Penicilliran patulin» (Beck et at,
1990).

Inflfisstudy,ascarchofEMBLmrdGeankdambasesshowedflratthemnmoadd
sequawearwdedbyflie680-prIaUHmaIHDNAfiagnauwasalsosinfllarmflicB-
ketoacylsynthasefimctional domain ofother6PKS genes (35%to7l% identity overa216-
arnino-acidspan). ToillusuateanaminoacidsequencecomparisonoffluPtotwoPKSgenes
andonefattyacidsynthetase(FAS)geneisshowninFigm'eS-l. Northernblotanalysiswas
usedtoidentilytheappmrdmatesizeandlocationofthecodingregionoffluP. Initially,the
680-prIaI/HindlIIDNAfi'agmentwasusedasaprobetohybridizetototalRNAfi'omA.
maritime: and an approximately 6-kb transcript was detected. Several genomic DNA
fi'agnents (in vector B-E2 fi'om a phage A genomic DNA library of A. parasiticrw) flanking
bothsidesofthe680-prIaI/Hincfl11DNAfi'agrnentwerethenusedasprobesinNorthem

blot analysis (Figure 5.2) Each probe (0.9.rtb SacI/SphI, 0.3-1d) SohI/Psll, 3.0-kb PstI, and

166

Figures-1. AminoacidsequencecomparisonofflquithtwoPKSgenesandoneFASgene
AP,anfinoaddsequence(AAS)anodedbyflre680—bpflagnemofflreAwgilhrs
pw'as'ia'cusfluP gene; PP, AAS of the N-terminus of the Penicllr’ran patulran 6-methylsalisylic
acid synthase gene (PKS gene); AN, AAS of the N-termimrs of the developmentally regulated
A. WwAgeneQKS-like gene); YSC, AASoftheN—terminus oftheSacdrmanyc-es
cerevia’aefattyacidsyntbasedenefiASgene).Theidentityandsimilarityare72%and86%
betweenAPandPP,35%and58%betweenAPandAN,and13%and39%betweenAPand
YSC.

 

167

Figure 5-1.

 

168

 

 

 

 

Clal
SphI Pstl SphI
Sacl Pstl SM Pstl HindlII SacI
_l__ 61 | Genomic DNA
Probes
I II III IV V VI

Figure 5-2. Aschernatic ofthe 9.1-kaachenomicDNAfi'agment containingtheflngene
inAmergillrerrasiricus. DNA‘probesusedinNorthernanalysisto definethetranscribed
region of fluP: I, 0.9-kb SacI/Sphl; II, 0.3-kb SphI/Pstl; III, 3.0-kb PstI; IV, 1.3-kb Pstl; V,
0.68-kb CIaI/Hr'nrflll; VI, 2.9-kb HindIII/Sacl. The arrow represents the proposed direction
of transcription of fluP.

1.3-kb PstI fi'agment) except the 2.9-kb HindIII/Sacl fi'agnent detected the same size
nanscriptasthatdetectedbythe680-prIaIlHindflIDNAfiagment. Sincethededuced
amino acid sequence ofthe 680-bp CIaI/Hindlll DNA fragment was similar to the B-ketoacyl
synthasefimctioml domain locatedintheN-termirmsofMSAS inP.parqun (Becketal,
1990),itwaspredictedthatfluPshouldbetranscribedflomrighttolefiasshowninFigm'eS-
2. The 6.1-kb .Sohl DNA fragnerrt, which contains the 0.3-lib SphI/Pstl, 3.0-kb Pstl, 1.3-kb
P511, and 1.5-1d) Par/5pm fi'agments, was predicted to encompass the 5’-end plus its flanking
region and most ofthe 3’-end offluP, and therefore could be used for disruption vector
construction.

Construction of a Disruption Vector pPKS. The 6.1-kb SphI DNA fiagnent was cloned
into the .Sphl site of pUC19 resulting in plasmid pUCl9—6. The disruption plasmid, pPKS, was
constructed by insertion of a 6.5-kb PvuII fi'agment from pUNH (Figure 5-3), containing a

functional copy of the nitrate reductase gene, niaD, into the SmaI site of pUCl9-6.

169

Fignn5-3.TlnfhrP-dimrpfionveampPKSandascbanaficdesaipfionofflwameaed
resnictionmapofgenomicDNAinthefluP-disruptedu'ansfonmnt. Thesolidblacklineisthe
6.1-kb .Sohlfi'agmentcontainingmost offluP. A6.5-kbfragnent,whichwascutofl‘fl'om
plasrnideNHwasinsertedintothemiddleofthefhrchne.Theshadedline(2.7kb)inthe
6.5 kb fi'agnent is afirnctional copy of the nitrate reductase gene (niaD). Abbreviations: Sp,
SuhLScSacLB,Banm;H,Hmn;smsnaL

 

170

Figure 5-3.

SphI 15.0 kb

HindIII 0.8 kb
BamHI 0.9 kb

PstI 1.5 kb

   
 
  
 
 
  
 
 
 
   

12.3 kb BamHI
SphI HindIIIZ-B kb

11.? kb PstI

HindIII 4.0 kb

BamHI4.5 kb

9.8 kaacI
9.0 kl) BamHI

pPKS was cut with Sphl and the linearized disruption vector was
transformed into NR-3

c = * thch-I

niaD

P

\

      

 

 

Double cross-over

is?" m 1 P Sir

 

 

 

The fluP gene in the genome
11E ip Ecfi l lirScrI fl :lfiSp SjE
niaD —

The disrupted fluP gene in the genome

 

2kb

171

Figure 5-4. Southern blot analysis of genonric DNA in A. parasiticus NR-3 and the quP-
- disrupted transformant. Ten ug ofgenomic DNA from each sample was digested with SacI
(Sa), SphI (Sp), Hindm (Hi), BamI-II (Ba), separated by agarose gel electrophoresis, and
transferredtoaNytranmembrane. A3.0-kastIintemal DNAfragmentofflquasusedasa
probe. A: disrupted, sample fiom the fluP-disrupted transformant (fluP). B: and D: wid,
samplefi'omthewild-typeUIuP‘). C: mixed, samplefiomthecolonywiththefluP/fluP“
genotype. Themnnbersontheleflsideofeachpanelisfliemoleuflarsizestandardladder
(unit: kb).

 

172

Figure 5-4.

Sa Sp Hi Ba Sa Sp Hi Ba Sp Ba Hi Sp Ba Hi

 

“1:56”: ”1in

1‘ l
A: disrupted B: wild C: mixed D: wild

173

Transformation of A. parasiticus NR-3 and Disruption of fluP. Plasmid pPKS was
linearized by cutting with the restriction enzyme Sphl to generate an approximately 12-kb
DNA fiagment. The linear disruption vector was transformed into protoplasts of A.
paramicus NR-3 (Figure 5-3). Nitrate-utilizing transformants (niaD‘) were selected on
Cmpdc-Doxagar(CDA)comahnngsodhunmuateasthesolemnogensomceandthen
nansfaredomococomnagmmedimn(CAM)mwnduaaqualhafivesaemforaflatordn
accrmmlation. All colonies (more than 300) transferred to CAM produced aflatoxins. The
selection oftrarrsformants containing the disruptedfluP gene was then conducted by Southern
blot analysis.

Southern blot analysis of genomic DNA isolated from more than 200 111'er+ transformants
identified two transformants (isolates 10 and 11) in whichfluP was disrupted (Figure 5-4).
Wild-type colonies gown on CAM (Figure S-SA) or on potato dextrose agar (PDA) (Figure
5-5B) gew with normal morphology of hyphae and spores. However colonies of disrupted
msfommmsgewwhhnmchlessaaialhyphaemidhadudddydispasedsporesonPDA
(Figure S-SC) or with cotton-like (flufl'y) hyphae and no spores on CAM (Figure S-SD).
Thereforethisgene fiomApmudtiaaswastentativelynamedfluP for_fl__ufl‘ygene. Wild-type
hyphae and cotton-like hyphae of the disrupted transformant gown on CAM are shown in
Figures S-SE and 5-5F. When the hyphae ofisolates 10 and 11 were transferred onto CAM
(secondary isolates), about 10% of the colonies recovered the wild-type phenotype and wild-
type genotype (fluF, nondisruptedfluP gene) (Figure 5-43 and 5-4D). Another 20 % of the
secondaryisolatesgewasamixtureofnormalphenotypeandabnormal phenotype(cotton-

like hyphae with no spores) and contained both the non-disruptedfluP allele and the disrupted

174

Figure 5-5. Morphology of A. maritime: NR-3 and the fluP-disrupted transformants gown
on coconut agar medium (CAM) or on potato dextrose agar (PDA). Pictures: Al
(magrification, 0.7 x), a colony of NR-3 on CAM; A2 (magnification, 1.4 x), an enlargement
of part of A1; B1 (magrification, 0.7 x), a colony of NR-3 on PDA; B2 (magnification, 1.4 x),
an enlargement of part of B1; C1 (magnification, 0.7 x), a colony of the fluP-disrupted
transformant on PDA; C2 (magnification, 1.4 x), an enlargement of part of C1; D1, a colony
of the fluP-disrupted transformant on CAM, D2 (magnification, 1.4 x), an enlargement of part
of D1; E (magnification, 20 x), hyphae of the wild-type (NR-3) on CAM; F (magnification, 20
x), hyphae of the fluP-disrupted transformant on CAM.

 

Figure 5-5.

175

 

 

176

Figure 5-5 (cont’d).

D1 DZ

 

177

Figure 5-6. Transcript accumulations of fluP, the B-tubulin gene, and the ver-I A gene in
A. parasiticus. Total RNA was isolated from cells gown in Reddy medium (Reddy er al,
l971)for24h,60h,and72h,respectively. ThirtyugoftotalRNAineachlanewas
separatedbyagarosegel electrophoresisandtransferred onto aNytranmembrane. PKSO‘IaP):
a ls-kadIhnernalfiagnerrtofflquasused asaprobe. var-I: a0.6-kb CIaI/EcoRI
internal fragment of the Mar-IA gene was used as a probe. B-Tubulin: a 2.0-kb
HiszH/Bamm internal fi'agnent ofthe B-tubulin gene was used as a probe.

 

178

Figure 5—6.

24h 60h 72h 24h 60h 72h

&Ol(b—>

21)Kb—>

1A Kb—>

 

PKS ver-1 B-Tubulin

 

179

Figure 5-7. Transcript accumulations of fluP in A. parasiticus NR-3 and the fluP-disrupted
transformant. Thirty ug of total RNA was separated by agarose gel electrophoresis, and
transferred onto aNytran membrane. A 1.3-kastI internal fragment offlquasused asa
probe. Lane panel: A: l, 2 and 3, genomic DNA isolated from fluP-disrupted isolate ll, 10,
and fi'om NR-3, respectively, gown on CAM for 72 h; B: l, genomic DNA isolated fiom
fluP-disrupted isolate gown on YES agar for 4 days; 2 and 3, genomic DNA isolated fiom
NR-3 gownonYES agarfor2 and4 days, respectively, 4and5, genomic DNAisolatedNR-
3 gowninYES liquidmediumforZand4days, respectively.

 

180

Figure 5-7.

<—6 kb-*

 

181

fluP allele (fluP‘lfluP) (Figure 5-4C). About 70 % the secondary isolates maintained only the
abnormal phenotype (cotton-er hyphae with no spores) and genotype (disrupted-fluP allele
only). The pattern of phenotype and genotype in the secondary isolates on CAM (the CAM
pattern) was repeatable from one transfer to the next transfer. Interestingly, when the hyphae
of isolates 10 and 11 were transferred onto PDA, the colonies gew with much less aerial
hyphaeandhadwidelydispersedsporesmgure 5-5C). ThispatterniscalledthePDApattem.
When the spores produced on PDA were inoculated onto either CAM or PDA, all the colonies
showed normal phenotype and normal genotype. When the hyphae gowing on PDA were
inoculated onto CAM or PDA, the CAM or PDA pattern appeared again.

fluP Transcript Accumulations in the Wild-Type Strain and the fluP-D’mrupted
Transformant. Total RNA extracted fi'om wild-type mycelia gown in selective Reddy’s
liquid medium (Reddy et al., 1971, an aflatoxin-inducing chemically defined medium), was
subjected to Northern hybridization analysis. A 6.0-kb transcript was detected in the 24-h, 60-
h, and 72-h samples using a 1.3-kb Ps'tI internal fragment of fluP as a probe (Figure 5-6, PKS).
In contrast, a 1.1-kb transcript was only detected in the 60-h, and 72-h (not in the 24-h)
samples using a 0.6-kb ClaI/EcoRI internal fi‘agment of var-1A gene (directly involved in
aflatoxin biosynthesis in A. mariticus) as a probe (Figure 5-6, ver-l). This pattern of
transcript accumulation of ver-IA was similar to that seen in previous studies (Trail et at,
1995). As a control, a 2.0-kb transcript was detected in the 24-h, 60-h and 72-h samples using
a 2.0-kb HirrdIH/BamHI intemal fi‘agnent of the B-tubulin gene (a house keeping gene) as a
probe (Figure 5-6, B-Tubulin). The transcript of the B-tubulin gene appeared in geater

abundance in the 24—h sample than that in the 72-h sample, a pattern seen in previous studies

182

(Trail etal, 1995). In contrast, the transcript offluP appeared in less abundance in the 24-h
sample than that in the 72-h sample. Also, the 6.0-kb transcript was not detected in the two
fluP-disrupted transformants (figure 5-7A, lanes 1 and 2), but was detected in the wild-type
cells gown on CAM for 72 h (Figure 5-7A; lane 3). Normally, sporulation can occur only on
solid gowth media, therefore if fluP is associated with sporulation the corresponding transcfipt
onsofidmediaiserpeaedtoappearingeaterabundanceflunfliatinfiqiudmedia Total
RNA ofthefluP-disrupted transformant was extracted fiom the firngus gown in YES liquid
mediumandYES solidmedium. Attwotimepoints, thefluPtranscriptisolatedfi'omcells
gown on solid media appeared in approximately 3-fold geater abundance (relative intensity on
Northern blot analysis is 100) than transcript (relative intensity on Northern blot analysis is 35)
isolated fi'om cells gown in liquid media (Figure 5-7B).

Aflatoxin Bi Accumulations in the Wild-Type Strain and the fluP-Disrupted
Transformant. In order to determine if fluP had an efl‘ect on aflatoxin accumulation, the
hyphae of the fluP-disrupted transformant and the wild-type (NR-3) were inoculated onto
YES agar. YES agar was used to measure aflatoxin accumulation in this study for two
reasons: (1) the fluP-disrupted transformant gew on YES agar with similar morphology
to the CAM pattern (fluffy-colony with no sporulation); (2) YES is the primary medium
used to study aflatoxin accumulation in this dissertation (Chapter 2, 3, and 4).

The hyphae of NR-3 took about 6 days, while the hyphae of the fluP-disrupted
transformant took about 18 days to coverthe surface ofthe gowth mediumin aPetri dish
Thequantityofhyphaetransferreddidnotappearto afl‘ectthehyphalgowthrateinthis
experiment. Mycelial samples were collected according to the specific temporal and regional

schedule as described previously (Materials and Methods). The dry weight of the mycelia

183

Table 5-1.

.mtmz 0996:? 05 .Lfix. ”fiancee“: voqummvtma§05 2 $3.39
.88. 838.82.. 8....» mo 5% .88 23:8... H 58:. o... 2...? seem Am.

.8882 ...a nausea. <28 .... Began :3 .928 8.33888 2... 5
A8282 28 8.882
... 288.. 83 coca—.82.. .38 93.. w. 98 .996 m. ..3 996 o ...... .98.. m comma. 88in»... 2.. 80.... 882.8 £28.30...
8...... 98.8 m. 98 .98.. m. .9§o N. .3 9.3 e vs. .98.. m .98.. v comma. 882... o... :8... 388:8 £38.80... 8%. A998 w.
.28 .98.. m. .953 N. .98.. o ..3 98.. 0 can .33 m .938 v .98.. N comma. .8280 85 89c 888:8 883 Amaiuggomce.
evasamfitmak. o... .«o ..3 mamz 25.2...» o... «o 8.9.8... 389?. 8.7.... t8m ... 8w“ mm.» co ESE 8.83 88200 A:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.882
...3... 3...: 883.62 33.8 2.2.... 23...... e 2 as...
m .. a o ... e ... a . .n . .2 a e .93 an n a .2 e a n ... .8 e .m a e .... e 2 .8889.
.....36 ...3: c.2342 33.2 «.33... 8.3.? n 2 3.2.8....
8..."... ...3: 33.8. ”.28.". 3...... 3.3... e ... 28.2
«..3... ...3..." 123.8 ...3... 2:5... ...3; n 2 28.2
“..3... 3...: 23.2 3:": “.3: 3.3.. e N. 38.2
e..“: 2...: 3:3... «.23.... «...3... 3.3.. o 2 .380
e..“: ...3: ...3”... ...3...“ Team... «...3... m 2 .880
...3”: ...3...“ ”...3...” ...3; You: ...3... e N. .838
...3; 3:”: «.3: 2:3... ...32... ....«m... N o .880

......x .....x ......x .....x ......x .....x ......x .....x 8.8.8
.32. .5 use... .92 .229»... 8...... as. 2.92. an En... on... 8.8.30 ease .o 8.8...

came mm; ..o gew

28.53.28: Baafivdak. o... 98 642 «acuteg .V ... 88838.88 .m 5.68%. 98 2.383 Ev .2832 .—.m 03:.

184

and. the accumulation of aflatoxin B1 (AFB1) (from the growth medium and mycelia) of
each mycelial sample were determined (Table 5-1).

The overall accumulation of AFB1 (per Petri dish) in NR-3 was approximately lO-fold
higher than that of the fluP-disrupted transformant, while AFB: accumulation per gram of
mycelium of NR-3 (7.0 ug/mg) was 2-fold higher than that of the fluP-disrupted
transformant (3 .2 ug/mg). The overall growth rate of the wild-type was 3-fold faster than
the fluP-disrupted transformant if the time to cover the Petri dish was used as the
indicator, and 4-fold faster if the dry weight per Petri dish was used as the indictor
suggesting that fluP may directly or indirectly control hyphal grth rate. The AFB:
accumulation per gram of dry weight of mycelium was nearly the same (3.2 ug/mg) in all
areas from a colony of the fluP-disrupted transformant which did not sporulate. However,
in NR-3, AFB: accumulation per mg of dry mycelium (3 .2 ug/mg) in the central region 2
days after inoculation (no sporulation) was almost the same as that of the fluP-disrupted
transformant, but increased quickly in the same region when sporulation appeared [for
example 4 days (5.8 pig/mg) and 5 days (7.4 ug/mg ) after inoculation in the central
region]. It was observed that the dry weight of mycelium and aflatoxin accumulation of
the fluP-disrupted transformant started to decrease 15 days after incubation in the central
region. These data suggested that mycelia and aflatoxin degraded after long incubation

times.

DISCUSSION

Based on the data collected in this study, it is reasonable to conclude that fluP is

185

associated with hyphal growth, hyphal development and sporulation, and in turn indirectly
influences aflatoxin accumulation in A. parasiticus. The disruption of fluP resulted in
cotton-like hyphae with no spores and reduced growth rate, and the recovery of the wild-type
allelefluPresultedinnomial hyphaewithnormal sporesandnonnalgrowthratesupportingthe
proposed fianction of fluP in A. parasiticus.

Other evidence which supports the association of fluP with hyphal cell development and
sporulation is derived form the comparison of fluP transcript level in liquid and solid
media. Normally, sporulation occurs only on solid media. Therefore, if fluP is associated with
sponflafionflreconespondhgnansaiptlevdonsofidmediamaybeerpectedtoappearin
greaterabtmdancethanthatinliquidmedia. Atthetwotimepointsanalyzed,thetranscripts
fiomsolidmediadidappearingreaterabundancefllanthoseinfiqtndmedia However,the
factthatfluPisexpressedinliquidmediamayalsomggeststhattlfisgeneisassociatedwith
growth in general.

Thepresenceorabsenceofthem’aD selectablemarkerhadnoobservableinfluenceonthe
growth rate ofthefluP—disrupted transformant on YES or CAM. When hyphae, derived from
SU-l (niaDYfluP‘) and the niaD mutant NR-3 (m'aDlfluF) of A. parasiticus were inoculated
onto YES or CAM, there was no observable differences in growth rate between the two
strains. ItislikelythatbecauseCAMandYES containmanydifl‘erentaltemativenitrogen
sources, the two strains did not have to use nitrate reductase (encoded by niaD) for normal
growth. However, when hyphae derived fi‘om SU-l (muDYfluP‘) and the fluP-disrupted
transformant (m’aD‘lfluP) of A. parasiticus were inoculated onto YES or CAM, there was a

great observable difl'erence in growth rate between the two strains. Therefore, the reduced

186

growth rate observed for the fluP-disrupted transformant (niaDY/IuP) on nonselective YES or
CAM is the result of disruption of the fluP gene.

The pattern of expression of the fluP gene and the B-tubulin gene were different during
sporulation. Microtubules are one of the 3 types of filamentous structures (cytoskeleton)
found in all enkaryotic cells (the other two are microfilarnents and intermediate filaments).
In filamentous firngi, not only are microtubules necessary for the formation of
cytoskeleton in general, but also involved in the formation of the mitotic spindle during
mitosis. Microtubules consist of or-tubulin and B-tubulin. May et al. (1987) May and
Morris (1988) suggested that B-tubulin participates in conidial development in Aspergillus
nidulans by demonstrating that the expression of B-tubulin gene was developmentally
regulated and directly correlated with the appearance of conidiating cell types. A large
increase occurred in the rate of B-tubulin synthesis and there was an accumulation of the
corresponding mRNA biosynthesis during sporulation in the filamentous firngus
Blastocladiella emersonii (Da Silva and Juliani, 1988). Temperli et al. (1990)
demonstrated that cytoplasmic microtubules arise from a nucleus-associated center (their
density was highest near the nucleus) and extend into the proximal cytoplasm during
mitosis by in situ immunolocalization using anti-B-tubulin monoclonal antibody. These
data suggest that the increased accumulation of B-tubulin during sporulation is the result
of the increased activity of mitosis in which B—tubulin is involved in the formation of the
mitotic spindle.

As mentioned above, fluP is also thought to be associated with sporulation in A.

parasiticus. A comparison of the transcript level between the B-tubulin gene and fluP may

187

reveal more information about the function of fluP. For example, in liquid media, the
accumulation ofthetranscript ofthe B—tubulingene decreased fi'om 24 to 72 h ofincubation,
wlfiledm'hrgthesmnefimepaiodflreacanmflafionoffluPUanscfiptmcreased Thereappear
to be at least two reasonable explanations for this observation. First, this experiment was
conducted in liquid media and sporulation was inhibited. Therefore the requirement for a large
ammefB-MmaymtbenecessmybemuseB-Maasasflnhrfldingmfitofmnofic
spindleduringsporulation (notasasignal). Secondandmoreimportantly,notonlymayfluP
(nuyencodeaéngbeassodMedwhhsponflafithdsonmybeassodfiedwithotha
biologicalprocessesintheproducingfimgus. 'I'hereforeeventhoughsporulationisinhibited
otherbiologicalprocessesstillneedtheexpressionoffluP. ThesedatasuggestthatfluP
firnctions at an early stage of vegetative cell difl‘erentiation.

The fluP gene isolated from A. parasiticus is firnctionally difl‘erent fi'om brIA, fluG, and
flbA in Aspergillus nidulans. The developmental regulatory gene brIA in A. m'dukms is likely
involved in controlling the switch from growth to development (Clutterbuck, 1969; Boylan er
al., 1987; Lee and Admas, 1994b). But DNA sequence analysis predicted that brIA
encodes a protein similar to a number of plant phytochromes (Grifith et al., 1994) which
are very different fi'om polyketide synthase. Lee and Adams (1994a) described the fluG
gene which is required for the activation of brlA expression and conidiophore development.
TheFlquroteinispresentatnearlyconstantlevelsthroughouttheA. m’dulcmslifecycle.
Sequence analysis of fluG predicted that a 864-codon fluG open reading frame shares
28% identity with prokaryotic glutamine synthetase I (Lee and Adams, 1994a). The M

meducesflufiyhyphaewimmsporeslheflqumphmotypeiswppressedwhm

188

M mutant colonies are grown adjacent to colonies ofthe wild-type strain even ifthe two
shainsareseparatedbydialysismembranewitha6—8kDacutofi‘(Adams etal, 1992).
Another flufi‘y gene, flbA was found to be required in conidiophore developmart prior to
activation of brIA. A 3.0-kb mRNA corresponding to flbA is present at low levels throughout
the A. m’dukms asexual life cycle (Lee and Adams, 1994b). flbA encodes an Aspergillus
nidrdam' regulator of G protein signaling (RGS) domain protein (Y u at al., 1996). The
phamtypicdefeasoftheflbAddefionnmtamscouldbepmfiaflyranediedbygmwth onhigh
osmolarity medium (e.g. 0.8 M NaCl) (Lee and Adams, 1994b). However, the fluP-disrupted
phenotype of A. paran’ticus could not be suppressed whar being grown next to the wild-type
orberemediedbygrowthonhighosmolaritymedium. ThereforefluPappearstobedifl‘erent
fiom brIA, fluG, and flbA in Aspergillus nichdans with regard to their association with
sporulation suggesting that fluP may be a new class of genes involved in fungal
development.
'IheproteinerrcodedbyfluPirrprasiticusislikelyaPKSandthecorrespondingend
product is likely a polyketide. Beck et a1. (1990) cloned the MSAS gene together with its
flanking sequences (total 7131 bp) fi'om Penicilliwn patulum. Vifrthin this sequence the
MSAS gene was identified as a 5322-bp long open reading flame. The transcript of fluP
is about 6.0 kb in length which is similar in size to the transcript derived from the MSAS
gene in P. patulum. A comparison of the deduced amino acid sequence of the 680—bp
DNA fiagment of the fluP gene showed a great deal of similarity with B—ketoacyl synthase

firnction domain of MSAS gene and other PKS genes.

189

The disrupted-fluP allele is not stable. Twenty percent of the colonies derived from cotton-
like hyphae “reverted” to the wild genotype and phenotype suggesting that the disrupted
fluP allele was genetically unstable. It is possible that reversion to the wild-type fluP allele
couldoccurbycertaingeneticmechanisms(suchasasimpledeletionoftheinsertedDNA
fiagment containing m'aD). It is also possible that the transferred hyphae were contaminated
with hyphae and/or spores which contained wild-type nuclei although the data would
argue that this scenario is unlikely. For example, if the transferred hyphae were
contaminated with hyphae and/or spores which contained wild-type nuclei, the initial
colony would predominately consist of wild-type hyphae with wild-type spores because of
the faster growth rate of the wild-type hyphae. Similarly, the possibility that the
transferred hyphae were heterokaryons is also not consistent with the data.
Heterokaryons may exist immediately afler transformation, but serial nrycelial transferring
should reduce the possrbility of a heterokarybn significantly. In- the unlikely event that a
heterokaryon survived serial transfer, the colony which originated fi'om a heterokaryon
would have normal hyphae and spores at the center of the colony. The normal hyphae
(with spores) would continue to appear during colony growth. The cotton-like flufiy
mycelium would only form a sector. The actual morphology of the colony observed
suggests that neither of these possibilities is likely to be true. Therefore, the most
reasonable explanation for the genetic instability of the knockout fluP appears to be
genetic recombination within the fluP—disrupted allele to restore the wild-type allele.

CAM and PDA had difi‘erent influences on the recovery of the wild-typefluP phenotype in

the fluP-disrupted transformant. When hyphae of the fluP-disrupted transformant were

190

transferred onto CAM, 10% of the resulting colonies recovered the wild-type morphology and
contained only the wild-typefluP allele WP); 20% gew as a mixture ofnomral hyphae plus
cotton-like hyphae and contained the wild-type fluP allele and the disrupted fluP allele
(fluP‘lfluP); 70% gew with only the cotton-like hyphae and contained only the disrupted fluP
allele(fluP). Incontrast,whenhyphae ofthefluP-disrupted transformantweretransferred
ontopotato dextroseagar(PDA), onlyonetypeofcolonyappeared. Such coloniesgewwith
nmchlessaerialhyphaeandhadwidelydispersed flquporea IhaeforethefluPallelein
disrupted transformants could “revert” to the wild-typefluP allele on either CAM or PDA, but
in difl‘erent patterns.

The fluP gene does not appear to be directly involved in aflatoxin biosynthesis. In liquid
media (Reddy’s medium), the fluP transcript was detected in the 24-h sample while the
transaiptofwr-lA(aswellasothergenesintheAFBr pathway), whichisdirectlyinvolvedin
aflatoxin biosynthesis, was not detected in the 24-h sample (but detected in the 60-h sample).
Thisdifi‘erencesuggeststhattheflngeneisnotdirectlyinvolvedinaflatoxinbiosynthesis.

The decrease in AFBr accumulation of the fluP-disrupted transformant gown on YES
agar could result from lack of sporulation and reduced gowth rate (decreased cell mass).
Even though the overall AFB1 accumulation (per Petri dish) of the wild-type was
approrn'mately lO-fold higher than that of the fluP—disrupted transformant, the
accumulation of AFB1 per mg dry weight in the central region after 2 days incubation
(which lacked sporulation) was essentially the same as the accumulation in any region of
the fluP-disrupted transformant. In the wild-type, AFB1 accumulation per gam dry

weight increased quickly only when sporulation appeared. Conidiophores and developing

191

conidia have been found to contain the highest concentration of Nor-1 protein (Chapter 4)
and Ver-lA protein in a gowing colony of wild type on solid medium (Liang and Linz,
unpublished data). These two proteins are directly involved in AFB1 biosynthesis.
Therefore, fluP may directly influence vegetative cell differentiation and sporulation,
which in turn influences aflatoxin biosynthesis in A. parasiticus.

Future studies onfluPirrAparasiticusmayinclude: (a) acompleteanalysisofthe
mdeofidesequenceofflqududingthededucedamumacidsequammdcompmison
bawearflqudotherPKngeshNohedinhyphalgowthmdsponflafiminoflrafimgm
narrow down the possrble product of fluP; (b) identification of the protein product of M; and
(c) a study of the mechanism ofthe regulation of expression offluP. These studies may
provide useful information for the development of new firngicides, improved agonomic
practices, resistant crops (by genetic engineering or classical breeding), and biocontrol
agents (such as nontoxigenic competitive fimgi) for control of aflatoxin contamination.

In summary, disruption of fluP resulted in slow gowth with cotton-like hyphae, no
sporulation, and reduced aflatoxin accumulation. The data suggest that fluP is associated
with vegetative cell gowth, sporulation, and in turn indirectly reduces aflatoxin

accumulation in A. parasiticras.

CONCLUSIONS

The data presented in this dissertation provided a further understanding of the mechanisms
of regulation of aflatoxin biosynthesis and the general biology at the molecular level in the
toxigenic filamentous fimgus Aspergillus parmiticus. These information may lead to the
development of new fungicides, the improvement of agonomic practices, the development
of resistant crops (by genetic engineering or classical breeding), and the development of
biocontrol agents (such as nontoxigenic competitive fungi ) for control of aflatoxin
contamination.

'I'hedataconfinnedthattheNor-lcprotein, derivedfromthenor-l cDNA, isakcto—
reductase which converts the AFB1 pathway intermediate norsolorinic acid (NA) to averantin
(AVN) in the presence of NADPH plus a factor(s) obtained fiom Eschreichia loci DHSor. The
dataalso arggestthat anunidentified cofactor(s) mayalsobenecessaryforthenativeNor—l
proteinfimctionandthatthenor-l geneisinvolvedinonlyonebranchoftheproposed
alternative pathways for conversion ofNA to averufin (AVF).

Polyclonal antibodies (PAb) raised against the Nor-1c protein were proven to be usefirl
to monitor the accumulation of the native Nor-l protein. A nor-l/GUS reporter construct
was also used to monitor the activity of the nor-1 promoter in A. parasiticus. The data
liquid media, and to temporal and spatial regulation on solid media; and that the activity of

the nor-l promoter is subjected to temporal and spatial regulation on solid media.

192

193

The data obtained by cell fractionation/Western blot analysis and in situ immrmolocalization
suggest that the Nor-l protein is mainly localized in the cytosol of the fungal vegetative cell;
the highest level of the Nor-l protein is associated with the developing irmnature conidium,
high levels are associated with the conidiophore, lower levels are associated with the vegetative
hyphalcelLandnoneisassociatedwiththematureconidium. Indicatedbyanor-l/GUS
reporter construct, the localization of the activity of the nor-l promoter follows a temporal and
spafialpatterninafugalcolony. Thedataalso arggestthatthelocalizationofNor-l protein
accunmlationandthelocalizationofnor-l promoteractivityarecorrelatedtotheprocessof
sporulation in A. parasiticus.

Disruption of fluP, a putative polyketide synthase gene, resulted in reduced gowth
rate, flufi‘y-mycelium (cotton-like hyphae), no sporulation, and reduced aflatoxin
accumulation suggesting that fluP is associated with vegetative cell gowth, sporulation,

and in turn indirectly influences aflatoxin synthesis in A. pwasiticus.

APPENDICES

APPENDIX A

APPENDIX A

CLONING OF THREE EeoRI DNA FRAGMENTS FROM COSMID Nor-A AND
ANALYSISOFTHEIRTRANSCRIPTS

Three difl‘erent but adjacent EcoRI DNA fi'agments (in the order of 2.8-kb, 4.0-kb and 4.6-
kb) fi‘om an aflatoxin gene cluster (cloned in cosmid Nor-A, Figure 1-23) in Aspergillus
pwasiticushavebeentriedtobeclonedandmapped. Onlytwo(the2.8-kbEcoRIfiagnent,
andflre4.0-kbEcoRIfiagnem)offlremweresuccessfifllyclonedandmapped Butthethird
one(a4.6-kbEcoRIfiagment)wasunabletobeclonedforunknownreasoninatleastfive
roundsofexperiments.

In Northern blot analysis, a 7.5-kb transcript was detected using the 2.8-kb anRI DNA
fragmentasaprobe,anda7.5-kbtranscriptplusa6.5-kbfianscriptweredetectedusingthe
4.0-kbEmRIDNAfiagnemasapmbeinApmusifiarsgowninanaflatordn-indudng
medium. Theacanmrlafionsofthetwouanscriptshadthesamepattemasthatofthe
accrrnudafionsofmr—larrdver-lfiarrscriptsinApm'asiticus. Thesedatasuggestthatthetwo

transcripts were related to or involved in aflatoxin biosynthesis.

194

APPENDIX B

APPENDIX B

IDENTIFICATION OF VERSICONAL CY CLASE GENE

Isolation and identification of the gene encoding versiconal cyclase (converting versiconal to
versicolorinBintheAFBrpathway)havebeentriedforaperiodoftime. Thisexperimentwas
startedwithaknownaminoacidsequenceofversiconalcyclase. Amixtureofl8-mer
mrcleotidesandambrtureofl7-mermrcleotideswerepreparedaccordingtoapartofthe
aminoacidsequenceofversiconalcyclase. Boththe18mer'sandthel7merswereusedas
probestoprobetheAFBrgeneclusterclonedincosmidNor-A. A400-prcoRI/X7ral
fiagment(ina4.0-kbEcoRIDNAfiagment)incosmidNor—Awasfoundtohybridizethe18-
merprobesinSouthernblotanalysis. This400—bpfiagnentwassequenced. Sequencedata
slwwedmaflfisfiagnanwmainedasirmursequaweasomofthe18maprobesThedaa

suggest the gene encoding versiconal cyclase was not included in cosmid Nor-A.

195

APPENDIX C

APPENDIX C

A MODIFIED PROCEDURE FOR CRUDE EXTRACT PREPARATION FROM
FUN GAL CELLS
CertainProteinsinvolvedinAFBrbiosymhesisareenremelyunstable. Thereforethe
qualityofcrudeermactfiomtoxigenicfimgalcellsisumallyone ofthemostimportantfirctors
whichinfluenceflreremltsofenzymaficassayorWesternblotanalysis
Grinding firngal cells under liquid nitrogen followed by addition of extraction bufl‘er
containing protease inhibitors is a common protocol used by many researchers. Two ofthe
commonly used protease inhibitors are phenol methylsulfonyl fluoride (PMSF) and
aprotinin. PMSF is quickly inactivated in the aqueous solution, and aprotinin can be
precipitated at low temperature. Grinding fungal cells usually cannot destroy all
organelles and most proteases are enclosed in organelles. Therefore, the procedure
described above cannot get all proteases inhibited. The crude extract prepared in this way
is stored in the freezer (at -80°C) and later is taken out and thawed for use. The freeze-
and-thaw procedure will destroy the intact organelles and proteases inside will be released.
This phenomenon has been ignored in most of published literature. To get a better result
of enzymatic assay or Western blot analysis, protease inhibitors can be added again afier

each fieeze-and-thaw step as soon as possible.

196

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